he Canine Estrous Cycle: Staging Using Vaginal Cytological Examination

alerie R. Beimborn, DVM; Heather L. Tarpley, DVM; Perry J. Bain, DVM, PhD; and Kenneth S. Latimer
DVM, PhD

lass of 2003, Ross University, School of Veterinary Medicine, St. Kitts, West Indies (Beimborn) and
Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA
0602-7388 (Tarpley, Bain, Latimer)

Introduction

aginal cytology is a simple technique that can be used by practitioners to help characterize stages of
he reproductive cycle of the bitch or to evaluate certain diseases of the genital tract. Vaginal cytology
sually is used in conjunction with the physical examination, clinical history, vaginoscopy, and
ormonal assays to determine the stage of the reproductive cycle. This is especially important if
rtificial insemination is to be performed. Other uses of vaginal cytology and ancillary testing include
etermination of the whelping date, diagnosis of inflammation of the vagina, and identification of some
ypes of neoplasia involving the vaginal vault and urethral orifice.
Estrous Cycle of the Bitch

he estrous cycle of the bitch contains four distinct stages that can be followed cytologically (Table 1).
hese stages include proestrus, estrus, diestrus, and anestrus. For some simplification and indication o
which structures predominate throughout the cycle, these four stages may be grouped into the
ollicular phase (proestrus and estrus) or the luteal phase (diestrus and anestrus). The bitch frequently
as been classified as monocyclic or having a reproductive cycle that lacks frequent, recurring periods
f heat and has a long period of anestrus regardless of pregnancy status.
Table 1. Characteristics of the Canine Estrous Cycle
Cycle
Stage

Length

Proestrus

~9 days

Hormonal
changes

Estrogen

Predominant
Cell Types

Erythrocytes

Neutrophils

Behavior
of the
Bitch

Clinical
Signs

Early: mixed
population of
cells

Early: present

Early:
present

Late: large
intermediate
and
superficial

Late:

Attractive
to the
male, but
wont
stand for
mating

Vulvar
edema
and
swelling,
bloody
discharge

Late:
decreased

cells
Estrus

~9 days

LH (surge)
Estrogen

90%
superficial
cells

Absent

Accepts
male and
will stand

Less
edema,
discharge
becomes
clearer

Abrupt
change in
relative # of
epithelial
cells.
Superficial
cells decrease
by 20%

Few to none

Ceases to
accept
male

Little
discharge,
edema
decreased

Parabasal and
Intermediate
cells

Few if any

Few if
present

No
outward
signs

Scanty,
tenacious
secretions

Bacteria
usually
present

Progesterone
Diestrus

~60 days
Progesterone

Anestrus

Depends on
whether
pregnant or
not. 70-80
days for CL to
regress and
130 days to
repair
endometrium.

Progesterone

roestrus: Proestrus typically lasts about 9 days. This is the reproductive phase that immediately
recedes estrus. Estradiol concentration increases as ovarian follicules mature and the uterus enlarges
he vaginal epithelium proliferates accompanied by diapedesis of erythrocytes from uterine capillaries.
hus, erythrocytes account for most of the cells observed on vaginal smears at this reproductive stage
rossly, vulvar edema, hyperemia, and a serosanguineous discharge occur.7,8

strus: Typically, estrus lasts for about 9 days. Estrus is characterized by the period of acceptance of
he male by the bitch. The female will seek out males and stand for them prior to copulation. This
tage of the reproductive cycle is difficult to determine cytologically because distinguish features are
nly present microscopically at the beginning and the end of estrus. During estrus, glandular secretion
ncrease, the vaginal epithelium becomes hyperemic, and ovulation occurs. This stage of the
eproductive cycle is influenced mainly by estrogens. The interval between successive estrus cycles is
ypically seven months.

Diestrus: This is the period where corpora lutea (CL) are present. The CL are fully functional and
ecrete increased large quantities of progesterone. During this phase of the reproductive cycle, the
itch refuses to accept the sexual advances of the dog. Physiologically, the uterine glands undergo
ypertrophy and hyperplasia. The cervix constricts and vaginal secretions become tenacious. The
ength of diestrus is variable; however, regression of CL requires approximately 70-80 days.

Anestrus: Anestrus is a prolonged period of sexual rest where the reproductive system is quiescent.
ecretions are minimal and very tenacious or absent. The cervix is tightly constricted and the vaginal
mucosa is pale.
Collection and Preparation of Cytological Specimens

aginal cytology specimens are easily and quickly collected and prepared. A saline moistened cotton
wab is inserted through the vulvar lips into the vagina. The swab is angled craniodorsally to avoid the
itoral fossa. A clean otoscope may be used to guide the swab and to provide a light source for visual
uidance. Once the swab is located cranial to the urethral orifice, the swab is rotated slightly to
xfoliate and collect the cells.

fter the swab is removed from the vagina, the cells are transferred to a clean glass slide by rolling the
wab along the surface of the slide. Intact cells are obtained during transfer by rolling the swab. In
ontrast, back and forth smearing of the swab to transfer the cells will cause sever cellular distortion
nd rupture. Once the cells have been transferred, the slides are air dried, stained with Romanowsky
r new methylene blue stains, and examined microscopically.

n alternative method to collect cytologic material from the vagina is to use a plastic medicine dropper
nd saline solution. The dropper is passed into the vagina and the bulb is squeezed several times.
xfoliated cells are collected by saline lavage. A drop of fluid is placed on a glass slide and a blood-type
mear is made, air dried, and stained as above. The medicine dropper technique is more gentle but the
aline saline will distort the morphology of the cells and dilute the cell count.

arious stains have been used for vaginal cytology including Romanowsky stains (Wright, Giemsa,
eishman, Diff-Quik), new methylene blue, toluidine blue, trichome stains (Papanicolaou, Shorr,
ano) and hematoxylin and eosin (H&E). 1Personal experience, preference, cost, and ease of use
sually determine the choice of stain. In any event, the stain should be economical, easily stored, and
roduce consistent staining that is adequate for cytologic needs.6
Cytologic Features of Vaginal Epithelial Cells

he vaginal epithelium is influenced by hormonal changes during the estrous cycle, allowing cytologic
monitoring of the various reproductive stages. The morphologic features of the epithelial cells are
escribed below

Basal Cells: Basal cells are the youngest cells of the vaginal epithelium and serve as precursors of the

ther epithelial cell types. They are located along the basement membrane and rarely are seen in
xfoliative cytology. These cells have a high nuclear to cytoplasmic (N/C) ratio, a round nucleus, and a
mall amount of basophilic cytoplasm.

arabasal cells: These cells are the smallest cells vaginal epithelial cells seen in cytologic
reparations. Parabasal cells also have a high N/C ratio, round nucleus, and basophilic cytoplasm. They
re uniform in size and shape. Parabasal cells also may contain cytoplasmic vacuoles and are known as
oam cells. The function of the vacuoles in these cells is unknown. Large numbers of foam cells may be
ound in prepubertal samples and should not be confused with neoplastic cells.

ntermediate cells: These cells vary in size but are usually two times the size of parabasal cells. The
/C ratio is decreased and they have large amounts of blue-green, keratinized cytoplasm. Their
orders are round to irregular and folded. Intermediate cells also may be subclassified as superficial
ntermediate or transitional intermediate cells according to their stage in the reproductive cycle.

uperficial cells: Superficial cells are the oldest vaginal epithelial cells. They either have small, round
yknotic nuclei or lack a nucleus if they are cornified. Their cytoplasm is abundant, blue-green, and
eratinized. Cell margins are angular with folded edges.
Cytologic Staging of the Canine Reproductive Cycle

ach stage of the canine reproductive cycle has a distinct, predominant cell population. that is easily
dentified cytologically.

roestrus: Predominant cell populations in early to mid proestrus include nondegenerate neutrophils
nd a mixture of parabasal, intermediate, and superficial epithelial cells (Fig. 1). The background of th
ide contains an abundance of mucus. Variable numbers of extracellular bacteria represent normal
ora. In late proestrus, the number of neutrophils declines and superficial cells begin to predominate
Fig. 2).

Figure 1. Early proestrus with a

mixture of nondegenerate
neutrophils; parabasal,
intermediate, and superficial
epithelial cells, erythrocytes, and
mucus (Wright-Leishman stain).

Figure 2. Late proestrus with

erythrocytes, scattered superficial
cells, and absence of neutrophils
(Wright-Leishman stain).

strus: In estrus, the cell population consists of ~ 90% superficial cells and < 5% parabasal or
ntermediate cells (Fig. 3). Less mucus is present in the background of the preparation. Normal
acterial flora usually is present and organisms often are attached to the superficial cells.

Figure 3. Estrus characterized by

superficial cells that are
keratinized, largely anucleate, and
have angular, folded cell margins
(Wright-Leishman stain).

Diestrus: Cell populations change abruptly in diestrus (Fig. 4). Superficial cells decrease by 20% and
maller intermediate cells increase in number. Neutrophils often reappear and may contain
hagocytosed erythrocytes and bacteria. Cytologically, this stage of the reproductive cycle can look
ery similar to early proestrus. Therefore, serial cytologic sampling is required to make the distinction.

Figure 4. Diestrus characterized

by a decrease in superficial cells,
increase in smaller intermediate
cells, and presence of neutrophils.
Cytologically, diestrus may appear
similar to early proestrus (WrightLeishman stain).

Anestrus: Parabasal and intermediate cells predominate during anestrus. Superficial cells are not
resent. Erythrocytes and neutrophils may be present in low numbers or are absent. 10
Vaginitis

aginitis (vaginal inflammation) may occur in bitches regardless of age, breed, or reproductive status.
linically, the most common signs of inflammation are mucoid to purulent vulvar discharge, pollakiuria
nd licking of the vulva.5 Typically, vaginitis is secondary to noninfectious causes such as vaginal
nomalies (e.g,. strictures, persistent hymen, vaginal hypoplasia, double vagina), vaginal immaturity
i.e,. puppy vaginitis), chemical irritation from urine, or mechanical irritation (e.g, .foreign bodies,
aginal neoplasia).9 Primary bacterial infection of the vagina is less common. Occasionally, it can be
ifficult to distinguish pathogenic bacteria from normal vaginal flora. However, a heavy growth of a
niform bacterium is more suggestive of a pathogen.3 Infectious agents that may be observed
nfrequently include Brucella canis, Ureaplasma, Mycoplasma, and herpesvirus.

ytologic changes in vaginitis include the presence of many degenerate or nondegenerate neutrophils
Fig. 5). Mucus, lymphocytes, and macrophages also may be present and may indicate chronicity. If
aginitis is due to an infectious agent, phagocytosed bacteria or intracnuclear inclusions also may be
bserved. An abundance of neutrophils may be present in diestrus. Therefore, this normal stage of
eproduction must be distinguished from inflammation.2

Figure 5. Vaginitis demonstrating

the presence of many
nondegenerate to degenerate
neutrophils and scattered
epithelial cells (Wright-Leishman
stain).
Vaginal Neoplasia

aginal neoplasia is the second most common form of reproductive neoplasia in the bitch, following
mammary neoplasia.6 Vaginal neoplasia includes transitional cell carcinoma (TCC) of the urethra,
ransmissible venereal tumor (TVT), squamous cell carcinoma (SCC), leiomyoma, and fibropapilloma.
aginal tumors are usually found in the older bitch except for TVT which is more common in young
ogs. The mean age of occurrence of vaginal neoplasia in the bitch is ~10 years. 6

itches with vaginal neoplasia typically present because of a mass protruding from the vulva, vaginal
ischarge and dysuria, stranguria, tenesmus, urinary incontinence, or perineal swelling due to
mechanical interference by a tumor of neighboring structures.6,9 These tumors are diagnosed by a
ombination of physical examination, medical history, palpation (digital examination), vaginoscopy,
aginal cytology, and surgical biopsy. Vaginal cytology is most helpful in diagnosing TVT, TCC, and
CC.

ransmissible venereal tumor is transmitted sexually, by mucous membrane contact (i.e,. sniffing and
cking), or via bite wounds and tends to be common in areas where dogs roam freely. These
eoplasms are either benign or malignant in behavior (please see Clerkship paper Canine Round Cell
umors for more information on this neoplasm). Metastasis is rare but may involve regional lymph
odes, skin, and subcutanteous tissues. TVT cells have a round nucleus, single nucleolus, and a thin
m of light blue, vacuolated cytoplasm (Fig. 6). TVTs may resolve spontaneously without treatment.6
reatment with vincristine or irradiation is very effective. 11

Figure 6. TVT cells with a round

nucleus, single nucleolus, and a thin
rim of light blue, vacuolated
cytoplasm (Wright-Leishman stain).

ransitional cell carcinomas and SCC are common in the distal urethra of the bitch and may involve the
agina or vestibule or both. Digital examination, vaginoscopy, and surgical biopsy are best for a
efinitive diagnosis. Occasionally, fine-needle aspirates may provide a definitive diagnosis of TCC or
CC. Cells in TCC exhibit anisocytosis, anisokaryosis, and have an increased N/C ratio (Fig. 7; please
ee Clerkship paper Cytologic Diagnosis of Transitional Cell Carcinoma in Dogs and Cats for
more information). Neoplastic squamous cells are characterized by anisocytosis, anisokaryosis, and
waxy blue cytoplasm, occasionally with a perinuclear halo (Fig. 8) Cytologic evaluation of urine
ediment is helpful but is only diagnostic in ~ 40% of cases. 6

Figure 7. Transitional cell carcinoma. The

epithelial cells exhibit anisocytosis,
anisokaryosis, nucleolar rings, basophilic
cytoplasm, and have an increased N/C ratio
(Wright-Leishman stain).

Figure 8. Squamous cell carcinoma. The

neoplastic squamous cells exhibit
anisocytosis, anisokaryosis, and waxy blue
cytoplasm. Neutrophils and erythrocytes also
are present (Wright-Leishman stain).

References

. Baker R, Lumsden JH: Color Atlas of Cytology of the Dog and Cat. St. Louis, Mosby, Inc., 2000; p.
35-237.

.Cowell RL, Tyler RD; Meinkoth, J.H.: Diagnostic Cytology and Hematology of the Dog and Cat. 2 ed.
t. Louis, Mosby, Inc., 1999, Chapter 20. Veterinary Clinics of North America: Small Animal Practice,
May 1991; 21(3): 523-531.

. Freshman JL: Clinical approach to infertility in the cycling bitch. Vet Clin N Am: Small Anim Pract
1:427-435, 1991.

. Goodman M: Ovulation timing: Concepts and controversies. Vet Clin N Am: Small Anim Pract
1:219-235, 2001.

. Johnson CA: Diagnosis and treatment of chronic vaginitis in the bitch. Vet Clin N Am: Small Anim
ract 21:522-531, 1991.

. Manothaiudom K, Johnston SD: Clinical approach to vaginal / vestibular masses in the bitch. Vet Clin
N Am: Small Anim Pract 21:509-519, 1991.

. Noakes DE, Parkinson TJ, England GCW: Arthurs Veterinary Reproduction and Obstetrics, 8th ed.

hiladelphia, WB Saunders, 2001, pp. 3, 4, 33.

. Olsen et al: Vaginal Cytology. Part I. A useful tool for staging the canine estrous cycle, Compendium
or the Continuing Education for Practicing Veterinarians, 6:288-289, 1984.

. Olson et al: Vaginal Cytology. Part II. Its Use in Diagnosing Canine Johnson, C.A.: Diagnosis and
reatment of Chronic Vaginitis in the Bitch. Reproductive Disorders. Compendium for the Continuing
ducation of Practicing Veterinarians. 6:385-390; 1984.

0. Raskin RE, Meyer DJ: Atlas of Canine and Feline Cytology. Philadelphia, WB Saunders Co, 2001, pp
77-312.

1.Rogers KS: Transmissible venereal tumor. Compend Contin Educ Pract Vet 19:1036-1044, 1997.

2. Thrall MA, Olson PN: The vagina. In: Cowell RL, Tyler RD, Meinkoth JH: Diagnostic Cytology and
Hematology of the Dog and Cat, 2nd ed. St. Louis, Mosby, Inc., 1999, pp. 240-248. (from reference #2