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BIOCHEMISTRY

MOLECULAR BIOLOGY EDUCATION

Vol. 34, No. 2, pp. 125128, 2006

AND

Laboratory Exercises
Energetic Metabolism and Biochemical Adaptation
A BIRD FLIGHT MUSCLE MODEL*
Received for publication, November 2, 2004, and in revised form, December 2, 2005
Pierre Rioux and Pierre U. Blier
From the Laboratoire de biologie evolutive, and Departement de Biologie, Universite du Quebec a` Rimouski,
allee des Ursulines, Rimouski Qc Canada G5L 3A1

The main objective of this class experiment is to measure the activity of two metabolic enzymes in crude
extract from bird pectoral muscle and to relate the differences to their mode of locomotion and ecology.
The laboratory is adapted to stimulate the interest of wildlife management students to biochemistry. The
enzymatic activities of cytochrome c oxidase and lactate dehydrogenase are measured in pectoral muscle
of black duck and ring-necked pheasant. The black ducks have a high cytochrome c oxidase/lactate dehydrogenase (LDH) ratio, which reflects high aerobic capacity required for sustained and long distance flight. The
low cytochrome c oxidase/LDH ratio in ring-necked pheasants and high level of LDH activity suggest that this
bird can only support short bursts of flight, which may be related to his strategy of predator avoidance.
Keywords: Metabolic enzymes, cytochrome c oxidase, lactate dehydrogenase, bird pectoral muscle.

This experiment is currently presented to second year

students in our biology program as a part of the Energetic
Metabolism class. It has been devised especially for students oriented in wildlife management and/or environmental sciences [1, 2].
In a previous experiment [2], we studied some metabolic
responses of aquatic organisms to environmental constraints. The compensation of enzymatic activities of goldfish muscle to low temperature acclimations was examined. This introduced the students to the concept of
metabolic acclimation and acclimatization. The present
experiment involves metabolic adaptation by comparing
the muscle of two bird species differing in their ecology
and habitat exploitation strategies. The students should
then relate the metabolic organization of the principal muscle involved in power generation that generates lift to the
ecology of the organism. Our goal is to help students to
realize that some answers to ecological or behavioral challenge can be metabolic and that understanding the adaptive metabolic toolbox of vertebrates or animals can be of
great relevance even for ecologists. We estimated the
aerobic and anaerobic muscle capacity in two species of
birds: one adapted to long lasting flights and undertaking
seasonal migrations (the black duck, Anas rubripes Brewster L.) and one displaying short and rapid flight activities
(the ring-necked pheasant, Phasianus colchicus Linnaeus).
In birds, the large pectoral muscle controls the lowering
* Financial support for this work was provided by the Fonds de
developpement pedagogique from the Universite du Quebec a`
Rimouski.
To whom correspondence may be addressed. Fax: 418-7241849; E-mail: pierre_rioux@uqar.qc.ca.
To whom correspondence may be addressed. Fax: 418-7241849; E-mail: pierre_blier@uqar.qc.ca.
This paper is available on line at http://www.bambed.org

of the wing muscles. This muscle allows sustained or burst

flight. The biochemical organization of muscle fibers presets the capacity of the type of flight involved. Sustained
flight requires mobilization of the oxidative pathway [3] and
high proportion of red fibers in the muscle tissue. For
example, the pectoral muscle of the Peking duckling contains 84.3% red fibers [4]. On the other hand, in species
that have lost the ability to fly, such as chicken, the pectoral muscle can contain up to 96% [5] and 100% [4] white
fibers.
The black duck is a bird that can weigh up to 2 kg and
is generally found in shallow water areas (salt marshes,
lakes, etc.). These ducks that live in northeastern America
migrate to the south during the fall as foraging grounds
become unavailable and cold. The reverse northbound
migration begins in February. Two-thirds of black ducks
utilize corridors extending through the Atlantic flyway. The
single most important corridor extends along the Atlantic
coast from the Maritime Provinces to Florida. In the province of Quebec (Canada), this bird species is one of the
most valuable to hunters [6 8].
The ring-necked pheasant is a phasianidea resembling a
chicken. Originally from Asia, this species was introduced
to North America at the end of the 19th century. It is a
non-migratory species with fast and short term flights. This
species is used as game in sport hunting and is also raised
commercially, which facilitates procurement [9, 10].
The activities of two enzymes are measured in the pectoral muscle; lactate dehydrogenase (LDH)1 and cytochrome c oxidase (CCO) are associated with energy metabolism. LDH is an indicator of anaerobic glycolysis
1
The abbreviations used are: LDH, lactate dehydrogenase;
CCO, cytochrome c oxidase.

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BAMBED, Vol. 34, No. 2, pp. 125128, 2006

potential. In the last step of glycolysis, this enzyme catalyze the reduction of pyruvate to lactate:
Pyruvate NADH H 7 Lactate NAD
This reaction is the only strictly anaerobic reaction of
glycolysis in vertebrates, and its activity has been linked
previously to power requirements of tissues. For example,
in human muscle (gastroctemius), the anaerobic use of
glycolysis can lead to the production of up to 60 mol of
ATP per minute through the production of lactate, whereas
oxidation of pyruvate can support only half of the ATP
production [11].
The assay of this enzyme is quite simple and straightforward because only two substrates are needed and the
catalyzed oxidation of one of the substrate can be directly
measured with spectrophotometer (NADH has an absorption peak at 340 nm). Furthermore, most vertebrates tissues contain high activity in LDH, which limits the significance of a control of noise.
The reaction catalyzed by cytochrome c oxidase include
the vectoral proton translocation across the inner membrane and reduction of molecular oxygen [12] is as follows:
4 cyt c2 8 H inside
02 3 4 cyt c3 2 H20 4 H outside
The evaluation of electron transfer relied to the activity of
this enzyme may be measured by the rate of change in
oxygen or reduced cytochrome c concentration. This rate
of electron transfer from cytochrome c to O2 can therefore
be followed up by two different technical approaches. The
reduced cytochrome c has an absorption peak at 550 nm.
The oxidation rate of reduced cytochrome c can be followed spectrophotometrically when cytochrome c concentration does not exceed 100 M. The rate of reduction
of molecular oxygen also can be followed in closed cell with
a Clark type electrode connected to an oxymeter. Even
though this method is more precise, it is significantly longer
and requires higher aptitude from experimenter (for example, the system has to be calibrated at every utilization).
The experiments objective is to measure the maximal
activities of two metabolic enzymes, LDH and cytochrome
c oxidase, in bird pectoral muscle and to relate the differences to their specific mode of locomotion and ecology.
The ring-necked pheasant and black duck were chosen
because of their availability in the province of Quebec,
Canada, and their different flight adaptations.
EXPERIMENTAL PROTOCOL

The equipment required for laboratory experiment are: a

visible spectrophotometer capable to measure at the
wavelength of 340 and 550 nm, a homogenizer (for example Tekmar or Heidolph models), a centrifuge, and automatic micropipettes.
Stock SolutionsHomogenization buffer: 100 mM phosphate buffer adjusted to pH 7.4. 50 M cytochrome c
(purchased from Sigma, St. Louis, MO) in 100 mM phosphate buffer. 0.16 mM NADH reaction medium and 6.0 mM
pyruvate (in 100 mM potassium phosphate buffer, pH 7.4).
NADH and pyruvate were purchased from Sigma. Bradford reagent (Bio-Rad protein assay). Albumin from bovine
serum albumin 1000 g ml1.

AnimalsBirds (ring-necked pheasant and black duck)

were hunted in October. The pectoral muscle (Pectoralis
major) was immediately dissected and placed in liquid
nitrogen. Samples were then transferred to 70 C until
analyses.
Tissue ExtractionTissue was finely cut and homogenized with 9 volumes of ice-cold 100 mM phosphate buffer,
pH:7.4, using a Tekmar homogenizer (Cincinnati, OH).
The crude homogenate was centrifuged at 400 g for 10
min at 4 C; the resultant supernatant was extracted and
kept on ice.
Dissection of a sample from the deep portion of the
muscle is recommended because there is proportionally
more red fibers in deep muscle tissue. A corresponding
difference in enzyme activities could be observed [5]. We
have sampled the tissue at least 0.5 cm under the surface
of the muscle.
Cytochrome c Oxidase AssayThe cytochrome c solution contained 100 mM potassium phosphate buffer and 50
M reduced cytochrome c, pH 8. Reactions were run
against a 50 M cytochrome c solution oxidized with
0.033% (w/v) potassium ferricyanide. The cytochrome c
reduction was carried out by the addition of sodium hydrosulfite. The excess hydrosulfite was removed by gently
bubbling with air for 10 min [13].
1 ml of reduced cytochrome c solution was pipetted to
a 1.5-ml spectrometric cell (1-cm light path). The reaction
was started by the addition of the enzyme (10 l of supernatant) to the reaction mixture (The supernatant may occasionally be diluted 1:2.) The reaction medium and enzyme extract were mixed thoroughly and immediately
placed in the spectrophotometer. The absorbance was
recorded at 20-s intervals for 23 min at 550 nm. Enzyme
activities were measured at 20 C with a UV/vis spectrophotometer (Lambda 11, PerkinElmer Life Sciences). Before conducting this experiment with students, we recommend that instructors verify the results with the
spectrophotometers used in class. Spectrophometers with
low optical quality (large beam) may produce unstable
results.
LDH Assay1 ml of 0.16 mM NADH reaction medium
and 6.0 mM pyruvate (in 100 mM potassium phosphate
buffer, pH 7.4) was pipetted into a spectrophotometric cell
(1-cm light path). The reaction was initiated by adding the
enzyme (10 l of supernatant) to the reaction medium. For
the LDH assay, the enzyme was usually diluted (1:20 to
1:50). The reaction medium and enzyme extract were thoroughly mixed and immediately placed in the spectrophotometer (Spectronic 20). The activity was measured by
following NADH absorbance at 340 nm at 20-s intervals for
23 min. Enzyme activities were measured in triplicate at
20 C. All chemicals for enzymatic assays were obtained
from Sigma.
Protein DeterminationProtein content was measured
by the Bradford method [14]. A standard curve (0 1000 g
ml1) was prepared with bovine serum albumin. 0.1 ml of
each standard and sample was mixed with 5 ml of dye
reagent and incubated for at least 5 min. The absorbance
was measured at 595 nm.
Enzymatic ActivitiesThe activity of the original undiluted enzyme is expressed in international units (IU) per g

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TABLE I
LDH and CCO activities in the pectoral muscle of
A. rubripes and P. colchicus

a
b

Species

CC0a

CCOb

LDHa

LDHb

CC0/LDH

A. rubripes
P. colchicus

11.29
6.50

0.075
0.042

233.27
2,298.47

1.56
14.85

0.0480
0.0028

IU g1 tissue.
IU mg1 protein.

of tissue and mg of proteins. One unit corresponds to the

catalysis of one mol of substrate per minute.
Enzymatic activities were determined by:
V A/t (min)X EM
when A/t is the change of optical unit per minute and
EM is the molar extinction coefficient.
Note: For reduced cytochrome c f EM 29.5 103
liters mol1 cm1
For NADH f EM 6.2 103 liters mol1 cm1 [15]
Enzymatic activities should be expressed per g of tissue
or per mg of proteins. Therefore students should consider
the total dilution of the muscle homogenate that they used
for the assays as well as the protein concentration in the
homogenate.
Before they assays, the student have to demonstrate
that the assay conditions of the protocol allow activities
measurements that reflect the enzyme content of the tissue (which means conditions that allow to reach Vmax). To
do so they have to measure net activities of different
dilution of the homogenate and determine whether the
activities are directly proportional to the dilution level.

capacity for a short burst of activity for fast takeoff supported by rapid wing beats [16, 17]. The results were
similar to the results seen with pheasant.
This experiment allows one to relate bird ecology to
muscle physiology and energy metabolism. The series of
questions that we suggest educators provide to students
will increase the students comprehension of the evolutionary plasticity of muscle metabolism. For example they
will realize that one strategy to enhance long time flight
capacity could be the selection of energetically efficient
substrates (lipids instead of carbohydrates). They will also
have to suggest mechanism in terms of developmental
regulation that could partly explain the huge range of aerobic capacity of muscle in vertebrates. Finally the questions introduce students to the general concept of
adaptation.
QUESTIONS
1.
2.
3.
4.
5.
6.

Describe the energetic metabolism responses to migration in

birds (fat, protein, and carbohydrate metabolism) [18 31].
Describe the particularities of gluconeogenesis in birds [32].
Compare and contrast the metabolic particularities of bat and
bird pectoral muscle [33].
Explain the differences between acclimation, acclimatization,
and adaptation [11].
Describe in detail the heat balance and thermoregulation in
response to flight and running [18]
Suggest mechanisms of regulation of mitochondrial content
that could explain the wide diversity of metabolic organization
of muscle among vertebrates [34].

AcknowledgmentsWe thank Yves Lemay for the samples.

We also thank Alexander Strachan and Nathalie Lefrancois for
critically reviewing the manuscript.

RESULTS AND DISCUSSION

REFERENCES

The values of LDH and CCO enzymatic activities are

shown in Table I. The enzymatic activities of the pectoral
muscle vary greatly between the two bird species. The
CCO/LDH ratio is higher for the black duck than for the
ring-necked pheasant. The CCO enzyme may be an indicator of the volume and/or the quantity of mitochondria in
a tissue. Thus, the black ducks have metabolic qualifications for sustained and long distance flight, as their pectoral muscles possess high aerobic capacity and metabolize fatty acid oxidation at high rates. The low CCO/LDH
ratio in ring-necked pheasant pectoral muscle and the high
level of LDH activity suggests that they can only support
short bursts of flight [5].
Our university offers a wildlife management program; we
chose these species because of their importance in this
field in our locality. Species choice may however vary
according to the ease of procurement. As such, we may
compare all species that possess highly aerobic pectoral
muscles (e.g. duck, geese) with any species that mostly
rely on high glycolytic capacity (e.g. chicken, turkey). Certain domestic species can therefore be used and may be
easier to obtain.
In the past, we also conducted this experiment with
ruffed grouse, which is a tetraonidae resembling a chicken;
adults can reach 500 g. This species is primarily found in
the bushy woodlands of North America and is a nonmigratory bird adapted to the harsh winter conditions of
our latitudes. Once established, the ruffed grouse inhabits
a relatively small territory (a few acres) and exhibits high

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