WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2023 53 (CONTINUED ON PAGE 54) Indoor Broodstock System Design The indoor G2G broodstock system is designed to control temperature and photoperiod (both solar and lunar cycles) (Fig. 2). Recordings of the sounds of black grouper during spawning (Schärer et al. 2014; Fig. 3) were played for the broodfish about one week before the full moon to stimulate egg maturation. Male pheromones induce ovulation in female honeycomb groupers E. merra (Amagai et al. 2022). Therefore, once black grouper sperm or male gonad tissue is collected and cryopreserved, small amounts will be added to the system about three days before the simulated full moon. The fish will be monitored for egg development and hormones will be administered, if necessary, for oogenesis and ovulation. Although Nelson et al. (2011) recorded spawning behavior for red grouper, the PI was unable to acquire the recordings for use in the G2G broodstock system. This is the primary reason black grouper was chosen over red grouper for female broodstock. The IR cameras installed in the broodstock systems will monitor the broodfish (Fig. 4). The video will be broadcast live on the CFK Science & Research webpage under the G2G project page. The page will have icons that observers can check if they witness specific behavior or physiological changes like extended abdomen often associated with egg hydration. This will help encourage citizen science, help reduce the SMART Center staff effort, and increase observation frequency. Broodstock Collection Contract collections of grouper broodstock began in March 2022, resulting in female black grouper within the approximate size range of 50-70 cm (3 small, 3 medium, and 3 large; Table 1). In April 2022, fish were captured over a two-week period and quarantined and acclimated at Dynasty Marine Associates. Fish were transported in 376-L polyethylene tanks, each supplied with aeration, with three fish per tank. Transport tank water was at 30 ppt salinity, 27-28 C and pH 8.0. Transport tank water was treated with copper acetate (0.15 ppm) and nitrofurazone (6 ppm). Each broodstock holding tank was stocked at three or four fish per tank, each with one small, one medium and one large fish to keep the biomass relatively consistent among tanks. Each tank was equipped with habitat for each fish to reduce territorial behavior (Fig. 4). Nonetheless, during early acclimation, there was much aggressive behavior until dominance could be established. In Tank 1, the fish acclimated to each other faster than the other tanks. These fish seemed more comfortable congregating under the habitat structure and aggressive behavior was rarely observed. In Tank 2, dominance was finally established after about one month and each fish established its habitat. In Tank 3, the fish labeled R3 (formerly BLK2) was so aggressive that all other fish were removed and she was isolated. Two of the three fish subsequently died from their injuries. In retrospect, stocking each tank with various sizes may have contributed to aggressive behavior as larger fish tended to intimidate smaller fish, except for R3 which dominated all other fish regardless of size. Daily observations of Tank 3 revealed R3 claiming the entire structure and occupying whichever habitat she desired and the other fish cowering along the tank wall. It is difficult to completely understand the differences observed among tanks during acclimation. Tank 1 had the greatest size disparity (size range: 48-61 cm) and had the fewest aggressive interactions. Fish in Tank 2 displayed some infighting and dominance (size range: 52-61 cm). Fish in Tank 3 displayed fighting and total domination (size range: 51-61 cm), all exclusively by R3 which was about 10.5 cm larger than the smallest fish in the tank. Therefore, a recommendation for future efforts to bring black grouper into captivity would be to focus on fish of similar size, although it is uncertain how large black grouper of similar size would behave or respond. Another recommendation would be to use larger tanks and separate habitat structures like reef environments. Except for grouper R3 in Tank 3, which was already trained to feed, the onset of feeding among new black grouper broodstock required about two weeks. New broodstock were offered Atlantic herring without vitamin supplements or additives until routine feeding was established. Once regular feeding on herring was established, vitamin B-complex (250 mg) and garlic extracts (1000 mg) were implanted into herring and then offered to fish. About one month later, the diet was diversified by adding shrimp. The plan moving forward is to acquire bycatch from the local Key West shrimp industry and sort through the bycatch for fish, crabs, squid, etc. The bycatch will be combined with the Atlantic herring currently being used. The selected bycatch and herring will be minced using a meat grinder and mixed with B-complex vitamins, garlic and maturation stimulator10. The mixture will be extruded into edible sausage casing. The resulting broodfish maturation diet will be fed over the next several months and the fish will be periodically sampled for egg maturation. Project Results and Outlook Broodstock are not ready to spawn but it is anticipated that fish maturation protocols will result in egg development. Periodically broodfish will be sampled and gonadal tissue will be observed under the microscope. Females in an advanced stage of oocyte maturation (vitellogenic oocyte diameters of 325 μm) will be selected for ChorulonTM injection or OviplantTM implantation (Marino et al. 2003). Fish will be monitored and egg collectors will be checked hourly after hormone treatment. If broodfish do not produce eggs FIGURE 3. A schematic of the acoustic system designed for the G2G broodstock tanks. The system will transmit sounds into the tank by a lowfrequency tactile transducer placed directly on the external tank wall. The sound in the tank will be received with a hydrophone and analyzed. The sound will be calibrated with an audio interface to compensate for any impedance from the tank wall and matched with the spectrograms from the original recordings.
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