Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)).

Abstract

Simple SummaryFor scientific studies on the zebrafish model, simple and routine reproductive procedures should be used to ensure stable and repeatable results. When the milt is collected, spermatozoa are spontaneously activated by urine or excrement (low osmolarity) which routinely contaminates the samples, because of the minuscule size of the fish body. Therefore, whenever milt is collected from a zebrafish for short-term milt preservation and artificial fertilization, milt must be collected into an immobilizing solution, which because of its high osmolarity stops the movement of spermatozoa and keeps the sperm immobile until fertilization. Usually, the spermatozoa showed forward movement during the 35 s period following dilution in water. The sperm concentration ranged from 0.08 to 3.52 × 109/mL with a volume from 0.1 to 2.0 μL per male. The most suitable extender proved to be E400, which allowed storage of sperm for fertilization for 6 to 12 h at a temperature of 0–2 °C. To achieve a good level of fertilization and hatchability, a test tube with a precisely defined amount of sperm with extender, eggs and activating solution proved to be the most effective.The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0–2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40–60%, respectively, with milt stored for 1.5 h in the E400 extender at 0–2 °C.