Nuclear magnetic resonance -based metabolomics was applied to study effects of egg aging on ovarian fluid metabolites in rainbow trout, Oncorhynchus mykiss. The eggs of three females were pooled and then assigned to three plastic vials for 18 days in vitro storage at 4°C. Ovarian fluid samples were taken 0, 6, 12 and 18 days after storage. Three groups of metabolites including amino acids, osmolytes and energy metabolites were found to change during storage period. The glucose levels of ovarian fluid showed significant decreases on days 12 and 18 after storage (p < .05). For acetoacetate and acetate, significant increases were observed, respectively, on days 12 and 18 after storage (p < .05). The creatine levels of ovarian fluid increased significantly on days 12 and 18 after storage (p < .05). Lactate levels in ovarian fluid elevated during storage period (p < .05). Glycerol levels showed a significant increase on days 12 and 18 after storage (p < .05). The values of osmolytes in ovarian fluid (e.g., betaine, taurine, trimethylamine-N-oxide, N,N-dimethylglycine) showed a decreasing trend on day 12 which continued until the end of storage on day 18 (p < .05). Almost all amino acids elevated on days 12 and 18 after storage (p < .05). After an apparent elevation in isoleucine levels on days 6 and 12, this amino acid decreased on day 18 after storage (p < .05). The osmolytes might act as antioxidant against free-radicals produced as a result of over-ripening. Glucose can be used as energy resource for eggs and bacteria during ova storage. Also, change pattern of amino acids indicate hydrolysis of proteins as the time of storage increases.