Evaluation of sample preparation methods for NMR-based metabolomics of Atlantic salmon (Salmo salar) ovarian fluid

Average embryo survival in farmed Atlantic salmon (Salmo salar) has declined sharply from about 80% to below 50% in North America over the past two decades. The underlying causes remain unclear, and no effective solutions currently exist for this problem, posing a major challenge to the production of a leading aquaculture species in the USA. Traditional selective breeding programs typically assign "breeding values" to broodstock fish based on growth-related traits; however, incorporating reproductive success traits (e.g., embryo hatch rates, metabolic biomarkers) into breeding value calculations may improve the efficiency of broodstock selection and help identify individuals more likely to produce viable offspring. Ovarian fluid (OF), collected non-lethally during egg harvesting, offers a valuable biological matrix for metabolomics-based biomarker discovery for fish reproduction. This study tested seven sample preparation methods for nuclear magnetic resonance (NMR) metabolomics analysis of OF: (a) filtration; (b) protein precipitation; (c) lyophilization; (d) dilution; (e) lyophilization-filtration; (f) dilution-filtration; (g) filtration-concentration. The objectives were to confirm that OF contains measurable metabolites, and to establish a repeatable and practical OF sample preparation method for NMR metabolomic analysis. Among the methods tested, filtration-concentration produced the most consistent quality spectra with lower variability, higher signal-to-noise ratio, and ease of execution compared with alternative methods tested. In addition, this study demonstrates that blood contamination that may accidentally occur during ovarian fluid collection can significantly alter OF metabolite profiles, potentially affecting downstream analyses and data interpretation. These findings provide a foundation for standardized NMR metabolomics in salmon broodstock reproductive research.