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REPRODUCTIVE BIOLOGY OF THE COYOTE (CANIS LATRANS):
INTEGRATION OF MATING BEHAVIOR, REPRODUCTIVE
HORMONES, AND VAGINAL CYTOLOGY
DEBRA A. CARLSON*AND ERIC M. GESE
Department of Wildland Resources, Utah State University, Logan, UT 84322-5230, USA (DAC)
United States Department of Agriculture, Animal and Plant Health Inspection Service,
Wildlife Services, National Wildlife Research Center, Department of Wildland
Resources, Utah State University, Logan, UT 84322-5230, USA (EMG)
The reproductive biology of wild Canis species is often described as unique among mammals because an unusual
combination of behavioral and physiological characteristics including a seasonally monestrous cycle, copulatory
lock or tie, obligatory pseudopregnancy, social monogamy, and biparental care of the young. We investigated
social behavior, endocrine profiles, and vaginal cytology of female coyotes (Canis latrans) during 4 breeding
seasons, 2000–2003. Blood levels of estradiol, progesterone, prolactin, and relaxin were measured, and mating
behavior and changes in vaginal epithelium were documented. After aligning the data from each individual to her
estimated day of ovulation, we compared pregnant coyotes with nonpregnant females and evaluated temporal
relationships among hormone levels, behavior, and vaginal cytology. We found that patterns of proceptive and
receptive behaviors correlated with the secretion of steroid hormones, as did vaginal epithelial cytomorphosis. In
addition, although progesterone levels of pregnant and pseudopregnant coyotes were indistinguishable, prolactin
demonstrated a discernible intergroup difference and relaxin was only detectable in pregnant females. Although
this study included characteristics not previously published for this species, it also showed how key aspects of
reproduction were correlated temporally, and emphasized the importance of an integrated perspective when
addressing the reproductive biology of coyotes, or other wild species of canids.
Key words: Canis latrans, coyote, mating behavior, ovarian cycle, pseudopregnancy, reproductive hormones, vaginal
cytology, wild canid
Coyotes (Canis latrans) are medium-sized wild canids
indigenous to North America. They are seasonally monestrus
(Gier 1968; Hamlett 1938; Kennelly and Johns 1976; Stellflug
et al. 1981), socially monogamous, and territorial (Andelt
1985; Bekoff and Wells 1986; Bromley and Gese 2001;
Camenzind 1978; Gese 2001). Once bonded, a coyote pair
remains together for an indefinite number of years, sharing
responsibility for territory maintenance. Litters averaging 3–7
pups are typically born March–May in most North American
latitudes after a gestation of 60–63 days (Gier 1968; Hamlett
1938; Kennelly et al. 1977; Knowlton 1972) and both parents
participate in the care and rearing of young (Andelt 1985;
Camenzind 1978; Gier 1968; Hatier 1995; Mengel 1971; Silver
and Silver 1969).
Mature offspring may disperse or remain within their natal
territories, assisting in the defense of resources and infant pups,
but typically only the dominant male and female breed (Andelt
1985; Bekoff and Wells 1986; Gese 2001; Gese et al. 1989,
1996). Juvenile coyotes ,12 months of age can be reproduc-
tively active in their 1st winter, but available evidence suggests
that juvenile and yearling (12–24 months) females are less
fecund than adult females 2 years of age (Gier 1968; Green
et al. 2002; Hamlett 1938; Kennelly and Johns 1976; Sacks
2005; Windberg 1995). Older females 10 years of age
gradually pass into reproductive senescence (Green et al. 2002;
Sacks 2005), whereas a male coyote was reported to have sired
pups when 12 years of age (Gese 1990). Older coyotes may
continue to maintain territory residency or revert to a transient
lifestyle (Gese 1990; Windberg 1995).
The reproductive tracts of adult coyotes experience extensive
remodeling during the breeding season, and histological evi-
dence suggests a female coyote is incapable of serial ovula-
tions, even if she is not impregnated during her 1st estrus (Gier
1968; Hamlett 1938; Kennelly and Johns 1976). Ovulation is
* Correspondent: debra.carlson@usu.edu
Ó2008 American Society of Mammalogists
www.mammalogy.org
Journal of Mammalogy, 89(3):654–664, 2008
654
spontaneous, synchronous, and bilateral. The subsequent
corpora lutea crowd other ovarian tissue to such a degree that
the existence of additional tertiary follicles appears improbable.
Furthermore, ovarian retrogression is protracted, the corpora
lutea taking .9 months to degenerate, thereby inhibiting a new
wave of follicular recruitment (Hamlett 1938).
Hypertrophy of the uterus and vagina are also remarkable
with gross morphological differences between sexually mature
and immature females (Gier 1968; Kennelly and Johns 1976).
Juvenile females may experience up-regulation of reproductive
hormones (specifically estradiol) and concomitant physical
signs such as vulvar edema and a serosanguineous vaginal
discharge. However, they may not ovulate (Hodges 1990;
Stellflug et al. 1981), with follicular development arresting
before, or at, the tertiary stage (Kennelly and Johns 1976).
Alternatively, subordinate females may ovulate, but proestrus
and estrus in these individuals appears delayed relative to the
estrous phases of dominant female pack-mates, and typically
subordinate females fail to breed (Hodges 1990).
Among wild species, not dependent on human intervention,
successful reproduction relies on a progression of key elements
(Asa and Valdespino 1998; Kleiman and Eisenberg 1973).
Mate acquisition, conception, gestation, and parental care rely
upon effective synchronization of physiological processes,
anatomical modifications, and social behaviors. During 4
consecutive breeding seasons (2000–2003) we measured the
levels of estradiol, progesterone, prolactin, and relaxin in
coyote sera and plasma. Concurrently, samples of exfoliated
vaginal epithelium were collected in proestrus, estrus, and
diestrus, and examined microscopically; observations of
mating behavior also were documented. The data were then
aligned to each individual female’s estimated day of ovulation.
Females housed with their mates (and that became pregnant)
were compared to a nonpregnant cohort (sequestered females).
Herein, we describe our observations and findings, comparing
pregnant coyotes with pseudopregnant females, but also
examine associations among behavior, endocrine patterns,
and vaginal cytology. Examination of our data suggests that
important relationships exist between these factors, and
integrated examinations of complex systems can increase our
understanding to an extent that might not be accrued from
simple experimental constructions.
MATERIALS AND METHODS
Animals.—Coyotes were captive born or wild caught as
pups, and reared at the National Wildlife Research Center
facility in Millville, Utah (418689N, 1118829W). All animals
were housed in outdoor enclosures with natural lighting. Adult
(2 years of age) coyotes were randomly assigned an unrelated
mate before winter, and whenever possible, an established pair
remained together for several years. Mated pairs resided in
individual 0.1-ha pens with access to sheltered den boxes.
Three pens formed a clover-shaped cluster separated by double
fencing and concrete barriers. Although physically separated,
all pairs were within visual and audible range of other coyotes.
As required, females were sequestered from their mates during
the breeding season and served as nonpregnant controls. In
these cases, the coyotes were housed individually in sheltered
outdoor kennels with attached den boxes for privacy, and pair-
mates were kept near each other in adjacent kennels during
their separation.
Mean (6SD) ages for female and male coyotes in this study
were 4.7 62.0 and 5.6 63.2 years, respectively; and females
weighed an average 11.0 61.3 kg (weights of males were not
collected). The coyotes were fed a commercially prepared
carnivore diet (Fur Breeders Agricultural Cooperative, Sandy,
Utah) once daily, and fasted 1 day per week. Water was
provided ad libitum. Vaccinations were given annually against
canine distemper, hepatitis, leptospirosis, parvovirus, para-
influenza, type 2 coronavirus, adenovirus, and rabies. Routine
parasite control was administered as indicated. Research proto-
cols were approved by the Institutional Animal Care and Use
Committees at Utah State University and National Wildlife
Research Center, and were in compliance with guidelines of the
American Society of Mammalogists (Gannon et al. 2007).
Study design.—During 2000–2003 breeding seasons (January–
March), mating behavior of 32 pairs of coyotes was observed
and recorded. In 2000–2002, serial blood and vaginal cytology
samples were collected from a subset of 18 females, in 2 cohort
groups—8 sequestered during estrus and 10 that remained with
their mates.
Females were considered ‘‘pregnant’’ in years when they
resided with their mates, and were observed with live pups
after parturition, or tested positive for relaxin. Alternatively,
sequestered females were considered ‘‘nonpregnant.’’ Hormone
profiles of 2 coyotes were excluded when they experienced
idiopathic spontaneous abortions in mid- and late-term
gestation. In the 1st case, 2 expelled fetuses were recovered;
in the 2nd case, 1 fetus was seen but later was consumed by the
female. So although their pregnancies were confirmed, cause of
the abortions was unknown, and the hormone data were con-
sidered potentially misrepresentative of a normal pregnancy.
However, behavioral and vaginal cytology data for these indi-
viduals were included with those of the pregnant cohort.
Ovarian cycle.—The reproductive cycle of a female is
partitioned into phases based upon changes in physiology or
behavior. For coyotes in this study, estrus was the phase during
which the female was receptive and cooperative with her
mate’s attempts to copulate, and the beginning of estrus was
demarcated by the 1st observed copulatory tie (or sperm in the
vaginal smear). Proestrus precedes estrus and is characterized
by the presence of red blood cells in vaginal exudate. Diestrus
immediately follows estrus and includes pregnancy and the
protracted luteal phase of nonpregnant females. Luteal activity
is typically prolonged in nonpregnant canids; and because the
expression of progesterone is indistinguishable between
pregnant and nonpregnant females, the latter are commonly
described as pseudopregnant (Concannon et al. 1989; Feldman
and Nelson 2004). Accordingly, a coyote was described as
pseudopregnant if there was a marked and sustained elevation
of serum progesterone after ovulation. Pseudopregnancy was
assumed to be covert and subclinical (i.e., no mammary
development, lactation, or denning behavior) unless otherwise
June 2008 655CARLSON AND GESE—REPRODUCTIVE BIOLOGY OF THE COYOTE
noted. After parturition, or regression of the corpora lutea,
hormone synthesis diminishes and anestrus follows diestrus.
Specimen collection and handling.— Peripheral blood sam-
ples were collected weekly by venipuncture of the cephalic or
saphenous veins, or daily from an indwelling catheter in the
external jugular vein. Samples were collected between 0700
and 0900 h, before the animals were fed and without sedation
or anesthesia. In some cases sampling began as early as 4
weeks before a female was receptive to her mate’s attempts to
copulate, or 6 weeks before a sequestered female ovulated.
Blood sampling continued throughout estrus and diestrus with
the latest samples collected 3 weeks after the birth of pups. To
minimize investigator disturbance of mating activity, blood
collection from paired females was restricted to weekly
sampling until 3 weeks after the 1st copulatory tie.
For quantitative analyses of progesterone, estradiol, and
prolactin, whole blood was collected in an evacuated tube and
allowed to clot at room temperature (20–248C) for 30–120 min.
The serum was separated from the blood cells, divided
into aliquots, and stored at 208C until testing. Progesterone
levels were determined from 727 specimens, and sample
subsets also were used to measure estradiol (n¼405) and
prolactin (n¼205).
Specimens for qualitative assay of relaxin were collected
as whole blood in sodium or lithium heparin. Plasma was
separated as soon as possible and stored at 208C until
testing. Samples for relaxin were collected from the 32 coyote
females included in this study but also from additional animals
enrolled in other studies at the research facility. Collectively,
these data were used to validate relaxin as a diagnostic marker
of pregnancy in coyotes and the results have been discussed
elsewhere (Carlson and Gese 2007).
Exfoliated vaginal epithelial cells were collected weekly
(typically the same day as a blood sample) using a sterile swab
premoistened with normal saline. The swab was gently passed
into the vaginal vault, carefully avoiding the clitoral fossa, and
rotated against the lining of the vaginal lumen (Feldman and
Nelson 2004). Once withdrawn the swab was immediately
rolled along a clean glass slide in 2 or 3 rows. The sample was
allowed to air dry at room temperature then fixed and stained as
soon as possible.
Mating behavior.—Continuous scanning observations were
conducted daily throughout available daylight hours, January–
March, 2000–2003. The animals were habituated to low-level
human activity before data collection although all enclosures
could be viewed through binoculars or a spotting scope from
observation sites 100–500 m away. The observer would view
a pen, document any interactive behavior occurring between
pair-mates, then scan the next pen. Because this process rarely
took more than 30 s per pen, all pens were viewed at least once
every 5–10 min.
Characterization of mating behavior was standardized
between observers and documented (Bekoff and Diamond
1976; Golani and Mendelssohn 1971; Schenkel 1967).
Behaviors recorded included courtship (nonantagonistic play-
wrestling or play-chases; allogrooming face, ears, or back;
body-bumps; hip-pushes; or sleeping curled against each
other); olfactory sampling (sniff or lick of the female’s
anogenital region by the male, solicitation by females with
diverted tail, and sniff or lick of the male’s inguinal area by the
female); overt sexual activity (attempted mount usually
preceded by the male standing perpendicular to the female
with his head or bent foreleg on her shoulders or back, male
mounting the female, and copulatory tie or lock); and mate
guarding (male shadowing the female around the pen, or when
in view of neighbors the male would stand on the female with
stiff forelegs on her back, or stand over her as she lay on the
ground).
Observers avoided redundant documentation by recording
a mating behavior only once even if a pair of coyotes continued
the behavior for an extended period of time (e.g., playing might
last 15 min and through several scanning passes). Exceptions
were any behavior that was terminated then reinitiated (e.g.,
precopulatory mounts).
Reproductive hormone assays.— Quantitative measurement
of progesterone in coyote sera was performed by competitive
binding enzyme immunoassay according to the manufacturer’s
instructions (Progesterone EIA, DSL-10-3900; Diagnostic
Systems Laboratories, Inc., Webster, Texas). By this method,
horseradish peroxidase–labeled progesterone competed with
free progesterone in coyote sera for a fixed quantity of rabbit
anti-progesterone. Microtiter wells coated with goat anti-rabbit
immunoglobulin G captured the antibody-bound progesterone.
Extraneous material was rinsed from the well, and addition of
tetramethylbenzidine chromogenic solution permitted photo-
metric measurement (Benchmark microplate reader; Bio-Rad
Laboratories, Hercules, California) of reagent standards,
controls, and unknown samples. Unknown coyote samples
were compared to a standard curve generated for each run
using Microplate Manager/PC software (version 4.0; Bio-Rad
Laboratories), with the quantity of progesterone being in-
versely proportional to the intensity of color development.
Stated level of sensitivity for progesterone was 0.13 ng/ml.
Quantitative measurement of estradiol was performed by
competitive binding enzyme immunoassay (3rd Generation
Estradiol EIA, DSL-10-39100; Diagnostic Systems Laborato-
ries). In this assay, estradiol–biotin conjugate competed with
free estradiol in coyote sera for available rabbit anti-estradiol
sites fixed to microtiter wells. Streptavidin–horseradish perox-
idase was added, binding to the biotinylated estradiol, and
tetramethylbenzidine precipitated color development in the
reagent standards, controls, and unknown coyote samples.
Color development measured with a photometer (Benchmark
microplate reader) was inversely proportional to the quantity of
estradiol captured in each well, and unknown coyote samples
were compared to a standard curve generated for each run
using Microplate Manager/PC software (version 4.0). Level of
sensitivity for this assay was 1.5 pg/ml.
Coyote samples were not pretreated or extracted before
testing for progesterone or estradiol. Validation procedures
including linearity and recovery assessments were performed
and the assays were found acceptable for use in this species
(Carlson 2008). When possible, samples from the same cohort
were tested together to reduce reagent lot-to-lot variability.
656 JOURNAL OF MAMMALOGY Vol. 89, No. 3
Unknown samples were tested in duplicate and intra-assay
coefficient of variation (CV) was 10% for all results included
in the data set. For progesterone reagent standards and controls,
within-lot interassay mean CV was 9.6%, and interlot CV was
20.2%. For estradiol (single lot only) interassay mean CV was
11.2%.
Prolactin was quantitatively measured by double-antibody
prolactin radioimmunoassay at the Colorado State University
Endocrine Laboratory (Colorado State University, Fort Collins,
Colorado). In this assay, coyote prolactin competed with
125
I
canine prolactin for a fixed amount of rabbit anti-canine
prolactin antibodies. Anti-rabbit immunoglobulin G was added
and radioactivity of the precipitated pellet was measured.
Unknown coyote samples were compared to a standard curve,
with the amount of iodinated antibody–antigen complexes
detected being inversely proportional to the quantity of pro-
lactin in the coyote sera. Lowest detectable limit for prolactin
was 2.33 ng/ml.
Qualitative measurement of relaxin in heparinized coyote
plasma was performed by enzyme-linked immunoassay for
canine relaxin (ReproCHEK; Synbiotics Corporation, San
Diego, California). Free relaxin in an unknown sample was
captured between polyclonal anti-relaxin antibodies in solid
phase (microtiter wells) and monoclonal anti-relaxin antibodies
conjugated to horseradish peroxidase. Subsequent color de-
velopment was directly associated with presence or absence of
relaxin in the sample. Absorbance was measured photometri-
cally, and an optical density .0.050 (minimum threshold for
a positive result) was adopted to distinguish between preg-
nancy and pseudopregnancy in the coyote. It was also im-
portant to note that relaxin persisted in peripheral blood after
parturition and could not reliably predict abortion in coyotes
(Carlson and Gese 2007).
Vaginal exfoliative cytology.— Air-dried samples of vaginal
epithelial cells and uterine exudate on glass slides were fixed
with methanol and stained with a modified Wright–Giemsa
stain (Diff-Quik; Jorgensen Laboratories, Loveland, Colorado).
Slides were then examined under high dry magnification
(400) with at least 5 fields per row (10 fields per slide) of
stained material viewed. The observed epithelial cells were
characterized as parabasal, small intermediate, large interme-
diate, superficial, and keratinized (anuclear) superficial (Shutte
classification—Christie et al. 1972; Feldman and Nelson 2004),
and their relative representations in the sample were graded on
a semiquantitative scale of 1–5 (Bradley and Benson 1974). In
addition, inclusions such as white blood cells, red blood cells,
mucus, amorphous debris (degenerating blood and epithelial
cells), and spermatozoa also were noted.
Data analysis.—Data were aligned by the estimated day of
ovulation for an individual before it was compiled by study
group. This estimate was either back-calculated from the day of
parturition, or based upon changes in serum progesterone
levels. Examination of data presented by Kennelly and Johns
(1976) suggested that coyotes ovulate immature (primary)
oocytes, similar to domestic dogs (Canis familiaris). If true,
then fertilization probably does not occur until 2–3 days after
ovulation (domestic dog—Tsutsui 1989). In this study, there-
fore, gestation was standardized and assumed to be 62 days
from fertilization (Gier 1968; Hamlett 1938; Kennelly et al.
1977) or 64 days after ovulation. Alternatively, the day of
ovulation for nonpregnant females was inferred from the
change in daily progesterone levels; specifically, the day on
which progesterone concentrations approximately doubled
from previous samples.
Hormone and vaginal cytology data are presented herein as
weekly mean values of all females within a cohort, and results
obtained on the estimated day of ovulation are included in
‘‘Week 1.’’ In circumstances in which multiple hormone assay
results were available for an individual within a given week,
a weekly mean value for that individual was calculated in order
to normalize the influence of each individual’s contribution to
the cohort mean. Behavioral observations were categorized,
aligned by the day of ovulation for each individual, then
reported as cumulative daily or weekly data for the cohort.
Multivariate analysis of variance and repeated-measures
statistical procedures were used to analyze endocrine hormone
profiles and detect differences between study groups, and
between successive weeks (Statistical Analysis System, SAS,
version 8.2; SAS Institute Inc., Cary, North Carolina). Pearson
correlation coefficient and multiple regression procedures
were used to analyze relationships between hormones and
behavior, and between hormones and vaginal cytology. Unless
otherwise noted, we assumed a level of statistical significance
to be ,0.05.
RESULTS
Behavior.—Females in this captive population were natu-
rally synchronized; each individual began behavioral estrus
within a 4-week period, mid-January to mid-February. Before
estrus, intrapair activity consisted of courtship behavior
including allogrooming, play-wrestling, play-chases, body-
bumps, and hip-pushes (Fig. 1). Olfactory sampling (i.e.,
anogenital and inguinal sniff or lick) by both the male and
female, as well as mate guarding, also increased during pro-
estrus. Males began mounting attempts; however, the overtures
were usually rejected by the female using gentle admonition,
aggressive rebuff, or passive avoidance tactics (such as sitting,
lying down, or running away).
Mounting attempts became more frequent, and the 1st cop-
ulatory tie marked the start of estrus. In contrast to the response
observed in proestrus, females were tolerant and receptive to
the males’ mounting attempts in estrus, and often the female
would solicit attention by positioning herself in front of the
male and diverting her tail. Also, an increased frequency of
mounting was expected during estrus because this was the
antecedent posture to copulation, and it was common for a male
to mount and dismount several times before achieving
intromission and coital lock.
Copulatory ties generally lasted 5–45 min, with ties occurring
early in estrus lasting longer than those observed later toward
the end. The earliest a copulatory tie was observed during this
study was on day 9, and the latest on day 15; however, 98.4%
(179/182) of all ties occurred between day 8 and day 10. At
June 2008 657CARLSON AND GESE—REPRODUCTIVE BIOLOGY OF THE COYOTE
the individual level, behavioral estrus lasted (mean 6SD) 7.6 6
6.0 days, with 59.4% (19/32) of pairs of coyotes beginning
estrus before ovulation (day 2.2 63.9 days).
During preovulatory estrus, physical contact such as body-
bumps and hip-pushes continued to rise. Mate guarding
postures such as a stand-over within view of neighbors and
shadowing became more frequent. Olfactory sampling (male
and female) increased almost 3-fold; specifically, vaginal sniff
or lick by males doubled on day 6 from the previous day,
continued to increase, then peaked on the estimated day of
ovulation.
Immediately after the periovulatory pulse of activity there
was a brief quiescence before the sexual activities of the cohort
pairs peaked again on day 5, particularly copulations (Fig. 1).
Sixty percent (203/341) of mounts and 65% (119/182) of ties
occurred during postovulation estrus. When pairs of coyotes
were observed in .1 tie per day, the multiple ties most fre-
quently occurred on days 4–6. Mate guarding also showed
a postovulatory surge on days 5–6. Mounting attempts peaked
on day 7 although this was associated with a decline in the
number of successful copulations.
As estrus waned, females began to reject some (but not yet
all) of the males’ attempts to copulate. Physical bodily contact
remained high in late estrus, but play behavior was only
sporadically observed until the transition into diestrus. On day
11 postovulation, sexual activities abruptly declined and obser-
vation of copulatory ties (2/182), mounting attempts (4/341),
olfactory sampling (14/667), or mate guarding (2/163) were
relatively rare (1.1%, 1.2%, 2.1%, and 1.2%, respectively)
during the next 10 days (Fig. 1). In 2001 a pair was observed in
a single tie 17 days after the previously recorded copulation;
similarly in 2002, another pair tied 15 days after their previous
mating. Back-calculation from parturition suggested an earlier
date of fertilization in both cases, and because neither copula-
tion was associated with other sexual behaviors (the tie lasted
,2 min), these events were considered to have had some other
unexplained intrapair social function rather than a sign of
extended or split estrus.
Although there was a dearth of sexual activity, the coyotes
in diestrus continued to engage in physical contact such as
allogrooming, body-rubs, play-wrestling, and chasing. In addi-
tion, a previously unseen behavior emerged, begging (Fig. 1).
Beginning on day 10 and continuing through the end of the
observations, females were periodically observed in a juvenile-
like submissive behavior that sometimes provoked regurgita-
tion of food by the male. Specifically, the female would
approach her mate with her tail held low and wagging, then she
would lick his mouth or gently bite his lower jaw, matching his
movement if he tried to turn away. Sometimes the male would
admonish the female and escape. At other times, however, the
female would cease the behavior and appear to be eating off the
ground. In 7 of 55 cases line of sight allowed the observer to
confirm that the food being consumed by the female had just
been regurgitated by the male.
Reproductive hormones.—During proestrus and estrus,
spontaneous up-regulation of ovarian steroid synthesis was
evident in changes in peripheral blood levels of estradiol and
progesterone, regardless of a coyote’s social status (Fig. 2).
Sequestered females (Fig. 2A) experienced a pattern of steroid
expression (estradiol followed by progesterone) and periodicity
similar to that of mated females (Fig. 2B), and distinction
between groups was not discernible until pregnancy was
established.
In both study groups of coyotes, fluctuating serum estradiol
levels generally increased during proestrus (Fig. 3), and an
interweek comparison, week 3 to week 2, suggested a
significant incremental rise in mean values (F¼20.93, d.f. ¼
1, 8, P¼0.002). Also during this time, the rate of change
among females residing with their mates appeared different in
contrast to the sequestered females (F¼7.84, d.f. ¼1, 8, P¼
0.023), although the between-group weekly means remained
statistically indistinct (week 3, P¼0.917; week 2, P¼
0.245). In estrus, weekly preovulation (week 1) estradiol
levels peaked at (mean 6SD) 57.1 616.3 pg/ml (n¼5)
among mated females (pregnant cohort) and 44.2 621.1 pg/ml
(n¼5) in the nonmated (nonpregnant cohort), whereas
postovulation (week 1) levels subsequently declined in both
groups.
Estradiol levels continued to fall from late estrus to early
diestrus (week 1 to week 2). Although the decremental change
was similar between groups (F¼0.07, d.f. ¼1, 8, P¼0.801),
comparison of the between-group mean difference was border-
line (week 2, Pjtj
0.05(2),8
2.16 ¼0.063, F¼4.45, d.f. ¼4, 4).
From week 2 to week 3, however, the study groups demon-
strated a notable divergence (F¼6.41, d.f. ¼1, 8, P¼0.035)
in estradiol levels. Specifically, the pregnant cohort experi-
enced a transient spike in week 3 (38.9 615.0 pg/ml, n¼5)
that was different (Pjtj
0.05(2),8
2.70 ¼0.027, F¼1.80, d.f. ¼
4, 4) from the nonpregnant group (16.2 611.2 pg/ml, n¼5;
Fig. 3). Nevertheless, serum estradiol levels continued to fall in
both cohort groups, and fluctuations appeared to dampen as
pregnant females approached parturition and nonpregnant
females entered anestrus (Fig. 3).
FIG.1.—Social and mating behaviors of coyotes (Canis latrans)
shown as daily cumulative data (n¼1,757) from 32 mated pairs during
breeding seasons in 2000–2003. Observations represent 21 days before
and after the estimated day of ovulation (day 0 on the chart). Behavioral
estrus ranged from day 8 to day 10 (as shown by the solid bar).
658 JOURNAL OF MAMMALOGY Vol. 89, No. 3
Although estradiol was the predominant ovarian hormone in
proestrus, progesterone synthesis was detectable in female
coyotes during this period (Fig. 4). Immediately before estrus,
the incremental change in mean progesterone levels, from week
2 to week 1, was significant (F¼6.24, d.f. ¼1, 14, P¼
0.026). Furthermore, while the most notable change observed
was periovulation (week 1 to week 1: F¼27.94, d.f. ¼1, 14,
P,0.001), successive weekly levels rose or fell significantly
(P,0.05) from week 2 through week 7; exceptions were
week 1 to week 2 (F¼3.22, d.f. ¼1, 14, P¼0.094), and week
3 to week 4 (F¼0.93, d.f. ¼1, 14, P¼0.352).
During estrus, progesterone levels in the mated females
(pregnant cohort) rose from (weekly mean 6SD) 58.9 667.5
ng/ml (n¼7) to 89.3 694.4 ng/ml (n¼8), whereas levels in
the nonmated females rose from 18.5 623.2 ng/ml (n¼8) to
74.1 645.2 ng/ml (n¼8). However, the mean levels were not
different between groups (week 1, P¼0.179; week 1, P¼
0.208; and week 2, P¼0.687). In fact, no overall effect of
status was detected throughout the study period (week 2to
week 7: F¼2.34, d.f. ¼9, 6, P¼0.157), possibly because
of the degree of individual variability observed among the
coyotes. For example; among females in estrus and residing
with their mates (pregnant cohort), the progesterone minimum,
maximum, and CV, respectively, were: week 1¼2.8 ng/ml,
181.4 ng/ml, 1.1; week 1 ¼6.7 ng/ml, 266.5 ng/ml, 1.0; and
week 2 ¼10.3 ng/ml, 305.5 ng/ml, 1.1. Among sequestered
females in estrus (nonpregnant cohort) the same hormone
parameters were: week 1: 5.4 ng/ml, 75.1 ng/ml, 1.3; week 1:
13.3 ng/ml, 108.9 ng/ml, 0.7; and week 2: 15.8 ng/ml, 147.5
ng/ml, 0.6.
Regardless of the variation, the secretion pattern of pro-
gesterone was generally consistent between study groups.
Progesterone levels (mean 6SD) peaked between week 3
(pregnant, 104.6 697.8 ng/ml, n¼7) and week 4
(nonpregnant, 85.0 657.8 ng/ml, n¼8) in pregnancy and
diestrus. Subsequently, levels declined in both groups. The
pregnant cohort appeared to experience a transient surge in
week 7, but the groups remained statistically indistinct until
parturition and the end of sample collection (Fig. 4).
In contrast to progesterone, there was a distinctive overall
effect of status (F¼6.03, d.f. ¼6, 6, P¼0.023) on coyote
prolactin blood levels. During early pregnancy and diestrus,
week 2 through week 4, mean prolactin levels did not markedly
change within either cohort group; however, subsequent weeks
showed a pronounced elevation among pregnant females
(Fig. 5). Specifically, a significant change occurred between
weeks 4 and 5, both in mean prolactin levels (F¼41.26, d.f. ¼
1, 11, P,0.001) and intercohort rate of change (F¼13.34,
d.f. ¼1, 11, P¼0.004). Levels in pregnant coyotes rose from
24.8 64.5 ng/ml (week 4, n¼5) to 33.0 67.8 ng/ml (week 5,
n¼5), whereas among nonpregnant coyotes prolactin
FIG.2.—Temporal relationship of weekly mean blood levels of
estradiol (pg/ml), progesterone (ng/ml), prolactin (ng/ml), and relaxin
(exp(optical density) 10; for graphical representation only), aligned
to the estimated day of ovulation (day 0). A) Pseudopregnant females;
B) pregnant coyotes. Solid arrow indicates ovulation. Columns
indicate weekly number of copulatory ties observed during the
breeding seasons in 2000–2003; dashed arrow indicates day of
parturition (day 64 postovulation). Solid bars indicate phases of
ovarian cycle studied.
FIG.3.—Weekly mean (6SD) serum levels of estradiol (pg/ml)
from 5 pregnant (n¼117) and 5 nonpregnant (n¼288) coyotes
during the breeding seasons in 2000–2002, aligned to the estimated
day of ovulation (day 0). An asterisk (*) indicates statistical difference
detected between study groups. Missing columns are weeks for which
there were insufficient data available.
June 2008 659CARLSON AND GESE—REPRODUCTIVE BIOLOGY OF THE COYOTE
increased from 19.6 65.5 ng/ml (week 4, n¼8) to 21.8 66.2
ng/ml (week 5, n¼8). Thereafter, prolactin levels remained
elevated throughout pregnancy, parturition, and the 1st week of
lactation in those coyotes observed with live pups. Non-
pregnant females, meanwhile, continued to synthesize prolactin
but at lower levels (P0.006).
Relaxin was detectable in plasma of pregnant coyotes within
4 weeks after ovulation (Fig. 5); specifically, 10 of 11 pregnant
coyotes tested positive (optical density .0.050) on day 27
postovulation, and 20 of 21 females with pups were positive on
days 28–30. By comparison, relaxin was not detected (optical
density ,0.033) in samples collected from 2 females residing
with castrated mates, 7 nonmated females, or 8 male coyotes.
In addition, from week 5 through parturition, relaxin remained
detectable in all samples collected from pregnant females; and
although absorbance intensity weakened, postpartum females
did not revert to negative until several weeks after whelping.
Vaginal exfoliative cytology.— A serosanguineous discharge
from the vagina was not always apparent upon gross exami-
nation of a female coyote in proestrus, but red blood cells were
typically observed microscopically when the vaginal smear
was examined (Fig. 6A). Epithelial cells on these smears
were primarily of parabasal and intermediate cell types but
gradually, as the female progressed through proestrus, the ex-
foliated epithelial cells appeared larger and samples presented
as admixtures of parabasal, small and large intermediate, and
superficial epithelial cells (Fig. 7). Red blood cells, amorphous
debris, and mucus remained grossly obvious throughout
proestrus (Fig. 8). However, leukocytes were only occasionally
seen on smears from this stage, and their occurrence was likely
due to secondary passage with the high number of red blood
cells rather than by diapedesis.
A coyote’s vulva appeared turgid but relaxed in estrus, and
passing a swab into the vagina for collection of exudate
was easier than in proestrus. On vaginal smears, superficial
epithelial cells were the predominant cell type from approxi-
mately day 4 through ovulation to day 7; these cells were
either keratinized (anuclear) or retained pyknotic nuclei
(Fig. 7). Concurrently, appearance of red blood cells, mucus,
and amorphous debris were diminished, and white blood cells
were rarely seen (Fig. 8). Thus, the appearance of superficial
cells (nuclear and anuclear) against a clear background repre-
sented the characteristic vaginal smear during estrus in the
coyotes (Fig. 6B), particularly after ovulation.
Spermatozoa were sometimes viewed in the vaginal smears
(Fig. 6B), confirming that mating had occurred, but they were
an unpredictable and erratic element during estrus. In several
circumstances spermatozoa were not recovered although the
females were known to be actively breeding. Sperm deposition
in the coyote was assumed to be transcervical, as in the
domestic dog, and if true, would thus explain the inconsistent
findings. Nevertheless, among those samples that did contain
spermatozoa, most were collected during the period of frequent
copulations, days 3–6 postovulation.
Toward the end of estrus (days 7–10), vaginal epithelial cells
regressed to intermediate forms; eventually returning to a pre-
ponderance of parabasal cells as the coyotes entered diestrus
(Fig. 7). Also during diestrus, red blood cells, amorphous
debris, and mucus once again became abundant (Fig. 8). Most
notable in this phase, however, was the reappearance and dis-
proportionate number of leukocytes (Fig. 6C), particularly in
relationship to the number of red blood cells (Fig. 8). Thereafter,
a fluctuating mix of blood cells and cellular debris persisted into
anestrus. We observed no discernible difference between the
vaginal smears from pregnant and pseudopregnant coyotes.
Interrelationships among behavior, endocrine patterns, and
vaginal cytology.—During proestrus, significant relationships
between olfactory sampling and other behaviors were detected.
FIG.4.—Weekly mean (6SD) serum progesterone (ng/ml) levels
from 8 pregnant (n¼245) and 8 nonpregnant (n¼482) coyotes
during the breeding seasons in 2000–2002, aligned to the estimated
day of ovulation (day 0). No intergroup statistical difference was
detected. Missing columns are weeks for which there were insufficient
data available.
FIG.5.—Weekly mean (6SD) serum levels of prolactin (ng/ml)
from 5 pregnant (n¼85) and 8 nonpregnant (n¼120) female coyotes
during the breeding seasons in 2000–2002. Data are aligned to the
estimated day of ovulation (day 0). An asterisk (*) indicates statistical
difference detected between study groups. Also shown are plasma
relaxin (OD ¼optical density) readings from 82 pregnant coyotes
(n¼209); seasons 2000–2003. Relaxin was ,0.100 OD for
nonpregnant coyotes (data not shown).
660 JOURNAL OF MAMMALOGY Vol. 89, No. 3
Specifically, there was a strong positive correlation between
olfactory sampling and mate-guarding (r¼0.739, P¼0.0039).
Also, relationships between olfactory sampling and mounting
attempts (r¼0.581, P¼0.0374), as well as with courtship
(r¼0.567, P¼0.0432), suggested that olfactory sampling
may stimulate other behavior, or inform coyotes (both male and
female) of their mate’s physiological status.
Eighty-four percent (558/667) of olfactory investigations
occurred during estrus; with the apparent peak in activity on
day 3 (56 events) reflecting an increase in vulva sniff or
lick and female solicitations. Meanwhile, 88% (143/163) of
mate-guarding events also occurred during estrus, and as in
proestrus, mate-guarding and olfactory sampling maintained
a positive relationship (r¼0.550, P¼0.0146).
Twenty-three percent (42/182) of all copulatory ties occurred
between day 8 and day 1; a time when progesterone syn-
thesis was increasing and estradiol was reaching its preovula-
tion peak on day 4 (Fig. 9). Throughout estrus, however,
copulatory ties appeared to have a significant relationship with
progesterone (r¼0.554, P¼0.0139) yet a poor correlation
with estradiol (r¼0.084, P¼0.7324). Furthermore, an
initial spike in the daily number of ties (18 events) was
observed on the estimated day of ovulation, a day when mean
progesterone levels experienced the greatest incremental
FIG.6.—Representative examples of exfoliated epithelium and
other inclusions commonly seen on a coyote vaginal smear (480).
A) Proestrus, dotted arrow indicates a parabasal cell, dashed arrow
FIG.7.—Relative proportion of exfoliated vaginal epithelial cells
viewed on vaginal smears (n¼133) collected weekly from 18 coyotes
during the breeding seasons in 2000–2002. Data are aligned to the
estimated day of ovulation (day 0) including 5 weeks preovulation to
6 weeks postovulation. Kerat Superf ¼keratinized (anuclear) super-
ficial, Superf ¼nucleated superficial, Lg Intrmd ¼large intermediate,
Sm Intrmd ¼small intermediate, Parabasal ¼parabasal cells.
indicates an intermediate cell, and solid arrow indicates a red blood
cell. Amorphous debris and mucus is conspicuous in the background.
B) Estrus, superficial cells with pyknotic nuclei and spermatozoa are
shown; note the relatively clear background as compared to A and C.
C) Diestrus, dashed arrows indicate intact white blood cells
(neutrophilic leukocytes). Also, parabasal and intermediate epithelial
cells have reemerged, and red blood cells, mucus, and amorphous
debris are abundant.
June 2008 661CARLSON AND GESE—REPRODUCTIVE BIOLOGY OF THE COYOTE
change (CV
(day 1 to day 0)
¼0.275); and another major peak in
copulations (23 events, day 5) immediately followed a second-
ary surge in progesterone (CV
(day 3 to day 4)
¼0.247; Fig. 9).
We noted peaks in mate-guarding (11–13 events per day) on
day 5, day 2 through day 1, and day 5; days immediately
adjacent to peaks in steroid hormone activity and sexual
behavior. However, although mate-guarding and copulatory
ties appeared to be positively correlated (r¼0.521, P¼
0.0221), mate-guarding did not appear to be statistically related
to estradiol (r¼0.248, P¼0.3054) or progesterone (r¼
0.032, P¼0.8964) levels, either singly or as covariables
(
Adj
R
2
¼0.047, P¼0.5629).
As estradiol levels diminished, superficial cells disappeared
(Fig. 10), and a strong association was detected between
estradiol blood levels and the appearance of superficial cells
(r¼0.897, P¼0.0004). Although this evidence suggests
that vaginal smears might be used as a surrogate measure of
breeding condition, defining the specific day of ovulation was
not possible and changes in cytology did not appear to be
correlated with progesterone levels (r¼0.340, P¼0.3358).
DISCUSSION
This study describes for the coyote the secretion pattern and
temporal relationship between estradiol, progesterone, pro-
lactin, and relaxin, from late proestrus through estrus and
diestrus; mating behaviors observed during those same estrous
periods; and changes in vaginal cytology. Furthermore, it
compares reproductive endocrine patterns of females residing
with their mates with those of females sequestered during
estrus, thereby providing contrasting profiles of pregnancy and
pseudopregnancy in the coyote. Graphic and statistical
comparisons also provided insight into relationships between
various elements of coyote reproduction, emphasizing the
importance of viewing reproductive biology as a spectrum and
integration of physiology and behavior.
Proestrus.—Proestrus appears to be a crucial period of
preparation and staging as levels of estradiol and progesterone
rise coincidentally with alterations in coyote behavior and
reproductive tissue. Appearance of red cells in vaginal exudate,
and transformation of epithelial cells, suggested progressive
remodeling of the reproductive tract for the ensuing rigors
of mating and pregnancy. Meanwhile, physical and social
interactions between pair-mates increased, as well as defensive
displays. Positive relationships between olfactory sampling and
other behaviors suggested a possible mechanism by which
either sex might assess the physiological status of their mate,
FIG. 10.—Weekly mean blood levels of estradiol (pg/ml) and
progesterone (ng/ml) and the relative proportion of epithelial cells
observed on vaginal smears from coyotes during the breeding seasons
in 2000–2002, 4 weeks preovulation to 6 weeks postovulation. Data
are aligned to the estimated day of ovulation.
FIG.8.—Inclusions other than epithelial cells viewed on weekly
vaginal smears (n¼133) from 18 coyotes during the breeding seasons
in 2000–2002. RBC ¼red blood cells, WBC ¼white blood cells,
Amorph ¼amorphous debris, Mucus ¼mucus, and Sperm ¼
spermatozoa. Data are aligned to the estimated day of ovulation
(day 0) including 5 weeks preovulation to 6 weeks postovulation.
FIG.9.—Daily mean blood levels of estradiol (pg/ml) and
progesterone (ng/ml) overlaying sexually specific behaviors in coyote
pairs (olfactory sampling, mounts, and copulatory ties) observed
during the breeding seasons in 2000–2003. Data are aligned to the
estimated day of ovulation (day 0).
662 JOURNAL OF MAMMALOGY Vol. 89, No. 3
and ritualized agonistic behaviors may reinforce the pair-bond
or inform a coyote about its mate’s readiness to breed, defend
territory, or provide parental care.
Estrus.—As previously noted, the majority of copulations
occurred after ovulation, suggesting that the coyotes may have
concentrated their reproductive effort to coincide with the
optimal time for viable sperm to encounter mature ova. Yet
interestingly, most 1st ties were observed before ovulation.
Because coyotes ovulate spontaneously, there is no apparent
physiological function that can be ascribed to exceptionally
early copulations (although spermatozoa can survive for several
days within the female reproductive tract). We also observed
exceptionally late copulations (albeit with very short ties).
It is unclear if such sexual behavior exists among free-
roaming coyotes; if so, it would presumably be costly because
a pair would be especially vulnerable to predation while in
a coital lock. But possible social and future benefits gained
through employment of multiple copulations include mate
fidelity, guaranteed paternity, advertisement of a confirmed
pair-bond (thus discouraging competitors), or assured access to
resources (Gier 1975; Hunter et al. 1993; Sillero-Zubiri et al.
1996). The influence of steroid hormones on preovulatory
copulations also cannot be ruled out. Progesterone levels in
coyotes began rising before ovulation and were positively
correlated with copulations. Furthermore, estradiol followed by
progesterone has been linked to expression of sexual behavior
in the domestic dog (Concannon et al. 1979). Therefore, the
preovulation ascendancy of progesterone described in this
study may suggest a possible physiological stimulus for
seemingly premature copulations among coyotes.
Ironically, mate-guarding appeared correlated to copulatory
ties, but not to either steroid. However, these results are not
surprising when one considers the male’s role in mating
behavior (and the influence testosterone certainly exerts during
the breeding season). Regardless, the association between
rising progesterone levels and overt sexual behavior reflects the
female coyote’s sexual determination. A male may shadow
a female, investigate her anogenital scent, play, groom her, et
cetera, but copulations will not occur without her permission
and explicit cooperation.
Cytomorphosis of vaginal epithelium to keratinized or
nucleated superficial cells was transient, but correlated with
estradiol levels. Predominance of superficial cells was most
pronounced after the preovulatory peak in estradiol, and per-
sisted until estradiol was withdrawn in late estrus. Meanwhile,
other cellular inclusions were conspicuously rare or absent.
In cases when mating behavior is unobservable or protracted,
vaginal smears may serve as a qualitative assessment of estra-
diol synthesis, thereby predicting a period of receptivity, or
optimal fertility.
Diestrus.—Estradiol secretion generally dampened during
diestrus although pregnant coyotes experienced an estradiol
pulse during week 3—the period in which implantation occurs
(Gier 1968). Meanwhile, progesterone levels were indistin-
guishable between pregnant coyotes and nonpregnant females.
However, prolactin and relaxin emerged as salient hormones
in diestrus, differentiating pregnancy from pseudopregnancy.
Relaxin was pregnancy specific and became detectable in
week 4, followed shortly thereafter by an increase in prolactin.
Prolactin levels in pregnant coyotes then diverged from
pseudopregnant females in week 5. Both hormones remained
elevated thereafter until parturition, persisting for several weeks
after the pups were born. It has been hypothesized (but not yet
established) that relaxin may serve as a signal between embryo
and mother, possibly stimulating prolactin synthesis, which in
turn facilitates the persistence of progesterone (required for
the maintenance of pregnancy in canines—Concannon et al.
2001). But although the temporal associations between relaxin,
prolactin, and progesterone are suggestive in this data set,
under the conditions of this study we were unable establish a
definitive link in the coyote.
As sexual behavior waned, food-begging immediately
appeared in diestrus. Episodes of begging, and sometimes
regurgitation, were seen throughout pregnancy with greater
intensity occurring during weeks 4 and 5. Interestingly,
begging was not restricted to pregnant females. In a tangent
study, pseudopregnant females who remained housed with their
mates throughout the breeding season also were observed
begging food (and receiving regurgitate) from the males
(Carlson 2008).
In conclusion, this study showed that key elements of
reproductive behavior, endocrine profiles, and vaginal cytol-
ogy are discernible in coyotes. Biologists studying wild or
captive populations might find it helpful to focus on selected
criteria when assessing a breeding population. The value of
seeing a pair in a copulatory tie, viewing spermatozoa on
a vaginal smear, or detecting pups in a den are obvious.
However, in the absence of such indicators, investigators might
depend instead upon the observation of affinitive and
appetitive behaviors in coyotes. When blood samples can be
obtained, measurement of blood hormone levels can provide
evidence of ovulation, and distinguish pregnancy from
pseudopregnancy. Also, the relative prevalence of exfoliated
epithelial cells, red cells, leukocytes, and other inclusions on
a vaginal smear can help predict the breeding status or
reproductive potential of an animal. However, the use of
several of these elements together will increase the overall
confidence in determining the reproductive status of an
individual, or population of coyotes.
ACKNOWLEDGMENTS
We thank T. D. Bunch, F. F. Knowlton, R. T. Skirpstunas, M. L.
Wolfe, and 2 anonymous reviewers for their insightful comments on
an earlier version of this manuscript and their valuable suggestions
for improvement. Thanks also to D. A. Wannemacher. We appreciate
the following National Wildlife Research Center staff as well as
undergraduate and graduate students at Utah State University for
assistance in the handling and care of the study animals, behavior
observations, and specimen collections: K. Anderson, R. Bartel, S.
Brummer, K. Casper, P. Darrow, R. Harrison, J. Hedelius, D. Jones, R.
Kikkert, S. Kirshner, L. Minter, H. Phillips, J. Robinson, A. Seglund,
H. Smith, J. Tegt, K. Wenning, M. Wollbrink, and D. Zemlicka. We
are grateful to T. J. DeLiberto for arranging the funding and logistical
support provided by United States Department of Agriculture, Animal
June 2008 663CARLSON AND GESE—REPRODUCTIVE BIOLOGY OF THE COYOTE
and Plant Health Inspection Service, Wildlife Services, National
Wildlife Research Center, Logan Field Station, Utah State University,
Logan, Utah.
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Submitted 16 December 2006. Accepted 23 October 2007.
Associate Editor was Roger A. Powell.
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