Nitrogen excretion during marsupial development in the terrestrial isopod Armadillidium vulgare.

Marsupial embryos of Armadillidium vulgare (Isopoda: Oniscidea) were collected at different stages of development and assayed for products of nitrogen excretion. Stages were classified as early stage one, late stage one (clear embryo and somite differentiation), early stage two (chorion shed, prior to blastokinesis), late stage two (following blastokinesis), and mancae (vitelline membrane shed; second embryonic molt). Stage one and stage two embryos were primarily ammonotelic. Mancae showed a significant increase in stored uric acid and decrease in ammonia production, in most cases to undetectable levels. The increased metabolic rate of mancae, and the fact that they imbibe marsupial fluid prior to exiting the marsupium, may have favored a switch from ammonotely to uricotely to avoid ammonia toxicity. Protein metabolism, estimated from ammonia production, accounted for 7% of the measured catabolic rate in Stage 2 embryos. Newly emerged juveniles showed a >2-fold increase in metabolism relative to mancae, accompanying the transition from aquatic to aerial respiration. Following 48 h post-emergence, juveniles resumed ammonia excretion, volatilizing the base (NH3) as in later instars. Elevated ammonia excretion in early juveniles may derive from the catabolism of remaining yolk protein. A sharp increase in whole-animal glutamine in juveniles is consistent with its role as an intermediary nitrogen store during periodic ammonia excretion. Total ammonia concentration in the marsupial fluid fluctuated but did not increase significantly over time and ammonia was not volatilized across the oostegites, indicating that embryo ammonia is transported into the maternal hemolymph for excretion.

Behavioral and endocrine responses to season and social dynamics of captive male southern hairy-nosed wombats (Lasiorhinus latifrons).

Although southern hairy-nosed wombats (SHN wombats; Lasiorhinus latifrons) rarely breed in captivity, further knowledge of their reproductive physiology and behavior is likely to improve their breeding potential. This study examined the effect of seasonal variation and changes in social dynamics on the physiology and behavior of a captive population of male SHN wombats (n = 6). Seasonal changes in urinary testosterone metabolites (UTM), urinary cortisol metabolites (UCM), qualitative estimates of spermatorrhoea (QS), aggressive behavior and reproductive behavior were measured over an 11-month period. While there was no effect of month on QS (GLM ANOVA, P = 0.27), reproductive behavior (GLM ANOVA, P = 0.19) or aggressive behavior (Tukey pairwise comparisons), the secretion of UTM (GLM ANOVA, P = 0.051) was only marginally affected by season, compared to that reported for wild male SHN wombats. Mean UCM concentrations of July and August 2016 were significantly higher than those between October 2015 and January 2016 (Tukey pairwise comparisons). To examine social dynamics, two trials of animal positioning exchange with the enclosure system were implemented and behavioral data were examined for each trial over a six week period; UTM, UCM and general behaviors (n = 27) were measured for each trial. Neither UTM nor UCM concentration varied significantly (P ≥ 0.45) before and after the exchanges. "Scratching" decreased at the group level following the animal exchange in both trials, suggesting reduction in self-grooming may be a behavioral response to novel stimuli. UCM and UTM concentrations were both positively correlated with "standing still" and "body rub" behaviors. This may be evidence of a hormonal control of a "freezing behavioral response" to external stimuli and marking behavior, respectively. As there was no evidence that changing the social dynamics affected reproductive or agonistic behavior or hormone concentrations, it was concluded that captive male wombats in this study showed reduced reproductive seasonality compared to wild wombats and that animal exchange resulted in a behavioral response to novel stimuli but was not sufficient to affect testosterone or cortisol secretion, within the context of our study.

Measurement of testosterone and cortisol metabolites and luteinising hormone in captive southern hairy-nosed wombat (Lasiorhinus latifrons) urine.

This study reports the validation and use of enzyme immunoassays (EIA) to measure changes in plasma and urinary luteinizing hormone, testosterone metabolites (UTM) and cortisol metabolites (UCM) in captive southern hairy-nosed wombats (Lasiorhinus latifrons). GnRH agonist and ACTH agonist challenges were conducted to validate urinary testosterone (male wombat only) and cortisol (male and female wombats) EIAs. Following intra-muscular injection of 8-12µg buserelin (n=4 males), there was a significant increase in both plasma (P<0.001) and urinary testosterone concentrations (P<0.001) 60min and 21h after administration, respectively. Plasma LH levels were elevated (p<0.05) at 20min but there was no significant increase found in urinary LH concentrations after injection. Intra-muscular injection of Synacthen® Depot (250µg) (n=3 males, 3 females) resulted in a significant increase (p<0.05) in plasma cortisol secretion 15min and in urinary cortisol concentrations 3h post injection, respectively. Sex-related differences in cortisol secretion were also reported in this study. These findings indicate that (1) urinary LH might not be an appropriate index for describing the reproductive status in captive male L. latifrons, and (2) the UTM and UCM assays appear to be suitable for the assessment of the testicular steroidogenic capacity and the adrenocortical activity in captive southern hairy-nosed wombats, respectively.

Non-invasive urine collection in the female southern hairy-nosed wombat (Lasiorhinus latifrons) with the aid of classical conditioning.

We propose that regular urine samples can be used to monitor and characterize the reproductive cycle of the wombat, but this approach has never before been attempted in a marsupial. We conducted a three stage conditioning process for non-invasive urine collection in captive female wombats, which included (1) initial habituation and observation of urination patterns; (2) classical association of a stimulus with urination and (3) urine collection with the classically-conditioned stimulus. Four of the five female wombats selected for this trial were successfully conditioned for urine collection. During stage 2, the animals urinated in response to tactile stimulation 96 times from 208 attempts (46%). In stage 3, urine was successfully collected 399 times from 485 attempts (82%), with the majority of samples being collected in the morning (280/388). Hand-raised females that were previously conditioned for toileting purposes as pouch young responded more rapidly to the stimulus than juvenile females with no prior conditioning. This study is the first description of a successful method of urine collection by classical conditioning in a marsupial.

Development of an immunoassay to measure progesterone using printed biosensors, and its application to the assessment of ovarian function in the numbat (Myrmecobius fasciatus).

A biosensor system was developed to measure progesterone levels in the urine of female numbats (Myrmecobius fasciata) as an index of ovarian function. Screen printed sensors were coated with a monoclonal progesterone antibody, and incubated in a mixture of sample/standard and progesterone-3-CMO-horseradish peroxidase (HRP). The difference in potential between the working and reference electrode was measured, after exposure to an HRP substrate. EIA and biosensor standard curves showed parallelism, and the biosensor gave values similar (r = 0.83) to the conventional EIA. Progesterone concentrations at different stages of the oestrus cycle were not significantly different to those obtained by EIA.

Ultraviolet properties of Australian mammal urine.

The exploitation of predator signals by potential prey is well researched, but relatively little is known about how predators exploit chemical cues (either deliberate signals or waste by-products) produced by their prey. In Finland, the urine of some small rodents ( Microtus spp. and Clethrionomys spp.) is reflective in the ultraviolet range of wavelengths, and diurnal raptors with ultraviolet vision use these urine marks to track their rodent prey. This study examines the potential for such a phenomenon in Australian systems by studying the ultraviolet properties of urine from 13 native and introduced mammal species that are variously preyed upon by raptors. Urine from all 13 species displayed various levels of ultraviolet absorbance in their urine and fluorescence in the ultraviolet range. However, no signs of ultraviolet hyper-reflectance were detected, suggesting that the urine of European voles have unique ultraviolet properties. Ultraviolet-sensitive predators in Australia may be able to distinguish between species based on variation in the ultraviolet absorbance of their urine, but ultraviolet properties did not differ between prey and non-prey species, nor marsupial and placental groups. Moreover, because many natural surfaces are ultraviolet absorbing, it is unlikely that raptors could rely upon the ultraviolet properties of urine to target key prey species.

Longitudinal gonadal steroid excretion in free-living male and female meerkats (Suricata suricatta).

Slender-tailed meerkats (Suricata suricatta) are small, diurnal, cooperatively breeding mongooses of the family Herpestidae. A prerequisite to fully understanding the mating system of meerkats is the development of a normative reproductive-endocrine database. This study examined longitudinal gonadal steroid excretion in all adult and juvenile individuals of both sexes within a social group of free-living meerkats sampled across an entire breeding season. The specific objectives of this study were to (1) validate noninvasive (fecal and urinary) gonadal steroid hormone monitoring techniques in male (testosterone) and female (estrogens, progestagens) meerkats; (2) test the feasibility of using these noninvasive methods under field conditions; (3) characterize the endocrine correlates associated with the female reproductive cycle, including estrus, gestation, and postpartum estrus; (4) examine longitudinal androgen excretion in males; and (5) determine whether social status (i.e., dominant versus subordinate) affected gonadal steroid excretion. In females, the results demonstrated the physiological validity of noninvasive monitoring in meerkats by corresponding excretory hormone concentrations to major reproductive events (i.e., estrous, pregnancy, parturition). Hormone excretory patterns during estrous intervals suggested possible mechanisms whereby reproductive suppression may operate in female meerkats. In males, androgen excretion did not correspond to changes in reproductive and aggressive behaviors, suggesting that dominance, and hence breeding access to females, was not regulated strictly by gonadal steroid production. The consistency in androgen excretion among male meerkats indicated that reproductive suppression may be mediated by behavioral (i.e., intermale aggression) rather than physiological (i.e., depressed spermatogenesis) mechanisms.

Clinical pathology and sample collection of exotic small mammals.

The clinical pathology of some of the less common and newly emerging small mammal species is detailed in this article. The species covered here include the chinchilla, prairie dog, African hedgehog, and sugar glider. Venipuncture sites and sampling techniques are discussed in general and for each species. Detailed information on the hematology and serum biochemistry values of these animals is presented in numerous tables. Specific information is also provided for urinalysis, fecal analysis, dermatologic sampling, and cytology.

A field lab method to determine urine concentration in small mammals.

1. The concentrations of 136 urine samples from four species of small mammals were compared using osmometry, refractometry and a colorimetric test for urea concentration. 2. To obtain a wide range of concentrations (430-3950 mOsm/kg), urine samples were collected under normal and dehydration conditions. 3. Regression analyses of paired values indicate that measurements of total solids concentration (refractometric method) permit evaluations of urine osmolality and estimations of the concentration of urea with a high degree of confidence.