Serum metabolomics assessment of etiological processes predisposing ketosis in water buffalo through the 1H-NMR spectroscopy

Objectives: The transition period is critically important to health and profitability of dairy buffaloes. The metabolic adaptation to negative energy balance (NEB) induced by the considerable increment of energy and nutrient requirements is still one of the major concerns that may decrease the productivity and predispose to different disorders. An inadequate metabolic adaptation is characterized by elevated concentrations of β-hydroxybutyrate (BHB). Nevertheless, a specifically BHB threshold for dairy buffaloes is not established and dairy cows’ reference are often used. The metabolic processes can be investigated using the metabolomics approach, which reflects the animals’ health status. The aim of the current study was to use the metabolic approach, specifically with the 1H-NMR, to assess the metabolomic profile of Mediterranean buffaloes (MBs) to investigate the metabolic changes associated with different levels of energy deficit. Materials and methods: The current cross-sectional investigation received an institutional approval by the Ethical Animal Care and Use Committee of the University of Naples Federico II (n.PG/2017/0099607). Sixty-two Italian MBs were selected within the entire group of fresh buffaloes (< 50 days in milk) from a single high-yielding dairy farm. All the buffaloes received a complete clinical examination before sampling to exclude clinical ketosis or other pathological statuses. The blood samples were collected from jugular vein into tubes containing clot activator to obtain serum for biochemical and metabolomic analysis. According to serum BHB concentration, animals were divided into two groups: Group healthy (Group - H) consisting of 37 MBs with a level of BHB < 0.70 mmol/L and Group at risk of hyperketonemia (Group - RK) made by 25 MBs with a level BHB ≥ 0.70 mmol/L. The statistical differences for biochemical parameters and metabolite’s concentration were performed by one-way ANOVA and Wilcoxon test according to data distribution. A post hoc pairwise comparison among metabolite concentrations was performed using Bonferroni correction. A p-value<0.05 was accepted, whereas a 0.05≤p-value≤0.10 was considered as trend to significance. A robust principal component analysis (rPCA), a partial least squares-discriminant analysis (PLS-DA) with variable importance in projects (VIP), and the metabolic pathways overrepresentation analysis (ORA) were generated to summarize the structure of the data and to highlight the metabolic pathways influenced by BHB concentration. Results: Among biochemical parameters, only AST was significantly increased in Group - RK. A total of fifty-seven metabolites were identified in serum samples: 27 amino acids and derivates, 9 organic acids, 5 alcohols, 4 carbohydrates, 3 amine and derivates, 2 fatty acids, 2 ketone bodies, 1 sulfone, 1 vitamin, 1 imidazole, 1 nucleoside, and 1 guanidine. Six of the identified metabolites showed a statistically significance, specifically: glycerol, taurine, and creatinine showed a significant reduction in Group - RK, whereas acetone, acetate and 3-hydroxybutyrate showed a significant increase. In addition, six metabolites showed a trend toward significance: methanol, proline, and glycine were reduced in Group - RK, whereas formate, citrate, glutamate were increased. The rPCA analysis failed to cluster groups, while the PLS-DA showed two cluster principally related to acetate, 3-hydroxubutyrate, acetone, and glycerol (VIP > 1.5). The ORA analysis identified five metabolic pathways possibly responsible for changes in metabolome profile: glyoxylate and dicarboxylate metabolism; pyruvate metabolism; glycolysis / gluconeogenesis; glycerolipid metabolism and taurine and hypotaurine metabolism. Conclusions: Metabolomic analysis through ¹H-NMR is a useful tool to achieve knowledge about metabolic profiling related to serum BHB modifications in dairy buffaloes. The metabolic state of our animals at risk of hyperketonemia suggests an initial mobilization of body resources, subclinical inflammation and potential oxidative stress status, changes in ruminal fermentations, influence on urea cycle and thyroid hormone synthesis. This study demonstrates that the metabolomic approach identified potential relationships with the development of subclinical ketosis even if the BHB concentration did not exceed the threshold value.