Anesthesia is a common practice used in fish research and aquaculture. For both applications, it is important to understand anesthetics effects on the animal and tissues of interest to ensure the validity of data and to improve animal welfare. Captive fish production is only possible with artificial reproduction, and it is known that manipulation is a stressor stimulus in fish. The most common method of determining fish stress responses is measuring the circulating level of cortisol. Therefore, the purpose of this study was to evaluate the effects of different concentrations (100, 200, and 300 mg L-1) of the anesthetic tricaine methanesulfonate (MS-222) on cortisol levels and their influence on the sperm quality maintenance in Rhamdia quelen. After hormonal induction, 28 sexually mature males (average weight = 363.00 ± 71.24 g) were randomly distributed among treatments, and their semen and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol levels, and reproductive hormones concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Anesthesia with 100 mg L-1 MS-222 presented a longer induction time than that with 200 and 300 mg L-1 MS-222. Sperm motility rate was significantly higher in the control than in the 300 mg L-1 treatment but did not differ among the control, 100, and 200 mg L-1 treatments. Estradiol level was significantly higher in non-anesthetized than in anesthetized fish, but plasma cortisol levels did not differ significantly between treatments (182.50 ± 42.03 ng mL-1). MS-222 anesthetizes fish by blocking the sodium channels, preventing the development of nerve action potentials. However, MS222 at concentrations of 100, 200, and 300 mg L-1 did not prevent stress in South American silver catfish males. In addition, its use did not maintain sperm quality, as it impaired motility and decreased levels of plasma estradiol.