BIOLOGY OF REPRODUCTION 32, 36 1-368 (1985)

I mmunoreactive Luteinizing Hormone, Estradiol, Progesterone,

Testosterone, and Androstenedione Levels During the
Breeding Season and Anestrus in Siberian Tigers

ULYSSES S. SEAL,”2 EDWARD D. PLOTKA,3

JAMES D. SMITH,2 FRANK H. WRIGHT,4
NICHOLAS J. REINDL, ROSS S. TAYLOR4
and MARIALICE F. SEAL5

Research Service2
VA Medical Center
Minneapolis, Minnesota
and
Departments of Biochemistry and Wildlife
University of Minnesota
St. Paul, Minnesota

Marshfield Medical Foundation3

Marshfield, Wisconsin

Minnesota Zoological Garden4

Apple Valley, Minnesota

Bloomington, Minnesota5

ABSTRACT

Seasonal analysis of 1239 captive births of Siberian tigers (Panthera tigris altaica) indicated a
peak in April to June (P<O.001). Studies on seven animals in Minnesota indicated that behavioral
heat cycles and ovarian follicular phase cycles began in late January and ceased in early June.
Behavioral observation of 12 heat cycles in four tigers yielded an estrous length of 5.3 ± 0.2 days
and an interestrous interval of 25.0 ± 1.3 days. Hormone assays on weekly blood samples (N=180)
from three female tigers indicated 16 cycles in two breeding seasons. Peak estradiol-17p levels were
46.7 ± 6.0 pg/mI (N=17) and interestrous concentrations were 8.7 ± 0.66 pg/mI (N28) during the
breeding season. Anestrous estradiol levels were 4.2 ± 0.5 pg/mI (N=70). The interestrous interval
between estradiol peaks was 24.9 ± 1-. 3 days (N=9) with two outliers of 42 days. Serum progester-
one concentrations from February to June were 1.2 ± 0.15 ng/ml (N=32), providing no evidence
for ovulation or corpus luteum formation. Luteinizing hormone (LH) levels were 0.56 ± 0.04 ng/ml
(N180). Serum testosterone (r=0.71, P<O.001) and androstenedione levels (r=0.75, P<0.0O1) were
correlated with estradiol during the breeding season. The duration of anestrus was 8 mo in two of
these tigers. The interval was shortened in one tiger by exposure to a 16L:8D photoperiod. The
Siberian tiger appears to be a polyestrous seasonal breeder and an induced ovulator whose breeding
season may be synchronized by photoperiod.

INTRODUCTION of reproduction, including contraception (Seal

The large felid species-cheetahs, jaguars, et al., 1978) and artificial insemination (Bonney
leopards, lions, pumas, snow leopards, and et al., 1981; Dresser et al., 1982). However,
tigers-are declining in numbers and many numerous unsuccessful attempts at artificial
forms are considered either endangered or insemination in the big cats have focused
threatened (Goodwin and Holloway, 1972). attention on the paucity of information con-
The need for careful genetic management of cerning the endocrine aspects of the reproduc-
small captive populations (Seal and Foose, tive cycles of the females and males of these
1983) has stimulated interest in elective control species.
The behavioral aspects of the estrous cycle
Accepted October 10, 1984. of Bengal tigers have been described (Kleiman,
Received February 28, 1984.
1974; Sadleir, 1966), but no systematic endo-
‘Reprint requests: U.S. Seal, Bldg. 49, Room 207,
VA Medical Center, 54th St. & 48th Ave. S., Minne- crine studies of tigers have been reported. The
apolis, MN 55417. fact that the domestic cat has been found to be

361
362 SEAL ET AL.

an induced ovulator in about 95% of its cycles days, occurred after the first estrus of the year. On
two occasions, estrus appeared to last 1 day. Three
and the high frequency of copulation during an
isolated estrous periods, following termination of a
estrus in the big cats has led to the inference
pregnancy or removal of cubs, occurred from July to
that they also might be induced ovulators. September. No behavioral estrus was observed from
However, a recent endocrine study of three October to December. The gestation period counted
isolated female lions (Schmidt et al., 1982) from the last day of breeding (at the Minnesota
Zoological Garden) was 104 ± 0 days (mean ± SEM,
found luteal phase levels of progesterone that
N=4).
remained elevated for several weeks in 7 of 9
cycles, indicating this species might be a spon- Demography
taneous ovulator. The survey data on captive Siberian tiger births
The present study was initiated to determine were taken from the International Tiger Studbook
the changes in serum hormone levels during (Seifert, 1980). There was no difference in sex ratio
by month of birth, and the overall sex ratio for 1296
estrus, the interestrous period, and anestrus in
births was 0.986 males per female, which is not
Siberian tigers. We also wished to determine the significantly different from unity. Age of first repro-
frequency and seasonality of estrous and duction was reached at 2 yr. The average litter size was
ovarian follicular cycles, if ovulation was spon- 2.43 ± 0.09 cubs for 436 litters.
taneous or induced, and if estradiol and proges-
Sample Collection
terone or their conjugates were excreted.
into the urine in sufficient quantity for use in The tigers were immobilized each week with
600-900 mg of ketamine, 150 mg promazine, and
following ovarian hormone cycles.
10-20 mg of diazepam administered by blow dart
(Seal et al., in preparation). Additional ketamine was
given as needed to maintain anesthesia. The tigers were
MATERIALS AND METHODS
weighed, rectal temperatures were taken and a baseline
sample of 60 ml of venous blood was collected 35-80
Animals
mm after the first injection of the immobilizing drugs.
Three mature female tigers born in May and June A portion of the blood was placed into tubes con-
1977, lMinnesota zoo numbers 368 (studbook 1422), taining EDTA for hematology and into glass tubes
711 (studbook 1432), and 712 (studbook 1328)j with no additives for serum preparation. The serum
were started in the study in February 1982. All were was separated on the day of collection and frozen
nulliparous. They exhibited behavioral heat cycles in until used for assay. A second blood sample was
the year prior to selection for study, but had not been collected 15-30 mm after the first and was processed
allowed to breed. They were maintained for the study for serum. Hematology analysis indicated that hemo-
in a separate holding facility remote from the exhibit globin, red blood cells and hematocrit values were
area. Each animal was housed separately with free stable during the study and there was no cycle of
access during the day to an outside area (5.5 X 6.8 m) seasonal change, in contrast to the annual cycles
and locked in at night into a connecting indoor area observed in another seasonally breeding carnivore, the
(2.6 X 2.6 m). They were in adjacent cages but not in wolf (Canis lupis) (Seal and Mech, 1983). Rectal
visual contact. An adult male was housed across a temperatures ranged from 37.8 to 40.6#{176}C (38.4 ±

corridor (4 m) from the females where they could see, 0.10, mean ± SEM, N150).
smell and hear him. Water was available ad libitum.
They were fed daily, at 1530 h, 2-3 kg of a prepared Assays
carnivore diet (Nebraska brand Feline Diet) to maintain
Luteinizing hormone (LH) was assayed in a hetero-
an approximately constant body weight. They were
logous radioimmunoassay system utilizing rabbit no.
weighed periodically with an overhead spring balance
15 anti-LH serum supplied by Dr. Gordon Niswender.
that was calibrated with known weights. Body weights
A purified ovine LH from Calbiochem-Behring Corp.
ranged from 112-122 kg for Tiger 368, 108-119 kg
(La Jolla, CA;lot no. 389537) was used for iodination.
for Tiger 711, and 108-122 kg for Tiger 712 during
The assay has a sensitivity of 0.2 ng/ml of sample and
the study. a coefficient of variation (CV) of 7%. Linearity for
tiger LH and parallelism of tiger LH with the ovine LH
Behavioral Observations standard was established by serial dilutions of tiger
Four animals were observed independently of the serum samples and of a Siberian tiger pituitary extract.
hormone studies and notes were made by the same All LH results are expressed in terms of ng NIH-0LH-
person daily for 10-15 mm, prior to the start of S7/ml serum.
maintenance activities. The criteria for estrus (Kleiman, Estradiol was assayed by a modification of the
1974) included increased frequency of calling, rubbing method of Hotchkiss et al. (1971) using methylene
against the walls and bars, rolling onto the back, and chloride as the extractant as previously described
frequent approaches to the front of the cage. Selection (Plotka et al., 1977) and 2,4,6,7,16,17-[3H] estradiol
of times to breed other tigers in this collection based (160 Ci/mM) as the radioactive tracer. The antiserum
on these criteria has resulted in breeding and preg- used was prepared by immunizing rabbits against
nancies. The duration of estrus was 5.3 ± 0.2 days and estradiol-17j3-6-CMO-bovine serum albumin (Vaituka-
the interestrous interval was 25 ± 1.2 days for 12 iris, 1971). The antiserum is specific for estradiol-1713.
cycles. Two long interestrous intervals, 60 and 50 The relative cross-reactivities at 50% displacement are
 ESTROUS CYCLES IN SIBERIAN TIGERS 363

1.1% for estrone and <1% for estradiol-17a and accomplished by addition of an internal standard and
estriol. The assay has a sensitivity of 2 pg (0.5 ml of recounting. Percent quench was calculated from the
sample is used for assay and thus the sensitivity was 4 difference between counts added and counts obtained.
pg/ml serum) and a CV of 20% at 4 pg (Plotka et al., This assay approach includes unchanged steroid,
1980). metabolites, conjugates, and exchanged label and thus
Progesterone was measured by a modification of provides a maximum estimate of urinary excretion of
the method of Thorneycroft and Stone (1972) as administered steroid during the period of sample
described previously (Plotka et al., 1975). The proges- collection.
terone assay has a sensitivity of 10 pg (0.1 ml of serum
was used in the assay and thus the sensitivity was 100
pg/mi) and a CV of 17% at 10 pg. RESU LTS
Testosterone was measured as described by
The seasonal frequency of births in 530
McMillan et al. (1974) with a sensitivity of 50 pg (1 ml
litters (1239 young) of Siberian tigers has been
of sample was used in the assay providing a sensitivity
of 50 pg/mI) and a CV of 15% at 50 pg. highest in April to June (P<0.001), worldwide
Androstenedione was assayed using a kit from in the Northern Hemisphere (Fig. 1). This
Pantex (Santa Monica, CA); sensitivity and CV were analysis suggested that Siberian tigers are
standard for the kit. Statistical analyses were done by
seasonal breeders and provided impetus to
analysis of variance and the criterion of significance
used was P<0.01. design the present study to detect possible
seasonality of estrous and ovarian follicular
Urinary Excretion cycles and to determine their frequency.
Excretion of label in urine was measured in separate
anesthetized animals over 150 mm following the iv. Estradiol
injection of 10 zCi (400 pg) of either tritiated estradiol
or progesterone in 10 ml 0.9% saline. Urine flow was Serum immunoreactive estradiol-1 7 values
established and maintained by subcutaneous adminis- ranged from 5 to 115 pg/mI in 180 weekly
tration of 1.5 1 of 0.9% saline during the sample
samples collected over 15 mo from the three
collection period. Urine was collected via indwelling
tigers (Figs. 2-4). Values greater than 20 pg/ml
catheter every 20 mm. Total tritium in each sample
was measured by addition of 0.2 ml urine to 10 ml of of immunoreactive estradiol were more than 3
cocktail and counting. Correction for quenching was SD greater than the mean of the remaining

160 SIBERIAN TIGER BIRTHS

140 Male

o Female
120
U)
-c
100

80

60

40

20

J FM AM J JASON
n11ffl D
MONTH
FIG. 1. Number of births by month and sex for registered Siberian tigers in Northern Hemisphere zoos. Data
from the International Tiger Studbooks (1976-1982) and ISIS.
364 SEAL ET AL.

90
80

70
a’ ‘TIGER 368
60
0 -

a) 0
50 Os
22
(0
C
(0 40
C
0
0

3
20 2
0
I-.
I0 I a.
Sc

Sc
1982 1983

WEEK OF YEAR
FIG. 2. Serum concentrations of estradiol, testosterone and progesterone in weekly samples from Tiger 368.
The PMSG was given intramuscularly as a single dose of 1200 IU by blow dart on June 25.

.103

as C

as -
w -
C
0
-
a’.Q a’
C
02
4w
(I) C
e
2? 3
:1 0
:1 w
I 0
04 ‘2
a-
I Sc

Sc

WEEK OF YEAR
FIG. 3. Serum concentrations of estradiol, testosterone and progesterone in weekly samples from Tiger 711.
The PMSG was given intramuscularly as a single dose of 1200 LU by blow dart on June 25. The photoperiod was
extended to 16 h of light by use of a 240-watt floodlight mounted in the ceiling of the indoor cage. The animal
was shut into the cage at night to ensure exposure to the light.
 ESTROUS CYCLES IN SIBERIAN TIGERS 365

113 11.5

I
a)
C
0
a)
4-
In
a)
Cs
0
a-
Sc

Sc

WEEK OF YEAR

FIG. 4. Serum concentrations of estradiol, testosterone and progesterone in weekly samples from Tiger 712.
The PMSG was given intramuscularly as a single dose of 1200 lU by blow dart on June 25.

values or the anestrous values and were con- followed by 30 pg/ml in the second sample
sidered indicative of an active ovarian follicu- obtained 30 mm later. This was at the 17-wk
lar phase. During anestrus estradiol levels were mark (Fig. 4) and occurred at 28 days between
4.2 ± 0.5 pg/ml (N=70, range 0.5-9.3 pg/mI). two estradiol peaks separated by 42 days.
Peak estradiol concentrations were 47.6 ± 6.0 The intervals between the estradiol peaks in
pg/mi (N=17, range 23.7-115 pg/mI). Inter- the spring of 1982 and 1983 were 27, 21 and
estrous levels were 8.7 ± 0.7 pg/mI (N=28, 21 days for Tiger 368; 22, 28, 28, 28 and 28
range 1.7- 15.1 pg/mI). These hormone peaks days for Tiger 711; and 42, 42 and 21 days for
were associated with behavioral signs of estrus Tiger 712. The duration of elevated estradiol
on the day of sample collection. Peaks of values was 6-10 days. These three animals
estradiol concentration occurred from February cycled 2, 4 and 3 times, respectively, during the
to June with low values from June through spring of 1982 and were observed for 2, 3 and 2
January under natural photoperiod. Tiger 711 cycles in the spring of 1983. Excluding the two
was exposed to an extended light photoperiod outliers, the interval between peaks was 24.9 ±
(16L:8D) from November 7, 1982 to February 1.3 days (N=9).
17, 1983 and exhibited peaks of estradiol in Measurement of the urinary excretion of
early and late January 1983. tritium for 150 mm after administration of 10
Estradiol concentrations in the second jiCi (400 pg) of labeled estradiol to Tiger 368
sample drawn 15-30 mm after the baseline indicated that 1.3% of the label was excreted in
sample were usually similar to those in the first the urine during this interval.
sample. In a series of 6 samples, collected at
20-mm intervals over 2 h from Tiger 711 on
Progesterone
April 15, 1982 (Week 15) estradiol values
ranged between 50 and 82 pg/mI. Estradiol Serum progesterone concentrations ranged
values in another set of 6 samples, collected from 0.5 to 12 ng/ml in the same set of 180
over 150 mm from the same animal on May 6, samples. The progesterone values were less than
1982 (Week 18) ranged from 4 to 13 pg/mI. In 1 ng/ml in 145 of the baseline samples and 18
one instance on April 29, 1982 in Tiger 712, a values were between 1 and 2 ng/ml (Figs. 2-4).
value of 7 pg/mI in the baseline sample was Excluding the values greater than 2 ng/ml,
366 SEAL ET AL.

serum progesterone concentration was 1.2 ± occurred after administration of pregnant

0.15 ng/ml (range 0.2-1.8 ng/ml) in the mare’s serum gonadotropin (PMSG) (67 and 61
samples collected February through June. ng/dl) and one, in Tiger 712 during the spring
Values greater than 2 ng/ml were observed in of 1982, fell midway between two estradiol
17 of the 180 baseline samples: Ten of these peaks 42 days apart.
were in Tiger 712, our most excitable animal; 3 Androstenedione concentrations ranged from
were in Tiger 368, with none above 3 ng/mi; 0.20 to 5.60 ng/ml in the three tigers in the
and 4 were in Tiger 711, with 1 value (4.6 samples collected from February 12 through
ng/ml) above 3 ng/ml. Eleven of 17 estradiol July 8, 1982. Androstenedione concentrations
peaks were not associated with elevations of were correlated with estradiol (r=0.75, N46,
progesterone and none of the elevated proges- P<0.0001) during the estrous season, as were
terone levels persisted more than 2 wk. Six of testosterone values (r=0.71, P<0.0001). The
the values above 3 ng/ml in Tiger 712 occurred correlation of androstenedione with testosterone
during the apparent anestrous interval, with no was r=0.74 (P<O.0001).
hormonal or behavioral indications of ovarian
cycling. This animal was sick when the highest PMSG and Photoperiod Effects
value (11.5 ng/ml), on January 20, 1983 (Week
Intramuscular administration of 1200 IU of
3; Fig. 4); was obtained. PMSG in saline, on June 25, to each of the 3
Measurement of the urinary excretion of
tigers resulted in an increase of estradiol to 113
tritium for 150 mm after administration of 10 pg/ml in Tiger 712 on July 2 and no estradiol
pCi of labeled progesterone to Tiger 711 elevation in the other 2 tigers (Figs. 2-4).
indicated that 0.9% of the label was excreted
There were testosterone and androstenedione
during this interval. peaks in all three tigers on July 2 and 8 (Figs.
2-4).
LH Exposure of Tiger 711 to a 16L:8D photo-
LH values varied between 0.1 and 2.0 ng/ml period from November 7 to February 11 (Fig.
(one outlier of 3.4 ng/ml) with no peaks 3) resulted in estradiol peaks on January 6 and
consistently associated with either estradiol or January 28, about 6 wk earlier than the other
progesterone peaks. However, the mean of the two tigers. She also exhibited estrous behavior
baseline LH values during the estrous season in these 2 cycles.
(0.82 ± 0.07 ng/ml, N=46) was higher than the
mean during anestrus (0.39 ± 0.06, N=70) for DISCUSSION
all three animals (P<0.0001).
The three Siberian tigers studied were
Stimulation of Tiger 712 with gonadotropin- seasonal their estradiol
in cycles. This correlates
releasing hormone (GnRH) (10 /.Lg in an i.v. with the observed pattern of births in the
bolus) on July 15 during anestrus resulted in an Northern Hemisphere. The occurrence of
increase from the baseline LH of 0.6 ng/ml to
postovulatory, postpartum, and postlactational
6.0 ng/ml at 11 mm, 2.0 ng/ml at 42 mm and a estrous cycles, based upon behavioral observa-
return to baseline at 60 mm. In a control series
tions of other tigers at the Minnesota Zoological
of 6 samples, collected June 10, LH values
Garden, could account in part for births at
ranged from 0.1 to 0.6 ng/ml.
other times of the year. Our experience with
one animal exposed to long-day photoperiods
Testosterone and Androstenedione
in the late fall suggests that the seasonal cycle
Serum testosterone concentrations in the in these animals may be synchronized by
baseline samples (Figs. 2-4) ranged from 10 to photoperiod, as has been established for the
100 ng/dl. During anestrus testosterone concen- domestic cat (Scott and Lloyd-Jacob, 1959).
trations were 23.4 ± 1.1 ng/dl (N70, range This could also account for some of the off-
10-46 ng/dl). Peak testosterone levels were season births in zoo animals that might be ex-
73.9 ± 4.2 ng/dl (N=17, range 52-100 ng/dl). posed to artificially extended photoperiods as a
All of the 17 estradiol peaks greater than 20 part of exhibit or management practices.
pg/mi were accompanied by testosterone peaks The usual cycle length of 25 days, based
greater than 50 ng/dl. Three testosterone peaks upon both the behavioral observations and the
greater than 50 ng/dl were not associated with hormone data, was derived from seven animals
comparable estradiol peaks. Two of these peaks of different genetic lineages over three seasons
 ESTROUS CYCLES IN SIBERIAN TIGERS 367

of observations. A mean cycle length of 49 days indicating that the primary route of estradiol
has been reported for the Bengal tiger in four excretion in cats is via the gut.
institutions (Kleiman, 1974; Rowlands and The correlation of serum testosterone and
Sadleir, 1968). There are no reported hormone androstenedione levels with estradiol, their lack
data for Bengal tigers. We observed 2 cycles of of correlation with progesterone (r=0.06,
extended length in both the hormone and the P>0.2) and their increase after PMSG treatment
behavioral studies. These approximately double- suggest an ovarian rather than adrenal origin for
length cycles did not appear to be the result of these hormones in these tigers. The synthesis
ovulations in the hormone study, since there of testosterone by ovarian follicles from the
was no significant elevation of progesterone ovary of the domestic cat has been described
during the interval between estradiol peaks in (YoungLai et al., 1976). There do not appear to
the two 42-day cycles in Tiger 712. There was be any other data on serum testosterone or
evidence of possible follicular activity based androstenedione levels in female felids. It
upon an increase in testosterone. appears possible that the pathway of estradiol
In contrast to reported results for lions synthesis in the tiger is by aromatization of tes-
(Schmidt et al., 1979), the three tigers in this tosterone, as has been indicated for mares (Sil-
study did not appear to have spontaneous berzahn et a!., 1983), cattle and sheep (Herri-
ovulations based upon the lack of a consistent man et al., 1979; Wise et al., 1982) and rats
or sustained elevation of serum progesterone (Bogovich and Richards, 1982). The cyclic
following any of the estradiol peaks. The elevations in testosterone may be responsible
elevations in progesterone observed in these for some of the behavioral aspects of estrus
tigers were small and of short duration relative (Martensz and Everitt, 1982). The occurrence
to the ovulatory and pregnancy values reported of testosterone and androstenedione peaks
for lions (Schmidt et al., 1979), a jaguar (Wiidt following PMSG treatment in two animals
et al., 1979), pumas (Bonney et al., 1981), and without an accompanying estradiol elevation
domestic cats (Stabenfeldt and Shille, 1977; suggests that a progressive decline and loss of
Wildt et al., 1981b). Consequently, it would the aromatization step in the synthesis of
appear from our data that Siberian tigers are estradiol may be the primary ovarian regulatory
not spontaneous ovulators. event for the seasonal decline in folliculogenesis
The occasional elevations of progesterone in these tigers.
above 2 ng/ml may have an adrenal origin as a
response to handling and immobilization, as ACKNOWLEDGMENTS
observed for deer (Plotka et al., 1983). The We thank N. Manning, M. K. Twite, M. D. Lewis, J.
tiger with the most frequent occurrence of H. Champlin, M. J. Heindel and G. C. Schmoller for
these spikes, Tiger 712, was our most excitable stustained excellence in performing the laboratory
analytical procedures. We appreciate the assistance of
animal in terms of response to the drugging
the many volunteers and keepers who assisted us in
procedure and the amount of drug required for working with the tigers, including L. Glaser, R. Burns,
initial immobilization. This animal had the D. Keyler, C. Lessler, and R. Bennett. This study was
highest progesterone value (11.5 ng/ml on supported in part by the VA Medical Research Service,
January 20, 1983) accompanying an illness that the Minnesota Zoological Garden, and the Marsh-
field Medical Foundation.
responded to treatment with antibiotics. These
data suggest that elevated progesterone levels in
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