Body temperature of free-ranging koalas (Phascolarctos cinereus) in south-east Queensland.

The distribution of the koala (Phascolarctos cinereus) in Queensland is predicted to contract as a result of climate change, driven by the frequency, intensity and duration of heatwaves and drought. However, little is known about the physiological responses of this species to environmental extremes under field conditions. This study aimed to establish the efficacy of surgically implanted thermal radio transmitters and data loggers to measure the body temperature of free-ranging koalas across a range of environmental conditions and ambient temperatures. Five free-ranging koalas in southeast Queensland were implanted with thermal transmitters and data loggers waxed together as a single package. Body temperatures were recorded for variable periods ranging from 3 to 12 months. Diurnal rhythms in body temperature were detected irrespective of season. The long-term diurnal body temperature peak for all koalas occurred between 16:00 and 17:00 h and body temperature was 36.7-36.9 °C, the long-term nadir occurred between 07:00 and 08:00 h and body temperature was 35.4-35.7 °C. Koala body temperatures as low as 34.2 °C and as high as 39.0 °C were recorded. Thermolability became apparent when ambient temperatures were outside the deduced thermal neutral zone for koalas (14.5-24.5 °C): heat was accumulated during the day and dissipated during the cool of the night. While this study is the first to report on body temperature of free-ranging koalas in their normal behavioural context, further investigations are necessary to determine the physiological boundaries of the thermal niche for this species, in order to better equip models that will more accurately predict the impacts of climate change on koalas.

Malocclusions in the koala (Phascolarctos cinereus).

Malocclusions are a misalignment or incorrect positioning of the teeth when the upper and lower jaws close. These are poorly described in the koala and can result in irregular mastication which can have lifelong effects on body condition and oral health. A total of 370 koalas from two populations in Queensland (295) and one in South Australia (75) were examined for malocclusions. The prevalence of malocclusions in South Australian free-ranging koalas, captive Queensland koalas and Queensland free-ranging koalas was 39% (44), 30% (29) and 22% (29) respectively. Four types of malocclusion were identified based on severity of misalignment of the incisor/canine region, types 1, 2, 3 and 4. Maxillary overbite measurements of the molariform teeth were determined and these anisognathic values were then used to describe malocclusions within familial relationships in captive colonies. Captive koalas with a malocclusion had narrower mandibular width that ranged between 0.5 and 1% less than the normal measurements. The specific malocclusions reported in this study affected individuals by leading to tooth rotation, mobility and erosion with inefficient mastication of food and vegetation compaction. These changes increased the oral cavity pathology, by placing animals at risk of periodontal disease. There was evidence of familial links to malocclusion types in captive animals. Therefore captive breeding recommendations should consider known koala malocclusion traits to minimise their effect on future generations.

Recording body temperature in koalas (Phascolarctos cinereus): a comparison of techniques.

OBJECTIVE: Compare the use of four techniques to measure body temperature in koalas: intraperitoneal (thermal data logger and temperature sensitive radio transmitter), rectal (certified thermometer), tympanic (infrared thermometer), and hind foot (infrared camera). METHODS: The body temperature data collected concurrently from the intraperitoneal loggers were used as the benchmark in the analyses. RESULTS: The rectal, foot and tympanic methods consistently recorded lower body temperature when compared with the benchmark. There was a strong positive relationship (R2 = 0.79) between logger and rectal measurements, but no significant relationship between logger and foot or logger and tympanic measurements. CONCLUSION: Rectal measurements can be used to record internal body temperature, with the caveat that such measurements will generally register a temperature approximately 0.25°C lower than the actual intraperitoneal temperature.

Measurement of testosterone and cortisol metabolites and luteinising hormone in captive southern hairy-nosed wombat (Lasiorhinus latifrons) urine.

This study reports the validation and use of enzyme immunoassays (EIA) to measure changes in plasma and urinary luteinizing hormone, testosterone metabolites (UTM) and cortisol metabolites (UCM) in captive southern hairy-nosed wombats (Lasiorhinus latifrons). GnRH agonist and ACTH agonist challenges were conducted to validate urinary testosterone (male wombat only) and cortisol (male and female wombats) EIAs. Following intra-muscular injection of 8-12µg buserelin (n=4 males), there was a significant increase in both plasma (P<0.001) and urinary testosterone concentrations (P<0.001) 60min and 21h after administration, respectively. Plasma LH levels were elevated (p<0.05) at 20min but there was no significant increase found in urinary LH concentrations after injection. Intra-muscular injection of Synacthen® Depot (250µg) (n=3 males, 3 females) resulted in a significant increase (p<0.05) in plasma cortisol secretion 15min and in urinary cortisol concentrations 3h post injection, respectively. Sex-related differences in cortisol secretion were also reported in this study. These findings indicate that (1) urinary LH might not be an appropriate index for describing the reproductive status in captive male L. latifrons, and (2) the UTM and UCM assays appear to be suitable for the assessment of the testicular steroidogenic capacity and the adrenocortical activity in captive southern hairy-nosed wombats, respectively.

First report of a spermatic granuloma and varicocele in a marsupial: A Koala (Phascolarctos cinereus) Case Study.

This study reports the first documented clinical case of a spermatic granuloma and varicocele in a marsupial. Initial clinical presentation included gross morphological changes in the left scrotal cord, epididymis and testis. Ultrasonography of the scrotum and spermatic cord, and gross and histopathological examination after hemicastration, confirmed the condition as a spermatic granuloma affecting the left caput epididymis, with a varicocele in the left proximal spermatic cord, which was causing azoospermia and infertility. Semen quality and serum testosterone secretion following a GnRH challenge was assessed prior to, and following surgery. After hemi-castration, an increase in androgen secretion to within normal reference ranges for the koala was observed with a subsequent increase in semen production and sperm quality resulting in the sire of a pouch young, 12months later.

Use of the gonadotrophin-releasing hormone antagonist azaline B to control the oestrous cycle in the koala (Phascolarctos cinereus).

The present study examined the effectiveness of the gonadotrophin-releasing hormone (GnRH) antagonist azaline B to suppress plasma LH and 17ß-oestradiol concentrations in koalas and its potential application for oestrous synchronisation. In Experiment 1, single subcutaneous injections of azaline B successfully blocked the LH response to exogenous mammalian (m) GnRH in a dose-dependent manner; specifically, 0 mg (n = 4) did not suppress the LH response, 1 mg azaline B (n = 6) suppressed the LH response for 24 h (P < 0.05), 3.3 mg azaline B (n = 8) suppressed the LH response significantly in all animals only for 3 h (P < 0.05), although in half the animals LH remained suppressed for up to 3 days, and 10 mg azaline B (n = 4) suppressed the LH response for 7 days (P < 0.05). In Experiment 2, daily 1 mg, s.c., injections of azaline B over a 10-day period during seasonal anoestrus (June-July; n = 6) suppressed (P < 0.01) the LH response to mGnRH consecutively over the 10-day treatment period and, 4 days after cessation of treatment, the LH response had not recovered. Experiment 3 was designed to test the efficacy of daily 1 mg, s.c., azaline B over 10 days to suppress plasma LH and 17ß-oestradiol concentrations and ultimately synchronise timed return to oestrus during the breeding season. Although azaline B treatment did not suppress basal LH or 17ß-oestradiol, oestrus was delayed in all treated females by 24.2 days, but with high variability (range 9-39 days). Overall, the present study demonstrates that the GnRH antagonist azaline B is able to inhibit the LH response in koalas to exogenous mGnRH and successfully delay the return to oestrus. However, although azaline B clearly disrupts folliculogenesis, it has not been able to effectively synchronise return to oestrus in the koala.

The use of a synthetic progesterone, levonorgestrel (LNG), to control the oestrous cycle in the koala.

This study investigated the efficacy of a synthetic progestogen, levonorgestrel (LNG), to control koala ovarian activity for the purposes of oestrous synchronisation. Captive koalas were administered either saline control or a 70-mg LNG implant on Day 2 of oestrus. Urogenital cytology, oestrous behaviour and plasma oestradiol-17ß and LH concentrations were monitored over a 6-week period. After LNG implant removal females were monitored to determine if the return to oestrus was synchronised. LNG-treated koalas immediately ceased displaying oestrous behaviour, showed no evidence of cornified epithelial cells in smears of urogenital cytology and exhibited low plasma oestradiol-17ß concentrations throughout the implantation period. In contrast, oestradiol-17ß levels in control koalas showed evidence of continued cyclic activity associated with behavioural oestrus and increased cornified epithelial cells in urogenital smears on Days 33 to 35 after saline injection. After implant removal, LNG-treated koalas exhibited oestrus at 13, 14, 17 and 30 days after implant removal. Plasma LH concentrations varied throughout the study period with no significant time (P = 0.49) or treatment (P = 0.13) effect. Overall results from this study suggest that LNG implants in koalas can inhibit oestrous behaviour and reduce circulating oestradiol-17ß levels before oestrus, most likely by preventing development of the pre-ovulatory follicle. However, there was no evidence of LH suppression by the LNG implants. Removal of LNG implants resulted in the synchronous return to oestrus in three of the four treated koalas. Further studies on a larger population are required to validate these findings.

Plasma prolactin concentrations during lactation, pouch young development and the return to behavioural oestrus in captive koalas (Phascolarctos cinereus).

Plasma prolactin (PRL) concentrations in captive koalas during lactation were determined by serial blood sampling. PRL concentrations were low (1.3 ± 0.1 ng mL-1; n = 5) during early lactation until pouch young (PY) began to emerge from the pouch (around Day 130) before significantly (P < 0.05) increasing between Day 161 and Day 175 (5.3 ± 1.0 ng mL-1). A significant (P < 0.001) peak in PRL (7.7 ± 0.6 ng mL-1) coincided with maturing young between Day 189 and Day 231. All females failed to exhibit any signs of oestrous behaviour until Day 268.8 ± 8.5 (n = 4), some 102 ± 19 days before PY were weaned following achieving target weights of 2.5-2.7 kg. Throughout lactation, plasma LH concentrations were relatively high (range 4.9-8.7 ng mL-1) and LH responses to exogenous gonadotrophin-releasing hormone were observed in all koalas at all times during lactation.

Monitoring male southern hairy-nosed wombat (Lasiorhinus latifrons) reproductive function and seasonality in a captive population.

This study reports on the development and application of techniques used to assess the reproductive status of captive male southern hairy-nosed wombats (n=4) at Rockhampton Zoo. Initially, a GnRH agonist was used to establish a method for determining a reliable index of plasma and faecal testosterone secretion. Intra-muscular injection of buserelin (4 microg) resulted in an increase (P<0.05) in plasma and faecal testosterone concentration 90 min and 3 days after administration, respectively. Seasonal changes in faecal androgen, sperm production (spermatorrhoea) and testicular, prostatic and bulbourethral gland size were examined over a 18-month period, with prostate and bulbourethral gland cross-sectional areas being assessed by ultrasonography. Plasma testosterone secretion increased from early late winter and then decreased in spring (P<0.001); no seasonal variation (P=0.22) in faecal testosterone metabolite concentrations was apparent. Testicular volume showed no significant variation (P=0.29) over the sampling period. While there was no seasonal change (P=0.197, n=54) in prostate size, bulbourethral gland size increased in late-autumn, peaked in mid-winter and declined in early summer (P= or <0.001, n=55). Spermatozoa were found in the urine throughout the year. While, the captive population of SHN wombats at Rockhampton Zoo demonstrated significant changes in reproductive function, the extent of seasonality was less pronounced than that previously reported for wild populations in Southern Australia.

Reproductive physiology of the female greater bilby (Macrotis lagotis Thylacomyidae): evidence for a male-induced luteal phase.

Endocrinology of the oestrous cycle, pregnancy and early lactation was investigated in captive Western Australian greater bilbies (Macrotis lagotis). Initially, six females were monitored for changes in urogenital cytology, plasma progestogen, pericloacal and pouch morphology in the absence of a male. This was followed by the introduction of a male and a reproductive assessment through mating, gestation and early lactation. In the absence of a male, there was no cyclical pattern of urogenital cytology, pericloacal or pouch development, and progestogen concentrations remained basal. Within 5 days of the introduction of a male, all females had a karyopycnotic index of 100%. Spermatozoa were present in the urogenital smear within 3 days of male introduction in all five females that gave birth. Five to 9 days after the introduction of a male, there was an increase in plasma progestogen concentration that remained elevated for 14-19 days. Six of the seven females gave birth approximately 3 days after reaching peak plasma progestogen concentrations. Gestation length ranged between 14 and 17 days. Plasma progestogen concentrations of the postpartum and early lactation period were lower (P < 0.0001) than during gestation, but greater (P < 0.0001) than those recorded before the introduction of a male. One female that gave birth early in the study that was examined until weaning of the pouch young showed a cyclical pattern of plasma progestogen secretion that ended at weaning. This study provides evidence that the luteal phase in the greater bilby is induced by the presence of a male. Similar to female reproductive physiology in the Peramelidae, elevated progestogen concentration in the greater bilby was extended into lactation.

Assessment of reproductive status in male echidnas.

This study reports the development and application of techniques to assess the reproductive status of male echidnas. The pattern of testosterone secretion over a 24-h period in five echidnas was documented. Testosterone secretion after injection i.m. of either 1000 IU hCG (n=4) or 4 microg GnRH agonist (n=6) was determined to establish whether this could be used as a practical index of the prevailing steroidogenic capacity of the testes. hCG (1000 IU) was also used to assess seasonal changes in testosterone secretion in six echidnas over a 13-month period. Seasonal changes in testicular volume were examined by transabdominal ultrasonography. Electroejaculation was attempted to monitor seasonal changes in sperm production, which was also determined by spermatorrhea. There was no apparent diurnal pattern of testosterone secretion in echidnas and circulating concentrations of testosterone remained relatively low (maximum 1.2 ng/mL) and stable over 24h. Injection of hCG resulted in an increase (P<0.01; n=4) in testosterone concentration with a peak (2.9+/-0.3 ng/mL) approximately 4h after injection. GnRH also induced an increase (P<0.01; n=6) in circulating testosterone that was apparent after 1h (2.6+/-0.3 ng/mL) and concentrations remained elevated (3.4+/-0.3 ng/mL) for up to 8h after injection. Seasonal changes in testosterone secretion determined after injection of hCG, increased (P=0.03; n=6) from late-autumn, peaked in late-winter, and decreased by early-spring. Testicular volume followed a similar seasonal pattern (P<0.01; n=6) with an increase from late-autumn, peak in winter and a decline in mid-spring. There was no seasonal change in live weight. Electroejaculation was attempted throughout two breeding seasons but no semen was obtained. Spermatorrhoea in the echidna was described for the first time and was subsequently used to assess seasonal sperm production. Spermatozoa were found in the urine from June to September. This study has demonstrated that exogenous hormones can be used to obtain an index of the prevailing steroidogenic capacity of the testes in echidnas, which is not apparent with repetitive non-stimulated samples over 24 h. The assessment of testosterone secretion after injection of trophic hormones provides a valuable and practical procedure for the assessment of reproductive status. Testicular ultrasonography and spermatorrhea are useful in assessing reproductive status and in this study were successfully used to determine seasonal reproduction in captive echidnas.

Studies of the oestrous cycle, oestrus and pregnancy in the koala (Phascolarctos cinereus).

As an integral part of the development of an artificial insemination programme in the captive koala, female reproductive physiology and behaviour were studied. The oestrous cycle in non-mated and mated koalas was characterized by means of behavioural oestrus, morphology of external genitalia and changes in the peripheral plasma concentrations of oestradiol and progestogen. The mean (+/- SEM) duration of the non-mated oestrous cycle and duration of oestrus in 12 koalas was 32.9 +/- 1.1 (n = 22) and 10.3 +/- 0.9 (n = 24) days, respectively. Although the commencement of oestrous behaviour was associated with increasing or high concentrations of oestradiol, there were no consistent changes in the morphology or appearance of the clitoris, pericloacal region, pouch or mammary teats that could be used to characterize the non-mated cycle. As progestogen concentrations remained at basal values throughout the interoestrous period, non-mated cycles were considered non-luteal and presumed anovulatory. After mating of the 12 koalas, six females gave birth with a mean (+/- SEM) gestation of 34.8 +/- 0.3 days, whereas the remaining six non-parturient females returned to oestrus 49.5 +/- 1. 0 days later. After mating, oestrous behaviour ceased and the progestogen profile showed a significant increase in both pregnant and non-parturient females, indicating that a luteal phase had been induced by the physical act of mating. Progestogen concentrations throughout the luteal phase of the pregnant females were significantly higher than those of non-parturient females. Parturition was associated with a decreasing concentration of progestogen, which was increased above that of basal concentrations until 7 days post partum.

Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus).

An experiment was conducted in which female koalas were mated for different durations of intromission and ejaculation to confirm that the luteal phase of the oestrous cycle in koalas is induced by the physical act of mating. Results showed that induction of a luteal phase in the koala usually required a complete duration of penile thrusting behaviour from the male. It is proposed that induction of a luteal phase in koalas may involve a copuloceptive reflex, triggered by the thrusting of the male's penis into the female's urogenital sinus. Although interrupted mating in koalas may be used to induce a luteal phase in preparation for an artificial insemination programme, this study showed that there is a 12.5% probability that pregnancy will result from semen prematurely emitted by the teaser male. A dose of 250 iu hCG was administered intramuscularly to eight oestrous females to determine whether it was possible to induce a luteal phase artificially. In contrast to control females, which received sterile saline injections, all females injected with hCG showed a significant increase in progestogen concentration above that of basal values, indicating that a luteal phase had been induced successfully.

MRI in the evaluation of late bone marrow changes following bone marrow transplantation.

Measurements of MR spin-lattice (T1), and spin-spin (T2) relaxation times in lumbar vertebrae have been performed in a pilot study on six adult patients, treated for acute myeloid leukaemia (AML). All patients were treated with initial chemotherapy and then proceeded to bone marrow transplantation (BMT), conditioned with Melphalan and total body irradiation (TBI). MR measurements were made between 21 and 89 months after TBI. The relaxation times in the six patients were compared with those in six healthy age-matched volunteers to establish whether there were differences between the two groups. Average T1 values in the vertebrae of the treated patients are significantly shorter (p < 0.01) than in the healthy volunteers. This is consistent with the observation of a relatively hyperintense vertebral bone marrow in the T1 weighted images and is likely to be a consequence of treatment induced fatty replacement of marrow. Shorter T1 values tend to be distributed within the centre of the lumbar vertebrae compatible with observations, made by others, which suggest that the peripheral zone of the vertebral body has been repopulated with bone marrow cells whereas the central zone, around the basivertebral vein, is predominantly fat. Histogram displays of vertebral body relaxation time distributions (T1, T2) for both patients and healthy age-matched volunteers are similar in that both patients and volunteers give histograms that are only slightly skewed. This similarity is probably a reflection of the fact that the patients have been in remission for over a year and have generally healthy bone marrow.

CT evaluation of the resectability of gastric cancer postchemotherapy.

BACKGROUND: To determine the accuracy of CT in the postchemotherapy assessment of resectability of gastric cancer. METHODS: Thirty patients deemed to have unresectable gastric cancer on CT were studied. This was verified at laparotomy in 10 of these patients. Following initial assessment, all received three to eight cycles of chemotherapy aiming for disease control and potential resection. Serial CT examinations, endoscopy, and biopsy were performed after the fourth, sixth, and eighth cycle of treatment. The primary tumor and lymph nodes seen on CT were compared with operative findings. RESULTS: After completion of chemotherapy, CT findings were correct in 23 patients. Fourteen of them had operable tumors and nine were inoperable. However, the CT findings were either equivocal or incorrect in the remaining seven patients. CONCLUSION: Chemotherapy is now able to downstage a previously inoperable gastric cancer, and CT is an accurate method in identifying those patients who can proceed to resection.

Epirubicin, cisplatin, and protracted venous infusion of 5-fluorouracil for esophagogastric adenocarcinoma: response, toxicity, quality of life, and survival.

BACKGROUND: The results of chemotherapy for patients with esophagogastric carcinoma have generally been modest but regimens developed more recently have produced higher response rates, and rekindled interest in neoadjuvant chemotherapy. One such regimen is epirubicin, cisplatin, and 5-fluorouracil (ECF). This study evaluates its efficacy, toxicity, impact on quality of life (QL), and impact on survival in a large consecutive series of patients with metastatic and locally advanced disease (LAD). METHODS: Patients with histologically confirmed esophagogastric carcinoma were treated with ECF (epirubicin 50 mg/m2 and cisplatin 60 mg/m2 every 3 weeks with continuous infusion of 5-fluorouracil (5-FU) 200 mg/m2/d). Responses were evaluated with computed tomography (CT) scan and endoscopy. QL was assessed using the European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire. RESULTS: A total of 235 patients were treated, 173 with metastatic disease and 62 with LAD. The mean number of cycles delivered was 6 (range: 1-11) and patients were followed-up for a median of 8 months. Response was observed in 135 of 220 (61%) evaluable patients, with a complete response (C(R)), 11% of the patients and a partial response in 50% of the patients. Patients with moderately differentiated adenocarcinomas and LAD responded most favorably. Symptomatic improvement was achieved in the majority of cases (63-78% depending on the symptom). Toxicity was generally only mild to moderate, with severe non hematologic toxicity in less than 12% of the patients and only 6 (2.5%) treatment related deaths. QL assessment showed no significant negative impact on emotional functioning and good symptomatic control. Surgery following response to ECF was performed in 29 of the LAD patients, and in 19 cases (66%) a potentially curative resection was possible, with histologic CR in 32% of the patients. CONCLUSIONS: ECF is a highly active regimen with acceptable toxicity in patients with esophagogastric adenocarcinoma. In a proportion of patients with LAD, chemotherapy enabled potentially curative surgery to be performed. The results justify further investigation of this regimen in a neoadjuvant setting.

CT evaluation of treatment response in advanced gastric cancer.

Ninety-one patients with advanced gastric carcinoma were followed by serial computed tomography (CT) in a phase II study using multi-agent chemotherapy. Regression of primary tumour was seen in 43, with complete resolution in 5 and > 50% regression in 29. Involvement of loco-regional lymph nodes was seen in 43 patients, with complete resolution in 16, and > 50% regression in 21. Hepatic metastases were seen in 40 patients, with complete resolution in three and > 50% regression in 17. Regression in other less common sites in the chest and abdomen were also documented. The pattern of maximum response in all sites of disease was seen within the first 12 weeks of chemotherapy. The difficulties of disease management are discussed and recommendations for CT evaluation in this group of patients are made.