Overview Breeding And Seed Production

Breeding and Seed Production for Aquaculture in SE Asia Evelyn Grace T. de Jesus-Ayson SEAFDEC AQD

Constraints to Sustainable Aquaculture Development in SE Asia Availability of technology Reliable supply of good quality seeds Availability of suitable food organisms/feeds Diseases Availability of trained technical manpower

Marine Fish Breeding and Seed Production

Milkfish Groupers Snappers Sea bass Rabbitfish Age at sexual 5 (M/F) 3 (F) 4 (M) 2.5 (M) 10 mos (M) maturity >5 (M) 5 (F) 4 (F) 1 yr (F) Size at sexual > 3 kg 2-3 kg (F) 2.5 kg (M) 1.5 kg (M) > 200 g maturity > 6 kg (M) 4 kg (F) 3 kg (F) Spawning March-Nov year round Apr-Nov April-Oct year round season Sexes separate protogynous separate protandrous separate hermaphrodite hermaphrodite Egg pelagic pelagic pelagic pelagic demersal characteristics adhesive

Acquisition of breeders collection from the wild growing wild fry to broodstock in captivity hatchery-reared

Broodstock facilities Tanks with recirculating water system Canvass tanks with flow-through water

broodstock feeds Milkfish & Siganid broodstock feeds For groupers, red snapper and sea bass * trash fish/squid * formulated feed * combination

When feeding with trash fish: * Remove head: potential source of VNN * Consider supplementation with essential fatty acids (fish oils) and/or vitamin mixes

sex determination * Apply gentle pressure on the abdomen and check for the presence of milt or check for the presence of oocytes by using a fine PE tube

Spawning Natural spawning Induced spawning Luteinizing Hormone-Releasing Hormone analog (LHRH-A) or Human Chorionic Gonadotropin (HCG)

Induced Spawning LHRH-a injection or implants (100  g/kg BW) HCG injection Single dose of 1000 IU/kg BW or 2 injections of 500 IU/kg BW given 24 hours apart or 1 st injection of 1000 IU/kg BW and 2 nd injection of 500 IU/kg BW given after 24 hours

Egg collection spawning normally occurs at night and eggs are collected the following morning Wash eggs and remove debris Incubation Get sub-samples and determine fertilization rate

Egg collection Incubation and hatching Larval rearing Harvesting

Strategies in developing seed production practices understanding the reproductive biology of the species development of broodstock management strategies improvement of egg and larval quality determining the nutritional requirements of the larvae determining physiological requirements of the larvae

Timing of events related to the onset of feeding milkfish sea bass rabbitfish grouper total eye pigmentation (h) opening of mouth (h) complete yolk resorption (h) oil globule resorption (h) time available to initiate feeding (h) mouth width at opening (  m) 54 32 32 55* 54 32 36 55 120 60 70 75 120 72 90 66 88 36 35 500 250 125 180

endogenous feeding period larvae are dependent on their yolk reserves as source of energy yolk oil globule red snapper yolk oil globule sea bass yolk grouper milkfish yolk

day 1 within 24 hours from hatching: grouper no functional eyes mouth still closed stomach poorly developed yolk Newly-hatched larva

mixed feeding period larvae survive on yolk reserves and external food yolk oil globule rabbitfish grouper yolk oil globule critical period: between 55-90 hours transition from endogenous to exogenous feeding

exogenous feeding period larvae are dependent solely on external food grouper day 4 day 2 grouper

What to feed ? first feeding period: considerations size of food density of food Chlorella rotifer * small-sized or screened rotifers are given as initial food; the green microalgae serve both as food for the rotifers and water conditioner Chlorella culture Rotifer

What to feed ? * Copepod nauplii may also be introduced at first feeding * Adult copepods may be seeded into the larval rearing tank a few days before stocking the larvae * Mass production of copepods is difficult copepods

Criteria in choosing a suitable food easily perceived by the larvae of appropriate size to fit the mouth size of the larvae easily digested by the larvae satisfies the nutrient requirement available in large quantities

As the larvae grow: grouper day 4 Other food items are given including: * copepods * mysids * brine shrimps * trash fish * formulated feeds

Considerations for artificial diet particle size physical performance in water attractability digestibility nutritional composition cost effectiveness

Larval feeds Milkfish: before day 15 day 15 up Grouper: graded sizes

live food vs formulated diet deficit of essential substances in formulated diets dry diets are susceptible to leaching – loss of nutrients zooplankton remain suspended in water column

days of culture 0 10 20 30 40 50 60 Protocol for rearing grouper larvae feeding management Nannochlorum (1-3 x 10 5 cells/ml) rotifers (ind/ml) artificial diet Artemia (1-3 ind/ml) water management siphoning of tank bottom 20-30 % 50-70 % flow-through 3 10 20 1 2 3-5g/ton/day

days of culture 0 5 10 15 20 Protocol for rearing milkfish larvae feeding management Nannochlorum sp . (1-3 x 10 5 cells/ml) rotifers (ind/ml) water management siphoning of tank bottom water change 10-20 % 50 % 1-2 g/ton/day larval diet 2-3 10 10-20 20-40 %

Freshwater Fish Breeding and Seed Production

Easy to culture (ideal for most systems) Simple hatchery, nursery technology Wide consumer acceptance (Asia, Europe, N.& S. America) Used to control luminous bacteria in shrimp ponds Disease resistant Why Tilapia?

Investment opportunities in tilapia: Hatchery short turn-over (1.5 - 2 months) small farm area requirement more investment options (high,medium, small) low risk less feed feed requirement greater control on prices wider market opportunities e.g. market for saline-tolerant tilapia, improved strains etc.

TILAPIA HATCHERY MGMT. Broodstock dev’t, selection Hatching Size grading Nursery rearing Marketing

BROODSTOCK SELECTION Criteria Growth Body shape Body thickness Color No deformities Feeding efficiency sexual maturity resistance to diseases social behavior

Hatchery Systems Concrete tanks Hapa in ponds Hapa in lakes Ponds (Direct)

Hapa in Pond Hatchery Medium scale investment Minimal water management low feed requirement - 2% of fish biomass Stock movement Frequent size-grading Ideal sex ratio 1M:4F or 1M:5F 1M:7F – 1M:10F OK but M more prone to spawning fatigue

Harvesting Methods egg collection fry collection 75% eggs 25% fry Egg collection: day 14 total harvest Fry collection day 10 onwards 75% fry 25% eggs

Hapa in Lake (or SWBs) Hatchery Low to medium scale investment Water quality – nature dependent Feeding is minimal (2% of the fish biomass) Production less sustainable than other systems Broodstock quality/unreliable (entry of unwanted species) Low cost of fingerlings

Tank Hatchery High level investment Greater control on water quality Cleaner environment for young fish Requires life support system Nursery ponds - an integral part of the system 100% dependence on artificial feeds Ease in moving stocks i.e, grading, culling etc. Highest fry output

Earthen Pond Hatchery Traditional low input minimal water management relatively low seed yield problems w/ cannibalism shorter broodstock use once a month total harvesting

Crustacean Breeding and Seed Production

Life Cycle Adult Estuary Eggs Nauplius Protozoea Mysis Postlarva Juvenile Hatchery phase

Wild-caught gravid female Wild-caught female Nauplii dealers Nauplii from domesticated stocks Source of Nauplii

Growth Molting Hatchery Operations Broodstock Management Procurement of wild spawner or broodstock 2. Stocking in tank (5 broodstock/ ton) 3. Feeding with pellets, trashfish, mussel meat, and annelids 4. Ablation (if necessary) after a week of holding

Induced Maturation : ablation (endocrine manipulation) After ablation: Restocking in tanks Regular sampling to separate gravid females Spawners placed in separate tanks

Spawning (8 PM- 4 AM) Nauplius (1.5-2 days) After eggs hatch to nauplius stage, they are stocked in larval tanks at a density of 80 to 100 nauplii/ liter

Hatchery Operations Feeding Diatoms*( or Tetraselmis ) STAGE: N I -N VI Z I Z II Z III M I M II M III PL 1-5 PL 10-15 5,000-10,000 cells/ml Feeding Scheme Protozoea Stages (3-5 days) Z I Z II Z III

Growth Molting Hatchery Operations Natural food production in the hatchery Algae that are frequently used as food for the larvae: Skeletonema Chaetoceros Tetraselmis

Hatchery Operations Feeding Diatoms*( or Tetraselmis ) Artificial Diet Artemia Egg custard * Skeletonema or Chaetoceros STAGE: N I -N VI Z I Z II Z III M I M II M III PL 1-5 PL 10-15 5,000-10,000 cells/ml 0.5 1 2- 5 ind/ml Feeding Scheme as recommended Mysis Postlarva

Mud crab production (tons) from aquaculture (FAO Fishery Statistics, 2004) Country 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Indonesia 1,906 1,339 5,176 866 5,143 5,126 3,879 9,039 7,152 2,243 Malaysia 623 381 277 231 188 225 219 311 204 174 Philippines 2,782 2,463 3,7 59 4,033 4,826 4,968 4,608 4,747 4,809 6,245 Singapore 342 353 215 45 78 86 75 93 104 60 Sri Lanka 1 2 1 - - - - - - - Thailand 45 132 115 19 9 9 5 10 10 10 Taiwan 1,526 797 430 796 381 315 423 239 226 239 Total 10,006 5,465 9,972 5,990 10,625 10,729 9,209 14,440 12,507 8,972 Export revenue in million Php P was valued at P1, 050 - 2003; P1,590 - 2004; P1,693 -2005) Tremendous growth underscores the importance of hatchery for seed supply. Major source of crablets for farming is from the wild.

Different kinds of alimango in the Philippines: *Hatchery technology is feasible for bulik * Scylla serrata (Giant mud crab – ‘kinis’ or ‘bulik’ ) Scylla tranquebarica (Purple mud crab – ‘lawodnon’) Scylla olivacea (Orange mud crab – ‘pulang alimango’)

Life cycle of mud crab Coastal water Open sea Estuary Mangrove forest Juvenile Sub-adult Adult mating Spawner Eggs Zoea 1 Zoea 2 Megalopa Crab instar hatching spawning Etq rev 030401 Zoea 3 Zoea 4 Zoea 5 HATCHERY PHASE

Broodstock sourcing BROODSTOCK MANAGEMENT Water change 2-3x / week > 500g ♀ with ‘aligi’ Stocking in tank Acclimation & disinfection Feeding

Incubation 300 or 500 L tank/female Disinfection Sampling for berried females 0.8 to 5 million zoeae per female (450-750 g BW) Stocking of zoeae in tanks flowing seawater Zoeae Collection of newly hatched zoeae

Feeding Rotifers Artemia Mussel & fish 0.5 – 1/ml satiation 2x daily Addition of Microalgae (optional) Z1 Z2 Z3 Z4 Z5 Megalopa Crab instar Stage Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 45 Activities LARVAL REARING Water Management Salinity Water replacement Prophylaxis 10-15/ml 100,000 cells/ml 30-60% 30-50 % 28-32 ppt 24-28 ppt

• mesh size 1 mm • area 20 m 2 or bigger • bamboo poles as support • net bottom buried 3-5 cm into the soil • 80- 100 cm deep Net cages Tanks • 80- 100 cm deep • area 40 m 2 or bigger megalopa 5-7 d + 3 weeks Fly size crabs (0.35-0.40 cm) (0.40 – 0.45 cm) (0.8-1.0 cm)

Abalone Breeding and Seed Production

Abalone - Supply/Demand Aquaculture Department 7,000 mt deficit

Aquaculture Department Diatom Culture tanks Dry Lab/ Stockroom Overhead Reservoir Sand Filter Box Seawater Pump Air Blower Settlement tanks Spawning tanks Incubation tanks Broodstock Holding tanks A Typical Abalone Hatchery Lay-out Seaweed tanks Primary nursery tanks Working area Worker’s room Intermediate nursery tanks

HATCHERY OPERATION BROODSTOCK Source – wild or hatchery-bred Size – 4-5 cm shell length (initial) Optimum size for spawning - 6-8 cm Sex ratio – 1 male: 4 females Spawning technique – spontaneous, group spawning Mature Gonads male female SEAWEED Gracilaria CONDITIONING CAGES Aquaculture Department

Female Male Group spawning female male Female Male Group spawning Aquaculture Department

Group Spawning Periodicity and Fecundity in Hatchery-bred Abalone 2,494 + 304 224,150 + 37,760 1-8 44 74 87 HB-2B/6 7,671 + 1,461 186,610 + 35,371 2-5 33 48 25 HB-2A/6 4,324 + 782 572,630 + 100,000 1-2 94 81 135 HB-1/7 Ave. # eggs per g BW Ave. # eggs per spawn Spawning Days interval frequency/ between month spawning SL BW cm g Group/ n Aquaculture Department

Creeping larvae ready for stocking Settlement tanks LARVAE PREPARATION FOR SETTLEMENT Benthic diatoms as larval food INCUBATORS Artificial illumination Aquaculture Department

IMPORTANT CONSIDERATIONS DURING LARVAL SETTLEMENT & PRIMARY NURSERY Combined diatom-crustose coralline algal films on settlement plates attract the highest numbers of post-larvae 2. Supplemental feeding using diatom slurries can improve survival rate 3. Continuous lighting to attract settling larvae to the upper water layer and to prevent respiration of diatoms that may cause oxygen depletion 4. Stocking density 250 - 300 veligers per liter 5. Static water condition no flow-thru of UV-treated rearing water within first 5 days; flow-thru filtered seawater on 6 th day from stocking 6. Mild aeration upon stocking then increase intensity from 5th day onward Diatom culture cca-diatom complex 40W DL Aquaculture Department

PRIMARY NURSERY REARING “ Clearing of diatoms on surfaces of settlement plate indicates the presence of settled and metamorphosed post-larvae...” Crustose coralline algae- diatom complex Indoor settlement tanks Outdoor nursery tanks 10 days Aquaculture Department

Mesh basket for Intermediate nursery INTERMEDIATE NURSERY REARING Early juveniles (5-8 mm) ready to feed on seaweeds Indoor tanks Outdoor nursery tanks Raceway-type Flow-through Aquaculture Department

ABALONE NURSERY IN SEA CAGES CAGE NURSERY REARING: Material: Amazon screen Size: 30cm high x 60cm-diam. Shelter surface area: 1.24m 2 Stocking density/cage: 1000-1,500 Food: Fresh seaweed Gracilaria Feeding rate: 40-50% of bw/day Rearing period: 90 days Final size at harvest: 28-30mm SL Survival: 90-95% Black net cover Wooden or Bamboo raft Aquaculture Department

Artificial Feeding in Abalone Juveniles Disadvantages: limited to tank culture water quality problems variable growth response Advantages: replacement for seaweeds provides complete nutrition easy to store/readily available produce shell-marking for stock enhancement activity Fed formulated diet Fed seaweed Aquaculture Department

Commonly used farming techniques Spider web (‘ambian’) Hanging long line (‘palabad’) Hanging long line (single) Multiple raft (‘alul’) Fixed off-bottom (‘parasdas’)

Regeneration of E. denticulatum from callus-like structures 1 2 3 4 5 Young plants grown in a nursery cage Outplanted plants in a cage 6 7

Plantlet regeneration of Kappaphycus alvarezii var. adik-adik 4b 4a 4c 5 6 7a 7b 1 3 2

Sporulation/Tissue Culture Wild source Slides/ Plates/ Cell wells 50mL t25 350 mL culture flask 2.0 L culture flask Aquaria Tanks Summary Research Hanging longline (inside cages) Outplanting (Cabalagnan) After Typhoon Frank 2008 2007 Cylindrical cages

GRACILARIA grown from spores Tissue culture of KAPPAPHYCUS Nursery in tanks Grow-out

Recommendations: Way Forward Continuous refinement of developed technologies Domestication of other potential species for aquaculture Determination of physiological requirements for optimum reproductive performance Determine physiological requirements of larvae/fry

Overview Breeding And Seed Production

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Overview Breeding And Seed Production