The Institutional Animal Care and Use Committee of The Jackson Laboratory approved all procedures used in this study and all mice were maintained at The Jackson Laboratory (Bar Harbor, ME, USA) in accordance with all institutional protocols and the Guide for the Care and Use of Laboratory Animals.

Mice from the following strains were used: C57BL/6J (stock no. 000664), CByB6F1/J (stock no. 100009), and BALB/cByJ (stock no. 001026). All mice were housed in a non-barrier facility with a photoperiod of 14 hours of light and 10 hours of dark (lights on at 5:00AM). Mice were housed in polycarbonate duplex mouse cages, 5 per side with pine shaving bedding, and were provided food (LabDiet® 5K52, St. Louis, MO, USA) and water (acidified) ad libitum. Adult mice between the ages of 9–13 wks (C57BL/6J), 14–23 wks (CByB6F1/J), and 8–12 wks (BALB/cByJ) were used.

Representative photographs and micrographs for each stage of the estrous cycle were obtained following these steps: 1. a preliminary observation was made about the stage of the estrous cycle by assessing the vaginal opening of each mouse, 2. the stage of the estrous cycle was verified by vaginal cytology, 3. stage of estrous cycle was confirmed by mating mice overnight and checking for ovulation the following morning as described later.

These steps can also be used to learn the visual method and train the eye to identify each stage. Proestrus and estrus are easier to identify by visual observation than metestrus and diestrus. Coat color and skin pigmentation can make it more challenging to evaluate some strains. It is easier to observe changes in agouti and albino strains than in black strains where changes to the vaginal opening are more subtle.

To evaluate the stage of the estrous cycle by visual observation, each mouse was held by the tail with the forepaws resting on a cage lid. The vaginal opening of each female was evaluated based on the criteria described by Champlin, et al. A digital image of each mouse was taken using a DSCF707 Cyber-shot digital camera (Sony, Japan). Additional lighting was supplied for photographs by fiber optic lights (Fiber Lite MI-150, Dolan-Jenner Industries, Boxborough, MA).

When evaluating stage of the estrous cycle using the visual method, it is important to always evaluate animals in the same area with respect to room lighting. The table or workstation should always face the same direction and there should be sufficient light available. The light source is also important to consider because it can change the perceived color of vaginal tissues and make evaluation difficult. Portable lights can be purchased and attached to workstations and moved as needed. However, LED lights should be avoided because they have a purple hue that makes visual detection challenging. Battery operated 4W fluorescent lamps (Maverick, Edison, NJ) were used in the vivarium for this study. In the laboratory, 32W Sylvania Octron fluorescent ceiling lights (Sylvania, Danvers, MA) were used for lighting.

The vaginal opening of mice in proestrus is characterized by swollen, moist, pink tissue. The opening is wide and there are often wrinkles or striations along the dorsal and ventral edges. As the mouse enters estrus the vaginal opening becomes less pink, less moist, and less swollen. Metestrus is characterized by a vaginal opening that is not open wide, not swollen, and white cellular debris may be visible. In diestrus, the vaginal opening is small and closed with no tissue swelling.

A vaginal swab was collected using a cotton tipped swab (Puritan Medical Products Company, LLC Guilford, ME) wetted with ambient temperature physiological saline and inserted into the vagina of the restrained mouse. The swab was gently turned and rolled against the vaginal wall and then removed. Cells were transferred to a dry glass slide by rolling the swab across the slide. The slide was air dried and then stained with approximately 400 µL of stain (Accustain, Sigma-Aldrich, St. Louis, MO) for 45 seconds. The slides were rinsed with water, overlaid with a coverslip, and viewed immediately at 200× magnification under bright field illumination. The stage of the estrous cycle was determined based on the presence or absence of leukocytes, cornified epithelial, and nucleated epithelial cells according to Felicio, et al [9].

When the female is in proestrus, mostly nucleated and some cornified epithelial cells are present. Some leukocytes may be present if the female is in early proestrus. As the stage of the cycle advances to estrus, mostly cornified epithelial cells are present. If the cycle is not interrupted by pregnancy, pseudopregnancy, or other phenomena, metestrus will begin. Metestrus is a brief stage when the corpora lutea form but fail to fully luteinize due to a lack of progesterone. The uterine lining will begin to slough and evidence of this is seen in the form of cornified eipithelial cells and polymorphonuclear leukocytes present in vaginal swabs. Some nucleated epithelia cells will also be present in late metestrus. Diestrus is the longest of the stages lasting more than 2 days. Vaginal swabs during diestrus show primarily polymorphonuclear leukocytes and a few epithelial cells during late diestrus. Leukocytes remain the predominant cell type having removed cellular debris. The cycle then repeats.

After the stage of the estrous cycle was determined, each mouse was paired with one CByB6F1/J male and the following morning the presence or absence of a vaginal plug was noted and each mouse was euthanized by cervical dislocation. The contents of the oviducts were flushed using M2 media into a petri dish to look for oocytes as evidence that ovulation had occurred.

The estrous cycle identification tool was developed using qualitative data from the literature [5], [9], [10] for the proportion of each cell type in a smear. A graphical representation of the existing data was created to represent the typical changes in cell types that occur during the entire estrous cycle. The continuous changes in cell types (leukocytes, nucleated epithelial, and cornified epithelial) occurring during the estrous cycle result in the lack of clear demarcations between stages and can make it difficult to determine the stage of the estrous cycle. For example, the vaginal cytology of a mouse in estrus is characterized by many cornified epithelia cells. However, if the mouse is in early estrus, nucleated epithelial cells may also be present. Presented here (Figure 1) is an estrous cycle identification tool that shows the changes in cell populations during the entire cycle. The estrous cycle identification tool makes it clear what cells types are present at each point of the cycle, including the transitional phases between each stage.

The estrous cycle identification tool is a visual aid that shows the 4 estrous stages and the relative proportion of cells present in each stage. Each cell type is shown in a different color. The name of each stage of the estrous cycle is shown on the outside of the circle progressing clockwise from one stage to the next. The 4 quadrants are different sizes to represent a rough estimate of how much time is spent in each stage of the estrous cycle.

To use the estrous cycle identification tool, collect cells using the vaginal cytology method described and view them using a compound microscope. Identify the cell types present on the slide and note the relative proportion of each cell type. For example, there may be all leukocytes on the slide or there may be about half cornified epithelial and about half nucleated epithelial cells. Next, look at the estrous cycle identification tool (Figure 1) and place an imaginary arrow on the chart with the end on the center of the chart like a hand on a clock. The arrow is moved clockwise until the cell types and proportion appear under the arrow. Once the arrow is placed, it points to the corresponding stage of estrous.

This tool makes it easy to determine the stage of the cycle when vaginal cytology is used. The relative amount and type of cells present during early proestrus and late metestrus are similar. The nucleated epithelial cells in proestrus are often well-formed, but are often irregularly shaped and vacuolated in metestrus [3]. Alternatively, early proestrus and late metestrus can be distinguished using the visual method.