Lindsay A. Hogan

Non-invasive assessment of stress in captive numbats, Myrmecobius fasciatus (Mammalia: Marsupialia), using faecal cortisol measurement

Annual patterns of faecal cortisol metabolite (FCM) secretion were examined in six captive numbats (Myrmecobius fasciatus). The use of enzyme-immunoassay for the measurement of FCM in the numbat faeces was validated using an adrenocorticotropic hormone (ACTH) challenge and the resultant FCM measurements represent the first description of adrenal endocrinology in this species. Total overall, baseline and peak FCM mean concentrations varied according to individual, but not gender. For males, mean baseline and overall FCM secretion was higher in spring in summer (compared to winter and autumn) and was elevated during the breeding season. For females, mean baseline FCM secretion did not differ by season or breeding season, but mean overall FCM secretion was elevated during the breeding season. Thus, male (but not female) numbats display an annual change in FCM secretion that is strongly linked to their seasonal pattern of reproduction. Significant FCM elevations (n=178) were observed in response to 20 different stressors, with these stressors being allocated to one of six categories: ANIM, ENVIRO, HAND, HEALTH, MAN and UNK. The mean proportion of positive responses to each category varied according to category, season and breeding season, but did not vary by individual or gender. ANIM and HEALTH stressors elicited a higher response rate than all other categories and an increase in the number of ANIM, ENVIRO, and HEALTH stressors were observed during the breeding season. Although there were multiple stressors within the captive environment that the numbats reacted to, this did not translate into a welfare issue.

Non-invasive methods of oestrus detection in captive southern hairy-nosed wombats (Lasiorhinus latifrons)

In order to develop a reliable method of oestrus detection in captive southern hairy-nosed (SHN) wombats, the reproductive behaviour of four groups of adult animals (1 male:2 female) was monitored using video surveillance and activity using movement-sensitive radio transmitters for a period of 12 months. During this time faecal samples were collected every 3 days and subsequently analysed for progesterone and oestradiol-17beta metabolites. In an attempt to induce and characterise oestrus-specific behaviour, each female was administered a subcutaneous injection of either 0.01 (n=2), 0.1 (n=4) or 0.2mg/kg (n=2) of oestradiol benzoate in one of two hormone trials. Remote video surveillance was an effective tool for detecting the reproductive behaviour of the captive SHN wombat. Courtship (n=426) and mating (n=46) was observed in five wombats and consisted of 13 distinctive behaviours in six consecutive phases: (1) investigation, (2) attraction, (3) chase, (4) restraint, (5) copulation and (6) recovery. Female sexual receptivity occurred at night and lasted for approximately only 13-h. Faecal progesterone metabolite analysis proved to be a reliable method for mapping oestrous cycle activity, but was not useful for the prediction of oestrus. Six out of the eight female wombats displayed periods of elevated progesterone secretion, corresponding to a mean (+/-SE) luteal phase of 20.9+/-1.1 days (n=23). Oestrous cycle length, defined as the interval between two successive luteal phases separated by a follicular phase was 31.8+/-1.1 days (n=12) and consisted of a follicular phase of 11.6+/-0.6 days (n=12). Changes in the secretion of faecal oestradiol-17beta metabolites provided little instructive information on oestrous cycle activity and were not associated with oestrus. Administration of oestradiol benzoate resulted in a spike of oestradiol-17beta metabolites in the faeces 3-4 days later, but was not dose dependent nor did it facilitate reproductive behaviour in either sex. Activity was not linked to key events in the oestrous cycle and appears not to be suitable as a method for detecting oestrus in the SHN wombat. We therefore recommend the use of 24-h video surveillance as the most reliable method for oestrus detection in captive SHN wombats.

One size does not fit all: Monitoring faecal glucocorticoid metabolites in marsupials.

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animals response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125-4.5days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z⩾2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies.

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.

Remote monitoring of the behaviour and activity of captive southern hairy-nosed wombats (Lasiorhinus latifrons)

The nocturnal nature of wombats makes it difficult to learn about their behavioural ecology and activity rhythms in the wild. A facility was established at Rockhampton Zoo to house 12 adult southern hairy-nosed wombats in order to monitor their behaviour by means of video surveillance and provide detailed information on their activity patterns using movement-sensitive radio-transmitters. After one year of monitoring it was established that the wombats spent, on average, as a proportion of total time 69.9% sleeping, 8.8% lying resting, 5.2% feeding, 5.2% exploring, 4.3% performing stereotyped behaviour, 2.5% sitting resting and 4.1% in other activities. Feeding, lying resting and sleeping varied with season by ~5%. Temporal patterns were bimodal for 8 of the 12 behaviours described, with most ‘active’ behaviours being expressed between 1800 and 2000 hours and 0200 and 0500 hours. The activity pattern was characterised by a strong circadian rhythm, with most activity occurring nocturnally. Within active periods there was an alternating rhythm of active and rest periods and activity peaks at the beginning and end of each night. Comprehensive and reliable information on the behavioural ecology, as well as captive management, of southern hairy-nosed wombats was obtained from the remote video and radio-transmitter recordings.

The effect of environmental variables on the activity patterns of the southern hairy-nosed wombat (Lasiorhinus latifrons) in captivity: onset, duration and cessation of activity

The influence of scotoperiod, ambient temperature and humidity on the activity of captive wombats was examined to differentiate environmental influence from that pertaining to food availability. The activity of 12 wombats (Lasiorhinus latifrons) housed at Rockhampton Zoo was remotely monitored by digital video-surveillance for one year, with environmental variables simultaneously recorded via dataloggers. Mean proportion of total daily time spent active was 18.2 ± 1.8%. Mean daily time spent active varied seasonally according to changes in temperature and humidity. Mean daily activity was greater during winter (301 ± 17 min) and spring (295 ± 16 min), than summer (234 ± 16 min) and autumn (238 ± 15 min) with differences due to a decrease in the number and length of activity bouts in summer and autumn; presumably these results reflect a need to maintain heat balance associated with exposure to elevated temperatures. Activity was positively correlated with temperature during winter (r = 0.60) and spring (r = 0.52) but negatively correlated during summer (r = –0.58) and autumn (r = –0.49), suggesting the existence of a thermoneutral zone where activity is maximised. We conclude that activity in captive wombats is influenced by environmental variables.

Reproductive behaviour of the southern-hairy nosed wombat (Lasiorhinus latifrons)

The southern hairy-nosed wombat (Lasiorhinus latifrons) is under threat as its current distribution range and population size is declining. The low reproductive rates observed for this species, both in the wild and within captivity, are a cause for concern; as such this study was designed to provide a comprehensive account of reproductive behaviour in L. latifrons. The behaviour of four groups of captive animals (each 1♂ : 2♀) was remotely monitored for 14 months using a digital video-surveillance system. Using this technology, 13 distinctive reproductive-based behaviours were identified and recorded; each behaviour was allocated to one of two categories: courtship/guarding or mating. The courtship-to-mating ritual was made up of six consecutive phases: investigation, attraction, chase, restraint, coitus and recovery. In total, 423 courtship and 49 mating bouts were observed from five (2♂ : 3♀) wombats. Courtship behaviour was observed on 116 days, with a mean bout number of 3.6 ± 0.2 per day and a mean total daily time of 29.8 ± 1.8 min. Mating was observed on 24 days, with a mean bout number of 2.1 ± 0.3 per day and a mean total daily time of 100.1 ± 9.8 min. Courtship occurred over the entire enclosure area and three distinctive courtship locomotor patterns were identified: spiral-circles, straight-lines and random. Matings occurred only in closed spaces and were always accompanied by courtship behaviour. In contrast, only 68% of all courtship days were associated with a mating event. Receptivity in the female wombats lasted for 13 h and the behavioural signs of oestrus were so subtle that only the reaction/interest of the male could be used for its detection. The covert reproductive behaviours identified for female L. latifrons closely match those previously reported for captive common wombats and may relate to this species’ need to conserve energy.

Non-invasive monitoring of male and female numbat (Myrmecobius fasciatus: Myrmecobiidae) reproductive activity

The reproductive endocrinology of the highly endangered numbat (Myrmecobius fasciatus) is described for the first time. Patterns of faecal steroid secretion (progesterone [PM], oestradiol-17β [E2] and testosterone [TM] metabolites) were examined within a captive numbat population over 1 year and revealed a highly synchronized seasonal pattern of reproduction. TM secretion increased progressively from September to November, peaked in December and then decreased in February. All females displayed luteal phases (1-3), between late-November to late-March, in association with pregnant (Pr, n=4), non-productive mated oestrous cycles (NMEC, n=8) and non-mated oestrous cycles (NEC, n=6). The mean oestrous cycle length was 30.2 ± 1.1 d (n=11) and was comprised of a mean follicular (n=11) and luteal (n=18) phase length of 16.2 ± 1.6 d and 14.0 ± 0.8 d, respectively. No variation in mean luteal phase length or PM concentration according to cycle type (Pr, NMEC, NEC) or cycle number (1st, 2nd or 3rd cycle) was detected. Longitudinal profiling of PM secretion confirmed that the female numbat is seasonally polyoestrous and that the luteal phase occurs spontaneously. Changes in the secretion of E2 provided little instructive information on oestrous cycle activity. Mating success was 31%, with age and subject having no effect on mating success. Timing of introduction, of male to female, appeared to impact mating success, with paired animals introduced for a shorter time frame (≤14 d) prior to the first observed mating successfully producing young. Collectively, results of the present study confirm that PM and TM can be reliably used to index numbat reproductive activity.

Technique for faecal marking in group-housed southern hairy-nosed wombats Lasiorhinus latifrons

This study compared the efficacy of plastic glitter, with a mean particulate size of 1.18 mm, as a faecal marker in group-housed captive wombats Lasiorhinus latifrons. The wombats voluntarily consumed the glitter through the use of appetizing food vehicles. Over 40 different food treats were tested as possible vehicles for the oral delivery of the faecal marker and of these six were deemed highly palatable: (1) golden syrup with horse pellets, (2) golden syrup with weetbix, (3) pitted dates, (4) honey with kangaroo pellets, (5) nutrigel with rolled-oats, and (6) strawberry sauce with rolled-oats. Mean transit time of glitter particulates through the alimentary tract of L latifrons was 2.9 +/- 0.5 d, with maximal output occurring 4.2 +/- 0.3 d after administration. A marker dose of 1.6 g / 3 d was required to reach a steady and detectable state of marker output. Using this dosage > 2 particulates (i.e. flecks) of glitter were defaecated in > 90% of faecal pellets, allowing the accurate identification of individual samples. Reliable labeling was obtained using gold, silver, metallic red, metallic green, metallic blue and white glitter, i.e. digestion did not affect the integrity of these colours. There was no evidence that long-term feeding of glitter had any negative effects on the normal formation of faecal pellets, the clinical health, weight or appetite of the wombats.

Wombat reproduction (Marsupialia; Vombatidae): an update and future directions for the development of artificial breeding technology

This review provides an update on what is currently known about wombat reproductive biology and reports on attempts made to manipulate and/or enhance wombat reproduction as part of the development of artificial reproductive technology (ART) in this taxon. Over the last decade, the logistical difficulties associated with monitoring a nocturnal and semi-fossorial species have largely been overcome, enabling new features of wombat physiology and behaviour to be elucidated. Despite this progress, captive propagation rates are still poor and there are areas of wombat reproductive biology that still require attention, e.g. further characterisation of the oestrous cycle and oestrus. Numerous advances in the use of ART have also been recently developed in the Vombatidae but despite this research, practical methods of manipulating wombat reproduction for the purposes of obtaining research material or for artificial breeding are not yet available. Improvement of the propagation, genetic diversity and management of wombat populations requires a thorough understanding of Vombatidae reproduction. While semen collection and cryopreservation in wombats is fairly straightforward there is currently an inability to detect, induce or synchronise oestrus/ovulation and this is an impeding progress in the development of artificial insemination in this taxon.