World Aquaculture Magazine - September 2021

26 SEP TEMBER 2021 • WORLD AQUACULTURE • WWW.WA S .ORG In May 2015, successful reproduction of the goliath grouper was achieved for the first time, when four consecutive volitional spawns of the same female occurred, yielding more than 6,000,000 eggs. Fertile eggs were transferred to the CEINER laboratory and were cultured in incubators with filtered seawater and constant aeration until hatching. Newly hatched larvae were transferred to the CEINER larval culture tanks and to the CENIACUA laboratory located in Punta Canoa, Bolívar, which also has facilities and personnel specialized in marine fish culture. Larvae were successfully cultured in the two laboratories until they became adults, becoming the first generation of goliath grouper broodstock (F1). In September 2020, with the 5-year-old F1 broodstock raised from eggs at CEINER, CENIACUA and maintained by Benchmark Genetics Colombia at the Punta Canoa Laboratory in Cartagena (Fig. 5), mature male and female individuals were selected as candidates for continuing the artificial propagation efforts. These F1 broodstock were successfully induced to spawn, generating eggs and larvae that have been and continue to be raised, effectively closing the cycle of this species in captivity for the first time. Artificial Reproduction With the 5-year-old GG F1 broodstock held at the Punta Canoa laboratory, researchers from CENIACUA, Benchmark Genetics Colombia and CEINER sampled and selected a sexually mature female and male for induced spawning and artificial fertilization. This selection was carried out by cannulation sampling to observe and measure oocytes to determine developmental stage (tertiary stage ≥ 400 µm diameter) and sperm motility under the microscope. These individuals were implanted with a single dose of slow- release LHRH-a hormone at 75-100 µg/kg pellets (Fig. 6) that were provided by the Institute of Marine and Environmental Technology at the University of Maryland. When the female showed signs of maturation with abdominal bulging/swelling, she was biopsied by cannulation to analyze the developmental stage of the oocytes. After 34 hours following implantation of the hormone pellet, oocytes were either stripped or obtained by introducing a cannula through the genital pore. Fertilization tests were conducted by mixing sperm previously collected and kept in 1.5-mL Eppendorf tubes on ice (Fig. 7). Subsequently, 50 to 100 mL of oocytes were extracted from the female in each test, which were placed in 5-L containers, where they were mixed with semen for approximately three minutes (Fig. 7) prior to diluting eggs in filtered seawater. This procedure was performed every hour from 34 to 42 hours after hormone pellet implantation. Before 37 hours, no fertilization was evident in collected samples, as oocytes were not yet fully hydrated. However, after 42 hours, first cell divisions began to be observed in the fertilized eggs after 40 minutes of each fertilization test. This is an important observation because it is well known that, in artificial propagation of marine fish, timing is everything. This corroborated previous results for other species of grouper, such as with the seven-band grouper E. septemfasciatus in Japan (Soyano et al. 2008) and the Nassau grouper E. striatus in Miami, Florida (Daugherty 2021). In both species, mature oocytes and fertilization were only achieved at more than 42 hours after ovulation induction with LHRH-a implants. Embryo Incubation and Larvae Hatching Embryos were placed in 1-m 3 incubator tanks with a continuous flow of filtered seawater and continuous aeration (Fig. 8). Larvae hatched between 24-28 hours after fertilization at ambient temperatures ranging from 26-30 C. The development of the embryo was monitored using a microscope (Fig. 9). Incubator bottoms were siphoned to extract dead eggs and chorions of hatched eggs. Larvae (Fig. 10) remained in the incubators without feeding until the second day after hatching (2 DPH). In the first spawning attempt, only about 5,000 larvae were obtained. However, they were successfully raised through the juvenile stages and generated the first individuals of the second generation (F2) of goliath grouper produced in captivity in the world. This remarkable scientific advance for aquaculture research in Colombia is also important at a global level as it represents a first step towards developing a technology package for the controlled reproduction and spawning of this vulnerable and threatened species of enormous ecological and economic importance throughout its distribution range in the Atlantic Ocean. It also FIGURE 6. Hormone induction of F1 goliath grouper in the Punta Canoa laboratory. FIGURE 7. Sperm ready for use (left) for artificial fertilization (right) of goliath grouper eggs.