CRYOPRESERVATION OF SPERMATOZOA OF THE EDIBLE MUD CRAB SCYLLA-SERRATA(FORSKAL)

Biotechnique for programmed freezing of the spermatozoa of the edible mud crab Scylla serrata and the procedures for assessing viability of the frozen-thawed spermatozoa are described. Free spermatozoa recovere d from spermathecae and those released from the spermatophores were su bjected to cryoprotectant treatment at different physiological tempera tures (30 degrees C, 23 degrees C, 15 degrees C), frozen to subzero te mperatures in a rate-controlled programmable freezer Kryo 10/1.7 and s ubsequently plunged into liquid nitrogen. Post-thaw viability, after 8 hr of storage at -196 degrees C, was assessed by artificial induction of acrosome reaction using divalent cation ionophore A23187. Hypo/hyp erosmotic sensitivity tests were used to assess the membrane integrity of the spermatozoa before and after cryopreservation experiments. Via bility tests using dye exclusion methods were discontinued owing to va riations in incorporation of different stains into the spermatozoa. Th e limitations of the dye exclusion method as an indicator of sperm via bility are discussed. Dimethyl sulfoxide, ethylene glycol and glycerol offered cryoprotection at concentrations ranging from 5% to 15% v/v. Post-thaw survival was highest (52%) with 12.5% glycerol at a cooling rate of -5 degrees C/min. (C) l997 Wiley-Liss,Inc.