Sea Urchin Gamete collection Spawning can be induced in adult sea urchins by injecting 1 ml of a 0.5M KCl solution into several sites in the soft membrane around the mouth. Within minutes, the gametes should appear: the sperm are off-white, the eggs are tan to orange. Collect the sperm by inverting the male onto a small, dry petri dish. After several minutes, cover the sperm and put on ice. Collect the eggs by inverting the female onto a small beaker filled with ASW (room temperature for L. variegatus, and below 14ºC for S.purpuratus and L.pictus ). Keep urchins from falling in with aluminum foil supports. Allow eggs to settle, pour off ASW and replace with fresh ASW (at same temperature). Injection of S. purpuratus with 0.5M KCl Collection of sperm (left) and eggs (right) from L. variegatus Alternately, the gonads can be removed from the animal by dissection and the gametes isolated from them. The sperm can be transfered to a small tube and stored at 4° C for up to 2 weeks. Eggs should normally be used on the same day as they are collected. Consult the Sea Urchin Embryology web site for a protocol for collecting the eggs with antibiotics, which allows them to be stored for several weeks before use. Check to see if the population contains a large percentage of immature eggs; they will not fertilize. Instructor's prep sheet Proceed to Fertilization