Computational protocol: Omega-3 and alpha-tocopherol provide more protection against contaminants in novel feeds for Atlantic salmon (Salmo salar L.) than omega-6 and gamma tocopherol

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Protocol publication

[…] Subsequently the dried polar metabolite fraction was resuspended in 200 μl D2O with 1 mM TMSP and transferred to NMR tubes. All samples were maintained at 277 K and analysed within 48 h of resuspension. NMR was performed on a Bruker DRU 600 NMR spectrometer (600.23 MHz for 1H) fitted with a 5 mm CPQCI cryogenic probe (Bruker Corporation). Three mm NMR tubes were used with the Bruker Sampletrack autosampler in which the samples were kept at 279 K before (and after) analysis. The spectra were recorded at 300 K with suppression of the residual water resonance using the noesygppr1d pulse sequence from the Bruker pulse sequence library. A pulse width of 7.91 μs was used to collect 128 free induction decays with 32 K data points with a spectral window of 12,019 Hz (20 ppm). The acquisition time was 2.73 s and the interscan delay was 3 s. The noesy mixing time was 10 ms, the data were zero filled to 64 K and exponential line broadening of 0.3 Hz applied before Fourier transformation and the spectra were phased and baseline corrected. [...] The processed NMR spectra were imported into Matlab (The Mathworks, Inc.) using ProMetab v3.3 software . The region from 10 to 0.5 ppm was imported with a resolution of 0.02 ppm which resulted in 4750 data points, and transformed using a generalized log transformation. PCA and PLS-DA analysis was performed in PLS-toolbox v7.0.1 (Eigenvector Research, Inc.) on normalized and mean centred data prior to multivariate statistical analyses. The quantitative data from NMR were first subjected to the Shapiro–Wilk test for normality and the metabolites were subjected to the Kruskal–Wallis analysis of variance followed by Games–Howell post hoc testing in order to assess the comparisons of statistically significant changes between groups compared to the control. […]

Pipeline specifications

Software tools SampleTrack, ProMetab
Applications Miscellaneous, NMR-based metabolomics
Organisms Salmo salar
Chemicals Vitamin D