Cryopreservation ofNereis virens(Polychaeta, Annelida) Larvae: The Mechanism of Cryopreservation of a Differentiated Metazoan

The volumetric changes, structural behavior, and subsequent survival rate of juvenileNereis (Neanthes) virens(Annelida: Polychaeta) at the three chaetiger stage of development have been observed during equilibrium and nonequilibrium low-temperature dehydration in a controlled-rate freezer and on the stage of a planar cryomicroscope. Using a cooling rate 0.3°C/min and 1.4MMe2SO, substantial levels of larval survival were observed following transfer to liquid N from a temperature of −25°C when the equilibrium fractional water volume is expected to be 7.1%. The cryopreservation under equilibrium conditions is thought to indicate vitrification of the internal Me2SO which will have reached 54% at −25°C. When a cooling rate of 2.5°C/min was used, the long-term survival rate was substantially greater, reaching 86%, depending on developmental stage following transfer to liquid N from a temperature of −35°C.