Cryopreservation of Gametes and Larvae of the Eastern Oyster Crassostrea Virginica.

Abstract

Storage of gametes and larvae offers benefits for research and commercial oyster production and should be applicable in the hatchery at a commercial scale. We optimized refrigerated storage of eastern oyster sperm. Significant differences were found in the motility of sperm suspended in artificial sea water (ASW) of different osmotic pressures (P < 0.001). The highest motility was found in undiluted sperm and the lowest in 1:31 dilution. The greatest larval survival at 12 h (48%) was obtained with sperm diluted in calcium-free Hanks' balanced salt solution (C-F HBSS). Thus, for storage of longer than 1 d, it is best to leave samples undiluted. However, when sperm samples are diluted, it is best to maintain high sperm concentrations and to use C-F HBSS as an extender. Samples were frozen at -2.5°C/min, held for 5 min at -30°C, and plunged in liquid nitrogen using 10% propylene glycol (PG) as the cryoprotectant for sperm and 10% or 15% PG for trochophore larvae. Motility and fertilizing ability of thawed sperm were affected by cryoprotectant concentration and thawing temperature (P = 0.0001). Larval survival was affected by the concentration of larvae per straw (P = 0.0011). Frozen samples were transported to an oyster hatchery at Grand Isle, Louisiana. After 4 months, 1,000 oysters from the control group, 230 oysters produced from thawed sperm, and 850 oysters from thawed larvae were found. Oysters produced from thawed larvae developed normally in the hatchery, demonstrating opportunities for use of cryopreservation in research and aquaculture. Flow cytometry with the fluorescent dyes Sybr-14 and propidium iodide (PI) was used to assess membrane damage of thawed sperm, and rhodamine 123 and PI were used to assess mitochondrial function. Preliminary studies of cryopreservation of oyster eggs were performed. Fluorescein diacetate (FDA) was used to identify viable eggs. Dimethyl sulfoxide (0.88 M and 1.75 M) and sucrose (0.12 M and 0.25 M) were the least toxic cryoprotectants evaluated. The cooling rate yielding least damage to eggs was -1.5°C per min. However, only an average of 14 eggs (out of 200) were stained with FDA in thawed samples and none were fertilizable.