The sample was fractionated and chemical compounds were identified by gas chromatography–linked mass spectrometry(GC–MS; QP-5050, Schimadzu, Japan) (Rameshkumar et al., 2000;Achiraman and Archunan, 2006). Two microliters of extract were injected into the GC–MS system on a 30 m glass capillary column with a film thickness of 0.25 lm (30 0.2 mm i.d. coated with UCON HB 2000) using the following temperature programme: initial oven temperature of 40 C for 4 min,increasing to 250 C at 15 C/min, and then held at 250 C for 10 min. GC–MS was run under computer control at 70 eV. The solvent (i.e. DCM) peak was seen at 4.0 min during GC–MS analysis. The mass spectrometer was programmed for a mass scan of 33–300, which allowed for identification of compounds from C3 through C18. The compound identification was made by probability-based matching using the computer library (NICT 12) built within the system. The identified estrus-specific compounds were then compared with the standard compounds run under the same conditions, and confirmed on the basis of retention time shown in GC–MS.