The oestrous cycle and basal body temperature in the common

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The oestrous cycle and basal body temperature in the common
J. Reprod. Fert. (1979) 57, 453–460
Printed in Great Britain
The oestrous cycle and basal body temperature in the
common wombat (Vombatus ursinus)*
D. G. Peters and R. W. Rose
Zoology Department, University of Tasmania, Box 252C, G.P.O., Hobart, Tasmania,
Australia 7001
Summary. An oestrous cycle length of 33 days (N = 4, range 32–34) was obtained
for the common wombat from a number of parameters including vaginal smears,
vaginal biopsies, changes in pouch morphology and behavioural observation. All but
one of the successive periods of oestrus occurred during winter. Hourly measurements of body temperature by telemetry showed a rhythm typical of nocturnal
species. Superimposed on this diurnal rhythm was another rhythm which could be
correlated with the oestrous cycle.
Introduction
Common wombats are large (25 kg) herbivorous, burrowing marsupials found in south-eastern
Australia (Ride, 1970). Their phylogeny was in doubt until Kirsch (1968, 1977) demonstrated a
link with the koala (Phascolarctos cinereus), thus placing them in the diprotodont superfamily
Phalangeroidea, along with the Phalangeridae ('possums') and the Macropodidae (kangaroos).
So far all the members of this superfamily that have been studied have been found to be polyoestrous, although some are seasonal breeders, and wombats would therefore be expected to conform to this pattern. Members of the wombat genus Lasiorhinus inhabit the warmer plains of
South Australia and field studies on one species L. latifrons, confirm the above expectations
(Gaughwin & Wells, 1978).
The common wombat (Vombatus ursinus) is found mainly in mountainous country on the
Australian mainland and over much of Tasmania. The latter population is sometimes referred to
as the sub-species V. ursinus tasmaniensis (Green, 1973). The capture, handling and aspects of
the ecology of the common wombat have been described by Mcllroy (1976, 1977). No
systematic study of the reproduction of this species has been carried out even though its
economic importance as a competitor with domestic stock and a destroyer of fences has
increased with closer settlement and pasture improvement.
In this paper we report on cytological changes during the oestrous cycle of the common
wombat. The number of animals used was of necessity very small because of the difficulties
involved in the capture, maintenance and handling of wombats.
Materials and Methods
Eight females were captured in the wild with a hand-held net. Their mean weight was 19 . 4 kg
(range 12 . 0–26 . 0 kg). Three animals had pouch young which were removed during the course of
the study. Four others were mature and non-lactating and one was immature. Information was
also gained from a hand-reared mature female wombat. Two males were present but both
escaped within 2 weeks of capture.
Reprint requests to Mr R. W. Rose.
0022-4251/79/060453-08$02.00/0
© 1979 Journals of Reproduction & Fertility Ltd
454
D. G. Peters and R. W. Rose
The animals were maintained in an enclosed area (0 . 5 hectares) of bushland at the University
of Tasmania in Hobart. The animals were fed daily with freshly cut grass and provided with
sleeping quarters made out of tea chests which could be entered via a wooden tunnel 2 metres
long.
The vaginal smear
Smears were taken daily during the period of captivity. They were obtained from the
posterior vaginal sinus by the use of a cotton-wool swab threaded through a I mm bore cannula.
The swab was rotated 6 times after passing through a glass tube which was inserted into the
urogenital sinus to the depth of the posterior vaginal sinus. The smear was transferred to a clean
slide, and then stained with Shorr's (1941) stain after fixation.
The smears were evaluated under the light microscope. The cells identified were:
karyopycnotic epithelial cells, intermediate epithelial cells, parabasal cells and leucocytes. A total
of 100 individual cells was counted. The criteria adopted for distinguishing between epithelial
cells were those of Hughes & Dodds (1968). Two indices were then calculated: the
Karyopycnotic Index (KI), which is the proportion of epithelial cells (excluding parabasal cells)
which are mature, and the Leucocytic Index (LI) which is the proportion of leucocytes in the
whole count (including epithelial cells). Confidence limits for these indices were calculated by the
method of Riimke (1960).
Correlative studies
The pouch and the opening of the urogenital sinus were examined daily, qualitative changes
being noted. Biopsies of the posterior vaginal sinus were taken at 3-day intervals during the
oestrous cycle using 3 mm biopsy forceps. The mucosae were fixed and histological sections
made which were stained with Shorr's (1941) stain and compared with the vaginal smear.
Behavioural changes were also noted.
Body temperature measurements
The hourly body temperatures of 3 mature female wombats were recorded for a period of 3
months (August—October). Two of the animals were undergoing oestrous cycles as indicated by
vaginal smears. The third, a parous non-lactating animal, had shown no significant change in its
smear pattern during the 4 months preceding the experiment and was ovariectomized (by Dr B.
Wells, Veterinary Surgeon, Kingston) to provide a control. For these experiments the animals
were housed separately in steel cages which were installed in a constant temperature room (22
± 1°C) in which the photoperiod was 12 h light:12 h dark. Vaginal smears were taken daily.
Plastic drums were provided as burrows and did not affect the radio transmission.
Body temperatures were determined using commercially available telemeters (Mini-Mitters
Company Inc. Portland, Oregon, U.S.A.). The method for calibration and recording of body
temperatures is given by Guiler & Heddle (1974). The telemeters were calibrated in a water bath
before and after the experiment. The telemeters showed a linear response over the physiological
temperature range of the order of 6 pulses per min per °C. Thus the temperature could be read to
an accuracy of 0 . 2°C. Under ether anaesthesia, the telemeters were implanted beneath the
muscles of the abdomen, near the inguinal region. Equipment used to monitor the temperature
was similar to that used by Guiler & Heddle (1974). The signal was received by portable AM
receivers installed in each cage. The output of each receiver was tape recorded by use of a time
switch as a series of 2-min events every hour. The accuracy of the recordings was checked each
day by making a direct count as the last recording was being made.
Wombat oestrous cycles and body temperature
455
Results
Oestrous cycles
Qualitative changes of the vaginal smear. Three phases of the oestrous cycle could be
detected in the stained vaginal smears: pro-oestrus, oestrus and post-oestrus. Pro-oestrous
smears were distinguishable 4–5 days before oestrus by a reduction in the proportion of parabasal cells. The nuclei of the epithelial cells became pycnotic, the cytoplasm expanded and
became eosinophilic while the cell assumed a polygonal outline. During the final days of prooestrus the proportion of leucocytes decreased rapidly to zero but red blood cells appeared
sporadically.
The first occurrence of a fully cornified smear coincided with vaginal tumescence and
increased activity (see below) and was considered to represent the day of oestrus (Day 0). The
red cells could have corresponded to the time of ovulation. The cornified smear was found from
Day 0 until Day 12 of the wombat's 33-day cycle. It was composed entirely of mature squamous
cells, all of which had either a pycnotic or karyolytic nucleus. The absence of leucocytes and
1 00
75
50
25
0
1 00
75
50
25
0
-3
0
5
10
15
20
25
30
33
Days after oestrus
Text-fig. 1. Quantitative changes in the vaginal smear during one oestrous cycle of Wombat 2.
Error bars denote 95% confidence limits.
456
D. G. Peters and R. W. Rose
cellular debris gave these smears a clear background. Characteristic post-oestrous smears were
found from Day 12 after oestrus to the next pro-oestrus. General changes in the smear consisted
of the appearance of leucocytes and non-cornified epithelial cells as well as navicular cells
(elongate epithelial cells, similar to those described by Poole & Pilton, 1964) and basophilic
fibrous material. Precocious parabasal cells with a pycnotic nucleus and eosinophilic cytoplasm
were also found during post-oestrus. From about Day 24, the smear had few mature epithelial
cells, large numbers of leucocytes and immature epithelial cells.
Quantitative changes in the smear. Text-figure 1 shows the variation in KI and LI during the
course of a wombat oestrous cycle and is representative of all cycles observed in the present
study. There was a close relationship between the qualitative and quantitive interpretation of the
smears. Oestrus (Day 0) was taken to be the first day that KI rose significantly above its postoestrous value. This coincided with the day of `tumescence' and the behavioural and basal body
temperature changes described below. Changes in the Leucocytic Index, although measured
independently of KI, show a marked negative correlation with KI. Except during periods of
vaginal cornification, large numbers of leucocytes were present in the smears. Variation between
estimates of LI made from the same smear was often as great as 20% (number of cells counted =
150). It is concluded that the LI was only marginally quantitative, although at all times consistent with subjective evaluation of the smear.
Text-figure 2 summarizes the reproductive events observed during the study. Of the 9 females
held in captivity only 4 came into oestrus, noted on 8 occasions. The mean oestrous cycle length
was calculated as 33 days (N = 4, range 32-34). Anoestrus persisted in all animals in captivity
after August until the end of the study in December. During anoestrus, the few cells present were
mostly leucocytes and parabasal cells. The appearance of the latter in clumps distinguished
anoestrous smears from late post-oestrous smears. Anoestrous smears from lactating and nonlactating females were indistinguishable from each other or from smears from immature and
ovariectomized females. Anoestrous smears were found in all animals. In all but the
ovariectomized female there were changes in the smear pattern which were subjectively
discernible both in the overall smear and as trends in the indices. These periods could be
distinguished from pro-oestrous smears in that KI and LI changes were positively correlated.
IA—1
A
2
3
A
E
0
8
9
A
M
J
J
A
S
0
N
D
Month
Text-fig. 2.
Summary of reproductive events during the study: [—]
♦ = oestrus;
= lactation; ........... = after ovariectomy.
= period of observation;
Wombat oestrous cycles and body temperature
457
Without exception, lactating animals remained anoestrous, both during pouch occupancy
and when nursing young at foot. Neither experimental removal of pouch young nor the achievement of independence by young at foot resulted in a return to oestrus during the 8-week period
when smears were taken after lactation ceased.
Correlative studies
Pouch and external genitalia. Daily examinations of the pouch revealed that wombats, like
many other marsupials, have periods associated with oestrous cycles during which the pouch is
free from scale. In anoestrus these occurred randomly and irregularly. In cycling animals the
pouch became noticeably dirty by 10 days after oestrus and clean again 9–6 days before the
approaching oestrus. No apparent changes in the length, erection or capping of the two nipples
were noted, nor were there any visible changes in the underlying mammary tissue during the
oestrous cycle.
The external genitalia increased in size for a period not exceeding 15 h on the first day of
cornification. This was observed on 3 occasions in 2 animals for which complete cycles were
recorded. The urogenital sinus appeared to be partly everted and protruded and was engorged
and moist. A vaginal biopsy at this time revealed superficial capillaries within the epithelium. This
oestrous vascularization of the vaginal epithelium was also indicated in late pro-oestrous smears
by the presence of red blood cells and suggests that the epithelium may be susceptible to
mechanical damage at this time.
Biopsies. Biopsies of the posterior vaginal sinus showed that during the post-oestrous
`cornification ' the epithelium actually regressed, there were no superficial (cornified) cells and
leucocytes had infiltrated the epithelium. Sections of the lateral vagina of a female on Day 16
after oestrus showed a similar epithelium and revealed several cornified cells in the lumen.
Behaviour. Intermittent observations were made of the behaviour of the captive wombats.
Only those activities relevant to the present study are included. More details are provided by
Mcllroy (1976, 1977).
Wombats are active at night, when they feed and excavate. They spend the daylight hours
asleep in the burrows. At or about oestrus their behaviour changes and female wombats become
very active, continually pacing up and down for much of the day and night. The hand-reared and
extremely tame wombat became vocal and aggressive towards both handlers and an immature
male wombat. This behaviour pattern was repeated 33 days later.
Body temperature
Diurnal temperature rhythm. An underlying diurnal rhythm was observed in all 3 animals
throughout the study (Text-fig. 3). It was typical of that for a nocturnal species, with maximum
temperatures occurring at night. A midnight temperature dip was frequently observed and corresponded to a rest period after feeding. The maximum diurnal range observed was 2 . 4°C, and the
minimum, 0 . 8°C. Minimum temperature rarely occurred outside the period 10:00–16:00 h.
Maximum temperatures were restricted to the period 21:00–06:00 h.
Basal body temperature and the oestrous cycle. In cycling and anoestrous animals,
secondary rhythms or changes were superimposed on the diurnal rhythm. Text-figure 4 shows
the effects of the oestrous cycle on the amplitude of the diurnal temperature rhythm in 2
wombats. The daily maximum body temperature (TmaX) remained relatively constant although
there were substantial changes in daily basal body temperature. In both animals, BBT changed
by about 1 . 5°C over the course of the oestrous cycle.
An oestrous cycle phase-shift in the diurnal rhythm was also noted. The time of the day at
which T. occurred (acrophase) changed from about 22:00 h on Days 0–10, to about 04:00 on
458
D. G. Peters and R. W. Rose
.
33 5
20:00
24:00
04:00 08:00 12:00 16:00
Hours
128
Text-fig. 3. Example of the diurnal rhythm in body temperature showing the calibration of the
telemeter (Wombat 3).
36-
Wombat 3
Wombat 2
35
BBT
34
BBT
33
. LI
80
LI
KI
40
-15
-10
-5
0
5
0
15
-5
Days
0
5
0
15
20
25
Text-fig. 4. Body temperature and vaginal smear indices during the oestrous cycle of 2 wombats.
Day 0 = day of oestrus; T. = daily maximum temperature; BBT = basal body temperature;
KI = karyopyknotic index; LI = leucocytic index. Estimates of BBT on Days -1, 0 and + 1 may
not be as accurate as those at other times because both animals were very active on these days.
Days 20-30. The daily minimum temperature usually occurred in phase with the acrophase,
about 12 h earlier. It was noted that animals rose earlier during the first week after oestrus.
Basal body temperature after ovariectomy. In the ovariectomized wombat basal temperature
fluctuated irregularly over a range of 1 . 2°C during the 5 weeks after operation. The mean
amplitude of diurnal variation (1 . 4°C) was similar to that of cycling and anoestrous animals.
Diurnal acrophase occurred between 23:00 and 04:00 h (as for the anoestrous animal).
Discussion
The results of this study justify the conclusion that the common wombat is polyoestrous.
Many marsupials have a sustained post-oestrous period of vaginal cornification as detected
by vaginal smears. Hughes (1962) and Tyndale-Biscoe (1968) found that smears were dominated
by cornified cells for as long as 10 days after the onset of cornification in Potorous tridactylus
and Bettongia lesueur, respectively. In our study, oestrous-type smears were found up to 15 days
after oestrus. The explanation for this lengthy period of cornification can be deduced from the
Wombat oestrous cycles and body temperature
459
studies of Pilton & Sharman (1962) and Tyndale-Biscoe (1968). In marsupials, smears are taken
from the posterior vaginal sinus or the urogenital sinus, although the most pronounced
cornification of the vaginal complex occurs in the lateral vaginae. The influx of desquamated
cornified cells from the lateral vaginae may mask the changes occurring in the epithelium of the
posterior vaginal sinus.
The limited scale of the present study on body temperature changes precludes many conclusions. Nonetheless, it appears that reproductive events in the wombat are accompanied by
changes in body temperature. In women, basal temperatures are elevated within a day of
ovulation (Simpson & Halberg, 1974), but in wombats they remain relatively low during the first
week after oestrus. If it is assumed that elevated temperatures correspond to the luteal phase, this
could indicate that the wombat's luteal phase does not commence until about Day 5—10 after
oestrus. The absence of a discernible trend in BBT in the ovariectomized wombat provides
further confirmation that the temperature rhythm observed in cycling animals is associated with
the oestrous cycle. The diurnal phase-shift may be universal in oestrous cycle temperature
rhythms, and may explain the inconclusive results of studies which rely on single daily measurements of `basal' temperature. The correlation between leucocyte proportions and cornified cell
proportions (LI and KI, respectively) sharply distinguishes oestrous cycle smear patterns from
anoestrous smear patterns. During the period of observations only a few of the eligible wombats
entered oestrus. Captivity and the daily handling for the purpose of smearing may have increased
the occurrence of anoestrus. However, it is also possible that some wombats do not breed every
year. Gaughwin & Wells (1978) have shown that the hairy-nosed wombat (Lasiorhinus
latifrons) is a seasonal breeder in South Australia and that in their study area the frequency of
reproduction varied markedly over an 8-year period. Maximum rates of reproduction appeared
to be associated with high rainfall and plentiful vegetation. Regular rainfall is normal over much
of Tasmania and hence is not liable to affect reproduction in the wombat. Vegetation growth
during the winter months does slow down considerably due to reduced temperatures and this
could be a limiting factor in the length of the breeding season. A lengthy period of weaning the
young at foot could also act to restrict the length of the next breeding season of the wombat.
This study was made possible by a research grant from the University of Tasmania. Dr E. R.
Guiler, Dr J. Haight, Dr C. H. Tyndale-Biscoe and Dr W. Whitten made helpful criticisms and
Mrs T. Vasos kindly typed the manuscript. Mr G. Shaw prepared the figures. We thank the
National Park and Wildlife Service of Tasmania for permission to capture the wombats.
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Received 7 March 1979

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