AbstractOver the last decade, the African turquoise killifish,Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. However, rearing this fish in captivity can pose challenges, due to the short window of fertility, inbreeding problems, and the continuous maintenance of different strains and transgenic lines. To date, the main means of long term strain maintenance is to arrest embryos in diapause, a poorly understood and unreliable method. To solve these problems, we developed a robust protocol to cryopreserve sperm and to revive them forin vitrofertilization (IVF), as a better option for long term storage ofN. furzerilines. We tested a variety of extender and activator buffers for spermin vitrofertilization, as well as cryoprotectants to achieve maximal long term storage and fertilization conditions tailored to this species. Our optimized protocol was able to preserve sperm in a cryogenic condition for months and to revive an average of 40% upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~25% and peaks of ~55% fertilization. This technical advance will greatly facilitate the use ofN. furzerias a model organism.