19. Cryopreservation and fertility of geoduck (Panopea zelandica) sperm and oocytes

Abstract

Geoducks are large salt water clams. They are currently farmed in North America. In New Zealand, there is a small wild fishery for the native geoduck, Panopea zelandica . More recently, there is increasing interest in farming this high value species in New Zealand. Already a small batch has been reared and is ready to be experimentally on-grown on existing mussel farms. Cryopreservation can be a powerful tool for hatcheries and selective breeding programmes for aquaculture. In hatcheries, cryopreservation allows year-round spat supply without the need to condition broodstock for out of season production. This is particularly useful with new species where optimized conditioning regimes have not been established. Spawning techniques may also need development and cryopreservation can enable hatcheries to capitalize on single sex spawnings where gametes would otherwise be wasted. In selective breeding, cryopreservation enables breeders to make crosses on demand; providing them with complete flexibility and enabling faster genetic gains. Towards this goal, we investigated cryopreservation and fertility of gametes from the New Zealand geoduck, Panopea zelandica. Geoducks were induced to spawn during their natural spawning season using thermal shock and serotonin injection. Sperm was collected as concentrated as possible by aspiration and stored at 4 °C before being used in experiments. Oocytes were maintained at either room temperature or 4 °C. For both sperm and oocytes, three cryoprotectants, ethylene glycol, dimethyl sulphoxide and propylene glycol were made up in Milli-Q water and evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. For sperm, a simple freezing method utilizing a rack attached to a polystyrene frame which floated the rack 3 cm above the liquid nitrogen surface was evaluated as a method of freezing. Sperm were diluted 1:1 with cryoprotectant solutions and loaded into 0.5 mL straws and frozen on the rack for 10 min before plunging into liquid nitrogen. For oocytes, two cooling regimes were evaluated. Oocytes were diluted 1:1, loaded into 0.25 mL straws and placed into controlled rate freezers that were programmed to hold at 0 °C for 5 min then cool at 1 °C min −1 to −10 °C, hold for 5 min then cool at either 0.3 or 0.5 °C min −1 to −35 °C. Upon reaching −35 °C, straws were plunged in liquid nitrogen. Straws containing sperm were thawed at room temperature. Straws containing oocytes were thawed at 28 °C until the ice had melted. Overall, unfrozen sperm obtained from serotonin injection had slightly higher fertility with fresh oocytes than sperm obtained from thermal shock (70% vs 50% at sperm concentration of 10 5 mL −1 ) The addition of bovine serum albumin to the fertilization assay improved sperm fertility by 30%. Sperm frozen in either 10% dimethyl sulphoxide or 10% or 15% propylene glycol gave the highest post-thaw fertilization rates (>70% at sperm concentration of 10 6 mL −1 ). Oocytes were lysed upon thawing prior to dilution for fertilization. Further work will investigate: optimization and development of cryopreservation methods for both gametes and larvae; the longevity of geoduck gametes and a larval assay to assess subsequent development.