[Methods]
① Take 4.5ml of ice-cold sperm diluent in a 100 ml beaker.
② Add 1.5 ml of sperm and mix with the diluent by immediate shaking. Start the stopwatch at the time of sperm mixing.
③ Immediately draw into the dispenser and drop 100 μl each over the hole of dry ice. Complete the dropping in 1 minute after mixing the sperm.
④ Wait for the dropped sperm to freeze.
⑤ Remove the storage tube from the liquid nitrogen tank.
⑥ Flip the frozen sperm pellet over the dry ice and drop it onto the underlying paper. Immediately fold the paper to transfer the frozen sperm into the storage tube and transfer the tube to the liquid nitrogen tank.
NOTE) Experiments handling dry ice and liquid nitrogen wear gloves.
