Cold Storage and Cryopreservation Methods For Spermatozoa of the sea urchinLytechinus pictus

Abstract Sea urchins have contributed greatly to knowledge of fertilization, embryogenesis and cell biology. However, until now, they have not been a genetic model organism because of the long generation times of commonly used species, and lack of tools for husbandry and genetic manipulation. We recently established Lytechinus pictus , as a multigenerational sea urchin model, because of its relatively short generation time of 4-6 months and ease of laboratory culture. To take full advantage of this new multigenerational species, methods are needed to biobank and share mutant L. pictus sperm. Here, we describe a new extender based on sperm ion physiology before spawning of sperm into seawater. This extender maintains sperm capable of fertilization for at least 5-10 weeks when stored at 0 °C. We use the extender, and the cryoprotectant dimethyl sulfoxide (DMSO), to cryopreserve sperm of both L. pictus , and the widely used sea urchin, Strongylocentrotus purpuratus . The simple methods we describe work well for both species, achieving > 90% development and producing larvae that successfully undergo metamorphosis to juvenile sea urchins. Sperm of these two species can be frozen and thawed at least twice and still give rise to larvae that undergo metamorphosis. Main Points Sperm can maintain fertilizing capacity ex vivo for 5-10 weeks when stored at 0°C. When freezing in liquid nitrogen no stepwise addition of cryoprotectant, or stepwise drop in temperature are required. A standard fertilization assay is presented to score cleavage stage sea urchin embryos produced by cryopreserved sperm. Sperm frozen and thawed more than once can produce larvae.