Tina Janssen

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.

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.

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.

Non‐invasive urine collection in the female southern hairy‐nosed wombat (Lasiorhinus latifrons) with the aid of classical conditioning

We propose that regular urine samples can be used to monitor and characterize the reproductive cycle of the wombat, but this approach has never before been attempted in a marsupial. We conducted a three stage conditioning process for non-invasive urine collection in captive female wombats, which included (1) initial habituation and observation of urination patterns; (2) classical association of a stimulus with urination and (3) urine collection with the classically-conditioned stimulus. Four of the five female wombats selected for this trial were successfully conditioned for urine collection. During stage 2, the animals urinated in response to tactile stimulation 96 times from 208 attempts (46%). In stage 3, urine was successfully collected 399 times from 485 attempts (82%), with the majority of samples being collected in the morning (280/388). Hand-raised females that were previously conditioned for toileting purposes as pouch young responded more rapidly to the stimulus than juvenile females with no prior conditioning. This study is the first description of a successful method of urine collection by classical conditioning in a marsupial.

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.

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.

Validation of techniques to measure reproductive hormones in the urine of female southern hairy-nosed wombats (Lasiorhinus latifrons)

Southern hairy-nosed wombats (SHNW – Lasiorhinus latifrons) do not breed well in captivity (Hogan et al., 2013). The Australian captive population is currently not self-sustaining with an average of only one pouch young born per year (Skipper, 2013), many of which do not survive to weaning (V. Nicolson, 2013, personal communication, previous SHNW studbook keeper). In order to improve the captive breeding success of SHNW, there is an urgent need to have a thorough understanding of female reproductive physiology, but information regarding the SHNW female reproductive function is still limited. For example, characterisation of the full endocrinology of the oestrous cycle, especially in terms of the timing of ovulation, has yet to be elucidated (Hogan et al., 2013). While venipuncture may provide immediate information regarding hormones in systemic circulation, it is an impractical methodology for most captive wildlife species, as it typically requires repeated capture, restraint and sedation, which can result in chronic stress and interfere with normal hormone secretion (Waiblinger et al., 2006). Although, non-invasive faecal hormone analysis has proven to be useful for the monitoring of progesterone metabolites in female SHNW (Paris et al., 2002; Hogan et al., 2010a), the measurement of elevated oestrogens indicative of the follicular phase has been problematic (Hogan et al., 2010a). Additionally, depending on the species, protein hormones such as follicle stimulating hormone and luteinizing hormone (LH) are not typically detectable in biologically relevant quantities in faeces (Pukazhenthi and Wildt, 2003). Hormones and/or their metabolites can be secreted in urine within hours of their secretion in blood (Cano and Aliaga, 1995; Monfort et al., 1991; Munro et al., 1991). Urinary hormone immunoassays are a non-invasive analysis procedure which can be employed for the detection of timing of ovulation (LH); demonstrated in a limited range of mammals, such as the Indian rhinoceros (Rhinoceros unicornis) (Stoops et al., 2004), five species of callitrichid monkeys (Saguinus Oedipus, Leontopithecus rosalia, L. chrysomelas, Callithrix jacchus, Cebuella pygmaea; Ziegler et al., 1993), killer whale (Orcinus orca) (Robeck et al., 2004), and the Pacific white sided dolphin (Lagenorhynchus obliquidens) (Robeck et al., 2009). Therefore, the analysis of urine may provide a more comprehensive evaluation of reproductive hormones in the female SHNW. Four studies have investigated urinary reproductive steroid hormone detection methods in three marsupial species; urinary progesterone metabolites (P4M) in female numbats (Myrmecobius fasciatus) (Matson et al., 2008; Ditcham et al., 2009), urinary oestrogen and progesterone metabolites in two aged Tasmanian devils (Sarcophilus harrisii) (Crichton et al., 2003), and a single female koala (Phascolarctos cinereus) (Takahashi et al., 2009). Unfortunately, in all these studies, a biological validation associated with a reproductive event was not conducted, and while hormone metabolite concentrations were detected, it is unknown if they were secreted in biologically relevant concentrations. It is important to note that due to species-specific differences in hormone composition and excretion routes (Hodges et al., 2010), each immunoassay needs to be properly validated for each species and biological sample in question. To date, no study has reported the use of immunoassays for the measurement of urinary reproductive protein hormones in marsupials. Given that it is possible to collect urine from captive female SHNW using either classical conditioning methods and/or direct collection off the floor of their enclosure (Swinbourne et al., 2014), the detection of LH, oestrogen and progesterone metabolites in urine could be highly beneficial for SHNW reproductive assessment. The use of either a synthetic or natural sequence mammalian gonadotrophin releasing hormone (GnRH) to trigger an LH surge, has been successfully used to validate immunoassays for the detection of serum LH in a range of marsupials (Allen et al., 2008; Ballantyne et al., 2016a; 2016b; Bryant, 1992; Fletcher, 1989; Johnston et al., 2004; Matson et al., 2009; Moore et al., 1996; Rudd et al., 1999; Sutherland et al., 1980; TyndaleBiscoe et al., 1983; Wilson et al., 2013). To test the efficacy of urine as a suitable biological sample for the measurement of reproductive hormones (LH, oestrogen and progesterone metabolites) in female SHNWs, a series of biological and immunoassay validation trials were conducted using two exogenous hormones. In the first trial, a supermaximal dose of GnRH agonist (GnRHa) was used to challenge the anterior pituitary to secrete a surge of LH that could then be analysed in both blood and urine. The second trial involved two sequential but increasing