Male Koalas Research Articles

Control of the koala (Phascolarctos cinereus) anterior pituitary-gonadal axis with analogues of GnRH

The aim of the present study was to determine whether analogues of gonadotrophin-releasing hormone (GnRH) could be used to both induce an acute testosterone response and suppress anterior pituitary function in male koalas, and induce a luteal phase in female koalas. Experiment 1 characterised the steroidogenic response of male koalas to administration of 30 microg (4.3 microg kg(-1)) natural-sequence GnRH. Intra-muscular injection of natural-sequence GnRH induced the release of LH and testosterone with peak concentrations at 30 min (3.7 +/- 1.9 ng mL(-1)) and 2 h (5.4 +/- 0.5 ng mL(-1)), respectively. In Experiment 2, a single injection of the GnRH antagonist acyline (100 microg (14.3 microg kg(-1)) or 500 microg (71.4 microg kg(-1))) did not influence the testosterone response to subsequent injections of natural-sequence GnRH. In Experiment 3, 4 microg (~0.67 microg kg(-1)) of the GnRH agonist buserelin induced a luteal phase in five female koalas based on a LH surge, secretion of progestogen, and a normal-length oestrous cycle. The findings have shown that (1) natural-sequence GnRH can be used to test gonadotroph cell function and determine the testosterone-secreting capacity of male koalas, (2) the GnRH antagonist, acyline, at the dose rates used, does not suppress the pituitary-testis axis in male koalas, and (3) the GnRH agonist, buserelin, induces a normal luteal phase in female koalas.

Use of a GnRH Agonist and hCG to Obtain an Index of Testosterone Secretory Capacity in the Koala (Phascolarctos cinereus)

Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating testosterone after injection of a GnRH agonist or human chorionic gonadotrophin (hCG). Hence, the aims of the present study were to examine fluctuations in testosterone secretion in the koala (n = 6) over a 24-hour period and then characterise testosterone secretion after injection of the GnRH agonist buserelin (4 micro g) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 hours, but there was no apparent diurnal pattern of testosterone secretion (P > .05). Injection of buserelin and hCG resulted in an increase (P < .05) in blood testosterone concentration. After injection of exogenous hormone, near maximal concentrations of testosterone occurred at around 60 minutes. There was a tendency for plasma testosterone to decline after 90 minutes with buserelin, but concentrations remained close to the upper limit for 240 minutes with hCG. There were strong positive correlations between the average testosterone concentration over 24 hours and the maximum observed testosterone concentration after stimulation with GnRH and hCG (GnRH, r = .772; P = .07 and hCG, r = 1.0; P < .01). The findings in the present study confirmed that individual male koalas can show large fluctuations in blood testosterone concentrations over time and that a GnRH agonist and hCG can be used in the koala to obtain an index of the prevailing steroidogenic capacity of the testes.

EFFECT OF TOOTH WEAR ON THE RUMINATION-LIKE BEHAVIOR, OR MERYCISM, OF FREE-RANGING KOALAS (PHASCOLARCTOS CINEREUS)

Rumination-like behavior, or merycism, of 5 adult male koalas (Phascolarctos cinereus) with various degrees of tooth wear was investigated using acoustically sensitive radio telemetry. Increased tooth wear was associated with a significant increase in mean rate of merycism mastication, mean number of merycism mastications per bout per 24 h, and mean number of mastications per 24 h. The ratio of mean number of ingestive mastications to mean number of merycism mastications per 24 h remained approximately 17:1 with increasing tooth wear. These results suggest that in contrast to true rumination, alterations in merycism behavior may be important in compensating for reduced molar effectiveness.

Glucuronuria in the koala.

Glucuronuria is normal in marsupial folivores such as the koala (Phascolarrctos cinereus), which excretes 2-3 g glucuronic acid daily. Although this has long been attributed to the metabolites of Eucalyptus terpenes, we have found that these are mostly excreted in the unconjugated form. We now report on the aglycones that account for most of the glucuronic acid in koala urine. Urine (24 hr) was collected from six male koalas (8.8 +/- 0.4 kg, mean +/- SE) that were maintained on E. cephalocarpa foliage. Urine samples were analyzed by liquid and gas chromatography (LC and GC) coupled with mass spectrometry (MS). Glucuronides were readily identified by LC-MS/MS, which generated characteristic product ions at m/z 113 and 175. From the corresponding parent glucuronide ions, the masses of the aglycones were calculated. Confirmation of identity was by GC-MS after hydrolysis with beta-glucuronidase and comparison with standard compounds. Quantitation was by GC. The major non-terpene aglycones were 4-methylcatechol, resorcinol, salicyl alcohol, and two unidentified C7H8O2 phenols. Smaller amounts of benzoic acid, benzyl alcohol, orcinol, p-cresol, phenol, and phloroglucinol were detected. We have previously reported that terpene metabolites account for about 10% urinary glucuronides in the same koalas fed E. cephalocarpa. The present study found that an additional 60% urinary glucuronic acid is conjugated with non-terpene, mainly phenolic, aglycones. It seems likely that these phenolic compounds are present in leaves as glycosides and are chiefly responsible for the glucuronuria in koalas.

The association of tooth wear with sociality of free-ranging male koalas (Phascolarctos cinereus Goldfuss)

The sociality (as measured by the degree of bellowing and amount of tree use) of five adult male koalas and one sub-adult male koala (Phascolarctos cinereus), with varying degrees of tooth wear, were investigated using acoustically sensitive radio-telemetry. Initial increases in tooth wear that coincide with an increase in age to maturity were associated with an increase in reproductive effort. Advanced tooth wear was found to be associated with a decrease in reproductive effort. This trend in reproductive effort is consistent with life-history predictions, and suggests that tooth wear has the potential to impose limitations on the reproductive longevity and fecundity of free-ranging male koalas.

Tree use, diet and home range of the koala (Phascolarctos cinereus) at Blair Athol, central Queensland

Free-ranging koalas (Phascolarctos cinereus) were monitored by means of radio-relocation in the area of Blair Athol Coal Mine and surrounding properties. Daytime tree use, home range and diet of these koalas was determined in spring and autumn, as was the leaf moisture composition of potential fodder species. Koalas used on average 93 (male koalas) and 56 (female koalas) trees during the period of observation, occupying home ranges of 135 and 101 ha respectively. Mean sightings per tree were 1.19 for both males and females and home-range sizes were not significantly different between sexes or seasons. Koalas were observed returning to previously used daytime roosting trees infrequently (&lt;12%). Although koalas were observed roosting in trees of the species that they ate, proportional species representation in the diet of these koalas during spring and autumn did not accurately reflect concurrent observations of their daytime tree-roosting behaviour. Koalas were observed to utilise non-fodder species for daytime roosting, and patterns of daytime tree use and diet selection varied between individuals inhabiting adjacent communities within the site. Leaf moisture of tree species represented in koala diets was greater in autumn than spring.

The effects of tooth wear on the activity patterns of free-ranging koalas (Phascolarctos cinereus Goldfuss)

The free-ranging activity patterns of five adult males and one sub-adult male koala (Phascolarctos cinereus) with varying degrees of tooth wear were investigated using acoustically sensitive radio-telemetry. Increased tooth wear was found to be associated with a significant decrease in the amount of time spent sleeping and being inactive. Furthermore, compared with individuals with low degrees of tooth wear, individuals with high tooth wear were found to spend considerably less time moving within and between trees, and had home ranges an order of magnitude smaller. Hence, feeding compensations were found to come at the expense of other activities such as sociality and therefore have important consequences for reproductive output. Individuals with high tooth wear also spent longer feeding per bout, and spaced feeding bouts out more evenly throughout a 24-h period, thereby exhibiting reduced degrees of nocturnality. The sub-adult was found to range over a disproportionately large area and spend a relatively large amount of time moving between trees, indicative of a dispersing individual. Moreover, the activity patterns of this individual were found to differ slightly from the adult trends and were perhaps affected by the extra energetic demands associated with dispersal.

Metabolites of dietary 1,8-cineole in the male koala ( Phascolarctos cinereus)

The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53±0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4±1.1 mmol/kg (3.1±1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with β-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3±0.4 and 1.4±0.4 (mean±S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.

A playback survey of the koala, Phascolarctos cinereus, and a review of its distribution in the Eden Region of south-eastern New South Wales

Summary A survey conducted by playing pre-recorded calls of a male koala and listening for responses was used to further assess the distribution of koalas in south-eastern New South Wales. Fourteen koalas were recorded from 388 sample sites. This detection rate (4%) was low compared to detection rates in north-eastern New South Wales. Insufficient data were generated to allow analysis of koala distribution in relation to environmental variables. The koalas were scattered through the region on various land tenures. The results suggest that conclusions regarding the distribution of koalas, based on previous records, may reflect uneven survey effort. The data from this survey and from other contemporary sources show the continued presence of koalas at localities where they had previously been reported as extinct. It is concluded that a low density koala population persists in the regional forests and that purported evidence of decline in the population is unconvincing. There were no significant differences in detection rates between different land tenures. The detection rate of koalas was highest in the north of the region, at lower altitudes. There were significant differences in detection rates between modelled vegetation types, but the small number of detections precluded analysis of habitat preferences.

Fate of the dietary terpene, p -cymene, in the male koala

The fate of the dietary terpene, p-cymene, is reported in a specialist Eucalyptus leaf folivore, the koala (Phascolarctos cinereus). Six male koalas were given two single oral doses of p-cymene (0.37 mmol/kg and 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods after each dose and analyzed for metabolites by extraction, gas chromatography, and mass spectrometry. A total of six metabolites were detected in the urine. A novel, extensively oxidized metabolite, 4-(1,2-dihydroxy-1-methylethyl)-benzoic acid, was identified and its structure elucidated by high resolution and chemical ionization mass spectrometry. Minor amounts of two glycine conjugated metabolites were also detected. Five metabolites were present in sufficient amounts to quantify, using isolated urinary metabolites as reference standards. The mean fractional recovery of administered p-cymene was 0.77 ± 0.09 and 0.84 ± 0.12 for the low and high dose, respectively. The major metabolite excreted was the novel carboxy diol compound. No fecal metabolites were found. Thus, the koala employs a strategy in the metabolism of p-cymene that promotes the production of extensively oxidized metabolites that consequently have increased polarity and enhanced renal excretion. This strategy is compared with that employed by other Eucalyptus leaf folivores.

Development of the male urogenital system of the koala phascolarctos cinereus.

This paper described several developmental stages of the male urogenital system in the koala Phascolarctos cinereus, employing both light and scanning electron microscopy. There are few studies of the development of the urogenital system in male marsupials. Findings by White and Timms (1994) that male koalas can be infected with Chlamydia psittaci emphasise the importance of studies on male animals and in particular their reproductive system. Specimens in our study ranged in age from 15 days postnatal to adults. Due to the rarity of such specimens, details of each specimen are linked to the changes of the structures at each available stage. Light microscopy revealed that differentiation of the gonads had commenced by 15 days postnatal and that the cytological arrangements of the urogenital system are essentially the same as those of other mammals. Scanning electron microscopy revealed stereocilia and microvilli along the lumen of each ductus epididymis and cilia and microvilli along the lumina of the vasa deferentia and urethra. The development of these structures coincided with the onset of sexual maturation, sperm production and differentiation at about three years of age.

Identification and variation of volatile compounds in sternal gland secretions of male koalas (phascolarctos cinereus)

The content of the sternal gland secretion from four male koalas (Phascolarctos cinereus) was analyzed using gas chromatography–mass spectrometry. Volatile fatty acids, aldehydes, ketones, mono- and sesquiterpenes were identified, together with various volatile nitriles and oximes, some of which had not previously been described from any mammalian skin gland. Benzyl cyanide and phenylacetaldehyde oxime were identified as major components of the secretions, together with smaller amounts of the novel 3-methylthiopropanal oxime. The relative abundance of the identified compounds was highly variable between individuals and the scent composition was found to be partly determined by the scent of surrounding conspecifics. Principal component analysis revealed a high similarity between related individuals.

Seasonal-Variation in Water Flux, Field Metabolic-Rate and Food-Consumption of Free-Ranging Koalas (Phascolarctos-Cinereus)

Mass-corrected field metabolic rates of free-ranging male koalas in central Queensland, Australia, varied between 0.329 MJ kg0.75 day-1 in summer and 0.382 MJ kg0.75 day-1 in winter. Field water influx measured 50.8 mL kg-0.8 day-1 in winter, increasing to 59.9 mL kg0.8 day-1 in summer for the same koalas, and was positively correlated with values for leaf moisture of food. Winter rates of water influx for koalas from Springsure were lower than those recorded for koalas from Victoria for the same period of the year. Mass-corrected feeding rates were lower in summer than winter; wet food intake was significantly lower than reported for similar sized female koalas from Victoria. The preferred browse was Eucalyptus crebra in winter and E. tereticornis in summer. Our study indicates that in central Queensland seasonal changes in diet selection by male koalas reflect increased energy requirements in winter and increased water requirements in summer.

Variation in reproductive parameters in the captive male koala (Phascolarctos cinereus).

Plasma androgen concentration was measured in six mature male koalas over 23 months. During this time, four animals were housed isolated from females. Androgen concentration showed a seasonal change, with a nadir about the middle of the year, and began to increase sometime before the start of the breeding season. The concentration was greater when males were housed with females, even when the females were not yet cycling themselves. Changes in androgen concentration were not associated with changes in testicular volume. When plasma extracts were examined following separation by high pressure liquid chromatography, the major circulating androgen was testosterone with only trace amounts of 5 alpha-dihydrotestosterone present. Plasma androgen fluctuated during the day suggesting some form of diurnal rhythm of secretion. Two further animals were fitted with vascular access ports to allow more frequent blood collection and samples were taken every second day for two months, a period that included a major portion of the breeding season. Androgen concentration fluctuated widely from 0 nmol L-1 to 20 nmol L-1 over successive sampling and indicating an episodic pattern of secretion. Plasma cortisol concentration was measured in all animals in these studies to monitor stress and remained low except when animals were undergoing surgery, were being moved to different enclosures or were exhibiting reproductive behaviours. Overall, there was no significant correlation between the concentration of androgen and cortisol in plasma (n = 215). This contrasted with a group of wild-bled animals (n = 4) where there was a highly significant negative correlation.

Behavioural determination of visual function in the Koala (Phascolarctos cinereus).

A behavioural estimate of the koala's dependence on vision was made using fabric hoods to degrade the vision of ten male koalas. Koalas were placed on the ground in a square enclosure, with a centrally placed 'tree'. With unobstructed vision, the koalas quickly travelled across the ground to the tree and climbed it. However, with severely reduced vision, the koalas would not search for or climb the tree. When their vision was obscured, the koalas tended to raise their forelegs when walking, taking high, reaching steps. The koala appears to require a certain minimum level of vision before climbing trees. Ocular chlamydial infection, which can cause a significant visual loss, may result in similar changes in behaviour and thus contribute to mortality.

Dissociative anaesthesia in free‐ranging male koalas and selected marsupials in captivity

Forty seven free-ranging, adult, male koalas were captured and administered an intramuscular injection of the dissociative anaesthetic, Telazol (tiletamine HCl plus zolazepam HCl), at dose rates of 5.0 to 7.7 mg/kg body weight. Anaesthesia induction was rapid and smooth and resulted in a surgical plane of anaesthesia lasting 30 to 45 min. There was no depression of heart rate or respiration. Mild salivation occurred in most animals, but was not a problem because the swallowing reflex was retained. There was no mortality or morbidity and the anaesthesia level was sufficient to allow electroejaculation and multiple blood sampling with no apparent animal discomfort. For 10 of 19 males in which anaesthesia was required for a 90 min protocol, a supplementary Telazol injection (average, 2.5 mg/kg) was necessary. All koalas recovered completely within 3 to 4 h of the initial injection. The results suggest that the optimal Telazol dosage for the adult male koala is 7.0 mg/kg body weight. The retrospective analysis of 259 anaesthesia records involving 14 species indicated that Telazol was equally effective and safe in other captive marsupials.

Habitat Use and Tree Preferences of Koalas in a Mixed Eucalypt Forest

The use of specific vegetation associations and tree species by koalas in a forest containing six Eucalyptus spp. is described. There was a positive correlation between density of koalas and the density of E. viminalis, and koalas used this species in preference to other tree species. There were seasonal differences in the use of both vegetation associations and tree species. Associations dominated by E. viminalis were preferred in most seasons. E. viminalis was the most preferred species in all seasons except summer, when E. ovata was preferred. This was not reflected in a change in preference for vegetation associations. Male koalas showed a preference for E. macrorhyncha, and to a lesser extent E. viminalis and E, ovata. Females showed a consistent preference for E. ovata and E. viminalis. Koalas showed a strong preference for large trees in all seasons, and for specific trees within species.

Eucalyptus Digestibility and Digestible Energy Requirements of Adult Male Koalas, Phascolarctos Cinereus (Marsupialia).

Three adult male koalas were individually fed on a diet of Eucalyptus melliodora, E. robusta and E. sideroxylon plus trace mineral salt and water to appetite. Fresh browse was offered daily for 14 days before and 7 days during quantitative collection of faeces. Samples of browse representing plant parts consumed were collected, and acid lignin ratio techniques were used to estimate nutrient digestibility. Apparent digestibility of DM was 54-70%, gross energy 47-64%, crude protein 32-50%, ether extract 37-50%, neutral detergent fibre 23-57% and acid detergent fibre 9-55%. Total faecal acid lignin excretion was used to estimate digestible energy (DE) intake. Since bodyweights (W) were maintained during the study, daily DE requirements for adult maintenance were estimated to range from 257 to 411 kJ/W0.75. If an 87% recovery in the faeces of dietary acid lignin was assumed, estimated DE requirements for adult maintenance ranged from 312 to 439 kJ/W0.75. These values are lower than commonly accepted interspecific values for eutherians but are consistent with evidence that the koala has a relatively low basal metabolic rate.

Behaviour of the Koala, Phascolarctos cinereus (Goldfuss), in Captivity IV.* Scent-Marking

Male koalas scent-mark by grasping a vertical object, generally a tree, and rubbing the sternal gland against it. The behaviour first appears at 3 y of age, and reaches a peak a year or two later. It is frequently performed in conjunction with bellowing, especially in response to agonistic encounters or the bellow of a rival male, and often by itself in circumstances where a bellow might be expected. It is sometimes a response to unfamiliar surroundings or objects, and otherwise is performed by a male moving about a pen. Scent-marking may primarily be a response to unfamiliarity which, having become associated with displaced aggression, has been entrained to the bellowing drive.

Behaviour of the Koala, Phascolarctos cinereus (Goldfuss), in CAptivity V*. Sexual Behaviour

At the height of the breeding season male koalas frequently attempted copulation. These attempts were often apparently spontaneous, but many followed bellowing or agonistic interactions. Sexual behaviour began at 3 y old in males, except for penile erections, which sometimes occurred in younger males. Males performed no courtship behaviour. Behavioural oestrus was brief, and consisted of four distinct types of activity: jerking, bellowing, mild aggression towards the male, and pseudomale behaviour. Oestrous females could become very excited by the presence of a male, and the four activities were very flexible in their expression. Copulation itself was quite brief and consisted of mounting, thrusting, convulsions, and disengagement. The pair were always in a vertical position in a tree, the male grasping the female's neck in his jaws.