Hormone Measurement

Blood was collected from carotid artery within 1 h after last treatment and centrifuged at 3,500 rpm for 30 min at 4°C. Serum was separated and stored at −20°C until assayed for corticosterone and 17β-estradiol.

Small amounts of yolk (100 mg) were sampled from the inside of the ovarian follicles. Each yolk sample was diluted in 1 mL of deionized water and homogenized.

Serum and yolk concentrations of corticosterone were determined using a double-antibody RIA system with 125I-labeled radioligands as described previously (Taya, 1985). The antiserum against 17β-estradiol was sheep anti-17β-estradiol (GDN244), kindly provided by Dr. GD Niswender, Animal Reproduction and Biotechnology Laboratory (Colorado State University, Fort Collins, CO). The antiserum against corticosterone was goat anti-corticosterone (Qasimi et al., 2017).