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Keywords:

  • Metabolic enzymes;
  • cytochrome c oxidase;
  • lactate dehydrogenase;
  • bird pectoral muscle

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. REFERENCES

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.

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 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 Québec (Canada), this bird species is one of the most valuable to hunters [68].

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)11 and cytochrome c oxidase (CCO) are associated with energy metabolism. LDH is an indicator of anaerobic glycolysis potential. In the last step of glycolysis, this enzyme catalyze the reduction of pyruvate to lactate:

  • equation image(1)

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 μmol 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:

  • equation image(2)

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 μM. 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 experiment's 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 Québec, Canada, and their different flight adaptations.

EXPERIMENTAL PROTOCOL

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. REFERENCES

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 Solutions—

Homogenization buffer: 100 mM phosphate buffer adjusted to pH 7.4. 50 μM 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 μg ml−1.

Animals—

Birds (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 Extraction—

Tissue 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 Assay—

The cytochrome c solution contained 100 mM potassium phosphate buffer and 50 μM reduced cytochrome c, pH 8. Reactions were run against a 50 μM 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 μl 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 2–3 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 Assay—

1 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 μl 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 2–3 min. Enzyme activities were measured in triplicate at 20 °C. All chemicals for enzymatic assays were obtained from Sigma.

Protein Determination—

Protein content was measured by the Bradford method [14]. A standard curve (0–1000 μg ml−1) 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 Activities—

The activity of the original undiluted enzyme is expressed in international units (IU) per g of tissue and mg of proteins. One unit corresponds to the catalysis of one μmol of substrate per minute.

Enzymatic activities were determined by:

  • equation image(3)

is the change of optical unit per minute and EM is the molar extinction coefficient.

Note: For reduced cytochrome c [RIGHTWARDS DOUBLE ARROW] EM = 29.5 × 103 liters mol−1 cm−1

For NADH [RIGHTWARDS DOUBLE ARROW] EM = 6.2 × 103 liters mol−1 cm−1 [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.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. 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 non-migratory 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 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. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. REFERENCES
  • Describe the energetic metabolism responses to migration in birds (fat, protein, and carbohydrate metabolism) [1831].

  • 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].

Table Table I. LDH and CCO activities in the pectoral muscle of A. rubripes and P. colchicus
SpeciesCC0aCCObLDHaLDHbCC0/LDH
  • a

    a IU g−1 tissue.

  • b

    b IU mg−1 protein.

A. rubripes11.290.075233.271.560.0480
P. colchicus6.500.0422,298.4714.850.0028

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. REFERENCES

We thank Yves Lemay for the samples. We also thank Alexander Strachan and Nathalie Lefrançois for critically reviewing the manuscript.

  • 1

    The abbreviations used are: LDH, lactate dehydrogenase; CCO, cytochrome c oxidase.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROTOCOL
  4. RESULTS AND DISCUSSION
  5. QUESTIONS
  6. Acknowledgements
  7. REFERENCES
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