M. S. Harris

Artificial insemination in marsupials

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.

Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying

Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart (Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryans staining. After freeze drying, Bryans staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.

Dissociation and preservation of preantral follicles and immature oocytes from female dasyurid marsupials

The mammalian ovary contains numerous immature preantral follicles that are not dependent on endocrine support, unlike the more mature hormone-dependent antral follicles. Preantral follicles can be enzymatically dissociated to yield immature oocytes that survive sub-zero preservation better as they lack a temperature-sensitive meiotic spindle. These techniques are highly applicable to gamete banking, which is an urgent requirement for Australian carnivorous marsupials as several species have rapidly declining populations and risk extinction. The present study developed protocols for the transport, dissociation, preservation and culture of granulosa cell-oocyte complexes (GOC) from the ovaries of dasyurid marsupials. High viability of GOC following enzymatic dissociation is reported and it was demonstrated that GOC are of significantly better quality following refrigerated storage for 24 h compared with storage at room temperature. Oocytes from primary follicles were not damaged by cold shock or the toxicity of vitrification media and following vitrification in liquid nitrogen 69.42+/-2.44% of oocytes were viable. However, the surrounding granulosa cells demonstrated significant damage post-thaw. These granulosa cells proliferated during a 48-h culture period resulting in significant improvements in GOC quality. The present study is a valuable step towards cryostorage of dasyurid gametes and represents fundamentally important methods by which we can contribute to the conservation of Australias native predators.

Optimization, validation and efficacy of the phytohaemagglutinin inflammation assay for use in ecoimmunological studies of amphibians

Amphibians are in the midst of an extinction crisis, yet there are few tools available to study amphibian immunocompetence under conditions of changing environments, disease and stress. We developed, validated and optimised a practical assay for studying amphibian immunocompetence and tested its efficacy in a real-world scenario of varying environmental conditions.

Characterisation of fibrous sheath and midpiece fibre network polypeptides of marsupial spermatozoa with a monoclonal antibody.

In this study cytoskeletal antigens common to brushtail possum and tammar wallaby spermatozoa were characterised using a monoclonal antibody (PSA‐10). Using indirect immunofluorescence, the PSA‐10 antibody detected antigens predominantly associated with the midpiece and principal piece of mature, permeabilised marsupial spermatozoa. The principal piece determinant, shared by a variety of other species, was found to arise in the marsupial testis. Midpiece localisation of the PSA‐10 epitope was detected only in marsupial spermatozoa and shown to arise in the epididymis. Immunogold labelling demonstrated that the PSA‐10 antigens were predominantly associated with the fibrous sheath and midpiece fibre network of both possum and wallaby spermatozoa. Western blotting suggested that two major possum and wallaby sperm polypeptides of 158 and 182 kDa were associated with the midpiece fibre network, a cytoskeletal structure unique to marsupial spermatozoa. A 32 kDa polypeptide was associated with the principal piece fibre network and/or fibrous sheath. The finding that these marsupial sperm cytoskeletal proteins share a common linear epitope suggests that they share some sequence similarity. The midpiece fibre network of marsupial sperm, like the fibrous sheath, has been proposed to have a structural role in providing passive stiffening for the flagellum (Harding et al., 1975, 1979; Olsen, 1975). The PSA‐10 monoclonal antibody may provide a tool for comparative studies of mammalian sperm cytoskeletal proteins, particularly the marsupial midpiece fibre network. It may also allow the formation of this unique marsupial cytoskeletal structure, and its fate during the fertilisation process, to be followed by immunological means. Mol. Reprod. Dev. 50:461–473, 1998.

Comparison of the production, quality, and in vitro maturation capacity of oocytes from untreated cycling and intermediate phase equine serum gonadotropin-treated fat-tailed dunnarts (Sminthopsis crassicaudata).

This study describes ovarian changes during the natural and stimulated reproductive cycle of breeding (< or =12 month) and retired (>12 month) fat-tailed dunnarts, Sminthopsis crassicaudata. Increased urinary cornified epithelial cells and the influx of leukocytes defined day 0, at which time the naturally cycling females had already ovulated; at day 16 females had no antral follicles, but by day 20 antral follicles had begun to develop. There was no difference between naturally cycling breeding and retired females. Females were stimulated with 1 IU equine serum gonadotropin (eSG) during the intermediate phase on day 16 and killed 3, 4, or 5 days later. Stimulation resulted in a significant increase in the number of growing antral follicles but retired females demonstrated a reduced response. Upon collection from breeding females 4 days following eSG stimulation, 100% of oocytes were at the first polar body (PB1) stage, those collected from retired females were immature upon collection but within 48 h 98.2+/-1.9% were cultured to the PB1 stage. The rate of ovulation was high in breeding females 5 days following stimulation but retired females were less reliable, and in both groups all oocytes were degraded. This is the first study to describe a reliable technique, involving ovarian stimulation during the intermediate phase and segregation of age groups, allowing the collection of a large number of healthy PB1 stage oocytes from S. crassicaudata. This is important for the development of further assisted reproductive techniques for this species and threatened dasyurids.

Fertilization in Monotreme, Marsupial and Eutherian Mammals

There are three quite distinct evolutionary lineages of mammals which have been separated for many millions of years by time and place. The northern-origin (European, Asian, African and North American) eutherian mammals - often called placental mammals - and the southern-origin monotremes (Australasian) and marsupials (Australasian and American).

Development and evaluation of electroejaculation techniques in the Tasmanian devil (Sarcophilus harrisii)

Electroejaculation (EEJ) has been used successfully to collect samples suitable for genome resource banking from a variety of endangered wildlife species. Ejaculates can also be used to evaluate the reproductive potential of individuals and provide information on seminal characteristics to aid in the development of sperm cryopreservation techniques. Electroejaculation techniques used for marsupial and eutherian species were tested on Tasmanian devils (n=35). Spermic ejaculates were collected in 54% (19/35) of EEJ attempts. Spermic ejaculates were low in volume (3.9±6.5×10(2) µL, range 10-3000 µL) and contained low numbers of spermatozoa (3.3±7.8×10(3) spermatozoa per ejaculate, range 6-33000). The osmolality and pH of presumptive urine-free ejaculates were 389±130 mOsm kg(-1) (range 102-566) and 7.0±0.9 (range 6.0-8.0), respectively. Prostatic bodies were observed in 79% (26/33) of ejaculates. Episodic fluctuations in serum testosterone concentrations were not detected during the EEJ procedure (P>0.05). Increases observed in serum cortisol concentrations during EEJ were less (P<0.05) than those observed after an adrenalcorticotropic hormone challenge and diurnal variation suggested that cortisol concentrations are greater during the day than at night (P<0.05). This information can be used to provide range values for the future examination of basic endocrine responses and the adrenal-pituitary axis of this species. This study also demonstrated that spermatozoa-rich devil electroejaculates are more difficult to obtain and poorer in quality than those of other marsupials.