The Zebrafish Obesogenic Test (ZOT) assesses obesogenic or anti-obesogenic effects through a whole-organism short-term assay, providing relevant information for environmental and human risk assessments. The ZOT is a cutting-edge method developed by our team to assess the effects of diet, drugs, and environmental contaminants on white adipose tissue (AT) dynamics in live zebrafish larvae.

The ZOT uses Nile Red as a lipophilic fluorescent probe to reveal adipocyte lipid droplets, allowing for the measurement of their size before and after exposure to tested molecules or mixtures. By evaluating obesogenic or anti-obesogenic effects through a whole-organism short-term assay, ZOT provides valuable information for environmental and human risk assessments, adverse outcome pathways mechanistic studies, and screening processes.

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The development of ZOT is detailed in various scientific publications, including Tingaud-Sequeira et al. (2011) and Ouadah-Boussouf and Babin (2016). As part of the OBERON European project, our lab further optimized the ZOT protocol, as described in Kassir et al. (2025, Biological Methods and Protocols, Accepted). This optimization includes improvements in staining, image acquisition, and image processing, resulting in a more accurate and efficient assessment of adiposity in two sub-locations of subcutaneous adipose tissue (SAT) – anal fin ray SAT (AFRSAT) and ventral SAT (VSAT).

To enhance protocol reproducibility and image processing speed, we developed the ZOT analysis software (ZOTAS) a deep-learning-based program that automatically processes ZOT fluorescence images.