AMER. ZOOL., 23:377-381 (1983)
Mineralized Tissues and Exercise Physiology of Snakes'
JOHN A. RUBEN
Department of Zoology, Oregon State University,
Corvallis, Oregon 97331
SYNOPSIS. Previous studies indicate a tight correlation of predatory modes, morphology,
and activity physiology in snakes. Active foragers like the coachwhip {Masticophisflagellum)
exhibit great stamina and high rates of aerobiosis and anaerobiosis during intense exercise.
The western rattlesnake (Crolalus vindis), a sit-and-wait predator, is capable of bouts of
intense activity for limited periods of time before exhaustion. During maximal activity
the rattlesnake has moderate powers of anaerobiosis and limited powers of aerobiosis.
Superior blood buffering capacity in the coachwhip seems at least partially responsible
for its stamina. New evidence presented here indicates that low endurance in the rattlesnake may be associated with exercise-related bone dissolution, resulting in hypercalcemia.
Such hypercalcemia may function to further debilitate the already poorly developed blood
buffering capacity of the rattlesnake.
on "sit-and-wait," or surprise tactics to capture their primary prey, rodents. The
venom apparatus is used both for defense
and for killing prey.
Contrasting behavioral modes exhibited
by these taxa in thefieldcorrelate well with
differences in morphology and physiology
(Ruben, 1977a, b, 1979). The species differ
in their capacities for aerobic and anaerobic metabolism during intense exercise
(Fig. 1). Thus, high capacity for aerobiosis
in Masticophis probably permits increased
levels of normal activity and perhaps facilitates repayment of oxygen debt incurred
by extensive utilization of anaerobiosis
during bursts of maximal activity.
Crotalus seems capable of producing significant quantities of anaerobically-derived
energy over limited periods of time and
this feature undoubtedly facilitates generation of extremely fast movements when
the animal strikes its prey. Reduced capacity for maximal rates of oxygen consumption in this snake perhaps results in prolonged periods of oxygen debt following
extended bouts of intense exercise. This
feature may not be a problem for a species
as relatively inactive as the western rattlesnake.
The pattern of activity metabolism
exhibited by Masticophis, i.e., relatively great
stamina combined with simultaneously high
rates of aerobic and anaerobic metabolism
1
From the Symposium on Adaptive Radiation Within
during intense exercise, is somewhat sura Highly Specialized System: The Diversity of Feeding Mech- prising. Extensive reliance on anaerobiosis
anisms of Snakes presented at the Annual Meeting of
generally causes reduced blood pH which
the American Society of Zoologists, 27-30 December
is associated with decreased blood-oxygen
1981, at Dallas, Texas.
INTRODUCTION
Of all the extant terrestrial vertebrates,
snakes have possibly undergone the most
extensive adaptive radiation since the midTertiary Period; in contrast to extinction
and seeming evolutionary "stagnation" in
many other Reptilia, they may still be "on
the make" (Romer, 1966). As a result of
that radiation, snakes today occupy an
almost worldwide distribution, exist in a
broad range of ecological niches, and consequently exhibit a variety of predatory and
defensive behavior patterns. This range of
ophidian variation is exemplified, in part,
by the coachwhip, Masticophis flagellum
(Colubridae) and the western rattlesnake,
Crotalus viridis (Crotalidae). Coachwhips,
which occur over most of the southern
United States and northern South America, are slender, fast-moving and seemingly
inexhaustable. They are active foragers,
relying on speed and endurance to capture
prey. Prey is not constricted.
The western rattlesnake occurs widely
throughout the western U.S. It is a relatively heavy-bodied serpent that lacks the
capacity for extensive locomotory speed.
Nevertheless it is capable of moderate to
great levels of activity for limited periods
of time. Western rattlesnakes probably rely
377
378
JOHN A. RUBEN
1.25 -|
6"
i.oo
o
Z
g
H
2
0.75
0.50 •
0.25
Mastic ophis
2.25-
2.25
2.00-
-2.00
1-50 H
S
- 1.50
1.00-
1.00
-0.50
0.50-
Crolalus
Mosticophis
FIG. 1. Standard and active oxygen consumption
rates and whole body lactate concentrations at rest
and after 5 min of intense exercise in Masticophis and
Crotalus. Mean values are reported; vertical lines represent range of observations. Stippled boxes—resting
individuals; hatched boxes—post-activity (from
Ruben, 1976).
affinity, oxygen carrying capacity and heme
unit interaction. Such factors are often
associated with fatigue and may interfere
with oxygen uptake in the lungs (Bennett,
197S). Apparently exploitation of the Mns-
ticophis strategy of activity metabolism is
facilitated, at least in part, by that snake's
ability to avoid severe blood pH depression
during and after intense exercise. That
capacity, in turn, seems based on Masticophis' superior blood-borne bicarbonatecarbonate buffering system and a particularly well vascularized lung. In contrast,
Crotalus, which experiences severe exercise-related blood pH depression and
fatigue, possesses a poorly vascularized lung
and an unremarkable bicarbonate-carbonate blood buffering system (Ruben, 1979).
A most interesting aspect of activity
metabolism in Crotalus has recently been
described: intense exercise to exhaustion
is accompanied by marked blood hypercalcemia (Ruben and Bennett, 1981).
Hypercalcemia in other vertebrates has
previously been thought to impair a number of physiological functions associated
with exercise (e.g., cardiac, nervous and
muscular functions) (Frankenhauser, 1957,
Parfitt and Kleerekoper, 1980) and may
well be a factor associated with the severe
fatigue exhibited by Crotalus following
intense exercise. Certainly, the discovery
of activity-related hypercalcemia in Crotalus raises a number of questions regarding activity physiology in snakes, especially
as regards (i) the presence or absence of
exercise-related hypercalcemia in more
active, high-stamina species of snakes, as
Masticophis; (ii) the source and cause of the
excess calcium in Crotalus; (iii) effects of
hypercalcemia on the blood buffering
capacity of Crotalus.
To elucidate these questions, aspects of
blood physiology before and after activity
were examined in Masticophisflagellum and/
or Crotalus viridis. Parameters investigated
included simultaneous measurement of
blood pH and blood and/or muscle calcium and phosphate levels before and after
intense exercise. Additionally, in vitro
examination of possible effects of hypercalcemia on the buffering capacity of Crotalus blood was also carried out.
Recent studies have suggested that exercise-related hypercalcemia in many vertebrates, including reptiles, is the result of
slight skeletal dissolution resulting from
MINERALIZED TISSUES AND EXERCISE
systemic diffusion of lactic acid produced
during intense activity (Ruben and Bennett, 1981). Consequently, as bone consists
primarily of both phosphate and calcium
(as calcium hydroxy-apatite), one might a
priori postulate concurrent post-active elevation of blood-borne phosphate. Moreover, while hypercalcemia similar to that
described here has previously been interpreted as deleterious to vertebrates, there
are no observations of any such effects on
reptiles.
379
60i
MATERIALS AND METHODS
To determine resting blood pH, 75 p\ FIG. 2. Post-exercise plasma hypercalcemia and
blood samples were taken anaerobically in hyperphosphatemia in Mashcophis and Crotalus Verheparinized syringes from previously tical lines = +1 SE.
undisturbed individuals of each species.
Blood was collected from tail incisions.
Elapsed time between first handling and marked signs of tiring occurred. After a
sample procurement was <15 sec; strug- 10 min rest period, blood samples were
gling by animals was minimal during this collected and analyzed as described above
period. The pH of the sample was mea- for calcium and phosphate content and
sured immediately with a Radiometer- muscle samples (from Crotalus) were meaCopenhagen BMS mark III acid-base ana- sured for phosphate concentration. Each
lyzer. The temperature of the electrode species was maintained at an environmenwas regulated at the body temperature of tally realistic temperature (30°C).
the experimental subjects. Immediately
The effect of additional post-active
after withdrawal of the previous blood blood-borne calcium and phosphate on
sample, another sample of 200 /A was with- blood buffering capacity in Crotalus was
drawn from each individual in a non-hep- investigated in vitro. This was accomarinized syringe. Plasma from these sam- plished by measuring the pH of pooled,
ples was then diluted at a 1:12 ratio with resting blood samples (from five individua calcium-suspending solvent (containing als) through which CO2 was gently bub3.60 X 10-3 M La203; 5.0 X 10"2 M HCl) bled. The pH and pCO 2 of the sample was
and then analyzed for calcium concentra- measured at varying levels of pCO 2 utiliztion on a Jarrell-Ash atomic absorption ing the same apparatus as described above
spectrophotometer equipped with a lami- for in vivo blood samples. Following connar-flow burner. A second plasma sample struction of this pCO2-pH curve, sufficient
from each individual was obtained and ana- powered bone was added to the pooled
lyzed for total phosphate content accord- blood sample to bring the concentration of
ing to the method of Gindler and Ishizaki, bone in the sample to approximately the
1969. Samples of muscle tissue (longissi- concentration of bone in the vertebrate
mus dorsi) were also obtained from Cro- body (about 45 m/Vf). A second pCCypH
talus and analyzed for total phosphate curve was then constructed in a similar
content. This was accomplished by homog- manner.
enization of muscle tissue in a Sorvall
Omnimixer and analysis by the same
RESULTS
method as given for total plasma phosBlood
pH
of
resting
Masticophis and Crophate. Different individuals from each
talus
(approx.
7.4
for
both
species) was not
species were stimulated to maximal activity
significantly
different
(P
>
0.05, /-test),
by manual prodding until exhaustion or
however pronounced differences were evi-
380
JOHN A. RUBEN
76-
approximately 1.25X that of the hydrogen
ion concentration in the sample before
addition of ground bone.
*
30°
74"
\
I
in vitro
\
\
a.
\
-o 7.2
o
o
DISCUSSION
\
\
Ca a d d e d ^ * \
70
•
6.8
10
30
50
70
•
90
FIG. 3. The effect of the presence of ground bone
on the in vitro capacity of Crolalus blood to buffer
carbon dioxide.
dent after intense exercise. Thus, blood
pH in Masticophis fell approximately 0.31
(±0.06 SE) pH units after exercise compared to the fall of 0.55 (±0.04 SE) pH
units in Crotalus (P < 0.05, Mest).
In vivo blood sampling indicated that both
Crotalus and Masticophis experienced
marked post-active hypercalcemia as well
as significant elevation of blood-borne
phosphate {P < 0.05, t-test) (Fig. 2). Of the
two genera, Crotalus exhibited a hypercalcemia approximately 2X that of Masticophis
and plasma hyperphosphatemia approximately 2.5X that of Masticophis {P < 0.05,
Mest, for both observations).
Total phosphate concentration in Crotalus longissimus muscle rose slightly following intense activity (total phosphate =
30.11 mM ± 2.8 SE, at rest; 31.88 ± 3.1
SE, following activity. N = 5), but the elevation was not statistically significant (P >
0.05, /-test).
In vitro experiments indicate that bone
dissolution may have a deleterious effect
on the buffering capacity of the blood (Fig.
3). For any given pCO 2 , blood pH was
depressed approx. 0.1 pH units in the presence of ground bond (P < 0.01, Chi
square). The pH notation obscures the
magnitude of the observed differences: at
any given pCO2, blood hydrogen ion concentration in the pooled sample was
The magnitude of blood pH depression
following intense exercise in Crotalus and
Masticophis as well as the degree of exercise
related hypercalcemia in Crotalus are similar to previously reported data for these
species (Ruben, 1979; Ruben and Bennett,
1981).
In light of previous studies, elevation of
blood plasma calcium and phosphate levels
in these species can be interpreted to be
associated, at least in part, with the dissociation of bone according to the following
formula:
Ca10(PO4)6(OH)2 - - Ca+2 + (HPCV2)
+ (PCV3) + (OH"1).
Such dissolution is presumably associated
with the increased solubility of calcium
hydroxy-apatite following post-active blood
and tissue pH depression. However, the
possibility that other, non-osseous, sources
of phosphate may contribute to the increment in plasma-phosphate levels should not
be ignored. For example, phosphate may
be lost or dissociated from blood cells following acidification of the blood, but insignificant change in longissimus dorsi muscle
phosphate concentration following intense
exercise in Crotalus may indicate that muscle tissue is not a contributory factor to
exercise-related hyperphosphatemia in
snakes.
In vitro data presented here suggest that
bone dissolution may actually impair blood
buffering capacity. These findings are
somewhat unexpected: others have suggested that bicarbonate released into the
blood as a result of bone dissolution might
act to supplement blood buffering capacity
(Poyart etal., 1971) and one might reasonably hypothesize that phosphate anions
generated as a result of bone dissolution
might also be a potential buffer. Clearly,
in the in vitro system investigated here, such
is not the case. Instead, processes here
might involve replacement of bound
hydrogen ions by calcium ions released into
MINERALIZED TISSUES AND EXERCISE
the blood following dissolution of the bone.
The effect would then be to decrease the
buffering capacity of the blood by reducing
the number of sites to which hydrogen ions
could be bound. Such calcium ion "exchange" phenomena are well known in soil
titration systems (Brady, 1974) and similar
phenomena may be occurring here. In any
case, further, more refined, in vivo observations seem in order.
In light of new data presented here, it is
perhaps even less surprising than previously realized that Masticophis possesses far
greater stamina than Crotalus. As mentioned earlier, previous workers have generally interpreted hypercalcemia and
depression of blood pH as factors interfering with exercise support systems in vertebrates. Consequently, these parameters
may well have resulted in chronic selection
in Masticophis, as well as other active foragers, for changes in the structures of mineralized tissues that reduce activity-related
hypercalcemia to facilitate maintenance of
blood buffering systems as well as to avoid
direct effects of sudden systemic elevations
of calcium.
These studies present further evidence
that the skeleton in snakes is not the almost
inert, weight-supporting apparatus that it
has frequently been perceived to be.
Clearly, the skeleton is a dynamic, fast
responding organ system that seems closely
associated, for better or worse, with physiological functions associated with exercise.
ACKNOWLEDGMENT
This research was supported by NSF
grant No. DEB 78-10837 toJ.A.R.
381
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