Effects of Non-Feeding on Development in a Teleost, Minami-Medaka, Oryzias latipes: Identification of Eleutheroembryonic Stage for Potential Alternative Regulatory Toxicology Tests Along the 3R Principles.

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Fish in the eleutheroembryonic life stage are defined as embryos or hatched fry before external self-feeding begins, and this stage is not classified as a protected life stage according to the EU (Directive 2010/63/EU) because of its alignment with the 3R principles (replacement, reduction, and refinement). In Minami-medaka (Oryzias latipes), the eleutheroembryonic stage is considered to extend until hatching, according to OECD TG210, whereas no supporting evidence to identify this stage has yet been reported. To clarify the medaka eleutheroembryonic stage, we investigated the effects of non-feeding on survival, growth, and gene expression in the NIES-R, Hd-rR, and d-rR strains. Non-feeding did not affect survival up to 6 days post-hatching (dph) in any strain, with survival rates exceeding 80%. However, non-feeding beyond 8 dph reduced the survival rates to below 50% at 30 dph. Fish growth, measured as total length, was not significantly affected by non-feeding up to 6 dph, except for the Hd-rR. Analysis of differentially expressed genes (DEGs) in response to non-feeding revealed that autophagy-related DEGs (wipi2, wdr45, wipi1, atg14, and map1lc3b) were found from 43 autophagy-related genes. map1lc3b and the other DEGs were upregulated after 4 and 6 days of non-feeding, respectively. However, the effect of non-feeding up to 6 dph was rescued by feeding. Together, the medaka fry < 6 dph were considered to be in the eleutheroembryonic stage for at least up to 4 dph, suggesting that hatched fry can be used to evaluate chemical toxicity and endocrine-disrupting activity according to the 3R principles. This study indicated that non-feeding did not affect survival up to 6 dph in NIES-R, Hd-rR, and d-rR strains of Minami-medaka, and fish growth was not affected by non-feeding up to 6 dph, except in the Hd-rR strain. Additionally, autophagy-related gene expression upregulated by non-feeding but was rescued by feeding. Together, we demonstrate for the first time that fry < 6 dph are in the eleutheroembryonic stage for at least up to 4 dph in Minami-medaka (Oryzias latipes).