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Vitamin A values of wild-caught

Cuban tree frogs (Osteopilus
septentrionalis) and marine toads
(Rhinella marina) in whole body,
liver, and serum.

ARTICLE in JOURNAL OF ZOO AND WILDLIFE MEDICINE · DECEMBER 2014

Impact Factor: 0.32 · DOI: 10.1638/2013-0289.1 · Source: PubMed

6 AUTHORS, INCLUDING:

Frank Ridgley
Miami Dade County
5 PUBLICATIONS 16 CITATIONS

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Eduardo V Valdes
Disney Research
44 PUBLICATIONS 173 CITATIONS

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Available from: Frank Ridgley

Retrieved on: 31 August 2015
 VITAMIN A VALUES OF WILD-CAUGHT CUBAN TREE
FROGS (OSTEOPILUS SEPTENTRIONALIS) AND MARINE
TOADS (RHINELLA MARINA) IN WHOLE BODY, LIVER,
AND SERUM
Author(s): Kathleen E. Sullivan, M.S., Greg Fleming, D.V.M., Dipl. A.C.Z.M.,
Scott Terrell, D.V.M., Dipl. A.C.Z.M., A.C.V.P., Dustin Smith, Frank Ridgley,
D.V.M., and Eduardo V. Valdes, Ing. Agr., M.Sc., Ph.D.
Source: Journal of Zoo and Wildlife Medicine, 45(4):892-895. 2014.
Published By: American Association of Zoo Veterinarians
DOI: http://dx.doi.org/10.1638/2013-0289.1
URL: http://www.bioone.org/doi/full/10.1638/2013-0289.1

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 Journal of Zoo and Wildlife Medicine 45(4): 892–895, 2014
Copyright 2014 by American Association of Zoo Veterinarians

VITAMIN A VALUES OF WILD-CAUGHT CUBAN TREE FROGS

(OSTEOPILUS SEPTENTRIONALIS) AND MARINE TOADS
(RHINELLA MARINA) IN WHOLE BODY, LIVER, AND SERUM

Kathleen E. Sullivan, M.S., Greg Fleming, D.V.M., Dipl. A.C.Z.M., Scott Terrell, D.V.M., Dipl.
A.C.Z.M., A.C.V.P., Dustin Smith, Frank Ridgley, D.V.M., and Eduardo V. Valdes, Ing. Agr., M.Sc., Ph.D.

Abstract: Recent issues surrounding captive amphibians are often nutritionally related problems, such as
hypovitaminosis A. Although supplementation of frogs with vitamin A is a topic of investigation, the underlying
issue is understanding vitamin A metabolism in amphibian species. To develop a range of ‘‘normal’’ vitamin A
concentrations for captive amphibians, baseline vitamin A concentrations must be established in wild amphibian
species. In this study, two species, Cuban tree frogs (Osteopilus septentrionalis; n ¼ 59) and marine toads (Rhinella
marina; n ¼ 20) were collected from the wild as part of an invasive species control program at Zoo Miami, Miami,
Florida. Serum, liver, and whole body samples were analyzed for vitamin A content. The Cuban tree frogs showed
higher concentrations on average of vitamin A in serum (82.8 ppb), liver (248.3 IU/g), and whole body (5474.7
IU/kg) samples compared with marine toads (60.1 ppb; 105.3 IU/g; 940.7 IU/kg, respectively), but differences
were not significant (P ¼ 0.22). What can be considered ‘‘normal’’ values of vitamin A concentrations across
different amphibian species requires further investigation. Although all amphibians collected in this study
appeared healthy, a larger sample size of animals, with known health histories and diets, may provide stronger
evidence of normal expectations.
Key words: Amphibian, Cuban tree frog (Osteopilus septentrionalis), liver, marine toad (Rhinella marina), serum,
vitamin A.

INTRODUCTION range of vitamin A concentrations in serum, liver,

Carotenoids and retinoids have been character-
ized as present and necessary for amphibians.15,17
Vitamin A is the generic descriptor for com-
pounds exhibiting the biologic activity of retinol.4
Deficiencies of vitamin A in captive amphibian
species have been documented and have led to the
death of many species of amphibians.14,23,25,29,30
However, the etiology of this issue is poorly
understood. Although a captive diet is generally
considered low in vitamin A, especially by using
commercially bred insects as feeders, this may not
explain the widespread incidence of dis-
ease.3,5,7,9,10,20,26 Normal concentrations of vitamin
A in most amphibian species are not established
for serum, liver, or whole body. Although retinol
concentrations have been reported in liver in frog
species,11,17 as well as whole-body concentra-
tions,3,21 none established a normal range for
optimal health. Understanding what an expected
From the Department of Animal Health, Disney’s
Animals, Science and the Environment, Bay Lake, Florida
32830, USA (Sullivan, Fleming, Terrell, Valdes); and the
Zoo Miami, Miami-Dade County Parks, Recreation and
Open Spaces, Miami, Florida 33177, USA (Smith, Ridg-
ley). Present address (Sullivan): Disney’s Animal Programs,
P.O. Box 10,000, Lake Buena Vista, Florida 32830-1000,
USA. Correspondence should be directed to Ms. Sullivan
(Kathleen.E.Sullivan@Disney.com).
and the whole body should be essential for
developing dietary requirements. When vitamin
A deficiency presents itself as short tongue
syndrome or squamous metaplasia, the common
recommendation is to increase dietary vitamin A
via supplementation.13,23,22 However, having a
target range of vitamin A values in the amphibian
means less risk of over or under supplementation.
The nutritional needs of the wide range of
amphibian species have not yet been defined by
requirements and scientific study. The authors
aim to add to the knowledge base of the commu-
nity on wild values of amphibian retinol in liver,
serum, and whole carcass.
CASE REPORT
Wild Cuban tree frogs (Osteopilus septentrionalis;
n ¼ 59) and marine toads (Rhinella marina; n ¼ 20)
were collected live from Zoo Miami, Miami,
Florida (n ¼ 42 Cuban tree frogs in September
2009; all others collected June 2009). These
species are deemed invasive to Florida by the
National Invasive Species Management Council
of the U.S. Department of Agriculture, and culling
of the population is encouraged.18 All frogs and
toads were weighed prior to sampling but were
not sexed (Table 1). Animals appeared healthy
with no visible signs of disease. To determine if
serum vitamin A samples were representative of

892
 SULLIVAN ET AL.—VITAMIN A IN FROGS AND TOADS 893

Table 1. Body weight and analyzed vitamin A concentrations of serum, whole carcass, and liver of wild-caught
Cuban tree frogs (Osteopilus septentrionalis) and marine toads (Rhinella marina).

Body weight (g) Serum vitamin A (ppb) Whole carcass vitamin A (IU/kg) Liver vitamin A (IU/g)

Marine toads
n 20 20 10 10
Average 125.0 60.1 940.7 105.3
Median 120.0 48.0 892.5 74.3
Standard error 9.5 11.7 99.7 37.0
Maximum 253.0 264.0 1,668.0 429.6
Minimum 29.0 9.0 611.0 1.0
Cuban tree frogs
n 59 26 13 34
Average 9.9 82.8 5,474.7 248.3
Median 7.0 38.0 4,209.0 256.0
Standard error 0.9 5.1 959.8 20.3
Maximum 30.0 552.0 12,857.0 503.9
Minimum 2.5 12.0 1,555.0 13.7

body vitamin A concentrations, paired serum and
liver or paired serum and whole body vitamin A
concentrations were obtained. Blood samples
were obtained from the live amphibians to
determine serum vitamin A concentrations. A
sample volume of (0.5–1.0 ml) of blood was
collected from the ventral abdominal vein under
manual restraint. Due to difficulties based on the
small size of the Cuban tree frogs (average weight
¼ 9.9 g), not all frogs were successfully sampled
for serum prior to euthanasia. Frogs and toads
were then euthanized with MS222 (Tricaine-S,
Western Chemical Inc., Ferndale, Washington
98248, USA). These animals were then refriger-
ated for a minimal time (no more than 3 hr) before
gross necropsy and liver samples were obtained.
The liver was removed from 10 toads and 34 frogs
due to sample size requirement and size variation
of the individual animals. The small intestine and
a small portion of corresponding liver in these
individuals were used in a separate study of
provitamin conversion (,5 g).16 Liver and intesti-
nal samples were excised for analysis or the whole
body intact was frozen at 808C. All serum
samples and livers were sent frozen and analyzed
via high performance liquid chromatography for
vitamin A (retinol, specifically) at Michigan State
Diagnostic Center for Population and Animal
Health (MSDCPA; Lansing, Michigan 48910,
USA). Whole carcasses were sent and analyzed
after freeze drying on a FreeZone 6 Liter Freeze
Dry System (LabConco, Kansas City, Missouri
64132, USA). According to the internal proce-
dures of MSDCPA, serum vitamin A was ana-
lyzed without saponification or hydrolysis and
represents only retinol, in keeping with a lack of
expected retinyl esters in circulation.2 Liver
vitamin A was analyzed following a heated
saponification, which would convert any retinyl
esters to retinol, and thereby represents both in
one value.24 Whole carcass vitamin A values
represent both retinyl esters and retinol, as a cold
saponification and hydrolysis was used.6 All data
on vitamin A in both species are shown in Table 1.
All data were analyzed by using SYSTAT
version 13.00.05 (Systat Software Inc., San Jose,
California 95110, USA). Body weights and type of
amphibian comparisons to serum, liver, and
whole carcass were performed with a general
linear model procedure. Effects of serum vitamin
A on whole carcass and liver vitamin A were
analyzed by a pairwise Pearson correlation.
DISCUSSION
The Cuban tree frogs showed higher concen-
trations of vitamin A in liver, whole-body, and
serum samples than marine toads (Table 1), but a
similarly wide range in serum and liver vitamin A
as compared with the marine toads, creating an
overall lack of significance (P ¼ 0.22). This is likely
reflective of the variety of diet thought to be
consumed by these wild amphibians.1,8,28 Both
species are insectivorous, and it cannot be deter-
mined whether the wide range shown in value
stems from seasonal availability of dietary items
and nutrient content, feeding ecology differences,
individual variation, or possible health of the
animals at sampling. There was no significant
difference between sampling periods, based on
body weight alone, or the interaction of frog type
894 JOURNAL OF ZOO AND WILDLIFE MEDICINE
and body weight (P . 0.1 for all). Comparisons to
historic liver concentrations seen from frogs
(obtained from a commercial supplier, species
not reported and varied),11,17 appear to show
diverse concentrations of vitamin A compared
with this study (225.2 lmol/g;11 5.79 IU/g17);
however, the diet of the captive commercial frogs
is not described in this historic work. Whole
carcass vitamin A has been reported as 25,110
IU/kg (n ¼ 7) for captive green frogs (Rana
clamitans) and 38,261 IU/kg (n ¼ 4) in captive
adult southern toads (Bufo terrestris).27 These
concentrations are markedly higher than those
presented herein, which may be the result of
species or dietary differences, sample size, or
possibly methodology of analysis, as the samples
in that study were not freeze-dried prior to
analysis. The International Species Information
System does not report any vitamin A values in
the serum of frog or toad species.12
Inferring whole-body vitamin A concentrations
from a serum sample may not represent actual
vitamin A concentrations present in the liver or
whole body in these two species, as shown in this
study’s weak relationships between either pairing
(Pearson correlation product-moment correlation
coefficient; serum to liver: r ¼ 0.27; serum to
whole carcass: r ¼ 0.31). However, in diagnostic
antimortem sampling, serum samples are the
easiest and least invasive when compared with
obtaining a liver sample. The amount of liver
sample needed (5 g; MSDCPA) may also not be
practical to remove on a small amphibian, such as
a frog or toad. However, determining baseline
serum concentrations are critical in establishing
normal value range for Cuban tree frogs and
marine toads alike.22
Questions remain on the dietary needs of
amphibians, both in terms of definitive vitamin
A requirements and possibilities of carotenoid
supplementation. Although one study did not
find the enzyme for provitamin conversion, due
to small sample size and handling, it did not
conclusively show that amphibians cannot in-
terconvert carotenoids to vitamin A in all
tissues.16 Using the skin of frogs as a constant
due to its constancy, a historic study found
higher relative total concentrations of caroten-
oids in the skin and ovaries (1 and 2.65,
respectively), followed by the liver and intestine
(0.7 and 0.23, respectively), indicating sex may
also be a factor.17 Differences in feeder insect
efficiency of delivery of carotenoids and vitamin
A continues to be studied,10,19,20,26 however, the
whole animal’s needs for this nutrient still
require investigation.
Acknowledgments: The authors would like to
thank Zoo Miami for their collection of the frogs
and toads used for analysis. Thank you to Vicki
Sikorski for assistance with frog sampling and
storage. Thanks also to Shannon Livingston and
Dr. Shana R. Lavin for help in reviewing this
paper.
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Received for publication 5 December 2013