Development of cryopreservation methods for sperm of pinto abalone (Haliotis kamtschatkana) for conservation aquaculture.

The pinto abalone, Haliotis kamtschatkana, is an ecosystem engineer with cultural and ecological significance. Due to the dramatic 98% decline in populations, a ten-year recovery plan was enacted to bring levels of pinto abalone back from the brink of extirpation in the San Juan Archipelago, Washington. The focus of this study was to assist restoration efforts in creating self-sustaining populations by developing methods of cryopreservation for male pinto abalone sperm. My study evaluated three commonly used cryoprotectants, a series of freeze/thaw temperatures, and developed methods of quality assessment specific to pinto abalone. Animals were provided and cared for by Puget Sound Restoration Fund and all experiments were done at the NOAA Research Station in Manchester, WA. I evaluated sperm quality with a computer-assisted sperm analysis (CASA) system, for which I developed parameters to track pinto abalone sperm motility. I used sperm motility to analyze sperm quality in a series of experiments culminating in a final experiment to test the optimized cryopreservation methods by fertilizing pinto abalone eggs. Toxic effects of commonly used cryoprotectants, di-methyl sulfoxide (DMSO), glycerol (GLY), and propylene glycol (PG), were tested at concentrations from 5%-20%. DMSO at 5% was the least toxic, yielding the highest percent motile sperm after an exposure time of 10 minutes. Using a programmable freezer, a series of freezing rates and endpoint temperatures were evaluated for cryopreservation of sperm in 0.5 mL freezing straws at a density of 1.8 x 108 sperm mL-1. A freezing rate of -3 ℃ min-1 to an endpoint temperature of -60 ℃, preservation in liquid nitrogen (-196℃), then thawing in a 40℃ water-bath for 8 seconds yielded the highest sperm motility. Cryopreserved sperm successfully fertilized eggs, but with lower success than with untreated sperm. The highest percent fertilized was 12.2% using a concentration of 1 x 106 sperm mL-1 with 14.2% post-thaw motility. Through this study, I have outlined the first attempt into cryopreservation of pinto abalone sperm and provided a foundation for future research to optimize the methods developed. Developing cryopreservation methods will allow hatchery managers to build a genetic library to improve production and maintain genetic diversity of this vanishing species.