Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperminjection

To evaluate the potential for fertilization by sperm injection into fish eg gs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological chan ges of gametes on the possible performance of intracytoplasmic sperm inject ion (ICSI), four different combinations of injection conditions were conduc ted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval sta ges, and another 3 (2%) developed normally and hatched. The highest fertili zation rate (blastodisc formation) was achieved by injection of activated s permatozoa into nonactivated eggs (35%). Injections were most effective whe n performed within the first hour after egg collection. Flow cytometric ana lysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Ou r study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.