Resetting the paradigm of reproductive science and conservation.

In the application of reproductive science to conservation breeding, it has long been assumed that artificial insemination using frozen thawed sperm would be the default technology. This has always been problematic considering the wide range of tolerance to freeze thawing among vertebrate sperm. Furthermore, those providing leadership for genome banking should be proactive to preserve maximum genetic diversity, however, for many species there is little or no sperm motility after thawing of cryopreserved sperm.

Integrating biobanking could produce significant cost benefits and minimise inbreeding for Australian amphibian captive breeding programs.

Captive breeding is an important tool for amphibian conservation despite high economic costs and deleterious genetic effects of sustained captivity and unavoidably small colony sizes. Integration of biobanking and assisted reproductive technologies (ARTs) could provide solutions to these challenges, but is rarely used due to lack of recognition of the potential benefits and clear policy direction. Here we present compelling genetic and economic arguments to integrate biobanking and ARTs into captive breeding programs using modelled captive populations of two Australian threatened frogs, namely the orange-bellied frog Geocrinia vitellina and the white bellied frog Geocrinia alba .

Marsupials the alternative therians - From gametes to birth.

Marsupial reproduction shares many common features with the more familiar eutherian mammals but things are often done differently, in alternative ways. Like the eutherians marsupials are placental but the period and degree of development supported in the uterus is much shorter and the long growth phase of development is supported by lactation. But these different ways of achieving often similar outcomes are also seen in gamete formation and function, fertilization and early development.

Marsupials: Progress and Prospects.

The chapter provides a review of the application of reproductive science to technologies for marsupial conservation and population management and discusses prospects for the future. This includes the status of technologies such as sperm freezing, artificial insemination, and exogenous hormone treatments to stimulate ovarian activity and cycling in the female. Fertility-based population management for introduced pest species and over-abundant native marsupials remain an elusive goal.

Delayed return to estrus following treatment with the gonadotrophin-releasing hormone agonist, Lucrin Depot, in the tammar wallaby.

Gonadotrophin-releasing hormone agonists that induce a reversible contraceptive state in several marsupials have the potential to be used to synchronize estrus. We used a model macropod, the tammar wallaby (Notamacropus eugenii), to investigate whether Lucrin Depot (AbbVie), a GnRH agonist microsphere preparation, could (1) inhibit follicular development and estrus in a cycle reactivated by removal of pouch young (RPY) and (2) facilitate a synchronous return to estrus. Our results show that females reactivated with bromocriptine and RPY in early seasonal quiescence (July 2015) were inhibited by Lucrin Depot (0.

Breeding in the fat-tailed dunnart following ovarian suppression with the gonadotrophin-releasing hormone agonist Lucrin Depot.

Lucrin Depot (AbbVie), a 1-month microsphere gonadotrophin-releasing hormone (GnRH) agonist preparation, was investigated as a potential agent to synchronise cycling in the fat-tailed dunnart (Sminthopsis crassicaudata). Forty-eight randomly selected females were treated with 5 or 10mgkg-1 Lucrin Depot (n=24 per dose). Eighteen females per treatment had their reproductive activity scored at 4, 8, 12 and 16 weeks using two ovarian (Graafian follicle and corpus luteum status) and two reproductive tract (uterine and vaginal muscularity and vascularity) parameters that formed a reproductive activity score.

Recent advances in tools and technologies for monitoring and controlling ovarian activity in marsupials.

Components of assisted reproduction technologies (ART), such as sperm cryopreservation, artificial insemination, superovulation and pouch young surrogacy, have been developed for a range of Australian and American marsupials. However, methods to effectively control ovarian function, arguably the key limiting factors in applying and integrating ART as a practical tool in conservation management, remain poorly developed. This is largely due to unique characteristics of the marsupial corpus luteum and its failure to respond to agents used to synchronize ovarian function in eutherian mammals.

Ovarian suppression in a marsupial following single treatment with a gonadotrophin-releasing hormone agonist in microspheres.

The effect of treatment with Lucrin Depot (1 month), a microsphere gonadotrophin-releasing hormone agonist preparation, was investigated in the fat-tailed dunnart (Sminthopsis crassicaudata) as a potential strategy to synchronise cycling. The status of the ovaries (ovarian size, number and size of Graafian follicles and corpora lutea) and reproductive tract (weight, vascularity and muscularity) in twelve untreated females were assessed to establish the activity parameters for randomly selected cycling animals. Thirty-six females were treated with 1mgkg (n=12), 10mgkg (n=12) or 20mgkg (n=12) Lucrin Depot.

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein.

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months.

Mammalian hair as an accumulative bioindicator of metal bioavailability in Australian terrestrial environments.

The current study represents the first investigation of the suitability of marsupial and eutherian mammalian hair as indicator tissue for metal exposure and accumulation within contaminated Australian terrestrial ecosystems. A soil metal contamination gradient was established across 22 sites at increasing distances from a decommissioned Lead/Zinc smelter in NSW, Australia. Within each site, soil and small mammal populations were sampled.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals.

Towards a ZP-based contraceptive for marsupials: comparative analysis and developmental expression of marsupial ZP genes.

Fertility control in the form of a zona pellucida (ZP)-based immunocontraceptive has shown potential as a humane form of control for overabundant marsupials including the brushtail possum and macropods. Further refinement and development of a ZP-based vaccine requires detailed knowledge of the protein structure and expression in order to ensure maximum efficacy and specificity. Sequencing and comparative analysis of the ZP3 protein from three marsupial orders in this study found a high overall level of conservation; within order Diprotodontia, the ZP3 protein is 86.

Characterisation of an epitope shared by an acrosomal acrosin-like protein and the surface of tammar wallaby (Macropus eugenii) spermatozoa.

Monoclonal antibodies (mAb) have been raised against marsupial sperm proteins to provide insights into the molecular nature of marsupial spermatozoa, and the proteins that mediate sperm maturation and interaction with the oocyte. This study reports the production of a mAb, designated WSA-1, which bound acrosomal and surface determinants on tammar wallaby spermatozoa. The acrosomal antigen was first detected in the wallaby testis; however, ejaculated spermatozoa demonstrated whole cell WSA-1 immunoreactivity as a result of binding an epididymal protein.

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums.

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro.