Anecdotal reports state that wellness treatments for horses, such as massage therapy, relaxes the treated animal. Massage therapists and horse owners typically report an "improvement" without verifying or quantifying the treatment results. This paper shows that the effect of wellness treatment and stress release can be measured with pupillometry. One of the horse's pupils was photographed at the beginning and end of the treatment to determine the changes in the balance between the sympathetic and parasympathetic system activities. The owners assigned horses to two experimental groups: animals receiving a massage (N = 18) and horses standing with a person next to the horse for the time of a massage in the stable lane (N = 10). Six animals in the experimental group were excluded from the analysis because the pupils could not be traced. We opened the images of the pupil with Fiji (ImageJ) and used the elliptical selection tool to measure the pupils' and iris' areas. The ratio between the pupils' aperture and the iris' area was a normalized measure for pupil size. At the end of the experiment, we compared the normalized size of the pupils with a two-tailed paired t-test within groups and a two-tailed t-test between groups. For the experimental group, it was before and after the treatment, and for the control group, before and after the horse was placed in the stable lane. Comparisons between the experimental and control groups were made at the procedure's beginning and end. The treatment significantly decreased the normalized pupil area in the experimental group, on average, by a factor of 0.78 ± 0.15 (P = 0.042). For the horses in the control group, the pupil size increased, on average, by a factor of 1.14 ± 018. Changes were statistically not significant (P = 0.19). The initial pupil size of the horses in the experimental group was 1.88 times larger than that in the control group. After the treatment, the difference was reduced to a factor of 1.25. At the beginning of the experiment, the horses in the experimental group had, on average, larger pupil sizes than the horses in the control group, indicating that the horses in the experimental group were more stressed before the treatment than the control animals. The observed changes in pupil size in the experimental group likely resulted from enhanced parasympathetic and diminished sympathetic activity resulting from the treatment. Observed changes in pupil size agree with the anecdotal horse owner reports and the therapist's treatment notes.
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