THE ACTIVATION OF ZEBRAFISH SPERM CELLS IN A MICROMIXER

摘要

The freshwater fish, Danio rerio (zebrafish), have become widely used as a model organism for vertebrate development, DNA mutation, and human disease studies [1]. Maintaining live colonies of the numerous developed strains of zebrafish under investigation can be prohibitively costly. As such, there is a growing need to catalog their reproductive cells and have them available on demand [2]. Thus cryopreservation of model strain gametes has become an important endeavor, where evaluation of freezing and thawing techniques is currently a bottleneck to these procedures. Zebrafish egg fertilization occurs externally and therefore sperm must encounter a hypo-osmotic environment en route to the egg [3]. Once active, the cells maintain peak motile activity for ～60 seconds. Evaluation of cryopreservation protocols consists primarily of motility analysis, where viability is ascertained by the percentage of cells that swim following activation. Traditionally activation is performed by hand, where a technician adds water to a sperm sample on a microscope slide. This subjective process introduces errors in non-systematic dilution and bias in evaluating motility. Recent advances in computer assisted sperm analysis (CASA) have helped reduce this bias, but the process is slow and still suffers from a lack of reproducibility.