Fig 1.
Timeline of experimental design.
The timeline includes animal handling, habituation, and experimental treatment phases, indicating number of animals used in each phase.
Fig 2.
The one-handed tickling procedure (a) consisted of one-handed repeated pinning, while rapidly stimulating the underside with the fingertips. The two-handed tickling procedure (b) consisted of scooping and supporting the animal with both hands, while vigorously tickling the sides and nape of the neck with the fingertips.
Fig 3.
Examples of profile (a) and quarter (b) images.
Profile images were selected for the assessment of quantitative measures as they allowed to detect more subtle differences across animals and treatments. Quarter images were used for qualitative measures as they showed more of the face and their assessment was only minimally affected by the head pointing upwards or downwards.
Table 1.
Qualitative and Quantitative measures taken for image analysis.
Fig 4.
Areas of the ear to score for Ear Colour from quarter images.
Ear colour should be determined by looking at the leading edge of the ear flap (dotted line) and lighter skin on the interior side of the flap (solid line). Both ears should be observed for colour, with preference given to the more visible ear. The ear canal, if visible, should not be used to determine colour, and shadows within folded over ears need to be considered as they may darken the skin inside the ear.
Fig 5.
Examples of quantitative measures.
a) Ear Angle (i), Eyebrow Angle (ii), and eye height (iii) are illustrated. b) Eyebrow height (iv) is measured from the bottom of the eyeball to the middle of the brow. c) Eye width (v) was the diameter of the visible iris.
Fig 6.
Boxplot showing the frequencies of positive USVs emitted by the rats during Positive and Contrast Treatments.
The box represents the middle 50% of the data, while the upper and lower whiskers show the extreme data points, excluding outliers (starred dots). The horizontal line within the box represents the median, while the square dot shows the mean.
Table 2.
Summary of descriptive and inferential statistics for the qualitative measures.
Fig 7.
a) Stacked bar chart showing Ear Colour score percentages within Positive and Contrast Treatments. A score of 0 corresponds to “not present”, a score of 1 to “moderately present” and a score of 2 to “obviously present”. b) Example images representative of Ear Colour in the Positive (left) and Contrast (right) Treatments.
Table 3.
Summary of inferential statistics for the quantitative measures.
Fig 8.
a) Boxplot showing the Ear Angle of rats during Positive and Contrast Treatments. The box represents the middle 50% of the data, while the upper and lower whiskers show the extreme data points, excluding outliers (starred dots). The horizontal line within the box represents the median, while the square dot shows the mean. b) Example images representative of Ear Angle in the Positive (left) and Contrast (right) Treatments.