For the preliminary study, appropriate concentration and volume of methanol solution,
weight of feces, length of methanol extraction for fecal extraction were investigated. Fecal
sample (0.1–1 g) was put into 2–18 ml of 0–100% of methanol and shaken for 5–60 min.
Solutions were centrifuged at 1700×g for 5 min and then assayed for progesterone as described
below. The results showed that detected progesterone concentration was increased
in a methanol concentration depending manner. Moreover, extraction of 0.3 g feces with
6ml methanol for 15 min resulted in highest recovery rate. Therefore, 0.3 g feces was
extracted with 6ml of 100% methanol for 15 min in the present experiment. After extraction
of feces, supernatant of centrifuged feces solution was stored at −30 ◦C until
assay.