Optimization of activation, collection, dilution, and storage methods for zebrafish sperm

In the present study, we optimized activation, collection, dilution, and storage methods for zebrafish ( Danio rerio) sperm. Our findings revealed that zebrafish sperm was motile in Hank's balanced salt solution (HBSS), glucose, and sucrose at an osmolality range of 25–270 mOsm/kg with the highest motility observed at 150–210 mOsm/kg. We compared three activation solutions: 0.3% NaCl, de-ionized water, and HBSS at 170 mOsm/kg. Our results indicate that the longevity of sperm motility depends on the final osmolality of the sperm suspension medium (extender) and activation solution combined. Evaluation of sperm collection methods suggested that higher percent motility is obtained when collected through dissecting without crushing (89 ± 3%, mean ± SD) or abdominal massage (90 ± 4%) than dissecting with crushing (65 ± 13%). The total number of motile sperm was higher for dissecting without crushing (147.0 ± 102.3 × 10 5/male) than abdominal massage (7.1 ± 11.9 × 10 5/male). Sperm suspended in HBSS had higher motility than those suspended in buffered sperm motility-inhibiting solution (BSMIS). Sperm retained motility longer when samples were stored at 4 °C than those stored at room temperature (25 °C). Motility which could be activated after storage decreased with increased dilution ratio and it is the resultant sperm density rather than the dilution factor that determines sperm motility. Our findings provide useful information on handling procedures of zebrafish sperm.