To confirm that ovarian somatic cells are not depleted by heat treatment, ovaries from each treatment group were immunohistochemically examined using the antibody against a steroidogenic enzyme, 3 b-HSD, that locates to ovarian interstitium and theca cells and catalyzes the conversion of pregnenolone to progesterone and 17-OH pregnenolone to 17-OH progesterone (Bhandari et al., 2006; Kobayashi et al., 1996).