Temperature-dependence of Early Development of Zebrafish and the Consequences for Laboratory Use and Animal Welfare

Zebrafish ( Danio rerio ) is widely used as a model organism in biological research. However, the impact of different incubation temperatures on specific developmental endpoints is still insufficiently studied.

This study quantifies developmental differences of zebrafish embryos incubated at two commonly used temperatures (26 °C and 28 °C), focusing on key developmental endpoints (onset of heartbeat, hatching time, eye size, yolk sac consumption, 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 swim bladder size.

Embryos incubated at 26 °C consistently exhibited delays in reaching developmental stages compared to those reared at 28 °C. Significant differences were apparent from the earliest stages of development, with the delays becoming more pronounced as the embryos progressed to more complex stages. Yolk sac consumption was delayed by approximately 10 hours at 26 °C by 120 hours post fertilization, indicating a delayed onset of independent feeding. These data suggest that exclusively time-based regulatory limits for rearing zebrafish eleutheroembryos, such as the 120 hours post fertilization threshold commonly interpreted from the German regulation TierSchVerV (Tierschutz-Versuchstierverordnung), do not adequately account for the temperature-dependency of zebrafish development.

The results highlight the need for guidelines that link incubation temperatures to developmental progress.