Viability of cryopreserved spermatozoa of the horseshoe crab,Limulus polyphemusL.

Summary Semen of the horseshoe crab, Limulus polyphemus L., was frozen and stored at −74°C for periods up to 50 days in a glycerol/sea water medium (phosphate buffer). After collecting in glass centrifuge tubes, untreated semen was cooled between 15 and 5°C at a rate of −0.5°C/min. After 30 min of equilibration, semen was transferred to 2-ml glass ampoules and diluted with three volumes of diluent (25 ml of 0.4 M NaCl/0.1 M glycine, 4 ml of 0.028 M NaH2P04/0.072 M Na2HP04(pH. 7.2), and 5 ml of glycerol). Each sample was then cooled−6°C/min between 5°C to −35°C and quick-frozen to −79°C by immersion in a solid C02/acetone bath. After varying storage periods, cryopreserved semen samples were thawed under running tap water (20°C) and their viability tested. Compared to unfrozen control semen, viability tests (control = 100%) indicated that 73% of the spermatozoa showed eosin dye exclusion, 40% showed sperm-egg attachment, and 17% of fertilized eggs showed embryonic development.