Mortality rates attributable to nutritional and housing-induced disorders historically have been extremely high in captive tortoises.1–3 Information in several reports4–6 and the lack of evidence in necropsy surveys7,8 suggest that nutritional disorders such as calcium deficiencies and subsequent metabolic bone diseases are rare in wild chelonians. Although a considerable amount of evidence-based information has been obtained on reptile physiology-biology9–11 and management,12,13 practitioners often offer a vast number of recommendations to reptile owners that are based on anecdotal information because of the lack of specific data. In juvenile chelonians, inadequate diets or inappropriate husbandry conditions often result in mineral deficiencies.14 However, pathophysiologic processes are still unknown15,16 and are usually extrapolated from information for other species.14,17
Several authors5,18–21 have suggested that mineral deficiencies can lead to nutritional secondary hyperparathyroidism, a pathological condition in which calcium resorption from bones is promoted by the action of parathyroid hormone as a consequence of a chronic hypocalcemic status. Common predisposing dietary factors include insufficient calcium or vitamin D3 and an improper calcium-to-phosphorous ratio, whereas the most important predisposing husbandry factor is inadequate exposure to UV radiation in the range of 280 to 315 nm.14,21–24 Although the causal relationship between lack of exposure to UVB light, insufficient calcium intake or absorption, and metabolic bone disease needs further investigation, there is recent evidence that UVB light influences 25-hydroxyvitamin D3 concentrations in Hermann's tortoises (Testudo hermanni).25 Clinical signs of calcium deficiency in terrestrial chelonians include lethargy, anorexia, loss of rigidity of the shell,14,26 shell deformities, and pathological fractures.6,14,21
The highest calcium requirements in chelonians may be related to growth and reproductive needs,15,17 and diet can lead to variations in bone mineralization.15 For these reasons, dietary calcium supplementation is a common practice in tortoise husbandry,27 and daily provision of supplemental calcium to recently hatched chelonians is recommended by some authors.17,28–33 Because precise calcium requirements have not been determined for most reptiles, anecdotal extrapolations are usually made on the basis of results of studies on birds and domestic mammals and adapted to herbivorous chelonians on the basis of each clinician's personal experience.14,17
In contrast, some studies9,15,19,34 have suggested that dietary calcium supplementation may be unnecessary or even detrimental because it may induce metastatic calcinosis and pathological accumulation. In addition, excess vitamin D provided by owners can have toxic effects in reptiles,35,36 birds,37 and mammals,38–40 including humans.41,42
Hematologic variables, including total calcium, ionized calcium, and phosphorus concentrations, generally are used to assess mineral status in reptiles.43,44 Although several venipuncture techniques have been described,45–48 it can sometimes be challenging to obtain blood samples from recently hatched chelonians.49 In addition, depending on the size of the hatchlings and the volume of blood needed, collection of an adequate blood sample could be lethal for small chelonians.50
Dual-energy x-ray absorptiometry is considered the criterion-referenced standard for in vivo determination of bone density in humans and several animals.51–65 The DXA technique involves the use of 2 x-ray beams with different peak kilovoltages that allow the subtraction of the soft tissue component.57,65 Currently, there are few reports about the use of this technique in reptiles,52–55 and the authors are aware of only 2 reports15,56 of the use of DXA in chelonians.
Hermann's tortoises are herbivorous, mediumsized chelonians that are found commonly along the Mediterranean coasts of Europe.6,66 In the wild, the diet of Hermann's tortoises consists almost exclusively of leaves, flowers, and fruits, although roots, mushrooms, and mussels are also eaten occasionally.57,66,67 The objective of the study reported here was to evaluate the effects of dietary and environmental management on BMD in Hermann's tortoises during the first year after hatching.
Materials and Methods
Animals—Twenty-six Hermann's tortoises within 1 month after hatching were obtained from a breeder in September 2009. All tortoises were considered healthy on the basis of results of a physical examination. The owner gave informed consent to allow participation of the tortoises in the study. The study was performed in accordance with regulations established by directive 2010/63/EU of the European parliament.
Housing—The tortoises were allocated into 3 groups (randomization was performed via a table of random numbers without stratification). Groups 1 (n = 9 tortoises) and 2 (10) were housed indoors in two 79 × 59 × 16.8-cm plastic containers with paper bedding. A combination of a compact UVB fluorescent bulba (lighting source) and an infrared lampb (heating source) was used in both vivaria. The fluorescent bulb was placed diagonally with the end of the longitudinal axis at a height 21 cm from the floor of the vivarium. The infrared lamp pointed at the same area as that of the fluorescent bulb. At the start and end of the study, UV radiation in the range of 280 to 320 nm was measured with a digital portable radiometerc at various distances from the fluorescent bulb and infrared lamp.
The lamps were controlled by a timer and active for 14 h/d, which simulated the summer photoperiod at Mediterranean latitudes between 38°N and 45°N. A day-night temperature gradient was established by a digital thermostat located in the room; mean ± SD environmental temperatures were maintained at 22°C ± 1°C during the day and 19°C ± 1°C during the night. The temperature in the basking zone never exceeded 37°C.
Relative humidity was recorded with an analogue hygrometer and oscillated between 65% and 75%. Shelters were placed in both warm and cold zones of the vivaria. Water was provided ad libitum.
Group 3 (n = 7 tortoises) was housed in a seminatural, sunlight-exposed enclosure. The enclosure was in an area located within the geographic range of Hermann's tortoises.57 Group 3 tortoises naturally hibernated for 6 months (from the middle of October until the middle of March).
Study design—At the end of the 10-month study period, a complete physical examination, including measurement of body weight and assessment of eventual pyramidal growth of the central part of the carapace scutes, was performed on each tortoise. Quantitative evaluation of pyramidal growth via a published index16 was considered infeasible because of the size of the tortoises. Pyramidal growth was assessed on a binary (present or absent) categorical scale. Assessment of pyramidal growth was performed by an investigator (NDG) who had extensive experience; that investigator was not aware of the group assignment of each tortoise.
Diet—Groups 1 and 3 consumed naturally growing vegetation. To simulate natural conditions, tortoises of group 1 were fed plants collected daily within the immediate vicinity of the outdoor enclosure, whereas tortoises of group 3 foraged on the naturally growing vegetation within their enclosure. Thus, the dietary differences between groups 1 and 3 were minimal or nonexistent. The diets for these 2 groups consisted of dandelions (Taraxacum officinale), clover (Trifolium spp), mallow (Malva spp), ribwort plantain (Plantago lanceolata), wood sorrels (Oxalis spp), and creeping cinquefoil (Potentilla reptans).
Group 2 was fed a diet composed of 50% chicory (Cichorium intybus), 35% red radish (Cichorium intybus var foliosum), 7.5% endive (Cichorium endivia var crispum), and 7.5% escarole (Cichorium endivia var latifolium).33 The vegetables were purchased at a market and were intended for human consumption.
No supplemental minerals were provided to any of the groups. The tortoises were fed ad libitum; food was provided at 8 AM each day.
DXA—The BMD of the shell, AAS, vertebral column, and pelvis were measured via DXA at the end of the study (Figure 1). Investigators were not aware of the group assignment of each tortoise. During DXA scans, the tortoises were positioned in sternal recumbency and immobilized by affixing their limbs and head in a retracted position with tape, as described elsewhere.15 A small animal densitometerd consisted of a cone-beam x-ray source that could generate energies of 35 and 80 keV and a flat 100 × 80-mm detector; it had a high spatial resolution (pixel size, 0.18 × 0.18 mm), which allowed accurate measurement of BMD in small rodents.59,68 The acquisition of DXA images required approximately 5 min/tortoise. Results were normalized on the basis of BSA and calculated in accordance with the following equation58: BSA (in m2) = 0.007184 × body weight (in kg)0.425 × straight shell length (in cm)0.725.
Statistical analysis—The primary outcome was BMD for each of the 3 groups. Secondary outcomes were body weight and frequency of pyramidal growth. Data were expressed as mean ± SD and minimum and maximum values. The BMD values were normalized on the basis of body size as recommended in clinical practice.69 Significance was determined via the paired nonparametric Kruskal-Wallis test or the Mann-Whitney 17 test for intergroup comparisons. Variations in the frequency of pyramidal growth among the 3 groups were tested with the Freeman-Halton extension of the Fisher exact test. Values of P < 0.017 (Bonferroni-Dunn correction) were considered significant. All statistical analyses were performed with appropriate statistical programs.e,f
Results
The UV radiation in the range of 280 to 320 nm was measured with a digital portable radiometer at various distances from the fluorescent bulb and infrared lamp at the start and end of the study (Table 1). Both the bulb and lamp had decreases in UVB radiation by the end of the 10-month study.
The UVB emission of a fluorescent bulb and infrared lamp positioned in the vivaria of Hermann's tortoises (Testudo hermanni) at the start and end of a 10-month study.
Distance (cm)* | ||||||
---|---|---|---|---|---|---|
Light structure | Time of study | 10 | 15 | 20 | 25 | 30 |
Fluorescent bulb | Start | 82 | 54 | 43 | 33 | 25 |
End | 62 | 32 | 20 | 13 | 9 | |
Infrared lamp | Start | 86 | 55 | 43 | 32 | 24 |
End | 63 | 31 | 18 | 13 | 9 |
Values reported are μW/cm2.
Distance between a digital portable radiometer and the fluorescent bulb or infrared lamp.
Body weights of tortoises recorded at the beginning and end of the study were reported as the mean ± SD, minimum value, and maximum value (Table 2). Body weights differed significantly (P = 0.016) between groups 2 and 3.
Body weight (grams) of Hermann's tortoises at the start and end of a 10-month randomized controlled study.
Start | End | |||
---|---|---|---|---|
Group* | Mean ± SD | Range | Mean ± SD | Range |
1 (n = 9) | 8.9 ± 1.1 | 7.0–10.5 | 51.1 ± 22.9 | 15.4–79.7 |
2 (n = 10) | 11.4 ± 1.06 | 8.0–11.5 | 65.4 ± 30.9 | 30.7–123.0 |
3(n = 7) | 9.2 ± 0.7 | 8.0–10 | 35.7 ± 7.1 | 27.4–44.8 |
Group 1 was housed in an artificial setting and fed naturally growing vegetation, group 2 was housed in an artificial setting and fed vegetables intended for human consumption, and group 3 was maintained in an outside enclosure and fed naturally growing vegetation.
Mean ± SD BMD of the shell was 0.05 ± 0.03 g/cm2•m for group 1 and 0.09 ± 0.15 g/cm2•m for group 2; mineralization of the shell could not be detected with this technique for group 3. Analysis of the data revealed that mineralization of the shell differed significantly (P < 0.001) among the 3 groups. Intergroup comparisons of mineralization of the shell revealed significant differences between groups 1 and 3 (P < 0.001) and between groups 2 and 3 (P = 0.001).
Mean BMD of the AAS was 0.076 ± 0.22 g/cm2•m for group 1, 0.08 ± 0.33 g/cm2•m for group 2, and 0.09 ± 0.15 g/cm2•m for group 3. Mean BMD of the thoracolumbar vertebrae was 0.08 ± 0.02 g/cm2•m for group 1, 0.10 ± 0.06 g/cm2•m for group 2, and 0.06 ± 0.01 g/cm2•m for group 3. Mean BMD of the pelvis was 0.05 ± 0.01 g/cm2•m for group 1, 0.05 ± 0.02 g/cm2•m for group 2, and 0.05 ± 0.01 g/cm2•m for group 3. No significant differences were found among groups for BMD of the AAS, thoracolumbar vertebrae, and pelvis (Table 3).
The BMD values at the end of a 10-month randomized controlled study conducted to evaluate the effect of management condition and diet on Hermann's tortoises.
Group* | Region | BMD (g/cm2•m) |
---|---|---|
1 | AAS | 0.08 ± 0.22 |
Vertebral column | 0.08 ± 0.02 | |
Pelvis | 0.05 ± 0.01 | |
Shell† | 0.05 ± 0.03 | |
2 | AAS | 0.08 ± 0.33 |
Vertebral column | 0.10 ± 0.06 | |
Pelvis | 0.05 ± 0.02 | |
Shell† | 0.09 ± 0.15 | |
3 | AAS | 0.09 ± 0.15 |
Vertebral column | 0.06 ± 0.01 | |
Pelvis | 0.05 ± 0.01 | |
Shell† | 0 |
Values for this variable differed significantly (P < 0.001) among groups.
See Table 2 for remainder of key.
Physical examination at the end of the study revealed the presence of pyramidal growth in 10 of 10 tortoises of group 1, 2 of 9 tortoises of group 2, and 0 of 7 tortoises of group 3. Frequency of pyramidal growth differed significantly (P < 0.001) among groups.
Discussion
The DXA analysis indicated that in the 2 groups of Hermann's tortoises maintained in captivity in similar microclimatic conditions, the 2 diets did not induce significant differences in mineralization of the shell or AAS. In contrast, the groups housed in captivity had higher BMD values than did the control group housed in a natural setting. This finding supported the contention that tortoises raised in adequate captive conditions do not develop calcium deficiency and do not require supplemental calcium. Furthermore, because no calcium deficiency was detected during the first year after hatching when there is massive growth in tortoises, it appears unlikely that deficiencies would develop later.
To our knowledge, few studies have been conducted to explore provision of supplemental calcium to chelonians. Investigators of 1 study15 suggested that the calcium and phosphorus content of most of the vegetables and fruits that comprise a conventional diet for captive herbivorous reptiles is lower than that of the plants these animals naturally eat in the wild. Those authors found that leopard tortoises receiving the recommended amount of supplemental calcium (2.9 g/kg of fresh weight of prepared food) had calcium deficiency in the shell but not in the skeleton.15 However, tortoises fed 3 and 9 times the dose of calcium recommended by the manufacturers developed multifocal calcification in the lungs, heart, kidneys, and subendothelial lamina of the arteries, with decreased growth apparent in the group given 9 times the recommended dose.15 Interestingly, investigators of another study34 reported similar findings in red-eared slider turtles (Trachemys scripta elegans). In turtles fed various calcium concentrations in that study,34 the group with the highest percentage of calcium (2.24% of dry matter) had a lower growth rate, compared with the growth rate for those groups receiving lower amounts of calcium. Considering the detrimental effect that provision of supplemental calcium can have (as indicated in studies), and considering the absence of calcium deficiencies in the captive tortoises in the present study, the best advice for dealing with tortoises that are artificially raised is to provide hatchling tortoises an adequate photothermal environment and a natural, plant-based diet without supplemental calcium.
In fact, the difference in BMD of captive-raised tortoises and control tortoises in the present study could have been secondary to the environment, apart from the diet. In the authors' opinion, the environment may have been responsible for the variations in BMD in 2 ways. The first hypothesis involves the hibernation period for the control tortoises during the trial. Tortoises that did not have a hibernation period should have been exposed to a higher quantity of calcium during the 10 months of the study. This hypothesis is speculative because evidence that the lack of hibernation increases BMD in hatchling chelonians is lacking. The second hypothesis is that there was an effect of the UVB fluorescent bulbs to which the captive-raised tortoises were exposed during the study. This second hypothesis appears unlikely because of the results of another study25 in which Hermann's tortoises exposed to natural sunlight had a higher plasma concentration of 25-hydroxyvitamin D3 than that in tortoises exposed to fluorescent bulbs.
The first hypothesis was also supported by the difference in body weight observed at the end of the study. The significant differences in body weight between the seminatural control group and the captive group fed vegetables intended for human consumption suggested that captive-reared chelonians should be fed a more natural diet to achieve growth similar to that of free-ranging tortoises, as has been mentioned in other studies.70–73 In fact, distinctive differences in the growth of captive and free-ranging tortoises have been reported,74 with the intrinsic growth rate of each captive Geochelone sulcata being 1.4 to 2.6 times as high as that of a typical free-living tortoise. Whether faster growth rates are linked to health problems70,73 or to a shorter life expectancy has been debated. More recently, growth-related disorders, especially pyramiding, have been detected more frequently in younger tortoises, possibly indicating a limitation in the life expectancy of tortoises with such pathological conditions.74 Nevertheless, the authors of that study74 suggest that this should not be considered conclusive evidence and that controlled clinical studies are necessary to assess the health risks related to fast growth in tortoises.
In the present study, it was likely that hibernation did not influence the growth rate because nonhibernating captive tortoises continued to eat and grow during the winter period. The present study was not conceived to assess the effects of housing or diet conditions on health. Nevertheless, the presence of a higher incidence of pyramidal growth in tortoises fed a diet intended for human consumption and maintained in artificial conditions was alarming. These factors should be carefully considered by clinicians who promote evidence-based health care of those patients.
Plasma calcium concentrations were not evaluated in the present study, primarily because of practical reasons (ie, collection of blood samples from a hatchling Hermann's tortoise that weighs only a few grams is invasive and can be dangerous to the tortoise's health).49 It should be mentioned that hatchling tortoises were used in the study because they are more likely to develop metabolic bone disease than are adult tortoises.14 Furthermore, interpretation of plasma calcium concentrations, especially in tortoises, is not a straightforward process. Plasma calcium concentrations may not adequately reflect a tortoise's bone mineralization,15 and they may fluctuate depending on season and sex, at least in mature tortoises.43,44 In contrast, DXA can be used to measure the mineral condition of the AAS and shell during the growth period in a fast, accurate, and noninvasive manner. The precision and accuracy of DXA measurement in red-eared slider turtles has been evaluated and was found to be satisfactory.56Although season-dependent variation in BMD has been described in female chelonians,75 it appears unlikely that a similar factor should be considered in immature 1-year-old tortoises.
An important advantage of DXA analysis is the ability to examine the entire skeleton as well as specific regions of interest. This aspect is particularly useful for the accurate examination of the mineral density in chelonians because of their peculiar body structure, compared with that of animals without a bony exoskeleton. Therefore, in the present study, we customized the densitometric analysis for chelonians by measuring BMD in the shell at a region of interest without the interposition of other bony structures and soft tissues (eg, the third intercostal space or lung projection area) at high density. We then subtracted the BMD of the shell from the BMD of the AAS or from the specific areas of interest (vertebral column and pelvis).
During the past several years, the projected BMD has replaced BMC. This appears to have been accepted because the biological variation of BMD is smaller than that of BMC, with the latter not being dependent on body morphometric variables. Moreover, conversion of BMC to BMD (by dividing the BMC value by the projected bone area) may be an appropriate procedure for normalization. Because BMD is susceptible to bone size and BSA is the variable that has the highest correlation with BMD,59,76–78 we normalized the BMD value to the square root of the BSA by use of the following equation58: BSA (in m2) = 0.007184 × body weight (in kg)0.425 × straight shell length (in cm)0.725. In tortoises, DXA has been used to compare the effect of various dietary regimens on bone density.15 In that study,15 there was no significant increase in the amount of calcification when tortoises were provided 0.4 and 2.9 g of calcium/kg, whereas tortoises fed 8.0 and 23.3 g of calcium/kg had a significant increase in BMD.
One unexpected finding of the present study was that the differences in mineralization among groups were found in the shell and not in the AAS. The natural-housed control group had the shell with the lowest BMD. Two hypotheses could explain this finding. The first hypothesis is that the control group was inadequate and had a generalized mineral depletion that was only evident in the shell. As previously stated, hibernation could explain the lower mineralization of the tortoises. The second hypothesis is that rearing the tortoises in an artificial environment led to an increase in mineralization attributable to several possible causes, including excess nutrients, lack of hibernation, or effect of UV light, and the excess calcium was deposited primarily in the shell. For this second hypothesis, the deposition of excess calcium would be expected to result in abnormal pyramidal growth of the scutes. We believe that the selection and management of the control group was appropriate and that no objective reason for mineral depletion in the control group can be provided. Thus, we suggest that the higher mineralization detected in group 2 reflected excess mineral deposition. This finding was supported by the observation of a higher frequency of pyramidal growth in the groups maintained in artificial conditions. Considering that the difference in mineral deposition was evident in the carapace, we speculated that excess calcium in Hermann's tortoises is primarily deposited in the carapace bones. This exaggerated deposition could result in pathological morphological consequences (ie, pyramidal growth). A definitive histologic confirmation of these hypotheses was not considered ethically acceptable. Further studies should involve the use of wild tortoises as control groups, although this adds an intrinsic difficulty for age determination.
One limitation of the present study was that DXA scans were performed on conscious tortoises to ensure animal well-being and avoid possible anesthetic-related complications. This factor may have affected the accuracy and precision of DXA measurements.56 Nevertheless, the correct placement of tape15 should effectively prevent movements that interfere with DXA scanning.
The results reported here provided evidence that provision of an adequate photothermal habitat and a balanced plant-based diet without supplemental calcium resulted in sufficient mineralization in captive-raised Hermann's tortoises. In addition, the higher BMD of captive-raised tortoises was morphologically associated with a higher incidence of pyramidal growth in captive-raised animals.
ABBREVIATIONS
AAS | Axial and appendicular skeleton |
BMC | Bone mineral content |
BMD | Bone mineral density |
BSA | Body surface area |
DXA | Dual-energy x-ray absorptiometry |
Repti Glo 10.0, 24-W fluorescent bulb, Exo Terra, Rolf C. Hagen Inc, Montréal, QC, Canada.
Heat Glo, 75-W infrared heat lamp, Exo Terra, Rolf C. Hagen Inc, Montréal, QC, Canada.
Solarmeter 6.2, Solartech Inc, Harrison Township, Mich.
Lunar Piximus, GE Medical Systems, Madison, Wis.
SPSS 12.0.2, SPSS Inc, Chicago, Ill.
VassarStats, Richard Lowry, Poughkeepsie, NY. Available at: faculty.vassar.edu/lowry/VassarStats.html. Accessed Jul 2, 2012.
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