Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation.

The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9-GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 µg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.

Observations on spindly leg syndrome in a captive population of Andinobates geminisae.

Amphibian health problems of unknown cause limit the success of the growing number of captive breeding programs. Spindly leg syndrome (SLS) is one such disease, where affected individuals with underdeveloped limbs often require euthanization. We experimentally evaluated husbandry-related factors of SLS in a captive population of the critically endangered frog, Andinobates geminisae. SLS has been linked to tadpole nutrition, vitamin B deficiency, water filtration methods, and water quality, but few of these have been experimentally tested. We tested the effects of water filtration method and vitamin supplementation (2017) and the effects of tadpole husbandry protocol intensity (2018) on time to metamorphosis and the occurrence of SLS. We found that vitamin supplementation and reconstituted reverse osmosis filtration of tadpole rearing water significantly reduced SLS prevalence and that reduced tadpole husbandry delayed time to metamorphosis. A fortuitous accident in 2018 resulted in a decrease in the phosphate content of rearing water, which afforded us an additional opportunity to assess the influence of phosphate on calcium sequestration. We found that tadpoles that had more time to sequester calcium for ossification during development had decreased the prevalence of SLS. Taken together, our results suggest that the qualities of the water used to rear tadpoles plays an important role in the development of SLS. Specifically, filtration method, vitamin supplementation, and calcium availability of tadpole rearing water may play important roles. Focused experiments are still needed, but our findings provide important information for amphibian captive rearing programs affected by high SLS prevalence.

Spindly leg syndrome in Atelopus varius is linked to environmental calcium and phosphate availability.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

The relationship between spindly leg syndrome incidence and water composition, overfeeding, and diet in newly metamorphosed harlequin frogs (Atelopus spp.).

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

Evaluating group housing strategies for the ex-situ conservation of harlequin frogs (Atelopus spp.) using behavioral and physiological indicators.

We have established ex situ assurance colonies of two endangered Panamanian harlequin frogs, Atelopus certus and Atelopus glyphus, but observed that males fought with each other when housed as a group. Housing frogs individually eliminated this problem, but created space constraints. To evaluate the potential stress effects from aggressive interactions when grouping frogs, we housed male frogs in replicated groups of one, two, and eight. We measured aggressive behavioral interactions and fecal glucocorticoid metabolite (GC) concentrations as indicators of stress in each tank. In both small and large groups, frogs initially interacted aggressively, but aggressive interactions and fecal GCs declined significantly after the first 2 weeks of being housed together, reaching the lowest levels by week 4. We conclude that aggressive interactions in same-sex groups of captive Atelopus may initially cause stress, but the frogs become habituated within a few weeks and they can safely be housed in same-sex groups for longer periods of time.