Successful fertilization of Varicorhinus macrolepis eggs with sperm subjected to two freeze–thaw cycles

Although sperm from several fish species have been successfully cryopreserved, few studies have been done in small and/or endangered species. The aim of the present work was to develop a method of freezing and refreezing Varicorhinus macrolepis semen in 1.8 mL cryovials. The effect of extenders and cryoprotectants on the motility of post-thaw sperm was examined. The motility of frozen–thawed sperm in extender D-15 was higher than that in MPRS and fish Ringer solution (P < 0.05). Dimethyl sulfoxide (DMSO) and glycerol provided greater protection to sperm than methanol during freezing and thawing; the most effective concentration of DMSO and glycerol was 10%. The fertilization rate of frozen–thawed sperm was not significantly different from that of fresh sperm. Furthermore, mean (±S.D.) hatching rate did not differ significantly between frozen–thawed (82.7 ± 12.4%) and fresh sperm (90.7 ± 4.5%). Although frozen–thawed sperm that was immediately refrozen had 0% post-thaw motility, frozen semen that was refrozen after dilution with D-15 (containing DMSO at a ratio of 1:2) had post–thaw motility of 38.3 ± 2.9%. Motility was lower for refrozen than for frozen sperm (P < 0.05). Furthermore, fertilization and hatching rates of refrozen sperm were 42.9 ± 6.7 and 34.1 ± 10.5%, respectively, which were lower than that of fresh sperm (P < 0.05).