125
The Right-to-Left Shunt of Crocodilians Serves Digestion
* Corresponding author; e-mail: farmer@biology.utah.edu.
Physiological and Biochemical Zoology 81(2):125–137. 2008. 䉷 2008 by The
University of Chicago. All rights reserved. 1522-2152/2008/8102-70162$15.00
DOI: 10.1086/524150
C. G. Farmer
1,2,
*
T. J. Uriona
1
D. B. Olsen
2
M. Steenblik
1
K. Sanders
3
1
Department of Biology, University of Utah, Salt Lake City,
Utah 84112;
2
Utah Artificial Heart Institute, 803N 300W,
Suite 180, Salt Lake City, Utah 84103;
3
Department of
Radiology, Musculoskeletal Division, University of Utah
Health Sciences Center, Salt Lake City, Utah 84132
Accepted 7/17/2007; Electronically Published 1/14/2008
ABSTRACT
All amniotes except birds and mammals have the ability to
shunt blood past the lungs, but the physiological function of
this ability is poorly understood. We studied the role of the
shunt in digestion in juvenile American alligators in the fol-
lowing ways. First, we characterized the shunt in fasting and
postprandial animals and found that blood was shunted past
the lungs during digestion. Second, we disabled the shunt by
surgically sealing the left aortic orifice in one group of animals,
and we performed a sham surgery in another. We then com-
pared postprandial rates of gastric acid secretion at body tem-
peratures of 19⬚ and 27⬚C and rates of digestion of bone at
27⬚C. Twelve hours after eating, maximal rates of gastric acid
secretion when measured at 19⬚ and 27⬚C were significantly
less in the disabled group than in sham-operated animals.
Twenty-four hours postprandial, a significant decrease was
found at 27⬚C but not at 19⬚C. For the first half of digestion,
dissolution of cortical bone was significantly slower in the dis-
abled animals. These data suggest the right-to-left shunt serves
to retain carbon dioxide in the body so that it can be used by
the gastrointestinal system. We hypothesize that the foramen
of Panizza functions to enrich with oxygen blood that is des-
tined for the gastrointestinal system to power proton pumps
and other energy-demanding processes of digestion and that
the right-to-left shunt serves to provide carbon dioxide to gas-
trointestinal organs besides the stomach, such as the pancreas,
spleen, upper small intestine, and liver.
Introduction
The specific aim of this study is to test the hypothesis that the
right-to-left shunt of blood past the lungs in American alligators
serves digestion by enabling high rates of gastric acid secretion
and that this process is highly sensitive to temperature. The
broad, long-term objective is to contribute to our understand-
ing of the selective factors that have shaped the cardiopul-
monary system of amniotes.
A central problem of the study of the circulatory arrangement
of amniotes is understanding the factors leading to the fully
divided circulatory systems of birds and mammals and under-
standing the reasons for the incompletely divided systems of
all other members of the group (Goodrich 1930; Ewer 1950;
Foxon 1950). One advantage of the divided circulatory system
is that high systemic blood pressures can be generated without
high pulmonary pressures (which can damage the vasculature
in the lungs), and high pressures deliver more blood to tissues
for a given resistance to flow. Furthermore, the divided system
prevents the mixing of oxygen-rich and oxygen-poor blood.
Because a primary function of the cardiorespiratory system is
to deliver oxygen to tissues and to carry away carbon dioxide
and other “waste” products of metabolism to sites where they
can be eliminated from the body (e.g., the lungs, gills, skin,
kidneys), the completely divided system seems to have many
advantages over the undivided system; so why then is complete
separation found in only two groups, both of which are en-
dothermic? Animals with an undivided system can reduce blood
flow to the lungs, the primary site of gas exchange in amniotes,
and divert this blood back into the systemic circulation, the
right-to-left shunt. Are there advantages to this pattern of blood
flow, and are there trade-offs or constraints in the evolution
of these systems?
Many functions for the shunt have been proposed. Shunting
may keep CO
2
out of the lungs during diving, facilitating the
uptake of oxygen from the lungs and saving the better-oxygen-
ated blood of the left ventricle for the brain and heart (Green-
field and Morrow 1961; Webb 1979; Grigg 1989). The shunt
may suppress metabolism and extend dive times (Hicks and
Wang 1999); it may serve thermoregulatory needs (Webb 1979);
it reduces pulmonary edema (Burggren 1982); it may facilitate
digestion (Jones and Shelton 1993); and it has been hypothe-
sized to speed recovery from a metabolic acidosis by seques-
tering hydrogen ions (H
⫹
) into the stomach and restoring blood
bicarbonate stores (Farmer 2000).
Carbon dioxide is the substrate for the formation of acid in
the oxyntic glands of the stomach, and an increase in the partial
pressure of CO
2
on the basolateral side of these cells can increase
the rate of gastric acid secretion (Kidder and Montgomery 1974;
Glauser et al. 1995). Work by Kidder and Montgomery (1974)
126 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
suggests that CO
2
diffusion to oxyntic cells is the rate-limiting
factor in maximal gastric acid secretion. Thus, theoretically,
shunting hypercapnic blood to the stomach could increase the
maximal rates of gastric acid secretion over what would be
possible from glands perfused with arterial blood. Importantly,
gastric acid can be secreted when the oxyntic cells are perfused
by the O
2
-rich, CO
2
-poor blood of the left ventricle, as occurs
in all birds and mammals, albeit theoretically at a slower max-
imal rate.
The crocodilian cardiovascular system is highly suitable for
studies of the shunt, which can be stopped by sealing the left
aortic orifice. The left aorta is a vessel that arises from the right
cardiac ventricle (Figs. 1, 2). The left cardiac ventricle gives rise
to a large vessel that subdivides to form the right aorta, the
common (primary) carotid, and the right subclavian. After
leaving the pericardium, the aortas loop over the primary bron-
chi and then run caudad to unite by a short, wide connection
craniad to the stomach in such a way that the right arch seems
to continue on as the dorsal aorta while the left arch seems to
continue on as the celiac artery. The celiac branches to supply
blood to the stomach, spleen, liver, pancreas, and upper small
intestine (Reese 1915). The dorsal aorta supplies blood to the
mesenteric artery, the lumbar arteries, the urogenital arteries,
and the arteries of the pelvis, hind legs, and tail. Thus, the
majority of blood vessels of crocodilians originate from the
right-dorsal aorta complex, while the left aorta appears to pri-
marily serve the stomach and other organs of digestion. The
left and right aortas have another site of communication, an
aperture in the interaortic septum just distal to the bicuspid
valves, the foramen of Panizza (Fig. 1A). The function of this
foramen is unknown.
Crocodilians have significant control over the shunt. The sem-
ilunar bicuspid valves are leaflets, and when ventricular pressures
exceed arterial pressures, the valves open and blood is ejected
into the aortas. However, in addition to these passive, pressure-
operated valves, the pulmonary artery of crocodilians has an
actively controlled cog-teeth valve. Epinephrine opens the valve,
and then right ventricular blood is ejected into the pulmonary
artery (Franklin and Axelsson 2000). Acetylcholine, gastrin-re-
leasing peptide, sotalol (an antagonist of b-adrenergic receptors),
substance P, and histamine cause a portion of the blood in the
right ventricle to be ejected into the left aorta, rather than into
the pulmonary artery, by either inducing closure of the cog-teeth
valve or causing constriction of intrapulmonary vasculature
(White 1956; Greenfield and Morrow 1961; White 1969; Grigg
and Johansen 1987; Axelsson et al. 1989, 1991; Grigg 1989, 1992;
Holmgren et al. 1989; Jones 1996; Hicks 1998; Franklin and
Axelsson 2000). Because the blood that crocodilians shunt into
the left aorta from the right ventricle has not passed through the
lungs, it is rich in H
⫹
and CO
2
but poor in O
2
, compared with
blood that has passed through the lungs. Thus, the left aorta
appears to serve as a direct and substantial conduit to shunt CO
2
-
and H
⫹
-rich blood past the lungs and to the stomach, pancreas,
liver, spleen, and upper small intestine (Reese 1915; Webb 1979;
Grigg 1989; Jones and Shelton 1993; Jones 1996). The evolution
of this highly specialized cardiovascular system and the great
degree of control that crocodilians have over this shunt suggests
that this system is uniquely tailored by natural selection to serve
important functions, but these functions remain poorly un-
derstood.
Material and Methods
Several groups of animals were used in a series of experiments
aiming to test the hypothesis that the right-to-left shunt serves
digestion and to gain insight into why the ability to shunt is
found in ectothermic but not endothermic amniotes. The first
step was to determine whether a right-to-left shunt occurs dur-
ing the postprandial period, because if the shunt does not occur
at this time, then it is impossible for it to serve digestion. Five
juvenile American alligators were used in these experiments,
referred to as the blood flow study. In this study, a single blood
flow probe was placed on the left aorta, because in crocodilians
it is possible to recognize a right-to-left shunt by this blood
flow trace (Shelton and Jones 1991; Jones and Shelton 1993;
Jones 1996; Fig. 2F). Once it had been established that a shunt
occurs throughout the postprandial period, the shunt was dis-
abled to assess the effects of the shunt on gastric acid secretion
and, therefore, on digestion and to examine the importance of
temperature in these processes.
To disable the shunt, a suture was placed around the left
aortic orifice, afferent to the foramen of Panizza (Fig. 1B).
Catheters of polyethylene tubing were placed in either the right
atrium or the right ventricle, and metal sutures were loosely
placed around the right and left aortas so that the vessels could
be identified under fluoroscopy. The sham surgery entailed
placement of the atrial or ventricular catheters and the marking
of the aortas, but the left aortic orifice was not sealed. The
effect of this occlusion was studied in two ways: (1) by direct
measurement of rates of gastric acid secretion and (2) by ra-
diographically following the digestion of bone.
To study preferred body temperature (T
b
), programmable
temperature data loggers (iButton Thermochrons, DS2422,
Maxim Dallas Semiconductors) were placed by oral route into
the stomachs of six juvenile alligators ranging in size from 2.3
to 4.1 kg. The temperature sensors have a range of ⫺40⬚ to
⫹85⬚C and are accurate to Ⳳ0.5⬚C. Each logger was 1.4 cm
in diameter and 1 cm thick. The animals were provided a
thermal gradient ranging from 15⬚ to 40⬚C. These animals had
not undergone surgery. After implantation of the probes, al-
ligators were returned to their cage, which was kept in a green-
house with an air temperature of approximately 23⬚C. Exper-
imental housing consisted of two tanks joined together; each
tank was 1.2 m. Fresh tap water constantlym # 2.4 m # 0.6
flowed through one tank, with a thermal gradient from the top
to bottom of the tank ranging from 15⬚ to 19⬚C. The second
tank was dry, and heat lamps created a thermal gradient ranging
from 23⬚ to 45⬚C. The basking areas were large enough for all
of the animals to bask simultaneously. The alligators were
housed together and were free to move about these thermal
Figure 1. Ventral view of the crocodilian heart, great vessels, and splanchnic circulation. A, Schematic of the heart and great vessels (after
Greenfield and Morrow 1961). The inset shows an enlargement of a cutaway view of the left aortic orifice. In this illustration, a leaflet of the
valve has been split and retracted to reveal the foramen of Panizza. This foramen allows communication between the right and left aortas. B,
Photograph of a dissection of the heart and great vessels of an American alligator. The yellow dots indicate the approximate location where a
suture was placed to occlude the left aorta afferent the foramen. C, Schematic of the splanchnic circulation (after Reese 1915). D, Photograph
of the splanchnic circulation. The left aorta arises from the right cardiac ventricle and can therefore carry O
2
-poor, hypercapnic blood, illustrated
by blue coloring. The right aorta arises from the left cardiac ventricle and carries O
2
-rich but CO
2
-poor blood, illustrated by red coloration.
The left and right aortas loop over the primary bronchi and then dive dorsad to unite by an anastomosis (A) just dorsad to the apex of the
cardiac ventricle. Beyond the anastomosis, the vessels align in such a way that the right aorta seems to continue on as the dorsal aorta (DAo),
while the left arch seems to continue into the celiac artery (C). The celiac gives rise to the spleno-intestinal artery, which carries blood to the
128 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
spleen and part of the small intestine. The celiac then divides to give rise to three arteries: the gastro-hepatico-intestinal, carrying blood to the
stomach, liver, and small intestine; the pancreo-intestinal, carrying blood to the pancreas and small intestine; and the gastric, carrying blood
to most of the stomach. The rest of the gastrointestinal system is supplied blood by the dorsal aorta, through the mesenteric artery and other
branches of the dorsal aorta. carotid, pulmonary artery, artery, aorta,CC p common LPA p left PA p pulmonary LAo p left RAo p right
aorta, atrium, RA-V atrial-ventricular aperture, pulmonary artery, subclavian,RAt p right aperture p right RPA p right RS p right RV p
ventricle, , .right H p heart S p stomach
gradients. Data collection was postponed for 1 wk after han-
dling. Subsequently core body temperature was recorded every
10 min until the buffer was full, which occurred after 2,048
samples were obtained, 14.2 d after programming. The animals
had fasted for 2 wk before being instrumented. Seven days after
data collection commenced, the animals were fed ad lib. to
satiety on whole mice. At the end of the experimental period,
the data loggers were retrieved by gastric lavage. The last 3 d
of the fasting period and the first 3 d of the postprandial period
were selected for analysis.
Animals
In the blood flow study, American alligators were used that
had been hatched in the lab and reared in an animal care facility.
In contrast, in the occlusion study and the preferred body
temperature study, the animals had been caught from the wild
at the Rockefeller Wildlife Refuge by the Louisiana Freshwater
Fish and Game Commission. They were transported to Utah.
All animals were maintained on a diet of mice in -m2.4 # 4.9
tanks containing areas for swimming and basking. They ex-
perienced a natural photocycle. The masses of the animals can
be found in Table 1.
Surgery
Before all surgeries, the animals fasted for 1 wk. They were
then weighed, anesthetized with isoflurane, intubated, and ven-
tilated (SAR-830, CWE, Ardmore, PA) with air that had passed
through a vaporizer (Dra¨ger, Lubeck, Germany). The vaporizer
was initially set to 4% isoflurane to induce a surgical plane of
anesthesia but was reduced to 0.5% and maintained at this level
throughout the majority of the surgery. The site where the
incision was to be made was scrubbed with Betadine, and the
rest of the animal was covered with a sterile drape. The surgical
procedures differed between the studies and thus separate de-
scriptions are given below; however, the recovery procedure
was the same. The animals were given 2 mo to recover from
the surgery. During recovery, the site of incision was treated
with topical antibiotics (Neosporin), kept dry, and covered with
clean bandages. The animals were also given antibiotics (Bay-
tril) intramuscularly until the incisions were completely healed.
There was no sign of infection in any of the animals. The
indwelling vascular catheters were flushed every other day with
a sterile saline solution containing heparin (10 IU mL
⫺1
).
Blood flow through the left aorta. The left aorta was located
as it exits the pericardial sac. Here it was cleared of surrounding
connective tissue, and a loose-fitting ultrasonic flow probe
(Transonic Systems, Ithaca, NY) was placed around the vessel.
The caudal end of the sternum was cut approximately 2.54 cm
to access the vessel.
Occlusion of the left aorta. Fourteen American alligators were
divided into two groups: five alligators underwent sham sur-
geries, and nine underwent experimental surgeries. The sham
surgery consisted of a midline ventral incision to expose the
heart and great vessels. The caudal end of the sternum was cut
approximately 2.54 cm to expose the vessels. The left and right
aortas were cleared, and a metal suture (B&S 22 surgical steel
monofilament, 316L stainless steel, Ethicon) was placed loosely
around each vessel as a marker. Polyethylene atrial or ventric-
ular catheters (PE-50 with flared end) were placed and secured
with purse-string sutures. The catheters were filled with a mix-
ture of heparin (10 IU mL
⫺1
) and saline, tunneled subcuta-
neously, and exteriorized in a dorsal location. The body wall
was closed with absorbable suture (0 Dexon II green braided
polyglycolic absorbable; Sherwood Medical, St. Louis, MO).
The skin was closed with individually placed silk sutures.
In the experimental group, the right aorta was marked with
a metal suture, but the left aorta was occluded at two sites.
First, the left aorta was cleared efferent from the point at which
the left, right, and pulmonary vessels share common walls and
occluded with a suture. Second, the fibrocartilaginous supports
of the aortic and pulmonary valves were identified visually. A
tapered needle was used to place a second silk suture on the
cardiac side of this support such that the left aorta was occluded
(Fig. 1B). Next, the right atrium or right ventricle was randomly
selected for catheterization. The catheters were tunneled sub-
cutaneously and exteriorized in a dorsal location. The body
wall was closed with absorbable suture (0 Dexon II). The skin
was closed with individually placed silk sutures.
Protocol for the Assessment of the Surgical Technique
To assess the effectiveness of the surgical procedure that sealed
the left aortic orifice, a 1–3-mL bolus of radio-opaque material
(Omnipaque 300 [iohexol]; Novaplus, Amersham Health) was
hand injected through the catheters under fluoroscopy. Then an
intracardiac injection of acetylcholine (0.2 mg kg
⫺1
) was admin-
istered via the same catheters, and the flow patterns were studied.
Protocol for the Measurement of Gastric Acid Secretion
Alligators consumed a meal of chopped steak weighing 5% of
their mass and were maintained at T
b
of 27⬚C. Steak was chosen
The Right-to-Left Shunt Serves Digestion 129
Figure 2. Blood flow through the crocodilian heart without (A, C, E)
and with (B, D, F) a shunt. A, B, Schematic of blood flow. Without
a shunt (A), the right ventricle (RV) ejects O
2
-poor, hypercapnic blood
(blue) into the pulmonary artery (PA), while the left ventricle ejects
O
2
-rich blood (red) into the right aorta (RAo), which gives rise to
many other vessels of the body (e.g., right subclavian [RS], common
carotid [CC]). This oxygenated blood can traverse the foramen of
Panizza to flow into the left aorta (LAo; Jones and Shelton 1993; Karila
et al. 1995; Axelsson and Franklin 1996; Axelsson et al. 1997). With a
shunt (B), the RV ejects the hypercapnic blood into the LAo, which
is continuous with the celiac and gastric arteries. C, D, Radiographic
images of blood flow patterns. Both images show the vessels receiving
boli (1–3 mL) of radio-opaque material (Omnipaque 300, iohexol;
Novaplus, Amersham Health) that had been infused into the RV. C,
The RV ejected contrast medium into the PA (no shunt). D, Infusion
of acetylcholine (0.2 mg kg
⫺1
animal weight; Jones and Shelton 1993)
into the RV induced a shunt and caused contrast material to be ejected
into the LAo. Animals that had the base of the LAo occluded showed
no sign of contrast medium in the LAo on infusion of acetylcholine
and contrast medium. E, F, Representative traces from a flow probe
(Transonic, Ithaca, NY) that was placed around the LAo (Q
LAo
) and
the electrocardiogram (bottom trace). E, The flat trace (no shunt) in-
dicates flow from the RAo into the LAo through the foramen of Panizza
(Jones and Shelton 1993). F, With a shunt, the trace consists of a large
pulse indicative of direct ejection from the RV into the LAo. S p
, in the left aorta.stomach Q p blood flow
LAo
instead of mice to avoid hair in the stomach. Although we
found that the postprandial preferred body temperature is 30⬚C,
pilot experiments indicated that the alligators would not sit still
for an hour at this temperature to have their rates of gastric
acid secretion measured. Thus, we chose a cooler temperature
to reduce their activity. Twelve and 24 h after the alligators ate,
we inserted a solid-state pH electrode (Medtronics) orally into
the stomach to measure rates of acid secretion for 1 h by
titration with NaHCO
3
(Fordtran and Walsh 1973). Two weeks
later, this same procedure was followed, except the body tem-
peratures of the animals were reduced to 19⬚C for the 1-h
measurement periods. During the measurement periods, the
body temperatures were recorded with a thermocouple secured
inside the cloaca ( ,26.64⬚C Ⳳ 0.78 mean Ⳳ SEM 18.91⬚C Ⳳ
). We computed the mean rate of acid se-0.056 mean Ⳳ SEM
cretion that occurred over the 1-h observation period. We then
selected the 10-min interval of this hour that had the highest
mean rate to determine maximal rates of acid secretion (Ford-
tran and Walsh 1973).
Protocol for Assessing Rates of Digestion,
Using Digital Radiography
Digital radiographs were used to monitor the effects of occlu-
sion of the left aorta on rates of digestion of bone. The alligators
were fed a meal containing a single defleshed, skeletally im-
mature bovine caudal vertebra weighing approximately 0.35%
of the body mass of the alligator (Table 1) and a mass of raw
hamburger containing 27% fat and weighing 5% of the body
mass of the alligator. After feeding, the animals were given 20
min in a water bath (30⬚C) to ensure that they were well hy-
drated. The animals were then housed separately at 27⬚C. The
first x-rays taken were on the day of feeding. Subsequent images
determined the rate of digestion of the bone on days 3, 6, 9,
13, 15, 17, 19, 21, and 23 following feeding. Twice a week the
animals were placed for 20 min into a tank of water held at
30⬚C so that they could drink.
A Siemens Axiom Aristos digital radiography unit was used
with standardized imaging parameters to include a subject-
object distance of 104 cm, and exposures using 50 kV and 2
mAs (milliamp seconds). The photoreceiver was kept horizon-
tal (standard extremity radiography configuration) so that the
experimental subjects could be placed directly on the surface
of the receiver. Animals were allowed to rest in the prone po-
sition. Collimation was restricted to the transverse width of the
animal and included the inferior (posterior) half of the chest
and entire pelvis. An experienced musculoskeletal radiologist
was present to assess proper positioning and exposure of each
image and to repeat the imaging as necessary. Before the transfer
of the digital images to the picture archive and communications
system (PACS), animal identification numbers were correlated
with time index data applied to the individual images by the
radiography unit. Once the images were transferred to the
PACS, the individual identifier codes were then manually ap-
plied to the images along with digital measurement data. The
130 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
Table 1: Mass of the animals and the caudal vertebrae used in the left aorta occlusion study
Animal
Mass (g)
Bovine Caudal
Vertebra (g)
Meal Mass #
Alligator Mass
⫺1
Days to Complete
Digestion
Experimental surgery:
E1 3,300 11.4 .0035 17
E2 3,300 11.5 .0035 15
E5 2,100 8.4 .0040 23
E6 1,500 5.5 .0037 21
E7 2,300 9.4 .0041 21
E8 2,000 7.0 .0035 21
Mean Ⳳ SE 2,417 Ⳳ 300 8.87 Ⳳ .98 .0037 Ⳳ .011 19.6 Ⳳ 1.23
Sham surgery:
C1 2,700 9.7 .0036 13
C2 2,500 9.7 .0039 21
C3 2,000 7.9 .0040 21
C5 1,700 6.2 .0036 17
Mean Ⳳ SE 2,225 Ⳳ 229 8.38 Ⳳ .84 .0038 Ⳳ .0001 18.0 Ⳳ 1.91
Note. No significant difference was found between the mass of the meals, expressed as a percentage of the body weight
of the animal, that were fed to the experimental and sham groups ( , two-tailed t-test assuming equal variance).P p 0.7
final annotated images were then transformed from digital im-
aging and communications in medicine format (DICOM) to
24-bit BMP and transferred to an individual storage file from
which they were finally burned to compact disc.
Data Analysis
Radiographic data from both studies were analyzed with the
same procedure. Measurements of the bone were made using
digital calipers at a Phillips Inturis PACS workstation that in-
cluded a high-resolution 4-megapixel monitor (1,536 # 2,048
pixels) made by NEC. The images were displayed two per
screen, with the sagittal plane oriented vertically. The images
were then magnified twice and run through an edge-enhancing
filter to improve the conspicuity of the bone margins. The
lightness/darkness as well as contrast of each image was inter-
mittently adjusted for optimum visualization. Because the in-
gested caudal vertebrae were not uniform cylinders, varying
degrees of obliquity and rotation affected their apparent di-
mension in the dorsal to ventral projection. Measurements were
therefore obtained parallel and perpendicular to the long axis
of the bones. The long axis was established by placing one of
the two Cobb angle lines along the central long axis. The second
line of the Cobb angle tool was then moved until the digital
angle measurement read 90⬚, thus setting a reference perpen-
dicular line to the long axis. As these lines could be moved to
any part of the image, accurate parallel measurements could
be made by placing subsequent linear measurement tools/cal-
ipers on or next to these reference lines. Figure 3 shows how
the maximum length, maximum width, and minimum width
of the ingested vertebrae were measured in relation to the ref-
erence lines. The maximum length was measured from the most
protuberant surfaces of the epiphyses parallel to the long axis.
Due to morphologic and orientational variances, this maximum
length was frequently not superimposed on the central long
axis. After fragmentation and separation of the epiphyses, the
maximum length was measured in the same manner from the
most protuberant visible part of the remaining metaphyseal
ends. The maximum width was measured from the outer cor-
tical surfaces of the wider metaphyses along the perpendicular
reference line. The minimum width was measured from the
outer cortical surfaces of the narrowest part of the diaphyses
along the perpendicular reference line. In the initial half of the
study, the limiting factor for measurement resolution was the
pixel dimension of the monitor, which yielded reproducible
measurements of Ⳳ0.1 mm. In the latter half of the study,
decreased conspicuity of the cortical surface from digestion was
a larger factor. As the length of the bone shortened, the position
of the minimum width measurement shifted. This had the effect
of increasing the minimum width measurement as time pro-
ceeded during the latter phase of digestion. If the bone assumed
an elliptical shape during digestion, the minimum width mea-
surement was dropped altogether and only maximum width
and length data were collected.
To minimize the effect of system performance variation on
measurement accuracy, we also obtained a maximum pelvic
width measurement to be used as a stable length reference.
Since this measurement was taken from between the most pro-
tuberant lateral acetabular margins, it is referred to as the trans-
acetabular width (TAW). The TAW line generally fell along the
intervertebral space of S1 and S2 (Fig. 3). Small degrees of
pelvic obliquity did affect the TAW measurement, and it is not
clear whether this may have introduced more or less variability
to the measurement accuracy than would have been seen with
normal fluctuation in the radiography system itself.
The ingested bone measurement data were then converted
to a percentage of the TAW measurement obtained on the same
day of imaging. Since each animal is similar in body proportion
The Right-to-Left Shunt Serves Digestion 131
Figure 3. A, Radiograph of an ingested vertebra illustrating the measurements taken: 1, long axis; 2, perpendicular axis; 3, maximum length;
4, maximum width; 5, minimum width. Note that the maximum length measurement is obtained parallel to but laterally displaced from the
long axis reference line in this vertebra with asymmetrical morphology. The arrow indicates an incidentally ingested tooth. B, The transacetabular
line extends between the most protuberant lateral margins of the acetabula. Note that the line falls over the intervertebral joint of S1 and S2,
and the spinous processes of the dorsal-sacral-caudal vertebra are centered on this well-positioned image. Arrows indicate scutes in the lateral
row. These scutes may overlie the acetabula and obscure the lateral margins.
despite variation in mass, reporting vertebral measurements as
a percentage of TAW had the additional advantage of elimi-
nating scale from the final data pattern.
The distribution of these measurements as percentages of
TAW was checked for normality, and a one-tailed Student’s t-
test was used to determine whether the differences seen between
the control and experimental groups were significant (P ≤
). Once an individual alligator had completed digestion,0.05
that animal was no longer included in the analysis.
Results
Preferred Body Temperature
Preferred T
b
was significantly greater and less variable after
feeding than when fasting; ,T p 26.0⬚Ⳳ2.02⬚C mean Ⳳ
bfast
,; ,SEM N p 6 T p 30.10⬚Ⳳ0.03⬚C mean Ⳳ SEM N p 6
bfed
( , two-tailed t-test).P p 0.0028
Blood Flow
In all five animals, the right-to-left shunt increased after con-
sumption of a meal. Figure 4 shows representative data collected
before and after eating from one animal weighing 2 kg. Each
value is the mean rate of shunted blood occurring over a 12-
h period.
Gastric Acid Secretion
The peak rates of gastric acid secretion (the maximum mean
value occurring during a 10-min interval during a 1-h period
of observation) are shown in Figure 5A, and mean rates mea-
sured over the entire observation periods (1 h each) are shown
in Figure 5B. ANOVA testing the effects of treatment (occlusion
of the left aorta) and temperature on rates of acid secretion at
24 h after eating showed a significant effect of treatment
( , ) and temperature ( ,F p 18.9 P p 0.0024 F p 9.14 P p
1, 8 1, 8
) as well as a significant interaction between the treatment0.0165
and temperature ( , ). At 27⬚C, the controlF p 9.3 P p 0.0158
1, 8
group had significantly greater rates of acid secretion than the
experimental group ( , Bonferroni pairwise compari-a p 0.05
son of all treatment by temperature effects). No difference be-
tween the control ( ) and experimental ( ) animalsN p 3 N p 3
was found at 19⬚C ( ). There was a significant decreasea p 0.05
in rates of acid secretion in the control group at 19⬚C compared
with 27⬚C and in the experimental group at 19⬚C compared
with 27⬚C ( ). The sharp decrease in rates of gastrica p 0.05
acid secretion at 19⬚C for the control group suggests this group
stopped shunting at the cool temperature.
Left Aorta Occlusion
Assessment of surgical technique. Spot fluoroscopy demonstrated
profound bradycardia/transient asystole within 5–20 s of in-
jection of acetycholine, at which time a second contrast bolus
was hand-injected. Closure of the pulmonary cog valve and
132 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
Figure 4. Blood flow through the left aorta (LAo) before and after
eating, from one alligator that is representative of the five individuals
studied. Each symbol is a mean of 12 h of shunted blood flow. Open
; filled .circle p fasting circles p postprandial
Figure 5. Rates of gastric acid secretion. Measurements were made for
1 h, beginning at 12 (black bars) and 24 (striped bars) h after the
animals consumed a meal of chopped steak. Except during the hour
of measurement, the animals were housed at 27⬚C. The first set of
data was collected at 27⬚C. The animals were given 2 wk rest, and
then the experiments were repeated, but during the hour of mea-
surement, body temperature was lowered to 19⬚C. Body temperature
was recorded with a thermocouple secured inside the cloaca
(, ).A,26.64⬚Ⳳ0.78⬚C mean Ⳳ SE 18.91⬚Ⳳ0.056⬚C mean Ⳳ SE
of the 10-min periods that gave the largest average rateMeans Ⳳ SEs
were included in this analysis. ANOVA testing the effects of treatment
(occlusion of the left aorta) and temperature on rates of acid secretion
at 24 h after feeding showed a significant effect of treatment (F p
1, 8
, ) and temperature ( , ) as well18.9 P p 0.0024 F p 9.14 P p 0.0165
1, 8
as a significant interaction between the treatment and temperature
( , ). At 27⬚ C, the sham surgery group had sig-F p 9.3 P p 0.0158
1, 8
nificantly greater rates of acid secretion than the occluded group
( , Bonferroni pairwise comparison of all treatment-by-tem-a p 0.05
perature effects). After 24 h, no difference between the sham and
occluded animals was found at 19⬚C ( ). There was a signif-a p 0.05
icant decrease in rates of acid secretion in the control group at 19⬚C
compared with rates at 27⬚C and in the occluded group at 19⬚C com-
pared with rates at 27⬚C ( ). The sharp decrease in rates ofa p 0.05
gastric acid secretion at 19⬚C for the shams at 24 h after feeding suggests
this group stopped shunting due to the prior exposure to the cool
temperature at 12 h postfeeding. B, of the mean ratesMeans Ⳳ SEs
of acid measured over the entire hour. The solid and striped bars
represent measurements made 12 and 24 h after feeding, respectively.
Sham surgery group: black bar (),diagonally striped bar (N p 3 N p
); occluded group: white bar (),horizontally striped bar3 N p 3
().N p 3
pulmonary outflow tract spasm/constriction was observed in
all cases. Successful control subjects showed a distinct jet of
contrast into the left aorta (shunting; Fig. 2D), while successful
experimental animals showed pooling of contrast in the right
ventricle with intermittent emptying through the pulmonary
cog valve and no visualization of the left aorta (left aortic shunt
obstructed). Due to the delayed and partial right atrial emptying
in subjects with atrial catheters, a greater volume of contrast
and more runs were required in those animals with atrial cath-
eters compared to those with ventricular catheters. Atrial runs
showed no significant improvement in right ventricular outflow
opacification in the supine versus prone position. Right ven-
tricular catheters yielded superior-quality angiograms. Images
were captured with a Siemens Axiom Sireskop SD fluoroscope
set at 4 frames s
⫺1
in angiography (iodine detection) exposure
mode. Iohexol doses ranged from a minimum of 5 mL to a
maximum of 20 mL. There was no perceivable reaction/irri-
tation to the animal subjects associated with the contrast in-
jections. Experimental animals showing signs of ejection of the
radio-opaque material into the left aorta were not included in
the study. At the end of the study, the animals were imaged a
second time to confirm that the left aorta remained occluded
throughout the study.
Out of the nine animals that underwent the experimental
surgery, the left aortas of six were successfully occluded, and
these animals were used in the study (Table 1). All five of the
animals that underwent a sham surgery were initially included
in the study. However, toward the end of the study, one of the
controls (C-4) bit off and ate the tip of the tail of another
alligator during a period in which they were allowed to swim
together in the tank of water. Thus, this animal was excluded
from the study.
Assessment of rates of digestion. The dissolution and subse-
quent fragmentation of the ingested vertebrae followed a ste-
The Right-to-Left Shunt Serves Digestion 133
Figure 6. Radiographic appearance of the typical digestion sequence of ingested bovine caudal vertebra. The freshly ingested vertebra displays
a distinct and intact cortical surface as indicated by the arrow in A. At the end of the first week, cortical erosion is evident along the wider
proximal metaphysis (arrow) and to a lesser extent in the smaller distal metaphysis (arrowheads), as shown in B. C shows the distinct cortical
loss in both metaphyses (proximally, arrow, and distally, arrowheads), seen at 9–13 d of digestion. At this point, loss of mineralization at the
physeal plates is evident and epiphyseal separation is imminent. In D, the proximal epiphysis has separated, and there is accelerated erosion
of the cortex because the medullary cavity is now exposed to digestive fluids. This radiographic appearance correlates with the rapid increase
in digestion rate that marks the slope transition in the last third of the digestion sequence, as depicted by maximum length/width per time
graph. E, The last visible remnants of the cylindrical vertebral diaphysis and a ghost of the separated distal epiphysis (arrow), as seen in the
last days of digestion.
reotypical sequence (Fig. 6). The first radiographically observ-
able changes included circumferential cortical thinning. As the
wider metaphysis anatomically had the thinnest cortex, it was
the first point of cortical perforation/fraying. This was closely
followed by fraying of the smaller metaphysis. The cartilage-
covered epiphyses were least affected in the initial stages of
digestion, and maximum length stayed relatively stable for the
first half of the digestion sequence, while both maximum and
minimum widths decreased at a constant rate. In the last third
of the digestion sequence, separation of the epiphyses with
fragmentation and continued cortical dissolution resulted in
an accelerated decrease in all three dimensions. The shapes of
the morphometric curves of this digestion sequence are similar
between control and experimental animals as well as between
rapid and slow digestors within these cohorts.
No significant differences were seen in the masses of the
vertebrae ingested with respect to the alligators’ body masses
(Table 1). Furthermore, no significant differences in any of the
measurements of the vertebrae (maximum width, minimum
width, length) were seen on the first or third days after feeding
(Fig. 7). Thus, the meals ingested by each group were not
significantly different. However, the ways the meals were pro-
cessed differed between the experimental and control groups.
By day 6, a highly significant ( , one-tailed t-test)P p 0.002
difference was found in the maximum width of the vertebrae.
Thinning of the bone occurred faster in the control group of
animals compared to the experimental (Fig. 7). Significant dif-
ferences were seen in either the maximum or the minimum
measurements from day 6 through day 17 of digestion. How-
ever, by day 19, significant differences could no longer be de-
tected (Tables 2, 3, and 4).
The total time required to completely digest the bone was
highly variable in each group (Table 1). There was no significant
difference between the sham-operated animals and the exper-
imental animals ( ). The experimental animals wereP
1 0.05
slightly larger on average than the controls, and there was a
trend for the larger animals to finish digestion more quickly
than smaller animals. However, ANCOVA of the mass of the
alligator and the time to digest the bone still produced of a P
value
10.05.
Discussion
The preponderance of these data indicates that the right-to-
left shunt of crocodilians serves digestion. The mechanism re-
mains to be demonstrated, but the shunt probably serves to
carry CO
2
to the gastrointestinal tract, where it is then used in
the formation of gastric acid. However, all amniotes produce
gastric acid, so it is not clear why the shunt is found in ec-
tothermic but not endothermic amniotes. One possibility is
that rates of gastric acid secretion are highly dependent on body
temperature (T
b
), and since ectotherms cannot always maintain
preferred T
b
, they may need to secrete large amounts of acid
while at the appropriate temperature; time is of the essence. In
crocodilians, competition for basking sites can be fierce, with
larger individuals dominating the sites (Grigg and Seebacher
2000). Basking may also increase small crocodilians’ risk of
predation, which is extremely high in the first two years of life
(Woodward et al. 1987). Thus, it may be especially critical for
younger animals to rapidly secrete acid when the opportunity
to obtain the requisite T
b
arises. In contrast, endothermic lin-
eages can secrete acid independently of environmental tem-
perature fluctuations and basking resources. This hypothesis
predicts the following: (1) after feeding, reptiles will bask to
increase T
b
, (2) rates of gastric acid secretion will be highly
sensitive to T
b
, and (3) maximal rates of acid secretion will be
greater in ectotherms than endotherms when at their respective
preferred T
b
.
134 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
Figure 7. of the following measurements expressed as aMeans Ⳳ SEs
percentage of the transacetabular width: the maximum width of the
ingested vertebrae, the minimum width of the ingested vertebrae, and
the length of the ingested vertebrae (see Fig. 3). There was no significant
difference in these measurements between the control (squares) and
experimental (diamonds) groups when the bones were initially ingested
or 3 d after eating. However, by 6 d after feeding, a highly significant
difference ( , unpaired t-test) between the experimental andP p 0.0025
the control groups was seen in the first part of the bone to show signs
of digestion, the thinning of the maximum width of the vertebrae.
During this first phase of digestion, the width of the bones thinned
at a constant rate. Thus, there was less overall variability in the mea-
surements, and significant differences ( , unpaired t-test) be-P ≤ 0.05
tween the groups were detected in one or both of the width mea-
surements on days 6, 9, 13, 15, and 17. However, when the epiphases
separated, which occurred around day 13, the bones began to fragment,
and some of the animals rapidly finished digestion at this point. By
day 19, only two animals of the control group retained bone (Tables
1–4), limiting the power of statistical analysis, and on days 21 and 23,
no control animals remained, eliminating the ability to statistically
analyze the data.
Table 2: of maximum width of theMean Ⳳ SE
vertebrae as a percentage of transacetabular width in
the left aorta occlusion study
Day
Postprandial
Experimental
Control
Mean Ⳳ SE N Mean Ⳳ SE N
0 33.9 Ⳳ 1.10 6 33.0 Ⳳ 1.47 4
3 30.6 Ⳳ .67 6 29.1 Ⳳ 1.12 4
6 26.4 Ⳳ .80 6 22.7 Ⳳ .56 4
9 22.3 Ⳳ 1.14 6 18.4 Ⳳ 1.95 4
13 17.1 Ⳳ 1.37 6 16.6 Ⳳ 1.12 3
15 15.9 Ⳳ 1.71 5 11.7 Ⳳ 1.30 3
17 18.5 Ⳳ 1.57 4 13.2 Ⳳ .37 2
19 15.6 Ⳳ 2.06 4 13.1 Ⳳ .65 2
21 17.8 Ⳳ .00 1
23 16.4 Ⳳ .00 1
We found that in American alligators, preferred T
b
was sig-
nificantly greater and less variable after feeding than when fast-
ing. Although our results differ from those in prior research,
which found no change in preferred body temperature with
feeding in a crocodilian (Diefenbach 1975a, 1975b), in the for-
mer study, the animals were handled daily to introduce them
to the thermal gradient. If the animals were unaccustomed to
this handling, it could have altered their behavior and may
explain the discrepancy between the studies. We also found a
pronounced decrease in rates of gastric acid secretion with
decreasing body temperature. Although we initially attempted
to study rates of gastric acid secretion at the postprandial pre-
ferred body temperature of 30⬚C, at this temperature, the an-
imals would not sit still for a 1-h period for observation, and
thus we chose to use 27⬚C. Nevertheless, we measured signif-
icantly greater rates of gastric acid secretion at 27⬚C in both
groups of animals. The effect of temperature is more complex
than a simple reduction in the turnover rates of enzymes with
temperature, because we saw less effect of temperature 12 h
into digestion than at 24 h. We suspect the prior cooling at 12
h shut down digestion, and this became apparent in our mea-
surements at 24 h. The exact mechanisms by which this occurs
merit further study. Nevertheless, as predicted by the hypoth-
esis, rates of gastric acid secretion are highly sensitive to body
temperature. Finally, we found that maximal rates of gastric
acid secretion are approximately an order of magnitude greater
in our control group of alligators than maximal rates reported
for endotherms (postprandial humans with mEqulcers p 0.9
kg
⫺1
h
⫺1
; Fordtran and Walsh 1973). Thus, all three predictions
of this hypothesis have proven correct.
However, other factors may also be important and may merit
investigation. For example, extremely high rates of gastric acid
secretion may be especially important for animals that consume
large meals. While endotherms tend to eat small but regular
meals, many ectotherms consume large meals; for example,
American alligators will voluntarily eat meals that weigh 23%
of their body mass (Uriona and Farmer 2006). High rates of
The Right-to-Left Shunt Serves Digestion 135
Table 3: of minimum width of theMean Ⳳ SE
vertebrae as a percentage of transacetabular width in
the left aorta occlusion study
Day
Postprandial
Experimental
Control
Mean Ⳳ SE N Mean Ⳳ SE N
0 22.4 Ⳳ 1.39 6 20.4 Ⳳ .46 4
3 18.9 Ⳳ .78 6 18.0 Ⳳ 1.27 4
6 17.2 Ⳳ .76 6 15.7 Ⳳ .72 4
9 15.7 Ⳳ .96 6 13.8 Ⳳ 1.10 4
13 12.7 Ⳳ 1.03 6 10.8 Ⳳ .44 3
15 12.2 Ⳳ .90 4 8.8 Ⳳ .66 2
17 10.2 Ⳳ 2.04 3 9.8 Ⳳ .38 2
19 9.3 Ⳳ .99 3 11.1 Ⳳ 01
21 17.0 Ⳳ 01
Table 4: of the length of the vertebrae as aMean Ⳳ SE
percentage of transacetabular width in the left aorta
occlusion study
Day
Postprandial
Experimental
Control
Mean Ⳳ SE N Mean Ⳳ SE N
0 82.2 Ⳳ 2.70 6 78.6 Ⳳ 4.49 4
3 80.8 Ⳳ 2.38 6 79.5 Ⳳ 3.30 4
6 78.45 Ⳳ 2.64 6 75.7 Ⳳ 2.80 4
9 74.33 Ⳳ 2.73 6 55.0 Ⳳ 15.41 4
13 61.2 Ⳳ 5.54 6 64 Ⳳ 3.60 3
15 53.0 Ⳳ 12.12 5 43.3 Ⳳ 19.57 3
17 56.4 Ⳳ 15.83 4 47.0 Ⳳ 4.22 2
19 41.6 Ⳳ 9.70 4 22.2 Ⳳ 13.04 2
21 44.7 Ⳳ .0 1
23 38.6 Ⳳ .0 1
acid secretion may be important for maintaining an acidic gas-
tric environment to prevent putrefaction.
Implications. The role of the shunt in digestion may explain
some of the novel and curious features of the crocodilian cir-
culation, including the function of the foramen of Panizza and
the allosteric regulation of hemoglobin. Once the acid-producing
cells of the stomach have created hydrogen ions, the ions are
pumped into the gastric lumen against one of the greatest con-
centration gradients known to exist in the body of an animal.
These pumps require ATP, and thus, the acid-producing cells
require oxygen in addition to carbon dioxide. The foramen of
Panizza enables boli of O
2
-rich blood to flow from the right
aorta into the left aorta and thus can enrich left-aortic blood
with this much-needed oxygen. Furthermore, unique features
of the blood will help unload this O
2
in the gastric circulation.
Crocodilian hemoglobin is unusual in decreasing its affinity for
O
2
on binding bicarbonate ions ( ), rather than on bind-
⫺
HCO
3
ing molecules of CO
2
; chloride ions (Cl
⫺
) competitively bind
to the same site (Perutz et al. 1981). Because gastric acid se-
cretion depletes the blood of Cl
⫺
but enriches it with ,
⫺
HCO
3
this allosteric regulation is suited to unloading O
2
to the acid-
secreting glands of the stomach. A digestive function of the
shunt also explains the chemical regulation of this blood flow
pattern. Many of the neural and endocrine molecules that in-
duce a shunt also promote gastric acid secretion (e.g., acetyl-
choline, gastrin-releasing peptide, histamine) or have other
roles in digestion (e.g., substance P; White 1956, 1969; Green-
field and Morrow 1961; Grigg and Johansen 1987; Axelsson et
al. 1989, 1991; Grigg 1989, 1992; Holmgren et al. 1989; Jones
1996; Hicks 1998; Franklin and Axelsson 2000).
Our results have several other implications. First, the right-
to-left shunt is probably essential for maximal rates of base
production, which are predicted to be equal to the high rates
of acid secretion found in our study. The egesta of alligators
are not highly acidic (C.G. Farmer, personal observation), and
therefore, the large volumes of gastric acid produced must be
neutralized in the gastrointestinal system by base. As is the case
for gastric acid secretion, maximal rates of base secretion have
been shown in studies of mammals to depend on blood-borne
CO
2
and cannot occur with the CO
2
available solely from en-
dogenous metabolism of the base-producing cells themselves
(Flemstrom and Isenberg 2001; Furukawa et al. 2005). Recall
that the left aorta is continuous with the celiac artery, which
in turn gives rise to the spleno-intestinal artery (carrying blood
to the spleen and part of the small intestine), the gastro-
hepatico-intestinal artery (carrying blood to the stomach, liver,
and small intestine), and to the pancreo-intestinal artery (car-
rying blood to the pancreas and small intestine). Thus, maximal
rates of base production in the pancreas, liver, and small in-
testine are probably dependent on the right-to-left shunt.
Furthermore, we hypothesize that this shunt serves to pro-
vide carbon to the liver, small intestine, and spleen for the
synthesis of glutamine, lipids, uric acid, and hemoglobin. Car-
boxylation of pyruvate to form oxaloacetate and the subsequent
formation of a-ketoglutarate (Delluva and Wilson 1946; Coul-
son and Hernandez 1983; Stryer 1995) is important to protein
metabolism; the a-amino group of many amino acids is trans-
ferred to a-ketogluterate to form glutamate. Glutamate can
then combine with ammonia to form glutamine. Glutamine
plays a key role in integrative metabolism; it is important to
acid-base homeostasis and the synthesis of uric acid (Campbell
1991), as a precursor in nucleic acid and nucleotide biosyn-
thesis, in the synthesis of amino sugars, in intraorgan transport
(Krebs 1980), and in red blood cell metabolism (Nihara et al.
1998). Coulsen and Hernandez (1983) report that the ability
of the liver of lizards to convert CO
2
and pyruvate to glutamine
is “without precedence” in that liver glutamine concentrations
were increased 30-fold when the animals were given pyruvate.
Furthermore, the fixation of CO
2
with pyruvate and the for-
mation of oxaloacetate can be stopped by giving a carbonic
anhydrase inhibitor in caimans and lizards (Coulson and Her-
nandez 1983). Thus, carbonic anhydrase appears to be required
for this reaction to occur in vivo. Finally, carbon dioxide is
requisite for fatty acid synthesis, a process that begins with the
carboxylation of acetyl CoA to form malonyl CoA (Wakil 1989;
136 C. G. Farmer, T. J. Uriona, D. B. Olsen, M. Steenblik, and K. Sanders
Stryer 1995). This irreversible reaction is the committed step
in fatty acid synthesis. In summary, CO
2
is requisite to nu-
merous biochemical processes carried out by the stomach, pan-
creas, spleen, liver, and small intestine. It is probable that all
of these processes are facilitated by blood-borne CO
2
. Thus,
the hypothesis that the right-to-left shunt of reptiles serves these
digestive functions warrants further investigation.
Acknowledgments
We thank J. Hicks and J. Moore for insightful conversations
regarding experimental design. We thank F. Adler, M. Butler,
D. Feener, and especially I. Terry for advice on the statistical
analysis of the data and R. Elsey and staff at the Rockefeller
Wildlife Refuge for providing animals. We are grateful to M.
Heath and Medtronics for assistance with gastric pH mea-
surements. This work was supported by National Science Foun-
dation grant IBN-0137988 to C.G.F.
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