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Deslorelin acetate is a gonadotropin-releasing hormone agonist formulated in a controlled-release subcutaneous implant and designed for reversible suppression of testosterone production in dogs. It has also been demonstrated to be effective in other animal species, but no data on its effectiveness in male land tortoises are available. This study aimed to evaluate the effect of a 4.7-mg deslorelin acetate implant on serum testosterone concentrations in male Hermann's (Testudo hermanni sp.) and Greek (Testudo graeca sp.) tortoises. Twenty adult male tortoises housed under the same environmental conditions were enrolled for the study and randomly assigned to a treatment (D, n = 10) or a control (C, n = 10) group. Starting in May, males from the D group were implanted with a 4.7-mg deslorelin acetate device, whereas males from the C group did not receive any treatment. Blood samples were collected once immediately before implant application (S0-May) and at 15 days (S1-June), 2 (S2-July), and 5 (S3-October) months after application. Serum testosterone at each sampling time was measured through a solid-phase, enzyme-labeled, competitive chemiluminescent immunoassay. Median serum testosterone concentrations were not significantly different between the two groups in all sampling times, and no interaction between treatment and sampling time was observed. The present study, therefore, suggests that a single treatment with a 4.7-mg deslorelin acetate implant has no effect on testosterone circulation in male Hermann's and Greek tortoises during the following 5 months., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)