C-18: Zebrafish cryobiology: Sperm, oocytes, embryos and stem cells

Abstract

Cryopreservation is a proven method for long-term maintenance of genetic material, nevertheless current protocols for fish are not standardized and yield inconsistent results, threatening the efficacy of large-scale genetic screening and stock centers. Previous reports have dealt with sperm cryopreservation in >200 fish species from marine and freshwater species with the most common observation from these publications being the inconsistency in the post-thaw results. Consortia are working to identify processes in zebrafish protocols to try and improve post-thaw variability. Although a great deal of work has been done examining the cryobiology of zebrafish oocytes at various stages of maturation, the successful cryopreservation, maturation and subsequent fertilization of these oocytes has not been proven. Similarly, zebrafish embryo cryopreservation has been extensively examined for the past 25 years, and several aspects of standard cryopreservation methods have made it a challenging system to develop successful protocols for. These problems include: (1) a large overall size resulting in a low surface to volume ratio that can slow water cryoprotectant efflux and influx; (2) the presence of compartments, such as the blastoderm and yolk; with different permeability properties, (3) susceptibility to chilling injury; and (4) extreme chilling sensitivity at early relatively undifferentiated developmental stages necessitating vitrification. New laser-warming protocols may help change how we understand this complex suite of challenges in zebrafish embryo cryopreservation, especially the relative need for cryoprotectant and the potential for prevention of damaging intracellular ice crystal formation. To diversify and expand our resource collections, however, cryopreserved zebrafish spermatogonial stem cells may provide one of the most important strategies for resource centers. Researchers have successfully developed simple effective cryopreservation methods for zebrafish testes, allowing the rapid and effective preservation of mutant and transgenic lines.