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

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.

KEYWORDS:

• ¹H-NMR
• metabolomics
• serum
• ruminal fluid
• protocol
• livestock

Acknowledgements

The authors acknowledge the funding provided by FAPESP (2020/08845-3), Fundect-MS and CNPq as these entities played a crucial role in advancing our study and facilitating the necessary resources for data collection, analysis, and interpretation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical statement

This study was conducted at the Ruminant Nutrition Facility and Animal Nutrition Laboratory of the School of Agrarian Sciences of Federal University of Grande Dourados, MS, Brazil. All procedures were approved by the Institutional Animal Care and Use Committee Guidelines at the Ethics Committee from Federal University of Grande Dourados (approval protocol: 023/2015 CEUA/UFGD).

Additional information

Funding

This research project received financial support from the São Paulo Research Foundation (FAPESP) grant number 2020/13939-7, 2020/08845-3, and 2021/12694-3; the Foundation for the Support of Teaching, Research, and Extension of Mato Grosso do Sul (Fundect-MS) 71/07187/021 - Chamada FUNDeCT n 12/2021; and the National Council for Scientific and Technological Development (CNPq) - PDPG CAPES/CNPQ - 310837/2020-0. The generous funding provided by these entities played a crucial role in advancing the study and facilitating the necessary resources for data collection, analysis and interpretation.