EVALUATION OF AROMATASE INHIBITOR LETROZOLE TO INDUCE MASCULINISATION IN ADULT DUSKY GROUPER Epinephelus marginatus

Introduction

The dusky grouper (Epinephelus marginatus) is a cosmopolitan species of importance for fisheries, aquaculture, and ecological interest. Like many other species in this family, dusky groupers are monandric protogynous hermaphrodites, characterized by slow-growth, late maturation and long lifespan, with natural sex inversion from female to male occurring around 80 cm total length and above 7 years old (Condini et al., 2018). These biological characteristics, combined with intense fishing pressure experienced since the last century and increasing habitat destruction, led to this species being classified as Vulnerable on the IUCN Red List in 2016. Considering its economic value, especially in Asian and Mediterranean countries, this species has long been of interest for capture-based aquaculture. However, given the high feed conversion ratio and the conservation status of wild stocks, this practice seems to be unsustainable. Furthermore, sudden mass mortality events, such as those caused by Betanodavirus in 2023 and 2024 in the Mediterranean populations (Kersting et al., 2024), have highlighted the urgent need to establish aquaculture breeding programs, allowing therefore to satisfy the market demand without impacting on the wild population, as well as to provide readily available stock for reintroduction in case of worsening conservation status. The main limiting factor in dusky grouper breeding is currently the difficulty in inducing sex inversion to males, which results in lack of sperm for fertilization. To address this issue, aromatase inhibitors have already been used in other congeners with successful and permanent results. However, in dusky groupers they have rarely been tested alone, and results were often limited to very low milt production or temporary inversion. The objective of this research is, therefore, to test letrozole, a non-steroidal aromatase inhibitor, in order to evaluate its effectiveness in inducing masculinization in individuals who are already within the natural range of sex inversion.

Materials and methods

Fifteen dusky groupers of average weight 6.3 ± 0.9 kg (mean ± SD) and around twenty-five years old were maintained in two rectangular tanks of 5 m³ in the broodstock facility of Aqualabs at the Institute of Marine Biology, Biotechnology and Aquaculture, in Crete. Starting on 11^th of March and every 28 days, letrozole (InvivoChem, China) has been injected in fish dorsal muscle in two different experimental doses, both diluted in 50 µl fish oil kg¯¹ of fish. Fish were randomly divided in three groups receiving respectively effective dose of 1.07 ± 0.02 mg kg¯¹ letrozole (n=5), 10.45 ± 0.18 mg kg¯¹ letrozole (n=5) and fish oil only as control (n=5). Fish were reared together in similar stocking density. On day 0, before starting the treatment, no sperm was found in the groupers as well as no gonadal biopsies were possible to be obtained with the catheter, probably due to underdeveloped gonads, as fish were outside their reproductive season. On the same day of injection and every 14 days, the groupers were checked for sperm production and approximately 1.5 ml of blood was collected with heparinized syringes. Masculinization and spermiation will be examined every 14 days, by applying gentle abdominal pressure and collecting sperm. Sperm quality will be analyzed with Computer Assisted Sperm Analysis (CASA) for sperm density, spermatozoa motility (%), progressive motile spermatozoa (%), rapid motile spermatozoa (%), straight-line velocity (VSL, µm sec¯¹), curvilinear velocity (VCL, µm sec¯¹), average path velocity (VAP, µm sec¯¹) and straightness (STR, %). Plasma levels of 17-β Estradiol (E₂), Testosterone (T), 11-Ketotestosterone (11-KT) and 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-P) will be quantified using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Results and discussion

The experiment is currently ongoing and the results are expected to be obtained around mid of June, at the end of letrozole treatment and in correspondence of the beginning of the natural spawning season. An update of this abstract will follow at the end of the experimental trial. Considering the inhibiting action of letrozole on the enzyme responsible for testosterone aromatisation into 17-β estradiol (CYP19A1), in letrozole-injected groupers the plasma concentration of 17-β estradiol is expected to decrease compared to control fish, whereas testosterone and 11-ketotestosterone should increase. The maturation inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one, instead, is likely to remain constant or slightly increase in treated groupers, therefore stimulating spermiation and sperm motility. Furthermore, hypothesizing a complete masculinization of groupers, a second experiment will be later conducted to evaluate their effective capability to fertilize eggs under natural spawning conditions.

Acknowledgements

This study is funded by the internal project ELEBOOST-Fakr awarded to IF.

References

Condini, M.V., García-Charton, J.A., Garcia, A.M., 2018. A review of the biology, ecology, behavior and conservation status of the dusky grouper, Epinephelus marginatus (Lowe 1834). Rev. Fish Biol. Fish. 28, 301–330. https://doi.org/10.1007/s11160-017-9502-1

Kersting, D.K., García-Quintanilla, C., Quintano, N., Estensoro, I., Ortega-Villaizan, M.D.M., 2024. Dusky grouper massive die-off in a Mediterranean marine reserve. Mediterr. Mar. Sci. 25, 578–585. https://doi.org/10.12681/mms.38147