For extraction, 1 mL of 100% methanol was added to the powdered sample and rotated slowly on a benchtop tube rotator (13 rpm; Thermo Scientific, Radnor, PA) for 24 hours at room temperature (Macbeth et al., 2010; Hunt et al., 2014). Samples were then centrifuged at 10 500 g, 10°C for 13 minutes and the supernatant was transferred to a new polypropylene tube. The remaining pellet was rinsed with 0.2-mL methanol, agitated and centrifuged and the supernatant was combined with the previously removed supernatant. Methanol extracts were stored at ≤−80°C until assayed. Methanol extracts were centrifuged again and a subsample was transferred to a borosilicate glass tube, dried under forced air and reconstituted in an assay buffer specific for each hormone EIA kit.

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