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The aim of the present study was to evaluate the effectiveness of human chorionic gonadotropin (hCG) in stimulation of second spermiation in already reproduced pikeperch males during the same spawning season. Fish (mean weight 919.9 ± 236.9 g) were divided into the control saline-injected group, and two groups injected with 200 and 400 IU hCG/kg body weight. Forty-eight hours following injection (at a temperature of 16.7 °C) the percentage of fish producing useable amounts of semen increased from 30% in the control group up to 80% in the treated fish and the highest volume of semen was recorded in fish treated with 400 IU/kg BW hCG. Spermatocrit (50.7 ± 9.1%) and sperm concentration (17.5 ± 5.0 × 109 spermatozoa/mL) were significantly higher in control fish than those recorded in hCG-treated fish (24.8 ± 7.7% and 8.1 ± 2.4 × 109 spermatozoa/mL in 200 IU/kg; 32.3 ± 17.6% and 10.9 ± 7.5 × 109 spermatozoa/mL in 400 IU/kg). No significant differences were observed between the groups with respect to sperm motility (as a spermatozoa activity time and percentage); however, a change in plasma alkaline phosphatase activity was found in the group injected with 400 IU/kg of hCG. The lack of significant differences in plasma metabolites and cortisol between control and hCG-treated fish indicated that the hCG had no effect on the stress response. Testosterone levels were significantly increased in the hCG-treated fish, whereas no significant differences in 17β-estradiol were recorded. No differences between the groups in plasma levels of thyroid hormones suggested lack of hCG-induced effect on hypothalamus-pituitary-thyroid axis. The results of this study demonstrated that the spermiation of already reproduced pikeperch males could be induced by hCG during the same spawning season. Based on the results on semen volume and concentration, injection with hCG could improve the efficiency of pikeperch reproduction to reduce the number of brooders and costs of juvenile pikeperch production. • hCG effectively stimulated second spermiation in pikeperch at the end of spawning season. • Producing useable amounts of semen increased up to 80% in the treated fish. • Plasma testosterone levels were considerably increased in the hCG-treated fish. • Neither hCG nor saline-treated groups showed stress response in pikeperch males. [ABSTRACT FROM AUTHOR]