The effects of osmolality and accidental urine contamination on spermatozoa velocity and motility were studied, combined with an examination of the biological characteristics of stripped and testicular sperm. Analysis of Northern pike sperm showed higher ionic concentrations of Na+ (123 ± 9 mM), Cl- (127 ± 7 mM), and K+ (35 ± 5 mM) in the seminal fluid of testicular sperm (TS), than in that of stripped sperm (SS): Na+ (116 ± 9 mM), Cl− (116 ± 7 mM) and K+ (25 ± 4 mM). Highest osmolality of seminal fluid was observed in TS with a value of 358 ± 77 mOsmol kg−1 compared with 273 ± 21 mOsmol kg−1 for SS and 68 ± 36 mOsmol kg−1 for urine. A significantly higher spermatozoa concentration was observed in TS (34 ± 5 × 109 ml−1) than in SS (23 ± 4 × 109 ml−1). Spermatozoa concentration per male and per kg body weight was 22 ± 17 × 109 for TS and 18 ± 2 × 109 for SS, respectively. Both TS and SS showed significantly higher spermatozoa velocities and motilities after dilution in urine than after dillusion in distilled water during the activity period. In conclusion, the results obtained from the present study provide information on northern pike sperm physiology that be used to improve sperm management efficiency for this species.