Plasma 25-hydroxyvitamin D3 concentrations

in Hermann’s tortoises (Testudo hermanni)

exposed to natural sunlight and two
artificial ultraviolet radiation sources

Paolo Selleri, DMV, PhD, and Nicola Di Girolamo, DMV

Objective—To determine the effect of various UVB radiation sources on plasma 25-
hydroxyvitamin D3 concentrations in Hermann’s tortoises (Testudo hermanni).
Animals—18 healthy Hermann’s tortoises.
Procedures—Tortoises were exposed to sunlight in an outdoor enclosure located in the
natural geographic range of Hermann’s tortoises (n = 6 tortoises) or a self-ballasted mercu-
ry-vapor lamp (6) or fluorescent UVB-emitting lamp (6) in an indoor enclosure for 35 days.
Plasma samples were obtained from each tortoise on the first (day 0) and last (day 35) days
of the study, and concentrations of 25-hydroxyvitamin D3 were determined. Amount of UVB
radiation in enclosures was measured.
Results—Mean ± SD plasma 25-hydroxyvitamin D3 concentrations for tortoises exposed
to the mercury-vapor and fluorescent lamps were significantly lower on day 35 (155.69 ±
80.71 nmol/L and 134.42 ± 51.42 nmol/L, respectively) than they were on day 0 (368.02
± 119.34 nmol/L and 313.69 ± 109.54 nmol/L, respectively). Mean ± SD plasma 25-
hydroxyvitamin D3 concentration for tortoises exposed to sunlight did not differ significantly
between days 0 (387.74 ± 114.56 nmol/L) and 35 (411.51 ± 189.75 nmol/L). Mean day 35
plasma 25-hydroxyvitamin D3 concentration was significantly higher for tortoises exposed
to sunlight versus those exposed to mercury-vapor or fluorescent lamps. Sunlight provided
significantly more UVB radiation than did the mercury-vapor or fluorescent lamps.
Conclusions and Clinical Relevance—Plasma 25-hydroxyvitamin D3 concentrations dif-
fered between tortoises exposed to sunlight and those exposed to artificial UVB sources.
Exposure to sunlight at a latitude similar to that of the natural geographic range is recom-
mended for healthy and calcium-deficient tortoises. (Am J Vet Res 2012;73:1781–1786)

T he primary function of vitamin D3 in vertebrates is

maintenance and regulation of calcium homeosta-
sis.1 Vitamin D3 aids bone mineralization via increas-
D.6 The importance of vitamin D3 in reptiles is indicat-
ed by results of studies7–9 in which captive and wild rep-
tiles voluntarily exposed themselves to UVB radiation.
ing uptake of calcium from the intestinal tract.2,3 This Animals can obtain vitamin D3 from food or via
function of vitamin D3 is especially important in cap- synthesis in the skin.10–13 Photolysis of 7-dehydrocho-
tive reptiles because of the high incidence of calcium lesterol to previtamin D3 in skin is dependent on UV ra-
deficiency–related pathological changes (ie, metabolic diation with a wavelength between 280 and 320 nm.1,14
bone disease) in these animals.4,5 In addition, chame- Previtamin D3 undergoes successive temperature-
leons with adequate circulating concentrations of vi- dependent isomerization steps to form vitamin D3.1,15
tamin D have better reproductive success than those Vitamin D obtained from food or via synthesis is con-
without adequate circulating concentrations of vitamin verted by sterol 25-hydroxylase to 25-hydroxyvitamin
D3 in the liver.16–18 The active form of vitamin D (1,25-di-
Received October 24, 2011. hydroxyvitamin D3) is synthesized via 1-hydroxylation
Accepted January 17, 2012. of 25-hydroxyvitamin D3 in the kidneys.19 Among as-
From the Clinica per Animali Esotici, Centro Veterinario Specialistico says for detection of vitamin D and its metabolites, as-
(CVS), Via Sandro Giovannini 51–53, 00137 Rome, Italy.
Presented in abstract form at the 18th Annual Conference of the As-
says for determination of serum 25-hydroxyvitamin D3
sociation of Reptilian and Amphibian Veterinarians, Seattle, August concentrations are the most useful for determination of
2011. the vitamin D status of an animal because that metabo-
The authors thank Professor Gloria Isani and Dr. Annunziata Can- lite has a longer half-life than other metabolites.20
navacciuolo for assistance with performance of assays, Dr. Raffa- Reptiles bask in UV light for thermoregulatory
ele Melidone and Dr. Alessandro Di Girolamo for assistance with purposes and to increase vitamin D production.8,21 In
study design, and Dr. Elena Marini, Eleonora Malerba, and Lorenzo
Gaspari for technical assistance. captivity, plasma concentrations of 25-hydroxyvitamin
Address correspondence to Dr. Di Girolamo (nicoladiggi@gmail. D3 increase in basking reptiles of several species when
com). they are exposed to UVB radiation.7,13,22–26 To the au-

AJVR, Vol 73, No. 11, November 2012 1781

thors’ knowledge, this response has not been identified EU of the European Parliament and the Council of the
for any terrestrial chelonian species, despite the natural European Union. The owners gave written informed
basking habits of tortoises27 and the large number of consent for participation of tortoises in the study and
tortoises kept as pets worldwide. Moreover, metabolic for collection of plasma samples.
bone disease may be the most common medical disor- Each tortoise was allocated to 1 of 3 groups (6
der of captive chelonians.28 Exposure of veiled chame- tortoises/group) via a restricted randomization pro-
leons (Chameleo calyptratus) to adequate UVB radiation cedure by use of a table of random numbers. The
is important for prevention of metabolic bone disease13; UVB radiation sources for the tortoises were a self-
exposure to adequate UVB radiation and provision of ballasted mercury-vapor lamp (n = 6 tortoises), a
adequate dietary calcium are likely important for pre- fluorescent UVB-emitting lamp (6), or natural sun-
vention of this disease in other reptiles. light (6). Tortoises housed indoors (self-ballasted
Most reptile curators and experienced herpetocul- mercury-vapor lamp and fluorescent UVB-emitting
turists believe that natural lighting is the best type of lamp groups) were placed in open-top plastic box-
light for reptiles.29 Various sources of UVB radiation esa (vivariums; 120 X 60 X 18.5 cm). A commercially
are available for captive reptiles. Some of these UVB available self-ballasted mercury-vapor lampb was the
sources (eg, self-ballasted mercury-vapor lamps) also source of light and heat for tortoises in that vivar-
produce substantial amounts of thermal energy. Many ium. That lamp was oriented vertically and placed
anecdotal accounts suggest use of this type of lamp 30 cm above the floor of 1 side of the box, in accor-
can reverse nutritional metabolic bone disease in rep- dance with the manufacturer’s instructions. For the
tiles.30 Mercury-vapor lamps produce different quanti- vivarium-housed tortoises exposed to the fluorescent
ties of UVB radiation than do UVB-emitting fluorescent UVB-emitting lamp, the UVB fluorescent lampc was
lamps.31 Unfortunately, equal irradiance values of dif- the light source and an infrared lampd was the heat
ferent types of light sources do not indicate those light source. In that vivarium, the fluorescent bulb was
sources have the same potential for increasing vitamin oriented diagonally with the end of the longitudinal
D synthesis in reptiles.32 Results of a recent study31 in- axis 21 cm above the floor of the vivarium. The in-
dicate bearded dragons (Pogona vitticeps) exposed to a frared lamp was oriented so that it heated the same
compact UVB fluorescent bulb producing low amounts portion of the vivarium that was exposed to UVB ra-
of UVB radiation had higher circulating concentrations diation via the fluorescent lamp. For each vivarium,
of 25-hydroxyvitamin D3 than did bearded dragons ex- light and heat were provided for 12 continuous hours
posed to light sources producing high amounts of UVB each day (8:00 AM to 8:00 PM). Shelters were available
radiation. That finding suggests the biological effects of for tortoises in each vivarium so that the animals could
lamps regarding vitamin D production in reptiles can- easily regulate their exposure to UVB radiation and heat.
not be predicted on the basis of values of radiometric
The authors observed the tortoises at 8:00 AM, 12:00 PM,
variables. Determination of plasma 25-hydroxyvitamin
4:00 PM, and 8 PM during 5 randomly selected days of
D3 concentration may be a more appropriate method
for evaluation of the biological effects of UVB radiation the study to verify that they had physiologic bimodal
sources; such biological effects could be influenced by basking pattern behavior.33 Environmental temperature
variables other than irradiance of lamps (eg, production in the indoor area mercury-vapor and fluorescent lamp
of a pattern and area of thermal radiation similar to that vivariums were kept was regulated so that it was similar
of natural sunlight). to the temperature in the outdoor area where the tor-
The objective of the study reported here was to de- toises exposed to natural sunlight were housed. In the
termine whether circulating concentrations of plasma indoor area, environmental temperature was maintained
25-hydroxyvitamin D3 in terrestrial chelonians are in- at 22 ± 1°C during the day (8:00 AM to 8:00 PM) and at
fluenced by the type of UVB radiation source to which 19 ± 1°C during the night (8:00 PM to 8:00 AM). Environ-
those animals are exposed. The hypotheses were that mental temperatures in basking zones (UVB radiation
plasma 25-hydroxyvitamin D3 concentrations in Her- coverage areas) were recorded at 12:00 PM each day with
mann’s tortoises (Testudo hermanni) would be influ- an infrared thermometere; temperatures in the basking
enced by exposure to UVB radiation and that Hermann’s zones never exceeded 37°C during the study.
tortoises exposed to natural sunlight or a mercury- The tortoises exposed to natural sunlight were
vapor lamp would have higher plasma concentrations housed in an outdoor, sunlight-exposed enclosure
of 25-hydroxyvitamin D3 than would Hermann’s tor- located in the natural geographic range of Hermann’s
toises exposed to a fluorescent UVB-emitting lamp. tortoises.27 To simulate natural conditions, these tor-
toises foraged on vegetation naturally growing in the
Materials and Methods enclosure. The tortoises kept indoors were fed that
same vegetation; these plants were collected each day
Animals—Eighteen client-owned healthy subadult in the vicinity of the outdoor enclosure to minimize
(age range, 3 to 8 years) Hermann’s tortoises were used differences in diet among the 3 groups of tortoises.
in the study. All of the tortoises had hibernated out- The diet consisted of Asteraceae (dandelions [Taraxa-
doors during the winter of 2010 to 2011. At the end cum officinale]), Fabaceae (clover [Trifolium spp]),
of the hibernation period, the tortoises were placed in Malvaceae (mallow [Malva spp]), Plantaceae (ribwort
an outdoor enclosure for 20 days prior to the start of plantain [Plantago lanceolata]), Oxalidaceae (wood
the study (acclimatization period). For all tortoises, the sorrels [Oxalis spp]), and Rosaceae (creeping cinque-
study began on April 25, 2011 (day 0). The study was foil [Potentilla reptans]). Water was provided to the
performed in compliance with the directive 2010/63/ tortoises ad libitum.

1782 AJVR, Vol 73, No. 11, November 2012

Table 1—Mean ± SD (range) 25-hydroxyvitamin D3 concentrations in plasma samples obtained on study days 0 and 35 from Hermann’s
tortoises (Testudo hermanni) exposed to a self-ballasted mercury-vapor lamp (n = 6 tortoises), UVB-emitting fluorescent and infrared
heat lamps (6), or natural, unfiltered sunlight (6) as light and heat sources and amounts of UVB radiation emitted by each of those
sources on days 0 through 35.

Plasma 25-hydroxyvitamin D3 concentration

Light and heat source UVB (µW/cm2)* Day 0 (nmol/L) Day 35 (nmol/L)
Self-ballasted mercury-vapor lamp 11.8 ± 1.2 (10–14)† 368.02 ± 119.34 (205.12–523.06) 155.69 ± 80.71 (77.23–304.88)‡
Fluorescent UVB and infrared heat lamps 24.7 ± 2.4 (18–29)† 313.69 ± 109.54 (145.98–423.98) 134.42 ± 51.42 (50.14–179.6)‡
Natural sunlight 205.3 ± 53.6 (87–278) 387.74 ± 114.56 (200.76–541.28) 411.51 ± 189.7 (119.36–667.04)§
*Amount of UVB radiation was determined at the basking site in each enclosure at 9:00 AM (1 hour after lamp activation; mercury-vapor
and fluorescent lamps) or 1:30 PM (natural sunlight) on days 0 through 35, and mean ± SD values were calculated. †Amount of UVB radiation
is significantly (P < 0.01) different from the value for natural sunlight. ‡Within a light and heat source group, the plasma concentration of
25-hydroxyvitamin D3 on day 35 is significantly (P < 0.05) lower than the value on day 0. §Value on day 35 is significantly (P < 0.01) higher than
values for other groups of tortoises on day 35.

Experimental procedures—On day

0, a blood sample (0.3 mL) was collected
from a jugular vein of each tortoise. Blood
samples were obtained from the subcara-
pacial sinus when blood could not be
obtained from the jugular vein within 2
attempts. Blood samples were collected
in tubes containing lithium heparinf and
were centrifuged (1,650 X g for 5 minutes)
within 30 minutes after collection. Plasma
was harvested and stored immediately at
–25°C. Another plasma sample was ob-
tained from each tortoise on day 35 of
the study by use of that same technique.
Plasma 25-hydroxyvitamin D3 concentra-
tions were estimated by use of an enzyme
immunoassay.g
During the study, the amount of UV
radiation with a wavelength between 280
and 320 nm to which tortoises were ex-
posed was measured by use of a digital Figure 1—Mean amount of UVB radiation in the basking area in an outdoor enclosure
housing 6 Hermann’s tortoises (Testudo hermanni) exposed to natural, unfiltered sunlight
portable radiometer.h The amount of UV from 6:00 AM through 8:30 PM on days 0 and 35 of the study. Error bars represent range.
radiation was measured at the basking site
in each enclosure.9 To determine variations in amount Results
of UVB radiation at the basking areas in the enclosures
during the daytime, measurements were obtained every Mean ± SD day 0 plasma 25-hydroxyvitamin D3
30 minutes on days 0 and 35 and mean ± range values concentration for all tortoises was 356.49 ± 112.32
were calculated. To determine variations in amount of nmol/L; the distribution of values was normal (P <
UVB radiation at the basking areas in the enclosures 0.01), and no significant (P = 0.52) differences in day
during the 35-day study period, measurements were 0 plasma 25-hydroxyvitamin D3 concentration were
obtained once every day in the outdoor enclosure at the detected among the 3 groups of tortoises. Mean ± SD
time of peak UVB radiation (1:30 PM) and once every plasma 25-hydroxyvitamin D3 concentrations were
day in the indoor vivaria 1 hour after lamp activation significantly lower on day 35 than they were on day
(9:00 AM). 0 for tortoises exposed to the self-ballasted mercury-
vapor lamp (day 0 value, 368.02 ± 119.34 nmol/L; day
Statistical analysis—Statistical analysis was per- 35 value, 155.69 ± 80.71 nmol/L; P = 0.017) and tor-
formed with commercially available software.i Plasma toises exposed to the fluorescent UVB-emitting lamp
concentrations of 25-hydroxyvitamin D3 were evalu- (day 0 value, 313.69 ± 109.53 nmol/L; day 35 value,
ated for normality via the Shapiro-Wilk test. Plasma 134.42 ± 51.42 nmol/L; P = 0.007; Table 1). Concen-
concentrations of 25-hydroxyvitamin D3 on days 0 and trations of 25-hydroxyvitamin D3 in plasma samples
35 and daily UVB measurements among groups of tor- obtained on days 0 (387.74 ± 114.56 nmol/L) and 35
toises were analyzed via a 1-way ANOVA and a Tukey (411.51 ± 189.75 nmol/L) from tortoises exposed to
honestly significant difference post hoc comparison natural sunlight were not significantly (P = 0.64) dif-
test. A t test for correlated samples was used to deter- ferent. The ANOVA results indicated day 35 plasma 25-
mine differences between day 0 and day 35 plasma 25- hydroxyvitamin D3 concentrations differed significantly
hydroxyvitamin D3 concentrations for tortoises in each (P = 0.002) among the 3 groups of tortoises; results of
group. Values of P < 0.05 were considered significant. post hoc analysis indicated values for tortoises exposed

AJVR, Vol 73, No. 11, November 2012 1783

to natural sunlight were significantly (P < 0.01) higher the author’s knowledge, no studies have been conduct-
than they were for tortoises exposed to mercury-vapor ed in which this technique was used for determination
or fluorescent lamps. of vitamin D concentrations in reptiles. Results of other
Mean ± SD UVB radiation outputs of the mercury- studies13,24 indicate certain reptiles (eg, red-eared slid-
vapor lamp (at 9:00 AM on days 0 through 35), the fluo- ers [Trachemys scripta elegans] and veiled chameleons)
rescent lamp (at 9:00 AM on days 0 through 35), and meet their need for vitamin D via both dietary sources
natural sunlight (at 1:30 PM on days 0 through 35) were and production in skin. Although the ability of reptiles
11.8 ± 1.2 µW/cm2, 24.7 ± 2.4 µW/cm2, and 205.3 ± to regulate dietary vitamin D intake is not known,9 re-
53.6 µW/cm2, respectively (Table 1). The ANOVA re- sults of a recent study26 indicate bearded dragons with
sults indicated the amount of UVB radiation emitted by low circulating 25-hydroxyvitamin D3 concentrations
the 3 UVB sources differed significantly (P < 0.001); preferentially eat food that is rich in vitamin D. Despite
results of post hoc analysis indicated natural sunlight this finding, excessive dietary vitamin D intake has
provided significantly (P < 0.01) more UVB radiation toxic effects in reptiles,39,40 birds,41 mammals other than
than did mercury-vapor or fluorescent lamps. Amounts humans,42–44 and humans.45,46 Plasma 25-hydroxyvita-
of UVB radiation emitted by the mercury-vapor and min D3 concentrations in tortoises in the present study
fluorescent lamps were not significantly different. The that were kept outdoors were higher than those pre-
amount of UVB radiation in the basking area of the out- viously reported for other chelonians.24,34,47 The high
door enclosure exposed to natural sunlight on days 0 plasma 25-hydroxyvitamin D3 concentration in tor-
and 35 from 6:00 AM through 8:30 PM was determined toises in the present study might have had toxic effects
(Figure 1). The mercury-vapor and fluorescent lamps or have led to development of hypervitaminosis D, but
provided constant amounts of UVB radiation (ie, sub- this seemed unlikely because healthy tortoises were
stantial variations during the day were not detected). included, tortoises were exposed to sunlight in their
The 3 groups of tortoises had similar basking patterns; natural geographic range, tortoises were provided pro-
none of the tortoises avoided the basking zone. tective shelters against UVB radiation, and endogenous
production theoretically does not cause an excess of vi-
Discussion tamin D3. Previtamin D3 can undergo thermal isomeri-
Results of this study supported the hypothesis zation to form vitamin D3 or can undergo photochemi-
that circulating 25-hydroxyvitamin D3 concentrations cal isomerization to form biologically inert products
in Hermann’s tortoises are influenced by exposure to (ie, lumisterol and tachysterol), which limits synthesis
UVB radiation. However, the hypothesis that tortoises of previtamin D3 in skin during prolonged exposure
exposed to natural sunlight or a mercury-vapor lamp to UVB radiation.48 Because exposure to UV radiation
would have higher circulating concentrations of 25- is a more efficient method for increasing circulating
hydroxyvitamin D3 than tortoises exposed to a fluores- 25-hydroxyvitamin D3 concentrations in reptiles ver-
cent UVB-emitting lamp was not completely support- sus supplementation of the diet with vitamin D26,49 and
ed. To the authors’ knowledge, this is the first study because reptiles seem to regulate exposure to UV light
in which plasma 25-hydroxyvitamin D3 concentrations better than they regulate dietary intake of vitamin D,
were determined for terrestrial chelonians exposed to stimulation of endogenous vitamin D3 production via
natural sunlight in an environment within their natural exposure to UV light may be preferable to addition of
geographic range. Mean circulating 25-hydroxyvitamin supplemental vitamin D to food for reptiles. However,
D3 concentrations in Testudo spp tortoises maintained exposure to UVB radiation can cause adverse effects in
outdoors in the United Kingdom without an additional reptiles50; therefore, use of appropriate UVB lamps and
source of UVB radiation,34 adult desert tortoises (Go- careful adherence to the manufacturer’s instructions are
pherus agassizii) housed in outdoor pens in the Mojave suggested.
desert, and African spurred tortoises (Geochelone sul- Results of the present study indicated significant
cata) and juvenile desert tortoises kept in indoor enclo- differences in circulating vitamin D concentrations be-
sures are 28.41, 20.5, and < 12.5 nmol/L, respectively.35 tween tortoises exposed to natural sunlight and those
Compared with chelonians in those other studies, the exposed to artificial UVB sources. This finding was at-
tortoises in this study had a higher mean concentra- tributed to differences in UVB emission of natural sun-
tion of 25-hydroxyvitamin D3 (356.49 nmol/L) at the light versus that of artificial lamps; it was considered
start of the study. Although this difference may be at- unlikely that other factors caused this result because
tributable to species differences, the tortoises in the the conditions in which the groups of tortoises were
present study may have been exposed to more UVB ra- kept were similar, other than the UVB radiation sources
diation (from sunlight) prior to the start of the study for the 3 enclosures. The amount of UVB radiation in
versus tortoises in those other studies. In addition, the outdoor enclosure exposed to sunlight was approxi-
the diet for tortoises in the present study (naturally mately 8 times as high as it was in the vivarium with
growing vegetation) may have contributed to the high fluorescent UVB-emitting and infrared lamps and 17
circulating 25-hydroxyvitamin D3 concentrations in times as high as it was in the vivarium with the self-
these animals. Plants of several types, such as alfalfa ballasted mercury-vapor lamp. Differences were de-
and perennial ryegrass, contain ergosterol (provitamin tected between the 2 types of artificial lamps regarding
D2).12,36 Theoretically, ingested vitamin D and vitamin D amount of UVB radiation emitted, but values were not
produced in animals via exposure to UV radiation can significantly different. Plasma 25-hydroxyvitamin D3
be differentiated via chromatographic separation of 25- concentrations were not significantly different between
hydroxyvitamin D2 and 25-hydroxyvitamin D3.37,38 To tortoises exposed to each type of artificial lamp. Results

1784 AJVR, Vol 73, No. 11, November 2012

of this study suggested a fluorescent UVB-emitting metabolic bone disease should be exposed to natural,
lamp and a thermal source (eg, infrared lamp or ceram- unfiltered sunlight at a latitude similar to that of their
ic heater) stimulated production of 25-hydroxyvitamin natural geographic range to maximize dietary calcium
D3 in tortoises as well as a mercury-vapor lamp did. uptake.
In the present study, distances between the floors
of the vivariums and the lamps were selected in ac- a. Tontarelli S.p.a., Castelfidardo (AN), Italy.
cordance with the minimum distances indicated in the b. Solar Glo, 125-W mercury-vapor bulb, Exo Terra, Rolf C. Hagen
manufacturer’s instructions; a decrease in those dis- Inc, Montréal, QC, Canada.
c. Repti Glo 5.0, 24-W fluorescent bulb, Exo Terra, Rolf C. Hagen
tances would likely have increased the UVB radiation Inc, Montréal, QC, Canada.
received by the tortoises but could possibly have caused d. Heat Glo, 75-W infrared heat lamp, Exo Terra, Rolf C. Hagen
ocular and dermal lesions.50 Although minimum ade- Inc, Montréal, QC, Canada.
quate circulating concentrations of 25-hydroxyvitamin e. Minitemp MT4, Raytek, Santa Cruz, Calif.
D3 have not been determined for tortoises, the manufac- f. Sarstedt Ag & Co, Numbrecht, Germany.
turer’s recommendations for safe use of lamps may not al- g. 25-Hydroxy Vitamin D EIA, AC-57F1, Immunodiagnostic Sys-
tems Ltd, Boldon, England.
low adequate 25-hydroxyvitamin D3 synthesis in tortoises. h. Solarmeter, model 6.2, serial No. 03787, Solartech Inc, Harrison
Further studies would be necessary to determine whether Township, Mich.
changes in lamp location and distance from vivarium i. SPSS, version 16.0, SPSS Inc, Chicago, Ill.
floors would increase UVB radiation received by tortoises
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1786 AJVR, Vol 73, No. 11, November 2012