Motility of fresh and aged halibut sperm

Preliminary studies of freshly collected halibut (Hippoglossus hippoglossus) sperm by light microscopy indicated that sperm motility, i.e. the percentage of motile sperm and the duration of fonvard progressive movement, was optimized under conditions of osmotic pressure ranging between 400 and 1100 mOsmol/kg and pH for 6.5-8.5. The quality of sperm, stored in vitro on ice, deteriorated rapidly within a few hours, a charactenstic particularly evident in sperm after freezing and thawing tests. Studies of several cryoprotectants indicated that halibut sperm could be successfully preserved frozen when diluted with 3 parts extender (sucrose: 150 mM, CaCl2: 1.7 mM, MgSO4: 7 mM, glycine: 86 mM and Tris: 30 mM at pH: 8), 10% of 1-2 propanediol and one part sperm (3:l). Additional studies of the motility of seawater-activated halibut sperm were conducted by dark field microscopy under stroboscopic illumination. These observations indicated that fast forward-moving sperm motility, which lasts 60-70 s, is correlated with propagating waves on the full length of the sperm flagellum with an initial beat frequency of 45-50 Hz which abruptly drops to about 10 Hz after 60-80 s. The beat frequency is blocked by KCN (5mM) and NaN3, (1.2mM), on intact sperm. The flagellum beat frequency of demembranated/reactivated sperm is dependent on the ATP concentration in the reactivation solution.