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

Abstract

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.