1Miyazu Station, Japan Sea-Farming Association, Miyazu, Kyoto 626-0052, 2National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan
Mature males of red tilefish captured by longline fishing for bottom fish were transported to the laboratory from five to eight hours after death. Since the milt could not be taken from their genital pore, we developed effective methods to obtain potent testicular spermatozoa from the testis and store them in a refrigerator for up to seven days for advantageous artificial fertilization. Testicular spermatozoa obtained by homogenization of the testis showed lower percent motility than those obtained from a minced testis. Testicular spermatozoa were immotile in isotonic NaCl or artificial seminal plasma (ASP; NaCl 135mM, KCl 2mM, MgCl2 2.3mM, CaCl2 1.4mM, NaHCO3 20mM buffered with HEPES-NaOH at pH8.0). Motility was initiated when osmolality was increased, and percent motility was highest in the 450mM NaCl solution buffered with 20mM HEPES at pH8.0. Duration of movement was over 20min in the solution. Spermatozoa maintained at 4°C, diluted 50-250 times with ASP buffered at pH6.0-8.5 showed high motility during seven days' storage.