Establishment of a Long-Term Germ-Free Medaka Model Reveals Microbiota-Dependent Regulation of Growth, Immunity, and Metabolism

Abstract Germ-free (GF) animal models are indispensable for dissecting host-microbiota interactions and their roles in health and disease. The small teleost fish medaka ( Oryzias latipes ) provides unique advantages for establishing GF models across developmental stages, yet the functions of its intestinal microbiota and metabolites remain poorly characterized. Here, we developed both early-life and chronic GF medaka models to systematically characterize host biology in the absence of microbiota and evaluate the contribution of gut-derived metabolites to growth and immune development. Using a refined sterile feeding and verification protocol, we successfully maintained GF medaka for up to 57 days post-fertilization (dpf). As anticipated, GF fish displayed developmental delays, impaired organogenesis, reduced immune competence, and metabolic dysregulation. Supplementation with sterile gut-derived metabolites partially alleviated these deficits, as evidenced by enhanced locomotor activity and immune responses. Mechanistically, recovery was associated with improved ribosome biogenesis, tricarboxylic acid cycle activity, and histidine and pyruvate metabolism, suggesting enhanced protein synthesis and immune maturation. However, metabolite supplementation also elevated oxidative stress and inflammatory responses and failed to fully restore long-term survival or organ development. Our findings support the use of GF medaka as a versatile platform for investigating microbiota-host interactions across life stages. By integrating metabolite interventions, this model provides critical insights into the functional roles of gut microbiota and offers a valuable tool for advancing microbiome research in health and disease.