A sampling method for differentiating breath and ruminal exhaled volatile organic compounds in dairy cows using methane as a marker

Frequent eructation in ruminant animals results in an exhaled blend of ruminal eructed and breath volatile organic compounds (VOC). The physiological distinction between the gas sources can limit the applicability of breath metabolomics (or breathomics) in describing the metabolic phenotype of cows. The objective of this study was to establish a benchmark sampling method for collecting breath samples in dairy cows while they were not eructating. Twelve multiparous mid-lactation Holstein cows were enrolled to collect (1) breath (BR; bloodborne VOC exchanged at the lungs) and (2) ruminal exhaled (RE; a mixture of VOC from ruminal eructation and breaths during eructations) samples. Gas samples were collected using a head chamber (GreenFeed system) with real-time CH₄ readings. By monitoring eructation events, a threshold of <150 mV CH₄ was set to sample breath, and >250 mV was used to collect BR and RE. Both samples were analyzed using secondary electrospray ionization-high resolution MS (SESI-MS) and GC. Implementing CH₄ as a marker resulted in 80% lower CH₄ concentrations in BR compared with RE. Analysis using SESI-MS revealed a total of 324 and 242 features consistently identified across all periods of the study in [M-H]⁻ and [M+H]⁺ MS ion mode, respectively, for BR and RE. In BR, 18 features exhibited greater concentrations, whereas 8 had a tendency to have greater concentrations compared with RE. In contrast, RE revealed 51 features with greater concentrations, and 13 with a tendency for greater concentrations compared with BR. Ruminal VFA acetate, propionate, and butyrate were 20.9%, 27.4%, and 32.7% greater in RE compared with BR, respectively. Lower CH₄ levels in BR and the greater VFA concentrations in the RE validated the ability of the method to differentiate breath from ruminal eructed VOC. Our study established a method to distinguish and separately collect BR and RE samples in dairy cows. This advance shows the potential to use breathomics as a reliable and noninvasive tool for metabolic assessments in ruminant research.