Probiotics are believed to contain active components that affect fish reproduction. This study aimed to evaluate the metabolite compounds contained in the probiotic Bacillus sp. NP5 and its effect on female catfish broodstock rematuration. The first step was to analyze the metabolite content of probiotic using liquid chromatography with tandem mass spectrometry LCMS/MS. Furthermore, feed supplementation was implemented with probiotic at concentrations of 106 CFU g-1 (P6), 108 CFU g-1 (P8), and control (C). Parameters observed included metabolite content of Bacillus sp. NP5, gonadosomatic index (GSI), hepatosomatic index (HSI), gonad histology, gonad maturity rate, fecundity, number of matured eggs, distribution of egg diameter, fertilization rate (FR), hatching rate (HR), larval survival (SR), rematuration period, and percentage of matured broodstock. The results showed that metabolites such as resveratrol (22.45%) and indoline (7.11%) are probably directly affecting reproduction. Other metabolites such as oxyresveratrol (22.95%) and vestitol (4.14%) are also thought to play an indirect role. Supplementation results showed the best reproductive performance in 108 CFU g-1 feed treatment with an increase in GSI (14.74%), HSI (1.65%), gonad maturity rate, percentage of mature eggs (86.00%), rematuration period (4 weeks), percentage of matured broodstock (100%), fecundity (243605.02 eggs/broodstock), FR (98.74%), HR (80.62%), and SR (87.31%). Application of probiotic Bacillus sp. NP5 can accelerate rematuration and improve reproductive performance of female catfish broodstock. Probiotics contain metabolites that have a positive effect on reproduction and can be an alternative in improving reproductive performance in female catfish broodstock. Keywords: Bacillus sp. NP5, female catfish, metabolites, probiotics, rematuration
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