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The oestrous cycle and basal body temperature in the common (Vombatus ursinus)

D. G. Peters and R. W. Rose Zoology Department, University of , Box 252C, G.P.O., Hobart, Tasmania, 7001

Summary. An oestrous cycle length of 33 days (N = 4, range 32\p=n-\34)was obtained for the from a number of parameters including vaginal smears, vaginal biopsies, changes in morphology and behavioural observation. All but one of the successive periods of oestrus occurred during winter. Hourly measure- ments of body temperature by telemetry showed a rhythm typical of nocturnal . Superimposed on this diurnal rhythm was another rhythm which could be correlated with the oestrous cycle.

Introduction

Common are large (25 kg) herbivorous, burrowing found in south-eastern Australia (Ride, 1970). Their phylogeny was in doubt until Kirsch (1968, 1977) demonstrated a link with the ( cinereus), thus placing them in the diprotodont superfamily Phalangeroidea, along with the ('possums') and the (). So far all the members of this superfamily that have been studied have been found to be poly- oestrous, although some are seasonal breeders, and wombats would therefore be expected to con¬ form to this pattern. Members of the wombat inhabit the warmer plains of 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 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.

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access 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 1 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 (I960).

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 , had shown no significant change in its smear pattern during the 4 months preceding the experiment and was ovariectomized (by Dr . 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.

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access 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 para¬ basal 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 pro- oestrus 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

100

10 15 20 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.

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access 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 post- oestrous 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 con¬ sistent 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 non- lactating 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.

Text-fig. 2. Summary of reproductive events during the study: |-1 = period of observation; = oestrus; 1 I = lactation; .= after ovariectomy.

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access Without exception, lactating animals remained anoestrous, both during pouch occupancy and when nursing young at foot. Neither experimental removal of pouch young nor the achieve¬ ment 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 corre¬ sponded 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 Tmax occurred (acrophase) changed from about 22:00 h on Days 0-10, to about 04:00 on

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access Text-fig. 3. Example of the diurnal rhythm in body temperature showing the calibration of the telemeter (Wombat 3).

Wombat 3 Wombat 2

Text-fig. 4. Body temperature and vaginal smear indices during the oestrous cycle of 2 wombats. Day 0 = day of oestrus; Tmax = 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

Downloaded from Bioscientifica.com at 09/24/2021 09:48:34PM via free access 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 con¬ clusions. 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 measure¬ ments 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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