Sexual dimorphism in baseline urinary corticosterone metabolites and their association with body-condition indices in a peri-urban population of the common Asian toad (Duttaphrynus melanostictus).

Field endocrinology research through the quantification of glucocorticoids or stress hormones in free-living wildlife is crucial for assessing their physiological responses towards pervasive environmental changes. Urinary corticosterone metabolite (UCM) enzyme-immunoassay (EIA) has been validated for numerous amphibian species as a non-invasive measure of physiological stress. Body-condition indices (BCIs) have also been widely used in amphibians as an indirect measure of animal health. Field endocrinology research on amphibian species in Asia is limited. In this study, we validated a UCM EIA in a peri-urban sub-population of the common Asian toad (Duttaphrynus melanostictus) in Pune, Maharashtra, India. We determined the baseline levels of UCMs in male (n=39) and female (n=19) toads. Secondly, we used a standard capture handling protocol to quantify changes in UCMs during short-term captivity. We also determined BCIs in the male and female toads using Fulton's index (K) and residual condition index (RCI). The results showed that mean baseline levels of UCMs were significantly higher in male toads than in females. There was no significant change in mean levels of UCMs of males and females between capture and captivity (0-12h). This highlights plausible habituation of the species to the peri-urban environment. Associations between UCMs with BCIs (K and R) were positive in male toads but negative in females. In conclusion, our UCMs EIA can be applied with BCIs to assess health of the Asian toads. We also suggest that direct fitness parameters such as sperm and oocyte quality, reproductive ecology and immunocompetence measurements should be applied in combination with these conservation physiology tools to quantify the fitness consequences of pervasive environmental changes on native amphibians.

Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models.

The use of hormonally induced spermatozoa expressed in urine (HISu) is a valuable component of reproduction technologies for amphibians. Five protocols for sampling HISu from the European common frog (Rana temporaria) were compared: (1) pituitary extracts, (2) 0.12 µg g⁻¹ luteinising hormone-releasing hormone analogue (LHRHa), (3) 1.20 µg g⁻¹ LHRHa, (4) 11.7 IU g⁻¹ human chorionic gonadotrophin (hCG) and (5) 23.4 IU g⁻¹ hCG (g⁻¹ = per gram bodyweight). From 1 to 24h after administration we assessed the number and concentration of spermatozoa in spermic urine and in holding water, and in urine the percentage of motile spermatozoa and their progressive motility. The protocol using 1.20 µg g⁻¹ LHRHa gave the highest total sperm numbers (650 × 106) and the highest percentage (40%) of samples with sperm concentrations above 200 × 106 mL⁻¹. The percentage motility and progressive motility was similar from all protocols. Considerable amounts of spermatozoa were expressed by R. temporaria into their holding water. We tested hormonal priming and spermiation in the common toad (Bufo bufo) using 0.13 µg g⁻¹ LHRHa administered 24h before a final spermiating dose of 12.8 IU g⁻¹ hCG. No spermatozoa were expressed in holding water. Priming resulted in 35% more spermatozoa than without; however, there were no differences in sperm concentrations. Primed B. bufo produced spermatozoa with significantly higher percentage motility, but not progressive motility, membrane integrity, or abnormal spermatozoa than unprimed males.