Two samples of mane hair from the top of the neck were collected from each horse, one sample before the start of the treatment period in September 2020 and one after the treatment period in December 2020. The samples were cut by the experimenter using scissors, taking care to cut as close to the skin as possible.

Approximately one centimeter from the bottom of the hair shafts closest to the skin was cut up into about 1-2 mm long pieces and weighed, with weights ranging between 6-8 mg. The samples were frozen in liquid nitrogen and pulverized with steel beads for two minutes in a Tissuelyser II, then 1 mL of methanol was added to each sample. The samples were left in a tube shaker overnight. The following day they were centrifuged, then 800 μl of the supernatant extracted into new tubes and evaporated in a Savant Speed-Vac Plus for about one hour until only a small pellet of cortisol was left. This pellet was dissolved in 150 μL RIA buffer and then 50 μL of the dissolved solution was mixed with 100 μL of primary anti-cortisol rabbit antibody. The samples were then incubated for 48 hours. Then 100 μL of tracer (125I) was added to each sample and the samples left to incubate for another 24 hours before 75 μL of Anti-Rabbit IgG SAC-CEL was added to each sample. After 30 minutes the reaction was stopped by adding 2 mL of water. The samples were then centrifuged for 15 minutes at 3000 rpm and 4C, decanted and analysed in a PerkinElmer 2470 Wizard gamma counter.