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\p=n-\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 measure- ments 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 poly- oestrous, although some are seasonal breeders, and wombats would therefore be expected to con¬ form 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. 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 animal, 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.