Introduction

Green iguanas (Iguana iguana) are lizards belonging to the Iguanidae family, which can vary from small to large, have arboreal habits, and have a predominantly herbivorous diet (Bauer and Bauer, 2014). The geographic distribution of these animals is primarily in tropical and subtropical regions of the American continent. Iguanas can be found in the wild, in zoos, in conservation centers, and in private collections as exotic pets (Bauer and Bauer, 2014).

When kept in captivity, iguanas are at risk of developing problems related to food and environmental management (Araújo et al., 2019; Salles and Boyer, 2021). One of the main problems in iguanas presented in veterinary practice is metabolic bone disease (MBD), which is usually diagnosed in lizards and testudines (Klaphake, 2010; Araújo et al., 2019; Salles and Boyer, 2021). In lizards, clinical signs include pliable mandibles, rounded skull, pathologic fractures, reluctance to move, and fibrous osteodystrophy of the long bones. In advanced cases, paresis, muscle tremors, and seizures may be present (Hedley, 2012; Bauer and Bauer, 2014; Salles and Boyer, 2021). MBD occurs mainly due to an unbalanced diet or insufficient exposure to ultraviolet rays, and it is characterized by an increase in bone resorption that can lead to osteopenia and osteoporosis (Klaphake, 2010; Araújo et al., 2019; Salles and Boyer, 2021).

Bone mineral density (BMD) in animals can be estimated through some imaging techniques, including dual emission x-ray absorptiometry (DEXA) (Zotti et al., 2004) and quantitative computed tomography (QCT) (Souza et al., 2018; Araújo et al., 2019; Woods et al., 2021), the two most frequently used methods that allow the diagnosis of demineralization in its initial stages. QCT is considered a highly accurate and effective technique for early detection of bone changes in humans and animals (Souza et al., 2018; Araújo et al., 2019; Woods et al., 2021). Studies have been carried out in several species of reptiles in attempts to establish reference intervals for bone density in free-ranging animals to enable the diagnosis of possible early changes in bone mineral metabolism (De Oliveira et al., 2012; Bonelli et al., 2013; Souza et al., 2018; Araújo et al., 2019). In green iguanas, only two studies report osteodensitometric evaluation, but they were carried out in iguanas living in in managed care (Zotti et al., 2004; Soroori et al., 2018). Therefore, the aim of this study was to obtain reference intervals for BMD using the QCT technique in healthy adult free-ranging green iguanas.

Case Series

A retrospective study was carried out on 10 free-ranging green iguanas, eight females and two males, from the Wild Animal Screening Center (CETRAS, Tangará), located in the city of Recife, Pernambuco, Brazil (8°03′14″S 34°52′51″W). All iguanas were rescued by CETRAS in urban areas close to conservation areas. The clinical and diagnostic imaging exams carried out were part of the CETRAS protocol to verify the health status of the animals to facilitate reintroduction procedures into the wild. During their brief stay in captivity at CETRAS (a period varying between 1 and 3 days), the iguanas were kept in a closed enclosure with bushes and access to sunlight and shaded areas, and were fed mainly with fruits and leaves.

Computed tomography (CT) studies were performed on 10 green iguanas. For this, a helical tomograph (Hi-Speed FXi single slice CT scanner, General Electric, Fairfield, CT, USA) was used, which was calibrated immediately before image acquisition. The tomographic images were acquired in transverse sections of 1 mm, craniocaudal direction, with a slice thickness of 1 mm, pitch 0.8, 120 kV, and auto mA, using a bone tissue reconstruction algorithm. The CT scan was performed without chemical restraint, with each iguana placed inside a plastic box (Fig. 1A). Immediately before the CT scans, vagal stimulation was induced by gently applying digital pressure to both eyes of the iguanas for a short period of time. In some cases, if the animal was restless or aggressive, physical restraint of the limbs was performed with adhesive tape (Fig. 1B). To acquire the images, a calibration phantom for quantitative computed tomography for bone tissue (“QCT phantom”) was used, which contained calibration objects with densities of 0, 100, and 200 mg/cm³ of calcium hydroxyapatite.

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After acquiring and digitizing the images, the spine was analyzed using primary images and multiplanar reconstruction (Figs. 2–3). For the quantitative analysis of the radiodensity of bone tissue, a region of interest (ROI) restricted to the trabecular bone of the central portion of each vertebral body of five thoracic vertebrae (T10–T14) was selected, with the attenuation values first obtained in Hounsfield units (HU). Radiographic attenuation values were also measured in the areas corresponding to the “water” (HUw) and “bone” (HUb) phantom, enabling correction and conversion into BMD values expressed in milligrams per cubic centimeter using the equation BMD = 200 HUt/HUb – HUw (Table 1). No eggs were observed in the coelomic cavity of the female iguanas during the tomography scans.

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Table 1.Individual values and measures of central tendency for sex, weight, and bone mineral density (BMD) of 10 free-ranging green iguanas (Iguana iguana) submitted for computed tomography.

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Measures of central tendency (mean ± standard deviation) were obtained from the BMD values of the iguanas. The Shapiro-Wilk test was used to evaluate the hypothesis of normal distribution of the sample. Information regarding the weight, length, sex, and BMD of the iguanas is described in Table 1. Venipuncture was performed in the ventral coccygeal vein and the blood count and biochemical analysis of serum levels of sodium, potassium, phosphorus, urea, ionized calcium, albumin, total protein, globulin, gamma-glutamyl transferase, aspartate aminotransferase, and alanine aminotransferase revealed no changes or signs of pre-existing disease.

Discussion

Radiographic examinations only allow a late diagnosis of demineralization, making an early diagnosis of changes in bone mineral metabolism impossible, meaning that many animals are only diagnosed when they are presented with pathological fractures (Hedley, 2012; Salles and Boyer, 2021). DEXA and QCT are considered noninvasive techniques capable of characterizing the initial stages of bone demineralization. Densitometric analyses by QCT have been the target of studies in humans and in domestic and wild animals (De Oliveira et al., 2012; Bonelli et al., 2013; Souza et al., 2018; Araújo et al., 2019; Woods et al., 2021), confirming the precision and clinical applicability of this technique. In animals, most studies are aimed at standardizing normal values for an early diagnosis of bone demineralization, and in reptiles normal values have already been described for boa constrictors (Boa constrictor) and green sea turtles (Chelonia mydas) (De Oliveira et al., 2012; Souza et al., 2018).

Another recent study on Amazonian turtles (Podocnemys expansa) comparatively analyzed healthy turtles and turtles with MBD (Araújo et al., 2019). QCT, unlike DEXA, has the advantage of allowing isolated analysis of cortical and trabecular bone, which contributes to the early diagnosis of changes in bone mineral metabolism (De Oliveira et al., 2012; Soroori et al., 2018; Araújo et al., 2019). Trabecular bone has a larger contact surface with drugs and hormones, making this bone more metabolically active and consequently more sensitive to processes leading to bone mass loss (Souza et al., 2018; Araújo et al., 2019).

In green iguanas, there are only two studies that performed densitometric analysis of the bone tissue, one using the QCT technique (Soroori et al., 2018), and the other using the DEXA technique (Zotti et al., 2004). Both studies analyzed iguanas bred in captivity, unlike the group of animals evaluated in this study, which was composed exclusively of free-ranging iguanas. Soroori et al. (2018) analyzed the mineral density of the trabecular bone in two green iguanas larger than 35 cm in length. However, the osteodensitometric values they reported (197.91 ± 18.45 mg/cm³) were significantly lower than those observed in our study of free-ranging iguanas. Several studies have documented a high incidence of MBD in reptiles in human care, which is generally due to inadequate management for long periods, especially with regard to nutritional and environmental management (Araújo et al., 2019; Salles and Boyer, 2021). Therefore, we believe that iguanas in human care, due to differences in their management and environmental conditions in comparison to their natural habitat, are not in the same condition as free-ranging iguanas in the wild. Consequently, the analysis of BMD in free-living iguanas shown in Table 1 may better represent the goal for osteodensitometric values for this species.

The tomographic examinations of the green iguanas in this study were carried out without chemical restraint. Tomographic examinations without chemical restraint have also been carried out successfully in other reptiles, allowing densitometric analysis (Souza et al., 2018; Araújo et al., 2019). The absence of chemical restraint made the examination faster and less invasive for the iguanas, and allowed the procedure to be safe for patients and the team involved in the examination. Physical restraint of the iguanas allowed for good positioning for densitometric analyses, without causing injuries such as fractures or dislocation. Despite the apparent risk of osteoarticular injury, this same restraint methodology is routinely performed by the CETRAS team, and no injuries have ever been reported. Despite this, caution is suggested with this type of restraint, especially in cases where the iguanas are aggressive or restless.

Another important aspect of the methodology is the use of a phantom for osteodensitometry, which enhances the precision of the results. This approach is crucial because multiple variables—such as kV, mA, reconstruction algorithms, and slice thickness—can affect the degree of radiographic attenuation between exams, even when using the same tomographic techniques (Bonelli et al., 2013; Souza et al., 2018; Araújo et al., 2019).

This study has some limitations. Because of the inclusion of only 10 iguanas and the disproportionate number of males and females (eight females and two males), no statistical analysis of effects of sex was possible. Another aspect to consider is that, despite being considered adults, the precise age of each specimen analyzed was unknown. We did not analyze sexual hormones or reproductive status, both of which can influence bone mineral metabolism in reptiles. On the other hand, the period in which the examinations were performed did not coincide with the reproductive phase, and it is important to note that the iguanas were apparently not gravid on CT.

To the authors’ knowledge, this is the first study to report the average BMD of the thoracic vertebrae trabecular bone in free-ranging healthy iguanas (624.12 ± 109.73 mg/cm³). These values may serve as reference standards for the species and aid in the early detection of bone mineral metabolism changes in captive-bred iguanas.