Intracytoplasmic sperm injection (ICSI) in zebrafish eggs

Direct injection of sperm into the cytoplasm of oocytes was first reported in sea urchin and amphibians more than 30 years ago. More recently, intracytoplasmic sperm injection (ICSI) has become an area of intensive research due to its potential for stock improvement in domestic animals. In humans ICSI is used to overcome problems related to male infertility. The value of this technique has also been recognized for the propagation of endangered species with reproductive difficulties in captivity. However, aside from the original studies of sperm injection in echinoderms, no other work has been reported in aquatic organisms. The goal of this study was to evaluate ICSI for application to finfish. Using wild-type, pigmented zebrafish Danio rerio as a model we were able to inject sperm into eggs and obtain phenotypically normal larvae. As with other organisms, we found that injection of sperm can cause lethal damage. However, unlike mammals, in which non-viable embryos only undergo early development, zebrafish developed beyond gastrulation despite altered phenotypes. From 84 eggs injected with single sperm cells, 7 embryos (8.3%) developed to the gastrula stage, of these 4 (4.8%) developed abnormally and 1 (1.2%) developed normally and hatched. Measurement of the DNA content of the hatched fish (by flow cytometry) showed it to be diploid. However, with these experiments we were not able to verify the contribution of male and female gametes. To overcome this we used a zebrafish line possessing a recessive phenotype for pigment (gold) as an egg donor, together with sperm from zebrafish carrying the dominant phenotype for pigment (wild-type). From 93 eggs injected, 9 (9.7%) of the embryos developed to the gastrula stage, of these 5 (5.4%) developed abnormally and 2 (2.1%) developed normally and hatched. All of the embryos that developed beyond 48 hr displayed pigmentation which verified fertilization by the injected sperm. These results show that ICSI is possible in fishes and opens the door to new areas of research, ranging from basic reproductive biology to the production of hybridization or heterozygous androgenesis (all-paternal inheritance) that otherwise would not be possible. Another potential application is the use of sperm preserved by methods other than freezing, which would reduce storage costs.