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Background: The sterile male release technique (SMRT) is a useful method applied for controlling invasive and pest species. However, the use of X-rays can lead to negative effects on the survival and health conditions of sterilized males., Results: This study was set up to evaluate the functional integrity of physiological, morphological and behavioural responses in males of the red swamp crayfish, Procambarus clarkii (Girard, 1852), exposed to a dose of 40 Gy of ionizing radiation. Concerning physiological responses, the results showed that the irradiation dose, tested at 5, 12, 28, 35, 65, 99, 132 and 193 days after treatment, has no effects on glycaemic and plasmatic total protein levels measured as biomarkers for general stress indexes. Nevertheless, the significant reduction of circulating haemocytes and the basal levels of phenoloxidase (PO) activities recorded in 40-Gy irradiated crayfishes indicate that the exposure shrinks their capability to mount a rapid nonspecific response, and higher levels of plasmatic total PO activity indicate the ability to compensate and maintain an inducible response. Histological analyses performed at the end of the experiment showed no morphological damage in the testicular acini of irradiated males. Moreover, behavioural responses to two different water stimuli (vaporization and jet), measured at 15 and 45 days after the irradiation, were not modified in exposed crayfishes compared to the control group., Conclusions: These results confirm the validity of SMRT on young males when the breeding season is less than 4 months but exposure to X-ray should be repeated at mid-breeding season when temperatures allow a longer breeding season. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)