Cryopreservation and in vitro fertilization at the zebrafish international resource center

In recent decades, laboratories throughout the world generated several thousand mutant, transgenic, and wild-type zebrafish lines and more lines continue to be produced. At the same time, relatively little effort has been expended to develop reliable, high-throughput, standardized, long-term cryopreservation storage methods, even though laboratories and the research community as a whole struggle to maintain the large number of lines alive. Safe and reliable methods for maintaining these valuable genetic resources are vital for future biomedical research.Cryopreservation is the most efficient method for large-scale, long-term storage of important genetic materials. It extends the time offspring can be produced from individual fish, reduces the need to maintain live populations, and can prevent catastrophic loss of irreplaceable research lines. Cryopreservation is also the most cost-effective alternative for maintaining genetic resources because it reduces costs for animal and facility maintenance, personnel, and space. In addition, it provides novel opportunities to develop new types of research using large numbers of lines. For example, several genetic strategies, such as TILLING-or enhancer and gene trapping-depend on the use of cryopreservation to bypass generations of live organisms until a strain is revived for research.This chapter describes and discusses the current cryopreservation method used at the Zebrafish International Resource Center. This method is derived from the initial protocol developed for zebrafish over 20 years ago that has recently been refined.