WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2018 65 (CONTINUED ON PAGE 66) generally breeds from March to September, but can be induced to spawn year round via hormonal stimulants and photothermal manipulation. During offseason spawning, there is a greater possibility of obtaining poor-quality eggs. Meeting broodstock nutritional requirements and providing appropriate environmental manipulation ensures that obtained spawn is of highest quality. Fish were inspected periodically to check for maturation stage of oocytes (Fig. 2). When oocytes are more than 600 µm diameter (> 700 µm is ideal) and males are actively producing milt, broodfish were ready for induced breeding. Selected broodfish were injected with luteinizing hormone-releasing hormone (LHRH) at 20 μg/kg for female and 10 μg/kg for male fish (Nhu et al. 2011, Samraj et al. 2011). Broodfish can be induced to spawn using intramuscular injection of human chorionic gonadotropin hormone (HCG) at 500 IU/kg for females and 250 IU/kg for males (Gopakumar et al. 2011). Fish were anesthetized during handling and horomone injection to avoid stress. Typically, a sex ratio of two males to one female at a stocking density of 1-1.9 kg/m3 provides consistent success (Benetti et al. 2008). Spawning occurs 39 h after hormone injection. Eggs and collection. Eggs were positively buoyant, regulated by oil globules in the egg. Eggs were cream-colored, transparent, and spherical, between 1.0-1.1 mm in diameter (Gopakumar et al. 2011). A 35-kg broodfish released about 3.5 million eggs (Samraj et al. 2011) and a 23 kg fish released about 2.1 million eggs, with a fertilization rate of up to 90 percent (Gopakumar et al. 2010, 2011). Eggs can be collected with the use of an egg collector using an air lift or with a hand net with a mesh size of 500 µm (Gopakumar et al. 2010, Samraj et al. 2011). Collected eggs were disinfected with a dip of 5 percent iodine for one minute to avoid fungal infection (Samraj et al. 2011). Volumetric methods were used to calculate the total volume of eggs spawned. Water parameters of spawning tank. Sterilized broodstock tanks, filled with de-chlorinated seawater, were maintained at an ideal water temperature of 26-29 C with adequate dissolved oxygen of 7 mg/L. Chillers were used for temperature maintenance, while air was supplied from an oil-free compressor and supplemental oxygen was supplied from a compressed oxygen cylinder. Salinity was maintained between 25 and 35 ppt. Other important water quality parameters such as pH, hardness and alkalinity were kept within desirable limits (Gopakumar et al. 2011). Egg Hatch-out and Larval Feeding Practices Tank preparation and egg hatch-out. The tank was filled with dechlorinated seawater to about 60 percent of the total water holding capacity of the tank and probiotics (INVE at 5 ppm) were added to improve environmental conditions. Hatching tanks were stocked with 10-15 eggs/L. The perivitelline space was thin and the embryo was pigmented. The incubation period depended on water temperature and egg size but required about 22 h at 28-30 C. Newly hatched fry were 2.2-2.7 mm (Gopakumar et al. 2010). Live Feed and Feeding Protocol. Production of highquality fry represents the greatest constraint to the development of cobia aquaculture around the world. Existing rearing technologies, feeding protocols and survival remain poor. There is a major larval dieoff during metamorphosis, when fish shift their feeding strategy, highlighting our inadequate knowledge of cobia larval nutrition. Studies have been conducted on larval stocking density, feeding quantity and feeding frequency. Water quality management and feeding protocols have been an underlying theme to attain greater survival of cobia fry (Fig. 3). Microalgae, rotifers and Artemia nauplii were used as live feeds in the cobia hatchery (Radhakrishnan et al. 2017). Typically, Nannochloropsis salina or Isochrysis galbana (at 1 × 105 cells/ mL) were used to maintain the greenwater technique from 0 to 20 days post-hatch (dph). Newly hatched larvae nourished themselves with the yolk sac for the first three days. The rotifer Brachionus plicatilis was then introduced and maintained at 10-15/mL up to 11 dph. Rotifers were enriched with N. salina or I. galbana or other commercial products, including Algamac 3050. Newly-hatched Artemia nauplii (7-13 dph) and enriched Artemia nauplii (11-28 dph) were fed to larvae at 1-3/mL. Cobia larvae weaning started with 200-300 µm size feed at 14 dph and then gradually increased to 300-500 µm, 500-800 µm, and 1200 µm up to 30 dph (Samraj et al. 2011). Cobia larvae were fed ad libitum using a pulse feeding technique (five times per day) and size grading is needed to reduce cannibalism and enhance survival at this stage. The first siphoning takes place during 23-30 dph, depending on the degree of accumulation of debris and dead larvae. Under a natural environment, a cycle of 13-14 h of light and 10-11 h of dark is needed throughout the nursery phase. Water Quality Maintenance. During the larval rearing phase, utmost care must be taken to preserve excellent water quality. Dissolved oxygen concentration was maintained between 7-12 mg/L. Strong aeration was avoided has it can cause stress to the larvae. Hence, mild diffused aeration from the tank bottom and pure oxygen should also be provided if possible. The concentration of ammonia should be < 0.1 mg/L and pH of 8 is desirable. Water samples for water quality assessment should be collected prior to water exchange of larval rearing tanks. To maintain water quality, probiotics were applied at 103-105 CFU/mL (5 percent) The predetermined quantity of probiotics was mixed with low-salinity or fresh water, aerated for one hour for multiplication of bacteria and slowly added to the tank. Water exchange started between 15-20 dph, depending on water quality, especially ammonia concentration. FIGURE 3. Feeding protocol for cobia larvae.
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