A. Lèche

Physiological stress in captive Greater rheas (Rhea americana): Highly sensitive plasma corticosterone response to an ACTH challenge

Up to the present no studies have been conducted either on baseline concentrations of adrenal hormones or on hormonal responses to stress in Greater rhea (Rhea americana) and most ratite species. The aims of this work were to assess the presence of corticosterone in plasma of Greater rhea, to validate a corticosterone (125)I-radioimmunoassay for determining corticosterone levels in plasma samples and to study the activation of the adrenal gland after an adrenocorticotrophic hormone (ACTH) challenge. Six captive Greater rhea juveniles of 10 months of age received an intravenous ACTH injection. Blood samples were taken at 0min (baseline pre-ACTH levels), and post-injection at 15, 30, 60min and at 24 and 48h. The high pressure liquid chromatography (HPLC) analysis of pooled plasma showed that corticosterone is the glucocorticoid found in the plasma of Greater rhea. Biochemical assays of standard validation (e.g., parallelism, exogenous corticosterone recovery) showed that measurements of corticosterone present in the plasma of the Greater rhea provided by commercial corticosterone (125)I-radioimmunoassay were accurate and precise. ACTH challenge induced a more than 40-fold increase in plasma corticosterone at 60min post-ACTH (from 4.0 to 166.5ng/ml, on average). The corticosterone response to ACTH in Greater rhea was higher than is usual in birds, an apparently typical characteristic of ratites.

Corticosterone stress response of Greater Rhea (Rhea americana) during short-term road transport

The effect of transport stress on blood corticosterone levels in captive Greater Rheas was investigated. Twelve adult individuals (7 males; 5 females) were loaded in pairs inside wooden crates and transported along a paved road for 30 min. Blood samples were taken before the individuals were introduced into the crate (baseline value) and immediately after they were unloaded (30 min after capture). To assess whether corticosterone levels were affected by the blood sampling procedure per se, another 6 (nontransport) control birds (3 males; 3 females) were also captured and sampled at the same times as their transported counterparts. Plasma corticosterone concentrations were measured using a commercially available corticosterone (125)I radio-immunoassay kit. Baseline corticosterone levels were similar in the control and transported birds (9.0 ± 1.6 and 10.4 ± 0.8 ng/mL, respectively). Transportation induced a highly significant (P < 0.001), more than 40-fold increase in the corticosterone levels (433.6 ± 35.4 ng/mL) that was about 5 times higher (P < 0.001) than in their nontransported counterparts (88.4 ± 14.8 ng/mL). The present findings suggest that Greater Rhea is a species highly sensitive to stressful manipulations. Both blood sampling and transportation induced highly significant adrenocortical responses. Considering that transportation is one of the unavoidable common practices in the management of Greater Rheas and, as shown in the present study, that it induces a significant 40-fold corticosterone stress response, efforts should focus on helping to generate management transport standards for optimization of the welfare of this ratite.

Seasonal changes in plasma levels of sex hormones in the greater Rhea (Rhea americana), a South American Ratite with a complex mating system.

Seasonal rhythm in sex hormones has been extensively studied in birds, as well as its relationship with the type of mating system. The Greater Rhea (Rhea americana), a South American ratite species, reproduces seasonally and has a complex mating system: female-defense polygyny and sequential polyandry. The present study aimed at analyzing the endocrine basis of reproduction in this species and its relationship with its mating system. We used HPLC and electrochemiluminescence techniques to identify and measure plasma testosterone and estradiol levels. Annual oscillations in sex hormones, testosterone and estradiol, in adult males and females were observed. Lower levels of these hormones were exhibited during the non reproductive season (February to July), whereas their maximum values were reached in September for males and November-December for females. These fluctuations reflect the seasonal changes in gonadal function. By contrast, no significant sex hormones oscillations were observed in juvenile males and females (negative control of seasonal changes). Greater rheas maintain high testosterone and estradiol levels throughout the reproductive period. The high testosterone levels during incubation and chick rearing did not inhibit parental behavior in males, which appears not to conform to the “Challenge Hypothesis”. In females, the high estradiol levels throughout the reproductive season would be needed to sustain their long egg-laying period.

Influence of breeding season on fecal glucocorticoid levels in captive Greater Rhea (Rhea americana)

Sex hormones and stress-related changes can be seasonally influenced. We investigate whether fecal glucocorticoid metabolite (FGM) levels can differ between male and female captive Greater Rheas during the breeding and non-breeding seasons. Over a 3-year-period, fresh fecal samples from 10 individuals (five of each sex) were collected during the breeding months (October, November, and December) and non-breeding months (April and June). A total of 960 samples were assayed using a commercial radioimmunoassay. Results showed that FGM levels (mean ± SE) were affected by the breeding season in a sex-dependent way. Male Greater Rheas showed significantly higher FGM levels in the breeding months than in the non-breeding months (13.44 ± 0.37 vs. 7.92 ± 0.1 ng/g feces, respectively). By contrast, females did not show FGM seasonal changes throughout the same sampling periods (7.55 ± 0.14 vs. 7.26 ± 0.73 ng/g feces). Moreover, during the breeding season months, males showed higher average FGM levels than females (13.44 ± 0.37 vs. 7.55 ± 0.14 ng/g feces, respectively), and no differences were found between sexes during the non-breeding season (7.92 ± 0.1 vs. 7.26 ± 0.73 ng/g feces, respectively). Our findings suggest that male Greater Rheas have a higher adrenocortical activity during the breeding season, which is probably indirectly related to the increased testosterone levels and agonist interactions that are also observed during that phase. Studies aimed to determine the appropriate sex ratio for captive rearing should be developed to minimize male agonist encounters and therefore improve welfare of the captive group.

Efficiency of the cloacal sexing technique in greater rhea chicks (Rhea americana)

1. The feasibility and accuracy of the cloacal sexing technique in greater rhea chicks was assessed using chicks of two captive populations of greater rhea in Córdoba, Argentina. 2. A total of 46 greater rhea chicks of 2 to 3 months of age were randomly arranged into three groups and the members of each group were sexed by a different operator. 3. A feather of each chick was plucked for sexing through a molecular method and results were used as controls. 4. Sex was correctly assigned by cloacal inspection in 98% of the cases. Chick manipulation was easily performed and no infections or traumatic lesions were observed a posteriori. 5. Cloacal sexing of rhea chicks up to 3 months of age does not affect animal welfare and should be considered an efficient alternative to molecular methods.

State of the art knowledge in adrenocortical and behavioral responses to environmental challenges in a threatened South American ratite: Implications to in situ and ex-situ conservation

The Greater Rhea (Rhea americana) is an endemic ratite to South America, whose wild populations have undergone a remarkable decrease due to habitat degradation and fragmentation by the expansion of the agricultural frontier, poaching and predation by dogs. Anthropogenic perturbations in wild environments, as well as the management in captivity, can generate different stress responses in this species, thus, the monitoring of adrenocortical and behavioral activities are considered primary assessment tools with both conservation and welfare implications. In this review we analyze and integrate the different measurements of glucocorticoids (in plasma, feces, and yolk) carried out in different captive and wild populations, taking into account the diverse predictable and unpredictable conditions to which the Greater Rhea responds in each of those environments. In addition, the translocation of this bird is presented as an application of stress physiology in field ecology for conservation purposes, in which we evaluated how this species responds when it is released into a novel environment. Our results indicate that this ratite has a striking high sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis compared to that of other bird species and shows a wide variety of adrenocortical responses depending on the environment in which it lives. This suggests that its HPA axis has a phenotypic plasticity that enables the rhea to cope with the environmental challenges. In this sense, we propose that one of the routes of this plasticity could be mediated by the maternal transfer of steroid hormones to the egg. Finally, we discuss the importance of integrating the monitoring of the adrenocortical response along with the environmental variables that define the life history of the species, in management and conservation programs ex-situ and in situ.