WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2014 31 diet. When all fish were actively feeding, they were fed shrimp and thread herring to satiation daily at a 1:1 ratio until the first sampling. The diet was based on feeding requirements and spawning research conducted at Mote with common snook (Neidig et al. 2012). After the first sampling, the daily food ration was adjusted on the basis of tank biomass and feeding behavior. In May 2014, the diet was supplemented with vitamin B1 (40 mg/kg) to prevent potential thiamine deficiency (Amcoff et al. 1998, Brown et al. 2005). Photo-thermal Manipulation To influence reproductive cycles, it is necessary to identify the appropriate environmental, hormonal and behavioral cues required by a particular fish species (Zohar and Mylonas 2001, Mylonas et al. 2010). There is no published information on the timing of wild spawning for black snook and the only observations of mature fish in Golfo Dulce that we are aware of are from April 2009 and July 20141. Using the average photo-thermal conditions for southwestern Costa Rica, we estimated a target photothermal range for maturation at 13 h of light and a water temperature of 29.3 C. Fish were maintained under a stepwise photo-thermal progression to identify the requirements to induce maturation (Fig. 5). Starting at an initial resting water temperature of 24 C and 12 h of light, temperature was slowly raised by 2 C before the first sampling (24 January 2014). Following each sampling, water temperature was increased by 2 C. Fish were allowed to recover for six weeks between sampling events. During the first three sampling events, no progression to maturation was observed and oocytes in all females were at the primary growth stage (Fig. 6a). Following sampling on 23 April 2014, temperature was increased to 30 C and day length to 13.5 h (Fig. 5). While sampling on 4 June 2014, two females had oocytes that had advanced to secondary growth, full grown (SGfg, Fig. 6c) stage. Oocytes were sufficiently mature that hormone induction would likely induce spawning. Three of the other five females also had positive progression in oocyte maturation, with oocytes ranging from ringed to late secondary growth (SGl, Figs. 6b and 6c). Photo-thermal conditions were maintained at 30 C and 13.5 h light and on 16 July 2014, six of seven females had oocytes that were staged at secondary growth (SG) with three at SGfg. One more sampling is planned before the fish will be cycled into resting conditions. Based on observations from earlier sampling periods, 26 C and 12 hr of light will be the resting conditions. At these conditions, fish will not be using energy for maturation and at 26 C black snook continues to feeds vigorously. Sampling and Hormone Induced Spawning To sample broodstock, tanks were drawn down and two dividers made from plastic mesh secured to a polyvinyl chloride (PVC) pipe frame were positioned in the tank to confine all fish into a small area. Fish were captured individually from the restricted area of the tank using a softmesh net and placed in a floating, oval polyvinyl tank of seawater where they were anesthetized using 300 mg/L pH-buffered tricaine methanesulfonate (MS-222™) for 1-2 min. Fish were then scanned to obtain the PIT tag number and weight and fork length measured. The reproductive status of each individual was identified by adapting the classification system for common snook (Neidig et al. 2000, Rhody et al. 2013). To assess the level of maturation in females, a cannulation biopsy was performed with an 8 fr, premature infant, silastic feeding tube (Fig. 7). An oocyte sample was used to assess the stage of maturation using light microscopy. If females had advanced late secondary growth (SGl) or more mature oocytes, they were implanted with gonadotropinreleasing hormone analog (GnRHa) at 50 μg/kg and returned to the tank. Immature females, those with oocytes at early secondary growth (SGe) or less mature, were given a dose of 25 µg/kg. GnRHa was delivered using an ethylene and vinyl acetate delivery system (Zohar and Mylonas 2001). Implants were administered with a 12-gauge needle and implanted into the dorsal muscle directly behind the second dorsal spine. All GnRHa implants used in these trials were made by the Institute of Marine and (CONTINUED ON PAGE 32) ABOVE, FIGURE 6. Black snook oocytes at different levels of maturation. a) Oocytes of a regressed female with oocytes only at the early stages of primary growth. b) Oocytes from a female that was maturing, with ring oocytes and the early steps of secondary growth. c) Mature oocytes from a female black snook.
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