Determination of the passage rate in saltwater
crocodiles (C. porosus) using beads, flagging tape
and fluorescent pigment as digestive markers
Student: Eveline Dijkstra
Student number: 3258882
Supervisors:
Dr. M.J.L. Kik
Dr. C.M. Gienger
Utrecht University - Faculty of Veterinary
Medicine, July 2014
Abstract
The saltwater crocodile (Crocodylus porosus) is extensively farmed for their skin and meat. In order
to optimise growth rates and production crocodiles need to be fed an optimal diet, based on their
nutritional physiology. In this study two experiments were conducted to determine the passage rate
of food in juvenile and sub-adult crocodiles. Plastic beads, pieces of flagging tape and a fluorescent
pigment were used as digestive markers, to evaluate whether these markers are suitable as digestive
markers in C. porosus.
Results showed differences in excretion patterns between the type of markers. Large percentages of
beads and pieces of flagging were retained in the stomach, not passing the digestive system. The
fluorescent pigment was excreted by juvenile crocodiles almost continuously during the experiment.
Despite the fact that the fluorescent pigment did pass the digestive tract, the obtained data is
inaccurate.
It can be concluded that the used markers are not useable as digestive markers in crocodiles, since
they were not successful in determining the passage rate in juvenile and sub-adult C. porosus. More
precise markers need to be used as digestive markers in crocodilians to achieve more accurate
knowledge about the crocodile digestive system to eventually improve crocodile nutrition.
Introduction
The saltwater crocodile (Crocodylus porosus) is extensively farmed for their skin and meat1,2. Similar
to sectors of extensive animal production systems, nutrition is considered to be of major importance
in crocodile farming to optimise growth rates of individuals, and therefore production1,3. In the past
few decades many attempts have been done to improve the diet of farmed crocodiles. However, in
order to compose an optimal diet for farmed crocodiles it should be based on their nutritional
physiology. Surprisingly only little accurate data is known about the digestive physiology of any
crocodilian species.
As part of a larger study, to provide data about the digestive efficiency of several crocodile species,
this study was designed to define the rate for food to pass the gastro-intestinal tract of juvenile and
adult saltwater crocodiles. The passage rate defines the rate at which residues of the diet run
through the digestive tract, the time from ingestion to defecation4. The passage rate is a commonly
measured variable in digestion studies, since it is an estimation of the retention time. The retention
time is a term for the period in which the digesta remain in the absorptive regions of the digestive
tract. It reflects the gut evacuation and is therefore related to the digestion rate. Consequently the
retention time can be an indication of the time and energy required to digest food5.
The gut passage rate between species is extremely variable6. Compared to ectotherm species,
endotherms have relatively short and consistent intestinal passage times7. The physical performance
of ectotherms is greatly influenced by external factors. The passage rate in reptiles is affected by
their body temperature8,9, diet quality10, diet quantity9, type of dietary marker8,11, and the
morphology of the digestive tract. For example, among the herbivorous reptiles the proximal colon
of iguanid, agamid, and scincid species contains transverse valves. The number of these valves is
positively correlated with the adult body size, and may as well have impact on the passage rate12,13.
Furthermore, just like in humans, simply individual variation is a possible cause for variability, even
under controlled conditions6.
Most studies used markers to determine the passage rate. Along with the large number of digestive
studies on different species, an extensive variety of markers came to existence since not all markers
are suitable to the morphology of every digestive tract. Besides, there are some other practical
considerations which may decline their usability, for example the type of data that needs to be
collected or the environment in which the animal lives.
In the next paragraphs several types of markers, used in reptilians over the past decades, are
reviewed. This review will give a structured overview of the markers concerning their effectiveness
during the trials, their advantages and disadvantages. For a quick overview and comparison of the
discussed studies, the data of the reviewed articles are recorded in table 5 (appendix, p. 32).
Digestive markers
In order to define the gut passage rate, the faeces of the experimental diet needs to be
distinguishable from other meals. In most cases a diet is mixed or labelled with a marker that
eventually will reappear in the faeces. The rate at which the marker reappears is the rate for
undigested residues of the diet to pass the digestive tract4. To ensure the reappearance of the
marker, a marker has to meet to certain characteristics. The ideal dietary marker is non-absorbable
and has the same particle size and density as the natural diet. This way, the marker will be
recoverable qualitatively and quantitatively in the faeces, and will the marker behave similarly to the
diet along the digestive tract. Also the passage of digesta should not be influenced by the marker4.
There are two options to mark an experimental diet; either by adding an inert marker to the diet, or
by feeding a diet that is different in some identifiable way, for example by colour or texture4. In the
scenario the marker will be called an external marker, since it is artificially added to the diet. In the
second option the identifiable parts of the diet can be defined as internal markers, as the markers
are a natural en integral part of the diet.
In a study of Greenwald et al. (1979)14 the faeces from a given meal could be identified by the
undigested hair of mice which were fed to corn snakes (Elaphe guttata guttata). Sadeghayobi et al.
(2011)15 used the seeds of three different types of fruit as a marker to determine the digesta
retention time in Galapagos tortoises (Geochelone nigra). By just feeding an animal’s natural diet,
without adding a foreign material to the experimental diet, all possible side effects of a marker are
eliminated. However, quantification is not possible using this technique4.
Most studies used a technique in which they added an external marker to the experimental diet.
Based on the literature of digestion studies in reptilians, basically four groups of inert faecal markers
can be defined; pigments and dyes, particulate markers, chemical markers, and radiopaque markers.
Pigment and dyes
The first category ‘pigments and dyes’ includes products which are added to stain the experimental
diet. Carmine red is a frequently used marker in humans16, mammals17, fish18,19 and reptiles1,20. Other
examples are the vital dye indigo carmine8,21 and fluorescent pigment5,22,23. A food colouring gel was
used in a study with Komodo dragons (Varanus komodoensis)24. Food colouring products are often
used as a marker since they are easy to obtain and are generally safe to use.
All of these studies used their marker to determine the time between the food intake and the first
appearance of the coloured diet in the faeces. Some of them also defined the last appearance in the
faeces1,5,20. Because of the small particle sizes, stains and dyes will easily mix with the diet, causing no
mechanical disruption of the passage of the food along the gut4. According to Zimmerman and Tracy
(1989)4 the use of a stain or a dye is not a quantifiable technique, since they are prone to partial
absorption. Also secondary staining of food material inside the gastrointestinal tract makes the
interpretation of the staining technique complicated8. This could be an explanation for the wide
range between the first and last appearance of the markers in the studies by Garnett (1988)1 and
Liesegang et al. (2001)20. Even though Hatch and Afik (1999)5 used a spectrophotometer to
determine the percentage of fluorescent marker present in each faecal sample, most findings are
merely based on macroscopic visual detection. Therefore data concerning the appearance of these
markers in the faeces should be carefully interpreted.
In addition, not every stain or dye sticks to the same fraction of the diet. Beaupre et al. (1993)23 and
Hatch and Afik (1999)5 observed a difference in transit time between a water soluble dye, such as
carmine red25, and a fluorescent powder. The water soluble dye appeared in the faeces long before
the fluorescent powder. From this they concluded that the soluble dye stained the liquid phase of
the diet and the fluorescent powder the solid phase. However, no statistical results are presented in
the study of Beaupre et al. (1993)23 and the study of Hatch and Afik (1999)5 reported no significant
differences in transit times between the used markers. It is argued that the difference in transit time
for each phase depends on the ability of a certain species to separate the liquid from the solid phases
in the gut5,23. These results point out another difficulty of comparing gut transit times among several
studies using different markers.
Particulate markers
The following group of inert markers are the particulate markers. These markers are defined as
synthetic indigestible, non-absorbable organic substances, which are clearly recognizable in faeces.
Particulate markers can be subdivided in non-quantifiable and quantifiable markers.
Examples of non-quantifiable markers are glitter and fluorescent polyacrylamide beads. Both
markers are generally larger in diameter than pigments and dyes, but still too small to use them as
quantifiable markers. These markers are mostly used for the same purpose as stains and dyes.
Because of their considerably small particle size they still easily blend with the experimental diet.
Kummrow et al. (2011)26 used crickets coated in glitter (< 1 mm) as a digestive marker in chameleons
(Chamaleleo calyptratus). Faecal samples were visually examined for the excretion of glitter.
Fluorescent polycrylamide beads, used by Waldschmidt et al. (1986)9, are beads of 100 μm in
diameter which are chemically bonded to a fluorescent dye. They used this marker in the
insectivorous side-blotched lizard (Uta stansburiana) and were able to detect the beads in the faeces
by examination with a UV microscope. Zimmerman and Tracy (1989)4 used fluorescent
polyacrylamide beads to determine the passage rate in chuckwallas (Sauromalus obesus), but
weren’t able to detect the markers in the faeces. They discussed that this could be either because
the beads never appeared in the faeces, perhaps because they retained in the deep colic valves, or
the beads no longer fluoresced after passing through the digestive tract. The first reason would imply
that the beads aren’t immiscible with the food, which would affect the passage time results.
Quantifiable particulate markers are frequently used as digestive markers in studies with reptiles,
especially in field studies since they are easily identifiable. Beads and pieces of plastic tape are the
most commonly used markers in this category. However, because beads and plastic pieces have a
relatively large particle size, these markers are immiscible with the food. Therefore reported transit
times in studies using these types of markers actually measured the passage rate of the marker itself.
In a study of Harwood (1979)8 two markers were used; carmine red and pieces of vinyl tape. The
vinyl tape usually passed through the digestive tract more slowly than the stained food. He states
that this increase in residency could have been caused by temporary adhesion of the vinyl tape to
the intestinal mucosa.
A study of Troyer (1984)27 showed that glass beads of 2 mm in diameter and 4 mm discs of vinyl
flagging tape moved at the same rate in green iguanas (Iguana iguana). She also noticed that the
initial experimental diet and its markers appeared together in the faeces, what would assume that
the markers moved at the same rate as the diet. Wikelski et al. (1993)28 found similar results in a
study with marine iguanas (Amblyrhynchus christatus).
Valente et al. (2008)29 used very-low-dense coloured dishes of flat foam (5 mm in diameter), which
appeared at a faster rate in the faeces than a simultaneously used marker with a higher density. They
assumed that the colour markers travelled with the fluid-phase of the digesta.
In a study of Hailey (1998)30 a combination of loops of polyester thread (2cm in circumference) and
radiopaque Ballotini microbeads (0,65-0,75mm) was used to determine the passage rate. The
recovery of beads was higher than the number of loops found in all cases. He states that this might
be due to fact that fibrous material passes more slowly through the gut than particulate material
such as microbeads. Additionally, the size of the marker might influence the passage rate. In a study
of Spencer et al. (1998)31 the 25mm2 plastic pieces had a longer transit time than the 1mm2 pieces of
plastic.
Since these type of markers are quantifiable, other options for defining the transit time are possible.
Just like with a pigment or a dye the transit time can be defined as the time between feeding and the
first appearance of a marker, the minimum transit time31,32. This definition is generally used in studies
using a single particulate marker. In experiments using a multiple number of markers the maximum
transit time (the time till last the appearance of the marker)15, the modal transit time (time till the
day on which the maximum number of markers reappear), and the median transit time (time till 50%
of the markers is excreted) can be determined30. Others defined the gut transit time as the time
between feeding and the time a certain percentage of the marker has appeared in the faeces. A
minimum of 50% is frequently used27,33. Amarocho and Reina (2008)34 defined the total passage time
as the time at which at least 73% of the markers was recovered and Valente et al. (2008)29 at 85%.
Often this terminology misused or given a different interpretation. For example, tmax is used as the
time till last appearance of the marker but also as the time at which the maximum concentration of
marker appeared10.
Chemical markers
In order to find the most accurate marker, researchers developed techniques using chemicals as a
digestive marker. Several chemicals can be used to bind specific elements in the feed and their
concentrations can easily be measured in the faeces.
Chromium is by far the most frequently used chemical marker in animal digestion studies3,10,35-37.
However, the use of chromic oxide is often criticised because it does not associate specifically with
either the particulate or the fluid phase of the digesta35. Also, dietary chromic oxide is not always
totally recovered from the faeces and therefore it needs to be used at relatively high concentrations,
which may affect the absorption and metabolism of nutrients35,36,38. Mixing a diet with chromic oxide
will colour the feed and eventually the faeces green. This way in some studies it is used as
pigment36,37. On the other hand, the alteration in colour of the feed may influence the voluntary
ingestion. Finally, the use of chromic oxide may be a health hazard, since it may be toxic even at low
concentrations35,38.
In order to nip problems concerned with marking the right components of the digesta, a dual-phase
marker technique is developed. This is a technique in which several markers are used simultaneously
to mark the fluid-phase and the solid-phase of the faeces35. Polyethylene glycol, Co-EDTA and CrEDTA are frequently used fluid-phase markers, while chromium and rare earth elements are
commonly used as particulate-phase markers35,39.
The dual-phase marker technique is frequently used in studies concerning herbivorous species, since
especially rare earth elements have a strong affinity for plant cell walls and can be used in their
soluble form to label indigestible plant fibre40. Despite these characteristics, recently a couple of
studies is published using this technique successfully in carnivorous mammals41-43. Apparently the
binding capacity of rare earth elements is related to the cation exchange capacity of the feedstuffs to
which labelling takes place. Carboxyl, hydroxyl and amino acid nitrogen groups seem to be the
functional groups concerning the rare elements binding39.
These markers can be measured in feed and faeces at low concentrations in the mg kg-1 range.
Because of the accuracy of these markers and the high percentages of recovery, these markers are
commonly used to determine the mean retention time of various components in the feed and to
estimate the apparent digestibility of nutrients in the feed.
Barboza (1995)10, Hatch and Afik (1999)5, and Tracy et al. (2006)44 performed this technique in their
studies, using a herbivorous or omnivorous reptile species. They were able to determine the flow
rate of the liquid-phase and the particulate-phase of the digesta. In all studies the liquid-phase
appeared before the particulate-phase. Also different flow rates were found for particles with
various lengths. The larger the particle, the longer the retention time.
In the study of Hatch and Afik (1999)5 polyethylene glycol was used as an aqueous marker and
glycerol triether as a lipid-marker. A lack of significant difference was found between the lipid marker
and the aqueous marker. They also did not find a significant difference between these markers and
the fluorescent powder marker they used, making the fluorescent powder a plausible marker for
reptiles who don’t measurably separate the liquid from the solid phases in the intestinal tract.
Radiopaque markers
Radiopaque markers are radiodense materials which can be used to outline internal organs and body
fluids in CT-scans or X-ray images. Unlike the previous marking techniques, it is possible to follow
these markers through the digestive system real time. Consequently, the gastrointestinal motility and
the behaviour of digesta in the digestive tract can be analysed for different anatomical
compartments.
Barium sulphate suspensions are frequently used in passage studies7,29,37,45. However, it is semiquantitative and evaluates the gastric emptying time of the liquid but not the solid fraction of the
ingesta. Unless the food is ground to fine particles, barium sulphate will separate in a liquid phase
and move at a different rate from the solid fraction. The gastric emptying time is known to be the
major variable in affecting the orocolonic transit time in cats46. Radiographical and sectional studies
in reptilians have shown that in omnivorous and carnivorous species the stomach is where the holdup is longest47. To intercept this problem Miller et al. (2012)7 defined the intestinal transit time from
the initiation of gastric evacuation till the last movement of digesta from the large intestine into the
rectum. However, just as in caiman (Caiman crocodylus)37, unexpectedly fast passage times were
measured in rainbow skinks (Lampropholis delicate). Despite a non significant difference between
their control and barium sulphate studies, the results of the beads control study were overall slower
than the barium results. Similar results were reported by Beaupre et al. (1993)23 and Hatch and Afik
(1999)5. This might imply that the barium sulphate moves, not only with gastric evacuation, but also
in the intestines at the same rate as the fluid-phase of the digesta.
Schumacher and Toal (2001)48 discussed the fact that barium sulphate might slow intestinal passage
times. Recently a study of Miller et al. (2012)7 did not found such an effect. This could be due to the
fact they used only a 0,15 ml dosage instead of the frequently used 10-15 ml/kg dosage7,49.
Scintigraphy is a quantitative and non-invasive method of determining the liquid-phase and solidphase of the digesta, but it requires expensive equipment and the use of radio-labelled markers46.
Alternatively, Valente et al. (2008)29 used barium-impregnated polyethylene spheres (BIPS®) to
measure the passage rate in loggerhead sea turtles (Caretta caretta). The BIPS® are inert, white and
have a density similar to food, but are sufficiently radiodense to show up clearly on abdominal
radiographs. However, the specific gravity of the spheres turned out to be higher than of the turtle
food, and therefore might travel with the solid phase of the digesta. They also described some
practical limitations using BIPS® in turtles. Hailey et al. (1998)50 reported difficulties counting the
microbeads because a large number of beads was used to outline the gut, and so some beads must
have overlapped in X-ray photographs. In Caiman crocodylus and the Crocodylus Porosus the
spheroids were separated in the stomach from the digestible food and most stayed in there till 24
days after feeding, long after the digestible food and barium meal had been eliminated36,37.
An overall major disadvantage using radiopaque markers is the intensive animal handling because of
the frequent radiographic imaging, resulting in higher stress levels of the animals and affected transit
times29,51.
Digestive
Markers
Internal
markers
External
markers
Pigments
and dyes
Particulate
markers
Chemical
markers
Quanifiable
Nonquantifiable
Figure 1: Schematic overview of the type of markers used in reptile digestion studies
Radiopaque
markers
Marker selection
According to the overview carmine red, chromic oxide, barium sulphate, barium polystyrene beads,
and lead glass beads have been used to determine the passage rate in Crocodiliae. The use of these
markers, however, resulted in a wide range of transit times. Besides most of these techniques are
not easy to recover, especially in non-laboratory conditions, or include intensive animal handling.
Since in many reptile studies fluorescent powder, pieces of tape, and beads have successfully been
used for determination of transit times, they are incorporated in the present study design. These
markers are selected because they are easy to detect, quantitative, inexpensive, do not require
extensive lab procedures and are therefore easy to use. A combination of plastic beads, fluorescent
powder and pieces of flagging tape is used as digestive markers for determining the minimum and
maximum transit times. By using a combination of these markers, which are all different in size and
texture, we attempt to get a better understanding of the difference in behaviour of these markers in
the digestive tract of C. Porosus.
Beads are used in the experimental designs, despite the fact that Davenport et al. (1990)36 and
Davenport et al. (1992)37 reported inconvenient results after using beads. However, by repeating the
experiments with beads we will be able to compare our results with those reported in the literature.
In these articles the main reason for the retained markers in the stomach is thought to be caused by
the anatomical and physiological functions of the crocodile stomach. However, in the following
experiments juvenile crocodiles are used with a larger body mass than used in previous studies and
for the first time sub-adult (culling size) crocodiles are used in a digestion study. From another
unpublished study, concerning sub-adult crocodiles (N = 11), it is known that the body size (snoutvent-length) is positively related to the size of the stomach, r = .59, p < 0.05. Therefore it is suspected
that the beads will behave differently. We expect that the size of the crocodile, and thus the size of
the gastro-intestinal organs, might influence the behaviour of beads within the gastro-intestinal
tract.
Material and Methods
Experiment 1: Digestive passage rate in yearling crocodiles
In this experiment 18 yearling crocodiles (mean body weight = 532 g, SD = 97 g; mean SVL = 296 mm,
SD = 15 mm) were selected from a captive population (Crocodylus Park, Darwin, Australia). Each
individual was randomly assigned to one of three treatment groups (N = 6 per group) that were fed a
meal of chopped red meat equivalent to 2% of their body mass1. An inert marker (<1% of meal mass)
was mixed with each meal to track the progression of food through the digestive tract. The first
group was fed a meal mixed with 15 plastic beads (1mm diameter), the second group was given a
meal mixed with 15 pieces (6 mm circles) of plastic field flagging, and the third group was given a
meal mixed with 250 mg of finely milled inert plastic pigment (Glow-Mark Fluorescent Pigment; 5-35
μm particle size; Scientific Marking Materials, Seattle, WA).
Table 1: Statistical descriptive of Experiment 1
Marker Group
N
SVL (mm)
Sig.*
Beads
6
292,5 ± 16,1
504,0 ± 81,8
Flags
6
294,8 ± 14,0
536,8 ± 104,5
Powder
6
300,2 ± 16,4
555,2 ± 113,8
Total
18
295,8 ± 14,9
0,687
BW (g)
532,0 ± 97,3
Sig.*
0,679
* Significance measured between groups
The meals were administered via gavage by inserting a lubricated syringe barrel behind the gular
valve, which is a structure consisting of the dorsal flap of the tongue and the palatal flap (velum
palati) extending from the soft palate52. Food was slowly pushed into the stomach by depressing the
syringe plunger. A second and a third meal (2% of body mass), not containing a marker, were given
every 3 days to simulate the feeding habits of wild-caught individuals and the consumption of small
meals every few days2.
The yearling crocodiles were individually housed in plastic tubs (40,0 cm * 30,0 cm) and kept inside a
30 °C constant temperature room. Each tub contained 1.0 L of fresh water, which was changed daily,
and included a resting platform to allow crocodiles to climb out of the water or to hide beneath it.
Every 24 hours the tubs were checked for the appearance of faeces and the digestive markers for
nine days after initial feeding. The number of beads and pieces of flagging that had passed through
the digestive tract were counted and a semi-qualitative scale was used to score passage of digesta in
the group fed a meal containing finely milled pigment. The pigment reference scale ranged from 0 to
6, corresponding to concentrations of respectively 25 mg pigment diluted into 1 L water, 50 mg L -1,
100 mg L-1, 150 mg L-1, 200 mg L-1, and 250 mg L-1. Scoring of pigment passage was facilitated by using
a high power black light as the pigment fluoresced brightly, even at the lowest concentrations.
On the ninth day post-feeding, stomach contents were flushed by using a common flushing
technique evaluated by Fitzgerald (1989)53, which is near 100% effective in recovering crocodilian
stomach contents54. A 10 mm o.d. piece of silicon tubing was inserted behind the gular valve and into
the stomach. A constant low velocity flow of tap water was used to fill the stomach cavity until visibly
extended. Crocodiles were held in a vertical position while a gentle massaging motion was used to
flush the stomach until all stomach contents were collected for enumeration.
Experiment 2: Digestive passage rate in culling size crocodiles
For this experiment 24 male sub-adult crocodiles (mean SVL = 956 mm, SD = 92 mm) were selected
from the same captive population as described above. After being immobilised, by a technique
described by Franklin et al. (2003)55, each crocodile was gavage fed an intact chicken head containing
30 beads and 30 pieces of flagging (identical to those used above). These markers were inserted into
a size 00 gelatine capsule, which was then placed into the oropharynx of the chicken. To prevent the
capsule from falling out of the chicken head, the upper and lower beak were glued together with 2-3
drops of cyanoacrylate glue, and the oesophagus was tied close with a knot. For 12 of these
crocodiles, the feeding was accompanied with the insertion of a temperature data logger (TidbiT v2;
Onset Comp.) programmed to record core body temperature every 10 minutes. Crocodiles were
given a follow up ad libitum meal of 2-3 chicken heads every 2 days following the initial feeding.
These crocodiles were housed in individual cages (0.75m * 0.75m * 2,0 m) that were partially
immersed into the water of commercial raising ponds. The unitised cages are standard practice for
housing farmed crocodiles, and they allow individual feeding, as well as prevent agonistic
interactions. The cages also permit free thermoregulation by providing individuals the choice
between water and a basking platform.
During the experimental period of six days post feeding the inert markers, animals of the
corresponding day post feeding group were sacrificed and dissected every 24 hours. The digestive
tract was dissected out from the oesophagus to the cloacae, and recorded the location of beads and
pieces of flagging along the tract. The digestive tract was separated into five discrete sections;
stomach, proximal-intestine, mid-intestine, distal-intestine, and colon. Sections were occluded with
cable ties before separation, and the plastic markers were filtered from the digesta using tap water
and a series of fine mesh sieves with 0.5-3 mm screen openings.
Table 2: Statistical description experiment 2
SVL
Days post feeding
N
Mean
Std. Deviation
1
4
957,00
77,02
2
4
928,75
80,04
3
4
952,50
80,39
4
4
977,50
88,13
5
4
940,00
79,97
6
3
986,67
200,08
Total
23
955,78
92,36
Sig.*
0,972
* Significance measured between groups, SVL = Snout Vent Length in mm
Statistical analysis
For evaluation of the collected data of experiment 1 and 2 statistical analyses were conducted using
IBM SPSS statistics 20. De used test for each analysis will be discussed in the following paragraphs.
Results
Experiment 1
An one-way ANOVA showed no significant differences in snout-vent-length (SVL) (F(2, 15) = 0.394, p
> 0.05), or body weight (BW) (F(2, 15) = 0.397, p > 0.05) between the three different marker
treatment groups either in experiment 1.
Markers were recovered as shown in figure 2. The means and standard deviations of the recovered
markers for each day post feeding are presented in table 2.
15
14
13
Average recovered markers
12
11
10
9
8
beads
7
flags
6
5
powder
4
3
2
1
0
Days post feeding
Figure 2: Excreted concentrations for each type of marker
The collected data of the beads, flags, and powder were checked for normality before running any
statistical model. According to the Shapiro-Wilk test of normality not all data were normal
distributed. Beads, as well the flags, were non-normal distributed for all days, with exception of the
stomach flush. A standardised residual plot was performed showing that all z-scores were within the
–1.96 and 1.96 limits. Therefore these data are assumed to be normally distributed data, and an
ANOVA model is allowed to use.
No flags were recovered during the nine days post feeding. By performing a stomach flush a total of
99% of the flags were recovered. An one-way ANOVA showed that the mean of recovered flags after
performing a stomach flush was significantly different (p < 0.01) from the previous days.
A single bead was recovered two days post feeding together with some undigested food in the
water. Two beads were recovered at day 7, 1 at day 8, and 3 at day 9 post feeding. Each time beads
were found between day 1 and day 9 they were found in the same cage, concerning a single
crocodile. The results of an one-way ANOVA for repeated measures show that the amount of
markers recovered was not significantly affected by the time post feeding, F(8,40) = 0.199, p > 0.05.
After performing a stomach flush beads were recovered from all crocodiles, ranging from minimal 1
to a maximum of 14 beads. The means of recovered beads of day 1 till day 8 were all significantly
different (p < 0.05) from the stomach flush. Day 9 was not significantly different with a p-value of
0.54. During this experiment 57% of the beads were recovered, where 49% was recovered by
performing a stomach flush.
Table 3: Average results of recovered markers for each day post feeding
Days post feeding
Marker
Group
1
2
3
4
5
6
Beads
0
0,17 ± 0,41
0
0
0
0
Flags
0
0
0
0
0
0
Pigment
0
1,83 ± 1,67
7
8
9
0,33 ± 0,81 0,17 ± 0,41 0,50 ± 1,22
0
0
0
0,96 ± 1,24 0,50 ± 0,79 0,42 ± 0,56 0,67 ± 0,66 0,17 ± 0,20 0,46 ± 1,01 0,08 ± 0,13
Stomach flush
7,33 ± 5,06a
14,83 ± 0,41b
1,83 ± 0,49c
a) Mean of recovered beads after the stomach flush was significantly different from day 1 – 8 post feeding (p < 0,05), b)
Mean of recovered flags after the stomach flush was significantly different from day 1 – 9 post feeding (p < 0,01), c) The
concentration of pigment is the stomach was significantly different from the concentrations of beads and flags (p < 0,01).
Fluorescent powder was first recovered 2 days after feeding within the cages of all six individuals.
The concentrations of powder excreted varied greatly among all individual animals during the
experimental period. All crocodiles excreted the highest concentrations of powder between day 2
and day 4 followed by another lower peak concentration a few days later. At day 4 post feeding 50%
of the initial amount of marker was excreted.
The results of the powder-group were mainly non-normal distributed, except for day 2 and the
stomach flush. Because of the large variation in data a non-normal distribution is less easy to rule
out. Therefore, these data were transformed by calculating the square root of these values. After
running a Shapiro-Wilk test of normality with the transformed values, the normality was improved
for each day, still day 8 and day 9 remained significant. Also, the Levene’s test for homogeneity of
variance changed from significant difference between the variances F(8, 45) = 3.662, p < 0.05 to
equal variances for the different days post feeding F(8, 45) = 1.397, p = 0.224. To check whether
these values were truly non-normal distributed the standardised residuals were plotted against the
standardised predicted values of days post feeding by performing a regression model. The plotted zscores contained a minimal absolute value of -1.013 and a maximum value of 2.622. Since only two
standardised residuals (3.33% of the sample cases) have a value greater than 1.96, there is evidence
that this model is representative for the actual data set and the data are normally distributed. Based
on the assumptions made above, a one-way ANOVA for repeated measures was allowed to evaluate
the data.
The results of the ANOVA showed that the time post feeding (day 2 – day 9) did not significantly
affect the recovered concentration of marker, F(7, 35) = 0.686, p > 0.05. A correlation analysis
showed a significant relationship between the recovered concentration of powder and the time post
feeding, r = -.472, p < 0.01. The concentration of powder recovered after the stomach flush was not
significantly different from the previous days (p > 0.05)
To rule out a non-normal distribution of the powder data a non-parametric Friedman’s ANOVA was
conducted to evaluate whether there would be major differences in outcome compared with a one-
way ANOVA for repeated measures. A post-hoc Wilcoxon signed-rank test was used with a
Bonferroni correction to correct for the number of tests.
6
Concentration scale
5
Crocodiles
4
1
3
2
3
2
4
1
5
0
6
Days postfeeding
Figure 3: Excretion patterns of the fluorescent pigment for all six individuals
The concentration of powder did not significantly change between day 2 and day 9 according to the
results of the Friedman’s ANOVA χ2(7) = 13.042 p > 0.05. To compare the results found after the
stomach flush with the previous days another test was performed, χ2(8) = 18.332 p < 0.05. A posthoc Wilcoxon test was used to follow up this finding. A Bonferroni correction was applied and so all
effects are reported at a 0.5 / 8 = 0.00625 level of significance. Similar to the parametric test it
appeared that the concentration of powder did not significantly change between day 2 and the
stomach flush, since none of the comparisons had a significance level smaller than 0.00625.
To compare the amount of marker that was recovered after the stomach flush between treatments,
percentages were used. In order to work with normally distributed data these values were
transformed using a Log transformation. An univariate analysis of variance showed that the
covariate, type of marker, was significantly related to the percentage of marker that stayed in the
stomach, F(1, 14) = 23.86, p < 0.01, r = 0.65. A post-hoc pairwise comparison with an adjusted
Bonferroni correction revealed that a treatment with fluorescent powder resulted in significant lower
concentration of marker in the stomach compared to treatments with flags (p < 0.01) and beads (p <
0.05). No significant differences were found between treatments with flags and beads (p > 0.05).
To evaluate whether a type of marker had an effect on the time of first appearance an univariate
analysis of variance was used. Results showed that the time of first appearance was significantly
affected by the type of marker that was used, F(1, 14) = 15.0, p < 0.01. A post-hoc pairwise
comparison with an adjusted Bonferroni correction revealed that a treatment with fluorescent
powder appeared significantly earlier compared to treatments with flags (p < 0.01) and beads (p <
0.01).
It was not possible to analyse a relation between the appearance of faeces and the appearance of
markers, since in none of the groups faeces could be detected.
Experiment 2
An one-way ANOVA was constructed to evaluate whether groups were equally distributed. The test
showed no significant differences in SVL between the various days post feeding groups, F(5, 17) =
0.166, p > 0.05. Unfortunately one crocodile was excluded from the experiment because of the
appearance of a parasite trail on the skin scutes.
About 72,9% of the beads were recovered, and 72,5% were located in the stomach. A single bead
was found in the proximal-intestine in a crocodile belonging to the 4 days post feeding group. In one
crocodile, from the 3 days post feeding group, two beads were found in the distal-intestine. In three
cases beads were found in the oral cavity.
Data were checked for normality by performing a Shapiro-Wilk test for normality and Q-Q plots of
the standardised residuals. Because data were normally distributed an independent one-way ANOVA
was applied to evaluate whether there is an effect of time post feeding on the location of beads in
gastro-intestinal tract. Results showed that the time post feeding did not significantly affect the
location of beads in the gastro-intestinal tract, F(15, 51) = 0.768, p > 0.05. According to the results of
an one-way ANOVA for repeated measures the location of the gastro-intestinal tract had a significant
effect on the number of recovered beads, F(1.00, 22.16) = 194.50, p < 0.01. A post hoc test with a
Bonferroni correction showed that the numbers of recovered beads in the stomach were significantly
different from other intestinal sections for all groups (p < 0.01).
Mean recovered beads
30
Days post feeding
25
1
2
3
4
5
6
20
15
10
5
0
Gastro-intestinal sections
Figure 4: This figure shows the mean numbers of beads found within the different gastro-intestinal sections for each day
post feeding
A higher recovery percentage of flags (88,3%) was found, and about 87,1% was recovered within the
stomach. Three flags were found in the proximal-intestine; one at 5 days post feeding, two at 6 six
days post feeding. Two flags were found 4 days post feeding in the mid-intestine. Three flags were
recovered in the distal-intestine at 3,4, and 6 days post feeding. The effect of time post feeding on
the gastro-intestinal location was evaluated with an independent one-way ANOVA. Results showed
that the time post feeding had no effect on the gastro-intestinal location, F(20, 86) = 1.109 p > 0.05.
A one-way ANOVA for repeated measures was used to evaluate the effect of location on the amount
of recovered flags. Results showed that the mean numbers of recovered flags in the stomach were
significantly different from all other intestinal sections, F(1.03, 22.74) = 714.18, p < 0.01. A pair wise
post hoc comparison test with a Bonferroni correction showed that the number of recovered beads
in the stomach were significantly different from other intestinal sections for all groups (p < 0.01).
Mean recovered flags
30
Days post feeding
25
1
2
3
4
5
6
20
15
10
5
0
Gastro-intestinal sections
Figure 5: This figure shows the mean numbers of flags found within the different gastro-intestinal sections for each day
post feeding
An one-way ANOVA showed no significant differences in mean numbers of recovered markers in the
stomach between the several groups post feeding for flags F(5, 17) = 1.557, p > 0,05, and beads F(5,
17) = 0.484, p > 0,05. However a correlation analysis showed that there was a significant relation
between the number of days post feeding and the amount of recovered flags in the stomach, r = -.48;
p < 0,05.
Gastro-intestinal section
Stomach
Proximal-intestine
Mid-intestine
Distal-intestine
Colon
Days post
feeding
Beads
Flags
Beads
Flags
Beads
Flags
Beads
Flags
Beads
Flags
1
25,00 ± 6,9a
30,00 ± 0b
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
2
20,75 ± 10,1a
27,75 ± 4,5b
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
3
20,00 ± 4,3a
24,00 ± 3,9b
0,00
0,00
0,00
0,00
0,5 ± 1,0
0,25 ± 0,5
0,00
0,00
4
20,25 ±
5,0a
24,00 ±
6,1b
0,25 ± 0,5
0,00
0,00
0,50 ± 1,0
0,00
0,25 ± 0,5
0,00
0,00
25,25 ±
5,1a
24,25 ±
3,8b
0,00
0,25 ± 0,5
0,00
0,00
0,00
0,00
0,00
0,00
b
0,00
0,67 ± 1,2
0,00
0,00
0,00
0,33 ± 0,6
0,00
0,00
0,04 ± 0,2
0,13 ± 0,5
0,00
0,09 ± 0,4
0,09 ± 0,4
0,13 ± 0,3
0,00
0,00
5
a
6
18,33 ± 14,2
Total
21,74 ± 7,4a
23,67 ± 4,0
25,70 ± 4,4b,c
Table 4: a) significant difference (p < 0,01) within the same row for beads, b) significant difference (p < 0,01) within the same
row for flags, c) significant relation between the number of days post feeding and the number of recovered flags (r = -,48; p <
0,05).
A paired sample t-test was used to determine whether there was a difference in the number of
markers recovered in the stomach between the two type of markers. Results showed that more flags
were recovered in the stomach (M = 25.70, SE = 4.42) than beads (M = 21.74, SE = 7.44), t(22) = 2.42, p < 0.05, r = .46. Results of paired sample t-tests showed that there were no significant
differences between the numbers of recovered beads and flags in the stomach for the several days
post feeding groups (p > 0.05).
Beside markers, various stomach contents as stones, glass and pieces of plastic were recovered
within the stomachs of all crocodiles during the dissections. One animal even contained 66 foreign
objects. The weight of the recovered corpora aliena varied between 0.5 and 66 grams, with an
average of 17 grams. The temperature data loggers showed body temperatures ranging between
23˚C and 30˚C.
Figure 6: Body temperatures (mean ± SD) of large sub-adult crocodiles, measured by data loggers in the stomach on a
typical day during experiment 2.
Discussion
The aim of the present research was to determine whether beads, pieces of plastic flagging, and
fluorescent pigment can be used as digestive markers in C. porosus. The behaviour of these markers
was studied in two experimental designs.
In the present study each type of marker clearly behaved differently within the gastro-intestinal tract
of the crocodiles. Results showed differences in excretion patterns. The most obvious outcome is
that the fluorescent pigment was excreted almost continuously during the experiment, while beads
and flags were only excreted sporadically and large percentages were retained within the stomach.
In the following paragraphs the results will be discussed for each type of marker and compared with
results of the existing literature.
Particulate markers
Since the numbers of recovered beads did not significantly differ between day 1 and day 9 it can be
concluded that the time range of nine days post feeding had no effect on the excretion of beads. The
statistical results showed that the number of beads recovered after the stomach flush was
significantly different from the previous days, with the exception of day 9, and therefore suggest that
about 49% remained in the stomach. Despite the fact that nearly half of the beads stayed in the
stomach, some beads passed the digestive tract. Although, the single bead found on the second day
post feeding was probably recovered because one animal regurgitated a part of its meal, since little
undigested food was found in the water as well. The beads excreted from day 7 till day 9 post feeding
only included two individuals. Nevertheless, after performing a stomach flush 57% of the beads were
recovered, presuming that 43% have passed the stomach.
The number of recovered flags after the stomach flush was significantly different from day 1 to day 9
including. A convincing 99% of the flags were recovered after performing a stomach flush, assuming
that just a single flag passed the pylorus into the small intestine.
The results of the first experiment were more or less established by the findings of the second
experiment, since the time post feeding did not affect the location of beads or pieces of flagging tape
in the gastro-intestinal tract, and again, nearly all markers were recovered within the stomach. Only
a few markers were found in the intestinal tract. The recovery percentages of the second experiment
were prominent. During this trial only 72,9% of the beads and 88,3% of the flagging tape were
recovered. The fact that not all markers were recovered assumes that the remaining percentages of
markers were not in the digestive tract. This could be caused either because markers moved through
the digestive tract before culling, meals were partially regurgitated or markers fell out the animal
during bleeding and skinning. During the latter procedures animals are hung up, head down, by their
tail. In three animals a couple of beads were found in the oral cavity. Because the animals were
housed in individual cages, which were connected to an open pond, markers could have easily
disappeared, especially because the beads were transparent and uncoloured.
Based on the recovery percentages, the pieces of flagging were more prone to stay in the stomach
and were easy to recover. However, a trend was noticeable showing that less pieces were recovered
the more time had passed after feeding. Combined with the fact that pieces of tape were found in
other parts of the intestine suggests that some movement through the intestinal tract took place.
These findings, that large amounts of particulate markers retained in the stomach, resemble with the
results reported in previous crocodile digestion studies. But what causes these markers to function in
most reptile species and not in crocodilians?
The main reason of particulate markers having difficulties with passing the stomach is thought to be
caused by the anatomy and physiologic functions of the stomach. Most reptilians have a simple
tubular stomach, however it is larger and more outpocketed in crocodilians56. The cardiac sphincter
of the stomach occurs at the junction of the oesophagus, with the cardiac sac at the left anterior
corner of the stomach. The pyloric region is divided into left and right halves by a thick collar muscle
and spongy tissue, which may operate as a gizzard. The pyloric region is much smaller and opens into
the duodenum via a powerful pyloric sphincter, which is placed next to the cardiac sphincter on the
cranial site of the stomach, forming a large pouch57. Because of the pouch-form stomach, particulate
dense material might sink to the bottom away from the sphincter. Also, in crocodilians the pyloric
sphincter sorts the stomach contents to make sure only fluid and fine material passes into the small
intestine37. In the literature it is discussed to what extent this sorting function plays a role in the
collection of particulate materials within the stomach of crocodilians. The presence of gastroliths, a
hard object of no caloric value (e.g., a stone, natural or pathological concretion) which is retained in
the digestive tract of an animal, is a common finding in crocodile digestion studies. In the present
study all stomachs of sub-adult crocodiles contained gastroliths with an average weight of 17 grams.
The possible function of gastroliths are reviewed by Taylor et al. (1996)49 and Wings (2007)58.
However their actual function in crocodiles remains uncertain. They might contribute to buoyancy
control, food processing or both49.
Apparently the pyloric sphincter is able to filter
small particles like the used markers from the other
stomach contents. A study of Davenport et al.
(1990)36 reported a similar stasis of particulate
markers in the stomach. In this study none of the
particulate markers (1 mm) did pass the stomach
for up to 24 days in juvenile crocodiles (C. porosus)
with an average weight of 154 grams. Another
study of Davenport et al. (1992)37, using juvenile
Caiman crocodiles of 240-600 grams, a few
particulate markers reached the rectum after 52
hours and about 25% of the particulate markers
reached the colon after 74 hours. In the latter study
two types of markers were used; 1 mm barium
polystyrene spheroids and 0,4 mm lead glass beads.
Both markers were found in the colon, assuming
that the pyloric sorting mechanism couldn’t
59
differentiate between the spheroids and the beads. Figure 7: Stomach of the crocodile
According to the results of the present study the
pyloric sphincter of the juvenile C. porosus was able to differentiate between the fluorescent
pigment and the particulate markers, and therefore the differences found in excretion patterns are
primarily caused by the ability of the marker to pass the pyloric sphincter. So, it can be concluded
that a very small particle size is needed to pass the pyloric sphincter.
In the present study was hypothesised that the size of the crocodile, and thus the size of the gastrointestinal organs, would have an effect on the filter function of the pyloric sphincter. It was expected
that pyloric sphincter in larger crocodiles would let through relatively larger particles. However,
according to the results of the present study there were no differences in marker passage between
the two experiments, neither compared with previous reported studies in which smaller animals
were used. Apparently animal size has no influence on the filter function of the pyloric sphincter and
even sub-adult animals are still able to select particles < 1 mm.
The beads in the present study behaved similarly to the way Davenport et al. (1992)37 described the
movement of beads in Caiman crocodiles, except for the fact that a few particles could already be
detected in the rectum after 54 hours. Even though the appearance of the markers in the faeces was
not recorded, it seems that markers travelled at a faster rate compared with the present results.
They reported that the pyloric sphincter was unable to differentiate between 0.4 or 1 mm beads, so
the size of the beads used in the present study shouldn’t have caused the delay in transit time of the
marker. However, the feeding frequency could play a part in this, since Davenport et al. (1992)37
noticed that each time a new (non-labelled) meal was eaten the particulate markers were again
dispersed throughout the stomach, and a few particles left the stomach. In their study crocodiles
were fed unlabelled food every day. In the present study the crocodiles were fed an unlabelled meal
every three days at day 3 and day 6. This could explain the fact that beads were excreted after 7 days
post feeding, just like the fact that 43% of the beads passed the stomach. In both studies two
unlabelled meals were necessary prior to the passage of beads through the pyloric sphincter.
Another factor that might have had an impact on the passage rate of the markers is the frequency of
animal handling. In the present study animals were gavage fed, because a previous trial with
voluntarily feeding was not successful. Crocodiles didn’t immediately take in their experimental diet
which made it hard to control whether they had consumed the whole diet including all markers.
Besides the gavage feeding animals were handled every day when their tubs were cleaned during the
first experiment. In order to gavage feed the crocodiles in the second experiment the crocodiles
needed to be immobilised. A comparative study of Franklin et al. (2003)55 showed that crocodiles (C.
porosus) returned at their physiological baseline levels approximately 8 hours after manual restraint.
The stress response was significantly reduced compared to stunned animals. Guilliette et al. (1997)60
reported an approximate 30-fold increase in plasma corticosterone levels in juvenile alligators
subjected to two hours of capture and restraint. It is known from avian literature that corticosterone
slow down the passage rate of food in the alimentary tract51. It is uncertain to what extent the way
and frequency of animal handling in the present study had impact on the passage rate, but it might
have contributed to the current results.
Also, the environmental temperature has a major influence on the digestive system of ectotherm
species. Digestion in crocodiles is directly affected by temperature; increasing temperatures result in
increased appetite, gastric contraction frequency and amplitude, and peptic activity61. Efficient body
temperatures for ingestion and digestion for crocodiles are estimated between 25˚C and 35˚C.
Temperatures higher than 35˚C cause an undue amount of stress and inappetence62, however
Coulson et al. (1981)63 reported ceased feeding in Alligator mississippiensis at temperatures below
22˚C. Caiman crocodiles even regurgitate their food when thermal regimes are unsuitable for
digestion64. According to the data loggers used in experiment 2 body temperatures ranged between
23˚C and 30˚C. During the day time temperatures were high enough to support digestion. Also, since
no major differences were found compared to experiment 1, wherein the environmental
temperature was controlled at a constant 30˚C, it is unlikely that the environmental temperature
affected the excretion rate of beads. Therefore, regurgitation is unexpected to be the main reason
for the missing markers in experiment 2.
In the study of Davenport et al. (1992)37 only two crocodiles were used to compare the effect of
particulate markers. The way these markers behaved were extremely different for both individuals.
One of them excreted all markers after 168 hours, while the other animal had still substantial
numbers of markers within the stomach after 192 hours. This is in agreement with the findings of
experiment 1, wherein two animals still contained 14 beads in the stomach after nine days, one
animal 10 beads, and the other crocodiles 3, 2, and 1 beads. So apparently not all juvenile crocodiles
selected stomach contents to the same extend, so individual variation is a major opponent when
using this marker, even under controlled conditions.
The pieces of plastic flagging tape obviously did not work as digestive markers in this study. Despite
the fact that the material is pliable and light, it is also prone to adhesion to the intestinal wall8, or in
this case the mucosa of the stomach. Especially under wet circumstances the tape becomes sticky, so
it is also very likely that the several pieces of tape clumped together within the stomach, making it
even more difficult to pass the pyloric sphincter. When looking at the overview of digestive markers,
it is remarkable that this type of marker has been mostly successful in herbivorous or opportunistic
omnivorous reptiles. The reason for the fact that mainly herbivores are able to excrete this type of
marker might be explained by their diet. Among herbivorous species some have teeth (sauria) and
others don’t (chelonie)65. In both cases animals are anatomically designed to tear leaves and
preclude mastication of food66. The lack of particle size reduction results in digestion of large food
particles, such as entire leaves13. In most species the stomach is not capable to fully break down
these particles. Therefore relatively large particles pass the pylorus into the small intestine to be
further fermented in the hindgut. In contrast to the crocodile stomach it is more likely for markers to
pass through.
Fluorescent pigment
In contrast to the particulate markers the fluorescent pigment was the only marker that passed the
stomach in large amounts. It was first detected two days post feeding within the tubs of all six
individuals, and 50% of the marker was recovered at day 4. Although the concentrations of powder
excreted varied greatly among the individual animals a trend in excretion was noticed. According to
the statistical results, the time post feeding seemed not to affect the concentration of excreted
pigment, since there were no significant differences in excretion between day 2 and day 9. However,
the time post feeding was significant negatively related to the excreted concentration of pigment.
This trend is also visible in figure 3. Besides, for almost all crocodiles their maximum concentration of
excreted marker was detected on day 2 or day 3, followed by another peak concentration a few days
later. In comparison with the other markers, the concentration of pigment recovered after the
stomach flush did not significantly differ from the previous days. Nevertheless, the fact that some
pigment was retained by the stomach flush indicates that not all pigment had left the stomach after
nine days.
Yet, what do these results mean? The fluorescent pigment is thought to travel at the same rate as
the solid fraction of the diet. In a study of Beaupre et al. (1993)23 the liquid phase appeared long
before the with pigment stained faeces. However, in a study of Hatch and Afik. (1999)5 it travelled at
almost the same rate as the liquid fraction in Six-lined racerunners (Cnemidophorus sexlineatus).
They concluded that the ability to separate the liquid fraction from the solid fraction differs among
species. In the literature it is unknown to what extent crocodile species are able to separate these
fractions of the diet. It is known that barium sulphate is evacuated from the stomach at the same
rate as the liquid phase. Davenport et al. (1992)37 noticed that barium sulphate labelled material first
left the stomach after 4 hours post feeding and was detected in the rectum in as little as 12 hours in
Caiman crocodiles, which probably represented the liquid fraction. After 52 hours all barium sulphate
labelled material filled the rectum. A first appearance of faeces stained with chromic oxide, which is
known not to travel with any fraction of the diet, was reported around 49 hours post feeding and a
mean last appearance after 136 hours. Davenport et al. (1990)36 reported a first appearance of
faeces stained with chromic oxide after 68 hours and mean last appearance after 97 hours in C.
porosus. Garnett (1988)1 recorded the first appearance of carmine red after 36 hours and the last
appearance at 96 hours in C. porosus using a diet of lean pork. He reported a first appearance after
24 hours and a last appearance after 48 hours when using a fatty pork diet.
The fact that the pigment was first detected after 48 hours seems plausible compared to other data.
Apparently it took at least 24 hours for the pigment to pass the digestive tract to be expelled.
Unfortunately the excretion of the pigment could not be related to the defecation pattern, since the
faeces could not be collected. The faeces was easily diffused within the water which made it
impossible to detect and the clarity of the water was affected by the repellence of skin. However,
when looking at the excretion patterns of the pigment it seems likely that most of the experimental
diet was excreted within the first few days. After that, only small concentrations were excreted,
which are probably caused by internal staining of the following meals, as described by Harwood
(1979)8. Therefore the pigment might be a usable marker to determine the first appearance, but not
to estimate the maximal transit time. However, because of the large time intervals in this study
design, the pigment might be excreted between 24 and 48 hours. Additionally, the fact that the
pigment concentrations were scaled and estimated by visual detection makes the data even less
accurate. Consequently, the present data can only be used as rough estimations of the passage rate.
Future research
Nevertheless, up till now most results reported in crocodile digestion studies have been rough
estimations of food passage rates. Besides, all study designs differ in ways of the used species,
experimental diet, feeding frequency, time interval, housing, recovering the marker, etc. This way
there are too many biases involved to compare the results from the previous studies and get a clear
view of the rate at which food is processed in crocodiles. However, with the present collection of
data and knowledge a basis of information about the food passage rate in crocodiles is formed. It can
be concluded that crocodiles are not the easiest and well willing species to work with, and not just
because they bite!
Because of the anatomical and physiological restraints of the crocodile alimentary tract more
advanced digestive markers are needed compared to other reptiles in order to obtain accurate data.
Since the size of the marker seems to be the main limitation for a marker to be successful in
crocodilians, the ideal marker needs to be small enough to pass the pyloric sphincter. To make sure it
moves at the same rate along the intestinal tract as the diet ideally it should be attached to a certain
feedstuff. Therefore a chemical marker might be a solution. Although the detection will require
special equipment it has some major advantages like the fact that they are quantifiable markers, and
by using several chemical markers for the different fractions of the digesta, it will be possible to
provide knowledge about the ability of crocodiles to separate the solid phase from the liquid phase.
With these markers it is possible to strip an experimental diet and estimate passage rates for several
nutrients.
Also, animal handling is still minimal involved since only the faeces needs to be collected. In the
present study it was not possible to collect the faeces. So attention is needed when designing the
experimental environment for future experiments. In terms of animal handling, in the present study
animals were gavage fed, however to reduce the influence of stress on the passage time, future
research should aim for voluntary feeding designs. Additionally the animal handling should be
minimised during the experiment.
Other implications for future research concern the individual variation in digestion among crocodiles.
Previous studies worked with very small population sizes. In order to intercept the large impact of
individual variation on test results, experimental populations should be large enough and the
environment should be controlled. Also by standardization of certain procedures within future
experimental designs will enhance the ability to compare data and indirectly enlarge the population.
By increasing the frequency of control during the experiment, not only data will become more
accurate, it will also reduce the differences between individuals
Conclusion
The used particulate markers in this experiment, beads and pieces of plastic flagging, are not
appropriate digestive markers for C. porosus. Large percentages of both markers were retained
within the stomach. The anatomy and physiological functions of the stomach are thought to have a
major role in this occurrence. Because of the small particle size of the fluorescent pigment, it was
able to pass the pyloric sphincter and was excreted almost continuously. However, because it is
unknown which fraction of the diet was measured and the inaccurate way of recovering the marker,
the results can only be interpreted as rough estimations of food transit time.
For years crocodile digestion studies are based on rough estimations of passage rates. In order to
provide more accurate data concerning the passage rate of food, a more precise markers needs to be
used in crocodilians. Over the last few years more accurate digestive markers have been developed
and more knowledge about their behaviour and effectiveness is available in various animal species.
By using chemical markers as digestive markers other doors in crocodile digestion and nutrition
research will be opened.
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Beatty & Sons. Norton, NSW; 1987.
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Appendix
Table 5: Overview reptile digestion studies
Author
Species
Food habit
Marker
Garnett (1988)1
Crocodylus
Porosus
Carnivore
Carmine red
suspension
Liesegang et al.
(2001)
20
Kanui et al.
(1990)21
Harwood (1979)8
Size of
marker
Dosage
Administration
1,0 ml in a meal
(2% of body
weight)
Behind palatal flap
before feeding
Geochelone
nigra
Herbivore
Carmine red
66 mg/ Kg body
weight
Mixed in food
Crocodylus
niloticus
Carnivore
Indigo carmine
solution
Concentration
solution
unknown
2 g beef soaked in
indigo carmine
solution
Sceloporus
occindentalis
Carnivore
Indigo carmine
solution
Concentration
solution
unknown
Injected in
mealworms
Tb ˚C
Transit
time
Def. of
passage
time
Lean pork
30
36-96 hours
t0-tmax
Fatty pork
30
24-48 hours
t0- tmax
23
8-18 days
t0- tmax
25
34,8h ± 1,5
t0
25-30
42,3h ± 1,0
t0
30
44,0h ± 0,8
t0
20,8
>9 days
t50
26,2
2,8 ± 0,2 days
t50
29,2
2,9 ± 0,2 days
t50
32,5
2,9 ± 0,2 days
t50
33,4
2,4 ± 0,2 days
t50
36,6
3,4 ± 0,5 days
t50
Experimental
diet
Beef
Tenebrio larvae
Gerrhonotus
multicarinatus
Carnivore
Indigo carmine
solution
Vinyl
Cnemidophorus
tigris
Carnivore
Concentration
solution
unknown
< 2mm in
diameter
Indigo carmine
solution
Vinyl
1
Concentration
solution
unknown
< 2mm in
diameter
1
Injected in
mealworms
Force fed with
mealworms
Injected in
mealworms
Force fed with
mealworms
Tenebrio larvae
Tenebrio larvae
Tenebrio larvae
Tenebrio larvae
14,9
>11 days
t50
18,1
7,6 ± 1,8 days
t50
23
5,2 ± 0,3 days
t50
27,7
5,9 ± 0,7 days
t50
29,9
5 ± 0,3 days
t50
32,4
5,1 ± 1,3 days
t50
14,9
>11 days
t50
18,1
>11 days
t50
23
6 ± 0,5 days
t50
27,7
7,5 ± 1,0 days
t50
29,9
5,2 ± 0,3 days
t50
32,4
4,9 ± 1,4 days
t50
25,1
3,5 ± 0,4 days
t0
28,3
2,9 ± 0,5 days
t0
34,3
2,8 ± 0,3 days
t0
38,4
2,7 ± 0,7 days
t0
25,1
3,6 ± 0,6 days
t0
28,3
3,5 ± 0,3 days
t0
34,3
2,4 ± 0,4 days
t0
38,4
2,9 ± 0,5 days
t0
Bjorndal et al.
(1989)22
Geochelone
carbonaria
Geochelone
denticulata
Beaupre et al.
(1993)
23
Hatch and Afik
(1999)5
Hailey (1998)30
Sceloporus
merriami
Cnemidophorus
sexlineatus
Kinixys spekii
Herbivore
Herbivore
Insectivore
Insectivore
Omnivore
Fluorescenting
pigment dye
Concentration
unknown
Fluorescenting
pigment dye
Concentration
unknown
UV-powder
0,02 ml per
cricket
Dyed guava fruit
Psidium guajava
22-32
2,6 ± 0,1 days
t0
Dyed mango fruit
Mangifera indica
22-32
6,6 ± 1,2 days
t0
Dyed lantana foliage
Lantana
urticifolia
22-32
9,5 ± 1,7 days
t0
Dyed guava fruit
Psidium guajava
22-32
3,6 ± 0,9 days
t0
Dyed mango fruit
Mangifera indica
22-32
4,8 ± 1,3 days
t0
Dyed lantana foliage
Lantana
urticifolia
22-32
8,7 ± 1,9 days
t0
Injected in crickets,
free feeding
Acheta
domestica
31
104,23 h
tmax
34
86,54 h
tmax
36
113,78 h
tmax
28-36
23.3 ± 1,65;
23.3 ± 1,65 h
t0; MRT
UV-powder
0,02 ml per
cricket
Injected in crickets,
free feeding
Polyethylene
glycol
0.5 mCi [(U)14C]
Gavaged with 30 µl
of a solution
28-36
23,7 ± 3,34;
28.8 ± 5.81 h
t0; MRT
Glycerol
triether
2 mCi [3H]
Gavaged with 30 µl
of a solution
28-36
20,1 ± 2,4;
26,4 ± 6,64 h
t0; MRT
50 or 100 loops
Sewed into fungi
Brassica
oleracea
30
2,8; 3,7; 3,3
days
t0; t50; tmax
Sewed into leaves
Brassica
oleracea
30
2,2; 4,7; 4,5
days
Polyester
thread
Loops 2,0 cm
in diameter
Crickets
Tied around
millipedes
Hailey (1998)30
Kinixys spekii
Omnivore
Brand et al.
(1999)33
Xerobates
Chelonia mydas
agassizii
Herbivore
Omnivore
30
6,5; 7,2; 7,22
days
Radiopaque
Ballotini
microbeads
0,65-0,75
mm beads
0,5 g (1300)
beads
Mixed in food, free
feeding
Polyester
thread
2,0 cm in
diameter
100
Sewed into leaves
Brassica
oleracea
Field
5 days
t50
Sewed into fungi
Agaricus
bisporus
Field
5 days
t50
Tied around
millipedes
Alloporus sp.
Field
9 days
t50
Mixed with leaves
and millipedes
30
3,0; 4,2; 4,2
days
t0; t50; tmax
Mixed with leaves
30
2,2; 4,7; 4,5
days
t0; t50; tmax
Chromic oxide
Meienberger et
al. (1993)67
Alloporus sp.
Plastic marker
strips
Cylindrical
polythene
beads
0,5 % of wet
mass diet
30 * 1-2 mm
2,0-2,5 mm *
1 mm,
packed per
40 into
gelatine
capsules
8
5 capsules
30
No complete
results
Force fed begin
study
Erodium
cicutarium
16,433,6
17,8 days (1133)
t0
Force fed begin
study
Schismus
barbatus
16,433,6
21,8 days
(14,5-33)
t0
Force-fed
Field
15-28
6,5-13,5 days
t50
Amorocho and
Reina (2008)
34
Van Damme et
al. (1991)68
Bjorndal et al.
(1991)32
Spencer et al.
(1998)
31
Chelonia mydas
agassizii
Omnivore
Cylindrical
plastic beads
2,0-3,0 mm
*1 mm,
packed per
20 into
gelatine
capsules
3-5 capsules
Force-fed
Lacerta
Vivipara
Insectivore
Pieces of plastic
3*2*0,1 mm
1
Placed in abdomen
cricket, free feeding
Trachemys
scripta scripta
Emydur
macquarii
Omnivore
Omnivore
Plastic flagging
Vinyl tape
Size of
duckweed
1x1 mm
1
40
Placed in mouth
turtle
Put in small sample
of fish
Leaves and fish
28,3
22 ± 6,3 days;
24,7 ± 6,0 days
t0; tmax
20
18,8 ± 1,9
hours
t0
25
13,5 ± 1,3
hours
t0
27,5
14,0 ± 1,4
hours
t0
30
11,2 ± 0,8
hours
t0
32,5
10 ± 0,8 hours
t0
35
13,5 ± 0,7
hours
t0
Duckweed
24-31
72 ± 26 hours
t0
Tenebrio larvae
24-31
71 ± 15 hours
t0
Duckweed/
Tenebrio larvae
24-31
85 ± 7 hours
t0
Fish
20
89 ± 6; 115 ± 9
hours
t0, MRT
Fish
30
70 ± 3; 89 ± 51
hours
t0, MRT
Valisnaeria
20
260 ± 10; 310
± 30 hours
t0, MRT
Vinyl tape
Valido and
Nogales (2003)69
Waldschmidt et
al. (1986)
9
Kummrow et al.
(2011)
26
Staton et al.
(1992)3
5x5 mm
20
Valisnaeria
30
118 ± 8; 158 ±
12 hours
t0, MRT
Fish
20
154 ± 12 hours
MRT
Fish
30
123 ± 11 hours
MRT
Valisnaeria
20
418 ± 25 hours
MRT
Valisnaeria
30
180 ± 12 hours
MRT
Adult crickets,
Coleoptera
larvae,
Plocama endula,
Solanum
lycopersicum,
Solanaceae
28-30
6,9 ± 3,8 days
tmax
Gallotia
atlantica (Teno
Bajo)
Omnivore
Glass beads
3mm in
diameter
0,02g
2
Mixed in food, force
fed
Gallotia
atlantica
(Izaña)
Omnivore
Glass beads
3mm in
diameter
0,02g
2
Mixed in food, force
fed
28-30
6,1 ± 5,3 days
tmax
Gallotia galloti
(Tetir)
Omnivore
Glass beads
3mm in
diameter
0,02g
2
Mixed in food, force
fed
28-30
2,4 ± 1,5 days
tmax
Uta
stansburiana
Insectivore
Polyacrylamide
beads
100 µm in
diameter
Unknown
Injected in cricket
Acheta sp.
22,
24,
28,
32, 36
-
t0
Chamaeleo
calyptratus
Insectivore
Glitter
<1 mm in
diameter
Live crickets coated
in honey and glitter
Acheta sp.
26-30
4 days
tmod
Alligator
mississippiensis
Carnivore
Chromic oxide
Voluntarly fed
Experimental
diet
28/32
Unknown
0,1% of the diet
Davenport et al.
(1990)
36
Crocodylus
porosus
Carnivore
Chromic oxide
Unknown
minced with fresh
fish (2% by weight)
Sprattus
30
68h;92-113h
t0; tmax
Crocodylus
porosus
Carnivore
Barium
sulphate
Unknown
Minced fish with
barium sulphate
(20% by weight)
Sprattus
30
see article for
detailed gut
movement
tV *
100
Minced fish with
barium sulphate
(10% by weight),
beads mixed in 2 g
of fish
Sprattus
30
-
Barium
sulphate
0,3 ml
Injected in whole
fish and live crabs
Sprattus and
crab
30
66 h
Barium
polysterene
spheroids/
barium
sulphate
Davenport et al.
(1992)
37
Tracy et al.
(2006)
44
1 mm in
diameter
Caiman
crocodylus
Carnivore
Chromic oxide
Unknown
Mixed in minced ox
liver (2% w/w)
Minced Ox liver
30
136 h
tmax
Caiman
crocodylus
Carnivore
Barium
sulphate
Unknown
Mixed in minced ox
liver (2% w/w)
Minced Ox liver
30
>12 h
tV *
Gopherus
agassizii
Herbivore
Barium
polystyrene
spheroids
1 mm in
diameter
Unknown
Mixed in minced ox
liver (2% w/w)
Minced Ox liver
30
-
Lead glass
beads
0.4 mm in
diameter
Unknown
Mixed in minced ox
liver (2% w/w)
Minced Ox liver
30
-
Ytterbium (Yb)
Yb for fiber fraction
Guinea pig chow,
chick-starter
chow
30
13 days
t50
Cobalt (CoEDTA)
Co-EDTA for liquid
fraction
30
7 days
t50
Barboza (1995)10
Xerobates
agassizii
Herbivore
Chromic oxide
Ytterbium (Yb)
Cr -mordanted
Cobalt (CoEDTA)
Franz et al.
(2011)70
Testudo graeca
Herbivore
Cr -mordanted
Cobalt (CoEDTA)
150 mg Cr in 1,5
g dose
3 mg Yb in 1,5 g
dose
38 mg Cr in 1,5 g
dose
62 mg Co
Placed into the back
of the mouth by a
syringe
Placed into the back
of the mouth by a
syringe
Placed into the back
of the mouth by a
syringe
Single intra-gastric
dose
Labelling particles
<2mm
Herbage
31
9,57 ± 1,48;
9.55 ± 1.31
days
Low-fiber pellet
31
5,29 ± 0.01;
6.73 ± 0.22
days
Grass
31
5,2 ± 1,38;
9.60 ± 1.06
days
High-fiber pellet
31
9,66 ± 3,75;
13.25 ± 3.09
days
Grass
31
14,95 ± 2,36;
14.22 ± 1.83
days
High-fiber pellet
31
12,67 ± 5,56;
14.77 ± 4.80
days
Grass
31
4,81 ± 1,84;
6.78 ± 0.82
days
High-fiber pellet
31
6,92 ± 3,60;
7.80 ± 3.82
days
Grass hay/
lettuce
27-30
89-238 hours
Grass hay/
lettuce
27-30
-
tmax; MRT
Range of MRT
Testude hermanni
Herbivore
Cr -mordanted
Labelling particles
<2mm
Cobalt (CoEDTA)
Geochelone nigra
Herbivore
Cr -mordanted
Labelling particles
<2mm
Cobalt (CoEDTA)
Geochelone
sulcata
Herbivore
Cr -mordanted
Labelling particles
<2mm
Cobalt (CoEDTA)
Draco dussumieri
Herbivore
Cr -mordanted
Labelling particles
<2mm
Cobalt (CoEDTA)
Miller et al.
(2012)
7
Trachylepis
margaritifer
Insectivore
Glass beads
2 per kricket
Implementation in
dead kricket
Grass hay/
lettuce
27-30
90-150 hours
Grass hay/
lettuce
27-30
-
Grass hay/
lettuce
27-30
131-197 hours
Grass hay/
lettuce
27-30
65-139 hours
Grass hay/
lettuce
27-30
209-556 hours
Grass hay/
lettuce
27-30
99-340 hours
Grass hay/
lettuce
27-30
202-210 hours
Grass hay/
lettuce
27-30
125-149 hours
Acheta domstica
25
53,52 ± 21,00
hours
27
61,89 ± 10,69
hours
32
22,47 ± 12,4
hours
tII – tV **
Barium
sulphate
1:1 BaSO4/H20
supsension, 0,15
ml per kricket
Barium
sulphate
Valente et al.
(2008)
1:1 BaSO4/H20
supsension, 0,15
ml per kricket
Caretta caretta
Carnivore
Coloured dishes
of flat foam
5 mm in
diameter
Caretta caretta
Carnivore
BIPS® capsules
1,5 mm
(30U),
5 mm (10U)
5 mm in
diameter
29
Coloured dishes
of flat foam
10-20
Injected in dead
kricket
Injected in dead
kricket
Placed inside fish at
first feeding
Placed inside fish at
first feeding
10-20
Placed inside fish at
first feeding
Acheta domstica
Acheta domstica
Merluccius
merluccius/
Sardina
pilchardus
sardina
25
49,47 ± 7,76
hours
27
56,46 ± 13,00
hours
32
16,21 ± 7,76
hours
25
21,75 ± 4,17
hours
27
31,85 ± 8,57
hours
16,2723,86
9,05 ± 3,05;
12,00 ± 4,53;
13,19 ± 4,64
days
t0; t50; t85
16,2723,86
13,25 ± 4,86
days
t0
8,5 ± 2,73 days
t0
* tV = Time at which the digesta reached the rectum
** tII – tV = The time from when gastric emptying was initiated until movement into the large intestine was complete (phase II through V) was defined as the intestinal passage time
Legend
Pigments and dyes
Particulate markers
Chemical markers
Radiopaque markers