WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2023 51 advanced so that they are now applicable for aquaculture production and establishment of sperm banks. With regard to the Guts to Glory project, there are several distinct advantages associated with protocols for in-vitro fertilization of grouper eggs with cryopreserved sperm: 1) inability to procure mature male grouper broodstock, 2) the ability to synchronize gamete availability of both sexes, 3) effectiveness with low quantities of semen, 4) simplifying broodstock maintenance, 5) ability to transport gametes, 6) avoiding aging of sperm — a result of sperm deterioration with age during the course of a spawning season and 7) conserving genetic variability during the grouper domestication process (Suquet et al. 2000). Sperm motility is affected by the availability of internally stored adenosine triphosphate (ATP) and the capacity of sperm to manufacture ATP is limited. Therefore, during cryopreservation, extender solutions are used to inhibit sperm motility. The most common extender solutions used in the cryopreservation of marine fish sperm are saline (1-10 percent) or sugar (5-10 percent). Modern extender protocols employ cryoprotectants that stabilize cell membranes during freezing (Suquet et al. 2000). For example, the best results for sperm cryopreservation of Pacific black grouper Epinephelus malabaricus have been obtained using an extender solution of 150 mM NaCl with 20 percent DMSO and a freezing rate of approximately 154 C/min, consistent with the use of liquid nitrogen (Gwo et al. 1993). The study described in this article focused on 1) developing cryopreservation protocols for sperm or semen of red grouper Epinephelus morio and black grouper Mycteroperca bonaci to create a sperm bank for long-term storage, 2) the capture and acclimation of female grouper broodstock, 3) induction of egg production through environmental manipulations, hormone injections or both and 4) use of cryopreserved sperm for in-vitro fertilization of grouper eggs. To the best of our knowledge, this study represents the first attempt to domesticate red grouper and black grouper, and especially at From Guts to Glory: Supporting Aquaculture with Discarded Grouper Gonads from the Florida Capture Fishery Patrick H. Rice and Caeley V. Flowers Although far behind other countries with grouper aquaculture, the United States remains a substantial contributor to global aquaculture research and technology development, especially regarding hatchery technology for new species. Grouper aquaculture research in the US is minimal relative to the rest of the world. The primary constraint to any aquaculture operation is a reliable source of fingerlings. Exacerbating the issue is the lack of hatcheries for any grouper species in the United States, so the ability to conduct research is critically limited. To address this shortfall, the College of the Florida Keys (CFK) established the Southernmost Marine Aquaculture Research and Training (SMART) Center that is designed to use undergraduate marine aquaculture research as a training tool and provide professional experience through internships and assistantships. Groupers (Family: Serranidae) are protogynous hermaphrodites, meaning that mature alpha females transition into larger mature males. Traditional grouper aquaculture requires capture and acclimation of male and female broodstock, often requiring years. In addition, capturing male grouper broodstock is problematic (Gwo 2009) because large males experience high fishing pressure and are thus more rarely captured. Despite this, large grouper are regularly harvested as part of the commercial capture fishery in the Florida Keys. Grouper gonads, representing valuable genetic resources, are routinely discarded as part of fish processing for market. Dr. Patrick Rice, the Chief Science and Research Officer at CFK, has partnered with local recreational and commercial fishers from the Florida Keys Commercial Fisherman’s Association to collect grouper gonads discarded during fish cleaning. Discarded gonads provide the male genetic material for sperm cryopreservation and subsequent in-vitro fertilization trails to evaluate their potential as a resource for grouper hatcheries, hence the project name “From Guts to Glory” (G2G). The science of sperm storage and cryopreservation was first reported in marine fish in 1953 (Blaxter 1953) and is now quite mature. Sperm cryopreservation techniques for marine fish have (CONTINUED ON PAGE 52) FIGURE 1. A female black grouper Mycteroperca bonaci ( 76 cm, 5.2 kg), identified as BLK-1, in captivity in the outdoor aquaculture systems at the College of the Florida Keys Southernmost Marine Aquaculture Research & Training Center (Photo: Patrick Rice). ~
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