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Laboratory Animal Science
Copyright 1997
by the American Association for Laboratory Animal Science
Vol 47, No 6
December 1997
632
Immunization strategies for diseases affecting the sys-
temic organs of humans have been available for many
years and have reduced considerably the incidence and
mortality of these diseases. There has, however, been a
remarkable lack of success in vaccination strategies used
to control mucosal diseases (1), such as those caused by
Salmonella and Vibrio spp., and human immunodefi-
ciency virus.
Chlamydia trachomatis is a particularly good example
because, despite the fact that this obligate intracellular
bacterium is a leading cause of sexually transmitted dis-
ease in humans, affecting millions of people throughout
the world (2), there is no effective vaccine for this mu-
cosal pathogen (3). Local mucosal immunization of labo-
ratory animals with antigen has been shown to induce
an increased mucosal immune response at the site of
exposure, compared with the response elicited after im-
munization at distant mucosal sites (4, 5). Because
chlamydiae invade the body via mucosal surfaces, such
as the female reproductive tract (FRT), an important
requirement for an effective vaccine against these bac-
teria is that it must stimulate the mucosal immune sys-
tem (6). Consequently, the body site used for immuniza-
tion of individuals with antigen preparations is an im-
portant consideration in attempts to raise immunity to
chlamydial and other infections.
The most common animal model used to investigate
mucosal immune mechanisms involved in chlamydial in-
fections of the genital tract is the guinea pig, in which
The Guinea Pig Estrous Cycle:
Correlation of Vaginal Impedance
Measurements with Vaginal Cytologic Findings
Kenneth G. Lilley, Ronald J. Epping, and Louise M. Hafner
Abstract | Immunologic responses elicited after vaginal immunization of laboratory mammals are affected
by the stage of the animal’s estrous cycle at which antigen is delivered. The study reported here documents
vaginal impedance as an improved means of determining accurately precise stages of the estrous cycle of
guinea pigs. Impedance changes were correlated with cytologic changes observed in vaginal smears taken
from the animals. The start of the impedance peak corresponded with proestrus, the rising side of the peak
with estrus, and the apex of the peak with metestrus; diestrus was seen approximately 3 days after the peak
apex. These results contrast with those of previous studies. Various dosage regimens of estradiol (1 to 1,000
g/animal over 1 to 6 days) were used in guinea pigs in an attempt to invoke an extended (>6 h) estrogen-
induced mucosa. Parenteral administration of 1,000 g of estradiol to guinea pigs daily for 6 days induced a 2-
to 3-day estrus stage in these animals.
Centre for Molecular Biotechnology, School of Life Sciences, Queensland
University of Technology, GPO Box 2434 Brisbane Q 4001, Australia
infection has been shown to closely parallel genital in-
fections of humans with respect to pathogenesis and
pathologic and immunologic findings of disease (7, 8).
A current focus of world-wide mucosal vaccinology involves
endocrinology of the estrous cycle, especially because concen-
trations of the female sex hormones (particularly estrogen)
also have been shown to have pronounced effects on the in-
tensity and duration of chlamydial infection in guinea pigs
(9, 10). The guinea pig has an estrous cycle of 15 to 17 days’
duration that consists of four sequential stages: estrus, met-
estrus, diestrus, and proestrus (11). The stage of the estrous
cycle affects the extent of antigen uptake in the FRT of mice
(12) and the presence of antigen-presenting cells (lowest dur-
ing estrus) in rats (13). Estrus is estrogen dominated, diestrus
is progesterone dominated (14), and concentrations of sex
hormones have been reported to affect the pathogenesis of
infection (15). The stage of the estrous cycle also is emerging
as an important consideration when planning the timing of
antigen delivery for mucosal vaccination; however, a conven-
ient method to identify accurately and monitor the stages of
the guinea pig estrous cycle is unavailable.
Vaginal cytologic examination commonly is used to moni-
tor the guinea pig estrous cycle, but it is time consuming and
requires experience with the technique. The guinea pig has a
vaginal closure membrane that covers the vaginal orifice
throughout the estrous cycle and that degenerates spontane-
ously at estrus (16). The absence of a vaginal closure mem-
brane in guinea pigs also may be used to indicate estrus (17),
although correlation is not 100% (18). It has been suggested
that vaginal impedance (rather than cytologic examination
of vaginal smears) may better correlate with stages of the
estrous cycle (19).
633
We investigated the use of vaginal impedance measure-
ments to monitor the guinea pig estrous cycle. We also in
vestigated the correlation between vaginal cytologic fea-
tures and vaginal impedance measurements throughout the
cycle. The impedance meter also was used in conjunction
with estradiol administration to the a nimals to investi-
gate potential induction of prolonged estrus in these ani-
mals to facilitate vaginal immunization.
Materials and Methods
Animals: Virgin female random outbred English short-
haired (ROES) strain guinea pigs were obtained from
Monash Animal Services (Melbourne, Victoria, Australia).
The animals were sexually mature (>60 days old), and each
weighed 500 to 900 g. Microchips (Trovan, Victoria) were
injected subcutaneously (dorsoscapular) into the animals,
and a scanner (Trovan LID500; Central Animal Register,
Victoria) was used for individual identification. Animals
were housed in an environmentally controlled room (21
1 C, 12/12-h light/dark cycle) at the University of
Queensland Medical School Animal Facility. Standard
guinea pig pellets (Norco Cooperative Ltd., Lismore, New
South Wales, Australia) and tap water supplemented with
ascorbic acid (4 mg/20 L; Melrose Laboratories, Victoria)
were available ad libitum.
This study was done in compliance with all relevant regu-
lations regarding safety, ethics, and use of animals, and all
procedures were approved by the Queensland University
of Technology Biomedical Ethics Committee (BEC no. 662).
In the case of the guinea pigs, procedures also were ap-
proved by the University of Queensland Animal Experi-
mentation Ethics Committee (AEEC approval no. QUT/060/
95/NHMRC).
Estrous cycle studies. Vaginal cytology: Vaginal smears
were prepared from specimens obtained daily at 1600 h from
29 female guinea pigs over 135 days, using the following pro-
cedure. To obtain a vaginal swab specimen, the anogenital
area of a restrained guinea pig was cleansed, using cotton
wool moistened with sterile phosphate-buffered saline solu-
tion (PBSS). If the vaginal membrane was present, it was
gently perforated with a sterile cotton-tipped aluminum swab
(Disposable Products, South Australia). Smears were collected
from the proximal third of the vaginal epithelium by gently
inserting a moist, sterile swab 2 cm into the vagina and twice
rotating it vigorously against the vaginal wall. The swab im-
mediately was rolled onto a glass microscope slide, and the
cells were fixed by spraying with Cytospray (SEA Trading
Pty Ltd, Queensland, Australia). The smears were stained
(20) and permanently mounted, using Depex (Labtek,
Queensland). Vaginal smears were evaluated, using standard
cytologic criteria for diagnosis of stage of the estrous cycle
(21), and were examined, using an Olympus CH2 microscope
at magnifications of 100x and 400x. Cell types and their dis-
tribution were assessed on a 1+ to 4+ scale, and stage of the
estrous cycle was determined (11, 16). Vaginal smears were
prepared from specimens collected from 29 guinea pigs over
135 days and were stained by use of the Papanicolaou method
to identify the cell types and their relative frequencies through-
out the estrous cycle. The stained slides were examined in
coded experiments to avoid bias, and these data were used to
differentiate stages of the estrous cycle in these animals.
Vaginal impedance measurements: Electric impedance
of the vaginal mucous membrane was measured daily at
1600 h in 43 female guinea pigs over 135 days as de-
scribed by Bartos and Sedlacek (19). Briefly, a probe con-
taining two electrodes was inserted 2 cm into the va-
gina, and the impedance was measured by the attached
impedance meter. The meter was a transistor indicator
with a 2-kHz oscillator that was based on a meter con-
structed by Petran (22). The probe was cleaned in 70%
alcohol before each guinea pig measurement.
Daily vaginal impedance measurements were obtained
from the 43 guinea pigs over a period of 135 days to as-
certain whether these varied throughout the estrous cycle
and whether they correlated with cytologic changes seen
in the animals. To further delineate cytologic changes
and vaginal impedance changes where impedance
peaked, impedance measurements were obtained and
smears were prepared every 3 h for 48 h.
Induction of estrus by estradiol administration: Estra-
diol benzoate (Intervet, New South Wales) was adminis-
tered subcutaneously (s.c.) to 29 female guinea pigs, us-
ing 100-
l volumes of various dosages (1 to 1,000 g,
Table 1); arachis oil diluent for the estradiol benzoate
(Health Mindery, New South Wales) was given to con-
trol animals. Six groups of five guinea pigs (groups 1–4,
and 6) and one group of four guinea pigs (group 5) were
used. Animals were given estradiol benzoate at 1700 h
daily for 6 days, except for those of group 4, which were
given 100
g of estradiol benzoate once only, and those
of group 6 (controls), which were given 100
l of arachis
oil daily for 6 days.
Results
Correlation between vaginal cytologic findings
and impedance measurements taken during the
estrous cycles of guinea pigs. Vaginal cytologic ex-
amination: Vaginal cytologic features at estrus consisted
Table 1. Induction of estrus by estradiol administration.
Twenty-nine female guinea pigs each were given 0 to 1,000
g of estradiol over a 6-day period in an attempt to induce a prolonged estrus mucosa
Group N Induction Dosage Result
1 5 Multiple 1 g/animal per day for 6 days Delayed subsequent cycle by 2 days
2 5 Multiple 10 g/animal per day for 6 days Enhanced epithelium maturation
3 5 Multiple 100 g/animal per day for 6 days Irregular cycle, enhanced epithelium maturation
4 5 Single 100 g/animal Normal cycle, enhanced epithelium maturation
5 4 Multiple 1,000 g/animal per day for 6 days Induced estrus
6 5 Multiple 100 l of arachis oil/animal per day for 6 days No effect
N = number of animals in each experimental group.
Identification of Estrous Cycle Stages Using Vaginal Impedance
Vol 47, No 6
Laboratory Animal Science
December 1997
634
predominantly of non-nucleated superficial squamous
cells (SSC[nn]), whereas those seen during metestrus
were a mixture of cell types, including SSC(nn), nucle-
ated superficial squamous cells (SSC[n]), and parabasal
cells (PB). Metestrus was characterized by a marked in-
flux of polymorphonuclear neutrophils (PMN, Figure 1).
Cells seen during diestrus consisted mainly of PBs and
PMNs; generally SSCs were lacking. Proestrus was con-
firmed by presence of intermediate squamous cells (ISC);
SSCs (nucleated and non-nucleated) and PBs also were
seen during proestrus, and PMN numbers were reduced.
There was overlap in cell types observed between each
of the stages of the cycle; however, differentiation of each
stage was facilitated by adherence to a quantitative as-
sessment of the cell types and their frequencies for each
stage, using a scale of 1+ to 4+ (Table 2).
Investigation of vaginal impedance: A cyclic increase in
electric impedance was evident in 98% of the guinea pigs
Figure 1. Representative photomicrographs of Papanicolaou-stained vaginal smears from guinea pigs illustrating the relative cellular
composition of the four stages of the estrous cycle. Estrus (upper left); metestrus (upper right); proestrus (lower left); diestrus (lower
right). SSC = superficial squamous cell, may be nucleated (n) or non-nucleated (nn); ISC = intermediate squamous cell; PB = parabasal
cell; PMN = polymorphonuclear leukocyte. Central arrows indicate progression of the estrous cycle. Magnification = x200.
635
tradiol administration. In group-3 (100 g of estradiol)
guinea pigs, commencement of the next cycle was irregu-
lar (i.e., 1 to 2 days in 3/5 animals) and started 5 days ear-
lier in 2/5 animals. All animals in this group had enhanced
vaginal epithelium maturation for 3 to 5 days after estrus.
In group-4 (single dose of 100 g of estradiol) guinea pigs,
the following two cycles were of normal duration, but en-
hanced vaginal epithelium maturation was evident for 1
to 2 days after estrus. In group-5 (1,000 g of estradiol)
guinea pigs, enhanced epithelium maturation was evident
from days 13 to 14 after the first estradiol injection, culmi-
nating in extended estrus, lasting 2 to 3 days from days 21
to 23. Thus, unlike the 1-, 10-, and 100-
g doses of estra-
diol per animal (which caused enhanced vaginal epithe-
lium maturation, but not complete estrus induction), the
1,000- g dose given daily to each animal for 6 days resulted
in induced estrus. Numbers of PMNs observed at estrus usu-
ally were low (often zero), whereas numbers of PMNs observed
during “estradiol-induced” estrus were scored as 1+ to 2+.
For all groups of animals, enhanced maturation of the
vaginal epithelium preceded the increase in vaginal im-
pedance by 1 to 2 days. In animals of the 1,000 g/animal
dosage group, the vaginal closure membrane was absent
(a sign of estrus) from the start of “induced estrus” and
failed to re-form until 7 days after the commencement of
“induced estrus” (c.f., re-forming of the membrane the day
after noninduced or “normal” estrus). In animals of the
lower-dosage groups, there was no consistent correlation
between induced vaginal epithelium maturation and ab-
sence of the vaginal closure membrane.
Administration of arachis oil to the control animals in
group 6 (Table 1) did not affect the estrous cycle.
Discussion
Cell types (and their frequencies) in vaginal smears pre-
pared from guinea pigs at various stages of the estrous cycle
were in accordance with published findings (11, 16), ex-
cept for the terminology used to describe a type of SSCs
found in the stained smears. Previous studies referred to a
cell type termed SCC(n), whereas in the study reported
here, further differentiation of this type of cell was made.
Using cytologic criteria, principally nuclear morphology, the
SSC(n) was differentiated from another cell type, the in-
termediate squamous cell (ISC) (23). This is an important
distinction, because proestrus is characterized cytologically
by the presence of 2+ to 3+ ISCs.
in this study; data from a typical guinea pig response are
shown in Figure 2. One guinea pig appeared to lack an
estrous cycle; it remained in cytologic diestrus, and an im-
pedance peak was not evident.
For all 42 guinea pigs exhibiting cyclic vaginal imped-
ance, the periodicity of the impedance peak corresponded
with an estrous cycle duration of 15 to 17 days (mean
2
SD, 16.1 1.1 days). Mean amplitude of the vaginal im-
pedance peak was 905 480 ( 2 SD), and mean imped-
ance for the remainder of the cycle (nonpeak) was 293
52 ( 2 SD); the difference was statistically significant
(P < 0.01, t test).
Vaginal impedance correlated with cytologic changes in the
estrous cycle of all guinea pigs monitored. A typical guinea
pig response is shown in Figure 3. Proestrus was cytologi-
cally evident 3 days prior to the start of the peak and for 2.5
days into the rising side of the peak. Estrus was evident for
approximately 12 h as impedance was increasing. Metestrus
was observed at the highest point of the impedance peak and
for 2.5 days after return of impedance to nonpeak values.
Diestrus was cytologically evident after metestrus, commenc-
ing 2.5 days after the impedance peak.
Induction of estrus in guinea pigs by estradiol adminis-
tration: Various dosages of estradiol were administered to
five groups of guinea pigs (five animals in groups 1, 2, 3,
and 4; four animals in group 5) in an attempt to evoke a
prolonged (2- to 3-day) estrogen-induced mucosa instead
of the typical 6 to 11 h, thereby facilitating immunization
at that stage of the cycle (Table 1). The stage of the guinea
pig estrous cycle was monitored by vaginal impedance mea-
surements and by examination of Papanicolaou-stained
vaginal smears prepared from specimens taken daily at
1600 h. Data were screened for signs of estrus induction,
such as increase in vaginal impedance, enhanced matura-
tion of the vaginal epithelium (i.e., increased presence of
cell types usually present at later stages of the estrous
cycle), or absence of the vaginal closure membrane.
Changes in the estrous cycle of animals in group 1 (1
g
of estradiol/animal daily for 6 days) included delayed com-
mencement of the next cycle by 2 days (3/3 animals) and
enhanced maturation of the vaginal epithelium for 5 days
after the next stage of the estrous cycle (5/5 animals). For
5 of 5 animals in group 2 (10 g of estradiol) the subse-
quent cycle was not delayed, but maturation of the vaginal
epithelium was enhanced from the onset of estrus and con-
tinued in an irregular manner to the second cycle after es-
Table 2. Morphologic cell types and their relative frequencies seen in
vaginal smears prepared from specimens collected from guinea pigs at the four stages of the estrous cycle
Cell types and relative frequencies
Stage of Duration Superficial Intermediate Parabasal Polymorphonuclear
estrous cycle of stage squamous squamous cells leucocytes
cells cells
Estrus 6–11 h 4+ 1+ 1+ 1+
Metestrus 2–4 day 2+ to 4+ nn 1+ 1+ to 2+ 2+ to 4+
Diestrus 8–10 day 1+ 1+ 2+ to 3+ 2+ to 3+
Proestrus 2–4 day 1+ to 2+ nn 2+ to 3+ 1+ to 2+ 1+
1+ to 2+ n
Vaginal smears were stained, using the Papanicolaou method; cell types in the smears were identified and their relative frequencies were determined in
relation to the other cell types and their distribution over the entire smear. Relative frequencies of cell types ranged from 1+ (0 to 10%) to 4+ (80 to 100%).
Superficial squamous cells (SSCs) were further classified into nucleated (n) and non-nucleated (nn) SSCs. The data were compiled from the analysis of
3,915 smears.
Identification of Estrous Cycle Stages Using Vaginal Impedance
Vol 47, No 6
Laboratory Animal Science
December 1997
636
Duration of the estrous cycle was determined cytologi-
cally; however, differentiating the stages of the cycle was
difficult because the stages merge together. Because es-
trus generally occurs between 1800 and 0600 h (19), it
often was missed when the smears were prepared from
specimens taken at 1800 h. By strictly following descrip-
tions of stages of the estrous cycle, as described in Table
2, it was possible to identify the different stages by cyto-
logic examination; however, the divisions between each
cycle stage were not easy to define. To predict confidently
the onset of an estrous stage, serial smears had to be
prepared from specimens collected daily over the dura-
tion of at least one complete estrous cycle (16 days). As-
sessment of vaginal cytologic features in smears is time
consuming and requires cytologic knowledge and expe-
rience. Vaginal impedance measurements, however, take
only seconds to obtain and are less amenable to subjec-
tive interpretation.
The cyclic increase in impedance seen in the guinea
pigs of this study was similar to that seen previously
(19); however, the magnitude of the readings in this study
differed. Bartos and Sedlacek (19) used a vaginal probe
with ring electrodes of separation (3 mm) different from
those used in this study (1 mm); this may account for
the approximately threefold higher values reported in
their study. The 16 (
1)-day duration of the estrous cycle
of the guinea pigs of this study is in full accordance with
the 16.3 days of one report (24) and the 16.6 days of an-
other report (19). Bartos and Sedlacek (19) made certain
assumptions about impedance values and estrous stages
in guinea pigs on the basis of previous work by Bartos
(25), using rats.
The study reported here is the first to investigate the rela-
tionship between cytologic and vaginal impedance changes
in the guinea pig over the estrous cycle, and our findings dis-
agree with the assumptions of Bartos and Sedlacek (19). Those
authors assumed that the highest impedance values corre-
sponded to proestrus, whereas the results of this study clearly
indicate that proestrus occurs prior to and during the vaginal
impedance peak and that the highest impedance value actu-
ally corresponds to late estrus and metestrus (Figure 3).
Proestrus can be detected by noting the start of the imped-
ance peak (i.e., 400
). Estrus was difficult to detect because
of its short duration of 6 to 11 h (26), but metestrus could be
identified by the highest impedance value of the cycle. Be-
cause metestrus lasts 2 to 4 days (26), diestrus easily was
identified as occurring 5 days after peak impedance.
To monitor and distinguish all stages of the guinea pig
estrous cycle, vaginal impedance changes, like vaginal cy-
tologic changes, need to be measured daily and followed
for at least one complete estrous cycle. Impedance mea-
surements are easy to perform, faster to assess, and more
effective for identifying stages of the estrous cycle for im-
munization than is examination of vaginal smears; there-
fore, impedance measurement is a valuable method to use
in place of examination of vaginal smears. Nevertheless,
due to the comparatively short duration (6 to 11 h) of the
estrus stage of the cycle, a confirmatory vaginal smear also
should be prepared, stained, and examined to confirm the
estrous stage.
Apart from an early report (27) containing “irregular re-
sults,” studies involving administration of estradiol to guinea
pigs for the purposes of investigating hormonal effects on dis-
ease processes have not assessed effects on the estrous cycle
after administration of the hormone. Administration of es-
tradiol (1,000
g/animal per day for 6 days) to a group of guinea
pigs (A. McCracken, unpublished results, this laboratory)
resulted in enhanced maturation of the vaginal epithelium
and an “induced estrus” in these animals that parallelled re-
sults of this study. The increase in PMNs observed during
the “induced estrus” may have been due to a physiologic re-
sponse to the prolonged absence of the vaginal closure mem-
brane. Lower dosage regimens of estradiol tested in this study
affected the duration and maturation of the estrous cycle;
however, they did not induce estrus for an extended period.
Administration of 1,000
g of estradiol/animal over 6 days
was successful in causing an “induced estrus” for an extended
period of 2 to 3 days.
Figure 3. Correlation of vaginal impedance with stages of the
guinea pig estrous cycle, as determined by vaginal cytologic ex-
amination. Vaginal impedance measurements and vaginal smears
were taken daily at 1600 h for 135 days. To further investigate
the peak region, smears were examined and impedance measure-
ments were obtained from 5 guinea pigs every 3 h for 48 h in the
region of the impedance peak. Cytologic changes consistent with
the estrous cycle stages were observed at the intervals noted: D =
diestrus, P = proestrus, E = estrus, and M = metestrus.
Figure 2. Changes in vaginal impedance in one guinea pig over 6
estrous cycles. Vaginal impedance measurements were obtained
from guinea pigs daily at 1600 h. The cyclic impedance relation-
ship for this guinea pig also was evident for 42 of 43 animals in
the study.
637
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From results of this study, it now is possible to adminis-
ter estradiol at a dosage known to induce estrus for a pe-
riod during which guinea pigs can be immunized with
chlamydial or other antigen for the purpose of eliciting a
local immune response. Alternatively, serial vaginal imped-
ance measurements may be taken in nonestradiol-induced
animals, and when impedance starts to increase, true es-
trus can be confirmed by vaginal cytologic examination,
and the vaccine can be delivered. Further research is re-
quired to ascertain whether this “induced estrus” differs
from “true” estrus in type and distribution of immunologi-
cally active cells, such as antigen-presenting cells, because
this may have a profound effect on the efficacy of vaccine
preparations delivered intravaginally.
Acknowledgements
The authors thank R. Dickson, Laboratory Manager of the Uni-
versity of Queensland Medical School Animal Facility, and his
technical staff, Ms. S. Deitrich and Ms. C. Smith, for assistance
with the maintenance and care of the animals. Mr. Dickson also
is acknowledged for provision of the impedance meter used in these
studies.
This research was supported financially by a grant from the
National Health & Medical Research Council of Australia to
P. Timms, L. Hafner, R. Epping, and M. O’Brien.
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Identification of Estrous Cycle Stages Using Vaginal Impedance
