Room-Temperature Storage of Zebrafish and Medaka Sperm Using Lactic Acid-Stabilized L-15 Medium

Zebrafish offer numerous advantages as a vertebrate model because of their rapid development, high fecundity, transparent embryos, and ease of genetic manipulation. A wide variety of transgenic and mutant fish lines have been generated, and efficiently sharing these resources is crucial for advancing research. Zebrafish lines have typically been exchanged as early embryos, adult fish, or cryopreserved sperm, making transportation costly and logistically challenging. Here, we provide a protocol for preserving functional zebrafish sperm for more than 7 days at room temperature and subsequent in vitro fertilization using the preserved sperm. In this protocol, sperm collected either from the cloaca of an anesthetized male or from dissected testes is stored in L-15-based storage medium. Importantly, the storage medium, originally developed for zebrafish, is also applicable to medaka, another widely used vertebrate model. This sperm storage method allows researchers to ship sperm using low-cost methods and to investigate key factors for motility and fertilizing ability in those sperm. Key features • Provides conditions for room-temperature preservation of zebrafish and medaka sperm for at least one week, based on Takemoto et al. [1]. • Introduces an in vitro fertilization method to achieve high fertilization rates with sperm samples preserved at room temperature. • Enables the easy exchange of fish lines across countries via low-cost postal shipping.