REPRODUCTIVE ENDOCRINOLOGY OF MARSUPIALS AND MONOTREMES
5.1. Non-invasive hormone monitoring
Reproduction is driven by complex endocrinological events and processes. To gain an understanding of the endocrine changes that occur during normal and abnormal reproductive function, it is essential to collect suitable biological samples; typically, this involves a series of samples collected over time to quantify and map changes in reproductive hormone concentrations and patterns.
Non-invasive (urine and faeces) hormone analysis techniques have been validated for a small range of marsupials. Despite the growing use of non-invasive hormone monitoring in wildlife species, its use in Australian mammals has been limited (Table 5.1). When establishing managed care breeding programs, collection of behavioural and hormone data for both males and females is important. Establishing normal baseline parameters of reproductive function and biology for a species through non-invasive hormone monitoring provides valuable data that can be used to guide reproductive management decisions and assist with investigation of reproductive failure. Examples include: predicting or confirming the timing of pairings for mating; estimating parturition dates; identifying acyclic periods or individuals; investigating infertility; evaluating seasonality; identifying the sex of an individual (e.g. echidnas [Oates et al. 2002]); monitoring efficacy of hormonal contraception in males and females; and establishing age of puberty and senescence for a species (Table 5.1).Enzyme-immunoassay is the most commonly used analysis technique for non-invasive sample evaluation. It uses either metabolite-specific antibodies or antibodies with broad cross-reactivity that detect the parent hormone and its specific metabolites. Regardless of the type of test used, species and sample type, validation is required before use for diagnostic purposes or to establish baseline values (Schwarzenberger 2007; Kersey and Dehnhard 2014).
The International Society of Wildlife Endocrinology (ISWE, http://www.iswe-endo.org) maintains a database of researchers and experts and information on previously validated studies or techniques, as well as recommendations for sample collection, storage, validation and analysis.5.2. Evaluating male reproductive endocrinology
For male monotremes and marsupials, reproductive failure and fertility are not necessarily inter-related. A male may be fertile, producing adequate testosterone to develop accessory sex glands, produce spermatozoa and drive sexual behaviour, yet fail to reproduce successfully. This may be caused by physical or behavioural incompatibility, stress, body size (in relation to the female), competition from conspecifics, overaggression or lack of appropriate sexual behaviour or experience. Although testosterone is needed for spermatogenesis, the amount required is typically minimal (as measured in the peripheral circulation) and mediated through the hypothalamic-pituitary- gonadal (HPG) axis. Seasonal testosterone changes influence accessory gland development (for seminal plasma and copulatory plug fractions) and reproductive behaviours in seasonal breeders. Monitoring changes in circulating or excreted testosterone may provide information about seasonality, but is unlikely to confirm the reproductive potential of an individual without additional measures of reproductive fitness.
A GnRH stimulation test evaluates the central activation and activity of the HPG axis, allowing for evaluation of the prevailing steroidogenic capacity of the testes. Therefore, a GnRH stimulation test is most useful to identify precocious or delayed sexual maturity in both males and females or to diagnose infertility because of hormone insufficiency in male marsupials (Allen et al. 2006).
In general, reproductive hormone evaluation in male marsupials or monotremes should be paired with other diagnostic tests for fertility such as the evaluation of spermatorrhoea or semen collection.
5.3. Evaluating female reproductive endocrinology
There is significant diversity in the reproductive biology and endocrinology of female marsupials and many species have reproductive strategies that further define their reproduction (Table 5.1) (Johnston and Keeley 2015). This diversity, coupled with the lack of detailed descriptions of the endocrinology of oestrous cycles and pregnancy for many species, means monitoring and evaluating the fertility of female marsupials can be challenging.
In marsupial species examined thus far, the endocrinology of pregnant and non-pregnant cycles is very similar. Compared with eutherian species, placental contributions to pregnancy are minimal, if at all, and typically restricted to the very end of the short gestation. Unlike eutherians, pregnancy in marsupials is accommodated within the luteal phase, with the exception of embryonic diapause pregnancies, which are broken up by a period of embryo dormancy. The time between oestrous cycles varies considerably, ranging from 1 yr or more if a species is monoestrous or seasonally monoestrous, to overlapping where succeeding periods of oestrus are separated only by the duration of the luteal phase or pregnancy, resulting in a follicular phase that overlaps with gestation or the luteal phase resulting in a pre- or postpartum oestrus (e.g. macropods; Table 5.1). For many species however, knowledge is still limited or non-existent.
For female marsupials, longitudinal non-invasive hormone monitoring can be useful to confirm appropriate timing of male to female introductions for mating, ascertain normal cycling and in koalas, to ensure ovulation has occurred after mating (Keeley and Johnston, unpublished). Non-invasive hormone monitoring cannot be used to confirm pregnancy, as a suitable hormone metabolite or change in reproductive hormone levels has yet to be identified to confirm fertilisation or implantation.