CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence.

Course-based undergraduate research experiences (CUREs) can reduce barriers to research opportunities while increasing student knowledge and confidence. However, the number of widely adopted, easily transferable CUREs is relatively small. Here, we describe a CURE aimed at determining the function of poorly characterized Saccharomyces cerevisiae genes. More than 20 years after sequencing of the yeast genome, nearly 10% of open reading frames (ORFs) still have at least one uncharacterized Gene Ontology (GO) term. We refer to these genes as "ORFans" and formed a consortium aimed at assigning functions to them. Specifically, over 70 faculty members attended summer workshops to learn the bioinformatics workflow and basic laboratory techniques described herein. Ultimately, this CURE was adapted for implementation at 34 institutions, resulting in over 1,300 students conducting course-based research on ORFans. Pre-/post-tests confirmed that students gained both (i) an understanding of gene ontology and (ii) knowledge regarding the use of bioinformatics to assign gene function. After using these data to craft their own hypotheses, then testing their predictions by constructing and phenotyping deletion strains, students self-reported significant gains in several areas, including computer modeling and exposure to a project where no one knows the outcome. Interestingly, most net gains self-reported by ORFan Gene Project participants were greater than published findings for CUREs assessed with the same survey instrument. The surprisingly strong impact of this CURE may be due to the incoming lack of experience of ORFan Project participants and/or the independent thought required to develop testable hypotheses from complex data sets.

Role of DNase Activity in Human Sperm DNA Fragmentation.

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

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.

Gut microbiota in the short-beaked echidna (Tachyglossus Aculeatus) shows stability across gestation.

Indigenous gut microbial communities (microbiota) play critical roles in health and may be especially important for the mother and fetus during pregnancy. Monotremes, such as the short-beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where the young feeds. Since both feces and eggs pass through the cloaca, the fecal microbiota of female echidnas provides an opportunity for vertical transmission of microbes to their offspring. Here, we characterize the gut/fecal microbiome of female short-beaked echidnas and gain a better understanding of the changes that may occur in their microbiome as they go through pregnancy. Fecal samples from four female and five male echidnas were obtained from the Currumbin Wildlife Sanctuary in Queensland and sequenced to evaluate bacterial community structure. We identified 25 core bacteria, most of which were present in male and female samples. Genera such as Fusobacterium, Bacteroides, Escherichia-Shigella, and Lactobacillus were consistently abundant, regardless of sex or gestation stage, accounting for 58.00% and 56.14% of reads in male and female samples, respectively. The echidna microbiome remained stable across the different gestation stages, though there was a significant difference in microbiota composition between male and female echidnas. This study is the first to describe the microbiome composition of short-beaked echidnas across reproductive phases and allows the opportunity for this novel information to be used as a metric of health to aid in the detection of diseases triggered by microbiota dysbiosis.

Anatomy of the Female Koala Reproductive Tract.

The koala (Phascolarctos cinereus), while being an iconic Australian marsupial, has recently been listed as endangered. To establish an improved understanding of normal reproductive anatomy, this paper brings together unpublished research which has approached the topic from two perspectives: (1) the establishment of an artificial insemination program, and (2) the definition of Chlamydia spp.-derived histopathological changes of the female koala urogenital system. Based on the presentation and histological processing of over 70 opportunistic specimens, recovered from wildlife hospitals in Southeast Queensland (Australia), we describe the gross and microanatomy of the koala ovary, oviduct, uteri, vaginal complex, and urogenital sinus during the interestrous, proliferative, and luteal phases of the reproductive cycle.

Intra-individual variation of sperm DNA fragmentation in the Human ejaculate.

This retrospective study assessed the biological intra-individual variability of the percentage of sperm with DNA damage (SDF) observed in subsequent ejaculates of the same individual. Variation in SDF was analyzed using the Mean Signed Difference (MSD) statistic based on 131 individuals, comprising 333 ejaculates. Either two, three or four ejaculates were collected from each individual. With this cohort of individuals two main questions were addressed; (1) does the number of ejaculates analyzed influence the variability in the level of SDF associated with each individual? and (2) is the variability observed in SDF similar when individuals are ranked according to their level of SDF? Results showed that the variation observed in mean SDF was not different when 2, 3 or 4 ejaculates were analyzed; consequently, we suggest that the assessment of SDF based on two ejaculates is likely to be representative of the mean SDF expected for the individual. In parallel, it was determined that the variation in SDF increased as SDF increased; in individuals presenting with an SDF value of lower than 30% (potentially fertile), only 5% possessed levels of MSD that could be considered as variable as that presented by individuals presenting with a recurrent high SDF. Finally, we showed that a single assessment of SDF in individuals with medium SDF (20-30%) was less likely to be predictive of the SDF value in the next ejaculate, and therefore, less informative of the patient's SDF status.

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.

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.

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.

Diversity and transmission of koala retrovirus: a case study in three captive koala populations.

Koala retrovirus is a recently endogenized retrovirus associated with the onset of neoplasia and infectious disease in koalas. There are currently twelve described KoRV subtypes (KoRV-A to I, K-M), most of which were identified through recently implemented deep sequencing methods which reveal an animals' overall KoRV profile. This approach has primarily been carried out on wild koala populations around Australia, with few investigations into the whole-population KoRV profile of captive koala colonies to date. This study conducted deep sequencing on 64 captive koalas of known pedigree, housed in three institutions from New South Wales and South-East Queensland, to provide a detailed analysis of KoRV genetic diversity and transmission. The final dataset included 93 unique KoRV sequences and the first detection of KoRV-E within Australian koala populations. Our analysis suggests that exogenous transmission of KoRV-A, B, D, I and K primarily occurs between dam and joey. Detection of KoRV-D in a neonate sample raises the possibility of this transmission occurring in utero. Overall, the prevalence and abundance of KoRV subtypes was found to vary considerably between captive populations, likely due to their different histories of animal acquisition. Together these findings highlight the importance of KoRV profiling for captive koalas, in particular females, who play a primary role in KoRV exogenous transmission.

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.

Chlamydiosis and cystic dilatation of the ovarian bursa in the female koala (Phascolarctos cinereus): Novel insights into the pathogenesis and mechanisms of formation.

Infection with Chlamydia pecorum is one of the main causes of progressive decline of koala (Phascolarctos cinereus) populations in Eastern Australia. Pathological changes associated with the chlamydial infection in the genital tract of female and male koalas have been widely described with reports of acute and chronic lymphoplasmacytic inflammation and the description of the cystic dilatation of the ovarian bursa. Although these disease manifestations can result in severe chronic inflammation, structural changes and even sterility, only limited data is currently available on the organism's distribution and associated histopathological and ultrastructural changes within the upper genital tract of affected females. This study examined the pathogenesis of the most common pathological lesion associated with chlamydiosis in female koalas, the cystic dilation of the ovarian bursa starting from the evidence that Chlamydia spp. induces disruption of the intercellular junctions in the epithelium of the reproductive organs in humans. Histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to evaluate the structural features and the expression of epithelial cell and cellular junctions' markers in affected bursae from 39 Chlamydia-infected female koalas. Epithelial cells from the ovarian bursae of one affected animal examined by transmission electron microscopy showed severe widening of the intercellular space, as morphologic evidence of disrupted permeability of the epithelial barrier. The epithelial cell-cell junctions markers E-cadherin, ß-catenin and ZO-1 expressions were significantly reduced in samples from cystic bursae when compared to normal tissue samples (P < 0.0001). On the other end, a significantly higher expression of the proliferation marker Ki67 was observed in cystic bursae compared to control samples (P < 0.0001). As these proteins are required to maintain epithelial functional integrity and cell-cell adhesive interactions, their loss may permanently impair and affect female koala fertility and suggest the molecular basis of the pathogenesis of the cystic accumulation of bursal fluid within this tissue.

Modelling Genetic Benefits and Financial Costs of Integrating Biobanking into the Captive Management of Koalas.

Zoo and wildlife hospital networks are set to become a vital component of Australia's contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive colonies. Emerging evidence suggests that biobanking and associated assisted reproductive technologies could address these economic and genetic challenges. We present a modelled scenario, supported by detailed costings, where these technologies are optimized and could be integrated into conservation breeding programs of koalas across the established zoo and wildlife hospital network. Genetic and economic modelling comparing closed captive koala populations suggest that supplementing them with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower the required colony sizes of conservation breeding programs, and greatly reduce program costs. Ambitious genetic retention targets (maintaining 90%, 95% and 99% of source population heterozygosity for 100 years) could be possible within realistic cost frameworks, with output koalas suited for wild release. Integrating biobanking into the zoo and wildlife hospital network presents a cost-effective and financially feasible model for the uptake of these tools due to the technical and research expertise, captive koala colonies, and ex situ facilities that already exist across these networks.

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.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence.

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman's rho = - 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus).

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

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.

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.

The Unique Penile Morphology of the Short-Beaked Echidna, Tachyglossus aculeatus.

Monotremes diverged from therian mammal ancestors approximately 184 million years ago and have a number of novel reproductive characteristics. One in particular is their penile morphology. There are differences between echidna and platypus phalluses, but both are somewhat similar in structure to the reptilian phallus. The echidna penis consists of 4 rosette glans, each of which contains a termination of the quadrifurcate urethra, but it appears that only 2 of the 4 glans become erect at any one time. Despite this, only a few historical references describe the structure of the echidna penis and none provides an explanation for the mechanisms of unilateral ejaculation. This study confirmed that the echidna penis contains many of the same overall structures and morphology as other mammalian penises and a number of features homologous with reptiles. The corpus cavernosum is well supplied with blood, extends up to the base of the glans penis and is primarily responsible for erection. However, the echidna possesses 2 distinct corpora spongiosa separated by a septum, each of which surround the urethra only distal to the initial urethral bifurcation in the glans penis. Together with the bifurcation of the main penile artery, this provides a mechanism by which blood flow could be directed to only one corpus spongiosum at a time to maintain an open urethra that supplies 2 of the 4 glans to facilitate unilateral ejaculation.