Validation of the Measurement of Beta-Hydroxybutyrate and Non-Esterified Fatty Acids in Bovine Saliva: A Pilot Report

Abstract

Serum beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) are biomarkers of situations of negative energetic balance in bovine. However, knowledge about their possible measurement and use in saliva is limited. In this report, two commercially available methods for the measurement of BHB and NEFAs were validated for use in bovine saliva. Both methods showed good precision and accuracy. The BHB concentrations were correlated between the saliva and the serum, but not the NEFA concentrations. The cows with hyperketonemia (n = 17) had increased salivary BHB compared to the cows with no clinical signs and no hyperketonemia (n = 34) and those with clinical signs of metritis (n = 17). The salivary NEFA concentration increased in newborn calves (n = 10) on days 1 and 2 of life compared to the day of birth before colostrum intake. The calves with symptomatic bovine respiratory disease complex (BRD, n = 7) showed higher salivary NEFA concentrations than those without clinical symptoms (n = 6). Thus, BHB and NEFAs can be reliably measured in bovine saliva using easily automatable colorimetric methods. Salivary BHB increased in hyperketonemia and could be a potential biomarker of this condition. Further studies should be undertaken to clarify the mechanism and possible use of salivary NEFAs as biomarkers.

Keywords: biomarkers; bovine; energy balance; ketone bodies; saliva.

Figure 1

Linearity under dilution assays performed in saliva samples with high beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs), serially diluted with distilled water. Dotted lines indicate the 95% confidence interval. R²: coefficient of determination.

Figure 2

Regression plots between levels of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) in saliva from 73 adult cows and the color score of the samples. Dotted lines indicate the 95% confidence interval. R²: coefficient of determination.

Figure 3

Regression plots constructed with the values of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) obtained in paired serum–saliva samples from 68 adult cows. Dotted lines indicate the 95% confidence interval. R²: coefficient of determination.

Figure 4

Concentrations of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) in serum (left) and saliva (right) of cows with neither clinical signs nor hyperketonemia (NCS-NH), cows with no clinical signs but hyperketonemia (NCS-H), and cows with clinical signs of metritis without hyperketonemia (CSM-NH). Boxes show first and third quartiles, the line inside the box indicates median value, and whiskers indicate 10–90 percentiles. Statistical results: asterisks indicate significant differences between groups (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001); ns: non-significant. Eta² was 0.54 for serum BHB, 0.40 for saliva BHB, 0.36 for serum NEFAs, and 0.05 for saliva NEFAs (from 0.01 to < 0.05: small effect; from 0.06 to <0.13: moderate effect; ≥0.14: large effect).

Figure 5

Receiver operating characteristic (ROC) curves and area under the curve (AUC) obtained with beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) in serum (left) and saliva (right) for discriminating cows with hyperketonemia (NCS-H group) from those without hyperketonemia (NCS-NH and CSM-NH groups).

Figure 6

Evolution of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) in serum and saliva of newborn calves (n = 10) during the first week of life. Boxes show first and third quartiles, the line inside the box indicates median value, and whiskers indicate 10–90 percentiles. Statistical results: asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ns: non-significant). Kendall’s W was 0.08 for serum BHB, 0.26 for saliva BHB, 0.32 for serum NEFAs, and 0.58 for saliva NEFAs (from 0.1 to <0.3: small effect; from 0.3 to <0.5: moderate effect; ≥0.5: large effect).

Figure 7

Levels of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs) in serum and saliva of calves with no clinical signs (N-BRD, n = 6) and calves with clinical signs of Bovine Respiratory Disease complex (BRD, n = 7). Boxes show first and third quartiles, the line inside the box indicates median value, and whiskers indicate 10–90 percentiles. Statistical results: asterisks indicate significant differences (*: p < 0.05; ns: non-significant). Coefficient r was 0.04 for serum and saliva BHB, and 0.59 for serum and saliva NEFAs (≥0.56: small effect; ≥0.64: moderate effect; ≥0.71: large effect).