https://doi.org/10.1038/s41598-022-06507-x
“The mobilization of body reserves during the transition from pregnancy to lactation might predispose dairy cows to develop metabolic disorders such as subclinical ketosis or hyperketonemia. These conditions are not easily identifiable and are frequently related to other diseases that cause economic loss. The aim of this study was to evaluate the serum metabolome differences according to the β-hydroxybutyrate (BHB) concentration. Forty-nine Holstein Friesian dairy cows were enrolled between 15 and 30 days in milk. According to their serum BHB concentration, the animals were divided into three groups: Group 0 (G0; 12 healthy animals; BHB ≤ 0.50 mmol/L); Group 1 (G1; 19 healthy animals; 0.51 ≤ BHB < 1.0 mmol/L); and Group 2 (G2; 18 hyperketonemic animals; BHB ≥ 1.0 mmol/L). Animal data and biochemical parameters were examined with one-way ANOVA, and metabolite significant differences were examined by t-tests. Fifty-seven metabolites were identified in the serum samples. Thirteen metabolites showed significant effects and seemed to be related to the mobilization of body reserves, lipids, amino acid and carbohydrate metabolism, and ruminal fermentation.”
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Metabolomic analysis
The metabolomics investigation was carried out through an NMR analysis solution with 10 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) in D2O set at pH 7.00 ± 0.02 by means of 1 M phosphate buffer containing 2 mM NaN3. TSP was used as an NMR chemical-shift reference, while NaN3 avoided microbial proliferation, as suggested by56.
Serum samples were prepared for 1H-NMR by thawing and centrifuging 1 mL of each sample for 15 min at 18,630g and 4 °C. The supernatant (700 μL) was added to 100 μL of NMR analysis solution. Finally, each sample was centrifuged as previously mentioned.
1H-NMR spectra were recorded at 298 K with an AVANCE III spectrometer (Bruker, Milan, Italy) operating at a frequency of 600.13 MHz, equipped with the software Topspin 3.5. According to56, the signals from broad resonances originating from large molecules were suppressed by a CPMG filter comprised of 400 echoes with a τ of 400 μs and a 180° pulse of 24 μs for a total filter of 330 ms. The water residual signal was suppressed by means of a presaturation technique. This setting employed the cpmgpr1d sequence, part of the standard pulse sequence library. Each spectrum was acquired by summing 256 transients using 32 K data points over a 7184 Hz spectral window, with an acquisition time of 2.28 s and a relaxation delay of 5 s.
The spectral phase was manually adjusted in Topspin, while the subsequent adjustments were performed in R computational language by means of a script developed in-house57. After the removal of the residual water signal, the1H-NMR spectra were baseline-corrected by means of peak detection, according to the “rolling ball” principle58, implemented in the baseline R package59. The signals were assigned by comparing their chemical shift and multiplicity with the Chenomx software library (Chenomx Inc., Canada, ver. 8.3).
The molecules of the first serum sample analyzed were quantified by means of an external standard by taking advantage of the principle of reciprocity60. Differences in water content among samples were then taken into consideration by probabilistic quotient normalization61. Molecule quantification was performed by means of rectangular integration, considering one of the corresponding signals free from interferences.
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