A freezing technique that maintains viability of sperm from the starfish Pisaster ochraceus

Embryos and larvae of the starfish Pisaster ochraceus are excellent for studying different aspects of early development. They are clear, easily obtained, and easy to culture, making them useful for morphological studies. In addition, the adults are large, produce large numbers of gametes, and early development is synchronous so that sufficient material for biochemical work can also be obtained. One major drawback is that they have a short breeding season. Gravid adult females can be maintained in the laboratory almost year round by controlling light conditions. Unfortunately, even under carefully controlled light and temperature conditions, by late autumn, the testes of most males undergo involution making it difficult to obtain sufficient sperm to raise cultures in the autumn, winter, and early spring. In order to overcome this shortcoming, our laboratory has experimented with techniques for freezing the sperm. This has involved testing a number of different cooling rates and cryoprotectants (CPAs) in order to develop a combination that maintained viability of some of the sperms. This article presents a technique that includes a combination of cooling conditions and a cocktail of CPAs that preserves the viability of sufficient sperm such that, when combined with careful rearing techniques, it allows us to produce normal healthy larvae almost year around.