Egg-handling procedures

Collection When spawning occurs, eggs are collected from the overflow egg collector tank using a fine (400 m mesh) net (Figure 12). The fertilised eggs of grouper are non-adhesive and pelagic, and range from 0.8 to 0.9 mm in diameter.

Grouper eggs are sensitive to handling during the early developmental stages, and should only be removed from the collection nets once the embryo has developed optic vesicles, i.e. at the eyed stage (see Figure 15) (Caberoy and Quinitio 1998). Handling eggs before this stage will result in increased mortality and a higher incidence of deformities (Caberoy and Quinitio 1998 Disinfection To minimise the chances of vertical transmission of VNN, fertilised eggs of many marine finfish species are treated with ozone (Battaglene and Morehead 2006; Buchan et al. 2006) (see Box 2), and this treatment is also recommended for grouper eggs (Liao et al. 2001). Grouper eggs should be subjected to a concentration exposure time (CT) score of around 1.0 (Su et al. 2001)this means they should be treated with ozone at a concentration of 1 mg/L for 1 minute or equivalent (e.g. 0.8 mg/L for 1.25 minutes).

Incubation After washing in ozone-treated water, the eggs are rinsed with clean, disinfected (using ozone) sea water (Figure 13) and, for incubation, transferred into 0.51.0 m3 tanks with aerated sea water. Only floating eggs are used for larval rearing because these are more likely to be fertilised than sinking eggs, which are unfertilised or dead. Unfertilised eggs settle to the bottom of the broodstock tank and are removed by siphoning. If any unfertilised eggs are transferred to the incubation tanks, they should be siphoned out and discarded to prevent water-quality degradation.

Recommended values for conditions in the incubation tank are listed in Table 4.
Recommended values for physico-chemical parameters for incubation of tiger grouper eggs. Note that there is very little information available on the tolerances of grouper larvae to various environmental parameters.

Recommended Temperature Salinity Stocking density* Aeration*

Refers to other Epinephelus species

Reference Toledo et al. (2004) Toledo et al. (2004)

2830 C 3234 ppt 400 eggs/L 100 mL/min

Qualitative evaluation of the eggs Egg quality in marine finfish is generally evaluated using both qualitative and quantitative methods.

Fertilised eggs (Figure 14) are examined under a microscope (10 or 20 magnification is sufficient) for the following: > eggs should be regular in shape > during the early stages of embryonic development, the individual cells should be regular in size > eggs and embryos should be completely transparent, with no dark areas > chorions (eggshells) should be free of any parasites or fouling organisms.

If there is only a low proportion of eggs that are irregularly shaped, dark or with aberrant embryonic development, the eggs can be used in the hatchery because it is likely that the poor-quality larvae will simply die during the larval-rearing procedure. If there is a high proportion of eggs exhibiting abnormal characteristics (>10%), the batch should be discarded. If the eggs have parasites or fouling organisms, the batch should be discarded because of the probability that they will transfer pathogens to the hatchery. Following disposal, all tanks and equipment used should be cleaned and disinfected (see Appendix 1 for a list of disinfection procedures

Fertilised eggs of grouper have a well-developed larva curled around the yolk (e.g. centre and upper right). Unfertilised eggs have no visible larva (e.g. centre left and upper left

Quantitative estimation of fertilisation and hatching rates

Fertilisation and hatching rates are also used as indicators of egg quality. For grouper, both fertilisation and hatching rates should be higher than 50%, and preferably higher than 80%. Fish larvae from batches of eggs low survival and a high incidence of deformities and other health problems. Such batches are usually discarded. Records of fertilisation and hatching rates should be kept to allow an evaluation of broodstock performance and spawning success, particularly on an annual basis.

Stocking larval tanks Generally, grouper eggs are stocked at the eyed stage before hatching (Figure 15) because at this stage they are more robust than the newly hatched larvae. Newly hatched larvae (Figure 16) are very sensitive to physical shock or changes in water quality, and moving them to the larval- rearing tanks may result in high levels of mortality. Because the hatching rate is not known before the eggs are stocked in the larval-rearing tanks, the number of eggs to be stocked needs to be estimated using historical hatching rates for that hatchery. Accurate estimates of the number of larvae stocked can be back-calculated using data from the actual batch stocked, as described above. If hatching rates are low, the larvae in the larval- rearing tanks should be discarded, and the tanks cleaned and disinfected (Appendix 1

Larval-rearing procedures

Larval-rearing tanks At RIM Gondol, both round and rectangular tanks are used for larval rearing. For rectangular tanks, the corners of the tank should be rounded to avoid larval aggregation in the tank corners. The preferred size of larval- rearing tanks is about 10 m3 in volume with a depth of 1.2 m. Based on experience in rearing grouper larvae of several species at RIM Gondol, the preferred colour for larval-rearing tanks is bright yellow or pale blue (Figure 17). These colours allow the grouper larvae to discriminate prey (such as rotifers and brine shrimp) more easily, and make tank management, particularly cleaning, easier.

Aeration should be provided in a grid pattern to ensure even mixing of the water in the tanks and to ensure dissolved oxygen levels are maintained throughout the tank. Airstones should be placed in each corner of the tank to prevent stagnation. Aeration should be light during the early stages of larval rearing, to avoid physically damaging the larvae. It can be increased during the larval-rearing cycle, as the larvae become more robust.

Water to the larval-rearing tanks should, as a minimum requirement, be filtered through a sand filter (Figure 18). More complex filtration and water treatment systems, such as ultraviolet or ozone disinfection and cartridge filters, will help maintain biosecurity in the hatchery. Larval-rearing tanks should at least be roofed to avoid direct sunlight and rain. Better still is to enclose the larval-rearing tanks in a buildingthis will help maintain optimal water temperature, reduce diurnal fluctuations in water temperature and facilitate biosecurity.

The larval-rearing tanks should be maintained as a separate quarantined area within the hatchery, with entry only to authorised persons, hand and foot washes on entry and exit, and disinfection of all equipment before use. Following each production cycle, all tanks and equipment (such as nets, feeding buckets, airstones and airlines etc.) should be cleaned and disinfected. Details of disinfection procedures are provided in Appendix 1 Larval development The newly hatched larvae of tiger grouper measure 1.41.7 mm TL. The larval development of tiger grouper is shown in Figure 19. The mouth opens 23 days after hatching (DAH) and the yolk is completely absorbed by 45 DAH. By 1030 DAH, most tiger grouper larvae have the elongated dorsal and pelvic spines typical of larval serranids. When the larvae are reared at high

density, they often become entangled with each other via these spines. This can lead to high mortality between 10 and 30 DAH. Tiger grouper larvae show drastic changes in their shape as they grow from the newly hatched larva to the juvenile stage (Figure 19). A detailed description of the larval morphological development of tiger grouper can be found in Kohno et al. (1993). Until the larvae have completed metamorphosis to the juvenile stage, they are very sensitive to environmental conditions and substantial mortality can occur due to apparently minor stresses. Tiger grouper metamorphose to juvenile at about 4045 DAH (Figure 19), although this can be delayed due to low water temperatures or poor nutrition. Because of the sensitivity of larvae, careful management is required throughout the larval-rearing phase.

Rearing the larvae Important points to remember when rearing larvae are listed in Box 6. The sea water used in larvalrearing tanks should be pre-treated using a sand filter to remove particulates, and then sterilised using ozone (see above) or chlorine (see Appendix 1) to reduce the potential for pathogen introduction in the water supply. Recommended initial stocking density for tiger grouper is 10 larvae/L.

As discussed further below, oil can be added to form a thin film on the water surface (around 0.2 mL/m2) at 15 DAH to prevent surface aggregation mortality in early-stage grouper larvae. Live foods used for larval rearing comprise microalgae (Nannochloropsis sp.), super-small (SS-type, 60 100 m) and small (S-type, 120180 m) rotifers (Brachionus rotundiformis) and brine shrimp (Artemia) nauplii. Artificial diets are introduced before feeding Artemia nauplii. The larval- rearing protocol is summarised in Figure 20. Note that the protocol described here and summarised in Figure 20 is a guide only, and specific hatchery protocols will depend on a wide range of factors including operator preferences and experience. Production of live food is not covered in this publicationwe recommend Manual on the production and use of live food for aquaculture by Lavens and Sorgeloos (1996) for a thorough guide to this topic.

Microalgae (usually Nannochloropsis) are introduced to the larval-rearing tanks 2 DAH, i.e. 2 days after stocking the larvae. The algal cell density is maintained at 300500 103 cells/mL. SS-type rotifers are introduced at 2 DAH (afternoon) when the larvae have partly absorbed their yolk. The SS-type rotifer density in the larval-rearing tanks should be maintained at 57 individuals/mL during 25 DAH. Following the period of feeding with SS-type rotifers, small (S-type) rotifers are fed at a density of 810 individuals/mL from 6 to 10 DAH, increasing to around 15 individuals/mL from 11 to 30 DAH. Rotifer density gradually decreases as

the rate of rotifer consumption by the larvae increases and eventually rotifers disappear by around 30 DAH.

The use of calanoid copepods as live feed during the early larval rearing of groupers has been shown to improve larval growth and survival (Doi et al. 1997; Toledo et al. 1997, 1999) and larval grouper will actively select copepod nauplii in preference to rotifers (Toledo et al. 1997), suggesting that copepods are a more acceptable and nutritional prey than rotifers. However, copepods are not widely used in commercial hatcheries and, while their application to larval rearing holds much promise, there is still considerable research and development required before they can be reliably produced and used in hatcheries (McKinnon et al. 2003).

From 9 DAH, small-size commercially formulated diet with a particle size of 200400 m is used. The formulated feed is sprinkled onto the surface of the water in small amounts frequently (as often as hourly) throughout the day. Only small amounts of feed are added such that the feed is consumed within 5 or 10 minutes; excess feed should not be allowed to accumulate on the bottom of the tank where it will decompose and degrade water quality. The feed size is increased to 400800 m from 3045 DAH. Only high-quality microdiets specifically formulated for marine finfish should be used and these should be stored in a refrigerator or freezer to maintain their quality.

From 16 to around 40 DAH, Artemia are fed at a density of 0.20.5 individuals/mL. As noted below, the Artemia should be supplemented with a commercial enhancement product that will increase the levels of essential fatty acids.

Larval-rearing tanks are maintained statically until 7 DAH. Initially, water exchange is limited to only about 10%/day (712 DAH) to avoid sudden changes in water quality, increasing to 20%/day when both artificial diets and Artemia are being fed (1324 DAH). From about 12 DAH, faeces, dead larvae and uneaten food accumulating on the bottom of the tank are siphoned out at least once daily to maintain water quality. Initially, only one-quarter of the tank bottom is siphoned each day and this is gradually increased until the whole tank is siphoned daily. Water exchange increases to around 50%/day from 25 DAH, then to a slow flow-through equivalent to around 100%/day from about 30 DAH.

Towards the end of the larval-rearing cycle, the metamorphosed juveniles should be fully weaned to pellets. This is particularly important if the fingerlings are destined for nurseries or farms using pellet feed, to reduce mortality associated with weaning fish from wet diets (e.g. trash fish) topellets. This requires frequent feeding of small amounts of pellets during daylight hours. To reduce labour, belt feeder can be used to deliver the feed either as a constant stream or in small batches (Figure 21).

Recommended larval-rearing conditions for tiger grouper are listed in Table 5. It is important to regularly measure water quality in the larval- rearing tanks. If water quality degrades, it may be necessary to replace the water at rates higher than the rates recommended above. However, the water used should be of similar temperature and salinity to the water in the rearing tanks to avoid stressing the larvae. It is also important that records are kept of water quality, feeding and other management aspects of the hatchery. Some examples of data sheets to keep such records are given in Appendix 2.

Nutritional enhancement of live foods Larvae of the closely related green or greasy grouper (E. coioides) require high levels of the highly fatty unsaturated fatty acids eicosapentaenoic acid (EPA, or 20:5n-3), arachidonic acid (ARA, or 20:4n-6) and docosahexaenoic acid (DHA, or 22:6n-3) for proper development, and provision of these fatty acids in the diet, via incorporation in the live foods used for larval rearing, improves survival, growth and pigmentation of the larvae and fingerlings (Alava et al. 2004). Various commercial preparations have been developed for nutritional enhancement of rotifers and brine shrimp (Alava et al. 2004). Because groupers have a very high requirement for DHA, we recommend the use of products high in DHA, particularly for Artemia because Artemia retroconvert long-chain fatty acids to shorter-chain fatty acids, thus lowering the levels of the essential fatty acids such as DHA. These nutritional supplementation products are packaged as liquid or spray-dried products. Generally, preparation involves measuring the required quantity, blending either to hydrate (for spray-dried products) or emulsify (for liquid products) the material, then applying to the live-food culture tanks. The manufacturers provide technical information on the application of their products. Of particular importance is the need to maintain high dissolved oxygen levels in the culture tanks during the application period (usually <12 hours). This may require the use of pure oxygen, or oxygen-supplemented air, particularly if the live-food organisms are at high density.

Days after hatching Feed management Microalgae* SS-rotifer (57 ind/mL) S-rotifer (810 ind/mL) S-rotifer (15 ind/mL) Artemia (0.20.5 ind/mL) Artificial diet

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

200400 m

 400800 m Water management Water exchange 10%/day 20%/day 50%/day Flow-through (100%/day) Siphoning