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.

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.

Plasma progesterone secretion during gestation of the captive short-beaked echidna.

This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology was assessed three times a week. The pouch of the female echidna only develops during gestation and it was possible to create a four-stage grading system using the most distinguishable characteristics of pouch development. Maximum pouch development was associated with declining progesterone concentrations, with the pouch closing in a drawstring-like manner at oviposition. Control of pouch development in pregnant echidnas is not yet clear but later pouch development is associated with a decrease in progesterone and pouch closure and may be under mechanical influences of the egg or young in the pouch. The length of pregnancy was 16.7 ± 0.2 days with a 15.1 ± 1.0 days luteal phase followed by an incubation period in the pouch. Eggs could be detected in utero at least 4 days before oviposition. Plasma progesterone peaked at 10.5 ± 0.9 ng/mL within 12 days of mating but then declined to basal levels within 1 day of oviposition and remained basal throughout egg incubation, confirming that progesterone is elevated throughout pregnancy and that gestation does not extend beyond the luteal phase. After the loss of an egg or pouch young, most females entered a second oestrous cycle and ovulated, suggesting echidnas are seasonally polyoestrous. The duration of the luteal phase in the echidna corresponds with that observed in other mammals.

Blubber cortisol levels in humpback whales (Megaptera novaeangliae): A measure of physiological stress without effects from sampling.

Baleen whales are vulnerable to environmental impacts due to low fecundity, capital breeding strategies, and their reliance on a large amount of prey resources over large spatial scales. There has been growing interest in monitoring health and physiological stress in these species but, to date, few measures have been validated. The purpose of this study was to examine whether blubber cortisol could be used as a measure of physiological stress in humpback whales. Cortisol concentrations were initially compared between live, presumably 'healthy' whales (n = 187) and deceased whales (n = 35), which had died after stranding or entanglement, or washed ashore as a carcass. Deceased whales were found to have significantly higher cortisol levels (mean ± SD; 5.47 ± 4.52 ng/g) than live whales (0.51 ± 0.14 ng/g; p < 0.001), particularly for those animals that had experienced prolonged trauma (e.g. stranding) prior to death. Blubber cortisol levels in live whales were then examined for evidence of life history-related, seasonal, or sampling-related effects. Life history group and sampling-related factors, such as encounter time and the number of biopsy sampling attempts per animal, were found to be poor predictors of blubber cortisol levels in live whales. In contrast, blubber cortisol levels varied seasonally, with whales migrating north towards the breeding grounds in winter having significantly higher levels (0.54 ± 0.21 ng/g, p = 0.016) than those migrating south towards the feeding grounds in spring (0.48 ± 1.23 ng/g). These differences could be due to additional socio-physiological stress experienced by whales during peaks in breeding activity. Overall, blubber cortisol appears to be a suitable measure of chronic physiological stress in humpback whales.

Contraceptive efficacy and dose-response effects of the gonadotrophin-releasing hormone (GnRH) agonist deslorelin in Tasmanian devils (Sarcophilus harrisii).

Contraception is increasingly used to manage breeding opportunities in conservation-dependent species. This study aimed to determine the efficacy, duration of effect, optimal dose and potential side effects of Suprelorin contraceptive implants in Tasmanian devils, for use in the conservation breeding program. In our pilot study, Suprelorin was found to effectively suppress oestrous cycles in female devils, yet caused a paradoxical increase in testosterone in males. Therefore, we focussed on females in further trials. Females received one (n=5), two (n=5) or no (n=5) Suprelorin implants, with quarterly gonadotrophin-releasing hormone (GnRH) challenges used to test pituitary responsiveness over two breeding seasons. Both Suprelorin doses suppressed pituitary responsiveness for at least one breeding season, with a reduced effect in the second. There was a dose-response effect on duration rather than magnitude of effect, with high-dose devils remaining suppressed for longer than low-dose animals. There were no apparent negative effects on general health, yet captivity and contraception together may cause weight gain. Suprelorin contraceptive implants are now routinely used in the Save the Tasmanian Devil Program insurance metapopulation to meet the aims of maintaining genetic and behavioural integrity by controlling individual reproductive contributions in group housing situations.

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.

One size does not fit all: Monitoring faecal glucocorticoid metabolites in marsupials.

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125-4.5days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z⩾2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies.

Body fat and circulating leptin levels in the captive short-beaked echidna (Tachyglossus aculeatus).

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.

Investigating the Market Value of Brumbies (Equus caballus) in the Australian Riding Horse Market.

Feral horses, also known as brumbies, are widely distributed across Australia with some populations being managed largely by human intervention. Rehoming of suitable feral horses following passive trapping has wide community acceptance as a management tool. However, there is little information about the number and relative economic value of feral horses compared with cohorts in the riding horse market. We examined 15,404 advertisements of horses for sale in 53 editions of Horse Deals, published from February 2017 to July 2022. Despite the considerable media attention and public scrutiny surrounding feral horse management, rehomed feral horses represented only a tiny fraction of the horse market in the current study. Of the 15,404 advertisements examined, only 128 (0.0083%) were for feral horses. We recorded phrases used to describe behavioural characteristics and other variables. The following variables were found to be not independent: Ridden Status, Height, Age, Sex, Colour, and Warning terms/more work. Using descriptive statistics to describe basic features of the data, the average price for feral horses ($1408) was lower than that for domestic horses ($1790) with the maximum price for a domestic horse being nearly twice the maximum for a feral horse. Univariate analysis showed feral horses were over-represented among "Unbroken" horses and underrepresented among "Ridden", "Broodmare" and "Harness" horses compared with domestic bred horses (p < 0.001). Feral horses appeared over-represented at shorter heights, among younger age groups (3 years or younger and 3.1 to 6 years) (p < 0.001) and in the dilute colour category (p = 0.008). The multivariable mixed model on price revealed that for domestic horses, the highest estimated marginal mean price averaged across the colour categories was for ridden horses aged 6.1-10-year-old at $1657.04 (95% CI $1320.56-$2074.66). In contrast, for feral horses, the multivariable mixed model demonstrated the similar highest estimated marginal mean averaged was for green broken 3-6-year-old horses that have undergone foundation training under saddle at $2526.97 (95% CI $1505.63-$4208.27). Australian feral horses were valued differently tfromsimilar domestic horses in the recreational riding horse market and further research is warranted to determine appropriate target markets and boost the sustainability of rehoming as a feral horse management tool.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population.

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

Diagnosing pregnancy in free-ranging dugongs using fecal progesterone metabolite concentrations and body morphometrics: a population application.

Assessing reproductive status and monitoring reproductive rates is important in the effective management of vulnerable marine mammal species such as the dugong (Dugong dugon). Knowledge of the reproductive physiology of this species is limited, and determining reproductive parameters (e.g., sexual maturation, pregnancy, and reproductive senescence) has been restricted by a lack of non-lethal methods for assessing reproductive status in free-ranging individuals. The aim of this study was to develop a method to identify pregnant individuals in a wild dugong population. Using an enzymeimmunoassay, we quantified concentrations of fecal progesterone metabolites (fP) in 322 dugongs, including confirmed pregnant females (n=10), presumed non-pregnant adult females (n=25), juvenile females (n=24), subadult females (n=41), adult females of unknown pregnancy state (n=63), and males of all sizes (n=159). External body morphometrics of each dugong were measured, and confirmation of pregnancy in adult female dugongs was determined by ultrasonography or observation of subsequent neonates. Concentrations of fP were different between sexes and reproductive size classes (P<0.001), and ∼30-fold higher in confirmed pregnant dugongs (2017-7760 ng/g) compared to presumed non-pregnant females (30-221 ng/g), juvenile females (29-195 ng/g), and males (24-261 ng/g) (P<0.001). Body measures of maximum and anal girths, and teat length were all greater in confirmed pregnant females than presumed non-pregnant females (all P<0.05). We evaluated a Discriminant Function Analysis (DFA) to provide a model for predicting pregnant and non-pregnant dugongs. Cross-validated results showed that the DFA correctly classified 100% of pregnant and non-pregnant females using fP concentrations, body length, fineness ratio (an index of body shape), and teat length (a female reproductive trait). Using the DFA model, we classified the pregnancy status of all female dugongs and identified a total of 30 females as pregnant and 133 females as non-pregnant from the sampled population over the sample period. Pregnant dugongs in the Moreton Bay population are characterized by fecal progesterone metabolite concentrations > 1000 ng/g, body length ≥ 260 cm, maximum girth ≥ 215 cm, anal girth ≥ 126 cm, and teat length ≥ 5 cm long. In summary, analysis of fP concentrations in combination with body morphometrics may be used to diagnose pregnancy in free-ranging dugongs, and provides a new tool to monitor breeding rates of wild sirenian populations.

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.

Validation of an Enzyme Immunoassay to Measure Faecal Glucocorticoid Metabolites in Common Brushtail Possums (Trichosurus vulpecula) to Evaluate Responses to Rehabilitation.

Volunteer wildlife rehabilitators rescue and rehabilitate thousands of native animals every year in Australia. However, there is little known about how exposure to novel stimuli during rehabilitation could affect the physiology of wildlife. We investigated this question in a species that commonly enters rehabilitation, the common brushtail possum (Trichosurus vulpecula). We evaluated five enzyme immunoassays (EIA) to determine the most suitable for measuring faecal glucocorticoid metabolites (FGM) as a proxy for evaluating the response of brushtail possums to potential stressors during rehabilitation. An adrenocorticotrophin hormone (ACTH) challenge was conducted on wild-caught possums to determine the best-performing EIA based on the successful detection of FGM peaks in at least two of three possums. While a number of assays met these criteria, the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was selected as it had the largest amplitude of change in response to the ACTH challenge. This assay was then used to measure FGM concentrations in 20 possums during rehabilitation. There was high variation in baseline FGM concentrations and response to captivity between possums. Significant changes in FGM levels were detected in most possums during captivity, but were not reliably associated with potentially stressful events that were identified by rehabilitators. The probability of an FGM peak occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. Our study has demonstrated that injured and orphaned possums show changes in FGMs during captivity and rehabilitation and has identified events that can induce a physiological response in some individuals. We recommend that research now focus on the relationship between these responses during rehabilitation and pre- and post-release survival.

MHC diversity and female age underpin reproductive success in an Australian icon; the Tasmanian Devil.

Devil Facial Tumour Disease (DFTD), a highly contagious cancer, has decimated Tasmanian devil (Sarcophilus harrisii) numbers in the wild. To ensure its long-term survival, a captive breeding program was implemented but has not been as successful as envisaged at its launch in 2005. We therefore investigated the reproductive success of 65 captive devil pair combinations, of which 35 produced offspring (successful pairs) whereas the remaining 30 pairs, despite being observed mating, produced no offspring (unsuccessful pairs). The devils were screened at six MHC Class I-linked microsatellite loci. Our analyses revealed that younger females had a higher probability of being successful than older females. In the successful pairs we also observed a higher difference in total number of heterozygous loci, i.e. when one devil had a high total number of heterozygous loci, its partner had low numbers. Our results therefore suggest that devil reproductive success is subject to disruptive MHC selection, which to our knowledge has never been recorded in any vertebrate. In order to enhance the success of the captive breeding program the results from the present study show the importance of using young (2-year old) females as well as subjecting the devils to MHC genotyping.

Behavioral and Physiological Responses of Calves to Marshalling and Roping in a Simulated Rodeo Event.

Rodeos are public events at which stockpeople face tests of their ability to manage cattle and horses, some of which relate directly to rangeland cattle husbandry. One of these is calf roping, in which a calf released from a chute is pursued by a horse and rider, who lassoes, lifts and drops the calf to the ground and finally ties it around the legs. Measurements were made of behavior and stress responses of ten rodeo-naïve calves marshalled by a horse and rider, and ten rodeo-experienced calves that were roped. Naïve calves marshalled by a horse and rider traversed the arena slowly, whereas rodeo-experienced calves ran rapidly until roped. Each activity was repeated once after two hours. Blood samples taken before and after each activity demonstrated increased cortisol, epinephrine and nor-epinephrine in both groups. However, there was no evidence of a continued increase in stress hormones in either group by the start of the repeated activity, suggesting that the elevated stress hormones were not a response to a prolonged effect of the initial blood sampling. It is concluded that both the marshalling of calves naïve to the roping chute by stockpeople and the roping and dropping of experienced calves are stressful in a simulated rodeo calf roping event.

Cyclic changes in cortisol across the estrous cycle in parous and nulliparous Asian elephants.

In the context of reproduction, glucocorticoids (GCs) are generally considered to have negative effects. However, in well-studied model species, GCs fluctuate predictability across the estrous cycles, and short-term increases promote healthy ovarian function. Reproductive challenges have plagued captive elephant populations, which are not currently self-sustaining. Efforts to understand reproductive dysfunction in elephants have focused on the suppressive effects of cortisol, but the potential permissive or stimulatory effects of cortisol are unknown. In this study, we provide a detailed examination of cortisol patterns across the estrous cycle in Asian elephants (Elephas maximus). Time series analysis was used to analyze cortisol and progesterone data for a total of 73 cycles from eight females. We also compared cortisol profiles between females that successfully conceived and females that failed to conceive despite repeated mating attempts. Our results revealed that cortisol fluctuates predictably across the estrous cycle, with a peak during the second half of the follicular phase followed by low levels throughout the luteal phase. Furthermore, this pattern was significantly altered in nulliparous females; cortisol concentrations did not decline during the luteal phase to the same extent as in parous females. This study highlights the complexity of cortisol signaling and suggests future directions for understanding the role of cortisol in reproductive dysfunction.

Reproductive hormone monitoring of dugongs in captivity: detecting the onset of sexual maturity in a cryptic marine mammal.

Determining the reproductive status of long-term captive animals is essential because the onset of sexual maturity and reproductive activity may necessitate changes in husbandry requirements. This study reports on the first multi-year reproductive hormone monitoring program for captive dugongs of both sexes using feces. Fecal samples were collected from one male (Pig) over 9 years (4-13.2y of age; n=288 samples, 0.8±0.1 samples per week from July 2007 to February 2012) and one female (Wuru) over 7 years (from neonate to 6.9 y; n=171 samples, 0.5±0.1 samples per week from July 2007 to February 2012), and from one solitary female dugong (Gracie) over 10 months (10.5-11.3y of age; n=54 samples, 1.1±0.2 sample per week from September 2008 to June 2009). Using enzyme-immunoassay, fecal progesterone (fP) and estradiol-17ß (fE) concentrations were assayed in the two captive females, and testosterone (fT) concentration in the captive male, and compared these to concentrations in wild dugongs. Female Wuru exhibited increasing fP concentrations at 5+ y, indicating early onset of ovarian cycling typical of non-pregnant adult females. Female Gracie maintained basal fP concentrations consistent with wild immature dugongs, indicating that she had not reached puberty by 11y. Nutritional plane may account for differences in age at sexual maturity in these female dugongs. At age 3-4y, Wuru had fE concentrations 1.4 times greater than maximum concentrations recorded in all wild females, and these concentrations were coincident with a period of rapid weight gain. For the male Pig, increasing fT concentrations at 9y provided early indications of puberty. Pig's tusks erupted by 11y, and sexual maturity (indicated by spermatic semen) was confirmed by 12.8y. Identification of sexual maturation prompted two trials of a male contraceptive treatment using the GnRH agonist, deslorelin (9.4mg administered in 2010 and 15.6mg in 2011). Testosterone production was not significantly suppressed by these dosages, and treatment did not terminate sperm production at week 10-11 post-implantation, even at the larger dose tested. Routine analysis of fecal hormones was helpful for making reproductive management decisions regarding individual captives and in guiding the long-term captive management of this cryptic species.