Pathway mapping of exhaled volatile organic compounds associated with blood and ruminal fluid metabolites to describe the nutritional and metabolic status of lactating dairy cows

Exhaled breath offers an interesting matrix of low invasive sampling of potentially relevant information about the organism's metabolism in the form of volatile organic compounds (VOC). The VOC can be exhaled by the ructus (Islam et al., 2023) or passed the blood-lung barrier for expiration through the lungs. In this work, we consider exhaled breath as a mixture of VOC derived from the lungs and from the upper gastrointestinal tract. However, the informative value of exhaled breath in ruminants remains largely unstudied. The aim of this study was to identify exhaled VOC that could be used to assess the nutritional and metabolic status of dairy cows. To do so, we performed untargeted analysis of exhaled VOC from dairy cows, investigated their correlations with commonly analyzed blood and ruminal fluid metabolites and the calculated energy balance (EB), and explored the underlying pathways of correlated exhaled VOC. This was done as part of a feeding experiment in which 32 lactating Holstein dairy cows were assigned to 2 basal diets for 12 weeks. Half of the cows were fed a hay-based diet, and the other half were fed a silage-based diet. During experimental wk 1-8, half of the cows in each basal diet group were supplemented with a control concentrate, and the other half received an experimental concentrate containing essential oils. During experimental wk 9-12, all cows received the control concentrate. Exhaled breath, blood, and ruminal fluid samples were collected every 4 experimental weeks (W4, 8, and 12) on 3 consecutive sampling days. Exhaled breath was analyzed for VOC, ruminal fluid for VFA and ammonia, and serum samples for albumin, total protein, urea, glucose, cholesterol, BHB, and nonesterified fatty acid (NEFA) concentrations. Pearson correlations were calculated to assess the associations between exhaled VOC and concentrations of blood and ruminal fluid metabolites and the calculated EB. Fifteen correlations were found between exhaled VOC (tetradecanal and γ-hydroxybutyrate (GHB), 3-penten-2-one, 4-hydroxy-4-methylpentan-2-one, 2-ethylhexanal, 2-ethylhexan-1-ol, p-cymene) and ruminal fluid (acetate, butyrate, valerate, and ammonia) and blood metabolite concentrations (BHB, NEFA, glucose, urea, and cholesterol) across the cow groups. The underlying pathways of 3-penten-2-one, GHB, and tetradecanal were mainly related to fat and protein catabolism and therefore to the actual animal metabolism. The correlations with the other 4 exhaled VOC, 4-hydroxy-4-methylpentan-2-one, p-cymene, 2-ethyl-hexan-1-ol, and 2-ethylhexanal, were diet- or time-related, specifically due to differences in feed ingredients. The results demonstrate the associations of single exhaled VOC with the nutritional and metabolic status of healthy dairy cows. Their potential as new biomarkers should be further investigated in cows in various nutritional and metabolic states.