Noninvasive Ejaculate Collection from the African Turquoise Killifish Nothobranchius furzeri

Ejaculate collection from live animals is a fundamental technique used to perform repeated measures on the same individual-for instance, to test the effect of different treatments/conditions on the same animal or to conduct longitudinal studies. Moreover, collecting the ejaculate allows one to perform in vitro fertilizations (IVFs) or sperm cryopreservation from valuable individuals (e.g., mutant, transgenic animals) without the need to kill the animals. We developed a noninvasive protocol based on a stripping procedure for collecting the ejaculate from live males of the African turquoise killifish (Nothobranchius furzeri). The proposed procedure allows the retrieval of mature sperm along with the seminal fluid. The ejaculate can be used within 1 h of the collection to perform in vitro fertilizations and/or sperm quality measurements. Contamination by mucus, water, urine, or feces can activate the sperm and/or negatively affect sperm quality, so special care is needed to avoid contamination and thus prolong sperm viability. Since in small fish the amount of ejaculate collected is limited to a few microliters, in the present protocol we also provide recommendations on housing conditions to maximize the probability to obtain ejaculate samples.