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bioRxiv · 10.1101/2025.10.09.681431

Long Term Culture of Germ-Free Zebrafish Using Gamma-Irradiated Feeds

Okyere, L.·Di Fulvio, A.·Gaulke, C. A.
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Abstract

Host associated microbiota play essential roles in regulating digestion, nutrient acquisition, immunity, and xenobiotic metabolism. Disruption of these communities is linked to numerous diseases and health defects though causal mechanisms underpinning these associations remain unclear in most cases. Gnotobiotic zebrafish provide a scalable low-cost in vivo model that is increasingly used to resolve causality in host-microbiota interactions. However, reliance on live diets limits the use of gnotobiotic zebrafish to early life stages where body systems and microbial communities are incompletely developed. As a result, many important host-microbiota interactions may be unable to be studied in this model system. Here we tested a simple method for long-term husbandry of gnotobiotic zebrafish using gamma-irradiated chow diets and evaluated effects on growth, gene expression, and microbial community composition. In conventionally reared animals, gamma irradiated diets did not affect growth or survival and only modestly impacted microbial community composition and diversity. In contrast, gnotobiotic zebrafish maintained on sterile irradiated diets for 55 days post fertilization were smaller, weighed less, and exhibited aberrant genes expression profiles relative to controls. These genes were enriched for pathways, related to immune response, xenobiotic metabolism, organ development, liver function, and lipid metabolism, with many expression patterns linked to the abundance of specific microbial taxa. Together, these findings establish a practical protocol for long-term maintenance of gnotobiotic zebrafish and extend the utility of this model to study microbiome-dependent effects on host physiology, and development beyond early larval stages of life. IMPORTANCEWhile the gnotobiotic zebrafish have been a powerful model for interrogation of host-microbiota interactions, their use has been limited to early life stages due to complications of long-term husbandry. To address this limitation, we developed a simple protocol that enables rearing germ-free zebrafish well beyond larval stages. Germ free fish exhibit physiological and developmental defects that mirror those described in mammalian counterparts supporting a conserved role for microbiota in vertebrate development and physiology. Our protocol provides a method to investigate microbial influences on adaptive immunity, metabolism, and chronic disease processes in zebrafish not possible with current methodologies. Given the rapid and simple methods for gnotobiotic derivation and the large number of transgenic animal lines available for zebrafish we anticipate this model will accelerate mechanistic discovery of microbial impacts on host health.

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Okyere, L., Di Fulvio, A., Gaulke, C. A.. 2025-10-09. Long Term Culture of Germ-Free Zebrafish Using Gamma-Irradiated Feeds. https://doi.org/10.1101/2025.10.09.681431

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