Preparation and Administration of Gonadotropin-Releasing Hormone Agonist (GnRHa) Implants for the Artificial Control of Reproductive Maturation in Captive-Reared Atlantic Bluefin Tuna (Thunnus thynnus thynnus)

Abstract

Mature migrating Atlantic bluefin tuna (Thunnus thynnus thynnus) were captured in the Mediterranean Sea with a purse seine and reared in floating cages for 2 to 3 years. During the natural spawning period (June–July) of two consecutive years, fish were randomly implanted underwater with a controlled-release delivery system (implant) loaded with gonadotropin-releasing hormone agonist (GnRHa), in order to induce final oocyte maturation (FOM), ovulation/spermiation, and spawning. At the time of sampling, males were significantly larger than females (ANOVA, P < 0.001), having a mean (± SE) fork length and body weight of 190 ± 3 cm and 122 ± 5 kg, compared to 176 ± 3 cm and 94 ± 4 kg of females, respectively. All fish were reproductively mature, with their age ranging between 5 and 12 years and males being a year older, on average. After GnRHa implantation, fish were monitored for spawning and the release of eggs, and were sacrificed at different times after hormone treatment in order to examine the progressive effect of the treatment on gonad maturation. The in vitro GnRHa release from the produced implants was maximal during the first 2 d, with a mean (± SE) release of 525 ± 166 μ g GnRHa implant⁻¹ day⁻¹. The plasma GnRHa profile in vivo reflected the release in vitro, and statistically significant elevations in plasma GnRHa levels were measured until 7 d after treatment (ANOVA, P < 0.01). The underwater implantation procedure was improved between 2004 and 2005, requiring an average (± SD) of 3.1 ± 1.4 min for each fish, and was 64 and 84% successful in 2004 and 2005, respectively. There were no differences between the histological appearance of the testes of GnRHa-treated and control males, and almost all of them contained intra-testicular spermatozoa. However, the proportion of spermiating control males (n = 17) was only 12% compared to 26% for the GnRHa-implanted males (n = 19). Also, there were no differences between controls and GnRHa-implanted fish in sperm concentration, initial spermatozoa motility, or duration of forward motility, which ranged between 29.02–48.54 × 10¹⁰ spermatozoa ml⁻¹, 58–63% and 8–9 min, respectively. Final oocyte maturation (FOM) and post-ovulatory follicles (POFs) occurred in 63% and 88%, respectively, of the GnRHa implanted females (n = 16), compared to 0% and 21%, respectively, of the control females (n = 14). In addition, two GnRHa-implanted females in 2005 were found to be ovulated at the time of sacrifice, and their eggs were fertilized in vitro with sperm from spermiating males, which resulted in viable embryos and larvae. Finally, although spawning was not observed, fertilized eggs were collected from the cages. Larvae produced from these eggs were identified as Atlantic bluefin tuna, demonstrating that the present GnRHa implantation method can be used to induce FOM, ovulation/spermiation, and spawning in captive-reared Atlantic bluefin tuna.

Keywords:

• Thunnus
• bluefin tuna
• GnRHa implants
• induced spawning
• FOM
• spermiation

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Corrigendum

Acknowledgments

We would like to express our thanks to the personnel of Tuna Graso, S.A., for their assistance during the spawning induction experiments. Especially, we would like to acknowledge the assistance of Luis Garcia Givert, the diver responsible for implanting and sacrificing the fish, whose expertise and dedication were instrumental for the success of this project. Thanks are also due to the staff of the Instituto Español de Oceanografía, Centro Oceanográfico de Murcia in Puerto de Mazarrón for their hospitality and assistance during the annual field experiments. This work was undertaken as part of the research program “Reproduction of the bluefin tuna in captivity—A feasibility study for the domestication of Thunnus thynnus” (REPRODOTT) and was supported by a research grant from the European Community under the Quality of Life and Management of Living Resources program (contract Q5RS-2002-01355).

Notes

¹Males in “spermatogenesis” contained cysts at all stages of spermatogenesis, including a small number of spermatozoa. “Spermiogenesis” males had testes with a large portion occupied by spermatozoa and “Spent” testes contained hypertrophied somatic tissues, large number of spermatogonia and small pockets with spermatozoa.

²“Unyolked” females had only primary oocytes, or oocytes at the lipid vesicle or cortical alveoli stage as their most advanced oocytes. “Yolked-Vitellogenic” fish contained fully vitellogenic oocytes of various diameters. “FOM” (Final oocyte maturation) was characterized by migration of the germinal vesicle and lipid-droplet coalescence, at the early stage; followed by germinal vesicle breakdown and yolk-globule coalescence, at the advanced stage. “Ovulated” females contained eggs in their ovarian cavity at the time of sacrifice, and these eggs were inseminated artificially on-board.