42 JUNE 2017 • WORLD AQUACULTURE • WWW.WAS.ORG treatments used during quarantine are presented in Table 2. Only fast-growing fish in every lot were selected for use as candidate broodstock. In many cases, if massive mortalities occurred in cages associated with VNN or Nodovirus infection, some surviving fish were also use as candidate broodstock because it was assumed that surviving fish were tolerant of both viruses. Two and three generations of grouper broodstock were held for further genetic improvement. Larval Rearing Most farmers use round and rectangular tanks with yellow tank walls for rearing larval of groupers, as recommended by Sugama et al. (2008). For rectangular tanks, corners should be rounded to avoid larval aggregation there. The size of larval rearing tanks are varied — 3×3, 3×4, 3×6 m — with a depth of 1.2 m. Larval rearing tanks are equipped with aeration and covered to avoid direct sunlight and rain. Water for larval rearing was disinfected with sodium hypochlorite and neutralized by sodium thiosulphate before stocking fertilized eggs. A protocol for larval rearing of groupers was proposed on the basis of previous studies at IMRAD-Gondol (Sugama et al. 2001, Sugama et al. 2016). Newly-hatched larvae (0-DAH) are stocked at 10-15 larvae/L. Live food is provided, including microalgae Nannochlorpsis sp. or Chlorella sp., zooplankton, Brachionus plicatilis or Brachiounus rotundiformis (SS type of 60-100 µm, S-type of 100-120 µm), copepod nauplii Acartia sp. and Artemia salina nauplii. Grouper larvae are weaned prior to feeding with Artemia nauplii. The larval rearing protocol is presented in Figure 1. Nannochloropsis are introduced to larval rearing tanks at as females then change to males at a later age (Sugama et al. 2001). Observations have been done to understand the quantity and quality of commercially produced fertilized eggs supplied to seed producers. The culture environment for grouper broodstock was maintained to allow a high level of fish welfare, with a maximum stocking density of 20 kg/m3. Broodstock of various species of grouper from ten farmers, each with 30-60 spawners that produced 1.54-4.28 kg of fertilized eggs in one month. Broodstock were kept in circular or rectangular tanks of 100-300 t water capacity. A flow-through system was applied by all farmers, with a water exchange rate of 200 percent per day. Broodstock were fed by mixed trash fish (mainly Sardinella sp. mixed with squid and 1 percent vitamin mix) at a ratio of 1-3 percent of fish body weight per day. Broodstock spawned naturally in tanks after midnight, starting from 0100 to 0400 hr and 3-5 days before and after the new moon phase. The hatching rate of fertilized eggs ranged from 65-93 percent and egg diameter ranged from 0.78-0.92 mm. Some fertilized eggs as a sample were taken for virus diagnosis using reverse transcription polymerase chain reaction (RT-PCR). Only eggs free of Virus Nervous Necroses (VNN) and Iridovirus were used for further rearing. Fertilized eggs hatched 20-22 hr after fertilization at water temperatures of 27.5-28.5 C. Eggs normally hatched at a water salinity of 32-34 ppt. IMRAD-Gondol has successfully developed domesticated broodstock. At present, grouper broodstock for breeding are provided from hatcheries and reared in marine net cages. Before stocking into spawning tanks, broodstock were quarantined and checked carefully for diseases. The most common diseases and White spot (Cryptocaryon irritans) Oodiniosis (Amyloodinium ocellatum) Skin fluke (Benedenia sp./ Neobenedenia) Copepods: Calingus sp Lepeophtheirus sp Scutica: Scutia sp Trichodina: Trichodina sp Gill parasite: Haliotrema Pseudorhabdosynochus sp TABLE 2. Treatments used during quarantine of grouper broodstock. Pathogen to be controlled Treatments Dosages Administration None Formalin Freshwater Formalin - 200 ppm - 300 ppm Move fish to a clean tank twice at 3-d intervals 1 hour bath 5-min bath at 1-wk interval, 2 times 24-h bath at 7-d intervals; 2 times Broodstock management requires that biological characteristics are understood and used to create a culture environment to enable the fish to reach advanced stages of maturation. Once protocols to achieve adequate spawning have been established, aspects of reproduction and genetic traits of the broodstock need to be managed to ensure the progeny have improved characteristics for ongrowing and market. Most grouper broodstock are artificially produced from hatcheries and reared in marine cages until they attain mature size and then are moved to spawning tanks.
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