Phytogenic diet enhances the growth and disease resistance of the black-spotted frog, Pelophylax nigromaculatus, by stabilizing intestinal homeostasis and boosting innate immunity.

Amphibian aquaculture has rapidly expanded due to the increasing global demand for animal proteins. Among cultured species, black-spotted frog (Pelophylax nigromaculatus) has gained attention because of its high economic and market value. However, frequent disease outbreaks under intensive farming conditions considerably hinder its sustainable growth. Recently, plant-derived functional additives have emerged as promising alternatives to enhance disease resistance in aquaculture due to their immunostimulatory, antioxidative, and gut microbiota-modulating activities. This study evaluated the effects of different concentrations of a herbal supplement (HS) containing 0.32 % ginsenosides and 20 % ginseng polysaccharides on P. nigromaculatus growth performance and disease resistance over a 60-day feeding trial. Compared to the controls, frogs fed HS-supplemented diets showed significantly higher specific growth and weight gain rates (P < 0.05). Serum antioxidant enzyme activity was significantly elevated in the HS groups (P < 0.05). Intestinal barrier-related gene (zona occludens 1 and 2 and claudin 3 and 5) levels were upregulated, and antimicrobial peptide gene OL-CATH1 levels were significantly increased, whereas pro-inflammatory cytokine gene (interleukin-10 and -12) levels were markedly downregulated (P < 0.05), suggesting enhanced innate immunity without systemic inflammation. Moreover, 16S rRNA sequencing revealed a notable increase in beneficial gut bacteria abundance and reduction in pathogenic species abundance in HS-treated frogs, indicating a favourable shift in intestinal microbiota composition. In the pathogen challenge test with Aeromonas hydrophila, HS-fed frogs exhibited significantly higher survival rates than the controls (P < 0.05). Validation trial revealed that Lactococcus lactis dietary supplementation improved the frog growth performance and gut microbial stability. Overall, HS enhanced the growth performance and disease resistance by modulating the gut microbiota, boosting the antioxidant capacity, and promoting the innate immune responses of P. nigromaculatus. Our findings suggest 0.04 % HS as a safe, eco-friendly, and sustainable functional additive for amphibian aquaculture.