Preparation of ruminal fluid and serum samples from beef cattle for nuclear magnetic resonance based–metabolomics

Abstract

ABSTRACT We evaluated different sample preparation methods for one-dimensional proton nuclear magnetic resonance metabolomics analysis of beef cattle ruminal fluid and serum. After collecting and pooling samples from five bulls (all of which received the same diet and were subjected to the same management conditions), we prepared the samples with the following methods: metabolite extraction using methanol and chloroform (M + C); metabolite extraction using methanol, chloroform and acetone (M + C + A); filtering with a 3-kDa filter; and filtering with a 2-μm filter. We evaluated the methods based on the spectral quality, reproducibility, ease of processing, speed and relative cost. The use of 2-μm filters did not produce good-spectra quality for serum samples, but it was a promising method for ruminal fluid. The yields of metabolites were similar for the the solvent extractions (22 metabolites for ruminal fluid and 29 for serum samples), but lower compared with the filter methods (31 metabolites for ruminal fluid and 47 for serum samples). The filters resulted in a higher concentration of metabolites and better reproducibility. In conclusion, the 2-μm and 3-kDa filter methods represent a suitable way to prepare biological samples from beef cattle for nuclear magnetic resonance – based metabolomics.