Bone quality evaluation of experimental osteometabolic disease in Pantanal alligators (Caiman yacare) by High Resolution Computerized Microtomography (µCT) / Avaliação da qualidade óssea de doença osteometabólica experimental em jacarés do pantanal (Caiman yacare) por Microtomografia Computadorizada (µCT)

Computerized microtomography is the gold standard examination for the evaluation of the three-dimensional bone structure. This experiment was developed to evaluate the structure and bone quality of Caiman yacare with metabolic bone disease using high resolution computerized microtomography (μCT). The animals were distributed into four groups: G1 - hyperphosphatemic diet with sun exposure deprivation (n=4), G2 - hyperphosphatemic diet with sun exposure (n=4), G3 - balanced diet with sun exposure deprivation (n=4), and G4 - balanced diet with exposure to sunlight (n=4). The parameters for the trabecular bone (Trabecular Number, Trabecular Thickness, Trabecular Separation, Bone Pattern Factor, Fractal Dimension, Euler Number, Structural Model Index, Degree of Anisotropy, Eigenvalues 1, 2 and 3, and Centroides X, Y and Z), and cortical bone (Number of Closed Pores, Volume of Closed Pores, Surface of Closed Pores, Closed Porosity, Volume of Open Pores, Open Porosity and Total Porosity). The overall results showed that the structure and bone quality of group G3 and G4 were better than those of groups G1 and G2, and that the diet factor influenced more than the sun exposure factor. The computerized microtomography allowed to evaluate the quality of the cortical and trabecular bones of the Pantanal alligator tibia with osteometabolic disease. The diet and sun exposure factors influenced individually the results of the μCT parameters between the groups, demonstrating the functional and structural complexity. Thus, these parameters can contribute to the interpretation of the mechanical behavior of bones and correlate them with the risk of lesions and fractures associated with osteometabolic diseases.(AU)

Microtomografia computadorizada é o exame padrão-ouro para a avaliação da estrutura tridimensional do osso. Este estudo experimental foi desenvolvido para avaliar a estrutura e a qualidade óssea de jacarés-do-pantanal (Caiman yacare) com doença óssea metabólica utilizando a microtomografia computadorizada (μCT) de Alta Resolução. Os animais foram distribuídos em quatro grupos, G1 - dieta hiperfosfatêmica com privação de luz solar (n=4), G2 - dieta hiperfosfatêmica com exposição à luz solar (n=4), G3 - dieta balanceada com privação de luz solar (n=4) e G4 - dieta balanceada com exposição à luz solar (n=4). Avaliaram-se os parâmetros para o osso trabecular (Número de Trabéculas, Espessura Trabecular, Separação Trabecular, Fator do Padrão Ósseo, Dimensão Fractal, Número de Euler, Índice do Modelo Estrutural, Grau de Anisotropia, Autovalores 1, 2 e 3 e Centroides X, Y e Z) e osso cortical (Número de Poros Fechados, Volume dos Poros Fechados, Superfície de Poros Fechados, Porosidade Fechada, Volume de Poros Abertos, Porosidade Aberta e Porosidade Total). Os resultados gerais evidenciaram que a estrutura e a qualidade óssea dos grupos G3 e G4 foram superiores aos dos grupos G1 e G2, sendo que o fator dieta influenciou mais do que o fator exposição solar. A Microtomografia Computadorizada permitiu avaliar a qualidade dos ossos cortical e trabecular da tíbia de jacarés do pantanal com doença osteometabólica. Os fatores dieta e exposição solar influenciaram individualmente no resultado dos parâmetros do μCT entre os grupos, demonstrando a complexidade funcional e estrutural. Assim, esses parâmetros podem contribuir na interpretação do comportamento mecânico dos ossos e correlacioná-los com o risco de lesões e fraturas associadas às doenças osteometabólicas.(AU)

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure.

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

Environmental influence on yolk steroids in American alligators (Alligator mississippiensis).

The egg yolk serves as a significant source of maternally derived steroids that are available to the embryo during early development. Altered deposition of yolk steroids can change the developmental trajectory of the embryo and have long lasting or permanent consequences. Alligators from contaminated environments have shown significant reproductive and developmental dysfunction, and it is unclear if altered deposition of yolk steroids could be a contributing factor. Alligator eggs were collected from Lake Woodruff (a reference lake), Lake Apopka (a site of known agricultural contamination), and the Merritt Island National Wildlife Refuge (MINWR) (home of the Kennedy Space Center and a site of heavy metal contamination). The yolks of eggs at embryonic stages 12 (prior to sex determination) and 24 (post-sex determination) were evaluated for concentrations of progesterone, 17-beta estradiol, and testosterone. Yolk concentrations of progesterone were significantly lower at embryonic stage 12 in eggs from Lake Apopka and MINWR when compared to eggs from Lake Woodruff. Yolk concentrations of 17-beta estradiol were significantly lower at embryonic stage 12 in eggs from MINWR when compared to the other two sites. Reductions in yolk 17-beta estradiol concentrations from embryonic stage 12 to 24 were significantly attenuated in eggs from MINWR versus that of Lakes Woodruff and Apopka. This study suggests that altered deposition of yolk steroids, and possibly differential utilization by the embryo, could be a contributory mechanism in the reproductive and developmental abnormalities seen in alligators from contaminated locales.

Developmental effects of embryonic exposure to toxaphene in the American alligator (Alligator mississippiensis).

A variety of organochlorine pesticides have been shown to adversely affect embryonic development. A number of abnormalities have been documented in alligators (Alligator mississippiensis) from highly-contaminated Lake Apopka, FL, USA that are similar to the results of experimental studies exposing embryos to pesticides. In the current study, we exposed developing alligator embryos to varying concentrations of toxaphene, a broad-spectrum pesticide found in relatively high concentration in Lake Apopka alligator egg yolk. The toxaphene, dissolved in 50 microl of ethanol, was applied topically to the eggshell just prior to the sex-determining period of development. Shortly after hatching, we examined a number of morphological and physiological endpoints to determine the consequences of sub-lethal embryonic exposure to toxaphene. Our results indicate that toxaphene had little or no effect on the morphological endpoints examined including body mass (BM) and size, liver, thyroid, and gonad development. In addition, toxaphene failed to affect sexual differentiation, or in vitro thyroxin, testosterone (T), and estradiol production. However, male plasma T concentration was higher in animals treated with 10 and 0.01 microg toxaphene/kg (based on mean egg mass) than control males. Because in vitro T production was not different among control groups, we suggest the difference in plasma T could be due to differences in hypothalamic-pituitary stimulation of the gonad or hepatic steroid degradation. This study indicates that technical grade toxaphene, at the applied doses, does not induce the same developmental abnormalities associated with alligators living in Lake Apopka. Future studies should consider the effects of embryonic exposure to a mixture of chemicals, including toxaphene metabolites, on development in alligators to better evaluate the consequences of environmental contamination.

Ectromelia in Morelet's crocodile from Belize.

Two Morelet's crocodiles (Crocodylus moreletii) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatchling crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.

Ectromelia in Morelet's crocodile from Belize

Two Morelet's crocodiles (Crocodylus moreletti) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatching crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.(Au)