1Aquatic Life Conservation Center, Agricultural Food and Environmental Sciences Research Center of Osaka Prefecture, Neyagawa, Osaka 572-0088, Japan, 2Graduate school of Agriculture, Kinki University, Nara 631-8505, Japan
This study aimed to develop an effective method for the cryopreservation and artificial fertilization of cryopreserved spermatozoa of the endangered Itasenpara bitterling. Milt was mixed with a cryopreservation diluent (10% methanol plus 90% artificial seminal plasma for carp) and stood for 180 min; the percent motility of spermatozoa further diluted with buffered solution showed no significant change. Milt was diluted with the cryopreservation diluent (1:40), and each 80 μL of the dilution was dispensed into long capillary tubes. The tubes were cooled in liquid nitrogen (LN) vapor at various cooling rates to various temperatures and then immersed and stored in LN. The milt was thawed by immersing the tubes in water at 20°C for 7 seconds. Post-thaw motility was determined by the percentage of motile spermatozoa diluted with buffered solution. The tubes cooled at the rate of －14°C min−1 to －50°C and immediately immersed in LN demonstrated a significantly higher post-thaw motility (30.6±2.1%) than under other conditions. The post-thaw milt diluted 1:500 with tap water before insemination showed a high percentage of fertility (88.7%); however, the percentage decreased rapidly with high dilution ratios. In contrast, fresh milt showed an invariably high percentage of fertility regardless of the dilution ratio.