Abstract
BackgroundSalivary alpha-amylase (sAA) is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and also by changes in its isoforms, although this last way of evaluation has never been used in veterinary medicine. This research evaluated the changes of sAA by three different ways in which sAA can be evaluated in an experimental acute stress model in six pigs based in a technique of temporarily restraining. These ways of evaluation were 1) activity by a spectrophotometric assay, 2) concentration by a fluorometric assay, and 3) isoforms of the enzyme by a Western blot.ResultsAlthough salivary cortisol significantly increased due to the stimulus of stress and all the pigs manifested signs of stress by high-pitched vocalization, sAA activity showed an increase of different degree in the six pigs after the stress stimulus, while sAA concentration showed decreases in four of the six pigs. sAA activity did not correlate with sAA concentration or salivary cortisol, and a low correlation was observed between sAA concentration and salivary cortisol (r = 0.48, p = 0.003). The inter-individual variability was higher in sAA activity than in sAA concentration and salivary cortisol. Finally, three possible isoforms of sAA at 154–160 kDa, 65–66 kDa and 59–60 kDa were observed that showed different dynamics after the stress induction.ConclusionsAlthough this pilot study’s results should be taken with caution due to the low sample size, it reveals a different behavior between sAA activity and concentration in pig after an acute stressful stimulus leading to evident external signs of stress by high-pitched vocalization, and opens a new field for the evaluation of possible selected isoforms of sAA as potential biomarkers of stress.
Highlights
Salivary alpha-amylase is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and by changes in its isoforms, this last way of evaluation has never been used in veterinary medicine
Activity and concentration of alpha-amylase in saliva Salivary alpha-amylase (sAA) activity (Fig. 1a) showed a significant change between the times (χ2 = 12.76, df = 5, P = 0.026), but the multiple comparisons test did not show any significant increase at any time (P > 0.08 for all times compared). sAA concentration (Fig. 1b) did not show significant changes along the times (χ2 = 7.33, df = 5, P = 0.197)
Salivary cortisol significantly changed between the times (χ2 = 13.71, df = 5, P = 0.033), with an increase (P = 0.045) at T + 0B compared to TB (Fig. 1c)
Summary
Salivary alpha-amylase (sAA) is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and by changes in its isoforms, this last way of evaluation has never been used in veterinary medicine. This research evaluated the changes of sAA by three different ways in which sAA can be evaluated in an experimental acute stress model in six pigs based in a technique of temporarily restraining. These ways of evaluation were 1) activity by a spectrophotometric assay, 2) concentration by a fluorometric assay, and 3) isoforms of the enzyme by a Western blot. SAA can be evaluated by its enzymatic activity, a high inter-individual variability in response to stress has been reported in pigs [15], as well in humans [13] and horses [10]. Despite the interest and use of this enzyme, to the authorsknowledge there are no studies comparing the different ways in which the sAA response against a stressor could be assessed: activity, concentration and changes in isoforms
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