Australia's most iconic animal, the koala (Phascolarctos cinereus), faces significant population decline and while conservation has focused on assisted breeding technology and reproductive pathologies, koala reproductive biology remains poorly understood. In particular, the koala (P. cinereus) prostate has a vital role in the production of seminal plasma and facilitating successful reproduction. Furthermore, prostatitis regularly occurs as a consequence of chlamydiosis, a substantial factor in the ongoing decline of koala populations. Despite this, little is known about the koala prostate's molecular physiology. This study presents the first proteomic profile of the koala prostate, offering insights into its histological segmentation and broader functional significance. Prostatic tissue was collected from six mature male koalas, with samples taken from the anterior and posterior segments. Proteins were digested using filter-aided sample preparation and analysed via liquid chromatography-tandem mass spectrometry with Zeno-SWATH acquisition. Peptide spectra were processed using DIA-NN and evaluated in RStudio to identify differentially expressed proteins and compare the koala prostate proteome with those of other species. Functional annotation and pathway analysis revealed that whole prostate activity was primarily centered on protein translation and muscle function. Segment-specific proteomic profiles demonstrated slight proteomic differentiation, with secretory proteins contributing to segment-specific functions. Cross-species comparisons showed strong homology between the koala and human prostate proteomes. This proteome provides a foundation for future investigations into prostate-related pathologies in koalas. Furthermore, understanding the koala prostate at a molecular level helps advance wildlife conservation through a better comprehension of its role in male fertility and offers broader evolutionary insight into marsupial reproduction.

The sophisticated control systems that govern reproduction in mammals are usually aimed at ensuring that offspring are born at favourable times of year. Although recent trends in reproductive biology research have tended to become ever more reductionist and focused on cellular and molecular details, we have taken the view in this article that it is equally important to take a step back from this level of detail. We have therefore provided an overview, albeit rather brief, that links reproductive biology with the evolutionary and environmental backgrounds in which this diverse assemblage of species have had to survive and adapt.

While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context, we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by filter-aided sample preparation digestion were analyzed by liquid chromatography tandem mass spectrometry with sequential window acquisition of all theoretical acquisition. A total of 4304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus, and cauda spermatozoa had strong enrichment for tubulins and included four histone proteins. The most significant proteomic remodeling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias toward protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.

Each year, hundreds of female koalas are presented to koala hospitals suffering from a range of morbidities, many of which require euthanasia for animal welfare reasons. These koalas represent a possible resource for genetic recovery by means of oocyte retrieval for genome banking or use in assisted reproductive technology. To examine the feasibility of koala oocyte recovery, this study conducted a preliminary survey of follicular activity and disease presence in fixed ovarian tissues from koala cadavers in South East Queensland. Ovarian activity and pathology were assessed by gross examination and histology. Bursal pathology was categorized into koalas with no, small (<10 mm diameter), moderate (10–20 mm diameter), or large (>20 mm diameter) sized bursae, whereas uterine pathology was diagnosed by an experienced reproductive pathologist. Antral follicles were observed in 94.4% of ovaries recovered from koalas with no bursal or uterine pathology (n = 18/44), 95.2% of the ovaries of koalas with bursal but no uterine pathology (n = 11/44), 100% of the ovaries of koalas showing only uterine pathology (n = 4/4) and 89.5% of ovaries from koalas with both bursal and uterine pathology (n = 11/44). Of the fixed ovarian tissue suitable for PCR Chlamydia detection (35/44), none were positive. As proof of concept, oocytes were also collected and evaluated from six koala cadavers within 2 h post-mortem. Although further studies are required to determine the quality and viability of the retrieved koala oocytes, our preliminary survey provides strong evidence that ovarian activity mostly continues unabated, irrespective of reproductive pathology, and that oocytes can be recovered successfully.

The koala (Phascolarctos cinereus) is recognised as threatened across two thirds of its distribution and identified as particularly susceptible to climate change. The aim of this study was to assess the spatio-temporal variation in microclimate across koala home ranges and determine any tendency for koalas to exploit this variability. Temperature data loggers were set out in a grid pattern across the study site on St Bees Island, Queensland. Resident koalas were collared with GPS units recording location at night or during the day. Our results revealed that temperature variation across the landscape was greatest on the hottest days (~10 °C). During the day, koalas were found in areas of the landscape that recorded lower daytime temperatures, and during the night, they were found in areas that recorded the highest daytime temperatures. We postulate that koalas avoided the hottest areas of their range during summer days and were more likely to use cooler non-fodder trees but utilised them at night because these areas corresponded with the location of fodder trees. From our results, we suggest that the microclimate of non-fodder trees both (a) explains their selection by koalas during the day and (b) highlights their importance to koala persistence, in addition to the known fodder species.

Release or discard of captured fish commonly occurs in commercial and recreational fisheries and can result in immediate fish mortality during capture or delayed mortality upon release. This review synthesizes data from Pacific salmon (Oncorhynchus spp.) fisheries examining intrinsic and extrinsic factors affecting Pacific salmon individual release mortality (RM) across species and fishing sectors as adult fish mature and transit from marine, to estuarine, to fresh water. RM risk was high (26–45 % observed mortality) in all fisheries and environments when captured fish were bleeding, had high levels of scale loss, had fin or eye damage, and were exposed to low oxygen from net crowding and exhaustion. Highest RM risk (>45 % observed mortality) was associated with gill net and purse seine fisheries. Air exposure and handling duration contributed to high RM when water temperatures in any environment exceeded 18°C. Estuarine and lower river environments have elevated RM risk due to osmotic, maturation, and temperature changes. Short to medium term (≤24 h) observations were poor predictors of longer-term RM, and observations of at least 5–10 days were needed to assess more complete RM rates. RM mechanisms were environment, fishery sector, and life-stage specific. Our best practice recommendations for modifying current fishing practices are gear- and location-specific and aim to minimise stress, injury, and bycatch, which could result in improvements to fish welfare, reductions in RM, and associated conservation benefits.

The sex chromosomes of egg-laying mammals (monotremes), which lack the sex determining gene SRY, evolved independently to those of all therian mammals. Here we characterise the candidate monotreme sex determining gene, the Y-localised anti-Müllerian hormone gene (AMHY) and trace its expression during the period of sexual differentiation. Monotreme AMHX and AMHY gametologues have significant sequence divergence at the promoter, gene and protein level, likely following an original allele inversion in the common monotreme ancestor but retain conserved features of TGF-β molecules. Expression of sexual differentiation genes in the echidna fetal gonad were significantly different from that of therian mammals. AMHY expression was seen exclusively in the male gonad during sexual differentiation, whereas AMHX was expressed in both sexes. Experimental ectopic expression of platypus AMHX or AMHY in the chicken embryo did not masculinise the female urogenital system, a possible result of mammalian specific changes to AMH proteins preventing function in the chicken. Our results provide fundamental insight into the first steps of monotreme sex chromosome evolution and sex determination with developmental expression data strongly supporting AMHY as the primary male sex determination gene.

Objective: Hatcheries are used to help supplement populations of Pacific salmon Oncorhynchus spp. for conservation and fisheries purposes and to provide stewardship opportunities. In British Columbia, small community-run hatcheries typically focus on the latter and generally do not know the efficacy of their release approaches, unlike larger production hatcheries. Methods: We used acoustic telemetry to track the survival and behavior of 90 hatchery-reared Coho Salmon O. kisutch smolts, released from a community hatchery, through Burrard Inlet and into the Strait of Georgia. This is the first study to track Coho Salmon smolts released directly into a marine environment and one of very few studies to track them in an oceanic setting. Result: Smolt survival was lowest through the first 3 km of the migration at 37%, and the estimated cumulative survival to the final array (similar to 20 km from release) was 10-23%. The presence of numerous predators in the region suggests that predation is a possible explanation for poor survival over the relatively short migration distance. Travel rates ranged from similar to 4 to 18 km/day, depending on the migration segment, which is slower than the marine migration of smolts from other species, likely increasing exposure to predators. However, we found potential evidence of "predator swamping," as survival estimates across migration segments were highest for the final of our three release groups and poorest for the first release group, with intergroup survival estimates increasing by 7-12% in the final three migration segments. Releasing smolts at high densities and at night, as our partner hatchery currently does, likely improves smolt survival. Conclusion: Our work demonstrates how acoustic telemetry can be used to examine hatchery practices and propose potential improvements. Saltwater readiness and the release location are factors that our results suggest may warrant future attention to potentially improve marine survival of Coho Salmon smolts.

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

Background Double- and single-strand DNA breaks (DSBs and SSBs, respectively) in spermatozoa, which emerge from intrinsic and extrinsic degenerative processes, are likely related to the underlying male pathology. Aim To determine whether the incidence of DSBs in the human ejaculate is a consistent predictor of whole sperm DNA fragmentation (W-SDF = SSBs + DSBs). Methods A correlation between the proportion of spermatozoa that showed whole W-SDF and those displaying only DSBs in DNA. Two patient cohorts were established: W-SDF ≤30% (low SDF; n = 153) and W-SDF ≥30% (high SDF; n = 222). Results An increasing level of W-SDF is associated with an increased incidence of DSBs in the ejaculate. When data from both the low and high W-SDF groups were combined, a linear relationship was observed, with DSBs increasing by 0.799 units for each unit increase in W-SDF. However, when the cohorts were analyzed separately, the relationships differed. In the low SDF group, DSBs increased linearly by 0.559 units for each unit increase in W-SDF. In the high SDF group, DSBs increased exponentially by 0.602 units per unit of W-SDF. Furthermore, the data dispersion between the two variables was significantly different between the cohorts, with the high SDF group showing 0.8 times greater variability than the low SDF group. Conclusions While the presence of DSBs in sperm is correlated with the W-SDF present in raw semen samples, the biological mechanisms responsible for DSBs are expressed in different proportions and/or at different levels in ejaculates with higher levels of DNA damage.

Although monotremes diverged from the therian mammal lineage approximately 187 million years ago, they retain various plesiomorphic and/or reptilian-like anatomical and physiological characteristics. This study examined the morphology of juvenile and adult female reproductive tracts across various stages of the presumptive oestrous cycle, collected opportunistically from cadaver specimens submitted to wildlife hospitals during the breeding season. In adult females, ovaries had a convoluted cortex with follicles protruding from the ovarian surface. While protruding antral follicles were absent from the ovaries of juvenile echidnas, histological analysis identified early developing primordial and primary follicles embedded into the ovarian cortex. The infundibulum epithelial cells of the oviducts were secretory during the follicular phase but not at other stages, the ampulla region was secretory at all stages and is likely responsible for the mucoid layer deposited around the zona pellucida, and the isthmus region of the oviduct appeared to be responsible for initial deposition of the shell coat, as in marsupials. Female echidnas have two separate uteri, which never merge and enter separately into the urogenital sinus (UGS). This study confirmed that both uteri are functional and increase in glandular activity during the luteal phase. In the juvenile uteri, the endometrium was immature with minimal, small uterine glands. A muscular cervical region at the caudal extremity of each uterus, just before the cranial region of the UGS was defined by the absence of glandular tissue in all female echidnas, including the juveniles. There was no evidence of a definitive vaginal region. A clitoris was also detected that possessed a less developed but similar structural (homologous) anatomy to the male penis; urethral ducts while present did not appear to be patent.

Objective:To determine the relationship between teratozoospermia and sperm DNA fragmentation(SDF)in the human ejaculate. Methods:This retrospective study included 100 normozoospermic men as a control cohort(abnormal forms>14%),210 patients with a high level of abnormal forms(≤4%)and 65 patients presenting with a moderate level of abnormal forms(>4%to≤14%)based on the World Health Organization definitions.Sperm morphology was assessed using bright field microscopy.Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay.Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation;receiver operating characteristic(ROC)analyses were conducted to assess sensitivity and specificity of this relationship. Results:A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate,the higher the level of SDF(Spearman's Rho =-0.230;P<0.001).Significant differences in the proportion of SDF were found when all cohorts were compared(P<0.001);these significant differences were also retained when the different cohorts were compared pairwise.ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia. Conclusions:Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF,there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.

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.

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.

It is possible that the reproductive strategy of the short-beaked echidna is related to seasonal changes in fat deposition and energy availability, regulated by seasonal changes in endocrine function. We predicted that circulating leptin levels would be directly proportional to adiposity during most of the year, but that a change in this relationship would occur during the pre-breeding season to allow increased fat deposition. To test this hypothesis, we made use of a captive colony of echidnas to describe and quantify changes in fat distribution and the adipostatic hormone leptin. First we assessed seasonal changes in circulating leptin levels, body mass and adiposity for three male and three female adult echidnas maintained on a standard diet. Second, we explored the relationship between circulating leptin levels and increased caloric intake for an additional five adult female echidnas that were provided with supplemented nutrition. Third we visualised fat distribution in male and female adult echidnas using magnetic resonance imaging (MRI) before and after the breeding season, to determine where fat is deposited in this species. For echidnas maintained on the standard diet, there were no seasonal changes in body mass, body fat or plasma leptin levels. However, female echidnas provided with supplemented nutrition had significantly elevated plasma leptin levels during the breeding season, compared to the pre-and post- breeding periods. MRI showed substantial subcutaneous fat depots extending dorso-laterally from the base of the skull to the base of the tail, in both sexes. Pre-breeding season, both sexes had considerable fat deposition in the pelvic/rump region, whilst the female echidna accumulated most fat in the abdominal region. This study shows that male and female echidnas accumulate body fat in the pelvic/rump and the abdominal regions, respectively and that circulating leptin may promote fattening in female echidnas during the breeding season by means of leptin resistance. However, further research is required to evaluate the precise relationship between seasonal changes in leptin and adiposity.

Assisted breeding technology has important management implications for avian conservation and aviculture but species-specific reproductive information is lacking.This study aimed to develop a successful semen collection for the king quail (Synoicus chinesis) in order to establish baseline seminal characteristics and to explore the effect of flock density on seminal quality.A total of 19 quail consisting of 2 groups of 5 and 4 and 1 group of 10 were housed in 3 separate aviaries located at the University of Queensland, Gatton, Australia during Spring. Morphometrics and morphology (normal and abnormal) of the sperm cells were also described by phase contrast and differential interference microscopy.Semen was sucessfully collected by cloacal massage from 90% of attempts with only minor evidence of faecal or urate contamination. No significant differences in seminal characteristics were found with respect to bird housing density. Mean ± SEM seminal characteristics for 103 ejaculates included volume (1.6 ± 0.02 μL), sperm concentration (8.1 ± 0.5 × 109 mL–1), progressive sperm motility (50.0 ± 0.04%), rate of sperm motility, (2.9 ± 0.13), pH (6.7 ± 0.2), the percentage of live sperm (88.0 ± 0.01%) and the percentage of total abnormal sperm morphology (21.0 ± 0.03%).Cloacal massage proved to be a highly successful technique to collect king quail semen and the seminal characteristics reported here are the future basis of assessing fertility and the application of assisted breeding in the species.

Although many coho salmon (Oncorhynchus kisutch) caught in recreational fisheries are harvested, a substantial number of salmon are released after capture. Mortality rates of coho salmon released from recreational fisheries are largely unknown in the marine environment. This two-year study investigated factors associated with postrelease mortality and travel rate of coho salmon in a marine recreational fishery in British Columbia, Canada. Adult coho salmon were recreationally angled in the marine environment, affixed with acoustic tags, and tracked during their return migration to natal spawning streams using a network of acoustic receivers. We found postrelease mortality to the first point of detection (similar to 50 km from release) was 31.5% (95% CI: 26.1 - 37.4%; n = 279). Scale loss, eye damage, bleeding, and smaller body size of coho salmon were associated with increased odds of mortality. Scale loss and smaller body size were also associated with slower migration rate post-release. Air exposure up to five minutes was not found to be a driving factor in mortality or travel rate. These fishingrelated injuries can cause immediate physiological and behavioural disturbances, increase vulnerability to predation, infection, and disease, and delay migration as the fish recovers. Smaller coho salmon may also be less capable of overcoming capture stress. Our study highlights the importance of quantifying mortality of wild fish in their natural environment, and we suggest that small changes to fishing practices (e.g., smaller hook sizes, less handling, etc.) could make large differences in release survival thus encouraging a more sustainable recreational fishery.

The use of samples or scats to provide important ecological, genetic, disease and physiology details on free-range populations is gaining popularity as an alternative non-invasive methodology. Koala populations in SE Queensland and NSW have recently been listed as endangered and continue to face anthropomorphic and stochastic environmental impacts that could potentially lead to their extinction. This scoping review examines the current and potential utility of the koala scat to contribute data relevant to the assessment of koala conservation status and decision making. Although we demonstrate that there is great potential for this methodology in providing details for both individual wild animal and population biology (distribution, abundance, sex ratio, immigration/emigration, genetic diversity, evolutionary significant unit, disease epidemiology, nutrition, reproductive status and stress physiology), the calibre of this information is likely to be a function of the quality of the scat that is sampled.

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.

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.