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

A.M. Swinbourne a,⇑, S.D. Johnston a, T.M. Janssen b, A. Lisle a, T. Keeley a

a School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland, Australia

b Australian Animals Care and Education, Mount Larcom, Queensland, Australia



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.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.

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 doses of equine chorionic gonadotrophin (eCG) in order to stimulate gonadal steroid secretion. Further assay validation included urinary steroid extraction, the analysis of longitudinal sample to evaluate the urinary hormone profiles of a pregnant female and a mated non-pregnant female, as well as a comparison of hormone profiles between matched urine and faecal samples.