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56° Congresso Internazionale Multisala SCIVAC 73
Physiology and pathology of the estrous cycle
of the bitch
Konrad Blendinger
Med Vet, Hofheim (D)
Sound knowledge of the bitch reproductive cycle is essen- gonadotropin release. Mean levels of FSH are moderately
tial. Individual bitches may vary from normal, be presented elevated, and those of LH slightly elevated, during anestrus.
at variable times during their estrous cycles for evaluation, At late anestrus, the pulsatile release of LH increases, caus-
and sometimes exhibit pathologic variations in cycles. Each ing the proestrous folliculogenesis. Estrogen levels are basal
of these scenarios requires veterinary interpretation. The (2-10 pg/mL) and progesterone levels at nadir (<1 ng/mL) at
normal canine reproductive cycle can be divided into 4 phas- late anestrus.
es, each having characteristic behavioral, physical, and
endocrinologic patterns, although considerable variation
exists. Bitches with normal estrous cycles but unexpected Proestrus
patterns must be differentiated from those with true abnor-
malities. Detection of individual variation within the normal During proestrus, the bitch becomes attractive to male
range of events in a fertile bitch can be crucial to breeding dogs but is still not receptive to breeding, although she may
management. Evaluation of the estrous cycle for true abnor- become more playful. A serosanguineous to hemorrhagic
malities is an important part of the evaluation of an appar- vulvar discharge of uterine origin is present, and the vulva is
ently infertile bitch. mildly enlarged.
Vaginal cytology shows a progressive shift from small
parabasal cells to small and large intermediate cells, superfi-
PHYSIOLOGY OF THE ESTROUS CYCLE cial-intermediate cells, and finally superficial (cornified)
epithelial cells, reflecting the degree of estrogen influence.
Estrous Cycle RBC are usually, but not invariably, present. The vaginal
mucosal folds appear edematous, pink, and round. FSH and
The onset of the first estrous cycle (puberty) of an indi- LH levels are low during most of proestrus, rising during the
vidual bitch is expected between 6 and 10 month of age but preovulatory surge. Estrogen rises from basal anestrous lev-
may not begin until she has reached 2 years of age. The els (2-10 pg/mL) to peak levels (50-100 pg/mL) at late
interestrous interval is normally 4-13 month, with 7 month proestrus, while progesterone remains at basal levels (<1
the average. ng/mL) until rising at the LH surge (2-3 ng/mL). Proestrus
lasts from 3 days to 3 weeks, with 9 days average. The fol-
licular phase of the ovarian cycle coincides with proestrus
Anestrus and very early estrus.
Behavior correlates with decreasing estrogen levels and
The anestrus phase of the extrous cycle normally lasts 1- increasing progesterone levels. Serosanguineous to hemor-
6 month. It is marked by ovarian inactivity, uterine involu- rhagic vulvar discharge may diminish to variable degrees.
tion, and endometrial repair. An anestrous bitch is not attrac- Vulvar edema tends to be maximal. Vaginal cytology
tive or receptive to male dogs. No overt vulvar discharge is remains predominated by superficial cells; RBC tend to
present, and the vulva is small. Vaginal cytology is predom- decrease but may persist throughout. Vaginal mucosal folds
inated by small parabasal cells, with occasional neutrophils become progressively wrinkled (crenulated) in conjunction
and small numbers of mixed bacteria. with ovulation and oocyte maturation. Estrogen levels
The endoscopic appearance of vaginal mucosal folds is decrease markedly after the LH peak to variable levels,
flat, thin, and red. The physiologic controls terminating while progesterone levels steadily increase (usually 4-10
anestrus are not well understood, but the deterioration of ng/mL at ovulation), marking the luteal phase of the ovari-
luteal function and the decline of prolactin secretion seem to an cycle.
be prerequisites. The termination of anestrus is marked by an
increase in the pulsatile secretion of pituitary gonadotropins,
follicle stimulating hormone (FSH), and luteinizing hor- Estrus
mone (LH), induced by gonadotropin-releasing hormone
(GnRH). Hypothalamic GnRH secretion is itself pulsatile, Estrus lasts 3 days to 3 wk, with an average of 9 days.
its intermittent secretion is a physiologic requirement of Estrous behavior may precede or follow the LH peak—its
Proceedings of the SCIVAC Congress, Rimini, Italy, 2007
56° Congresso Internazionale Multisala SCIVAC 74
duration is variable and may not coincide precisely with the epithelial cells seen on a vaginal smear. Full cornification
fertile period. Primary oocytes ovulate 2 days after the LH continues throughout estrus until the “diestral shift”
peak, and oocyte maturation is seen 2-3 days later; the lifes- occurs 7-10 days after the LH surge, signifying the first
pan of secondary oocytes is 2-3 days. day of diestrus.
The vaginal smear then changes abruptly, with appear-
ance of neutrophils and epithelial cells changing from full
Diestrus cornification to 40-60% immature (parabasal and interme-
diate) cells over the next 24-36 hr. If vaginal cytology is
During diestrus, the normal bitch becomes refractory to performed until the diestral shift is observed, the LH surge,
breeding, with diminishing attraction of male dogs. Vulvar ovulation, and the fertile period can be analyzed retrospec-
discharge diminishes and edema slowly resolves. Vaginal tively.
cytology is abruptly altered by the reappearance of
parabasal epithelial cells and frequently neutrophils. The
appearance of vaginal mucosal folds becomes flattened and
Luteinizing Hormone
flaccid.
Estrogen levels are variably low, and progesterone levels
At the end of the follicular phase of the estrous cycle, a
steadily rise to a peak of 15-80 ng/mL before progressive-
marked increase in LH over usual baseline values devlops
ly declining in late diestrus. Progesterone secretion
over 24-48 hr, followed by a return to baseline values. This
depends on both pituitary LH and prolactin secretion. Pro-
surge is thought to occur in response to the decline in
liferation of the endometrium and quiescence of the
estrogen levels and increase in progesterone levels. The
myometrium develop under the influence of elevated prog-
LH surge triggers ovulation, making it the central
esterone levels. Diestrus usually lasts 2-3 mo in the
endocrinologic event in the reproductive cycle of the
absence of pregnancy.
bitch. Daily serial measurement of LH to identify the
Parturition terminates pregnancy 64-66 days after the
exact date of the LH surge is an accurate diagnostic tool
LH peak. Prolactin levels increase in a reciprocal fashion to
for timing breedings.
falling progesterone levels at the termination of diestrus or
Affordable semiquantitative in-house kits are available
gestation, reaching much higher levels in the pregnant
for measuring serum LH levels in the dog and for identi-
state. Mammary ductal and glandular tissues increase in
fying the preovulatory LH surge and thus the time of ovu-
response to prolactin levels.
lation and the true fertile period. Blood samples must be
drawn daily (at about the same time) for LH testing, as the
LH surge may last only 24 hr in many bitches. The kits can
Estrogens
be subject to variable interpretation, so the same person
should run the tests if possible.
Increased estrogen causes an increased turnover rate of
vaginal epithelial cells, resulting in the progressive corni-
fication seen on vaginal cytology. Progressive edema of
the vaginal mucosa also develops and can be visualized Progesterone
with endoscopic examination. Estrogen assays are per-
formed by many commercial laboratories; however, the Progesterone levels begin to rise at approximately the
information is of little value for ovulation timing because time of the LH surge (prior to ovulation). Rising proges-
peak estrogen levels vary from bitch to bitch, and even rel- terone acts synergistically with declining estrogen to
ative changes do not correlate to ovulation or the fertile reduce edema of the vulva and vagina, which can be seen
period. on vaginoscopic exam. Other observable clinical signs are
Estrogen is best assessed by serial vaginal cytologies minimal. Serial blood samples performed every 2 days may
and vaginoscopy. Estrogen levels do not indicate the fertile identify the initial rise in progesterone (usually >2 ng/mL),
period because ovulation is triggered by the LH surge, not which indicates that the LH surge has occurred. Proges-
an estrogen peak. Examination of the cells on the surface terone can be assayed by radioimmunoassay at most vet-
of the vaginal epithelium can provide information about erinary commercial laboratories. Several in-house semi-
the stage of the estrous cycle. Proper technique is impor- quantitative kits are also available.
tant so that the cells obtained are representative of the hor- No single absolute value of progesterone correlates to
monal changes occurring. The sample should be collected any particular stage of the cycle. Progesterone varies from
from the cranial vagina; cells from the clitoral fossa, 0.8-3.0 ng/mL at the point of the LH surge, from 1.0-8.0
vestibule, or caudal vagina are not as indicative of the ng/mL at ovulation, and from 4.0-20.0 ng/mL during the
stage of the cycle. fertile period. However, if accurate serial quantitative prog-
Under the influence of rising estrogen levels, the num- esterone assays are obtained, the LH surge may be estimat-
ber of layers composing the vaginal epithelium increases ed as the day a distinct increase in progesterone level is
dramatically, presumably to provide protection to the seen. While this is not as accurate as actual identification
mucosa during copulation. As estrogen rises during of the LH surge by assay, estimation by progesterone lev-
proestrus, the maturation rate of the epithelial cells els is still very useful and is often more widely available
increases, as does the number of keratinized, cornified and convenient.
56° Congresso Internazionale Multisala SCIVAC 75
Table - Some aspects of the estrous cycle in the bitch
Duration Progesterone Estrogen (E2) levels Notes
Anestrus 1-6 month Basal (<1 ng/mL) Basal (2-10 pg/mL)
Proestrus 3 days-3 weeks
(9 days average) Initial basal (<1 ng/mL); Rising to peak levels
At LH surge: 2-3 (0,8-3) ng/mL (50-100 pg/mL)
At day of ovulation: 4-10 ng/ml
(ovulation: range 1-8 ng/mL)
Estrus 3 days-3 weeks
(9 days average) Abrubt decrease at the Primary oocytes
day of LH peak to ovulate 2 days after
10-20 pg/mL LH peak;
Basal (2-10 pg/mL) Oocyte maturation
during the following finished 2-3 days
few days later; lifespan of
secondary oocytes:
2-3 days
Diestrus 2-3 month Peak at 15-80 ng/mL Basal (2-10 ng/mL)
(in the absence of
pregnancy)
Vaginal cytology
Parabasals
Small Intermediate Cells
Large Intermediate Cells
Partly cornified
Superficial cells
Completely Cornified
Figure 1 Figure 2
SOME CONSIDERATIONS Quantitative progesterone assays
ON PROGESTERONE ASSAYS
1) It is critical that the result is available at least the day after
Semiquantitative in-clinic progesterone collection.
assays 2) The laboratory must be familiar with the detection of
canine progesterone. Some of the human assays have the
When timing breeding using semiquantitative in-clinic same technical problems with canine serum as the semi-
progesterone assays, only a range of progesterone is quantitative in-clinic progesterone assays. The results of
obtained, this makes it difficult to accurately identify the most human assays have to be adapted to the canine species
day of the initial rise in progesterone or the true fertile by a factor which is done by a accurate laboratory.
period. Technical problems with these kits have also 3) A safe rule of thumb is that when progesterone is >2
been seen. Therefore, these assays should be used only ng/mL, breeding should begin. Optimal ovulation timing
for routine breedings in which a wider margin of error is should use quantitative progesterone assays from com-
acceptable. mercial laboratories—the cost difference is minimal.
56° Congresso Internazionale Multisala SCIVAC 76
Regardless of which assay is used, an additional test B) Shortened interestrous interval
should always be performed 2-4 days after the first rise is
detected to indicate that the cycle has progressed as Premature drop of progesterone
expected, a functional corpus luteum has been formed,
and ovulation has occurred. Split oestrus
Pubertal bitches are more likely to manifest a split or false
heat. The exact cause for split/false heat is unknown. One pos-
PATHOLOGY OF THE ESTROUS CYCLE sible explanation is that the increased levels of estradiol-17ß
from follicular development during anestrus, and prior to the
A) Prolonged interestrous interval onset of a “true” proestrus, may initiate the clinical signs. Dur-
ing a split heat the bitch shows some of the signs of a true
Primary anestrus in the bitch is the lack of estrous proestrus-estrus, such as a serosanguineous discharge passing
cycling by 24 month of age. from the vagina, vulvar swelling, and the attraction of male
Previous ovariohysterectomy may be the reason for a dogs. In some cases, bitches exhibiting split heats will even be
bitch not to cycle. Challenge testing of serum estrogen has receptive to mating. However, after a few days, true estrus
been described: A paired blood sample is taken before and begins in several more days or weeks. During a split heat, proe-
after 60 to 90 minutes of Buserelin administration intra- strous-estrous behaviour occurs in the absence of ovulation
venously at a dose of 0.02 to 0.03 µg/kg. Intact bitches, at during the first part of the “split”. However, conception occurs
any stage of the reproductive cycle, will show an elevation in during the second part of the split heat, or true estrus.
serum estrogen concentration to greater than 15 to 20 pg/ml.
Silent heat is defined as ovarian activity with no con- Anovulatory cycle
comitant vulvar swelling, exudation of serosanguineous vul- Anovulatory cycles, in which serum progesterone concen-
var discharge, or attraction of male dogs. It can be diagnosed trations never rose above 3.5 ng/ml, were reported in 11 of
by monthly assay of serum progesterone concentrations, 1152 (1%) bitches. Five of these 11 bitches (45%) had a nor-
with a value of greater than 2 ng/ml indicating presence of mal, ovulatory estrus at their next season.
functional luteal tissue, or by weekly assessment of vaginal
cytology, with increasing percentages of cornified vaginal Hypoluteoidism
epithelial cells indicative of rising serum estrogen concen- Luteal dysfunction is generally considered to be a primary
trations. Pubertal bitches are more likely than normal adults ovarian problem, but secondary luteal insufficiency has been
to manifest a silent heat. described in a Great Dane with a pituitary defect. Diagnosis
requires documentation of low serum progesterone concen-
Abnormalities of sexual differentiation. Female appear- trations in diestrus. If serum progesterone falls to less than 2
ing dogs may have a 78,X0; 79,XXX; 79,XXY; or ng/ml for greater than 48 hours, pregnancy may be terminat-
78,XX/78,XY karyotype. Male pseudohermaphrodites, that ed. From a more practical (but empirical) standpoint, serum
have male gonads and female external genitalia, also may progesterone values less than 10 – 15 ng/ml during the first
present for primary / prolonged anestrus. Diagnosis of abnor- 4 weeks after mating may be suspected as hypoluteoidism.
mality of sexual differentiation is made by visual inspection of Supplementation of progesterone may be progesterone in
abnormal external genitalia, histopathology of excised oil administered parenterally (2 mg/kg every 3 days to not
gonadal tissue, measurement of serum gonadotrophin concen- later than 58 days from ovulation or 52 days from the first
tration, and/or assessment of the karyotype. diestrous vaginal smear). Ally-trenbolone (Regumate) is
Drug induced anestrus may be present in bitches that administered at a dose of 0.088 mg/kg once dayly orally, to
received anabolic drugs, androgens, progestogens or gluco- not later than 61 days from ovulation or 55 days from the
corticoids. Such agents may be used to enhance performance first diestrous vaginal smear. Bitches receiving the oral
of show or working dogs or for other reasons. progestogen, ally-trenbolone, may have poor milk produc-
Hypothyreoidism may lead to anestrus, prolonged or tion in the early postpartum period.
irregular interestruous intervals, prolonged proestrus or oth-
er reproductive signs. Short anestrus syndrome
Bitches with systemic diseases, such as renal failure or In some breeds (e.g. German Shepherd) some individuals
hypercorticism or cancer cachexia, may be less likely to tend to have a shortened interestrous interval of less than 4
cycle than normal bitches. month. This condition is correlated with poor reproductive
Progesterone-secreting ovarian cysts with serum prog- performance. Heritability is assumed/discussed.
esterone concentrations greater than 2 ng/ml may inhibit the
ovarian cycle. Embryonal death/resorption
Ovarian aplasia is a rare congenital anomaly in dogs, in The exact incidence of embryonic and fetal loss is diffi-
which there is defective prenatal germ cell migration. Serum cult to determine. In 22 pregnant beagle bitches from 22 to
gonadotropin concentrations are elevated in affected dogs. 54 days postcoitum, 13 of 117 embryos and fetuses (11%)
Immune-mediated oophoritis has been described in the were found to be in different stages of resorpiton. In
dog with autoimmune destruction of the ovary. another study, 13 resorptions were present among 98 fetal
Hyperprolaktinaemia during prolonged pseudopregnan- implantation sites (13%) in 12 beagle bitches examined by
cy may lead to a prolonged interestrous interval. hysterotomy at 48 days of gestation.
56° Congresso Internazionale Multisala SCIVAC 77
In case, the whole litter is resorbed, the interestrus inter- stop, progesterone concentration will increase and the vagi-
val may be shortened. nal smear will show intermediate and parabasal cells, and
leukocytes. If the problem persists oestrus can be stopped by
oral administration of low doses of megestrol acetate
C) Prolonged estrus (Ovarid, Pitman Moore/ not available in Germany) (once
daily first week: 0,1 mg/kg; second week: 0,05 mg/kg).
A prolonged estrogen influence may be caused by one of Ovarian tumors (granulosa cell tumors)
the following conditions: Tumors should be removed.
Ovarian cysts (follicular cysts) Pathologic liver function (metabolism of estrogens is
Cysts can be treated with GnRH (Fertagyl, Intervet), reduced)
10µg/kg once or repeatedly, it probably is advisable to Exogenous estrogens
administer GnRH repeatedly, IM for example thrice daily
during two days. This treatment, however, does not always
Suggested readings
resolve the problem. If luteinisation of cystic follicles or fur-
ther luteinisation of luteinised cysts take place the estrus will Johnston et al. (2001): Canine and Feline Theriogenology, Saunders
