Effects of Freezing Rate and Lowest Cooling Pre-storage Temperature on Post-thaw Fertility of Amago and Masu Salmon Spermatozoa

Abstract

Semen of amago salmon Oncorhynchus masou ishikawae was diluted 6 times with extender solution (90% 300mM glucose+10% DMSO) and frozen on dry ice (the dry ice method) or on a stainless steel case floating on constant-temperature ethyl alcohol (the alcohol method). Pellets of various volumes (50-200 μl) that were frozen on dry ice invariably showed a high percent of fertility in spite of the difference in their freezing rates (FR; 50.3 to 29.9°C/min). The fertilityof pellets frozen by the alcohol method and exposed to -30, -50, -70, or -90°C before immersion in liquid nitrogen depended on the alcohol temperature. The highest fertility (87.5%) of 100 μl pellets wasobtained with samples exposed to-70°C (FR=92.6°C/min). Pellets frozen by the alcohol method at-30°C (FR=30.6°C/min) or-90°C (FR=125.6°C/min) showed low fertility rates (58.2% and20.4%, respectively). Two hundred μl pellets frozen by the alcohol method showed a similar percent of fertility to the 100 μl pellets of the same temperature, although their freezing rates were lower. These phemomena were also found in masu salmon O. masou masou spermatozoa. The present results indicate that for spermatozoa of both species, freezing rates in the range of 29.9 to 92.6°/min have little influence on the post-thaw fertility. The most critical factor for obtaining the highest percent of fertility in pellet methods is the temperature reached before immersion in liquid nitrogen, with approximately -70°C being the most suitable temperature for amago and masu salmon spermatozoa.