BIOLOGY OF REPRODUCTION 8, 191-494 (1973)

Determining the Stage of the Estrous Cycle in

the Mouse by the Appearance of the Vagina
ARTHUR K. CHAMPLIN1 AND DARROLD L. DORR
The Jackson Laboratory, Bar Harbor, Maine 04609

AND ALLEN H. GATES

Division of Genetics, University of Rochester Medical Center,
Rochester, New York 14642

Received July 27, 1972; accepted November 22, 1972

A simple method is described by which the various stages of the estrous cycle of
the mouse can be easily determined by the appearance of the vagina. Data are presented
which show that this method of determining estrous state is at least as accurate and
reliable as the customary method of vaginal smearing. The method is faster, and less
stressful to the animals than the vaginal smear procedure.

Since the initial study of the estrous vagina during the estrous cycle were first
cycle in the guinea pig by Stockard and mentioned by Allen (1922) in his classic
Papanicolaou in 1917, the vaginal smear study of the estrous cycle of the mouse.
technique has been the accepted procedure The examination of these vaginal changes
for determining the estrous state in various was originally refined at The Jackson
mammalian species. The technique in- Laboratory by M. N. Runner and his co-
volves an identification of the cell types workers. The method has since evolved
and their relative quantities present in into a visual method of identification of
preparations obtained from the vagina by the estrous stages in mice and is as reliable
either scraping or swabbing the vaginal as vaginal smearing. It is far more rapid
walls, or by vaginal washings. The tech- and permits numerous observations to be
nique is not without its disadvantages. made in a single day without mechanical
Emery and Schwabe (1936) have reported manipulation of the vaginal tissues, thereby
that frequent vaginal smearing of both cas- avoiding the chance of artificially induced
trate and intact rats using cotton swabs cornification of the vaginal epithelium. It
resulted in an abnormally high incidence has been used routinely in our laboratories
of cornified or estrus-like smears. Frequent for a number of years.
smearing has also been found to result in
MATERIALS AND METHODS
cornification of the vaginal epithelium in
mice (Wade and Doisy, 1935). Vaginal Forty (SJL/J x C57BL/10Dg)F virgin female

smearing, particularly during estrus, could mice, ranging in age from 63 to 86 days, were
removed from the groups of four females per
induce pseudopregnancy and result in ab-
cage in which they had been housed since wean-
normally long cycles. ing, and placed either alone (12 females) or in
Gross changes in the appearance of the new groups of four or eight females (12 and
16 females, respectively). The differential housing
Present address: Department of Biology, Colby
was done as part of another experiment designed
College, Waterville, Maine 04901 to test the estrus-suppressing effects of grouping

491

Copyright #{174}
1973 by The Society for the Study of Reproduction
All rights of reproduction in any form reserved.
492 F1AMPLIN, DORR AND CATER

TABLE 1 ticularly with respect to the dorsal lip, the color

APPEARANCE OF THE VAGINA AT VARIOUS
and moistness of the tissues, the size of the vaginal
STAGES OF THE ESTROUS CYCLE
opening, and the presence or absence of visible
cellular debris in the vagina are all factors which
Estrous are considered in identifying the stage of estrus.
Stage Appearance The characteristics of the various stages are de-
1)iestrus Vagina lsas a small opening and the
scribed in Table 1 and shown in Fig. 1.

tissues are bluish-purple in color

One should be aware of the fact that the estrous
and very moist (Fig. 1A). stages in the
mouse are not of equal duration.

Proestrus Vagina is gaping and the tissues are In a preliminary study the estrous cycles of 15
reddish-pink and moist. Numer- females were followed by the visual method for
ous longitudinal folds or striations the duration of one complete cycle. In a cycle
are visible on both the dorsal and of 4 days, the combined proestrus-estrus stage
ventral lips (Fig. 1B). lasts only about 24 hr.

Estrus Vaginal signs are similar to pro- In addition to the data comparing the two meth-
estrus, but the tissues are lighter ods of determining the estrus state, the vaginal
pink and less moist, and the stria- signs of proestrus were related to the behavioral
tions are more pronounced (Fig. and physiological correlates of this stage. A record
1C). was kept over a 7-month period of the number
Metestrus-1 Vaginal tissues are pale and dry. of first-night matings and, in some cases, the num-
Dorsal lip is not as edematous ber of fertile matings involving females from a
as in estrus (Fig. 1D). variety of strains that had been chosen from the
\[etestrus-2 Vaginal signs are similar to metes- colony in the late afternoon to be in proestrus
trus-1, but the lip is less edematous by the visual method and paired immediately with
and has receded. Whitish cellular males. A mating was considered to have occurred
debris may line the inner walls if a vaginal plug was found in the female on
or partially fill the Vagina. the morning after pairing. The fertility of the
mating was evaluated by flushing the contents
of the oviducts into a depression slide with saline
among females from a number The of strains. or culture medium (‘Whitten, 1971) 1 or 2 days
females were housed in stainless-steel cages, con- after mating and examining the recovered ova
taining a bedding of pine shavings, and were for the presence of pronuclei or for evidence of
kept in a constant-temperature room (25#{176}C) to cleavage.
provide a controlled environment which was free
of male mouse odors. Artificial illumination was RESULTS
provided between 5 AM and 7 PM.
The results of the study comparing the
After a 16-day adaptation period, the estrous
state of each female was determined daily in the
two methods for determining the estrous
early afternoon for 21 days by both the visual state were examined in several ways. The
method described below and by the lavage method number of metestrus periods experienced
of obtaining vaginal smears (Snell, 1941). The was taken as a measure of the number of
two procedures was performed independently, each
cycles completed during the 21-day period
by a different investigator.
The visual method utilizes the changes in ap-
of observation. An examination of the data
pearance of the vagina that occur during the es- presented in Table 2 shows that both meth-
troims cycle. The degree of vaginal swelling, par- ods yielded essentially the same results.

TABLE 2
NUMBER OF METESTRUS PERIODS OBSERVED IN 21 DAYS AS DETERMINED BY THE

APPEARANCE OF THE VAGINA AND BY VAGINAL SMEARS

Number of fe males per cage

1 4 8

Mean no. metestrus

± SE (appearance) 4.67 ± 0.189 2.83 ± 0.208 2.81 ± 0.187
Mean no. metestrus
± SE (smears) 4.75 ± 0.249 2.75 ± 0.249 2.63 ± 0.154
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 ESTROUS CYCLE DETERMINING BY VAGINAL APPEARANCE 493

TABLE 3
TEMPORAL CORRELATION BETWEEN ESTROUS STAGES CLASSIFIED BY APPEARANCE OF THE VAGINA
AND BY VAGINAL SMEARS

. Proestrus Metestrus
No. of days that diagnosis of a stage
by vaginal smear occurred after its No. of % of No. of % of
diagnosis by vaginal appearance stages total stages total

0 48 65.8 93 68.0
1 23 31.5 30 22.2
>1#{176} 2 2.7 12 8.9
Total 73 Total 135

#{176}Includes classifications with no obvious temporal relationship.

It is also apparent, although not essential the dangers associated with mechanical
to the comparison study, that (SJL/J X manipulation of the vaginal tissues can be
C57BL/ lODg) F1 females exhibited a sup- eliminated, as can the possible confusion
pression of estrous cycling when housed in that may result from the three-step process
groups of either four or eight per cage. of obtaining the cellular sample from the
Temporal correlation of the two methods vagina, its transfer to a glass slide, and
was examined by comparing the time of identification of the estrous stage from the
appearance of proestrus and metestrus by sample. In addition, the method eliminates
the visual method with the same events the chance of bacterial transfer from one
determined by vaginal smearing. These animal to the next, as well as the transfer of
two stages were selected because they dis- vaginal secretions and pheromones which
play clear-cut and distinctive characteris- could confound behavioral observations.
tics in both methods. The data presented The changes in the vagina are similar
in Table 3 show that there was exact tem- in all strains of mice examined. One should,
poral correlation 65-70% of the time. The however, be aware of the fact that slight
data also indicate that the estrous state de- differences do exist between strains in the
termined by vaginal smears tended to be size of the vagmal opening and in the de-
slightly delayed over that determined by gree of coloration observed at the different
the appearance of the vagina. estrous stages. The latter difference is par-
The determination of proestrus by the ticularly noticeable between albino and
visual method seemed to relate well to the pigmented strains. Pigmentation of the
behavioral and physiological correlates of vaginal tissue causes a slight masking of
the stage. Of 458 females selected from the color changes described in Table 1.
the colony in the late afternoon and identi- Of the numerous strains observed, the es-
fied as being in proestrus, 425 (92.8%) trous stages of the albino strains are the
mated on the first night after being paired easiest to identify by this method and those
with a male. Sixty-seven (94.4%) of the 71 of DBA/2J aninials seem to be the most
matings examined were found to be fertile. difficult, in that the vaginal opening is rela-
tively small throughout the estrous cycle
DISCUSSION
in these animals.
Estrous stages identified by an examina- One stage that the untrained observer
tion of the vagina appear to be at least could mistake for proestrus-estrus in some
as reliable as those determined by vaginal strains occurs between metestrus-2 and
smears. In addition, the visual method is diestrus. The vaginal tissues may be pink,
faster for the experimenter and less stress- being free of white sloughing cells, while
ful to the animals. By use of this method, the opening has not yet returned to its
494 CHAMPLIN, DORR AND GATES

smallest size. However, the distinctive fea- to Dr. W. K. \Vhitten (principal investigator) and
ture of this stage, in contrast to the proes- by the John Hartford Foundation, Inc. (Dr. R. A.
Doherty, principal investigator). The photographic
trus-estrus stage, is the absence of longi-
assistance of Mr. John Roths is greatly appreciated,
tudinal striations within the vagina. as is the financial assistance of the Committee on
The data presented here indicate that Research, Travel and Sabbaticals at Colby College.
estrous stages identified by vaginal smears The Jackson Laboratory is fully accredited by
tend to be slightly delayed in relation to the American Association for Accreditation of
those determined by vaginal signs. It is Laboratory Animal Care.

likely that this delay can be explained by

the fact that vaginal washings were used REFERENCES
to obtain the cellular material from the ALLEN, E. (1922). The oestrous cycle in the
vagina. Washings might be expected to mouse. Amer. I. Anat. 30, 297-371.
contain at least some of the cellular ma- EMERY, F. E., AND SCHWABE, E. L. (1936). The
terial that was a part of the vaginal tissues vaginal smear of rats as influenced by frequent
examinations. Anat. Rec. 64, 147-154.
on the previous day, thereby resulting in
EMMENS, C. W. (1950). Estrogens. In “Hormone
a slightly delayed identification of a par- Assay” (C. W. Emmens, ed.), pp. 391-417.
ticular stage of the estrous cycle. Perhaps Academic Press, New York.
the temporal correlation between the two SNELL, C. D. (1941). Reproduction. In “Biology
methods would have been better if vaginal of the Laboratory Mouse” (C. D. Snell, ed.).
1st edit., pp. 55-58. Blakiston, New York.
smears had been taken by a gentle scraping
STOCKARD, C. R., AND PAPANCOLAOU, C. N.
or swabbing of the vaginal walls, rather
(1917). The existence of a typical oestrous cycle
than by vaginal lavage, in order to obtain in the guinea-pig with a study of its histological
cells from the wall epithelium rather than and physiological changes. Amer. J. Anat. 22,
sloughed cells. Emmens (1950) recom- 225-283.
WADE, N. J., AND DOISY, E. A. (1935). Cornifica-
mends gently scraping the dorsal vaginal
tion of vaginal epithelium of ovariectomized
wall.
rat produced by smearing. Proc. Soc. Exp. Biol.
Med. 32, 707-709.
ACKNOWLEDGMENT
WHI1-FEN, W. K. (1971). Nutrient requirements
This study was supported by NIH Training for the culture of preimplantation embryos in
Grant CA 05013 from the National Cancer Insti- vitro. In “Advances in the Biosciences 6. Shering
tute to The Jackson Laboratory and by NIH Re- Symposium on Intrinsic and Extrinsic Factors
search Grant HD 04083 from the National Insti- in Early Mammalian Development” (C. Rasp#{233},
tute of Child Health and Human Devlopment ed.), pp. 129-141. Pergamon, Elmsford, NY.