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dissertation entitled

EFFECTS OF DIET COMPOSITION AND AMBIENT
TEMPERATURE ON DIGESTIVE FUNCTION
AND BIOENERGETICS OF THE GREEN IGUANA
(Iquana iquana)

 

presented by

DAVID JONATHAN BAER

has been accepted towards fulfillment
of the requirements for

Doctorate Animal Science

 

 

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EFFECTS OF DIET COMPOSITION AND AMBIENT TEMPERATURE

ON DIGESTIVE FUNCTION AND BIOENERGETICS
OF THE GREEN IGUANA (Iquana iquana)

 

BY

David Jonathan Baer

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1992

ABSTRACT

EFFECTS OF DIET COMPOSITION AND AMBIENT TEMPERATURE
ON DIGESTIVE FUNCTION AND BIOENERGETICS
OF THE GREEN IGUANA (Iguana iquana)

BY

David Jonathan Baer

The green iguana (Iguana iquana) is a folivorous
ectotherm that has been used as a source of food for humans
in Central and South America. Intensive production of
captive iguanas may help re-establish native populations
that have declined in the recent past. A component of
captive management is the development of diets that support
rapid growth and reproduction. Two studies were conducted
with individually—housed, ad libitum—fed, captive iguanas to
determine the relationship between diet composition,
temperature, digestive function, and bioenergetics. During
the first study, 21 iguanas were fed 3 diets (Latin—square
crossover design) that contained 3 levels of dietary fiber
(19%, 24%, and 27% of dry matter as neutral detergent fiber

(NDF)). Mean (:SEM) daily dry matter (DM) and metabolizable
energy (ME) intake (6.99, 7.57, 6.86 i 0.62 g/d/kg body
weight (BW); and 22.2:2.l, 23.2:2.2, l9.l:2.l kcal/d/kg BW)

were not different between the 3 diets, and mean daily

growth rate (GR) (2.22 and 2.35 [:0.21] g/d) was not
different between the low and medium level fiber diets,
respectively. Mean daily GR was lower (p<0.0037) when the
iguanas were fed the high fiber diet (1.42 g/d). Metaboliz-

ability of DM (65.8, 62.2, 57.8 [:1.4%]), and energy (70.7,
66.4, 62.3 [:1.2%]) were lowest on the high fiber diet

(p<0.0011 for DM, p<0.0001 for energy). NDF digestibility
followed a similar pattern, (47.1, 42.5, 38.5 [:1J8%]).
During the second study, 12 iguanas were housed (crossover
design) at 2 ambient temperatures (28 C and 35 C). Daily DM
intake (3.5, 12.7 [:.5] g/d), GR (0.6, 3.4 [:.3] g/d), and
energy expenditure [EE, determined by respiratory exchange

(RE: 5.5, 14.3 [1.9] kcal/d) and doubly-labelled water (DLW:
11.3:3.3, 33.4:3.0 kcal/d)] were lower for iguanas housed at

28 C than 35 C. Nutrient metabolizability, and the
efficiency of energy deposition above maintenance (kg) were
not different between the iguanas housed at the different

temperatures. Mean EEdlw (24.0:3.6 kcal/d) was higher
(p<0.001) than EEre (10.0:.9 kcal/d), and EEdlw may

overestimate EE. These studies demonstrate that in
captivity, herbivorous lizards will consume manufactured
diets ad libitum, and digestion and bioenergetic studies can
be conducted under controlled conditions. A diet containing
less than 27% NDF, and an ambient temperature of 35 C may
improve growth rate of captive iguanas, but ambient

temperature does not affect nutrient metabolizability.

Copyright by
DAVID JONATHAN BAER

1992

To my dearest Charlotte,

thank you for your steadfast support

ACKNOWLEDGMENTS

There are many people who have helped me complete this
project. The iguanas were provided by Dr. Dagmar Werner of
the Fundacién Pro Iguana Verde, and Dagmar’s spirit for the
iguana management project helped focus my research goals.

Partial funding for this project was provided by the
Smithsonian Institution’s Office of Scholarships and Grants
(Scholarly Studies Program), and the National Zoological
Park. Many people at the zoo assisted in various aspects of
these studies. Dr. Olav Oftedal paved the way for these
studies to be conducted at the zoo, and has provided me with
many invaluable research opportunities as my career has
developed. Mr. Michael Jakubasz provided logistical
support, and was always available, with a smile, to correct
problems, and to assist in whatever needed to be done. Mr.
Miles Roberts, Assistant Director for Research, and the
keeper staff, Mr. Frank Kohn, Mr. Mike Deal, Ms. Cathi
Mathis, and Mr. Jerry Harris, of the Department of
Zoological Research, helped in many aspects managing the
iguanas, including assistance in the care of the iguanas and

sample collection.

Vi

Ms. Charlotte Kirk provided help every step of the way,
from collecting, feeding, cleaning, bleeding, and weighing
to proof—reading. Her dedication and enthusiasm in helping
me complete this research was inspiring.

Dr. William Rumpler truly provided me with an
opportunity to complete this project. Not only did Bill
provide a flexible environment and training in calorimetry
techniques, but he provided me an opportunity to discuss my
research findings, and provided insight into understanding
bioenergetics. His support will not be forgotten and his
friendship will always be cherished.

I would also like to thank the members of my graduate
committee, Drs. Mel Yokoyama, Dale Romsos, and Rich Erdman
for their support and insightful comments. I would
especially like to thank my graduate committee chairman, Dr.
Duane E. Ullrey, for his support and help during my graduate
training.

I would also like to thank my parents, Leah and Ruben,
and my family who provided me with the encouragement and

support necessary to complete this project.

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . . . .

REVIEW OF THE LITERATURE . . . . . . . . . . . .
Natural History . . . . . .
Natural Diet and Herbivory in Lizards . . . .
Adaptations to Herbivory. . . . . . . . . . .

Behavioral Mechanisms . . . . . . . . . .
Dentition . . . . . . . . .

Digestive Organs and Digesta Retention.
Volatile Fatty Acids and Microbial

Populations. . . . . . . . .
Temperature and Digestion . . . . . . . . .
Bioenergetics . . . . . . . . .
Digestive Physiology and Function . . . . .

EFFECT OF DIETARY FIBER ON DAILY INTAKE, RATE OF
GROWTH, AND NUTRIENT AND ENERGY METABOLIZABILITY

Introduction. . . . . . . . . . . . .
Materials and Methods . . . . . . . .
Results . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . .

EFFECT OF AMBIENT TEMPERATURE ON DIGESTIVE
FUNCTION, RATE OF GROWTH, AND BIOENERGETICS.

Introduction. . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . .
Animals and housing
Diet and measurement of food intake
Total excreta and skin collections.
Indirect calorimetry. . . . . . .

viii

Indirect calorimetry: respiratory exchange . . . 67
Indirect calorimetry: doubly—labelled water . . . 72
Sample preparation and analyses . . . . . . . . . 78
Calculations and statistical analyses . . . . . . 79
Results . . . . . . . . . . . . . . . . . . . . 84
Discussion. . . . . . . . . . . . . . . . . . . . . .104
Intake. . . . . . . . . . . . . . . . . . . .106
Rate of digestion . . . . . . . . . . . . . . . .106
Rate of digesta passage . . . . . . .107
Nutrient and energy metabolizability. . . . . . .108
Bioenergetics . . . . . . . . . . . .110
Comparison of respiratory exchange
and doubly— labelled water. . . . .113
Analysis of respiratory exchange calorimetry. . .113
Analysis of the doubly— —labelled water method . .114

Comparison of RE calculated by different methods.121
Assessment of the difference between
respiratory exchange and doubly— labelled

water methodologies. . . . . . . . . .123
Comparison of EEre to other
respiratory exchange data. . . . . . . . .124
Comparison of EEre to predicted field
metabolic rate . . . . . . . . . . . .125
Maintenance energy requirement. . . . . .129
Use of energy above maintenance for growth . . .130
Summary . . . . . . . . . .131
CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . .132
LITERATURE CITED . . . . . . . . . . . . . . . . . . . .136

ix

LIST OF TABLES

Table 1. Ingredient composition of three diets
with three levels of dietary fiber. . . . . . . . . .

Table 2. Nutrient composition of three diets
with different levels of dietary fiber. . . . . . . .

Table 3. Mineral composition of three diets
with different levels of dietary fiber. . . . . .

Table 4. Body weight, rate of gain, and feed
efficiency of iguanas fed different levels of
dietary fiber . . . . . . . . . . . . . . . . . .

Table 5. Nutrient and energy intake of iguanas
fed different levels of dietary fiber . . .

Table 6. Nutrient and energy metabolizability
of diets with different concentrations of
dietary fiber when fed to iguanas . . . . . . . . . .

Table 7. Metabolizability of dry matter and
energy and digestibility of neutral detergent
fiber (NDF) in diets fed to herbivorous lizards .

Table 8. Diet composition of offered diet and 99%
confidence intervals of orts composition
(dry matter basis). . . . . . . .

Table 9. Carbon dioxide (C02) production,
oxygen (02) consumption, and gas recoveries from
ethanol combustion trials . . . . . . . . . . .

Table 10. Physical characteristics and daily rate
of gain of iguanas housed at different ambient
temperatures. . . . . . . . . . . . . . . .

Table 11. Nutrient and energy intake and
apparent metabolizability by iguanas housed
at different ambient temperatures . . .

32

38

39

4O

42

45

57

85

87

89

92

Table 12. Nitrogen and carbon intake and
balance, and protein and fat gain of iguanas
housed at different ambient temperatures. . . . . . . . 93

Table 13. Doubly-labelled water kinetics and
pool sizes of iguanas housed at different ambient
temperatures. . . . . . . . . . . . . . . . . . . . . . 96

Table 14. Respiratory exchange calorimetry
gas production rates, and energy expenditure
of iguanas housed at different ambient temperatures . . 98

Table 15. Retained energy, and ratios of CO2
production and energy expenditure measured by
respiratory exchange and doubly-labelled water
(DLW) of iguanas housed at different ambient

temperatures. . . . . . . . . . . . . . . . . . . . . .100
Table 16. Carbon dioxide production, energy

expenditure, and retained energy determined by

different methods . . . . . . . . . . . . . . . . . . .101

Table 17. Resting oxygen consumption and
carbon dioxide production of Iquana iquana
at different ambient temperatures . . . . . . . . . . .126

 

xi

 

LIST OF FIGURES

Figure 1. Schematic drawing of closed—circuit
respiratory calorimeter . . . . . . . . . . . . . . . . 68

Figure 2. Typical regression lines for 18O and
2H turnover for one iguana at two different ambient
temperatures. . . . . . . . . . . . . . . . . . . . . . 75

Figure 3. Regression of retained energy and
metabolizable energy intake of iguanas housed at
different ambient temperatures. . . . . . . . . . . . .103

Figure 4. Relationship between energy expenditure
and ambient temperature for endotherms and ectotherms .112

Figure 5. Comparison of predicted field metabolic
rate (FMR) with actual energy expenditure of
iguanas housed at different ambient temperatures. . . .127

INTRODUCTION

The green iguana (Iquana iquana) has been captured and
eaten by some peoples of Central and South American
countries since ancient times (Werner, 1986; Fitch et al.,
1982). Hunted animals are either eaten by compesinos (local
farmers) or taken to city markets where they are sold. Both
the meat and the eggs (a delicacy) from gravid female
iguanas are consumed (Fitch et al., 1982). These products
can be an important source of food (e.g., protein),
especially for the rural compesinos (Werner, 1986).
Unfortunately, populations of green iguana have diminished
over the past few years (Fitch and Henderson, 1977; Fitch et
al., 1982; Werner, 1986). Some populations of the green
iguana have become extinct (Fitch and Henderson, 1977), and
the species has been declared endangered in most countries
where it is hunted (Fuller and Swift, 1985).

There are at least two reasons for the decline in
population size: 1) habitat destruction by humans, and 2)
unmanaged hunting or overhunting. Slash and burn
agricultural practices destroy iguana habitat (Stoney,
1987). Regrowth of the habitat often takes many years, and
the new habitat may not be sufficient to support populations

of iguanas. The meat and eggs from hunted animals that are

 

2
sold in city markets generally costs more per unit weight
than fish, poultry, pork or beef, but consumers are
apparently willing to pay the additional price. Some
cultures claim a medicinal value (e.g., cure for impotence)
of the meat of iguanas or stew made from the meat (Fitch et
al., 1982). Villa (1968) estimated that 150,000 animals
were eaten annually in Nicaragua. Gravid females burrow in
ground nests to lay eggs. These slow—moving females are
especially easy prey, and their capture is particularly
devastating to future population growth.

In 1983, the Smithsonian Tropical Research Institute in
Panama undertook a project to develop an economically
feasible iguana management project based on the principles
of sustainable agriculture (Werner, 1986; Gradwohl and
Greenberg, 1988). In the late 19805, the project moved from
Panama to Costa Rica and the Fundacién Pro Iguana Verde was
established to continue the project. This project involves
raising iguanas in captivity and releasing them into
appropriate habitats. In release areas where habitats have
been previously destroyed, new and ecologically appropriate
habitats would be established by reforestation (Stoney,
1987). Concomitant with the release of captive—raised
animals into reforested areas are educational programs
designed to teach compesinos the basics of wildlife and
habitat management (Chapin, 1986). The goals of the iguana
management project are: 1) to develop methods for the

rational exploitation of a renewable natural resource, 2) to

promote the conservation of an endangered species, and 3) to
improve habitat by developing an incentive for preservation
or reforestation (Werner, 1986, 1989).

Vital to the success of this intensive animal
production project is an understanding of the nutritional
requirements of the iguana. Appropriately formulated diets
should improve growth rate and animal health, and minimize
food costs. The results of initial nutritional studies in
Panama indicate that such is the case, and increased growth
rate of hatchlings and yearlings in captivity has been noted
(D. Werner, O. Oftedal, M. Allen, unpublished data,
summarized by Allen et al., 1991). However, these studies
were preliminary, and few data are available on the
relationship between diet composition and ambient
temperature on the bioenergetics, nutrient metabolizability,
and growth rate of ectotherms, especially those that are
herbivorous. Thus, more rigorous scientific experimentation
is required.

The goals of the research described here were 1) to
determine the role of dietary plant fiber in modulating
energy intake, growth rate, and nutrient metabolizability,
2) to determine the effect of ambient temperature on
nutrient intake, growth rate, and nutrient metabolizability,
and 3) to estimate the maintenance energy requirements and
efficiency of energy utilization above maintenance of the

green iguana.

REVIEW OF THE LITERATURE

Natural History

The green iguana (Iguana iguana) is primarily an
arboreal species over most of its home range. While egg
laying and tree—to—tree movements occur on or across the
ground, feeding occurs primarily in trees (Rand, 1978).
Iguana iguana inhabits Neotropical forests ranging from
southern Mexico to northern South America, although some
populations are insular (Etheridge, 1982; Taylor, 1956).
Adults can weigh up to 6 kg, reaching a total length of 169
cm and a snout—vent length (SVL) of 51 cm (Swanson, 1950).
Females lay one clutch annually in a deep ground burrow
(Cohn, 1989; Fitch and Henderson, 1977). Clutch size is
dependent on several factors, including female body size.
Larger females produce more eggs. Therefore, by increasing
growth rate of females, egg production may be improved.
Clutch size of wild females ranges between 20—70 eggs, and
females can begin laying eggs in their third year of life
(Dugan, 1982; Wiewandt, 1982). Egg laying occurs between
late January and April, and hatching occurs between late

April and July. Seasonal and geographic variations

4

5
influence the timing of egg laying and hatching (Rand and
Greene, 1982; Harris, 1982; Fitch and Henderson, 1977;
Swanson, 1950).

Natural predators of the green iguana include birds
(e.g., hawks), mammals, other lizards (e.g., the basilisk)
and snakes (e.g., boas) (Van Devender, 1982; Greene et al.,
1978). Mortality of wild hatchling iguanas can be as high
as 95% (Cohn, 1989; Harris, 1982; Van Devender, 1982; Rand
and Dugan, 1980; Rand and Robinson, 1969). This high
mortality rate is an important impetus for captive rearing.
Animals raised in captivity and released at a larger size
may be better able to escape predators. One tactic iguanas
use to avoid detection by predators is to become immobile
when disturbed (Greene et al., 1978; Moberly, 1968a). If
disturbed further, iguanas can escape from predators by
climbing, running, or swimming away (Swanson, 1950). Prime
iguana habitats are trees with branches that overhang a
river or stream. When disturbed, the animals drop into the
water, submerge, and swim away. Moberly (1968a) reported
that animals can drop into the water from a height of 18 m.
It is difficult to determine how far the animals can swim
submerged because it is almost impossible to locate them in
the brush when they surface. Arendt (1986) observed one
iguana swimming submerged for approximately 20 m.
Furthermore, iguanas can remain submerged for extended
periods. Moberly (1968b) conducted diving studies with nine

iguanas and reported that they voluntarily remained

6
submerged for 50—270 min (mean: 82 min). This escape
behavior is possible, in part, because of a high capacity

for anaerobic metabolism (Moberly, 1968b).

Natural Diet and Herbivory in Lizards

It is difficult to determine the natural diet of the
green iguana, in part because of the limited time that is
spent feeding. Moberly (1968a) estimated that green iguanas
were inactive 90% of the time. The limited amount of time
spent feeding, their inactivity, and the difficulty in
observing animals in the field has restricted the amount of
information available regarding the natural diet and feeding
behavior. Anecdotal reports of field researchers have
provided some information (Swanson, 1950; Hirth, 1963;
Henderson, 1974), but a recent report on Iguana iguana
feeding habits from islands and from nearby mainland Panama
has provided some of the first quantitative data on the
natural diet (Rand et al., 1990). Focal animal observations
were made on Iguana iguana for 378 hr (Rand et al., 1990).
Approximately 145 min of the 378 hr of observations were
spent feeding (0.6% of the time). Stomach samples of 31
animals were analyzed and found to contain primarily leaves
from trees and vines. Stomachs from more than half the
animals contained leaves from only one plant species. Other

major components of the diet were blossoms, buds, and

fruits.

There are some sporadic reports of individual iguanas
eating atypical feedstuffs such as animal or insect matter
(Hirth, 1963; Arendt, 1986; Loftin and Tyson, 1965). These
observations appear to be isolated occurrences, but they
have led to confusion about the green iguana’s feeding
strategy. Iguana iguana currently are considered strictly
herbivorous and primarily folivorous (Rand et al., 1990;
Troyer, 1984a,b; Iverson, 1982; Van Devender, 1982).
Iverson (1982) defined truly herbivorous reptiles as those
species whose diet consists only of vegetation throughout
the year. Based on this definition, herbivory in extant
reptiles occurs primarily in the families of Agamidae and
Iguanidae (only three other extant herbivorous lizard
species exist outside these two families) (Zimmerman and
Tracy, 1989). Thus, of the 3,751 species of lizards
(Halliday and Adler, 1986), herbivory is limited to
approximately 50 species (Pough, 1973; Iverson, 1982;
Zimmerman and Tracy, 1989), and folivory is apparently
limited to the green iguana (Rand, 1978).

Several hypotheses have been set forth to explain why
herbivory has not had a greater radiation among lizards.
One hypothesis suggests that herbivorous reptiles are at a
higher predation risk as a consequence of the need to forage
(Szarski, 1962). Ostrom (1963) and Sokol (1967) suggested
that changes in the morphology of the jaw prevented the

evolution of an efficient grinding system to masticate plant

 

8
tissue and initiate plant digestion. Other hypotheses
relate to body size. Herbivory is a feeding strategy found
more frequently in larger lizards (Pough, 1973).
Therefore, Pough (1973) suggested that smaller lizards have
a higher weight—specific energy requirement than larger
lizards even though total energy requirements are greater
for larger lizards. Thus, a relatively low energy dense
herbivorous diet may restrict energy intake too severely for
smaller species. A higher weight-specific energy
requirement may explain the size—related ontogenetic shift
in diet from carnivory or insectivory to herbivory of some
species (Rand, 1978) but does not provide an energetic
explanation for the herbivorous diet apparently consumed by
hatchling green iguanas (Van Devender, 1982). Hypotheses
explaining the radiation of herbivory in lizards have not
been rigorously tested, partially due to the lack of
information on fiber digestion and energy requirements of
herbivorous lizards.

Although young iguana were originally thought to be
insectivorous (Swanson, 1950; Hirth, 1963; Henderson, 1974),
it is now generally accepted that the green iguana is
herbivorous, beginning immediately post—hatching (Van
Devender, 1982; Iverson, 1982; Rand, 1978; Troyer, 1984a,b).
Hatchling weight (approximately 10 g across several
incubation conditions [Werner, 1988]) relative to adult body
mass (perhaps 5000 g) is approximately 0.1—0.3%. Herbivory

in mammals does not become a viable feeding strategy until

9
the digestive system is functional. This occurs
peri—weaning. At this point, mammalian young are
approximately 30% of adult weight (Derrickson, 1986; Troyer,
1984b). The extraordinary 100—fold difference between the
timing of the onset of herbivory in mammals and in the green
iguana is further evidence for the unique adaptations of the

green iguana to its ecological niche.

Adaptations to Herbivory

The digestion of plant fiber by Iguana iguana requires
behavioral, physiological, and morphological specializations
and symbiotic microbial populations. As with all
herbivorous species, mechanisms for the acquisition of
fermentative microbial populations, an ability to tear and
ingest leaves, a capacious organ for fermentation, and a
mechanism for sufficient digesta retention are all important

factors.

Adaptations to Herbivory: Behavioral Mechanisms

Dispersal behavior of hatchlings emerging from the nest
site and social organization of Iguana iguana have been
associated with the acquisition of microbial populations
(Troyer, 1984c; 1984d). In mammals, there is close contact

between young and adults, especially the mother. In

10
reptiles, parental care is limited; therefore, there have to
be other behavioral mechanisms for the acquisition of
microbial populations from the adults. Consumption of older
conspecific feces by hatchlings has been reported (Troyer,
1982, Troyer, 1984c), and it is thought that fecal
consumption is essential to establish microbial populations
in the hindgut. Hatchlings must disperse from their nest
site to have contact with the adults and access to the adult
feces since adult feces are not found in nests (Troyer,
1982). The social contact between hatchlings and older
conspecifics is limited to a short time period since
hatchlings and adults generally occupy different ecological
niches (Troyer, 1984c). Troyer (1982, 1984c) suggested that
the dispersal pattern of hatchlings is influenced by their
need to contact older conspecifics for the acquisition of
microbial populations. However, fecal consumption by
hatchlings does not increase bacterial concentrations in the
hindgut and may increase growth rate only slightly (Troyer,
1982, 1984c).

Geophagy has been reported in Iguana iguana (Troyer,
1984c; Sokol, 1971). Troyer (1984c) suggested that this
behavior is critical for the acquisition of microbial
populations, especially in hatchlings from 0—3 weeks of age.
Hatchlings offered soil had a higher concentration of
bacteria in their hindgut and a slightly greater growth rate
than control iguanas. The significance of Troyer’s (1982,

1984c) findings regarding fecal and soil consumption are not

11
clear since the methods of sample collection and bacterial
counting were not described. There is further confusion
because the species of bacteria in the hindgut were not

identified.

Adaptations to Herbivory: Dentition

Insectivorous lizards can select prey that are small
enough to be swallowed whole (Throckmorton, 1976).
Folivorous species would have a difficult time swallowing
many of the items they ingest (e.g., large leaves). Iguana
iguana possess teeth that allow them to crop leaves, using
head movements around the atlantoocipital joint to assist in
cropping. The teeth are pleurodont (attached to the side of
the jaw), isodont (of equal length), and multi—cusped.

There are 15—20 cusps on the anterior and posterior sides of
the teeth, creating a very sharp serrated edge. The number
of teeth ranges from 19—32 per jaw; older animals with
larger skulls typically have more teeth (Throckmorton, 1976;
Montanucci, 1968; Ray, 1965). Tooth replacement occurs in a
wave pattern, starting with the teeth in the back of the
mouth and proceeding to the anterior of the jaw (Kline and
Cullum, 1984). In young animals, there are six replacement
waves annually, with four teeth added per wave (Kline and
Cullum, 1984, 1985). The fleshy tongue assists in
manipulating the food in the oral cavity, but no mastication

occurs (Throckmorton, 1976).

12

Adaptations to Herbivory: Digestive Organs and Digesta
Retention

A capacious digestive organ and digesta retention are
essential for microbial processing and the fermentative
extraction of energy from plant fiber. The primary site of
fermentation in Iguana iguana is the hindgut. The relative
capacity (defined as the percentage of the wet digesta mass
of the organ compared to empty body mass) of the stomach and
small intestine is higher in the iguana than in mammalian
herbivores (8.7% versus 2.6—3.2%, respectively), and the pH
of the stomach is very low (pH=1.5) (Troyer, 1984a; Parra,
1978). Although there appears to be some disappearance of
hemicellulose in the stomach, this disappearance may be a
result of hemicellulose hydrolysis as a consequence of the
high acid concentration (Troyer, 1984a). As might be
expected, the concentration of volatile fatty acids (VFAs)
is lowest in the stomach and highest in the hindgut (Troyer,
1984a). It is also unlikely that typical anaerobic microbes
would be able to survive at the low pH of the stomach. The
relative capacity of the hindgut (large intestine and
cecum/colon) is 11.8%. In large nonruminants (horse,
capybara) the relative capacity of the large intestine,
cecum and colon combined is 11.4%, and in small nonruminants
the relative capacity of the hindgut (large intestine,
cecum, colon) is 4.7% (Parra, 1978; Troyer, 1984a). The

relative size of the iguana hindgut is comparable to that of

13
mammalian nonruminant herbivores.

Some authors have described the hindgut of Iguana as
consisting of a cecum and colon separated by a valve or
mucosal folds (Parsons and Cameron, 1977; Guard, 1980),
although histological evidence has not been provided.
Iverson (1980, 1982) did not distinguish between these two
compartments and described the hindgut as a colon containing
two types of transverse valves (Iverson, 1980, 1982).
Presumably, these valves increase the absorptive surface of
the hindgut, and are important in regulating digesta
retention. The presence and number of each type of valve is
species—specific (Iverson, 1980; 1982). One type of valve,
the circular valve, contains a sphincter muscle in some
species. The circular valves, if present, are always
proximal to the other type of valve, the semilunar valve.
Iguana iguana has one circular valve and four to six
semilunar valves (Iverson, 1980). The circular valve can
occlude the lumen of the colon by 61—97% (n=20 animals) and
the semilunar valves can occlude the lumen by 14—54%
(Iverson, 1980). There are no significant ontogenetic
changes with respect to the number of valves (Iverson,
1980). This observation provides some evidence that
morphological adaptations for digesta retention, an
important component of herbivory, are present in hatchlings.

Mean transit time of digesta is reported to be 3.1 days
for hatchlings (n=7), 3.6 days for juveniles (n=5), and 5.5

days for adults (n=4) (Troyer, 1984b). These passage rates

14
were determined using glass beads or surveyor’s tape as
markers. The diet during these passage studies was leaves
of Lonchocarpus pentaphyllus. There was no apparent effect
of age of the leaf on mean transit time. However, the
leaves were force—fed. The effect of force—feeding on
digesta passage and nutrient metabolizability or
digestibility in iguanas is unknown. Digesta are retained
in the iguana foregut for less time and in the hindgut for
more time than predicted based on relative weight or length
of the specific organs (Troyer, 1984b).

Passage time for other lizard species has been
calculated by several methods (e.g., mean time, peak
appearance, 25%, 50% or 90% disappearance, transit time),
using different markers with animals fed a variety of diets
(all force-fed) as summarized by Zimmerman and Tracy (1989).
The range of mean retention times in six herbivorous species

is 3.1 days (Iguana iguana) to 5.5 days (Iguana iguana and

 

Sauromalus obesus (chuckwalla)).

Adaptations to Herbivory: Volatile Fatty Acids and

Microbial Populations

lg yitgg rates of volatile fatty acid production have
been measured in two male iguanas (Iguana iguana). Hindgut
contents were removed from the anesthetized males and placed
in plastic bags (the type of plastic was not reported) that
were flushed with oxygen-free carbon dioxide. Hindgut

digesta subsamples were withdrawn from the bags at 15 min

15
intervals for 45 min. For the two animals, the rates of
production (”moles/hr) of acetate were 184 and 168, of
propionate 68.4 and 11.6, and of butyrate 231 and 218.
These values are equivalent to 0.185 and 0.153 kcal of
metabolizable energy/hr (McBee and McBee, 1982). This
amount of energy is estimated to be 30-38% of the required
amount necessary for the animals to sustain themselves under
field conditions (McBee and McBee, 1982; Nagy, 1982a).
Concentrations of VFAs in the Iguana iguana hindgut (from 3
animals) are comparable to the concentrations of VFAs in the
rumen, and higher than in the hindgut of several species of
other nonruminants (Troyer, 1984a; Parra, 1978). These
measurements provide some evidence that fermentation of
fiber occurs in the hindgut, and fermentation of fiber may
be an important source of energy. Nevertheless, the data
represent a limited sample size.

Direct microscopic counts (clump-counts) of formalin
preserved bacteria from hindgut contents of Iguana iguana
(n=11) yielded concentrations of 30x109 clumps of bacterial
cells per gram of digesta. Colony counts from roll tubes in
anaerobic conditions yielded 3.3 to 23.5 x109 bacterial
cells per gram (McBee and McBee, 1982). It is interesting
to note that the dominant bacteria in three animals were
Clostridium species, and in eight animals the dominant

bacteria were Leuconostoc species (McBee and McBee, 1982).

 

Lachnospira species (McBee and McBee, 1982) and Lampropedia

merismopediodes (Troyer, 1982) were also identified. No

16
bacterial species from the genera Bacteroides,
Fusobacterium, or Ruminoccocus have been cultured from
hindgut contents (McBee and McBee, 1982). Ciliated protozoa
have been observed in some samples (Troyer, 1982) but not in
others (McBee and McBee, 1982).

The hindgut of some iguanine lizards (e.g., C clura
species) contains large populations of nematode worms
(Oxyuridae and Atractidae families). A single, apparently
healthy adult Cyclura carinata (ground iguana) had in excess
of 15,000 worms in the colon (Iverson, 1979). This large
infestation has not been observed in the gastrointestinal
tracts of carnivorous or omnivorous lizards. Therefore,
there has been speculation that these nematodes are not
necessarily parasitic but may be commensalistic or
mutualistic (Iverson, 1982). Iverson (1982) proposes that
nematodes may aid in: 1) the mechanical breakdown or mixing
of digesta, 2) producing useful waste products (e.g.,
vitamins, cellulase, VFAs), or 3) controlling the
composition or number of other gastrointestinal tract
microbes. McBee and McBee (1982) reported that nematodes
were not present in remarkable numbers in the gastro—
intestinal tract contents they obtained from 11 animals.
Thus, the role of nematodes in the gastrointestinal tract

remains unclear.

17

Temperature and Digestion

Ectothermic species regulate body temperature primarily
by absorption of heat from the environment (Pough and Gans,
1982), but physiological mechanisms may be important
(Bartholomew, 1982). Behavioral responses to changes in
environmental temperature are particularly important in
maintaining the mean activity temperature. This temperature
is defined as the arithmetic mean of body temperature at
which a free—ranging animal engages in its ordinary routine
(Pough and Gans, 1982). Many physiological processes are
temperature sensitive, and some of these physiological
changes can indeed help maintain a relatively stable body
temperature. Temperature influences metabolic rate
(Moberly, 1968a,b), ventilation rate (Giordano and Jackson,
1973), heart rate and cardiac output (Tucker, 1965; Baker
and White, 1970), peripheral blood flow (Baker et al.,
1972), pattern of glycogen utilization (Stolk, 1960),
acid—base balance (Wood and Moberly, 1970), and embryonic
development (Licht and Moberly, 1965).

The mean body (cloacal) temperature of adult Iguana
iguana was reported to be 36.1 C (range 34.0—37.2 C) when
mean ambient temperature was 29.6 C (range 29.0—32.0 C)
(Hirth, 1963). McGinnis and Brown (1966) reported a mean
cloacal temperature for 14 adults and 5 juveniles of 36.1 C
(31.0—39.0 C) when mean ambient temperature was 32.1 C

(29.0—33.5 C). Moberly (1968a) reported a mean cloacal

18
temperature of 33.8 C (26.0—40.4 C) but did not report the
ambient temperatures. Based on these data, the
self—selected body temperature is probably between 33 C and
36 C. This temperature is defined as the body temperature
maintained by an ectotherm in a temperature gradient,
including temperatures above and below the mean selected
temperature, under controlled laboratory conditions (Pough
and Gans, 1982). Panting begins when cloacal temperature
reaches 42 C (McGinnis and Brown, 1966). The approximate
lethal temperature is 46—47 C, and animals begin to move to
shade when cloacal temperatures are between 41 C and 44 C
(McGinnis and Brown, 1966).

Of particular interest is the effect of temperature on
digestion. It has long been thought that reptiles must
increase body temperature in order to digest their food
efficiently (Huey, 1982). Unfortunately, results have been
conflicting. In a study with Dipsosaurus dorsalis (desert
iguana), gross energy metabolizability increased with
increasing temperature (Harlow et al., 1976). However,
Zimmerman and Tracy (1989) measured gross energy
metabolizability in the same species and reported that it is
unaffected by temperature. In both studies,
metabolizability was determined at 33, 37 and 41 C, and
animals were force—fed. Zimmerman and Tracy estimated the
amounts to be force—fed based on the calculated field
metabolic rate at each temperature. Harlow's group fed the

same amount of food at each temperature. Therefore, Harlow

19

et a1. may have grossly overfed their animals, especially at
the lower temperature. Zimmerman and Tracy suggested that
the overfeeding of the lizards may have resulted in a
decreased metabolizability in Harlow's studies. In a study
with green iguanas, temperature was manipulated by setting
light cycles for either 4 hr or 8 hr of light per day
(Troyer, 1987). Iguanas housed with lights on for 8 hr
maintained higher cloacal temperatures, for a longer period
of time, than the iguanas housed with lights on for 4 hr.
Animals housed under the 8 hr cycle had higher dry matter
metabolizability coefficients than the other group (Troyer,
1987). These animals were also force—fed, and the diet
consisted of Lonchocarpus leaves. Differences in
metabolizability may have been related to differences in
temperatures but the results were confounded by differences
in light:dark cycle.

The degree of nutrient metabolizability is influenced
both by the rate of digesta passage and the rate of
digestion (Van Soest, 1982). Retention time (measured

using 51Cr-labelled fiber) was shorter for Sauromalus obesus

 

(chuckwalla), a herbivorous species, housed at 36 C than for
lizards housed at 32 C (Zimmerman and Tracy, 1989).
Zimmerman and Tracy also concluded that rate of digestion is
slower at lower temperatures. However, Troyer (1987)
reported that there was no significant difference in mean
transit time at two temperatures. One might expect

digestibility to be thermally stable since temperature

20
changes should influence both rate of passage and rate of
digestion in a similar manner. Troyer (1987) speculated
that fluctuations in body temperature may affect
fermentative microbes, perhaps by affecting the number or
the species of microbes present. Small sample sizes,
force—feeding, the use of nutritionally incomplete diets,
and short or no adaptation periods may all contribute to the
apparent confusion about the effect of temperature on

digestion.

Bioenergetics

The energetics of reptiles has received much attention
since the late 18008 (reviewed by Benedict, 1932). There
have been many publications on energy expenditure (average
daily, resting, field metabolic rates, etc.) in reptiles
since the early work. Several publications have summarized
these studies (Bennett and Dawson, 1976; Bennett, 1982;
Congdon et al., 1983; Andrews and Pough, 1985). Data on
energetics have been used to help explain activity patterns,
allometric relationships, feeding strategies, and other
ecological relationships. There has been no attempt to
develop a comprehensive system to describe energy
utilization and flow in reptiles. Such a system can be
critical for comparing animal performance and the value of

different feeds (Lofgreen and Garrett, 1968; Moe and

21
Tyrrell, 1973).

There are several problems associated with measuring
energy expenditure in reptiles (McDonald, 1976). Some of
these problems relate to changes in energy expenditure as a
function of ambient temperature, but temperature can be
easily adjusted under controlled conditions. Other problems
relate to the methods of measurement. Indirect methods of
calorimetry are commonly used to measure energy expenditure.
These methods include open— or closed-respiratory exchange
systems, and doubly—labelled water turnover. There are
inherent problems with measurement of energy expenditure
based on the turnover of doubly-labelled water (fing)
(Lifson et al., 1955; Nagy, 1980; Nagy and Costa, 1980).

However, the 2

HQBO technique is very useful, especially in
long—term studies with free—ranging animals. Oxygen
consumption has been measured by respiratory exchange under
a variety of conditions, and in some studies, energy
expenditure has been calculated based on carbon dioxide
production. Measuring CO2 production is an attractive
method because it is relatively simple, but it is also
inaccurate. Energy equivalents of oxygen consumed or carbon
dioxide produced vary according to the substrate being
oxidized. Measurement of energy expenditure of reptiles
based on CO2 production has additional drawbacks. Reptiles
can have variable respiratory quotients (RQs) that can be
below 0.7. Two possible explanations for the unusually low

RQs sometimes observed in reptiles are: 1) the retention of

22
CO2 as bicarbonate during gastric acid secretion and the
urinary excretion of bicarbonate, and 2) the nasal secretion
of bicarbonate from nasal salt glands (McDonald, 1976;
Schmidt—Nielson et al., 1963). The errors associated with
CO2 production are greater when measurements are made over
short periods of time. Because of these problems, it is
common to report results of energetic experiments directly
as the rate of oxygen consumption rather than to assign
energy equivalents. Another problem related to measuring
energy expenditure indirectly is that many species,
including Iguana iguana, have a relatively large capacity
for anaerobic metabolism (Moberly, 1968a,b). Thus, short-
term measurements may not provide an accurate estimate of
aerobic metabolism.

Field metabolic rate (FMR) is a measurement of average
daily energy expenditure. FMR has been measured in nine
iguanid species, and it is related to body mass as described
by the relationship FMR (kcal/d) = 0.0535body weight (g)°'80
(Nagy, 1982a). This relationship is based on CO2 production
measured using doubly—labelled water (Lifson et al., 1955).
Respiratory quotients (RQ) are used to calculate energy
expenditure from carbon dioxide production, but the ROS used
for the FMR relationship have been measured in only one
herbivorous lizard (Sauromalus obesus, desert iguana)
(RQ=O.93) (Nagy and Shoemaker, 1975) and one insectivorous
lizard (species not identified) (RQ=0.75) (Nagy, 1982). An

additional problem with this allometric relationship is the

23
relatively narrow weight range of the nine species used (0.5
g to 1,481 g; only three species were greater than 100 g and
there is only one observation over 1,000 g). In addition,
most of the nine species are desert—adapted animals (Troyer,
1983). Nevertheless, this equation is an important step in
understanding allometric energy relationships in lizards.

Moberly (1968a,b) measured 02 consumption directly in
Iguana iguana but used animals in a limited size range
(370-1220 9), and the animals were physically restrained.
The relationship between body size and energy expenditure is
important because of its influence on feeding strategy.
Pough (1973) emphasized that smaller animals have higher
weight-specific energy requirements. This fact may have an
impact on the evolution of herbivory in lizards since most
herbivorous species are large. However, even 10 g Iguana
iguana hatchlings are herbivorous (Iverson, 1982).
Therefore, Iguana iguana hatchlings may need to be more
selective feeders than adults to ensure that their diet can
be easily fermented and is high in digestible energy
(Troyer, 1987).

Mammalian arboreal folivores generally have atypically
low metabolic rates (McNab, 1986). Iguana iguana does not
appear to have an unusually low metabolic rate even though
it is an arboreal folivore (Andrews and Pough, 1985). This
analysis is based upon actual oxygen consumption, but the
data were not collected under standardized conditions (e.g.,

animals were physically restrained) (Moberly, 1968a).

24

Digestive Physiology and Function

The digestive physiology differences between
comparably—sized endotherms and ectotherms occur at several
levels, including differences in organ weight, small
intestine length and surface area, and in yitrg substrate
uptake capacity. Liver, kidney, heart and brain are smaller
(by weight) in Amphibolurus nuchalis (body weight = 34 g)
than in Mgg musculus (body weight = 29 g) (Else and Hulbert,
1981). In addition, the length of the small intestine is
approximately half as long in ectotherms (Dipsosaurus
dorsalis, Sauromalus obesus) than in endotherms (M.
musculus, Neotoma lepida) (Karasov and Diamond, 1985;
Ferraris et al., 1989). Furthermore, the total surface area
of the small intestine is smaller in ectotherms than in
endotherms. This difference in surface area may be
important in explaining the slower rate observed in
ectotherms than in endotherms of carrier-mediated transport
of D—glucose and L—proline (Karasov et al., 1985; Karasov
and Diamond, 1985; Ferraris et al., 1989). Ectotherms also
have lower mitochondrial volume density, and smaller cristae
and inner mitochondrial membrane surface areas than
endotherms (Else and Hulbert, 1981).

Metabolizability of nutrients has not been rigorously
studied in reptiles. Limited amounts of daily food
consumption and long rates of digesta passage make

metabolizability studies difficult to conduct and

25

time-consuming. Digestion of dry matter, energy, and fiber
has been determined in several species of lizards and
summarized by Zimmerman and Tracy (1989). There have been
some technical problems associated with the measurement of
digestibility of these nutrients. In most studies performed
to date, animals have been force—fed. In many studies,
animals have been fed nutritionally incomplete diets. For
instance, some diets used to measure digestibility have been
sweet potato tubers (Throckmorton, 1973), Opuntia fruit
(Christian et al., 1984), or dandelions (Ruppert, 1980). A
commercially available rabbit diet has been used in some
studies (Harlow et al., 1976; Zimmerman and Tracy, 1989).
Dry matter metabolizability ranges from 45—86% (Karasov et
al., 1986; Voorhees, 1981), gross energy metabolizability
ranges from 48-86% (Christian et al., 1984; Throckmorton,
1973) and neutral detergent fiber (NDF) digestibility ranges
from 21—81% (Karasov et al., 1986; Voorhees, 1981). These
values should be regarded as preliminary because of the
potential problems associated with force—feeding, inadequate
adaptation to diet, and potentially inappropriate diets.

Troyer (1984a) measured dry matter and gross energy
metabolizability of Lonchocarpus pentaphyllus leaves at
different stages of maturities in hatchling (n=9), juvenile
(n=10) and adult (n=4) Iguana iguana. The animals were
force-fed and there was no indication of an adaptation
period to the diet. Dry matter digestibility ranged from

46—53% and NDF digestibility ranged from 38-57%. There was

26
no effect of animal age (hatchling vs juvenile vs adult) on

digestibility.

EFFECT OF DIETARY FIBER ON DAILY INTAKE, RATE OF GROWTH, AND
NUTRIENT AND ENERGY METABOLIZABILITY

INTRODUCTION

The green iguana (Iguana iguana) is a herbivorous
lizard. This feeding strategy is unusual in extant lizards,
and it occurs primarily in two families. Furthermore, of
the 3,751 lizard species (Halliday and Adler, 1986), the
green iguana is probably the only truly folivorous lizard
(Rand, 1978). Thus, its ability to digest plant fiber is
interesting from an ecological and evolutionary perspective.
Aside from its unusual feeding strategy, the green iguana
has also been an important source of nutrients, particularly
protein, for some of the people in Central and South
America. People in these regions have hunted and consumed
this species for many years. Overhunting and habitat
destruction have decimated local populations and have
prompted the development of new techniques for captive
management and farming of this species.

Reptilian herbivores may be different from mammalian
herbivores in many respects, although it appears that the
process of fiber digestion is similar. Anaerobic microbial

species have been identified from the hindgut of the green

27

 

28
iguana (McBee and McBee, 1982), including cellulolytic
species. Volatile fatty acid production rates have been
measured in samples of digesta taken from the hindgut
(Troyer, 1984a; McBee and McBee, 1982). Furthermore,
morphological adaptations in the hindgut provide a mechanism
for digesta retention for fermentation (Iverson, 1980,
1982).

While there may be similarities in fiber digestion,
there are many metabolic and feeding differences between
herbivorous mammals and reptiles. Some of these differences
include metabolic rate, thermoregulatory mechanisms,
comminution patterns, and digestive capacity. Metabolic
rates are lower in reptiles and thermoregulation is achieved
primarily by behavioral responses to ambient conditions.
Comminution patterns are different in reptilians as a
consequence of jaw structure. In addition, digestive
capacity may be lower in reptilian herbivores than in
comparably—sized mammalian herbivores (Karasov et al., 1985;
Ferraris et al., 1989).

Diet metabolizability and digestibility has been
determined in some species of reptilian herbivores but
methodological problems with feeding may have biased
previously reported data. Diet formulation for captive
green iguanas, and its impact on animal performance are key
issues in developing diets for captive management of this
important source of nutrients for people of Central and

South America. The goal of this research project was to

29
determine if established methods of assessing diets, such as
digestion and metabolism trials, could be applied to the
green iguana. In addition, the relationships between plant
fiber intake and nutrient digestibility, metabolizability,
and growth rate are not known. Therefore, this study was
designed to determine the effect of three different levels
of dietary plant fiber on dry matter intake, fiber
digestibility, dry matter and energy metabolizability, and
growth rate of captive green iguanas fed a manufactured

diet.

 

30

MATERIALS AND METHODS

Twenty—one individually-housed green iguanas (Iguana
iguana) were used to determine the effect of dietary fiber
on nutrient intake, digestibility, metabolizability, and
growth rate. The animals were hatched in Panama as part of
a captive breeding program and brought to the National
Zoological Park (Washington, DC) for this study. The
iguanas were approximately 2.5 yr old at the beginning of
this study. The study began in May, 1988 and ended in
April, 1989. Many of the animals were infested with
protozoa or other flagellate parasites (observed either
directly or by flotation techniques) and all animals were
treated orally with ivermectin (MSD Agvet, Rahway, NJ) twice
prior to the start of the study. Animals were housed in
cages with plywood sides and backs, and hardware cloth tops
and floors. There was a shelf in each cage. The cage was
approximately 66 cm wide, 53 cm high, and 51 cm long.
Overhead fluorescent lights were on a 12:12 L:D cycle, and
suspended approximately 0.5 m over each cage was an infrared
light to provide heat. A sheet metal tray was placed under
the hardware cloth bottom of each cage to collect excreta.
All procedures used in this study were approved by the
Animal Welfare Committee of the National Zoological Park.

This study was designed as a Latin-square crossover

31
study. Three levels of dietary fiber were fed to each
iguana. The iguanas were blocked into homogeneous groups
based on body weight (a total of 7 blocks) and randomly
assigned to one of three diets, and fed each diet in a
random order. The iguanas consumed the diet for
approximately 12 wk, and then they were gradually switched
to a different diet (over a 7 to 10 d period). Thus, each
iguana received each one of the three diets during the three
different periods.

The diets were meal—type diets that the iguanas readily
consumed without the need for force-feeding. Diets were
purchased from a commercial supplier (Zeigler Bros. Inc.,
Gardners, PA) and stored in a freezer for the length of the
study. Two batches of diet were purchased. One batch was
used for periods 1 and 2 and the second batch was used for
period 3. Nutrient intakes were calculated based on diet
composition of each batch in order to control for small
changes in nutrient concentrations between the two batches.
The three diets were similar in chemical composition except
for the concentrations of plant fiber. Plant fiber levels
were manipulated by altering the relative amount of corn,
soybean meal and alfalfa meal. Small adjustments were made
in the limestone concentration of the diets to maintain an

appropriate calcium—to—phosphorus ratio (Table 1).

 

32

Table 1. Ingredient composition of three diets with three

levels of dietary fiber (% of total).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ingredient Low Medium High
fiber fiber fiber
Corn, ground 27.7 16.7 5.7
Soybean meal, 48.5% CP 36.5 33.0 29.5
Alfalfa meal, dehy, 17% CP 10.0 25.0 40.0
Wheat bran 15.0 15.0 15.0
Soybean oil 3.0 3.0 3.0
Meat and bone meal 2.0 2.0 2.0
Molasses, cane 2.0 2.0 2.0
Limestone 2.0 1.5 1.0
Calcium phosphate, dibasic 0.8 0.8 0.8
Vitamin premix1 0.4 0.4 0.4
Salt 0.4 0.4 0.4
Mineral premix2 0.2 0.2 0.2

 

 

 

 

1Formulated to contain 4,000,000 IU vitamin A/kg, 600,000 IU vitamin
D/kg, 48,000 IU vitamin E/kg, 1,200 ppm vitamin Bl, 2,000 ppm vitamin
82, 2,400 ppm vitamin 8,, 36,000 niacin, 9,600 pantothenic acid, 100
ppm biotin, 1,600 ppm folic acid, 6 ppm vitamin Bu, 600,000 ppm

choline chloride.

2Formulated to contain 40,000 ppm iron, 4,000 ppm cooper, 40,000 ppm

zinc, 52,000 ppm manganese, 160 ppm iodine,

80 ppm selenium.

 

 

33

Animals were weighed, and cloacal temperature and
snout—vent length were measured at the beginning and end of
each period. Animals were weighed on an electronic balance.
Snout—vent length was measured by placing the snout of the
iguana flush with the end of a board and marking the
location of the cloaca. cloacal temperature was measured
using an electronic thermometer, immediately after the
iguanas were removed from their cages to avoid a temperature
rise associated with agitation due to handling. The
thermocouple was lubricated with KY jelly and placed at
least 6 cm into the cloaca. The temperature was recorded
after approximately 1 min.

Iguanas were offered fresh diet three times per week
(Monday, Wednesday and Friday), usually in the morning.
Enough diet was offered to ensure that diet was always
available. Amounts offered were increased as the iguanas
grew, and their intake increased. Food consumption was
determined by weighing the amount of diet offered and
reweighing the uneaten portion 24 or 48 hr later. Any diet
that had spilled was collected and reweighed. The amount of
diet consumed was determined as the difference between the
amount offered and the amount reweighed.

Metabolism trials were started after the iguanas had
consumed the diet for approximately 8 wk. To determine the
starting and ending points of excreta collection, iguanas
were pulse—dosed with 0.15% brilliant blue (FD&C #1, Allied

Chemical Corp., Morristown, NJ) in their diet for 1 day.

 

34

Collections started when the marker appeared in the feces.
Fecal and urinary excreta were collected together and pooled
for an individual over the collection period, which lasted
approximately 4 wk. The samples were collected into pre—
tared containers. During the collection periods, fresh diet
was offered on a daily basis. Samples of at least 5 kg of
diet were collected and stored in a freezer for subsequent
nutrient and energy analyses.

Excreta samples were freeze—dried and immediately
reweighed. The samples were then ground in a Wiley mill
fitted with a 2-mm screen. Subsamples were dried in a
forced-air convection oven at 105 C to determine dry matter
content of the previously freeze-dried samples. The
corrected dry matter was determined as the product of the
freeze-dried weight and the weight of the oven—dried
subsamples. Diets and excreta were analyzed for ash,
neutral detergent fiber, acid detergent fiber, acid lignin,
and gross energy. Diets were also analyzed for crude fat
and minerals. Crude fat was determined by methylene
chloride extraction (FES 80, CEM Corp., Matthews, NC).
Minerals were analyzed by a commercial laboratory (New York
Dairy Herd Improvement Corp., Ithaca, NY). Ash was
determined by combustion at 650 C. Gross energy was
determined by adiabatic bomb calorimetry (Parr Instrument
Co., Moline, IL). The residue remaining after combustion of
the subsample in the bomb was titrated with 0.0725 N sodium

carbonate to correct for the heat of formation of nitric

35
acid and sulfuric acid. Fiber fractions were determined by
the methods of Van Soest (Robertson and Van Soest, 1981)
using a Fibertec system (System M, Tecator AB, Haganas,
Sweden). Heat—stable a-amylase (Sigma Chemical Co., St.
Louis, MO, catalog # A-3306, 200 ul/sample) and protease
(Sigma P-3910, 5 mg/sample) were used to facilitate
filtration during fiber analysis. Neutral detergent fiber
(NDF) samples were preincubated with a-amylase for 1 hr at
60 C and then incubated with the protease for 1 hr at 80 C
prior to refluxing with the neutral detergent. Cellulose
was calculated as the difference between acid detergent
fiber (ADF) and acid lignin (AL) and hemicellulose was
calculated as the difference between NDF and ADF.

The excreta was comprised of a combination of fecal and
cloacal wastes. The apparent digestibility of fiber
fractions could be calculated since there were no
quantitatively significant fiber components excreted in non-
fecal wastes. However, for other nutrients and energy,
metabolizability coefficients were calculated since the
excreta contained a non-fecal source of these nutrients and
energy. Metabolizable energy intakes were determined as the
difference between gross energy intake and gross energy of
the excreta. No correction was made for energy lost in skin
through ecdysis. Nutrient intakes were calculated on a
daily basis to correct for differences in the length of the
collection periods for different iguanas. Metabolizability

and digestibility coefficients were calculated using the

36
direct method (without internal or external markers) since
total excreta collections were feasible (Schneider and
Flatt, 1975). Apparent metabolizability or digestibility

coefficients were calculated as:

nutrient intake g or kcal) - nutrient excreted (g or kcal) 100
nutrient intake (g or kcal)

Mean daily growth rate was determined as the difference
between final and initial weight, divided by the number of
days between weighing.

Data were analyzed with PC—SAS version 6.03 (SAS
Institute Inc., Cary, NC). The data were first analyzed for
normality (PROC UNIVARIATE) and subsequently analyzed for
homogeneity of variance. Variance was analyzed by Spearman
correlation analysis of the absolute values of the residuals
and predicted values, and by graphical analysis of the plot
of the residuals and predicted values. If these two
assumptions of analysis of variance were met (normality and
homoscedasticity of variance), analysis of variance was used
to determine the effects of diets (PROC GLM). The
statistical model included square, iguana(square),
period(square), and diet. Least-squares means (LSMEANS) and
probability of the difference of the least—squares means
(PDIFF) were used to determine diet effects. Gross energy
of feces from one animal during one period (diet 2) was not
determined (insufficient sample size) so metabolizable
energy content and energy metabolizability was not

determined for that individual.

37

RESULTS

Mean cloacal temperature of the iguanas used in this

study was 31.8 x 0.2 C (n=48). Chemical composition of the

three diets is presented in Tables 2 and 3. Mean initial
body weight of iguanas is presented in Table 4. Although
the iguanas were randomly assigned to a sequence of diets,
at the beginning of each period, mean body weight of iguanas
on the medium fiber diet (606.3 g) was lower than mean body
weight when the iguanas started the low fiber diet (540.1 g,
p<0.0425). Mean initial body weight of iguanas fed the high
fiber diet was 601.4 9. Mean initial body weights were not
different between iguanas when fed the high fiber diet and
medium fiber diet, or when fed the high fiber and low fiber
diets.

Mean final body weight was lower when iguanas were fed
the high fiber diet (750.1 9) than when they were fed the
medium fiber diet (861.2 g, p<0.0041). Mean final body
weight was also lower when iguanas were fed the low fiber
diet (769.8 9) than when they were fed the medium fiber diet
(p<0.0155) (Table 4).

Rate of body weight gain was lowest when iguana were

fed the high fiber diet (1.42 g/d). This rate was lower

Table 2.

dietary fiber.1

38

Nutrient composition of three diets with different levels of

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nutrient Low fiber Medium fiber High fiber
Mean SE Mean SE Mean SE
Dry matter (%) 91.4 0.8 91.7 0.6 92.0 0.6
Crude protein (%)2 29.3 0.4 28.7 0.4 29.1 0.3
Crude fat (%) 6.2 0.1 6.4 0.2 5.8 0.2
Crude fiber (%)2 5.9 0.1 9.3 0.5 12.4 0.7
Acid detergent 11.0 0.2 16.0 0.3 19.6 0.6
fiber (ADF) (%)
Neutral detergent 18.6 0.6 23.6 0.6 27.3 0.7
fiber (NDF) (%)
Lignin (%) 2.5 0.1 4.5 0.1 6.5 0.8
Cellulose (’6)3 8.5 11.5 13.1
Hemicellulose (%)‘ 7.6 7.6 7.6
Ash ($3) 8.5 0.1 8.8 0.1 9.5 0.1
Gross energy 4.51 0.02 4.52 0.01 4.51 0.03
(kcal/g)

 

 

 

 

 

 

 

1 Mean of 2-6 analyses.
2 Analyzed by commercial laboratory.
3 Calculated as difference between ADF and lignin.

‘ Calculated as difference between NDF and ADF.

 

39

Table 3. Mineral composition of three diets with different levels of

dietary fiber.1'2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nutrient Low fiber Medium fiber High fiber
Mean SE Mean SE Mean SE
Calcium (%) 2.02 0.21 2.06 0.06 1.96 0.20
Phosphorous (%) 0.87 0.04 0.83 0.04 0.82 0.05
Magnesium (%) 0.30 0.01 0.31 0.02 0.33 0.00
Potassium (%) 1.61 0.03 1.80 0 01 2.05 0.05
Sodium (%) 0.20 0.02 0 22 0.02 0.20 0.01
Iron (ppm) 471 16 553 40 615 4
Zinc (ppm) 253 5 278 12 267 16
Copper (ppm) 33 2 35 3 35 2
Manganese (ppm) 293 2 327 23 317 25
Molybdenum (ppm) 2.9 0.4 3.9 0.4 3.7 0.1
Selenium (ppm) 0.38 0.00 0 41 0 03 0.43 0.02

 

 

 

 

 

 

 

1 Mean of 2 analyses

2 Analyzed by commercial laboratory

 

40

 

 

 

 

 

 

 

 

 

 

 

 

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41

(p<0.0037) than when the iguanas were fed the medium fiber
diet (2.35 g/d) or when they were fed the low fiber diet
(2.22 g/d, p<0.0106). There was no difference in rate of
body weight gain when the iguanas were fed the medium and
low fiber diets (Table 4).

Nutrient and energy intakes are presented in Table 5.
The minimum length of the excreta collection period was 7 d
and the maximum length was 25 d. As a consequence of the
difference in the length of the collection periods, these
data are presented on a daily basis. Daily dry matter
intake was also measured during the entire length of the
three periods as well as during the shorter excreta
collection period. The first period lasted between 88 and
99 d (mean 90 d), the second period lasted between 88 and
116 d (mean 104 d), and the third period lasted between 110
and 131 d (mean 122 d). Daily dry matter intake was not
different between the three diets. During the complete
period, the overall mean daily dry matter intake was 9.71
g/d/kg BW. During the excreta collection period, the
overall mean daily dry matter intake was also not different
between the three diets. The range in daily dry matter
intake was from 1.08 to 15.98 g/d/kg BW and the overall mean
daily dry matter intake during the fecal collection period
was 7.14 g/d/kg BW. Likewise, daily organic matter, gross
energy and metabolizable energy intakes were not different

between the three diets. NDF, ADF, hemicellulose, and

42

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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43
cellulose intakes were not different between the medium and
high fiber diets but NDF and ADF intakes were lower on the
low fiber diet than the high fiber diet, and lower on the
low fiber diet than the medium fiber diet. On the
otherhand, cellulose and hemicellulose intakes were not
different between the three diets.

Nutrient and energy metabolizabilities, and fiber
digestibilities are presented in Table 6. In general,
metabolizability of nutrients and energy decreased with
increasing concentrations of dietary fiber. Dry matter,
organic matter and gross energy metabolizability were
highest on the low fiber diet and lowest on the high fiber
diet. Mean dry matter digestibility was 65.8% on the low
fiber diet and 57.8% on the high fiber diet. Mean dry
matter digestibility of the medium fiber diet was 62.2%.
Dry matter metabolizability was not different between the
medium and low fiber diets (p<0.0680). Organic matter
metabolizability was 69.6%, 65.7%, and 60.7% for the low,
medium, and high fiber diets, respectively. Gross energy
metabolizability was 70.7%, 66.4%, and 62.3% for the low,
medium, and high fiber diets, respectively.

Neutral detergent and acid detergent fiber
digestibilities were not different between the medium and
high fiber or between the low and medium fiber diets.
However, NDF and ADF digestibilities were higher on the low
fiber diet than the high fiber diet. Mean NDF digestibility

of the low fiber diet was 47.1%, and was 38.5% on the high

44
fiber diet (p<0.0026). Mean ADF digestibility was 40.7% on
the low fiber diet and 27.8% on the high fiber diet
(p<0.0103). Cellulose digestibility was higher (p<0.0266)
on the low fiber diet (44.4%) than on the medium fiber diet
(34.4%), and was higher (p<0.0003) on the low fiber diet
than the high fiber diet (21.5%). However, cellulose
digestibility was not different between the low and medium
fiber diets. Hemicellulose digestibility was not different
between the medium and high fiber diets (58.5% and 58.6%,
respectively) but was lower on the low fiber diet (27.5%)

(p<0.0106 and p<0.0108, respectively).

 

45

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46

DISCUSSION

The digestive tract morphology of the green iguana is
well suited to support a symbiotic population of microbes
for fiber digestion and to retain digesta for fermentation.
In fact, the extent to which fiber digestion occurs has been
measured in the green iguanas as well as in several other
herbivorous reptilian species (Troyer, 1984a,b; Zimmerman
and Tracy, 1989). All species in the subfamily Iguaninae,
except one, possess two types of valves in the colon: 1) the
circular valve, and 2) the semilunar valves (Iverson, 1980;
1982). The number and types of valves present is species
specific (Iverson, 1980). It is thought that the function
of these valves is to control digesta retention. The green
iguana can retain digesta in the hindgut for fermentation as
assessed by VFA production and fiber fraction disappearance
(Troyer, 1984a). In fact, digesta is retained in the
hindgut longer than expected.(X?'test) based on the relative
volume and length of the hindgut as compared to the foregut
and small intestine (Troyer, 1984a).

Several theoretical models have been suggested to
explain and describe the relationship between the two
competing processes of 1) rate of digesta passage, and 2)
rate of digestion (especially fermentation), and how these

two processes may interact and influence daily intake and

 

47
animal performance. Most of these models have been
developed for mammalian herbivores and have not been applied
to non—mammalian species. The physical form of the diet is
one important factor in determining physiological responses
(e.g., food intake, nutrient digestibility, rate of digesta
passage) to dietary manipulations. The relationship between
particle size and voluntary feed intake has been known for
many years (Minson, 1963) and continues to be of interest
(Weston and Kennedy, 1984). The relationship between
changes in dietary plant fiber intake and nutrient intake,
metabolizability and growth rate has not been studied in
reptilian herbivores.

In this study, the iguanas were blocked by body weight
and randomly assigned to a sequence of three levels of
dietary fiber. Nevertheless, mean initial body weights were
different between iguanas at the start of each of the three
periods. The most extreme difference in mean initial body
weight (66 g) was between iguanas fed the low fiber and the
medium fiber diets. As a consequence of this difference,
nutrient and energy intakes were adjusted to a per unit body
weight (kg) basis for more appropriate comparisons between
iguanas fed different diets.

Daily dry matter, organic matter, gross energy, and
metabolizable energy intakes were not affected by changes in
the concentration of dietary fiber. However, there were
differences in the apparent metabolizability of these

nutrients. Increased concentrations of dietary fiber

48
decreased metabolizability of these nutrients. These diets
were formulated to contain different amounts of NDF and ADF,
but the ratio of these two fiber fractions was similar
between all three diets. Thus, the difference between NDF
and ADF (the hemicellulose) was not different between the
three diets. The difference in cellulose concentration was
not the same for all diets, but the magnitude of increase
from low to high fiber diets was not as great as that for
NDF or ADF. Thus, there was no difference in cellulose and
hemicellulose intake between the three diets. On the other
hand, NDF and ADF intakes were different between the high
and low fiber diets and the medium and low fiber diets, but
intakes were not different between the high and medium fiber
diets. The absence of a difference in NDF and ADF intake
between the medium and high fiber diets may be related to a
slight decrease in daily dry matter intake from the medium
to the high fiber diet even though this change in daily dry
matter intake was not significant. The increase in fiber
concentration did not offset the decrease in intake, so
daily fiber intakes were not different.

Fiber fraction digestibility (NDF, ADF, hemicellulose,
and cellulose) were affected by fiber concentration. In
general, fiber fraction digestibility increased with a
decrease in NDF and ADF concentration. However, fiber
fraction digestibility was not different between the medium
and high fiber diets (except for cellulose), nor between the

low and medium fiber diets (except for hemicellulose).

49
Fiber digestibility of the medium fiber diet was medial to
that of the low and high fiber diets. However, the pattern
of hemicellulose digestibility was different. Hemicellulose
digestibility was not different between the medium and high
fiber diets but was much lower on the low fiber diet than on
the medium and high fiber diets. This difference may be
related to analytical problems in determining NDF and ADF,
especially in feces, and because hemicellulose is determined
as a difference. Small errors in NDF and ADF analyses may
result in a relatively large error in estimates of
hemicellulose content (Van Soest, 1982; McAllan and
Griffith, 1984). Furthermore, the endogenous fecal
component may include compounds that are measured as part of
the ADF fraction, and these compounds may be proportionally
greater in the excreta from iguanas fed the low fiber diet
than the other diets.

Differences in fiber digestion between the diets,
especially the large difference between the low and high
fiber diets, may be related to non-selective retention of
feed particles in herbivorous lizards (Foley et al., 1987).
Thus, all feed particles would have a similar rate of
passage with little or no compensation for changes in diet
composition. There is evidence that even fine particles can
be selectively retained in the hindgut of mammalian
fermenters, such as the rabbit (Warner, 1981) and in the
ringtail possum (Pseudocheirus peregrinus) (Chilcott and

Hume, 1985). Foley et a1. (1987) reported that selective

 

50
retention of fine particles in the hindgut of Uromastyx

aeqvptius (a herbivorous Agamid lizard) did not occur, but

 

the method of determining particulate retention was not
described. If there is limited or no selective retention of
particles, then the higher concentration of fiber fed to the
iguanas on the high fiber diet in this study may have been
more refractory to microbial attack. Microbial attachment
requires time (Akin, 1986). Without additional retention
time relative to the lower fiber diets, fiber digestion may
be expected to be lower on the high fiber diet, as was
observed (Table 6). Furthermore, if there was a change in
digesta retention, it would be likely that daily dry matter
intake would change, and no change in daily dry matter
intake was detected (Table 5).

Another possible explanation for the observed decrease
in fiber digestibility on the high fiber diet is related to
the lignification index (percent lignin in the ADF) of the
three diets. This index shows a strong negative
relationship with NDF digestibility in_yiyg, presumably due
to lignin’s ability to resist microbial attack (Deinum,
1971; Van Soest, 1982; Jones and Wilson, 1987). The
lignification index of the three diets was approximately
23%, 28%, and 33%, for the low, medium and high fiber diets,
respectively (Table 2). The higher lignification index of
the high fiber diet would suggest that this diet would have
the lowest NDF digestibility, and that the low fiber diet

would have the highest NDF digestibility. These predictions

 

 

51
are supported by the data collected from these green iguanas
(Table 5).

Although dry matter intake and metabolizable energy
intake were similar for all diets, energy metabolizability
was different between all three diets, and it was lowest on
the high fiber diet. Thus, the ME density (kcal/g dry
matter consumed) was different in all three diets. The ME
density was lowest on the high fiber diet and highest on the
low fiber diet. Furthermore, the rate of daily gain was
lowest, and the ratio of feed-to—gain (feed efficiency) was
highest on the high fiber diet. There were no differences
between the daily rate of gain and feed efficiency between
the medium and low fiber diet but there were differences
between the high fiber diet and the low and medium fiber
diets.

Mammalian hindgut fermenters and ruminants generally
respond to a decrease in digestible energy (DE) density of
ground diets by increasing food intake. Thus, total energy
intake and gain can remain constant. Rabbits can compensate
with increased intake for decreased DE density from 3.51 to
2.08 kcal/kg DM to maintain a constant growth rate
(Spreadbury and Davidson, 1978). Similarly, horses can
compensate with increased intake for decreased DE density
from 3.39 to 2.61 kcal/kg DM and maintain a constant growth
rate (Laut et al., 1985). Foregut fermenters respond to
changes in energy density in a similar manner (Montgomery

and Baumgardt, 1965). The response to changes in DE density

52
from long or coarse forages is opposite to that of ground
diets. This has been documented in ruminants (Minson,
1982), foregut marsupial fermenters (McIntosh, 1966; Forbes
and Tribe, 1970), as well as in hindgut fermenters (e.g.,
horses) (Darlington and Hershberger, 1968).

The iguanas in this study were fed a meal—type diet
that consisted of relatively small particles. There was
little change in daily dry matter intake of the iguanas in
this study in response to a change in energy density (in
this case ME). However, the energy density change may not
have been severe enough to alter daily dry matter intake.
The change in ME density was only from 2.74 kcal/g dry
matter to 2.39 kcal/g dry matter. This difference is
smaller than the ranges that elicited a response in
mammaliandherbivores.

As a consequence of the relatively slow rate of digesta
passage and large amount of individual variability (e.g., 1—
8 d for the brilliant blue marker to appear in the feces),
it was not possible to quantitate the rate of digesta flow
of the iguanas in this study. Based on the observation that
the brilliant blue was usually excreted as a single bolus on
a single day, there was little evidence for much mixing or
selective retention of the digesta. There was a clear and
clean demarcation of the marked feces (e.g., those feces
that were blue/green) with other feces, even when unmarked
feces were excreted at the same time. This observation may

provide additional support for Foley’s conclusion based on

53
his observation in Uromastyx (Foley et al., 1987). However,
the observation of little mixing or selective retention in
the iguana may be a function of the type of diet fed, and
selective retention may be more important when the more
natural diet, such as fruits, flowers, and leaves, are
consumed.

Nutrient and energy digestibility has been measured by
others in herbivorous lizards, including the green iguana.
These data are summarized in Table 7 and are adapted from
Zimmerman and Tracy (1989). In many of these studies, the
animals were force-fed (Ruppert, 1980; Zimmerman and Tracy,
1989; Troyer, 1984a,b, 1987; Karasov et al., 1986). In
several studies, the lizards were fed only one item, such as
sweet potato tubers, dandelion leaves, or Lonchocarpus
leaves (Throckmorton, 1973; Ruppert, 1980; Troyer, 1984a,
1987). In the study reported by Throckmorton (1973), it
appears that the lizards were fed only sweet potato tubers,
and they were fed sweet potato tubers for at least four
months prior to the start of measurements. In other
studies, adaptation periods for these herbivorous species
were as short as four days (Ruppert, 1980). Adaptation
periods as short as four days may be inappropriate since
many of these lizard species have mean retention times as
long as 7 days (Zimmerman and Tracy, 1989). The effects of:
1) force—feeding, 2) monotypic diets, or 3) short adaptation
period may result in data that are difficult to interpret.

Dry matter metabolizability from previous studies ranged

 

54
from 45% to 86%, with an overall mean of 60% (Table 7). The
dry matter metabolizability from the current study ranged
from 58% to 68%, depending on the level of dietary fiber
(Table 6). Energy metabolizability of diets fed to lizards
in previous studies ranged from 48% to 83%, with an overall
mean of 65%. Gross energy metabolizability of diets fed to
iguanas in the current study ranged from 62% to 71%.
Neutral detergent fiber digestibility from previous studies
ranged from 21% to 81%, with an overall mean of 44%.
Neutral detergent fiber digestibility in the current study
ranged from 38% to 47%. Thus, despite potential
methodological problems with previous studies, the overall
mean from these studies agrees well with data collected from
iguanas fed ad libitum, a nutritionally complete diet, and
adapted to the diet for six to eight weeks prior to excreta
collections.

Unfortunately, dry matter intake from several of the
previous studies has not been reported. In several
instances, investigators that force—fed their lizards fed
the lizards an amount based on the estimated energy
expenditure from an equation published by Nagy (1982a)
(Ruppert, 1980; Troyer, 1987). Dry matter intake is not
reported in other studies where lizards were fed ad libitum
(Throckmorton, 1973; Christian et al., 1984).

The ability of green iguanas to digest fiber from these
diets was comparable to that of other mammalian herbivores,

including ruminants (Parra, 1978). Of course the absolute

 

55
amount of fiber digested on a daily basis was relatively low
and the growth rate was relatively slow. However, the feed
efficiency of these iguanas was comparable to that of
domesticated species used for productive purposes (NRC
1984a,b, 1985, 1988). Again, the absolute rate of gain was
relatively slow compared to production animals.

The data from these iguana provide important baseline
data on intake, growth rate, fiber digestibility, and dry
matter and energy metabolizability. This information is
critical to formulation of diets for captive management and
production. Daily intake and growth rate of iguanas when
fed the medium fiber diet (24% NDF, dry matter basis) was
not different from that when the same iguanas were fed the
low fiber diet (19% NDF, dry matter basis). When the same
iguanas were fed the high fiber diet (27% NDF, dry matter
basis), daily dry matter intake did not change but growth
rate and digestibility were depressed. However, these data
are based on diets that may not reflect the diet of free—
ranging green iguanas. Differences in the physical form of
the diet consumed by free-ranging iguanas (e.g., large
pieces of unmasticated leaves) may have a large impact on
intake, growth rate, and digestibility, even with leaves of
comparable nutrient composition. It is interesting to note
that dry matter and neutral detergent fiber digestibilities
of fresh picked Lonchocarpus leaves (47% NDF, dry matter
basis) were similar to those of the diets used in the

current studies (Troyer, 1984a, 1987) even though the NDF

 

56
content of the leaves was higher than the diets used in this

study.

57

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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58

 

 

 

 

 

 

 

 

 

 

 

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00 mm mm ~0050000 .0000000 0300005 m05000> 0500000500
on 3000 000000 mm 0050000 0500000500
av mm 3000 000000 mm m050000 0500000500
om mm 0000 000000 mm m050000 0500000500
00 00>000 000000000 hmlom o050000 0500000500
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0000000000000\>000000N0000000: 0000 00500000008 0000000

 

 

 

 

.0005000000v 0000000 05000>00000 00 000 00000 00 Am0zv 00000
000000000 0000500 00 0000000000000 000 000000 000 000000 >00 00 0000000000000002 .5 00009

 

EFFECT OF AMBIENT TEMPERATURE ON DIGESTIVE FUNCTION, RATE OF
GROWTH, AND BIOENERGETICS

INTRODUCTION

Ambient temperature is an important modulator of
physiological and behavioral responses that occur in
ectotherms, such as the green iguana (Iguana iquana).
Temperature affects many diverse physiological functions,
ranging from arterial oxygen concentration to water flux,
but the effects of temperature on digestive physiology and
bioenergetics are not well understood. Herbivory is
relatively rare in reptiles, so many studies of digestive
function have been performed using insectivorous or
carnivorous reptiles. Moreover, the evolution of herbivory
in lizards, and the ecology of herbivorous reptiles may be
related to constraints imposed by ambient temperature. In
addition, manipulation of temperature in captivity may be a
technique that can improve management and propagation.
Therefore, the relationship between temperature, herbivory,
and an ectotherm’s ability to extract energy from plant
tissue has theoretical and practical significance.

Previous nutritional research on herbivorous lizards

has been plagued with several problems. A serious problem

59

60
with many published studies is that the herbivorous lizards
were force—fed (Zimmerman and Tracy, 1989; Troyer, 1987;
Harlow et al., 1976). Force-feeding was necessitated by the
fact that many captive lizards would not eat ad libitum.
However, force-feeding may alter digestive processes and
create difficulty with data interpretation. In fact,
published data on the effect of temperature on fiber
digestibility, and dry matter and energy metabolizability,
are contradictory (Troyer, 1987; Zimmerman and Tracy, 1989,
Harlow et al., 1976; Karasov and Diamond, 1985). The extent
of nutrient and energy digestibility or metabolizability is
a function of two competing processes, the rate of digesta
flow, and the rate of digestion or fermentation (Van Soest,
1982). Changes in ambient temperature may affect both of
these processes and have little net effect on apparent
digestibility or metabolizability. The use of digested
nutrients, and the partitioning of energy between
maintenance functions and growth may also be modulated by
temperature in ectotherms.

Whereas the effects of temperature on metabolic rate
are well established in many reptilian species,
bioenergetics are not well understood. The primary measure
in many reptilian studies is the metabolic rate, usually the
rate of carbon dioxide production or oxygen consumption
(Bennett, 1976). The relationship between intake energy,
energy expenditure, and the efficiency of use of energy

above maintenance for growth is unknown. Manipulating

61
growth rate and maximizing efficiency of tissue deposition
are of great importance for successful captive management
and production of the green iguana as a source of food for
humans in Central and South America.

Different methodologies are often necessary to study
energy metabolism in the field as compared to a controlled
laboratory environment. The doubly-labelled water (DLW)
method is well suited for field studies. The principle of
the DLW method is the measurement of the difference in flux
between ?H excreted as 430, and 18O excreted as both C“Tb and
HQJO (Lifson et al., 1955). Once the isotopes are
administered, the animal has to be recaptured only once to
determine the average rate of carbon dioxide production.
Respiratory exchange calorimetry is well suited for
controlled studies in the laboratory. This method permits
the direct measurement of carbon dioxide production and
oxygen consumption. Few studies of lizards comparing these
methods have been published (Congdon et al., 1978).

The objectives of this research were to determine the
effect of two different ambient temperatures on nutrient
intake, growth rate, nutrient metabolizability and
bioenergetics, including the rate of energy expenditure and
the efficiency of use of energy above maintenance for tissue
deposition, in the green iguana. Furthermore, the study was
designed to compare energy expenditure measured by the DLW
method and by respiratory exchange indirect calorimetry.

The iguanas were housed at two different ambient

 

62
temperatures, 35 C and 28 C. The first temperature, 35 C,
was selected because it is near the iguana’s self-selected
body temperature (Moberly, 1968a). Since death can occur at
temperatures above 40 C, a temperature lower than 35 C was
selected as the second temperature. During tests at 25 C
the iguanas did not eat, but they did eat at 28 C.

Therefore, 28 C was selected as the second temperature.

63

MATERIALS AND METHODS

Animals and housing

Twelve individually-housed green iguanas (Iguana
iguana) were used in this study. Two groups of six iguanas
each were randomly assigned to either 28 C or 35 C ambient
temperature. Each group was equally balanced for sex, as
determined by femoral pore size. After the first period (54
d), the ambient temperatures were switched, and a second
period (37 d for iguanas at 35 C, and 39 d for the iguanas
at 28 C) was completed. The iguanas were hatched in Costa
Rica as part of a captive breeding program (Fundacion Pro
Iguana Verde). The iguanas were approximately 6 months old
in September, 1989, when they were brought to the
Smithsonian Institution’s National Zoological Park
(Washington, DC), where these studies were conducted. All
procedures were approved by the Animal Welfare Committee of
the National Zoological Park.

The iguanas were acclimated to environmental chambers
(Environmental Specialties, Inc., Raleigh, NC) for
approximately 8 months prior to the beginning of this study.
During the acclimation process, the iguanas were not
individually housed but were housed as two groups in two

environmental chambers. During this time period, the

64

iguanas were treated orally with ivermectin (MSD Agvet,
Rahway, NJ) for external and internal parasites.
Approximately 6 months prior to the start of data
collection, the iguanas were moved to individual cages that
were located inside the environmental chambers. The cages
were approximately 66 cm wide, 53 cm high, and 51 cm long.

One environmental chamber was maintained at 28 C and
the other at 35 C. Both chambers were maintained at 60%
relative humidity. The chambers provided a stable

temperature (2 0.1 C), and relative humidity (: 1.0%).

Overhead fluorescent-type bulbs (Chroma-50®, General
Electric Co.) were used for incidental lighting with a 12:12
lightzdark cycle throughout the acclimation process and
study. These bulbs may have also provided appropriate
wavelengths for the bio-conversion of vitamin.Eg precursors

to the active forms of vitamin D3 (Allen, 1988).

Diet and measurement of food intake

The ingredients and chemical composition of the meal-
type diet are presented in Tables 1, 2, and 3. The medium—
fiber diet was used for this study. The diet was
manufactured commercially (Zeigler Bros. Inc., Gardners,
PA). Fresh diet was stored in a freezer for the entire
length of the study. Only one manufactured lot of feed was

used. Iguanas were fed in ceramic crocks three times each

65
week (Sunday, Tuesday, and Thursday). On these days, the
diet remaining in the cage from the previous feeding (the
orts) was collected, weighed, and discarded. Fresh diet was
weighed into a ceramic crock to the nearest 0.1 g. An
amount of new diet was provided in excess of current intake
to ensure that there would be diet remaining the next time
the crock was removed and remeasured. Any diet that spilled
into the cage was collected. If an iguana had defecated in
the feed crock, food intake was estimated using the mean
food intake for the period immediately preceding and
following that day’s feeding. A subsample of approximately
30 g of fresh diet was subsampled from the manufactured lot
of diet each time the animals were fed. These subsamples
were composited by weight, and separately for each period.
Fresh water was provided in ceramic crocks, and the amount
of water was monitored each day to ensure fresh water was

available ad libitum.

Total excreta and skin collections

During periods of excreta (fecal and cloacal waste
combined) and skin collections for the balance and digestion
trials, the excreta and skin were collected in separate,
pre-tared containers. These containers were stored in a
freezer until needed for nutritional analyses. Collections

of excreta and skin were made from the metal tray located

66
beneath the hardware cloth floor of the cage and from the
cage surface. One day each week (Sunday), all cages, trays,
ceramic crocks, and environmental control chambers were
scrubbed with appropriate cleaning solutions.

To determine the starting and stopping point for the
collection of excreta samples for balance measurements, the
iguanas were fed, once at the beginning and once at the end
of the collection period, a measured amount of diet that had
been mixed with brilliant blue (0.15% w/w, FD&C #1, Allied
Chemical Corp., Morristown, NJ). When the feces appeared as
a green or blue color, the excreta collections started. At
the end of the collection period, the iguanas were again fed
a measured amount of marked diet, and when the feces
appeared green or blue, the collections stopped. Skin
collections started and stopped at the same time as the

excreta collections.

Indirect calorimetry

Indirect calorimetry was performed on all animals by
respiratory exchange, and by the doubly-labelled water (DLW)
method. Respiratory exchange calorimetry measurements were
performed for three consecutive days at the beginning and
end of each of the two periods. However, during the first
period, the iguanas housed at 35 C were measured only twice

at the beginning of the period, and three times at the end

67
of the period. The mean of the six (or five) measurements
was used in subsequent calculations and statistical
analyses. These measurements were performed in closed-
system calorimeters. Doubly—labelled water was administered
at the beginning of each period, after respiratory exchange

measurements were completed.

Indirect calorimetry: respiratory exchange

Six closed-system calorimeters were constructed for
respiratory exchange calorimetry by covering the inside of
plywood shells with polyester resin-coated fiberglass
(Figure 1). Following at least three coats of polyester
resin on the fiberglass, the interiors of the calorimeters
were painted with epoxy-based paint. Mean chamber volume
was 0.304 HP, based on measured linear dimensions. High
density urethane foam (0.64 cm thick x 2.54 cm wide) was
used to form a gasket between the fiberglass box and a 0.64
cm thick plexiglass top. The top was fastened to the box
with 14 bolts to create an airtight seal. A normally—closed
quick-connect sampling port (Swagelok Co., Solon, OH), and a
mixing fan were mounted on the inside of the plexiglass
tops. The mixing fan (1840 l/min capacity) was used to
circulate the air within the chamber prior to gas sample
collection. Rubber matting was placed on the floor of the

calorimeter to prevent slipping when the iguanas walked

68

 

 

 

E ‘\
U Gasket
m
m Bolt
L
.C
_9 Plywood side
0
I

l Length: 100 cm i

SIDE VIEW

E Pl‘l
o eXIg ass top
8 Bolt
E Sampling port
5 Circulating fan
; Gasket

 

TOP VIEW

 

 

 

Figure 1. Schematic drawing of closed—circuit respiratory

calorimeter.

69
across the floor. A piece of wood was also placed in each
calorimeter to provide a climbing surface for the iguanas.
When the iguanas were in the calorimeter, a piece of burlap
was placed over the top of the chamber to minimize excita-
tory visual cues. Separate ceramic dishes containing a pre—
weighed amount of diet and water were placed on the
calorimeter floor. A thermometer and relative humidity
meter were placed inside each calorimeter. To test the
accuracy of these calorimeters, 100% anhydrous ethanol
(Warner—Graham Co., Cockeysville, MD) was combusted.
Initially, background air samples were collected. Small
alcohol lamps were filled and weighed, placed into the
calorimeter, and ignited. The calorimeter top was bolted in
place and the ethanol was allowed to burn for approximately
1 hr. At the end of the hour, the mixing fan was turned on
(which blew out the lamp) and the air was allowed to mix for
at least 5 min. Subsequently, an air sample was pumped out
of the calorimeter with a diaphragm pump, through anhydrous
calcium sulfate (Drierite®, 4 mesh, W.A. Hammond Drierite
Co., Xenia, OH), and into an 86-1 polyvinyl fluoride bag
(Tedlar®, E.I. du Pont De Nemours & Co., Wilmington, DE).
The calorimeter top was then opened and the alcohol lamp
removed, and reweighed. The amount of ethanol combusted was
determined as the difference in weight. Recovery of oxygen
and carbon dioxide was measured and compared to the

theoretical values based on 3 moles of oxygen and 2 moles of

70
carbon dioxide produced per mole of ethanol combusted:

CH3CHZOH + 302 —> 2co2 + 3H20.

Prior to being placed in a calorimeter for respiratory
exchange measurements, the iguanas were weighed. The
iguanas were placed in a calorimeter with the plexiglass top
in place but not secured for approximately 36 hr. After 36
hr, the mixing fan was started and allowed to run for at
least five minutes. The plexiglass tops then were closed
and secured with bolts, and initial temperature and relative
humidity were recorded. Based on initial temperature and
relative humidity, initial dry air volume of the chamber was
calculated. After approximately 24 hr, the mixing fan was
started, and the air in the chamber was mixed for at least 5
min. A gas sample was then pumped out of the calorimeter,
through the Drierite®, and into a Tedlar® bag. Normally—
closed valves were used on the calorimeter top and on the
gas collection bag. The tops were then opened, and the fans
were left on to introduce fresh air into the calorimeters.
Prior to being resealed for the next 24-hr period, a
background gas sample was collected from inside the chamber.

The following day, the gases were pumped out of the
Tedlar® bag and analyzed. Oxygen was analyzed by
paramagnetic deflection using a Beckman Industrial (LaHabra,
CA) Model 755A analyzer. Carbon dioxide was analyzed by
infrared spectroscopy using a Beckman Industrial Model 880

non-dispersive infrared analyzer. These analyzers were

71

calibrated using two tanks of mixed gases whose
concentrations were determined after calibrating the ana-
lyzers with appropriate gas standards from the National
Institute of Standards and Technology (Gaithersburg, MD).
Hydrogen gas concentration was determined using a Quintron
Microlyzer Model 12 analyzer (Quintron, Inc., Milwaukee,
WI). Gas permeability of the Tedlar® bag was tested by
filling the bags, and analyzing the gas composition of the
bag over a 48-hr period. No changes in gas composition were
detected.

Respiratory exchange carbon dioxide production rate
(pCOhe, l/d) and oxygen consumption rate (p02.l/d) were

calculated as:

 

 

CO _ [C031final — [C0211n1tial (chamber volume (1))
P 2:9 ‘ elapsed time (min) I
and
P0 - [02] final - [0211n1tial (Chamber valume (1))
2 _ .

elapsed time (min)

These rates were converted to standard temperature and
pressure, and expressed on a 24-hr basis. Energy

expenditure from respiratory exchange (EE kcal/day) was

re!

calculated as (Weir, 1949):

FE“ = 3.9411302 + 1.106pC02.

72

Respiratory quotient was calculated as:

R0 = pC02(l/d)
p02(l7d) '

Indirect calorimetry: doubly-labelled water

In addition to respiratory exchange, energy expenditure
was measured by the doubly-labelled water technique. Stable
isotopes of oxygen (HQWO) and hydrogen (H50) were
administered to the iguanas at the beginning of each period,
immediately after the respiratory exchange calorimetry was
completed.

Prior to being infused with the isotopes, a baseline
blood sample of 8—10 ml was taken by venipuncture on the
ventral side of the tail, approximately 5 cm posterior to
the cloaca, while the iguanas were manually restrained. A
12—cc syringe fitted with a 22-g x 1" needle was used for
all blood collection. Samples of blood were placed in
lithium heparinized tubes and gently mixed. The tubes were
spun for 15 min in a refrigerated centrifuge at 2,000 RCF
(3,000 rpm). The serum was collected and frozen (-20 C) in
a sealed cryovial (#5000-0050, Nalge Co., Rochester, NY)
until needed for isotope analyses. Isotopes were infused by
gavage using a #12 stomach tube inserted into the stomach
through a plastic syringe casing. The syringe casing was

used to protect the gavage tube from being punctured by the

73

iguana's teeth, and it was wrapped with duct tape to
minimize damage to the iguana’s oral cavity. Deuterium
oxide (HBO, 99.9%, Cambridge Isotope Laboratories, Woburn,
MA) and 18oxygen (HQWD, 10.0% atom percent excess, (APE),
Isotec, Miamisburg, OH) were mixed together in the ratio of
0.602 g fibO/g solution and 0.398 g H;”O/g solution.
Approximately 5 g/kg body weight of the isotope solution was
administered. The amount of isotope administered was
quantitated by taring an empty syringe on an electronic bal—
ance, filling the tared syringe with the isotope solution,
and reweighing the syringe. This weight was recorded to the
nearest 0.01 g as the weight of the administered dose. When
the isotope solution was administered, the time was
recorded. After isotope administration and before the
syringe and gavage tube were removed from the iguana’s
mouth, they were rinsed with 3—4 ml of water. Blood samples
(8-10 ml) were drawn at day 2, day 11, day 21, day 34 and
day 55 post-dosing during period 1, and on day 2, day 10,
day 20, and day 41 during period 2. Each time a blood
sample was taken, the time of sampling was recorded, and the
iguana’s weight, snout-vent length, and cloacal temperature
were measured.

tame APE of the plasma was analyzed by isotope-ratio
mass spectroscopy by a commercial laboratory (Metabolic
Solutions, Inc., Acton, MA). Previous analysis of HQWD
standards by this laboratory indicated that the results were

accurate and precise (Seale et al., 1993). The 390

74

concentration was determined by infrared (IR) spectroscopy
(Miran 1FF, Foxboro Co., Foxboro, MA). Prior to analysis,
subsamples of plasma (approximately 3 ml) were vacuum
sublimed (Stansell and Mojica, 1968; Byers, 1979). An
autosampler was used to deliver each sample to the
spectrometer. The autosampler mixed the sample 1:1 with a
BRIJ® solution to prevent the formation of air bubbles. The
BRIJ® solution contained 1 ml octanol and 4 ml BRIJQ’2made
to a total volume of 4 l with distilled water (Sigma
Chemical Co., St. Louis, MO). The IR spectrometer was
fitted with a temperature—controlled (20 C), calcium
fluoride flow-through cell. The dc voltage output from the
spectrometer was measured by an integrator (Model 4270,
Spectra—Physics, San Jose, CA) and converted to peak height.
Ten standards ranging from 0.01% to 0.25% EEO (v/v) were
analyzed, and plasma sample concentration was determined by
inverse regression analysis (Gill, 1978). Linear regression
of the standard concentrations and their peak height
produced ar2 >0.999. Each sample was read in duplicate,
and a set of 10 standards was read 8 times during each day’s
run. Approximately 60 samples and 80 standards were
measured during each day's run.

18Oxygen elimination rates (k0, l/d) and.2hydrogen
elimination rates (kH, l/d) were determined by least squares
linear regression of the natural log of isotope
concentration and elapsed time from isotope administration

using PC—SAS (PROC REG) (Figure 2). The zero time

75

 

 

LN of isotope concentration

 

 

 

I l l l 1

~10 O 10 20 3O 4O 50 60

)—

 

—1O

Elapsed time (days)

 

 

 

Figure 2. Typical regression lines for 18O and 2H turnover

for one iguana at two different ambient temperatures.

 

 

76
intercepts were used to determine 2H pool size (ND, kg), and
180 pool size (Nb,lkg) at the time of dose administration.
For the DLW method, rate of carbon dioxide production
(pCOMMN l/d) was calculated based on the equation derived

by Schoeller et al. (1986a) as:

N
pcomw = (70°76) (1.011% — 1.041(3) — 0.0246r0,

where rm is the rate of water loss from fractionating

gaseous routes, and is estimated as:

r0, = 1.05No(ko-kH) .

Energy expenditure (EEMM, kcal/d) was calculated as:

C02 d1 w

EEdlw = 3'941(p RQ ) + 1-106pcozdlw

where RQ is the respiratory quotient (pCOyfiXk) measured
from respiratory calorimetry.

Water flux (pHJL.nfl/d) was calculated as:

Nok.
gmgo==
[1_ (1_f1) (2 - 3pC02r9)

2%. 1

 

where f1 = 0.941, and is the fractionation constant for the

77
exchange of H30(gas)/HQO(liquid) (Seale et al., 1989;

Schoeller et al., 1986a).

Lean body mass (LBM, kg) was calculated as:

 

LEM: 0.732

and body fat (%) was calculated as:

 

= body weight - lean body mass
body fat body weight 100.

No correction was made to LBM of body fat to correct for the
weight of the gastrointestinal tract contents.

During isotope solution administration, the isotope
dose was regurgitated by two iguanas in the first period
(both housed at 28 C), and by one iguana in the second
period (housed at 35 C). Therefore, isotope administration
could not be quantitated for these iguanas. Doubly-labelled
water data (energy expenditure, body composition) from those
three iguanas were not used in subsequent analyses, but data

on isotope elimination rates were used.

 

78

Sample preparation and analyses

Samples of feed, excreta, and skin were kept frozen (-
20 C) until the time of sample preparation for analyses.
Excreta samples were defrosted in a refrigerator overnight
and then mixed with water in a Waring blender. The samples
were homogenized for approximately 5 min. The entire
contents of the blender were quantitatively transferred into
a pre—tared plastic container that was then frozen, and
subsequently freeze-dried. Immediately after freeze—drying,
the pre-tared containers were reweighed. The difference in
weight was the uncorrected dry weight of the excreta. The
samples were then crushed with a mortar and pestle to
produce a homogeneous powder. Subsamples of this powder
were used for subsequent analysis for dry matter (DM), ash,
carbon (C), nitrogen (N), neutral detergent fiber (NDF),
acid detergent fiber (ADF), acid lignin (AL), and gross
energy content. Skin samples were freeze-dried in a pre—
tared container. The samples were then reweighed and ground
'in a small Wiley mill fitted with a 2 mm screen. Skin
samples were analyzed for nitrogen, carbon, and gross energy
content. Feed samples were analyzed for the same nutrients
as the excreta.

Dry matter was determined in a vacuum drying oven at 55
C for 6 hr. Absolute dry matter content of the excreta was
determined by correcting the freeze-dried excreta weight

with the dry matter determined by vacuum oven drying.

79
Nitrogen and carbon were determined by combustion using
approximately 100-mg subsamples (CHN 600, Leco Corp., St.
Joseph, MI). Crude fat was determined by methylene chloride
extraction (PBS 80, CEM Corp., Matthews, NC). Gross energy
content was determined by isoperibol dynamic bomb
calorimetry (Parr Instrument Co., Moline, IL). Gross energy
of the sample was corrected for acid formation by sodium
carbonate titration. Ash was determined by combustion at
600 C for 6 hr. Fiber fractions were determined by the
methods of Van Soest (Robertson and Van Soest, 1981) using a
Fibertec system (System M, Tecator AB, Hoganas, Sweden).
Heat—stable a-amylase (Sigma A-3306, 200 ul/sample) and
protease (Sigma P-3910, 5 mg/sample) were used to facilitate
filtration during fiber analysis. Neutral detergent fiber
(NDF) samples were pre-incubated with a—amylase for 1 hr at
60 C and then with the protease for 1 hr at 80 C prior to
refluxing with the neutral detergent. Cellulose was
calculated as the difference between acid detergent fiber
(ADF) and acid lignin (AL). Hemicellulose was calculated as
the difference between NDF and ADF. Organic matter was

calculated as the difference between dry matter and ash.

Calculations and statistical analyses

A preliminary study was conducted to determine if the

iguanas were able to feed selectively on the meal—type diet.

80
Five collections of offered diet and orts were collected at
five different times during a 5-wk preliminary period. The
five diet samples were pooled. The diet and ort samples
were analyzed for dry matter, ash, fat, nitrogen, carbon,
and organic matter. Protein was calculated from nitrogen
(Nx6.25) and percent total carbohydrate was calculated as
the difference between 100% and the sum of fat, protein, and
ash, expressed on a dry matter basis. Multivariate
statistics (Hotteling’s T—test) were used to determine if
the composition of the offered feed (organic matter,
protein, carbon, fat, ash, and total carbohydrate) was
statistically different from the orts collected during each
of the 5 weeks (Gill, 1978). On one occasion, one iguana
consumed all of the offered diet; therefore, there were no
orts.

Nutrient intakes were calculated as the product of
nutrient concentration in the diet and daily dry matter
intake. Since it was not possible to separate cloacal and
fecal excreta, apparent metabolizability of dry matter and
gross energy were calculated using the direct method
(Schneider and Flatt, 1975). There is no quantitatively
significant component of fiber excreted in the urinary
wastes; therefore, apparent digestibilities were calculated
by the direct method for NDF, ADF, cellulose, and
hemicellulose. Average daily growth rate was determined as
the difference between final weight and initial weight

divided by the number of days on study. Metabolizable

81
energy intake (NEH) was calculated as the difference between
intake energy (IE) and energy lost in excreta (FE+UE) and
skin (SE).

Nitrogen and carbon balance were calculated as the
difference between intake and combined urinary and fecal
excretion and skin ecdysis. Carbon balance was then
corrected for carbon lost as carbon dioxide based on carbon
dioxide production from respiratory exchange calorimetry
measurements (pCOuB).

Retained energy (RE) was calculated by three different
methods. For all three methods, RE was expressed on a daily
basis, and as a function of metabolic body size (body
weightm"U. By the first method, REre was determined as the
difference between.ng and EE measured by respiratory
exchange (EEm). Since food intake was depressed by
approximately 50% in the calorimeters, REm was adjusted to
correct for the decrease in IE. The depression in food
intake may be a consequence of the movement of the iguanas
from one environmental chamber to another for the
calorimetry measurements, or due to a change in housing
conditions during the calorimetric measurements. The
correction was based on the difference between mean daily
intake during the balance period, and intake when the
iguanas were in the calorimeter. The amount of
metabolizable energy associated with the decreased food
intake was determined as the product of the difference in

IE, and the measured ME density of the diet (3.18 kcal/g dry

82
matter). Fifteen percent of this difference (representing
the heat increment) was added to the measured EEm. This
correction represents an estimate of the heat increment
associated with the ingestion and digestion of food
(Blaxter, 1989). By the second method, REmy was determined
as the difference between MEiland the energy expenditure
measured by the DLW method (EEmM). By the third method,
RECNbal was calculated based on the sum of energy retained as
fat and as protein based on C and N balance (Brouwer, 1965).
The following assumptions were made: that the mean N and C
concentration of protein deposited was 16.0% and 52.0%,
respectively, and the mean gross energy content of the
protein deposited was 5.7 kcal/g. Furthermore, the mean C
concentration of fat deposited was 76.7%, and the mean gross
energy concentration of the fat deposited was 9.5 kcal/g.
In addition, it is assumed that there are no changes in the
size of the carbohydrate stores. Retained energy was
calculated as the sum of deposited protein energy and fat
energy.

The apparent partial efficiency of use of metabolizable
energy for growth (kg) was determined for each temperature
by the regression of REre and MEi (Van Es et al., 1984). The
slopes and intercepts of the two regression lines were
compared to determine the effect of temperature (Gill,
1978). The metabolizable energy requirement for maintenance
(th) was calculated from the parameters of the regression

lines of REre and MEizfldr iguanas at each temperature, when

 

83
REre was equal to zero (Van Es et al., 1984). Two iguanas
were in negative energy balance (both housed at 28 C). Data
from these iguanas were excluded in the calculation of kg
and MEm.

Statistical analyses were conducted using PC—SAS
version 6.04 (SAS Institute Inc., Cary, NC). Tests of the
assumptions of analysis of variance (ANOVA) were completed
for normality (PROC UNIVARIATE) and homogeneity of variance.
Homogeneity of variance was tested by plotting of residuals
and predicted values, and by the calculation of Spearman
correlation coefficients of the residuals and predicted
values (PROC CORR). No violations of the assumptions of
ANOVA were detected. Analysis of variance (PROC GLM),
least-squares means (LSMEANS), and probability of the
differences between least-squares means (PDIFF) procedures
were used to test the effects of ambient temperature and
sex. The ANOVA model included terms for sex, iguana(sex),
period, temperature, and the temperature by sex interaction.
Data were also analyzed using analysis of covariance
(ANCOVA) with initial body weight as the covariate. There
was no improvement in the coefficient of variation by ANCOVA
so ANOVA was used for subsequent analyses. Tukey’s
studentized range test (PROC MEANS) was used to compare
RE“, REmM, and REmmn. Paired t-tests were used to determine
differences between pCOZre and pCOmh" and between EEre and

EEdlw'

84

RESULTS

Crude protein, total carbohydrate, crude fat, ash,
carbon, and organic matter composition of the offered diet
and the 99% confidence intervals (CI) of orts composition
are presented in Table 8. The nutrient concentration of the
offered diet is within the 99% CI of the orts. Thus, there
was no difference for these nutrients between the offered
diet and the orts, for any of the 5 times that ort samples
were collected and analyzed. Therefore, there is no
evidence that the iguanas were able to select for these
specific nutrients from the offered diet; and, it is
possible to determine nutrient intake as the product of
nutrient concentration and the difference between the
offered and uneaten portion, provided that all of the
uneaten portion is collected.

Recoveries of carbon dioxide and oxygen were measured
four times in each of the six calorimeters during the course
of the study (Table 9). The mean amount of ethanol
combusted was 3.48 g. Carbon dioxide and oxygen recoveries
were used to test the accuracy of the calorimeters, and

recoveries were calculated based on the amount of ethanol

 

85

 

 

 

 

 

 

 

 

 

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86
combusted and the amounts of carbon dioxide produced and
oxygen consumed relative to the predicted values. Predicted
values were based on the stoichiometric relationship:

CH3CHZOH + 302 -> 2co2 + 3H20.

The mean amount of carbon dioxide recovered was 96%,
and ranged from 88% to 101%. The mean amount of oxygen
recovered was 97%, and ranged from 84% to 102%. The mean
respiratory quotient (RQ) was 0.66, and ranged from 0.64 to
0.76 (Table 9). The predicted RQ was 0.66 (each mole of
ethanol combusted uses three moles of oxygen and produces
two moles of carbon dioxide). On occasions when recoveries
were low, repairs were made to the calorimeters prior to use
for metabolic measurements of the iguanas. These repairs
generally consisted of replacing the top gasket or resealing
the top seams.

Due to differences in the length of the two balance
periods (54 d for period 1, and 37-39 d for period 2),
intake and growth data are presented on a daily basis. All
variables were tested to determine differences between males
and females, and there were no differences between the
sexes for any variable. For the variables analyzed, there
were no differences between the two periods, except for
initial and final body weight. For all variables, the

variances of the two treatment groups were not different.

 

87

Table 9. Carbon dioxide (Gib) production, oxygen (02)

consumption, and gas recoveries from ethanol combustion

 

 

 

 

 

 

 

 

 

 

trials.

Variable Mean 1 SEM Minimum Maximum
Ethanol combusted (g) 3.48 0.90 2.56 5.65
Final.CI5 concentration 1.25 0.33 0.84 2.05
(% of dry gas)

Final.Cb concentration 19.24 0.49 18.04 19.86
(% of dry gas)

(XE produced (1, STP) 3.25 0.84 2.19 5.14
(% consumed (l, STP) 4.96 1.38 8.06 3.12
Respiratory quotient 0.66 0.03 0.64 0.76
(RQ)

Recovery of CO2 (%) 96.03 3.13 87.86 101.89
Recovery of 02 (%) 97.24 4.65 83.51 101.84

 

 

 

 

 

1 Mean of 24 combustion trials.

 

88
Thus, there is only one standard error for the mean (SEM).
However, for some variables related to stable isotope
kinetics, the sample sizes were not balanced since some
iguanas regurgitated their dose. Therefore, each mean has a
different SEM. All means presented in the following tables

are the least-squares means 2 standard error of the mean

(SEM), except in Table 16.

Measurement of cloacal temperature confirmed that the
iguana’s temperature was near its ambient temperature.
Initial body weight (BW), final BW, snout—vent length,
metabolic body size (BWmnfl, and body composition of the
iguanas are presented in Table 10. Mean body weight was
942.4 g (ranging from 606 to 1393 g) and 882.5 g (ranging

from 500 to 2082 g) (i 38.9 g) for iguanas housed at 28 C

and 35 C, respectively. Mean metabolic weights were 0.97
and 0.99 kgmnifor iguanas housed at 28 C and 35 C,
respectively. Initial snout—vent lengths (SVL) were 26.7 cm

and 26.5 cm (i 0.30) for the iguanas housed at 28 C and 35

C, respectively. There was no difference in body weight or
SVL between the two groups at the beginning of each of the
periods. Body weight at the end of the period was lower for
the iguanas housed at 28 C than at 35 C, as was daily rate
of gain. Mean daily rate of gain was 0.57 g/d and 3.40 g/d

(:0.25) for the iguanas housed at 28 C and 35 C,

respectively.

Table 10.

of iguanas housed at different ambient temperatures.

89

Physical characteristics and daily rate of gain

 

 

 

 

 

 

 

 

 

 

 

Variable Units Mean 28 C (N=24) Mean 35 C (N=24) P
Initial body 9 942.4 882.5 0.3043
weight1
Final body 9 1009.4 1137.5 0.0093
weight2
Rate of gain3 g/d 0.57 3.40 0.0001
Shout-vent cm 26.7 26.5 0.7584
length‘
Mean kg"'75 0.97 0.99 0.4927
metabolic
body weights

Mean 28 C SEM Mean 35 C SEM P

(N=21) (N=21)

Lean body 9 756.8 30.0 757.1 26.9 0.9950
mass
Body fat % 14.8 1.0 16.2 0.9 0.3820

 

 

 

 

 

 

 

1SEM : 38.9.

2$EM : 27.5.

+
O
N
01

3SEM _

4SEM : 0.30.

‘SEM : 0.02.

 

90
Body composition was determined based on the kinetics
of 18O and its total water pool size at the time of isotope
dose administration (zero time intercept). Body fat was

14.8 i 1.0 and 16.2 i 0.9% of total weight at the beginning

of each period, for iguanas housed at 28 C and 35 C,
respectively. There was no difference in body composition
between the two groups at the beginning of each of the
balance periods.

Nutrient intakes and metabolizabilities (or
digestibilities) are presented in Table 11, and nitrogen (N)
and carbon (C) intakes are presented in Table 12. Nutrient
intakes were lower for iguanas housed at 28 C than for
iguanas housed at 35 C (p<0.0001); however, there were no
differences in nutrient metabolizability or digestibility
between the two groups of iguanas. The difference in
nutrient intakes between iguanas housed at the two
temperatures was approximately 3 to 4 fold.

Dry matter intake is expressed on an absolute basis
(g/d), and on a body weight basis (% of body weight/d).
Mean daily dry matter intake (:SEM) was approximately four
times greater (p<0.0001) for iguanas housed at 35 C than for
iguanas housed at 28 C (12.70 and 3.51 x 0.51 g/d,
respectively). Daily dry matter intake ranged from 1.36 to
9.48 g/d for iguanas housed at 28 C, and from 8.55 to 20.74

g/d for iguanas housed at 35 C. Daily dry matter intake as

a percent of body weight was also less for iguanas housed at

91

28 C than for iguanas housed at 35 C (0.35 and 1.33::0.05%

of body weight/d, respectively). Apparent dry matter
metabolizability was not different between the two groups

(67.0 and 67.2 x 1.1%). Mean daily metabolizable energy

intake (MEi) was lower (p<0.0001) for iguanas housed at 28 C

than for iguanas housed at 35 C (11.38 and 41.75 kcal/d :

1.99 kcal/d, respectively). Gross energy metabolizability

was 71.0 and 70.9 t 0.9% for the iguanas housed at 28 C and

35 C, respectively, and it was not different between the two
temperatures. The mean dietary ME density (kcal/g dry

matter) was 3.18 x 0.04 kcal/g dry matter, and was not

different between the two ambient temperatures. Fiber
digestibility was not different between the two ambient
temperatures.

As with the other nutrients, nitrogen and carbon
intakes (Table 12) were significantly greater at the higher

temperature (p<0.0001, 0.16 vs 0.58 i 0.03 g N/d, and 1.5 vs
2.54 x 0.14 g C/d for 28 and 35 C, respectively).

Furthermore, nitrogen and carbon balances were significantly
lower for iguanas housed at 28 C than for iguanas housed at

35 C (p<0.0001, 0.07 VS 0.24 z 0.02 g N/d and 0.30 VS 1.96 x

0.14 g C/d for iguanas housed at 28 and 35 C, respectively).
These temperature-dependent differences in C and N

balance correspond to a significantly lower deposition of

92

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Table 12.

and protein and fat gain of iguanas housed at
different ambient temperatures (N=24).

93

Nitrogen and carbon intake and balance,

 

 

 

 

 

 

 

 

 

 

Variable Units Mean 28 C Mean 35 C SEM P

Nitrogen g/d 0.16 0.58 0.03 0.0001

intake

Nitrogen g/d 0.07 0.24 0.02 0.0001

balance

Carbon g/d 1.50 2.54 0.26 0.0001

intake

Carbon g/d 0.30 1.96 0.14 0.0001

balance

Protein gain g/d 0.43 1.42 0.06 0.0001
kcal/d 2.48 8.07 0.32 0.0001

Fat gain g/d 0.09 1.58 0.15 0.0001
kcal/d 0.85 14.89 1.42 0.0001

 

 

 

 

 

 

 

94
protein and fat for iguanas housed at 28 C as compared to

those housed at 35 C (p<0.0001). Rate of protein (Nx6.25)

deposition was 0.43 and 1.42 z 0.06 g/d for iguanas housed

at 28 C and 35 C, respectively. The amount of protein

deposited corresponds to 2.48 and 8.07 z 0.32 kcal deposited

as protein/d for iguanas housed at 28 C and 35 C,

respectively. Fat deposition was 0.09 and 1.58 z 0.15 g/d

for iguanas housed at 28 and 35 C, respectively. The amount

of fat deposited corresponds to 0.85 and 14.89 i 1.42 kcal/d

for iguanas housed at 28 C and 35 C, respectively.

Isotope kinetics are presented in Table 13. Enough
isotope was administered to the iguanas that regurgitated
their isotope dose to determine turnover, and to calculate
isotope half life. Therefore, the mean isotope half life
from all iguanas is presented in Table 13. However, since
the amount of isotope administered was not quantitatively
known in the iguanas that regurgitated the dose, pool size,
carbon dioxide production rate, water production rate, and
energy expenditure were not calculated. Therefore, these
means have a different SEM as a consequence of the unequal
replication.

The half lives of ?H and 180 were approximately three
times longer for iguanas housed at 28 C than for those
housed at 35 C (p<0.0001). The mean.2H half life for

iguanas housed at 28 C was 32.25 $1437 d, and ranged from

18.70 to 52.96 d. The mean Hilufliflife for iguanas housed

95

at 35 C was 11.11 x 1.37 d, and ranged from 5.26 to 13.53 d.

As expected, the mean 180 half life was less than the 2H half
life. Mean 180 half life for iguanas housed at 28 C was

24.51 i 0.91 d, and the mean 180 half life at 35 C was 8.52
z 0.91 d. The range in 180 half life was 14.38 to 37.36 d

and 4.52 to 11.43 d for iguanas housed at 28 C and 35 C,
respectively.

Ambient temperature did not affect the estimates of
total body water pool size (TBW, 1) when measured by Hi
(p<0.5413) or 18O (p<0.9951). Mean TBW pool size measured
by'zH zero time intercept was 0.692 x 0.02 and 0.667 t 0.025
1 for iguanas housed at 28 C and 35 C ambient temperatures,
respectively. Mean TBW pool size measured by 180 zero time
intercept was 0.554::0.022 and 0.554 2 0.020 1 for iguanas
housed at 28 C and 35 C ambient temperatures, respectively.
TBW pool size based on.?H kinetics was approximately 20% to
25% greater than the pool size based on 18O kinetics. The
ratio of TBW pool size determined by the two isotopes

(Nwflk) was 1.25 z 0.01 and 1.20 x 0.01 for iguanas housed

at 28 C and 35 C, respectively. This ratio was independent
of ambient temperature (p<0.0616). Water flux (pHg3,:ml/d)
was less for iguanas housed at 28 C (10.19 i 4.94) than for

iguanas housed at 35 C (46.73 x 4.42) (Table 13).

The rate of carbon dioxide production (pCOkflw, l/d),

Table 13.

96

iguanas housed at different ambient temperatures.

Doubly-labelled water kinetics and pool sizes of

 

 

 

 

 

 

 

 

 

 

 

 

Variable Units Mean 28 C Mean 35 C P
(N=24) (N=24)
2H half life1 d 32.25 11.11 0.0001
180 half life2 d 24.51 8.52 0.0001
Mean SEM Mean SEM P
28 C 35 C
(N=21) (N=21)
2H pool size 1 0.692 0.028 0.667 0.025 0.5413
(ND)
180 pool size 1 0.554 0.022 0.554 0.020 0.9951
(No)
Ratio of N'D/No 1.25 0.01 1.20 0.01 0.0616
Carbon dioxide 1/d 1.92 0.56 5.31 0.50 0.0049
production rate
(pcozdlw) )
Water ml/d 10.19 4.94 46.73 4.42 0.0019
production rate
(pH20)
Energy kcal/d 11.34 3.33 33.35 2.99 0.0033
expenditure
(EEdlw)

 

 

 

 

 

 

 

1 SEM : 1.37.

2 SEM : 0.91.

 

 

97
measured by doubly—labelled water turnover, was lower for
iguanas housed at 28 C than for iguanas housed at 35 C

(p<0.0049) (Table 13). Mean pCOmu,was 1.92 x 0.56 and 5.31

_ 0.50 1/d for iguanas housed at 28 C and 35 C,

+

respectively. Using the RQ measured during respiratory
calorimetry, and the energetic equivalents of carbon dioxide
and oxygen (Weir, 1949), mean daily energy expenditure
(EEmM) was approximately one—third lower (p<0.0033) for

iguanas housed at 28 C (11.34 i 3.33 kcal/d) than for those
housed at 35 C (33.35 x 2.99 kcal/d).

Rates of carbon dioxide production and oxygen
consumption from respiratory exchange calorimetry are
presented in Table 14. These rates are based on the mean of
all six measurements (except for period 1 when the iguanas
housed at 35 C were measured 5 times) from each iguana.

Rate of carbon dioxide production and oxygen consumption was
approximately 2 to 3 times greater for iguanas housed at the
higher temperature (p<0.0001). Mean rate of carbon dioxide

production was 1.43 and 3.49 z 0.19 l/d, and mean rate of
oxygen consumption was 1.79 and 4.60 x 0.29 l/d for iguanas

housed at 28 C and 35 C, respectively. These rates of gas
consumption and production correspond to RQs that are higher

at 28 C than at 35 C (p<0.0252). RQs were 0.80 and 0.77

(:0.01) for iguanas housed at 28 C and 35 C, respectively.

Based on the caloric equivalents of carbon dioxide and

Table 14.

98

rates, and energy expenditure of iguanas housed at

Respiratory exchange calorimetry gas production

 

 

 

 

 

 

 

 

 

 

 

 

 

 

different ambient temperatures (N=24).
Variable Units1 Mean Mean SEM p
28 C 35 C

Carbon dioxide 1 STP/d 1.43 3.49 0.19 0.0001
production rate
(pCOZre)
Oxygen l STP/d 1.79 4.60 0.29 0.0001
consumption
rate (p02)
Hydrogen gas nd.STP/d 4.46 12.59 2.04 0.0201
production rate
(PH2)
Respiratory 0.80 0.77 0.01 0.0252
quotient (RQ)
Energy kcal/d 5.48 14.26 0.93 0.0001
expenditure
(EEre) 2

kcal/mbw/d 5.84 14.19 0.40 0.0001
1STP = Standard temperature and pressure.

2mbw

metabolic body weight.

 

 

99
oxygen, energy expenditure (EEm, kcal/d) was 2 to 3 times
greater (p<0.0001) for iguanas housed at 35 C than when the
same iguanas were housed at 28 C. Mean rate of daily energy

expenditure was 5.48 and 14.26 kcal/d (10.93) for iguanas

housed at 28 C and 35 C, respectively.

Rate of hydrogen gas (H2) production (Table 14) was
higher for the iguanas housed at 35 C (12.59 ml/d) than for
the iguanas housed at 28 C (4.46 ml/d) (p<0.0201).

Retained energy (kcal/mbw/d) was calculated by three
different methods, and the least-squares means are presented
in Table 15. RE“ and RECNbal (kcal/mbw/d) were lower for
iguanas housed at 28 C than for those housed at 35 C
(p<0.0001). However, there was no difference in RE,“w for
iguanas at housed at 28 C or 35 C. The SEM for REdlw was
higher than the SEM for REre and RECNbal (Table 15).

The three means (REE, REmM, and REmmfl) were compared
using Tukey’s test (Table 16). There was no difference
between the overall mean (independent of temperature) of
REre and REmmu, but REdlw was lower than REre and RECNbal
(p<0.05).

The ratio of pCOmn,to pCOhm, and the ratio of EEdlw to
EEre are presented in Table 15. There was no effect of
temperature on the magnitude of the ratio of the rate of
carbon dioxide production or energy expenditure between the
DLW method and respiratory exchange calorimetry (Table 15).

The mean ratio of carbon dioxide production rate

100

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102

(gKXme/pcohfi) was 1.60 z 0.21 for iguanas housed at 28 C
and 1.53::0.19 for iguanas housed at 35 C. The mean ratio
of energy expenditure rate (EEmM/EEm) was 2.39 i 0.31 and
2.19 z 0.28 for iguanas housed at 28 C and 35 C,

respectively.

A paired t-test was used to determine that the overall
mean (independent of temperature) of pCOMh,was higher than
the overall mean of pCOhB (p<0.01) (Table 16). The overall
mean of EEdlw was also higher than the overall mean of EEre
(p<0.001) (Table 16).

The apparent efficiency of use of metabolizable energy
above maintenance (kg) was not different for the iguanas
housed at the two ambient temperatures (Figure 3) but the
intercepts were different (p<0.001). The k.g (n=10) for

iguanas housed at 28 C was 0.79 z 0.09, and the kg (n=12)
for iguanas housed at 35 C was 0.69::0.08. The calculated

0&5 was 5.82 kcal/mbw/d and 8.82 kcal/mbw/d for the iguanas

housed at 28 C and 35 C, respectively.

103

 

 

3 5 I I T I 1

RE 282 0.79MEI — 4.60 /

3° - RE 35-.— 0.69MEI — 6.06/ ‘

      
   
  

 

 

 

 

 

Retained energy (kcal/mbw/d)

 

 

 

 

28 (3 :35 C
Symbol . A ‘
kg .79 1 .09 .69 a .08
ME 5 82 a 32 —
m
r2 030 037
1 1 1 1
O memlo 20 30 40 50 60 70

Metabolizable energy intake (kcal/mbw/d)

 

 

 

Figure 3. Regression of retained energy and
metabolizable energy intake of iguanas housed at

different ambient temperatures (see text for description

of symbols).

104

DISCUSSION

The relationship between ambient temperature and
digestive physiology of herbivorous lizards has been used as
a theoretical framework to explain several evolutionary and
ecological phenomena, including the evolution of large body
size (Pough, 1973), and the geographic distribution of
herbivorous lizards (Zimmerman and Tracy, 1989; Rand, 1978;
Szarski, 1962). A central component of these various
evolutionary and ecological theories is the effect of
temperature on rate of digestion (especially fermentation),
rate of digesta passage, and digestive efficiency, which
usually refers to metabolizability or digestibility. In the
Western Hemisphere, many herbivorous species of lizards are
distributed near the tropics. There is little species
radiation from the hot climate near the equator.

Herbivorous species not found near the tropics are found in
other hot environments. For example, Dipsosaurus dorsalis
and Sauromalus obesus are two herbivorous lizard species
that are found at high latitudes, but they are most widely
distributed in the warmest environments of temperate North
America (the Sonoran and Mojave deserts). Zimmerman and
Tracy (1989) discussed the relationship between geographical

distribution of herbivorous lizards and temperature, and

105
stated that ’ . . . herbivory may have more stringent
environmental constraints on its effectiveness compared with
constraints imposed by carnivory.’ Others suggested that
warmer temperatures are necessary for improving digestion,
and that the limited radiation of herbivory may be related
to temperature constraints. Rand (1978) stated ’ . . . the
possibility of putrefaction or fermentation proceeding
faster than digestion at low temperatures is a real risk and
zoo keepers report that this is a cause of death in tropical
herbivorous reptiles kept in temperate areas.’ Many
herbivorous lizards are relatively large (Pough, 1973), and
increased body mass may be a thermoregulatory strategy
(Spotila et al., 1973). Thus, the increased body size may
help stabilize the thermal conditions for the symbionts of
the gastrointestinal tract (Skoczylas, 1978). Skoczylas
(1978) stated that ' . . . this [improved thermal
conditions] may affect the degree to which plant food is
utilized and may determine the evolutionary success of the
species.’

All of these theories related to the interplay between
environment and digestive physiology, yet this relationship
is poorly understood. These relationships are important to
consider. For instance, understanding the effect of
temperature on digestion may provide insight on the
evolutionary aspects of reptilian herbivory, or may help
explain the limited geographic distribution of reptilian

herbivores. Moreover, ambient temperature is an

106
environmental factor that can be manipulated in captivity.
Appropriate alterations in temperature that improve rate of
growth and reproductive success in captivity may be

important tools for captive management of reptiles.

Intake

Daily dry matter and nutrient intakes of iguanas used
in this study were three to four times greater at 35 C than
at 28 C, yet there were no differences in nutrient or energy
metabolizability between the iguanas housed at the two
temperatures. Gut fill is finite since it is limited by
physical constraints of gastrointestinal tract volume.

Since gut fill can limit daily dry matter intake, there must
be an increase in rate of digestion, rate of passage, or
both in order to account for the observed increase in daily

dry matter intake.

Rate of digestion

There is some evidence from carnivorous reptiles that
the rate of gastric digestion may increase with increasing
temperature (Coulson and Hernandez, 1983; Parmenter, 1981;
Greenwald and Kantner, 1979; Diefenbach, 1975a,b; Skoczylas,

1970). Since all of these studies have been conducted with

107
carnivorous species, it is unknown if microbial fermentation
in herbivorous lizards would follow a similar pattern. In
ruminants, digestibility decreases in cold-stressed animals,
and there is also a decrease in microbial activity (Kennedy
and Milligan, 1978).

Rate of hydrogen gas production may be a good indicator
of microbial fermentation in hindgut fermenters (Marthinsen
and Fleming, 1982). Hydrogen gas production rate was lower
in the iguanas housed at 28 C than those housed at 35 C.
Thus, it is possible that there may have been a decreased
rate of digestion or fermentation in the iguanas housed at

28 C as compared to the iguanas housed at 35 C.

Rate of digesta passage

Concomitant with a change in rate of digestion there
may also be a change in rate of passage. An effect of
ambient temperature on rate of passage has been reported for
several species of reptiles, including herbivorous lizards.
Rate of passage was not quantitated for the iguanas used in
this study. However, it was observed that the iguanas
housed at 28 C generally required 1-2 d additional days for
the brilliant blue marked feces to appear. The iguanas in
this study were maintained at a constant temperature for the
entire period with no diurnal changes. Troyer (1987)

manipulated ambient temperature by exposing iguanas (Iguana

108
iguana) to incandescent bulbs for 4 hr or 8 hr. These bulbs
were suspended directly over the cages. Iguanas exposed to
the lights for 8 hr maintained higher cloacal temperatures
for a longer period of time than iguanas exposed to the
lights for 4 hr. Under both conditions, there was a diurnal
fluctuation in temperature that may better mimic iguanas in
natural conditions than does a constant ambient temperature.
Mean transit time of 2 mm glass beads was not different
between these two groups of iguanas (80 hr vs 75 hr; Troyer,
1987). However, these iguanas were force-fed. Marker
appearance (“Cr-mordanted fiber) in the feces of Sauromalus
obesus increased by approximately 1 day, from 4.7 d to 5.5
d, for force-fed chuckwallas housed at 36 C and 32 C,
respectively (Zimmerman and Tracy, 1989). A review of data
from other lizard species (Zimmerman and Tracy, 1989)
revealed that increased ambient temperature increased the
rate of digesta passage (Anolis carolinensis, Windell and
Sarokon, 1976; Uta stansburiana, Waldschmidt et al., 1986;

Cnemidophorus tiqris, Gerrhonotus multicarinatus, Sceloporus

 

occidentalis, Harwood, 1979).

Nutrient and energy metabolizability

If there is an increase in rate of digestion with an
increase in temperature, it may be offset by an increase in

rate of digesta passage at the higher temperature. The net

109
effect of these two competing processes may result in little
or no difference in digestibility, and probably
metabolizability, at different ambient temperatures. This
hypothesis would be supported by the data collected from the
iguanas in the present study, and by Karasov and Diamond
(1985) in studies with Sauromalus obesus. A similar
conclusion was reached by Zimmerman and Tracy (1989) in

studies with Dipsosaurus dorsalis. Conversely, Troyer

 

(1987) reported that iguanas maintained under incandescent
lights for 8 hr rather than 4 hr had a higher ’digestive
efficiency' (56% vs 49% energy metabolizability).
Furthermore, Harlow et al. (1976) reported that energy
metabolizability in Dipsosaurus dorsalis was 54%, 63%, and
70% for lizards housed at 33, 37, and 41 C, respectively,
and that these differences in metabolizability were
significantly different. In all previous studies, lizards
were force-fed, and this may have confounded the results.
In some cases (Harlow et al., 1976), lizards at the lower
temperature (33 C) may have been overfed by as much as 330%
of their estimated energy requirement for maintenance
(Zimmerman and Tracy, 1989). This degree of overfeeding may
dramatically increase fecal output and lead to an
underestimation of metabolizability. Based on data from
this study, using gg libitum-fed iguanas, it appears that
nutrient and energy metabolizabilities were not influenced
by differences in ambient temperature. However, the

increased nutrient and energy intake at the higher

110
temperatures may increase the amount of nutrients and energy
supplied to the iguanas at a given metabolizability. While
there may be an increased amount of energy available to
iguanas at a higher temperature, there may also be an

increase in the energy requirement for maintenance.

Bioenergetics

Energy metabolism has been studied extensively in
reptiles for many years (reviewed by Benedict, 1932; Bennett
and Dawson, 1976; Bennett, 1982). Studies have included the
measurement of metabolic rate, energy metabolizability, and
the influence of environmental factors that may influence
energy metabolism, such as temperature. Measurements of
metabolic rate have been performed in the field using the
doubly—labelled water technique, and in controlled
environments using respiratory exchange. Only one published
study has simultaneously compared these two techniques in
lizards (Congdon et al., 1978). With most studies of
reptilian energetics, metabolic rate is reported as the rate
of oxygen consumption or carbon dioxide production. There
has been little emphasis on the conversion of data collected
from respiratory gas exchange to energetic equivalents.
Therefore, it has not been possible to study the
bioenergetics of reptiles. Few studies have integrated the

aspects of energy intake and metabolizability, and the

111

partition of energy between expenditure and storage. No
studies have been reported that establish the effect of
temperature on the efficiency of use of energy for growth.

The effect of ambient temperature on energy expenditure
and food intake is different for reptiles (Coulson and
Hernandez, 1983) than for mammals and birds (Blaxter, 1989)
(Figure 4). Endotherms have a thermoneutral zone (TNZ).
Body temperature is maintained in the TNZ by behavioral
mechanisms, and temperature changes within the TNZ do not
change energy expenditure. Below and above the TNZ, body
temperature is maintained by physiological mechanisms, and
energy expenditure increases (Blaxter, 1989, NRC, 1981).
The response to changes in ambient temperature is much
different for ectotherms than endotherms. As temperature
decreases from the preferred body temperature, energy
expenditure in ectotherms decreases. As temperature
increases, energy expenditure increases, but the increase is
a non-linear function of temperature (Coulson and Hernandez,
1983). Food intake of endotherms increases below the TNZ
and decreases above the TNZ. Food intake of ectotherms
increases as temperature increases, and the increase in food
intake may be necessary to support the non-linear increase

in energy expenditure.

112

 

 

 

 

 

8 Ectotherm

3 Endotherm

+4

U

CI

(1)

Q.

>< l I

(D l l

g l l

e l I

E]; l l

g: i Thermoneutral :

1.1.1 Jl zone !
Lower Upper
Critical CriticaI

Temperature

 

 

 

Figure 4. Relationship between energy expenditure and

ambient temperature for endotherms and ectotherms.

113

Comparison of respiratory exchange and doubly-labelled water

In this study, energy expenditure was measured using
two methods of indirect calorimetry: 1) respiratory exchange
(EEm), and 2) doubly-labelled water (EEMM). In general,
these two methods agree very well under controlled
conditions (Seale et al., 1993). In field studies, EEdlw may
be higher than EEre as a consequence of the increased
activity of free-ranging animals in their environment. For
the iguanas used in this study, the magnitude of the
difference between EEre and EEdlw was large. The measurements
of pCOZleandEEdlw were 1.5 and 2 times greater than pCO2re

and EE These differences may be related to: 1) actual

re’
differences in energy expenditure under the different
measurement conditions, 2) problems associated with

measuring respiratory exchange, or 3) violations of

underlying assumptions, especially for the DLW method.

Analysis of respiratory exchange calorimetry

Potential problems with respiratory exchange
measurements with the iguanas used in this study may be a
consequence of the need to move the iguanas to a different
environmental chamber (but with the same ambient conditions)
for the measurements, and a decrease in mean dry matter

intake when they were in the calorimeters. In addition, it

114
was not possible to collect and measure excreted urinary
nitrogen from the calorimeters. Inability to collect this
source of N may result in errors when converting gas
production to caloric equivalents; however, the error would
be small (Seale et al., 1989; Farrell, 1974). Based on the
recoveries of oxygen and carbon dioxide from ethanol
combustion, it appeared that these closed-circuit
calorimeters were well suited for measurement of respiratory
exchange. They were relatively inexpensive to construct,
and if sealed properly, they provided accurate and precise

measurements.

Analysis of the doubly—labelled water method

The assumptions and problems with the doubly-labelled
water method have been discussed by several authors (Lifson,
and McClintock, 1966; Nagy, 1980; Speakman, 1990; Seale et
al., 1989; Nagy and Costa, 1980; Speakman and Racey, 1986).
Some assumptions of the DLW method are difficult or
impossible to control, such as the assumption that water and
(KL fluxes are constant. Thus, the isotope turnover rates
and the calculation of rate of Gig production is an average
measurement over the length of the particular study (Nagy,
1980). Other assumptions of the DLW method were minimized
in this study, such as changes in background isotope

concentrations due to changes in diet or water source

115

(Coward, 1991; Roberts et al., 1988; Klein et al., 1984).
For example, the iguanas in this study were offered diet
from the same manufactured lot and drinking water from the
same source throughout both periods. Thus, changes in
background isotope concentrations are unlikely.
Furthermore, with the large rate of air turnover in the
environmental chambers, it is unlikely that labelled CO2
could reenter the iguanas via pulmonary tissue exchange.

There were other potential problems associated with the
DLW technique applied to these iguanas. The problems may
include: 1) change in total body water pool size, 2) unknown
fractionation constants and the effect of temperature on
fractionation constants, 3) the loss of isotopes in forms
other than COzenuiIQO, and 4) unknown RQ during the DLW
measurement period. The RQ is necessary to establish the
relationship between pCO2 and p02, and to convert the rate
of pCOztx>a.rate of energy expenditure. Each of these
potential problems of the DLW method may have affected the
estimation of energy expenditure in these iguanas, and may
help explain the relatively large difference between EEre
and EEMM. Furthermore, there was a large difference in body
water pool size when it was determined by H50 space
compared to H2180 space (ND/No). The difference in 2H20 and
EQNO pool size, and the discrepancy between EEre and EEdlw may
be a consequence of violating different assumptions of the
DLW method. Four of these assumptions will be considered in

detail.

116

First, the equation used to calculate CO2 production is
based on the assumption that the body water pool size is
constant (Nagy, 1980). With these iguanas, body water pool
size may have increased as a consequence of the increase in
body size (Table 10). Furthermore, repeated blood sampling
may also result in an apparent increase in body water pool
size. However, for the iguana with the greatest weight
gain, who also had approximately 48 m1 of blood drawn, the
increase in water pool size would be less than 50%, and this
increase would result in an error of less than 5% in
calculation of CO2 production rate (Nagy, 1980). The ratio
of pCOZdIW/pCO2m is much greater than 5%. Furthermore, this
ratio was not different for the few iguanas that did not
change body weight, and presumably had little change in body
water pool size over the DLW measurement period. Thus, it
is unlikely that changes in body water pool size would
explain the large difference between EEre and EEflw.

Second, the isotopic fractionation constants used to
calculate pCO2 may not be valid for these lizards.
Fractionation constants are used to correct for potential
changes in isotopic concentrations that may occur for H4)
and.CI5 during changes in state from liquid to vapor. The

three constants are (Schoeller et al., 1986a,b):

1) f1:= 0.941 for HEO(gas)/HQO(liquid)
2) f2 = 0.992 for H2180(gas)/H2180(liquid)
3) f3 = 1.039 for C1802(gas)/H21°O(liquid).

 

 

 

117
The fractionation constants may be species—specific and
temperature dependent (Nagy, 1980; Nagy and Costa, 1980).
In some situations, potential errors associated with
isotopic fractionation (especially'fg and f2)‘will negate
each other, and produce little additional error in the
calculation of pCO2 (Nagy, 1980). For instance, if
temperature affects both H and O fractionation to the same
extent, then the difference between H and O turnover will
not be affected (Lifson and McClintock, 1966; Nagy, 1980).
There was no effect of temperature on the ratio of
pCOZdlw/ijO2re in this study. This observation suggests that
ambient temperature had little effect on isotopic
fractionation. Furthermore, the extent of the error
associated with isotopic fractionation, especially fl and
f2,:may be minimized in these iguanas since insensible water
loss is very limited in iguanas. Transcutaneous water loss
in humans is approximately 5 times greater than in iguanas
(Schoeller et al., 1986a,b; Bentley and Schmidt-Nielsen,
1966; Mautz, 1982), and this difference in water loss may be
related to a lipid barrier in reptilian epidermis (Roberts
and Lillywhite, 1980; Lillywhite and Maderson, 1982).

An incorrect value for the fractionation constant f3
[CFMN (gas)/H{”O (1iquid)] may introduce an error that could
explain the high flux of 180 observed in these iguanas.

This fractionation can occur if the enzyme carbonic
anhydrase can discriminate between 160 and ”0. If f3 is

higher than previously measured (Schoeller et al., 1986b),

118
then 180 flux may be increased. Isotopic discrimination
between 180 and 16O is known to occur in plants (Lou and
Sternberg, 1992; Bricout, 1979). Isotopic discrimination in
the green iguana could be associated with its own carbonic
anhydrase enzyme, or with enzymes associated with the
symbiotic microbes in the gastrointestinal tract.

Third, violations of the assumption that.2H and 18O
isotopes are lost from the body only as H20 and CO2 can lead
to an error in the calculation of pCOg (Haggarty et al.,
1991). Several sets of reported data suggest that hydrogen
can be sequestered in several forms in the body. Hydrogen
but not oxygen can exchange with the non-water pool during
fatty acid synthesis, and during peptide bond formation
(Haggarty et al., 1991; Humphrey and Davis, 1974). This
sequestration can result in an overestimation of total body
water pool, and an overestimation of water flux (Culebras et
al., 1977; Nagy and Costa, 1980). Consequently, pCOzvfiJJ.
be underestimated (Haggarty et al., 1991). The
overestimation of water flux and the underestimation of CO2
flux will be dependent on the relative size, and the
relative rate of exchange of theriH between the water pool
and the non-water pool. The exchange and sequestration of
2H in the non-water pool is generally used to account for
the larger body water pool size that is usually observed
when measured by 2H as compared to 18O (ND/No). However, the
difference in these pools is usually no greater than 10%

(Schoeller et al., 1980, 1986a). In these iguanas the

119
difference in pool size was 20% to 25%. This larger
difference may be a function of greater'?H exchange into a
relatively large non-water pool in the green iguana than has
been observed in Other species.

Few data have been reported on the sequestration of
oxygen, and the loss of oxygen from sources other than CO2.
Oxygen can exchange with the bicarbonate pool through the
action of carbonic anhydrase (Nagy, 1980; Haggarty and
McGaw, 1988). In fact, this is the metabolic basis of the
DLW method (Lifson et al., 1949). However, there may be a
disproportionate loss of 180 since HCO;‘or COf“ may contain
a disproportionate amount of 180 relative to H20 or CO2
(Nagy, 1980). In reptiles, the excretion of sodium,
potassium, and ammonium bicarbonate salts may be important
in regulating acid-base balance (Minnich, 1972, 1982;
Schmidt—Nielson et al., 1963; Coulson and Hernandez, 1983),
and the bicarbonate pool size may be larger in reptiles than
in mammals (Bickler, 1981; Thorson, 1968). The error
associated with 180 exchange with the bicarbonate pool is
estimated to be only 0.1% in humans (Haggarty and McGaw,
1988), and is much less than the error associated with 41
exchange (approximately 4-10%). In reptiles, the large
bicarbonate pool, and the excretion of bicarbonate as salts
through the nasal gland may increase the turnover rate of
18O. This increase in 18O flux could result in an
overestimation of the difference of the turnover rates of

18O and 2H, and an overestimation of CO2 flux. Thus, a large

 

120
bicarbonate pool that has a high flux in these iguanas may
help to explain the magnitude of the observed difference
between pCO2re and pCOmhv Furthermore, in reptiles, the
concentration of plasma bicarbonate is temperature
independent (Howell and Rahn, 1976), and in these iguanas,
the discrepancy between pCOhfi and pCOmn,was also
temperature independent. Overestimation of 18O flux may be
exacerbated by isotopic discrimination between 160 and 18O
and high rates of bicarbonate excretion.

Fourth, the DLW method provides an estimation of pCO2
and there may be errors in using RQ to convert from rate of
carbon dioxide production to the rate of energy expenditure.
The energy equivalent oflflk can vary, depending on the
combination of substrates being oxidized. The RQ (pcoflfixx)
will reflect the mix of substrates being oxidized. Thus, it
is important to have an estimate of the RQ, or preferably,
to have measured RQ by respiratory exchange from animals
under similar metabolic conditions. For the iguanas in
this study, the RQ measured by respiratory exchange was used
to determine pO2 from the pCO2 measured by the differential
turnover of the DLW. The overall mean RQ of 0.78 was used
(Table 14). In general, the RQ of reptiles is lower than
the RQ of ureotelic species. This difference is a
consequence of the lower RQ associated with uric acid
formation from protein oxidation as opposed to urea
formation (Brafield and Llewellyn, 1982). Even if the RQ of

the iguanas during the DLW measurement period was 1.0, EEdlw

121
would be approximately 17% less, and would still be more
than 100% greater than EEm. The use of RQ is important for
converting the rate of CIA production to EE. Ignoring the
RQ and estimating EE based solely on pCO2 can lead to errors
as high as 20% (Blaxter, 1989). However, errors in RQ alone
could not explain the large difference between EEre and EEdlw
in these iguanas.

There may have been systematic errors that have led to
an overestimation of EEdlw or the iguanas may have been much
more active outside of the respiratory calorimeters.
Calculation of RE by different methods may provide some
insight into which set of data (EEdlw or EEm) provides a more

accurate estimate of EE.

Comparison of RE calculated by different methods

In order to make comparisons between RE calculated by
different methods, it is first necessary to assign caloric
equivalents to pCOzénuigxb in order to calculate EE. Nagy
(1982a) has used a caloric equivalent of 5.19 kcal/1 CO2
from DLW experiments to calculate energy expenditure. This
energy equivalence of. CO2 was based on an estimated RQ of
0.93 for herbivorous lizards. The RQ was estimated based on
the composition of the diet consumed by Sauromalus obesus,
but it has been used for all herbivorous lizards. The

energy equivalents used in this study were those suggested

122
by Weir (1949). However, in this study there was no
correction for protein oxidation based on urate excretion.
For the iguanas in this study, it was not possible to
separate N excreted in feces from non—fecal sources
(urates). The correction for nitrogen excretion in birds,
who also excrete nitrogen as uric acid, is only 0.28 kcal/g

N. Urate N excretion of Sauromalus obesus may represent as

 

much as 90% of total N excretion (Nagy and Shoemaker, 1975).
Based on this assumption, the correction for urate nitrogen
excretion from the iguanas in this study would be less than
0.5% of EEm.

RE was determined by three methods: 1) the difference
between MEi and EEre (corrected for decreased dry matter
intake in the calorimeter) (REm), 2) the difference between
NEH and EEdlw (REmM), and 3) the sum of energy stored as fat
and protein, determined by the balance of carbon and
nitrogen (REamfl). REdlw and REre were calculated as a
difference whereas RECNbal was calculated as the sum of energy
stored as fat and protein. Therefore, REdlw and REre are
independent of REQmfl. REdlw is significantly lower than REre
and REQmfl, but REre and RECNbal are not different.

Furthermore, there are several iguanas that had negative
values of REMw but had positive growth rates. Given the
similarity between REre and REQmu, and the negative values
of REdlw of some growing iguanas, these data would suggest
that EEdlw may be an overestimate of actual energy

expenditure.

123
Assessment of the difference between respiratory exchange
and doubly—labelled water methodologies

{XXL and EE measured by the DLW method are greater than
when measured by respiratory exchange. In addition, REre
and RECNball were not different from each other but REdlw is
much lower than the RE calculated by the other two methods.
Furthermore, several iguanas had negative values of REM“,
even though those iguanas were growing. Thus, it appears
that the DLW method overestimated EE in these iguanas.

There are at least two inconsistencies that are
apparent from the DLW method: 1) there is a large
discrepancy between water pool size measured by'?H and 18O at
their zero time intercepts, and 2) pC02.is high relative to
respiratory exchange. Body composition estimates based on
2H pool size at zero time intercept result in estimates of
body fat that are negative whereas body composition based on
180 pool size at zero time intercept results in estimates of
body fat that may be reasonable. Thus, it appears that the
2H pool size at the zero time intercept may have been
overestimated. There may be a relatively large pool of H1
in a form other than HgD‘that is not being accounted for
when measuring from the blood pool. Water flux, a function
cflfZH flux, was somewhat lower than predicted for free-
ranging tropical reptiles (Nagy, 1982b), but this difference
may be related to the relatively dry diet that the iguanas
were fed in this study. Based on these observations, the

measurement of 2‘H flux may be valid, but the:2H zero time

124
intercept may be high.

Therefore, the overestimation of pCO2 may be a
consequence of a high rate of 18O flux, perhaps from a
disproportionate excretion of 18O-labelled bicarbonate. The
only reported direct comparison between respiratory exchange
and DLW in lizards (Sceloporus species) was done with four
animals for 7.5 days. DLW overestimated pCO2 measured by
respiratory exchange by only 3.2% in these lizards.
Therefore, the DLW method may be valid under other
conditions and for other species. Species-specificity may
be a function of species—specific metabolism of bicarbonate
that is reported to occur in reptiles (Howell and Rahn,
1976).

In these iguanas, there may have been more than one
assumption of the DLW method that was violated, and the
violations seem to be temperature independent. Indirect
calorimetry data from respiratory exchange may provide a
better understanding of energy metabolism for the iguanas in

this study.

Comparison of EEre to other respiratory exchange data

p02.and pCO2re of these iguanas is similar to other data
reported for green iguanas (Table 17). The similarity of
these data to previously reported data helps confirm the

suitability of the closed-circuit calorimeters used in this

 

 

125
study to measure EE, and the accuracy of EEm. The
similarity between published data and pCOhm also supports
the suggestion that pCO2re is a more accurate estimate of

actual pCO2 than pCOzdlw, in these iguanas.

Comparison of EEre to predicted field metabolic rate

Nagy (1982a) developed a regression equation to predict
field metabolic rate (FMR) of iguanid lizards as a function
of body weight. The DLW method was used in nine iguanid
species that ranged in weight from 0.5 g to 1,481 g. Data
from Iquana iguana were not available. The range in body
weight may appear large, but almost 90% of the observations
were from species whose body weight was from 1.6 g to 167 g,
and only one observation was from a species with a body
weight of over 1 kg. EEre from the iguanas used in this
study was compared with predicted FMR using Nagy's equation
(Figure 5). The EEre of the iguanas housed at 35 C was not
different from the predicted FMR (paired t-test). The mean

difference was 0.33::0.48 kcal/d. However, EEre of the

iguanas housed at 28 C was lower than predicted FMR
(p<0.001). The mean difference of the predicted FMR and

actual EEre was -8.78 x 0.93 kcal/d. Field metabolic rate

is the energy expenditure of free—ranging animals in their

 

 

126

Table 17. Resting oxygen consumption and carbon dioxide

production of Iguana iguana at different ambient temperatures.

 

 

 

 

 

 

 

 

 

 

 

Reference N Weight Temperature p02 pCO2

range (C) (ml/g/hr) (ml/g/hr)
(kg)

1 15 0.4-1.2 15 0.022

2 15 0.4-1.2 20 0.040

3 12 0.6-1.9 28 0.089 0.071

2 15 0.4-1.2 30 0.081

2 15 0.4-1.2 35 0.150

4 4 0.7-1.0 35 0.183

3 12 0.5-2.5 35 0.194 0.150

5 3 0.6-1.5 35 0.26 0.20

1 15 0.4—1.2 40 0.165

 

 

 

 

 

 

1 Moberly, 1964.

2 Moberly, 1968a.

3 This study, measurements from respiratory exchange.
4 Gleeson et al., 1980.

5 Gleeson and Bennett, 1982.

 

127

 

 

 

   
  
  
 

 

 

 

   

 

 

 

30/E/ll I I I
A

{5 Ambient temperature
\
6 25 _ C 28 C ._
O A 35 C
.x
g 20 — j
L1.
(1) 0.80
+6 15 _ FMR (kcal/d) = 0.05358W(g)
.9
6
g 10 '- A . '-
E
E . . . '

5 ~ . i
:3 T .0 " ‘. 5
I:

O/L/ll I l I

500 1000 1500 2000 2500

Body weight (BW, g)

 

 

 

Figure 5. Comparison of predicted field metabolic rate
(FMR, Nagy, 1982a) with actual energy expenditure of

iguanas housed at different ambient temperatures.

128
normal habitat. Presumably, in the field, reptiles can
self-select ambient temperatures. Therefore, the FMR of
reptiles should be a function of their self—selected body
temperature range. The self-selected body temperature of

Iguana iguana is thought to be near 35 C. Thus, the iguanas

 

housed near their self—selected body temperature range had
predicted FMRs that were not different from EEm.
Furthermore, the predicted FMR of iguanas housed below their
self—selected body temperature overestimated measured EEm.
Therefore, this prediction equation may be valid for iguanid
lizards over 1000 g, and in their self-selected body
temperature range.

Field metabolic rate was not different from the EB”,
yet FMR includes energy expenditure associated with some
activities that may have been limited for the captive
iguanas used in this study. For instance, some behaviors of
free—ranging iguanas were not exhibited in these captive
iguanas. Examples of these behaviors include foraging for
leaves, escaping predators, and climbing to the forest
canopy. Restricted activity in respiratory calorimeters may
result in differences between FMR and EE measured under
controlled conditions (Weathers et al., 1984). These
differences are likely to be a function of a species
activity pattern; and, the error may be species specific.
The similarity of FMR and EEre in this study may reflect the
rather limited activity of iguanas in the wild (Moberly,

1968a), and their relative inactivity in captivity. Thus,

129
the energy expenditure associated with activity in the field
may not be different from energy expenditure associated with
activity under the captive conditions experienced by these

iguanas, although the actual activities may be different.

Maintenance energy requirement

The relationship between RE and.NEh can be used to
determine the energy requirement for maintenance (MEm) using
regression methods. In general, calculation of MEIll by
linear regression of RE and MEi does not take into account
the non-linearity of energy storage above maintenance that
has been reported in many species, and assumes that there
are no measurement errors in MEi (Van Es, 1972). Even with
all of the apparent problems, the regression technique may
be accurate. In human subjects, theINEQ was not different
when determined by the use of regression coefficients as
compared to maintaining the subjects near maintenance and
correcting for small changes in energy balance (Van Es et
al., 1984). Ema was calculated to be 5.82 and 8.82 kcal/d
for iguanas housed at 28 and 35 C, respectively. There are
many factors that can affect MEm. Some of these factors are
weight, age, diet composition, and rate of growth (Blaxter,
1989) . The difference in MEm between the two groups of
iguanas may be a function of temperature or may be a

function of other variables that could confound the measure,

130
such as rate of growth. Other methods for estimating MEm
were not possible with the current experimental design. The
regression method used to estimate MEm of these iguanas may
be prone to many errors, but these data provide a framework

for future experimentation.

Use of energy above maintenance for growth

No other studies have attempted to determine the
apparent partial efficiency of use of metabolizable energy
for tissue deposition above maintenance (kg) for reptiles.
This measure is often determined by maintaining an
individual animal at two levels of food intake, one near
weight maintenance and one above weight maintenance
(Blaxter, 1989). This method was not possible under the
current design, but the estimates from this study may
provide a set of initial data for further validation.

The kfizmay be function of level of intake in relation
to the maintenance requirement (defined as RE = 0), and this
relationship may be curvilinear. However, there was no
curvilinear component detected by least—squares regression
for these iguanas at either temperature. Furthermore, for
these iguanas, there was no effect of temperature on the
efficiency of tissue deposition above maintenance. Thus, at
both temperatures, energy consumed above maintenance was

used for growth with similar efficiency. These efficiencies

131
are based on assumptions that were made to correct for the
decrease in food intake during the calorimetry measurements.
These efficiencies may be different for a different set of
assumptions, but the parallel relationship between the two

efficiencies would not necessarily change.

Summary

Daily food intake of male and female Iguana iguana

 

housed at 35 C was greater than for the iguanas housed at 28
C. However, the fiber fraction digestibility, and the dry
matter and energy metabolizability of the diet consumed was
not affected by ambient temperature. Even though metabolic
rate and maintenance requirements may have been higher at 35
C than at 28 C, daily rate of gain was also greater at 35 C
than at 28 C. Thus, the increase in food intake may have
been proportionally greater than the increase in MEm.
However, metabolizable energy consumed above maintenance was
stored with similar efficiency at both temperatures.
Maintaining iguanas at an ambient temperature that includes
their self-selected body temperature may promote growth and

improve reproductive success in captivity.

CONCLUSIONS

Diet composition and ambient temperature affect the
digestive function and bioenergetics of the green iguana.
This study confirmed that dietary fiber is digested in the
gastrointestinal tract of the green iguana. The range in
concentration of dietary fiber used in this study did not
affect daily dry matter intake but did affect nutrient and
energy metabolizability, and growth rate. To maximize
growth rate, a diet that contains less than 27% neutral
detergent fiber (% of the dry matter) is recommended. The
fiber study demonstrated that herbivorous lizards will eat a
manufactured diet, gg libitum, in captivity, and that
rigorous metabolizability and growth trials can be conducted
using standard techniques.

Nutrient and energy metabolizability were not affected
by the ambient temperature to which these iguanas were
exposed, although these temperatures affected energy
expenditure, daily dry matter intake, and growth rate.
However, with increasing temperature, the increase in energy
intake was greater than the increase in energy expenditure.
Since metabolizability was constant, greater amounts of
nutrients and energy were available for growth. Ambient

temperature did not affect the efficiency with which

132

133
metabolizable energy consumed above maintenance was used for
growth.

The agreement was poor between energy expenditure
measured by respiratory exchange and the doubly—labelled
water method. The rate of energy expenditure measured by
the doubly-labelled water method was approximately twice the
rate measured by respiratory exchange. Analysis of retained
energy computed from each method of measuring energy
expenditure, and comparison of the rate of oxygen
consumption to previously published data from a number of
sources, suggests that the discrepancy may be related to
errors in the doubly-labelled water method used in these
iguanas. The overestimates of the rate of carbon dioxide
production when using the doubly-labelled water method may
be related to a violation of the assumptions on which the
method is based. It is also likely that more than one
violation may have occurred. Mathematical adjustments that
improved the ratio of Nwflk led to an even larger difference
between pCOwh,and pCOmm. Thus, a single, systematic error,
is unlikely the cause of the discrepancy between respiratory
calorimetry and the DLW method.

Energy expenditure measured by respiratory exchange of
iguanas, in closed-circuit calorimeters, was accurate and
economical. A minimal amount of equipment was necessary for
these measurements, and measurements on several iguanas
could be made at one time, in a relatively small space.

The physiological responses of the iguanas used in

134

these studies are a function of the diets that were fed and
the environment in which the iguanas were housed. These
diets and the environment are much different from those
experienced by free-ranging iguanas in the tropics. These
diets consisted of relatively fine particles. Wild iguanas
would normally consume large pieces of leaves, fruits or
flowers. This difference in particle size may influence
digestive physiology. Iguanas in the wild also are likely
to experience diurnal and seasonal changes in temperature,
rainfall, and food availability. The iguanas used in these
studies were maintained in a stable environment. Thus, the
interpretation of the data from these studies must take
these differences into account. While the conditions used
in these studies may be different from those experienced by
free-ranging iguanas, they are similar to environmental
conditions in zoos that maintain this species. Thus, the
data may be applicable to systems of captive management.

The data collected from these studies provides a
framework for future research with the green iguana. Fiber
intake of free-ranging iguanas may be much higher than the
highest level of fiber used in these studies. There is
still little known about hindgut fermentation in this
species of iguana. In the wild, iguanas undoubtedly
encounter secondary plant compounds that may have wide
ranging effects, including effects on digestive processes
and food selection. Furthermore, only two ambient

temperatures were used in this study. Physiological

135
responses to changes in temperature are probably non-linear,
and studies of the response to more temperatures will be
necessary to better understand their effects. A primary
goal of the captive management plan for the green iguana in
Central America is intensive production. Defining the
nutritional requirements of this species is likely to be
important for successful intensive production.

Folivory in lizards is rare, and the green iguana has
been able to exploit this ecological niche. This species
has the potential to provide a source of nutrients to humans
in Central America. The intensive production of iguanas may
encourage an alternative agriculture that is less
destructive than traditional slash-and-burn techniques, and
the overgrazing associated with intensive beef cattle
production. Thus, reintroduction of captive-raised iguanas
into appropriately restored habitat has the potential to
provide a source of human food, and a monetary return,
without the adverse ecological impact of the more exploitive
and environmentally harmful practices currently in use in

some areas of the tropics.

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