Whistle blower

(diestrus I, diestrus II and diestrus III) respectively (Maeda, 2000). Review by other authors and publications, they considered the additional phase as anestrus stage which demonstrated the inactive ovarian activity (Westwood, 2008).

The ovulation occurs from the start of proestrus stage to the end of estrus stage (Young et al., 1941; Schwarts. 1964). The mean cycle length in the female rat is 4 days, predicted from the onset of sexual maturity up to the age of 12 months (Long & Evans, 1922; Freeman, 1988).

2.2 Vaginal Cytology

The type of cells present in vaginal smears has long been used to identify the stages of the estrous cycle in laboratory rats and mice by microscopic evaluation in aiming to indicate the functional status of the hypothalamic- pituitary- ovarian axis (Cora et al., 2015).

Before the establishment of vaginal cytology, the stages of estrous cycle were defined and characterized based on behaviour changes, for instance when animal accept or ready to copulate. Apart from that, record in Cora et al. (2015) said that the morphological and histological changes can be observed in the reproductive tract of cycling females. Additionally, the observation is made in macroscopic changes such as swelling in the vulva, congestion in the uterus and vaginal excretions such as haemorrhage and mucous. The microscopic changes in uterine tissue and ovaries is observed as well to characterize the stages of estrous cycle (Cora et al., 2015).

However, in small rodents like rats, the macroscopic observations were not always reliable. Tissue pathology was and still is an invasive procedure for staging in-life estrous cycle. The need of more reliable and non- invasive protocol to determine the stages of estrous cycle has led to Stockard and Papanicolaou (1917). This publication was setting up the characterization of vaginal canal changes during the estrous cycle that can be used for any animal species, macroscopically (histology) and microscopically (cytology).

Also from the findings, other researchers such as Joseph Long, Herbert Evans and Edgar Allen, pursued their works and researches and described the estrous cycle of rats of mouse (Long & Evans, 1922; Allen, 1922). Long & Evans (1922) in their publication, expressed the importance of Stockard and Papanicolaou (1917) publication articulately. Developed by Papanicolaou, the Pap smear test now used as a