Temperature-dependence of early development of zebrafish and the consequences for laboratory use and animal welfare.

Zebrafish (Danio rerio) are widely used in biological research, but the impact of incubation temperatures on developmental endpoints is still insufficiently studied. This study quantifies developmental differences in zebrafish embryos incubated at 26°C and 28°C, focusing on key endpoints (heartbeat onset, hatching time, eye size, yolk sac consumption, and body length). For this purpose, we recorded a high-resolution time series comprising hourly observations of early developmental stages and key events and bi-hourly observations of body length until 120 hours post fertilization. Additionally, we recorded a low-resolution time series at 72, 96, and 119 hours post fertilization for detailed measurements of eye size, yolk sac area, and body length. Embryos incubated at 26°C showed consistent delays in developmental stages compared to those at 28°C, with delays becoming more pronounced at later stages. Yolk sac consumption was delayed by about 19.8 hours at 26°C by 119 hours post fertilization, suggesting a delayed onset of independent feeding. These findings suggest that time-based regulatory limits for rearing zebrafish, such as the 120-hour threshold in German regulations (TierSchVerV), do not fully account for temperature-dependent development. The results emphasize the need for guidelines linking incubation temperatures to developmental progress.