Exhalomics as a non-invasive method for assessing rumen fermentation in dairy cows: Can exhaled breath metabolomics replace rumen sampling?

Previously, we utilized secondary electrospray ionization-mass spectrometry (SESI-MS) to investigate the diurnal patterns and signal intensities of exhaled volatile fatty acids (EX-VFA) of dairy cows. The current study aimed to validate the potential of exhalomics approach for evaluating rumen fermentation. The experiment was conducted in a switchback design, with 3 periods of 9 d each, including 7 d for adaptation and 2 d for sampling. Four rumen-cannulated original Swiss Brown (Braunvieh) cows were randomly assigned to 1 of 2 diet sequences (ABA or BAB): (A) low-starch (LS; 6.31% starch of DM) and (B) high-starch (HS; 16.2% starch of DM). Feeding was 1x/d at 0800 h. Exhalome (with the GreenFeed System), and rumen samples were collected 8 times to represent every 3-h of a day, and EX-VFA and ruminal VFA (RM-VFA) were analyzed using SESI-MS and HPLC, respectively. Further, the VFA concentration in the gas phase (HR-VFA) was predicted based on RM-VFA and Henry's Law constants. No interactions were identified between the types of diets (HS vs. LS) and the measurement methods on daily average VFA profiles [ruminal (RM) vs. exhaled (EX) or Henry's Law (HR) vs. exhaled (EX)], suggesting a consistent performance among the methods. Additionally, when the 3-h interval VFA data from HS and LS diets were analyzed separately, no interactions were observed between methods and time-of-day, indicating that the relative daily pattern of VFA molar proportions was similar regardless of the VFA measurement method used. The results revealed that the levels of acetate sharply increased immediately after feeding, trailed by an increase in the A:P ratio and a steady increase for propionate (2 h after feeding in HS, 4 h for LS), and butyrate. This change was more pronounced for the HS than the LS diet. However, there was no overall diet effect on the VFA molar proportions, while the measurement methods affected the molar proportions. Furthermore, we observed a strong positive correlation between the levels of RM and EX acetate for both diets (HS: r = 0.84; LS: r = 0.85), RM and EX propionate (r = 0.74), and RM and EX A:P ratio (r = 0.80). Both EX-VFA and RM-VFA exhibited similar responses to feeding and dietary treatments, suggesting that EX-VFA could serve as a useful proxy for characterizing RM-VFA molar proportions to evaluate rumen fermentation. Similar relationships were observed between RM-VFA and HR-VFA. In conclusion, this study underscores the potential of exhalomics as a reliable approach for assessing rumen fermentation. Moving forward, research should further explore the depth of exhalomics in ruminant studies, to provide a comprehensive insight into rumen fermentation metabolites, especially across diverse dietary conditions.