Abstract
Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability 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 of N. furzeri as a model organism.
Highlights
Abbreviations HBSS Hank’s Balanced Salt Solution BSMIS Buffered sperm motility-inhibiting solution FBS Fetal bovine serum DMSO Dimethyl sulfoxide DMF Dimethylformamide MetOH Methanol DMA Dimethylacetamide in vitro fertilization (IVF) In vitro fertilization
Over the last few years, the African killifish Nothobranchius furzeri has emerged as an important model system for the study of vertebrate biology
Overall we found that sperm cryopreserved using 10% DMSO as cryoprotectant maintained the highest level of activation (Fig. 4A), followed by methanol
Summary
Abbreviations HBSS Hank’s Balanced Salt Solution BSMIS Buffered sperm motility-inhibiting solution FBS Fetal bovine serum DMSO Dimethyl sulfoxide DMF Dimethylformamide MetOH Methanol DMA Dimethylacetamide IVF In vitro fertilization. N. furzeri husbandry requires considerable space, since they are often optimally grown when singly housed because of fish-to-fish aggression and food competition[7]. Given these constraints, it is essential to develop protocols to maintain stocks without constant breeding. As the number of strains and lines increase, this method becomes untenable at larger scales To solve these problems, fish researchers rely on sperm cryopreservation and in vitro fertilization techniques. There is no established protocol available for sperm cryopreservation and in vitro fertilization for killifish species. Our method should facilitate the husbandry and the usefulness of N. furzeri as a model organism for research
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