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Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.

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This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

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The density-related permanence test (DRP) is used to classify young birds (while in groups) according to their individual permanence in proximity to either a high or low density of conspecifics (HD or LD, respectively). The birds' performance in DRP was associated with underlying differences in the social responses of their individuals. Quails in homogeneous groups of LD residents responded with less compact groups and higher levels of agonistic interactions to the presence of an intruder, and showed higher levels of agonistic interactions among cagemates than the homogeneous groups of HD birds. This study deepen the characterization of DRP evaluating whether contrasting behavior during test is associated with birds' stress responses to challenging situations during rearing. Plasma corticosterone responses were individually assessed after submitting DRP categorized birds to a brief (10 min) partial restraint (mechanical stressor; experiment 1), or after introducing them during 5 min as intruders in homogeneous groups of 6 unknown same category (HD or LD) conspecifics (social stressor; experiment 2). Experiment 1 showed that basal corticosterone levels were similar in all groups and the partial mechanical restraint induced an increase in plasma corticosterone concentration also in all groups. However, the increase induced by the stressor was higher in the LD birds than in their HD counterparts. In experiment 2, compared to controls, social stressor showed no plasma corticosterone changes in the intruders that were introduced in an unfamiliar group of HD conspecifics. However, intruders (both HD and LD) that were visiting the LD residents showed an increased corticosterone response compared to their control counterparts. Results suggest that categorization of birds in the DRP test could have relevance for selection programs oriented to obtain birds better suited to intensive rearing conditions that includes high density of animals and exposure to unavoidable stressors.

Assuntos

RESUMO

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

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Parrots kept in zoos and private households often develop psychological and behavioural disorders. Despite knowing that such disorders have a multifactorial aetiology and that chronic stress is involved, little is known about their development mainly due to a poor understanding of the parrots' physiology and the lack of validated methods to measure stress in these species. In birds, blood corticosterone concentrations provide information about adrenocortical activity. However, blood sampling techniques are difficult, highly invasive and inappropriate to investigate stressful situations and welfare conditions. Thus, a non-invasive method to measure steroid hormones is critically needed. Aiming to perform a physiological validation of a cortisone enzyme immunoassay (EIA) to measure glucocorticoid metabolites (GCM) in droppings of 24 Blue-fronted parrots (Amazona aestiva), two experiments were designed. During the experiments all droppings were collected at 3-h intervals. Initially, birds were sampled for 24 h (experiment 1) and one week later assigned to four different treatments (experiment 2): Control (undisturbed), Saline (0.2 mL of 0.9% NaCl IM), Dexamethasone (1 mg/kg IM) and Adrenocorticotropic hormone (ACTH; 25 IU IM). Treatments (always one week apart) were applied to all animals in a cross-over study design. A daily rhythm pattern in GCM excretion was detected but there were no sex differences (first experiment). Saline and dexamethasone treatments had no effect on GCM (not different from control concentrations). Following ACTH injection, GCM concentration increased about 13.1-fold (median) at the peak (after 3-9 h), and then dropped to pre-treatment concentrations. By a successful physiological validation, we demonstrated the suitability of the cortisone EIA to non-invasively monitor increased adrenocortical activity, and thus, stress in the Blue-fronted parrot. This method opens up new perspectives for investigating the connection between behavioural disorders and stress in this bird species, and could also help in their captive management.

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The study evaluated whether different short-term endocrine testicular and adrenocortical responses to short photoperiod exposure can persist over time and particularly when birds exhibit spontaneous cloacal gland recovery. At 11 wk of age, 33 male Japanese quail exposed to long photoperiod were switched to short photoperiod (8L:16D). Another group of males was kept under long photoperiod (n = 11; LD quail). After 5 wk of short photoperiod exposure, quail were classified as nonresponsive or responsive to short photoperiod, depending on whether the cloacal gland volume was above or below 1,000 mm(3) and with or without foam production, respectively. Since 11 wk of age and during a 20-wk period, droppings of all quail were collected to determine corticosterone and androgen metabolites (AM) by enzyme immunoassays. Cloacal gland volume was also determined weekly. Both short photoperiod nonresponsive (SD-NR) and responsive quail showed overall significantly lower (P < 0.01) AM values (518.8 ± 11.9 and 248.6 ± 17.1 ng/g, respectively) than quail that remained under long photoperiod (814.3 ± 24.1 ng/g). However, nonresponsive quail showed a significantly smaller reduction in their AM levels than their responsive counterparts. During the first 6 wk of short photoperiod exposure, SD-NR quail showed similar corticosterone metabolites values than LD quail. Corticosterone metabolite profiles changed from 7 wk of short photoperiod exposure onward, with photoperiodic differences (P < 0.01) persisting up to the end of study (LD: 228.9 ± 22.4 > SD-NR: 133.1 ± 15.5 > short photoperiod responsive: 61.6 ± 17.9 ng/g, respectively). Testicular and adrenocortical glands showed different degrees of activity associated with cloacal gland photoresponsiveness to short photoperiod manipulation. Our findings suggest long-term effects of short photoperiod, both in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenocortical axis activity of quail, including males that exhibited spontaneous cloacal gland recovery.

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PURPOSE: to evaluate the reactivity of VEGF-A and cleaved caspase-3 in the adrenal gland cortex of female pinealectomized rats treated with melatonin. METHODS: forty adult female rats were divided into 4 groups (G) of 10 animals: GI - no surgical intervention, with vehicle administration; GII - sham pinealectomized with vehicle administration; GIII - pinealectomized with vehicle administration; GIV - pinealectomized with melatonin administration (10 µg/animal) during the night. After 60 days of treatment, all animals were anesthetized, and the adrenal glands were removed and fixed in 10% formaldehyde (phosphate buffered) for histological processing and paraffin embedding. Sections (5 µm thick) were collected on silanized slides and submitted to imunnohistochemical methods for the detection of cleaved caspase-3 (apoptosis) and of vascular endothelial growth factor (VEGF-A) in the adrenal cortex. The data obtained were submitted to analysis of variance (ANOVA) complemented by the Tukey-Kramer test (p≤0.05). RESULTS: reactivity to cleaved Caspase-3 was noted in the zona glomerulosa of the adrenal glands in all studied groups. There were no significant differences in the zona glomerulosa; however, the zona fasciculata (15.51±3.12*, p≤0.05) and the zona reticularis (8.11±1.90*, p≤0.05) presented the smallest percentage of apoptosis in the pinealectomized group (GIII). The reactivity to the VEGF-A was stronger in the zona glomerulosa and weaker in the zona reticularis in all groups. We found a stronger VEGF-A reactivity in the zona fasciculata in the pinealectomized group (GIII). CONCLUSIONS: the pineal gland affects the arrangement of the zona glomerulosa and reticularis of the adrenal glands, which are related to the production of sex hormones.

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OBJETIVO: avaliar os efeitos da melatonina na apoptose e atividade celular no córtex da adrenal de ratas pinealectomizadas. MÉTODOS: foram utilizadas 40 ratas adultas, divididas randomicamente em 4 grupos com 10 animais cada: GCont - sem intervenção cirúrgica e com ministração do veículo; GSham - sem retirar a pineal com ministração do veículo; GExpV - pinealectomizado e tratado com veículo; GExpM - pinealectomizado e tratado com melatonina (10 µg/animal, por dia) durante a noite. Após 60 dias de tratamento, todos os animais foram submetidos à eutanásia, as adrenais retiradas, fixadas em formol a 10 por cento e processadas para inclusão em parafina. Nos cortes histológicos com 5 µm de espessura, foram realizados métodos imunoistoquímicos para detecção da apoptose (Caspase-3-clivada) e da atividade celular (fator de crescimento endotelial vascular, VEGF-A). Para avaliar a percentagem de apoptose foram contadas as células reativas em 1.000 células de cada zona da região cortical da adrenal em cada animal; já o VEGF-A foi expresso em escores. Os dados quantitativos foram analisados pela análise de variância (ANOVA) e pelo teste de comparações múltiplas de Tukey-Kramer (p<0,05). RESULTADOS: na avaliação do índice apoptótico, não foram encontradas diferenças significantes na zona glomerulosa entre os vários grupos estudados. Na zona fasciculada (GExpV=15,51±3,12*, p<0,05) e na reticulada (GExpV=8,11±1,90*, p<0,05) houve redução no índice apoptótico no grupo GExpV em relação aos demais grupos de estudo. Na reatividade ao VEGF-A, houve maior positividade, indicando maior atividade celular na zona na fasciculada do GExpV em relação aos outros grupos. CONCLUSÕES: a melatonina atua na região cortical da adrenal, em especial nas zonas fasciculada e reticulada, que estão relacionadas com a produção de esteroides sexuais.

PURPOSE: to evaluate the reactivity of VEGF-A and cleaved caspase-3 in the adrenal gland cortex of female pinealectomized rats treated with melatonin. METHODS: forty adult female rats were divided into 4 groups (G) of 10 animals: GI - no surgical intervention, with vehicle administration; GII - sham pinealectomized with vehicle administration; GIII - pinealectomized with vehicle administration; GIV - pinealectomized with melatonin administration (10 µg/animal) during the night. After 60 days of treatment, all animals were anesthetized, and the adrenal glands were removed and fixed in 10 percent formaldehyde (phosphate buffered) for histological processing and paraffin embedding. Sections (5 µm thick) were collected on silanized slides and submitted to imunnohistochemical methods for the detection of cleaved caspase-3 (apoptosis) and of vascular endothelial growth factor (VEGF-A) in the adrenal cortex. The data obtained were submitted to analysis of variance (ANOVA) complemented by the Tukey-Kramer test (p<0.05). RESULTS: reactivity to cleaved Caspase-3 was noted in the zona glomerulosa of the adrenal glands in all studied groups. There were no significant differences in the zona glomerulosa; however, the zona fasciculata (15.51±3.12*, p<0.05) and the zona reticularis (8.11±1.90*, p<0.05) presented the smallest percentage of apoptosis in the pinealectomized group (GIII). The reactivity to the VEGF-A was stronger in the zona glomerulosa and weaker in the zona reticularis in all groups. We found a stronger VEGF-A reactivity in the zona fasciculata in the pinealectomized group (GIII). CONCLUSIONS: the pineal gland affects the arrangement of the zona glomerulosa and reticularis of the adrenal glands, which are related to the production of sex hormones.

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Runway tests are considered indicative of underlying sociality in birds and their ability to make social discriminations and establish interactions among conspecifics. Herein, social reinstatement behavior in male juvenile Japanese quail selected for a reduced (LS, low stress) or exaggerated (HS, high stress) adrenocortical response to brief mechanical restraint was evaluated. Individual males were given the choice to reinstate with either 2 (low density, LD) or 8 (high density, HD) unfamiliar randombred conspecifics placed in goal boxes at opposite ends of a two-choice runway (TCRW). Then, the same males were individually retested in a single goal box runway wherein they were exposed to a goal box containing either a LD or a HD of males. In the TCRW, a higher (P < 0.01) number of HS males started their ambulation toward the goal box containing the HD as opposed to LD of conspecifics. The HS males also spent more (P < 0.01) time in close proximity (within a 10-cm close zone; CZ) to the HD (218 s) rather than LD (57 s) of conspecifics. In contrast, the LS males did not differ in their initial direction of travel and they spent similar average amounts of times in the CZ of their stimulus LD (141 s) and HD (124 s) conspecifics. Similar to the TCRW results, in the single goal box runway, HS males spent more (P < 0.01) time in the CZ of HD rather than LD conspecifics, whereas LS quail spent similar amounts of time in the CZ of LD and HD males. Considering that runways are novel (and therefore frightening) environments, our findings suggest that HS quail may find better shelter (i.e., more comfort) in close proximity to a larger rather than smaller group of conspecifics, whereas LS birds find groups of varying conspecific density equally attractive. The results suggest that LS quail possess favorable social adaptive qualities because they appear to be better suited to cope with situations where the density of conspecifics is variable.

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CONTEXT: In some patients, PCOS may develop as a consequence of an exaggerated adrenarche during pubertal development. OBJECTIVE: The aim of the study was to assess adrenal function during childhood and pubertal development in daughters of women with PCOS (PCOSd). DESIGN: We included 98 PCOSd [64 during childhood (ages 4-8 yr) and 34 during the peripubertal period (ages 9-13 yr)] and 51 daughters of control women (Cd) [30 during childhood and 21 during the peripubertal period]. In both groups, an acute ACTH-(1-24) stimulation test (0.25 mg) and an oral glucose tolerance test were performed. Bone age and serum concentrations of cortisol, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), glucose, and insulin were determined. RESULTS: PCOSd and Cd were similar in age and body mass index. During the peripubertal period, basal and poststimulated DHEAS concentrations were higher in PCOSd compared to Cd. Among PCOSd, 12.5% of girls in childhood and 32.4% in peripuberty presented biochemical evidence of exaggerated adrenarche. Stimulated insulin was higher in PCOSd compared to Cd during childhood (P = 0.03) and peripuberty (P = 0.03). An advancement of 8 months between bone and chronological age was observed in peripubertal PCOSd compared to Cd. CONCLUSIONS: In PCOSd, basal and stimulated DHEAS concentrations were higher during the onset of puberty. Around 30% of the PCOSd demonstrated an exacerbated adrenarche, which may reflect increased P450c17 activity. In addition, a modest advance in bone age was observed, probably secondary to the hyperinsulinemia and/or adrenal hyperandrogenism.

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