**1.9 Criteria for estrous cycle staging**

Changes in estrous cycle can only be effectively predicted and identified if the investigator is adequately acquainted of the continuous variations and appearance in the vaginal cytology throughout the several stages of the animal's reproductive cycle (**Table 5**). There are numerous reports on the appearance of normal reproductive tract of all the different phases of the estrous cycle [14, 16], nevertheless, to attain consistency in method of staging the cycle, the following criteria should be followed.

1.To clarify the interval as well as the stages of the reproductive cycle, cytological samples of the vaginal cells should be collected for a minimum of 14 successive days (can also be done for up to 19 or 21 days). It has to be prepared at a particular time of the day preferably in the early periods of the morning between 7 am and 12 noon [15]. This is vital as Certain short phases may be "missed" particularly if samples are not consistently gathered for instance, in smears harvested too late or very early in the day, pro-estrus phase might be missed. Irrespective of what time the samples are harvested, they must be prepared at about same matching period of the day. This must be maintained throughout the collection

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#### **Table 5.**

*Similarities and differences in estrous cycle in mice and ovarian cycle in humans.*

period to diminish inconsistency. Vaginal lavage method is ideal for collection of the samples.


diestrus. Constant light causes persistent estrus and failure to cycle while housing in large groups causes persistent diestrus [25]. Nonetheless, mice in persistent diestrus will synchronize upon exposure to male urine.

9.If the outcome of drug or extract/compound expression on the cycle is even in a set (e.g., consequential persistent diestrus or estrous), so this may provide a valuable summary of the data. Conversely, if some animals exposed to the compound or extract display persistent diestrus, while others exhibit prolonged estrous, then such outcomes may possibly not reveal the alterations caused by the test compound. Therefore, the discrepancy in collection may perhaps result in a general belief that reproductive cycle remained unaffected. The procedure should be repeated in such cases.
