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.
① Write the date and group number in the stopper of the straw tube.
② Dispense 3 mL of cryoprotectant solution into a 15mL tube
③ 1 mL (5-fold diluted) of sperm mixed well
④ Insert a slightly cut blue tip attached to the pipetman at the plug side of the straw tube, and aspirate the sperm diluent until the straw is full (if it comes to the stopper, it cannot inhale naturally).
⑤ Push the powder into the mouth opposite the straw plug.
⑥ Perform the same procedure until the sperm diluent is exhausted.
⑦ Incubate at 4°C for 1-2 minutes and let sit on a holder on liquid nitrogen.
⑧ After 15 minutes or longer on the holder, put into liquid nitrogen.
After completion of all experiments, straws in liquid nitrogen were collected and stored in a tank.