MONOTREMES
1.1 Monotreme infertility and reproductive failure
Few institutions have successfully bred monotremes (Holland and Jackson 2002; Hawkins and Battaglia 2009; Ferguson and Turner 2013; Wallage etal.
2015; Dutton-Regester et al. 2021). Enclosure and burrow design, and for platypuses (Ornithorhynchus anatinus), individual animal temperament and pair compatibility, are important factors. There are no published reports of infertility or reproductive tract disease in short-beaked echidnas (Tachyglossus aculeatus) or platypuses. Broken or damaged egg shells and degenerating fetuses have been observed in zoo echidnas and may have been associated with mechanical damage rather than infectious or other processes (L Clark, S Johnston and A Roser, unpublished; Currumbin Wildlife Sanctuary, Monotreme Research Centre records). Twins and triplets have been observed on three occasions in short-beaked echidnas; however, in both cases the puggles did not survive to weaning (L Clark, S Johnston and A Roser, unpublished).Reproductive parameters and assessment of the platypus are less understood and developed compared to the short-beaked echidna (Hawkins and Fanning 1992; New et al. 1998; Holland and Jackson 2002; Higgins 2004; Johnston et al. 2007a; Hawkins and Battaglia 2009; Morrow et al. 2009; Nicol and Morrow 2012; Ferguson and Turner 2013; Wallage et al. 2015). For institutions breeding platypuses or short-beaked echidnas, a reproductive examination at the commencement of the breeding season that includes assessment of general health, measurement of testicular volume and recovery of spermatozoa in the urine is recommended (Johnston et al. 2007a).
1.2 Male reproductive assessment
Monotremes are testicond (Booth and Connolly 2008; Middleton 2008). The penis of the short-beaked echidna terminates in a bifid glans, with each side having two epidermal rosettes into which semen is released during ejaculation (Temple-Smith and Grant 2001; Johnston et al.
2007b; Fenelon et al. 2021). During erection, the echidna penis engorges with blood preferentially into one of either side of the penile shaft, so that only two of the four rosettes are fully dilated during erection and align in parallel along the shaft of the penis, while the other two are constricted and non-functional (Johnston et al. 2007b). The platypus penis possesses ‘rear-facing’ keratinised epidermal spines across most of its surface and terminates with laterally positioned, retracted rosettes consisting of a group of foliate papillae. The penile spines may assist in holding the penis in position within the female’s urogenital sinus during copulation in water. The platypus glans penis is asymmetrical, with the left side larger than the right (Temple-Smith and Grant 2001).Monotremes lack seminal vesicles and possess only a primitive prostate that is unlikely to contribute any significant volume to seminal plasma. There are reports of a single pair of bulbourethral (Cowper’s) glands in the short-beaked echidna (Griffiths 1968) and platypus (Temple-Smith 1973).
During the breeding season of the short-beaked echidna, spermatorrhoea (spermatozoa in urine) provides an indication of spermatogenesis and can be used to assess sexual maturity or the onset of breeding (Johnston et al. 2007a). Urine can also be readily collected via ultrasound-guided cystocentesis. Attempts to collect semen via electroejaculation (EEJ) in the short-beaked echidna have been unsuccessful, most likely because of complex erection and ejaculatory mechanics (Johnston et al. 2007b; Fenelon et al. 2021). Short-beaked echidna seminal characteristics have been described from a single zoo animal conditioned to masturbate on demand. The ejaculate is of small volume (~20 μL), appears to contain very little seminal plasma and sperm concentration is at the higher end of the scale for mammals (1-8.5 ? 109/ mL). Although a technique for the assessment of sperm DNA fragmentation has been reported in the short- beaked echidna (Johnston et al.
2009), reproductive tract disease and abnormalities of the spermatozoa have not been reported.Seasonal increases in testicular volume (over 4-fold in echidnas) are associated with increases in testosterone secretion and sperm production when coming into the breeding season (winter to early spring) (Temple-Smith and Grant 2001; Johnston and Keeley 2015). Crural glands also increase in size (Grant and Temple-Smith 1998; Morrow et al. 2016) and a large increase in androgens and cortisol before and during the breeding season is associated with aggressive behaviour and increased venom secretion in the platypus (Temple-Smith and Grant 2001). The testes and crural glands of both species are readily assessed by ultrasound examination (Johnston et al. 2007a; Morrow et al. 2016) (Fig. 5.1). Their size and volume can be determined and the health and presence of abnormalities assessed. The steroidogenic capacity of the echidna testis can be assessed readily by means of a GnRH or hCG stimulation test, but is complicated without the use of these exogenous hormones by low and variable circulating testosterone levels both in and out of the breeding season (0.24-0.62 ng/mL v. 0.09 ± 0.05 ng/mL, respectively), making accurate assessment of circulating testosterone levels without gonadotropin stimulation challenging (Johnston et al. 2007a). Non-invasive monitoring of testosterone and its metabolites in faeces may be a better choice for detecting seasonal change, but is yet to be thoroughly investigated (Oates et al. 2002).
1.3 Female reproductive assessment
Female reproductive anatomy is described in Middleton (2008), Booth and Connolly (2008) and Fenelon et al. (2024). Ultrasonography has been used to examine the development of follicles and CL on the short-beaked echidna ovary and eggs within the uterus (Higgins 2004;
Fig. 5.1. (a) Ultrasound images of normal left testis of a short-beaked echidna (Tachyglossus aculeatus) in December before testicular recrudescence being initiated, (b) left testis in June at the beginning of the breeding season, (c) right crural gland in December and (d) right crural gland in July.
All images were captured at the same scale and white crosses indicate linear measurements used to calculate volume. (Image reproduced with permission from Morrow etal. 2016, Interaction of hibernation and male reproductive function in wild Tasmanian echidnas Tachyglossus aculeatus setosus. Journal of Mammalogy 97, 852-860, Oxford University Press).Morrow and Nicol 2009; Dutton-Regester et al. 2021). Both the short-beaked echidna and platypus possess a clitoris and a pair of bulbourethral gland homologues (Hughes and Carrick 1978; Fenelon et al. 2024).
There is limited data on the endocrinology of the monotreme oestrous cycle and gestation (Carrick et al. 1975; Hughes and Carrick 1978; Handasyde et al. 1992; Nicol et al. 2005; Ferguson and Turner 2013; Wallage et al. 2015; Dutton-Regester et al. 2021). Female short-beaked echidnas have the potential to have at least two (sometimes three) reproductive cycles in a breeding season if young are lost early on, as well as the potential to ovulate up to three eggs per cycle (although typically one) and the capacity to successfully wean offspring in consecutive years (Wallage et al. 2015; S Johnston, M Renfree, L Clark and A Roser, unpublished). Previous estimates of gestation in the short-beak echidna ranged from 18 to 20 d and incubation of the egg in the pouch ranged from 9 to 12 d, although the most recent observations by Dutton-Regester et al. (2021, 2022) suggest a gestation of 16-17 d and an egg incubation of most commonly 10 d. For echidnas, observations of copulation or characteristic changes in the female behavioural activity patterns are the best indicators of reproductive potential and activity (Dutton-Regester et al. 2022). During echidna gestation it is possible to observe significant changes in pouch morphology, wherein the lateral margins thicken and pouch area deepens. By the time of egg lay, the pouch opening is capable of muscular constricture, to prevent subsequent loss of the egg and puggle (Dutton-Regester et al.
2021). Female echidnas undergo a period of non-activity directly following ovipo- sition where they incubate the egg in their temporary pouch. During this period, the female remains in the nest box or burrow with little or no evidence of feeding or movement (Dutton-Regester et al. 2021; Dutton-Regester et al. 2022). This incubation period is also characterised by precise control of body temperature (Nicol and Andersen 2006). Although free-ranging females as young as 5 yr of age have been reported to participate in courtship activity (Rismiller and McKelvey 2000), a 3-yr-old zoo-based female was recorded as giving birth and successfully raising a puggle (Wallage et al. 2015). There is no published information on platypus oestrous cycle dynamics or ovulatory patterns. The estimated gestation period ranges from 15 to 21 d (Holland and Jackson 2002; Hawkins and Battaglia 2009) after which the female lays her eggs (1-3, usually 2) in a nesting burrow for an incubation period of up to 12 d. It is thought that the eggs are placed onto the abdomen and primarily incubated with the female lying in a curled posture (Grant 2007).2.