Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

Getting out of a mammalian egg: the egg tooth and caruncle of the echidna.

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.

Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation.

Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.

Immunohistochemical characterization of the immune cell response during chlamydial infection in the male and female koala (Phascolarctos cinereus) reproductive tract.

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been investigated. Thirty-two female koalas and 47 males presented to wildlife hospitals with clinical signs suggestive of Chlamydia infection were euthanized with the entire reproductive tract collected for histology; immunohistochemistry (IHC) for T-cell (CD3ε, CD4, and CD8α), B-cell (CD79b), and human leukocyte antigen (HLA)-DR markers; and quantitative real-time polymerase chain reaction (rtPCR) for Chlamydia pecorum. T-cells, B-cells, and HLA-DR-positive cells were observed in both the lower and upper reproductive tracts of male and female koalas with a statistically significant associations between the degree of the inflammatory reaction; the number of CD3, CD4, CD79b, and HLA-DR positive cells; and the PCR load. CD4-positive cells were negatively associated with the severity of the gross lesions. The distribution of immune cells was also variable according to the location within the genital tract in both male and female koalas. These preliminary results represent a step forward towards further exploring mechanisms behind chlamydial infection immunopathogenesis, thus providing valuable information about the immune response and infectious diseases in free-ranging koalas.

Koala retrovirus genetic diversity and transmission dynamics within captive koala populations.

Koala populations are currently in rapid decline across Australia, with infectious diseases being a contributing cause. The koala retrovirus (KoRV) is a gammaretrovirus present in both captive and wild koala colonies that presents an additional challenge for koala conservation in addition to habitat loss, climate change, and other factors. Currently, nine different subtypes (A to I) have been identified; however, KoRV genetic diversity analyses have been limited. KoRV is thought to be exogenously transmitted between individuals, with KoRV-A also being endogenous and transmitted through the germline. The mechanisms of exogenous KoRV transmission are yet to be extensively investigated. Here, deep sequencing was employed on 109 captive koalas of known pedigree, housed in two institutions from Southeast Queensland, to provide a detailed analysis of KoRV transmission dynamics and genetic diversity. The final dataset included 421 unique KoRV sequences, along with the finding of an additional subtype (KoRV-K). Our analysis suggests that exogenous transmission of KoRV occurs primarily between dam and joey, with evidence provided for multiple subtypes, including nonendogenized KoRV-A. No evidence of sexual transmission was observed, with mating partners found to share a similar number of sequences as unrelated koala pairs. Importantly, both distinct captive colonies showed similar trends. These findings indicate that breeding strategies or antiretroviral treatment of females could be employed as effective management approaches in combating KoRV transmission.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.

In vitro transmission of Chlamydia using naturally infected koala (Phascolarctos cinereus) semen.

Transmission of Chlamydia pecorum infection has generally been assumed to be via the urogenital route and in an attempt to confirm this we investigated an in vitro method of Chlamydia infection using naturally infected koala semen to inoculate a cell line and attempt to estimate C. pecorum infectious load. A total of 57% of 122 koala semen samples had low C. pecorum copy number or no burden, while 18% of semen samples contained >10000 inclusion-forming units/mL, as determined by quantitative polymerase chain reaction. In vitro inoculation of a McCoy cell line resulted in successful infection from 4% of semen samples where C. pecorum burden was >105 inclusion-forming units/mL. Our preliminary study suggests that transmission of C. pecorum infectious dose may be restricted to peak bacterial shedding in semen associated with recent infection. Here, we report venereal transmission of C. pecorum in koala semen is possible; however, we speculate that antimicrobial factors and innate immune function receptors associated with semen may inhibit chlamydial growth. These mechanisms have yet to be reported in marsupial semen.

Reproductive behaviour before and after oestrus and oviposition in the captive short-beaked echidna (Tachyglossus aculeatus).

CONTEXT: Most of our current knowledge regarding echidna reproductive behaviour is based on qualitative measurements; therefore, it is unclear if specific behavioural cues could be utilised in their captive reproductive management. AIMS: This study aimed to identify quantitative changes in general and reproductive behaviour of echidna breeding pairs and pregnant females that might facilitate the detection of oestrus and impending oviposition and provide a summary of reproductive behaviour observed in a captive colony over a three-year observation period. METHODS: Three echidna breeding pairs and two trios were monitored daily for seven reproductive and eight general behaviours during the 2020 breeding season. After confirmed copulation, females were monitored for four egg-laying and eight general behaviours until egg incubation. General observations of reproductive behaviours during the 2018-2020 breeding seasons were recorded as part of routine husbandry. KEY RESULTS: For breeding pairs, there was a significant rate of change over time before and after copulation for the behaviours 'urogenital sniffing', 'rolling' and 'copulation attempt'. For pregnant females, time engaged in 'pacing' significantly increased while 'time eating' and the 'quantity of food eaten' significantly decreased on the day of oviposition. We were not able to identify oestrus from specific behaviours, but our observations suggest that the female echidna's period of receptivity is less than 24h. CONCLUSIONS: The frequency that males express 'urogenital sniffing', 'rolling' and 'copulation attempt' toward the female can be used to alert zookeepers that copulation has likely occurred. Increased pacing, reduced feeding time and quantity of food eaten can aid zookeepers to identify impending oviposition. IMPLICATIONS: This study demonstrates that there are quantifiable changes in specific echidna behaviours that can be incorporated into zoo husbandry practices to improve the reproductive management of this species.

Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus).

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

Assisted breeding technology in the saltwater crocodile Crocodylus porosus: a review and look to the future.

This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding sufficient volume and sperm concentrations for empirical and molecular analyses of sperm preservation and physiology. Nevertheless, there is still much to learn with respect to fundamental anatomy, physiology and behaviour in both sexes, but particularly in the female. Although lessons can be learned from successful AI in the alligator, the details of this research are not readily accessible. Future research needs to focus on the proximate factors of seasonality and the underlying control of the female's annual reproductive cycle; this will require novel and innovative ways to collect blood samples without causing stress or injury, and ideally a dedicated crocodile research breeding colony. Because the saltwater crocodile is a farmed species, there is likely to be sufficient impetus for the application of assisted breeding technology to drive future productivity in the industry. These developments will also have benefits for the genetic and reproductive management of endangered captive populations.

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: assessing the temporal acquisition of sperm motility.

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

Gross and microanatomy of the male reproductive duct system of the saltwater crocodile Crocodylus porosus.

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

Modification of Crocodile Spermatozoa Refutes the Tenet That Post-testicular Sperm Maturation Is Restricted To Mammals.

Competition to achieve paternity has contributed to the development of a multitude of elaborate male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes, termed capacitation, in the female reproductive tract before realizing their potential to fertilize an ovum. However, the evolutionary origin and adaptive advantage afforded by capacitation remains obscure. Here, we report the use of comparative and quantitative proteomics to explore the biological significance of capacitation in an ancient reptilian species, the Australian saltwater crocodile (Crocodylus porosus,). Our data reveal that exposure of crocodile spermatozoa to capacitation stimuli elicits a cascade of physiological responses that are analogous to those implicated in the functional activation of their mammalian counterparts. Indeed, among a total of 1119 proteins identified in this study, we detected 126 that were differentially phosphorylated (± 1.2 fold-change) in capacitated versus, noncapacitated crocodile spermatozoa. Notably, this subset of phosphorylated proteins shared substantial evolutionary overlap with those documented in mammalian spermatozoa, and included key elements of signal transduction, metabolic and cellular remodeling pathways. Unlike mammalian sperm, however, we noted a distinct bias for differential phosphorylation of serine (as opposed to tyrosine) residues, with this amino acid featuring as the target for ∼80% of all changes detected in capacitated spermatozoa. Overall, these results indicate that the phenomenon of sperm capacitation is unlikely to be restricted to mammals and provide a framework for understanding the molecular changes in sperm physiology necessary for fertilization.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Using the Koala (Phascolarctos cinereus) as a Case Study to Illustrate the Development of Artificial Breeding Technology in Marsupials: an Update.

The successful development and application of an assisted breeding program in any animal relies primarily on a thorough understanding of the fundamental reproductive biology (anatomy, physiology and behaviour) of the species in question. Surely, the ultimate goal and greatest hallmark of such a program is the efficacious establishment of a series of reliable techniques that facilitate the reproductive and genetic management of fragmented populations, both in captivity and in the wild. Such an achievement is all that more challenging when knowledge of the reproductive biology of that species is essentially rudimentary and without adequate models to compare to. Using the koala (Phascolarctos cinereus) as a case study, this chapter provides insights into the development of a concept that began as small undergraduate student project but that subsequently evolved into the first-ever successful artificial insemination of a marsupial. Apart from this historical perspective, we shall also provide a brief review of the current reproductive biology of the koala, discuss technical elements of current assisted breeding technology of this species, its application to the closely related wombat, and the potential role it might play in helping to conserve wild koala populations in the form of a live koala genome bank. There is little doubt that the unique reproductive biology and tractability of the koala has been a benefit rather than a hindrance to the success of artificial breeding in this species.

Reproductive biology of captive southern hairy-nosed wombats (Lasiorhinus latifrons). Part 1: oestrous cycle characterisation.

Southern hairy-nosed wombats (SHNWs: Lasiorhinus latifrons) do not breed well in captivity. To better understand their reproduction, daily urine samples were collected from nine captive females and analysed for volume (mL), specific gravity and a qualitative index of the number of epithelial cells, then stored at -20°C until samples could be analysed for progesterone metabolites (P4M). The mean oestrous cycle length was 35.1±2.4 days; however, individual cycle length ranged from 23 to 47 days. The mean luteal phase length was 20.8±1.3 days (range: 12 to 33 days). Urinary P4M was divided into four oestrous cycle stages: (1) early follicular phase, (2) late follicular phase, (3) early luteal phase, (4) late luteal phase, and analysed against urinary characteristics. During the late follicular phase, urine volume decreased (P=0.002) while urine specific gravity (P=0.001) and concentration of epithelial cells (P=0.004) both increased. The level of variability in oestrous cycle length suggests that some captive females may exhibit abnormal cycles; however, the changes in the urinary characteristics associated with the different stages of the oestrous cycle appear to offer a possible non-invasive means of monitoring the reproductive status of captive SHNWs.

Reproductive biology of captive female southern hairy-nosed wombats (Lasiorhinus latifrons). Part 2: oestrous behaviour.

The poor captive breeding success of southern hairy-nosed wombats (SHNWs; Lasiorhinus latifrons) has been attributed to the difficulty in accurately characterising oestrous behaviour and their relationship to circulating reproductive hormones. Over two wombat breeding seasons, the use of infrared cameras for 24-h remote behavioural monitoring and the analysis of urine samples collected from seven captive females, were investigated to determine the relationship between behaviour and changes in urinary progesterone metabolites (P4M). Urinary P4M was divided into two concentrations: (1) ≤ baseline P4M values and (2)>baseline P4M values and evaluated against urine volume (mL) and the duration (s) and frequency of 23 behaviours recorded for 8 days surrounding D0 of the luteal phase (D0: a sustained increase in P4M for three or more consecutive days). When P4M was ≤ baseline, the duration of urination and volume both decreased, whereas the duration and frequency of both pacing and rump bites by the female towards the male increased. These results suggest that there were detectable behavioural changes that can be mapped to the changes in the SHNW oestrous cycle, which may be used as behavioural indicators to identify the reproductive status of females.

Magnetic-activated cell sorting is not completely effective at reducing sperm DNA fragmentation.

PURPOSE: To determine whether there is a homogeneous reduction of sperm DNA fragmentation (SDF) in sperm samples recovered from the MACS procedure, compared to spermatozoa in the initial ejaculate (NEAT) and those retained in the column. METHODS: This study investigated the relative change in sperm DNA quality (SDF) of neat ejaculates (10 idiopathic infertile and 10 normozoospermic patients) to subpopulations of spermatozoa that had passed through the column (MACS-) and those retained (MACS+) by the annexin-V conjugated microbeads. RESULTS: While the MACS protocol was capable of reducing the mean proportion of SDF (59.2%; P = 0.000) and sperm with highly degraded DNA (SDD; 65.7%, P = 0.000) in all patients, the reduction was not homogeneous across the patient cohort. A significant positive correlation (r = 0.772, P = 0.000) was apparent between the level of SDF in the NEAT ejaculate and the efficacy of SDF reduction observed in the MACS- fraction. CONCLUSION: MACS is capable of reducing the proportion of SDF, especially spermatozoa with a highly degraded DNA molecule. However, this reduction did not preclude the presence of a small subpopulation of spermatozoa with damaged DNA in the MACS- fraction. The MACS protocol was two- to threefold more efficient when the SDF in NEAT ejaculate was equal to or greater than 30%. In 4 of 20 individuals, the level of SDF after MACS resulted in semen for ICSI with a higher or non-significant reduction when compared to SDF observed in the NEAT ejaculate.