MALE MARSUPIALS

2.1 Physical examination: reproductive tract and external genitalia

Marsupial moles (Notoryctes spp.) are the only truly testi- cond marsupial (Johnson and Walton 1989). In all others, the testes are located externally in a loosely pendulous or pendulous, pedunculated prepenile scrotum.

When assessing the scrotal contents of marsupials, it is important to palpate the testis for presence (fully descended), tone and evidence of fibrosis or adhesion to the tunica vaginalis. It is also good practice to evaluate the presence, relative size and completeness of the epididymis to exclude the possibility of congenital seg­mental aplasia.

Male marsupials lack seminal vesicles, so the majority of seminal plasma in the ejaculate is from the large pros­tate and bulbourethral glands. The marsupial prostate is entirely disseminate, with all the glandular tissue lying between the urethral lumen and the outer urethral muscle, with significant taxon variation in morphology (Rodger and Hughes 1973; Campbell et al. 2023). The glandular lumen of the caudal prostate also produces characteristic prostatic bodies, thought to be produced by ‘a pinching of the distal cytoplasm of the epithelial cells’ (Rodger and Hughes 1973), similar to those described in humans as protostomes. It has not been pos­sible to separate the prostatic bodies from the spermato­zoa by centrifugation or passive separation, which makes visualisation of the spermatozoa in an ejaculate more difficult. In some species, such as the common brush­tailed possum (Trichosurus vulpecula), southern

Fig. 5.2. Dual ultrasound images from a free-ranging koala (Phascolarctos cinereus) showing a normal left testis and a right testis that histologically had atrophy of the seminiferous tubules, reduced germ cells and lack of spermatozoa. The image of the right testis shows multiple hyperechoic foci, particularly at the margins, with more and larger hypoechoic areas in contrast to the comparatively homogeneous echotexture of the left. The epididymides of both were histologically and ultrasonographically normal. Credit: Rebecca Larkin, Moggill Koala Hospital

Fig.

hairy-nosed wombat and the koala, there are significant seasonal changes in prostate volume. Marsupials possess varying pairs (1-3) of bulbourethral glands surrounded by striated muscle, which empty via ducts into the base of the penile urethra. Ultrasonography has been used to describe the normal structure of prostate and bulboure­thral glands in the koala (Fig. 5.3) (Stalder et al. 2016; Larkin et al. 2018) and the southern hairy-nosed wombat (Hogan et al. 2010a).

Although there is significant diversity in the morphol­ogy of the marsupial glans penis, in most species it is tapered, furrowed or forked and remains retracted within a prepuce within the common vestibule when not erect. Keratinous cuticular penile spines are present distal to the glans penis in the koala, wombats, kowari (Dasy- uroides byrnei) and crest-tailed mulgara (Dasycercus cristicauda). Only in the koala is there evidence that these spines may have a role in inducing ovulation by means of a copuloreceptive reflex (Johnston et al. 2004). Dasyurus spp., dibbler (Parantechinus apicalis) and fat-tailed pseudantechinus (Pseudantechinus macdonnellensis) have a unique penile appendage, known as the accessory erectile body, that extends out from the dorsal aspect of the penis and is not believed to have a role in copulation (Woolley and Webb 1977). The penis of the sugar glider (Petaurus breviceps) is bifurcated along two-thirds of its length (Ward and Renfree 1988b; Bradley and Stoddart 1993). In the greater bilby (Macrotis lagotis) the distal penile urethra is bifurcated (Johnston et al.

In some marsupials, androgen-dependent scent glands are present on the chest and/or head (e.g. sugar gliders possess both, koalas only the former) and play a role in dominance and chemical communication and may reflect reproductive potential (Bradley and Stoddart 1993; Allen et al. 2010). Examination of the size and activity of scent glands should be a component of the reproductive assessment.

2.2 SPERMATOZOA

2.2.1 Sperm production

Spermiogenesis and extratesticular maturation of sper­matozoa in marsupials is significantly different to euthe­rians. Although there is taxonomic differentiation in both these phenomena, the primary contrast with euthe­rians is the dorsoventral condensation of the sperm nucleus during spermiogenesis and continued morpho­logical maturation of sperm during epididymal transit (Setchell and Carrick 1973; Temple-Smith and Bedford 1976; Rodger and Mate 1993). Post-testicular changes in marsupial spermatozoa include species-specific subcellu- lar modifications: reorientation of the sperm head to align parallel to the long axis often associated with a nuclear groove; elaboration of the mid-piece plasma membrane and/or mitochondrial network; restructuring and compaction of the acrosomal matrix to the dorsal surface; and loss of the cytoplasmic droplet. The recogni­tion of species differences in normal sperm maturation and morphology is fundamental for evaluating the qual­ity of sperm samples recovered from testicular or epididy- mal biopsies.

2.2.2 Sperm morphology and concentration

Marsupial spermatozoa are generally longer than those of eutherians and occur in lower overall concentrations, but there is considerable variation between taxonomic groups (Cummins and Woodall 1985; Temple-Smith 1994). The shortest spermatozoa are found in the koala and wombat and are ~80 μm; the longest occur in the honey possum at 342 μm. Dasyurid spermatozoa are also typically much longer than in other marsupials, appear to survive longer in the female reproductive tract and are ejaculated in low concentrations.

2.2.3 Semen collection

Electroejaculation under anaesthesia is the primary method used to collect semen in marsupials. Successful EEJ protocols have been described for the tammar wal­laby (Notamacropus eugenii), eastern (Macropus gigan- teus) and western grey kangaroos (M. fuliginosus), yellow-footed rock-wallaby (Petrogale xanthopus), Matschie’s tree-kangaroo (Dendrolagus matschei), south­ern hairy-nosed and bare-nosed wombats, koala and common brush-tailed possum and eastern ring-tailed possums (Pseudocheirus peregrinus) (Rodger and White 1975; Rodger and Pollitt 1981; Johnston et al. 1994; Tag­gart et al. 1995; Taggart et al. 1996; Johnston et al. 1997a; Taggart et al. 1998; Paris et al. 2005a; Paris et al. 2005b). EEJ has been unreliable in dasyurids, most likely because of the low overall numbers of spermatozoa produced, possible differences in ejaculation mechanics and the challenges of suitable probe size and positioning in the small dasyurid species (2004). Sperm abnormalities have been documented in the koala (Wildt et al. 1991; Johnston et al. 1994), wombat (MacCal- lum 2004) and Tasmanian devil (Keeley et al. 2011). Sperm abnormalities associated with the flagellum (e.g. broken neck, segmental aplasia of the mid-piece, severely coiled tails) are relatively easy to identify. Sperm quality stand­ards databases, such as those developed for humans and domestic species, are not currently available for marsupi­als. Basic assessment of sperm morphology should include an evaluation of the extent of morphological consistency within a sample, with relatively homogeneous sperm mor­phology representing a fundamentally normal sample. A technique for assessment of sperm DNA fragmentation (double-stranded DNA breaks) has been established for the koala (Johnston et al. 2009) and dunnart (Sminthopsis spp.) (Johnston et al. 2016) and is currently being devel­oped for the bare-nosed wombat, eastern grey kangaroo (Macropus giganteus) and eastern ring-tailed possum (S Johnston, unpublished).

The mode of sperm motility varies among the marsu­pial taxonomic groups and appears to be linked to sperm structural variation (Taggart 1994). As with eutherian spermatozoa, marsupial spermatozoa gain their capacity for motility during epididymal transit. In dasyurids, the number of motile cauda epididymal spermatozoa is typi­cally low compared with other marsupials (Keeley et al. 2011). For most marsupial species, the mid-piece and tail are circular in transverse section, facilitating a motility pattern that is similar to eutherian sperm, resulting in a rapid, rigid tail motion over a limited arc or essentially a standard effective and recovery stroke resulting in rapid forward movement. However, in the honey possum and all dasyurid spermatozoa, both the sperm tail and mid-piece have a radial displacement of their dense outer fibres and axoneme doublets, resulting in flattening of these struc­tures and translating to a sinusoidal mode of motility that may increase efficiency (Taggart 1994). Understanding that there are different patterns of motility of marsupial sperm is essential when assessing sperm quality.

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