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First published online October 18, 2006
Journal of Experimental Biology 209, 4313-4318 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02523
Contribution of the diaphragmaticus muscle to vital capacity in fasting and post-prandial American alligators (Alligator mississippiensis)
Department of Biology, 257 South 1400 East, University of Utah, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: uriona{at}biology.utah.eu)
Accepted 5 September 2006
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Summary |
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Key words: alligator, Alligator mississippiensis, CO2 breathing, diaphragmaticus, ventilation, post-prandial state, vital capacity, evolution, archosaur, ventilatory mechanics, crocodilian
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), and two
mechanisms that supplement inspiration
(Fig. 1). The first mechanism,
the hepatic piston, entails a decrease in pleural pressure caused by a caudal
movement of the lungs, liver and other viscera
(Boelert, 1942;
Gans and Clark, 1976). Pelvic
aspiration refers to an increase in abdominal volume and decrease in abdominal
pressure caused by rotation of the pubes and gastralia caudoventrad
(Farmer and Carrier, 2000). An
important element of the hepatic piston is the diaphragmaticus muscle. This is
a pair of two thin muscles extending in a caudocranial orientation
(Reese, 1915); the lateral
component of the muscle is attached by a short tendon in front of the hip
joint to the edge of the ilium, and the ventral portion originates partly on
the innermost side of the last (caudad) gastralium, which is embedded in the
rectus abdominus muscle [American alligator
(Farmer and Carrier, 2000)] and
partly on the border of the pubis [caimen
(Gans and Clark, 1976;
Reese, 1915)]. The
diaphragmaticus fans as it runs craniad to its place of insertion. Ventrally
and laterally the muscle inserts directly on the pericardium and on a fibrous
membrane that encapsulates the liver. Dorsally the muscle inserts on an
aponeurosis that passes over the dorsal border of the liver and that binds the
liver to the esophagus in the region of the lower esophageal sphincter
(Reese, 1915). Unlike the
mammalian diaphragm which partitions the body cavity obliquely into thoracic
and abdominal chambers, the crocodilian diaphragmaticus encases laterally and
ventrally most of the peritoneal viscera. Because the lungs adhere to the
hepatic membrane, contraction of the diaphragmaticus pulls the liver-lung
complex caudad and facilitates inspiration
(Gans and Clark, 1976).
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). Some
testudines (e.g. Lissemys and Trionix) also have a muscle called a
diaphragmaticus, which differs because the fibers have dorsoventral
orientation. In this group the diaphragmaticus (craniad) together with the
transversus abdominis (caudad) enclose the viscera. These muscles arise from
the inner lateral border of the carapace in the region of the first to seventh
ribs, proceed both laterally and ventrally, and, after becoming membranous,
insert on the plastron in the middle and on the dorsal aspect of the pubic
symphysis (George and Shah,
1959).
The selective factors that have favored the evolution of these structures
in some groups of tetrapods but not in others remain incompletely understood.
Insight into these selective factors comes from understanding the relative
importance of the muscles to different facets of the life history of the
animals. These muscles may facilitate inspiration
(Gans and Clark, 1976),
expiration (Gaunt and Gans,
1969; Snapper et al.,
1974), gastrointestinal functions
(Pickering et al., 2004;
Pickering and Jones, 2002), or
venous return (Keith, 1905).
They may be most important during exercise, while diving, basking, digesting,
or at some other time.
American alligators are highly aquatic in their lifestyle and tend to eat large meals. Theoretically, a large mass of food in the stomach could significantly increase the difficulty of inspiring large volumes of air into the lungs. This in turn could impact numerous facets of their life history, such as dive-time and exercise capacities. Thus, we undertook this study of the importance of the diaphragmaticus muscle to vital capacity during the postprandial period in American alligators to contribute to our understanding of when and how this muscle is used.
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Materials and methods |
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Experimental design
Similar experiments were conducted sequentially on two sets of animals and
will be identified from now on as study 1 or study 2. In study 1 very young
individuals were used. Shortly after complete absorption of the yolk sac, the
animals of study 1 were randomly divided into two groups. The first group
served as the control and in animals of the second group the diaphragmaticus
muscle was transected. The control animals did not undergo a sham surgery. At
the time of the surgery for transection the animals' mass ranged from 49 to 66
g with a mean mass of 54.2±7.3 g. A series of experiments was conducted
with these animals that yielded significant differences between the transected
and control groups. At the time of these experiments, the animals weighed
between 62 to 130 g, with a mean mass of 97.5±18.1 g.
To ensure that these differences were not due to effects of the surgery, other than the transection of the muscle, the experiments were repeated on a second set of animals, study 2. In this study 2 year old animals were randomly divided into two groups. The first group received a sham surgery and in animals of the second group the diaphragmaticus muscle was transected. At the time of the surgery the animals' weight ranged from 112 to 153 g with a mean mass of 128.2±15.3 g. At the time of the experiments, these animals weighed between 97 and 210 g, with a mean mass of 148.2±36.5 g.
Surgery
Animals were lightly anaesthetized by enclosure in a box with a rag soaked
in isoflurane. Once drowsy, they were intubated and ventilated with a small
animal ventilator (CWE Inc., Ardmore, PA, USA) using air that had passed
through an isoflurane vaporizer (Drager, Lubeck, Germany). The level was
initially set at 4%, but was reduced to 0.5% for the majority of the surgery.
All surgeries were performed using sterile technique. With the animal in the
supine position, the belly and sides were disinfected with betadine and the
animal was draped with a sterile cloth. An incision was made along the ventral
midline of the animal, caudad to the last long rib. The ventral portion of the
diaphragmaticus muscle was located along both the right and left sides of the
body. In the experimental animals the muscles were gathered with forceps,
cleared of veins and other organs, and transected. Two more incisions were
made on each lateral side of the animals, craniad to the pelvic girdle. The
pelvic attachment of the diaphragmaticus to the ilium was identified and
severed.
In the sham surgeries the same incisions were made and the muscles were identified but not transected. All incisions were sutured. The animals were treated both with a topical antibiotic and intraperitoneal injections of antibiotics (Baytril) until the incisions had healed. During healing, the animals were kept dry and were given water orally every day.
Ventilation
A pneumotach, which also functioned as a mask, was made using 2 cm of a 5
ml syringe. Two ports were drilled into the side to attach polyethylene tubing
(PE190). Stainless steel mesh (size 200 mesh) was placed between the two
ports. The tubing was then attached to a pressure transducer 30 cm away from
the mask. The top of the mask was only partly covered, allowing air to escape
the mask and ensuring low resistance of the pneumotach. A third port was
drilled at the bottom of the mask to push air up and out of the top of the
mask. Air-flow was set by a flow control unit (Bel-Art Products, Pequannock,
NJ, USA) at 50 ml min-1. The system was calibrated by injecting
known volumes of gas into the mask prior to placement on the animal. The mask
was sealed over the nares of the alligator using dental adhesive (Impregum F,
3M ESPE, St Paul, MN, USA).
Data collection and analysis
Analog signals from the pressure transducer (Omega PX 42.3±1.7 kPa;
Omega, Stanford, CT, USA) were amplified (0.3 V mV-1) and filtered
(10 Hz low pass) using a Grass amplifier (Grass Telefacter, West Warwick, RI,
USA). The analog signals were then converted to a digital form using an analog
to digital converter (Biopac Systems, Goleta, CA, USA) and stored on a
Macintosh computer. Signals were sampled at a rate of 50 Hz and analyzed with
Acqknowledge software (Biopac Systems, Goleta, CA, USA). Volume, time and
maximum velocity for both inspiration and expiration were then determined. To
account for differences in body mass between individuals, the relative vital
capacities were computed by dividing volume by body mass. Statistical
significance was checked using a t-test with statistical significance
set at P<0.05.
Experimental protocol
Study 1
These experiments were conducted on control (no surgery) and experimental
animals approximately 1 year after the animals underwent surgery. Animals were
fasted for 4 days prior to data collection. They were transferred into large
Rubbermaid (Rubbermaid Inc., Fairland, OH, USA) containers from their holding
tanks and taken to the experimental room at least 2 h before the experiments
commenced. The experimental room was kept at 20°C. The order of the
animals was randomly chosen for each trial. The tail and front legs were fixed
to the table with duct tape in such a way as to allow the animal to stand but
not run around on the table. A mask was attached and the ventilation trace
checked to ensure the nares were not occluded and that the mask was properly
sealed. Ventilation was then stimulated with a gas mixture (5% CO2,
balance N2) that flowed through the mask for a period of 3 min. The
largest single inhalation occurring during this period and the subsequent
expiration were selected for analysis. The same protocol was repeated on the
animals 24 h after consuming a meal to satiation of the gizzards and hearts of
chickens. The control and experimental animals voluntarily consumed an average
of 15% of their mass in food, with the meal size varying from 9-23% of the
controls' body mass and 9-20% of the experimental animals' body mass. There
was no significant difference (P>0.05 t-test) in the
average voluntary meal size consumed between the two groups. The same five
control and five experimental animals were used for each treatment.
Study 2
These experiments were conducted on sham operated and experimental animals
approximately 1 week, 2 weeks, 2 months and 6 months after the animals
underwent surgery. The same protocol for collecting data as in study 1 was
followed for this study with the exceptions given below. Ventilation was
stimulated with a gas mixture containing 5% CO2, balance
N2 or 5% CO2, 21% O2, balance N2,
which flowed through the mask for a period of 3 min. For the post-prandial
portion of the experiment the animals were force-fed an amount of homogenized
chicken liver weighing 11% of the alligator's body mass. The same three sham
and three experimental animals were used for each treatment.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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0.05) difference between the experimental and control groups of
animals in the following parameters: maximum inspiratory volume, expiratory
volume, inspiratory time, expiratory time. It is in the post-prandial state
that a difference in ventilation between the two groups was observed, even
though the groups ate equivalent meals. The control and experimental animals
voluntarily consumed an average of 15% of their mass in food, with the meal
size varying from 9-23% of the controls' body mass and 9-20% of the
experimental animals' body mass. There was no significant difference
(P>0.05, t-test) in the average voluntary meal size
consumed between the two groups. A sample of ventilation data is provided in
Fig. 2 illustrating the
differences between the experimental and the control groups in a post-prandial
state. The maximum inspiratory volume (vital capacity) in the group that
underwent transection was significantly less than in the control group (mean
49.6 ml kg-1 and 62.2 ml kg-1, respectively;
Fig. 3). On average, the
experimental group took 2.8 s to inhale the maximum volume, whereas the
control group took an average 2.1 s. The control group was able to reach a
higher volume in a shorter time by generating higher maximum flow during their
breath (Fig. 3). The maximum
flow measurements of the control group averaged 3042.2 ml min-1
kg-1 compared with 1620.0 ml min-1 kg-1 of
the experimental group. There was no significant difference between the
maximum rates of exhalation between the two groups.
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Study 1: comparison between fed and fasted states
Compared with the fasting state the vital capacity decreased
post-prandially for both the experimental and control groups (37.4 ml
kg-1 and 20.2 ml kg-1, respectively), however inhalation
time was not different (Fig.
3). Similarly, compared with the fasting state the volume exhaled
decreased post-prandially for both the experimental and control groups (34.0
ml kg-1 and 20.4 ml kg-1, respectively), and exhalation
time was 0.4 s less in both groups. Compared with the fasting state the
maximum flow reached during inhalation decreased post-prandially by 770.7 ml
min-1 kg-1 for the experimental group but there was no
change in the control group. Fig.
3 and Table 1
provide a summary of all ventilatory values analyzed in this study.
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Study 2: comparison between experimental and control groups
One week, 2 weeks, and 2 months after the surgery no difference in vital
capacity was found between the sham and experimental groups. However, 6 months
after the surgeries a difference between these groups became apparent in the
post-prandial condition, and yielded results in accordance with the data of
study 1. Six months after the surgery, in the fasted state, there was once
again no significant difference in vital capacity between the experimental and
sham groups of animals. However, in the post-prandial state a difference in
ventilation between the two groups was observed. The vital capacity in the
group that underwent transection was significantly less than the vital
capacity in the sham group (mean 53.5 ml kg-1 and 71.2 ml
kg-1, respectively; P<0.05 t-test;
Fig. 4). The post-prandial drop
in the maximum inspiration, averaged 12% and 28% for sham and experimental
groups, respectively.
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Study 2: comparison between fed and fasted states
Six months after the surgery, the vital capacity decreased significantly
from the fasting state to the post-prandial state for the experimental group
and was close to being significant (P=0.06) in the sham group
(Fig. 4). The experimental
group went from 75.1 ml kg-1 when fasted to 53.5 ml kg-1
when fed, or a drop in vital capacity equal to 21.6 ml kg-1.
Whereas, the sham group went from 81 ml kg-1 when fasted to 71.2 ml
kg-1 when fed, or a drop in vital capacity equal to 9.8 ml
kg-1. Furthermore, the vital capacity decreased significantly for
the average of all the experiments performed (1 week, 2 weeks, 2 months and 6
months after the surgery) in both groups when post-prandial. This represents a
drop in volume of 16.7 ml kg-1 for the sham group and a 25.1 ml
kg-1 for the experimental group.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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There are several possible explanations for the lack of any effect of
transection on vital capacity in fasting animals. It is possible that the
diaphragmaticus normally makes negligible contribution to inspiration in
fasting, standing animals. If this is the case, it raises the interesting
question of whether the same would be true during more taxing times, such as
while swimming or running, and there is some evidence that transection of the
diaphragmaticus reduces tidal volume during treadmill-exercise in fasting
alligators (Hartzler et al.,
2004). Alternatively, it is possible that over the course of the
year between when the animals underwent surgery and when the experiments were
done, the animals gained the ability to compensate for the loss of the
diaphragmaticus in the resting state, perhaps by increasing the capacity of
other components of their ventilatory mechanism, or by regaining partial use
of the diaphragmaticus due to the growth of scar tissue that mechanically
reconnected the transected muscle. Although the surgery may have eliminated or
reduced the ability of the animals to use the hepatic piston for inspiration,
costal and pelvic aspiration may have compensated for this loss. Our visual
observations of the animals suggest different breathing mechanics are being
used.
In order to address the possibility that over time the muscle may have grown back and this was responsible for the results obtained in study 1, or that these results were due to the type of control used (no surgery), or due to the type of gas used (5% CO2, balance N2) a second study was conducted. For this study, sham and experimental animals were used to look at the changes in the maximum inspiration 1 week, 2 weeks, 2 months, and 6 months after surgery and with both a hypercapnic-anoxic gas mixture and a hypercapnic-normoxic gas mixture. At 1 week, 2 weeks, 2 months after surgery, the vital capacity obtained were variable and no significant difference was measured. These initial results could be attributed to healing from the surgery, which interfered with the animals' ability to breathe. After 6 months, the results from study 2 matched those of the first study (study 1) remarkably well, in that there was a decrease in the post-prandial vital capacity for both the experimental and control groups (Fig. 4). Six months after the surgery a large meal significantly decreased the maximum volume of inspiration in animals with a transected diaphragmatic muscle when compared to sham operated animals by 21% and 12%, respectively. This represents a 16% difference between the two groups or a 17.7 ml kg-1 greater loss in vital capacity for the transected group. This was a slightly smaller loss in maximum inspiratory volume than was seen in study 1 (18% was found in the first study). This could be due in part to a change in the experimental design where the animals were force fed a homogenized meal that was slightly smaller then the average meal size that the animals chose to eat on there own. Despite this change in protocol the overall trends observed in the main study were again seen for vital capacity. Thus, we think the preponderance of the data indicate that the diaphragmatic muscle makes an important contribution to vital capacity in the post-prandial state.
The observation that the large meal voluntarily eaten by these animals
significantly reduced vital capacity in both the control and the experimental
groups has some interesting implications for the life history of these
animals. Juvenile crocodilians have very high rates of mortality. For example,
several studies on both American alligators and on saltwater crocodiles
indicate that less than 50% of hatchlings survive to the age of one year
[Woodward et al. (Woodward et al.,
1987) and references therein]. Because juvenile animals take
refuge from predators such as birds and raccoons by submergence, their
abilities to remain submerged may be directly related to their chances of
surviving. A decrease in vital capacity may reduce pulmonary oxygen stores and
presumably reduce aerobic dive-time. Furthermore, this reduction in lung
oxygen stores may occur at a critical time, during the post-prandial period,
when metabolic rate and thus the demand for oxygen is known to increase by
about fourfold (Busk et al.,
2000). An increase in demand for oxygen, concomitant with a
decrease in supply, could have significant ramifications. For example, if a
juvenile is able to stay submerged aerobically for 6 min in the fasting state,
then with a fourfold increase in metabolism during the post-prandial period
this would be reduced to about 90 s. A reduction in lung oxygen stores by 43%
presumably would reduce this time to about 50 s, a very short time for an
animal seeking protection by submergence. Thus an extension of dive-time may
be a selective advantage that accompanied the ability of the diaphragmaticus
to increase vital capacity during the post-prandial period.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Boelert, R. (1942). Sur la physiologie de la respiration de l'Alligator mississippiensis. Arch. Int. Physiol. 52,57 -72.
Busk, M., Overgaard, J., Hicks, J. W., Bennett, A. F. and Wang, T. (2000). Effects of feeding on arterial blood gases in the American alligator Alligator mississippiensis. J. Exp. Biol. 203,3117 -3124.[Abstract]
Farmer, C. G. and Carrier, D. R. (2000). Pelvic aspiration in the American alligator (Alligator mississippiensis). J. Exp. Biol. 203,1679 -1687.[Abstract]
Gans, C. and Clark, B. (1976). Studies on ventilation of Caiman crocodilus (Crocodilia: Reptilia). Respir. Physiol. 26,285 -301.[CrossRef][Medline]
Gaunt, A. S. and Gans, C. (1969). Mechanics of respiration in the snapping turtle, Chelydra serpentina (Linné). J. Morphol. 128,195 -228.[CrossRef]
George, J. C. and Shah, R. V. (1959). The structural basis of the evolution of the respiratory mehanism in chelonia. J. Anim. Morphol. Physiol. 1, 1-9.
Hartzler, L. K., Munns, S. L. and Hicks, J. W. (2004). Contribution of the hepatic piston to ventilation in the American alligator, Alligator mississippiensis. FASEB J. 18,A238.4 .
Keith, A. (1905). The nature of the mammalian diaphragm and pleural cavities. J. Anat. Physiol. 39,243 -248.[Medline]
Pickering, M. and Jones, J. F. X. (2002). The diaphragm: two physiological muscles in one. J. Anat. 201,305 -312.[CrossRef][Medline]
Pickering, M., Campion, D. and Jones, J. F. X. (2004). A gastrointestinal role for the amphibian `diaphragm' of Xenopus laevis. J. Zool. 264, 45-51.[CrossRef]
Reese, A. M. (1915). The Alligator and Its Allies. New York: G. P. Putnam's Sons.
Snapper, J. R., Tenney, S. M. and McCann, F. V. (1974). Observations on the amphibian "diaphragm". Comp. Biochem. Physiol. 49A,223 -230.[Medline]
Woodward, A. R., Hines, T. C., Aberrombie, L. and Nichols, J. D. (1987). Survival of young American alligators on a Florida lake. J. Wildl. Manage. 51,931 -937.
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Significant difference (P
significant difference
(P