Abstract
We examined a new method that used a sodium hydroxide (NaOH) solution to extract all sperm from spermatophores of the spiny king crab Paralithodes brevipes (H. Milne Edwards and Lucas, 1841). Spermatophores were put into 1ml microtubes containing 0.25ml of 20% NaOH solution. After soaking in the NaOH solution for 10, 30, 60 and 90 min, the microtubes were mixed for 30 sec. The mixed solution was then placed in a hemacytometer, and sperm were counted under an optical microscope. The total number of extracted sperm in each microtube was calculated: (number of sperm counted/volume of NaOH counted)×total volume. The number of extracted sperm increased with time soaked until 60 min. The number of extracted sperm showed no significant increase between soaking time of 60 min and 90 min, which indicates all spermatophores were completely dissolved by 60 min. Sperm cells kept the body but not spines of sperm in NaOH solution even after 180 min after mixing. Therefore, to extract sperm, spermatophores should be agitated after 60 min of soaking in 20% NaOH solution, and sperm should be counted within 3 hours after mixing.
