Evaluation of the aromatase inhibitor letrozole to induce masculinization in adult dusky grouper (Epinephelus marginatus)

The dusky groupers is a species of great ecological and commercial interest. Due to its high economic value, combined with its protogynous hermaphroditism and predictable spawning aggregations, the species is currently considered Vulnerable by the IUCN, primarily because of overfishing and severe viral outbreaks. Although efforts have been made in the past to establish full-cycle dusky grouper aquaculture programmes, one of the main challenges was represented by the limited availability of functional males, as well as the transient nature of sex reversal induced in juveniles with 17α-methyltestosterone (17α-MT). The aim of this study was to evaluate letrozole, a non-steroidal aromatase inhibitor, as a tool to induce, and possibly permanently maintain, sex reversal in captive-bred adult dusky groupers. After a preliminary study on letrozole pharmacokinetic in a model organism (gilt head seabream), fifteen twenty-five years old dusky groupers were divided in three groups receiving 1 mg kg¯¹ letrozole (n=5), 10 mg kg¯¹ letrozole (n=5) and vehicle only (n=5). The treatment was given to fish every twenty-eight days, from March to June 2026, with a total of four injections. To monitor treatment effectiveness, fish were checked for spermiation every fourteen days, and blood sample were collected to analyse steroid hormones in plasma, whereas gonadal biopsies were taken at the end of the experiment. Contemporary, three additional groupers of the same age were treated with 10 mg kg¯¹ letrozole, and, at the end of the transition, their ability to fertilise eggs under spontaneous spawning was evaluated. Letrozole was observed to persist in plasma for more than twenty-eight days after a single injection, while following doses were relevant to sustain the xenobiotic concentration in systemic circulation. Both doses were effective to induce sexual transition in all of treated fish in similar extent, although sperm was observed one month after the end of manipulations. The limited milt volume obtained from treated males and from the spontaneously sex-reversed male, together with the substantial absence of fertilised eggs and reproductive behaviour, suggests that letrozole could be an effective tool to increase the availably of male broodstock, although reproductive dysfunctions associated to spermiation and spawning require still further investigation.