Urinary samples were collected from 12 healthy heifer murrah buffalo (Bubalus bubalis) approximately 20–30 months old at Government Livestock Farm, Orathanadu, Tanjore, Tamilnadu, India. The animals were housed in sheds and paddocks, fed with a standard diet straw and adlibitum water. The phases of the estrous cycle were carefully determined for six naturally occurring consecutive cycles and confirmed by trans-rectal palpation by assessing the morphological changes in the internal reproductive organs. Further, the estrus phase (day ‘0’) was confirmed by fern pattern analysis from cervico mucus samples (Sukh Deo and Roy, 1971).The cervical mucus, collected by inserting a sterile swab into the cervical regions, was smeared between two glass slides and observed for the presence of fern like crystals.