Changes in alpha-amylase activity, concentration and isoforms in pigs after an experimental acute stress model: an exploratory study

Background: Salivary 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. Results: Although 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. Conclusions: Although 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.