52 JUNE 2023 • WORLD AQUACULTURE • WWW.WAS.ORG Partnership Program (AADAP) - Investigational New Animal Drug (INAD) Program Management System, and 5) procuring equipment, materials and supplies. There was very little effort dedicated to female grouper broodstock collection in the first year. During the second year of the G2G project, in an effort to expand grouper sperm collection, the PI worked with local and regional fishers and grouper researchers to collect sperm samples while fishing for red grouper and black grouper, as well as research focused on the goliath grouper E. itajara. During catch-and-release grouper fishing, the PI modified a handheld vacuum pump8 with an air pump needle. When fish were brought up to the vessel transom, the PI would insert the needle into the urogenital pore and create a vacuum to extract gametes from the grouper gonad. Gametes were typically extracted with a vacuum pressure between 7-10 psi. This method was also used to determine the sex and oocyte maturation level of female grouper captured for broodstock. Female Black Groupers BLK-1 and BLK-2 The CFK SMART Center’s outdoor flow-through system was used for temporary holding of the first two female black grouper for behavioral analysis and were used to train SMART Center staff on the nuances of black grouper husbandry. BLK-1 (76 cm, 5.2 kg) (Fig. 1) was the first black grouper broodfish at the SMART Center in May of 2020 followed by BLK-2 (61 cm, 2.5 kg) eight months later. Atlantic thread herring Opisthonema oglinum was the initial introductory food, supplemented with B-complex vitamins to stimulate the immune system9. Each herring was implanted with Super B complex vitamin (Nature Made Nutritional Products). To the extent possible, only one treated herring was fed during each feeding. Valenzuela-Gutiérrez et al. (2021) suggested that garlic extract boosts fish health and combats disease during aquaculture. Therefore, 1000 mg garlic extract (Nature’s Bounty) was inserted into each herring gut and subsequently fed to BLK-1 and BLK-2. The two black groupers were placed into the same holding tank in the SMART Center outdoor systems to observe natural interactions between the two female black groupers. BLK-2 was placed into the tank first without the use of any sedatives because of its small size and ease of handling with soft-mesh fish nets while BLK-1 was sedated due to its large size. Although BLK-1 was about 15 cm larger than BLK-2, the introduction of sedated BLK-1 into the tank with non-sedated BLK-2 offered a brief opportunity for BLK-2 to exert dominance over the larger BLK-1. However, once BLK-1 regained consciousness, the fish immediately exerted dominance by opening the mouth and flaring the gills, demonstrating the ability to completely engulf BLK-2, which caused the smaller fish to immediately retreat and seek refuge. Witnessing this event was extraordinary and provided insight towards methods for future black grouper pairing events. BLK-1 was treated for injuries sustained from BLK-2 aggression with povidoneiodine topical treatment; however, the injuries persisted, despite additional antibiotic treatment. Unfortunately, BLK-1 never fully recovered and died four days later. A major lesson learned is to avoid combining broodfish of significantly different size. The aggressive nature of this fish in captivity requires that all broodfish stocked into a tank are of approximately the same size. BLK-2 (later renamed “R3”) was the first broodfish to inhabit Tank 3 of the indoor recirculating system. cryopreservation of sperm for subsequent in-vitro fertilization of eggs produced from captive female broodstock. Male Gamete Sourcing The COVID-19 pandemic impacted every aspect of the G2G project as it was initially awarded during February 2020. The project began during the annual fishery closure for grouper species in the Atlantic (January-May). Early months were used to inform the fishing community about the project, primarily through the Florida Keys Commercial Fisherman’s Association (FKCFA). To communicate with as many fishers as possible (both commercial and recreational), the PI used local radio programs1 with an emphasis on fishing broadcasts2, news broadcasts3 and music/talk radio4. Fishers were informed that the project would be targeting red grouper and black grouper and encouraged them to contact the PI or Co-PI if any groupers were captured once the fishery opened on or after 1 May 2020. Fishers were incentivized by offering a cash reward for each viable gonad and/or sperm/semen sample. The typical method for gonad collection started with a call from a fisher indicating that there were fish and/or samples to be analyzed. Fishers were instructed to keep whole fish on ice if possible, and if not, to put the viscera into a plastic bag on ice. The PI kept a compound microscope5, a 10-L liquid nitrogen Dewar6 with sperm cryopreservation extender media7 available as needed. The PI would meet the fisher, collect the sample and if necessary, place a small sample on a glass depression slide for microscopic analysis and viability determination. Sperm viability was determined by observing sperm motility under a compound microscope at 400× magnification. Usually during the first visit, the PI would then train the fisher on 1) how best to store a sample, 2) gonad location in the grouper peritoneal cavity, 3) what gonads look like and 4) basic grouper reproductive biology (e.g., hermaphroditism, species-specific size at transition, reproductive behavior, etc.). The primary effort during 2020 was focused on 1) developing broodstock collection contracts with a third-party collector, 2) obtaining a broodstock collection Special Activity Licenses (SAL) from Florida Fish & Wildlife, 3) preparing a CFK Institutional Animal Care and Use Committee (IACUC) application, 4) applying for the U.S. Fish & Wildlife Service, Aquatic Animal Drug Approval FIGURE 2. A conceptual diagram of the CFK SMART Center indoor recirculating aquaculture life-support systems with environmental controls showing the flow of water from the sump through the heater/chiller system and UV sterilizer, into the broodstock tanks and back into the sump.
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