Citation
Fatigue in skeletal muscle

Material Information

Title:
Fatigue in skeletal muscle
Creator:
MacIntosh, Brian Robert, 1952-
Publication Date:
Language:
English
Physical Description:
x, 106 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Acidosis ( jstor )
Action potentials ( jstor )
Calcium ( jstor )
Dogs ( jstor )
Fatigue ( jstor )
Muscle contraction ( jstor )
Muscles ( jstor )
Nerves ( jstor )
pH ( jstor )
Skeletal muscle ( jstor )
Dissertations, Academic -- Physiology -- UF ( mesh )
Fatigue ( mesh )
Muscles ( mesh )
Physiology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 100-105.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Brian Robert MacIntosh.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
023787756 ( ALEPH )
20298213 ( OCLC )
AEK9518 ( NOTIS )

Downloads

This item has the following downloads:

EHCQKHKWY_GSY6ZJ.xml

fatigueinskeleta00maci.pdf

fatigueinskeleta00maci_0029.txt

fatigueinskeleta00maci_0007.txt

fatigueinskeleta00maci_0052.txt

fatigueinskeleta00maci_0032.txt

fatigueinskeleta00maci_0081.txt

fatigueinskeleta00maci_0093.txt

fatigueinskeleta00maci_0013.txt

fatigueinskeleta00maci_0020.txt

fatigueinskeleta00maci_0043.txt

fatigueinskeleta00maci_0110.txt

fatigueinskeleta00maci_0047.txt

fatigueinskeleta00maci_0084.txt

fatigueinskeleta00maci_0060.txt

fatigueinskeleta00maci_0005.txt

fatigueinskeleta00maci_0068.txt

fatigueinskeleta00maci_0067.txt

fatigueinskeleta00maci_0111.txt

fatigueinskeleta00maci_0012.txt

fatigueinskeleta00maci_0004.txt

fatigueinskeleta00maci_0079.txt

AA00009126_00001.pdf

fatigueinskeleta00maci_0017.txt

fatigueinskeleta00maci_0010.txt

fatigueinskeleta00maci_0080.txt

fatigueinskeleta00maci_0082.txt

fatigueinskeleta00maci_0031.txt

fatigueinskeleta00maci_0042.txt

fatigueinskeleta00maci_0059.txt

fatigueinskeleta00maci_0109.txt

fatigueinskeleta00maci_0019.txt

fatigueinskeleta00maci_0056.txt

fatigueinskeleta00maci_0076.txt

EHCQKHKWY_GSY6ZJ_xml.txt

fatigueinskeleta00maci_0083.txt

fatigueinskeleta00maci_0014.txt

fatigueinskeleta00maci_0025.txt

fatigueinskeleta00maci_0099.txt

fatigueinskeleta00maci_0090.txt

fatigueinskeleta00maci_0113.txt

fatigueinskeleta00maci_0000.txt

fatigueinskeleta00maci_0021.txt

fatigueinskeleta00maci_0026.txt

fatigueinskeleta00maci_0057.txt

fatigueinskeleta00maci_0094.txt

fatigueinskeleta00maci_0072.txt

fatigueinskeleta00maci_0003.txt

fatigueinskeleta00maci_0035.txt

fatigueinskeleta00maci_0001.txt

fatigueinskeleta00maci_0030.txt

fatigueinskeleta00maci_0041.txt

fatigueinskeleta00maci_0096.txt

fatigueinskeleta00maci_0015.txt

fatigueinskeleta00maci_0074.txt

fatigueinskeleta00maci_0091.txt

fatigueinskeleta00maci_0114.txt

fatigueinskeleta00maci_0055.txt

fatigueinskeleta00maci_0040.txt

fatigueinskeleta00maci_0033.txt

fatigueinskeleta00maci_0089.txt

fatigueinskeleta00maci_0037.txt

fatigueinskeleta00maci_0100.txt

fatigueinskeleta00maci_0117.txt

fatigueinskeleta00maci_0054.txt

fatigueinskeleta00maci_0011.txt

fatigueinskeleta00maci_0118.txt

fatigueinskeleta00maci_0063.txt

fatigueinskeleta00maci_0086.txt

fatigueinskeleta00maci_0023.txt

fatigueinskeleta00maci_0065.txt

fatigueinskeleta00maci_0053.txt

fatigueinskeleta00maci_0002.txt

fatigueinskeleta00maci_0049.txt

fatigueinskeleta00maci_0058.txt

fatigueinskeleta00maci_0064.txt

fatigueinskeleta00maci_0016.txt

fatigueinskeleta00maci_0073.txt

fatigueinskeleta00maci_0036.txt

fatigueinskeleta00maci_0069.txt

fatigueinskeleta00maci_0078.txt

fatigueinskeleta00maci_0050.txt

fatigueinskeleta00maci_0103.txt

fatigueinskeleta00maci_0107.txt

fatigueinskeleta00maci_0044.txt

AA00009126_00001_pdf.txt

fatigueinskeleta00maci_0061.txt

fatigueinskeleta00maci_0095.txt

fatigueinskeleta00maci_0087.txt

fatigueinskeleta00maci_0088.txt

fatigueinskeleta00maci_pdf.txt

fatigueinskeleta00maci_0119.txt

fatigueinskeleta00maci_0027.txt

fatigueinskeleta00maci_0085.txt

fatigueinskeleta00maci_0106.txt

fatigueinskeleta00maci_0048.txt

fatigueinskeleta00maci_0046.txt

fatigueinskeleta00maci_0104.txt

fatigueinskeleta00maci_0097.txt

fatigueinskeleta00maci_0066.txt

fatigueinskeleta00maci_0051.txt

fatigueinskeleta00maci_0115.txt

fatigueinskeleta00maci_0116.txt

fatigueinskeleta00maci_0028.txt

fatigueinskeleta00maci_0070.txt

fatigueinskeleta00maci_0008.txt

fatigueinskeleta00maci_0018.txt

fatigueinskeleta00maci_0092.txt

fatigueinskeleta00maci_0112.txt

fatigueinskeleta00maci_0098.txt

fatigueinskeleta00maci_0108.txt

fatigueinskeleta00maci_0071.txt

fatigueinskeleta00maci_0022.txt

fatigueinskeleta00maci_0006.txt

fatigueinskeleta00maci_0075.txt

fatigueinskeleta00maci_0045.txt

fatigueinskeleta00maci_0039.txt

fatigueinskeleta00maci_0009.txt

fatigueinskeleta00maci_0101.txt

fatigueinskeleta00maci_0105.txt

fatigueinskeleta00maci_0077.txt

fatigueinskeleta00maci_0062.txt

fatigueinskeleta00maci_0102.txt

fatigueinskeleta00maci_0034.txt

fatigueinskeleta00maci_0024.txt

fatigueinskeleta00maci_0038.txt


Full Text

















FATIGUE IN SKELETAL MUSCLE


BY


BRIAN ROBERT MACINTOSH
















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1979




FATIGUE IN SKELETAL MUSCLE
BY
BRIAN ROBERT MACINTOSH
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1979


ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to
Dr. W. N. Stainsby, Chairman of my Supervisory Committee,
for his valuable assistance and counsel over the past
four years. Acknowledgement is also due the other
members of my Committee: Dr. M. Fried, Dr. A. B. Otis,
Dr. P. Posner and Dr. C. W. Zauner. Each has unselfishly
contributed time and effort to provide me with the
guidance I needed to complete the requirements for this
degree.
Special thanks are expressed to Donna T. Dolbier, who
provided technical assistance and to Dr. L. Bruce Gladden
who collaborated with me on several research projects
during his Post-doctoral tenure with Dr. Stainsby.
Financial support for me during the pursuit of the
Ph.D degree has been provided by the following agencies
and departments:
NIH, grants to Drs. Stainsby, Otis and Cassin;
Department of Physiology (Teaching Assistantship);
College of Nursing (Teaching Assistantship).
The research reported in this dissertation has been
supported by The American Heart Association, Florida
Affiliate Grant # AG 7 and Sponsored Research Seed Grant
awarded to Dr. Stainsby.
I would like to thank Wendy Auerbach for doing an
excellent job of typing this manuscript.
li


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF FIGURES V
LIST OF TABLES vii
ABSTRACT viii
INTRODUCTION 1
EVENTS LEADING TO SKELETAL MUSCLE CONTRACTION .... 5
POSSIBLE FATIGUE MECHANISMS AND CURRENT THEORIES
OF FATIGUE 9
Central Nervous System 9
Neuromuscular Junction Failure 10
Attenuated Calcium Release 11
Reduced Capacity of the Contractile Apparatus . 13
GENERAL METHODS 16
02 UPTAKE AND DEVELOPED TENSION 22
Introduction 22
Methods 23
Results 26
Discussion 34
EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE 41
Introduction 41
Methods 4 2
Results 48
Discussion 59
iii


Page
EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH
CONTRACTION 6 8
Introduction 68
Methods 69
Results 70
Discussion 79
SUMMARY 89
O2 Uptake and Developed Tension 89
Time-Course of the Twitch Contraction in
Fatigue 90
Acidosis and the Twitch Contraction 91
PROPOSED HYPOTHESES FOR SKELETAL MUSCLE FATIGUE . 92
Depletion of Calcium at Lateral Sacs 92
Compartmentalization of Calcium Within the
Lateral Sacs 93
Attenuated Trigger for Release of Calcium ... 93
Reduced Binding Sensitivity for Calcium .... 94
CONCLUSIONS 9 5
APPENDIX 9 6
BIBLIOGRAPHY 100
BIOGRAPHICAL SKETCH 106
IV


LIST OF FIGURES
Page
1. Excitation-Contraction-Coupling 7
2. Gastrocnemius-Plantaris Muscle Preparation . 18
3. Sample Experiments; VOg versus Developed
Tension 28
4. VOg versus Developed Tension; all Data,
Normalized 29
5. Twitch and Twin Contractions: Developed
Tension and dP/dt 39
6. Twitch Characteristics from Fast Traces of
Developed Tension and dP/dt 43
7. Procedure for Fatiguing Contractions with
Periodic Fast Traces 46
8. Q, POg, PCOg and pH During and Following
Fatiguing Contractions 50
9. Developed Tension, Half Relaxation Time and
Contraction Time 53
10. Peak Rate of Force Development and Peak Rate
of Relaxation 57
11. Tetanic Contraction Before and After Fatiguing
Contractions 58
12. Twitch Developed Tension and dP/dt Before,
During and After Ischemic Fatigue 60
13. Half Relaxation Time versus Developed Tension
for Dantrolene, Reduced Stimulation Voltage
and Ischemic Fatigue 61
14. The Effects of Preceding Contractions on
Developed Tension 63
15. Procedure for Ventilation and Sampling Pattern. 72
v


Page
16. Arterial and Venous [H+] 76
17. PC>2 During Different Ventilatory States .... 78
18. Developed Tension, dP/dt and -dP/dt During
Different Ventilatory States 81
19. Contraction Time and Half Relaxation Time
During Different Ventilatory States 83
vi


LIST OF TABLES
Page
I 02 UPTAKE AND DEVELOPED TENSION BEFORE AND
AFTER FATIGUE IN SERIES 1 30
II RELATIONSHIP BETWEEN 02 UPTAKE AND
DEVELOPED TENSION IN SERIES 2-5 32
III PHOSPHORYLCREATINE ANALYSIS DURING
CONTRACTIONS AND FOLLOWING THE RECOVERY
PERIOD 55
IV STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP 74
V STATISTICS FOR TWITCH CHARACTERISTICS
VERSUS BLOOD GASES 84
Vll


Abstract of Dissertation Presented to the Graduate
Council of the University of Florida
In Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
FATIGUE IN SKELETAL MUSCLE
By
Brian Robert Macintosh
June 1979
Chairman: Wendell N. Stainsby, D.Sc.
Major Department: Physiology
The in situ dog gastrocnemius-plantaris muscle
preparation has been used to study fatigue. Skeletal
muscle fatigue (reduced force output for a given stimulus)
results from a thirty minute period of isometric
contractions at 2.5 to 20/sec. This fatigue is not a
result of failure of motor nerve propagation or transmitter
release. The ratio of oxygen uptake to developed tension
(total tension minus resting tension) is unaltered during
or following fatiguing contractions. The economy of force
production is unaltered by twin impulse stimulation,
relative ischemia or administration of moderate doses of
curare or succinylcholine.
vii i


When developed tension is reduced due to repetitive
stimulation for thirty minutes at 2.5, 5 or 10/sec
contractions, the time to peak tension and half relaxation
times are unaltered. The peak rates of force development
and of relaxation are reduced proportionally to the
reduction in developed tension.
Following a forty minute period of recovery, the
twitch developed tension remains greatly attenuated, but
tetanic (200 msec of 100/sec stimulation) developed tension
is virtually the same as it was before the stimulation
period. Phosphorylcreatine is restored to resting levels
within forty minutes of recovery. Also, at this time,
blood flow and oxygen uptake have returned to pre-fatigue
values and venous PC>2, PCC>2 and pH are at resting levels.
The fatigue observed in these experiments appears to
be due to a reduction in the intensity of activation
obtained with a single impulse. Energy sources are
available and with maximal activation the contractile
mechanism is capable of the same force output it had
before the fatiguing contractions.
Further experiments were conducted to determine if
intracellular acidosis could have been the cause of the
reduced intensity of activation. A sixty to ninety
minute period of hypoventilation with an air mixture high
in O2 (arterial PO2 was maintained at 75-100 mm Hg) resulted
in a reduction in arterial pH to 7.08. There was no
reduction in twitch developed tension associated with this
IX


acidosis. It is likely that intracellular pH fell as
much during the respiratory acidosis as it did during
fatiguing contractions at 10/sec. The fatigue observed
during contractions at 10/sec could not be a result of
intracellular acidosis.
It can be concluded from these experiments that
twitch fatigue is not a result of energy deficiency,
reduced capacity of the contractile elements, intra
cellular acidosis (induced by reduced ventilation for
sixty to ninety minutes) or neuromuscular junction
failure. By the process of elimination it appears
that twitch fatigue results from a reduced activation
of the myofilaments during a twitch contraction. This
may be due to either a reduced sarcoplasmic Ca^+
concentration during contraction or a reduced response
of the myofilaments at a given Ca^+ concentration.
x


INTRODUCTION
The word fatigue has been used in the past with
several various definitions. Some authors (1, 52) equate
fatigue with exhaustion. Others (47, 50) use the word
fatigue to represent an inability to maintain a particular
work output. More recently (23, 30) skeletal muscle
fatigue has been defined as a reduced capacity of the
muscle to develop tension. Edwards et al. (23) and Fitts
and Holloszy (30) have observed that a twitch contraction
can still be attenuated when the force generating capacity
of the muscle is fully recovered.
A single impulse does not maximally activate the
contractile apparatus (17) and therefore does not permit
the full contractile response of which the muscle is
capable. The capacity to develop tension must be
evaluated under conditions of maximal activation. This
can be accomplished with a caffein or K+ induced
contracture or a maximal tetanic contraction.
If fatigue is defined as a reduced capacity of the
muscle to generate force, then what is it called if there
is an attenuated response to a single impulse? It appears
to be a separate phenomenon and probably occurs by a
separate mechanism (since a muscle recovers full capacity
to develop tension before twitch tension recovers to
pre-fatigue values). Edwards et al. point out (23) that
1


2
this "twitch fatigue" may be associated with perception
of increased effort necessary to maintain a given workload
or force output- For the purposes of this dissertation,
fatigue will be used as a generalization referring to a
reduced response of the muscle to a given stimulation.
Twitch fatigue will refer to an attenuated response to a
single impulse.
Wilson and Stainsby (66) have reported that twitch
developed tension of the in situ dog gastrocnemius-
plantaris muscle remains attenuated for hours following
a series of contractions at 10-14 per second. Fitts and
Holloszy (30) have reported that tetanic developed tension
recovers quickly following a period of repetitive
stimulation. This may also be the case for the in situ
dog gastrocnemius-plantaris muscle. If this is so, then
the fatigue observed by Wilson and Stainsby is only twitch
fatigue. This preparation, then, would provide a model
for studying twitch fatigue independent of tetanic fatigue.
Very little is known of the fatigability of the canine
gastrocnemius-plantaris muscle or of the mechanisms
responsible for that fatigue. The purpose of the present
study was to provide further information regarding the
fatiguing effects of repetitive stimulation on the
gastrocnemius-plantaris muscle.
To facilitate the reader's understanding of skeletal
muscle fatigue, a brief description of pertinent muscle
physiology and current theories of fatigue precedes the
sections describing the experiments which have been done.


3
The dog in situ gastrocnemius-plantaris muscle group
has been used throughout the series of studies reported
herein. To avoid repetition, the general procedures and
description of the preparation appear as a separate
chapter before any of the studies. The specific
procedures used in each study are described separately
in a Methods section for that study.
In the first study, the relationship between oxygen
uptake and developed tension has been determined for
skeletal muscle before, during and after fatiguing
contractions. These experiments were done to determine
whether or not the energy used by a muscle relative to the
amount of tension developed is altered by fatigue. There
are reports that indicate a change in either direction can
be expected (7, 21, 28).
Further studies were conducted to determine whether
or not changes occur in the time-course of the twitch as
a result of repetitive stimulation. Changes in the rate
of force development and in the time-course of a twitch
contraction have previously been interpreted as indications
of changes in the duration and intensity of activation of
the muscle (17, 36). These measurements may facilitate
an understanding of the mechanism(s) responsible for the
fatigue.
A third series of experiments has been conducted to
study the effects of respiratory acidosis on the twitch
contraction. Acidosis has been claimed to be one of


4
the major causes of fatigue (29). If this is the case,
respiratory acidosis should reduce the developed tension
of a twitch contraction.
In the final chapter of this dissertation, a brief
discussion of the possible theories for the mechanism of
fatigue observed in these experiments is presented. It
is evident that further research will be necessary to
permit evaluation of these theories with respect to
fatigue of the canine gastrocnemius-plantaris muscle
group. However, considerable evidence has accumulated
as a result of my studies, which disputes several of the
current theories of fatigue.


EVENTS LEADING TO
SKELETAL MUSCLE CONTRACTION
Muscular contraction is the result of a sequence of
chemical and physical events beginning with activity in
the central nervous system (CNS)(or sensory input to the
CNS). Failure or impairment at any site in this process
will result in a reduced contractile response of the
muscle. Fatigue and twitch fatigue, then, are results
of such failure. The identification of the site(s) of
failure in fatigue would provide a better understanding
of the mechanism(s) effecting the fatigue. Below, a
brief discussion of the normal sequence of events leading
to contraction is presented. CNS control of motor nerve
activity is complex and will not be described. For
simplicity, this discussion is based at the cellular level.
This sequence of events is described in a number of text
books (45, 62) and is illustrated in Figure 1. Following
the presentation of events leading to contraction, each
step in the sequence is considered as a potential site
for a mechanism of fatigue.
The sequence of events occurring at the nerve
terminal may be susceptible to failure. The arrival of
an action potential at the nerve terminal triggers the
release of acetylcholine from the terminal bouton.
Synaptic vesicles fuse to the terminal membrane and
5


6
release their contents into the synaptic cleft.
Acetylcholine diffuses the short distance across the
cleft (500 A).
Binding of acetylcholine to specific receptors
causes a transient increase in permeability of the muscle
membrane to Na+ and K+. This results in depolarization
of the end plate. The resulting change in membrane
potential is called the end-plate potential. Destruction
of the acetylcholine is accomplished by acetylcholines
terase which is located among the receptors on the post-
synaptic muscle membrane. Reconstitution of synaptic
vesicles is accomplished by reuptake of choline and
subsequent acetylation in the Golgi apparatus (enzyme:
choline-acetyl-transferase). Portions of the Golgi
complex, containing acetylcholine, are pinched off,
forming new synaptic vesicles.
A single action potential on a motor neuron usually
generates an end plate potential large enough to bring
the adjacent membrane area to threshold. Propagation of
an action potential over the membrane ensues. Transverse
tubules, located at regular intervals along the length of
the muscle fiber permit rapid communication with deep
portions of the muscle. Depolarization of transverse
tubules triggers release of calcium from the lateral sacs.
Lateral sacs are the terminal portions of the sarcoplasmic
reticulum lying adjacent to the transverse tubules. The
Ca^+ released from the lateral sacs raises the sarcoplasmic


7
FIGURE
The sequence of events occurring in
excitation-contraction coupling are
listed below. The numbers refer to
numbered events shown in the diaqram
above.
1. An action potential travels along a
motor nerve.
2. Acetylcholine which has been released
from the nerve terminal binds to
receptors on the muscle membrane,
causing depolarization an end-plate
potential.
3. When the end-plate potential reaches
a threshold value an action potential
is fired. This action potential is
propagated over the entire muscle
membrane, and causes depolarization
of the transverse tubules.
4. Depolarization of the transverse tubules
triqgers release of Ca2 + from the lateral sacs.
5. Ca2 + which has been released, binds to troponin
which is associated with the thin myofilaments.
Contraction results.
Ca2+ is reaccumulated by an active transport
mechanism located in the longitudinal
tubules. Relaxation occurs.
6.


8
free Ca2+ concentration. The subsequent binding of Ca2+
to troponin results in activation of the contractile
proteins in the muscle. The amount of Ca2+ released in
response to one action potential propagated over the
muscle membrane is not sufficient to saturate the troponin
molecules and therefore, incomplete activation occurs (17).
For complete activation and therefore maximal force
production, a period of nerve activity at a high frequency
is necessary. Relaxation occurs as Ca2+ is sequestered
(active transport) by the longitudinal sarcoplasmic
reticulum. Following reuptake, calcium is translocated
along the longitudinal reticulum to the lateral sacs,
completing the Ca2+ cycle (67). The mechanism of this
translocation is unclear.


POSSIBLE FATIGUE MECHANISMS AND
CURRENT THEORIES OF FATIGUE
Central Nervous System Fatigue
Events initiating muscular contraction originate
from sensory input or directly in the central nervous
system. Any study of fatigue during exercise of the
whole animal must consider the possibilities of central
inhibition resulting in reduced muscular performance.
There are conflicting reports concerning the potential
for a central component in muscular fatigue. For example,
Merton (44) found that maximal voluntary effort was not
different from the response of the muscle to maximal
tetanic stimulation of the motor nerve. He was studying
brief contractions of the adductor pollicis of humans.
Conversely, Asmussen and Mazin (1) have reported that
"diverting activity" (visual stimulation) permits greater
muscular performance than that which is accomplished when
the eyes are closed. Further experiments demonstrated
that immediate recovery from exhausting exercise (with
eyes closed) occurred if the eyes were subsequently
opened.
It is apparent from the work of Asmussen and Mazin
(1) that central effects can alter muscular performance.
It is important to keep in mind though, that under some
9


10
circumstances (i.e., brief maximal effort) fatigue appears
to be due entirely to peripheral mechanisms (44).
Neuromuscular Junction Failure
In the normal sequence of events preceding a muscular
contraction, an action potential is propagated over the
muscle membrane. The occurrence of a normal muscle action
potential is dependent on transmitter release and muscle
membrane properties. Repetitive stimulation may alter
these properties, and this could result in alterations in
the contractile response. Merton (44) found no change
in fatigue in the electromyogram resulting from maximal
stimulation despite an attenuation of force output.
Bergmans (3) studying human extensor digitorum brevis
observed no change in the surface electromyogram during
fatiguing contractions. Electromyography is not the
most sensitive technique for measuring the membrane
response, but any large alteration in muscle action
potential generation and propagation would probably
have been detected.
Using small muscle bundles, and measuring intracellular
potentials, Hanson (37) noted only minor changes in the
rat soleus muscle resting potential and action potential
following repetitive stimulation. The amplitude of the
action potential was reduced in fatigue, but was restored
within a few minutes of recovery. Grabowski (35) noted
a reduced amplitude of the muscle action potential of
fatigued frog muscle fibers. A reduced amplitude could


11
also be produced in a rested muscle by reducing extra
cellular Na+ concentration. Under these conditions,
twitch height is not altered. It would appear from the
results of these experiments that following a period of
fatiguing contractions, the amount of neurotransmitter
released is sufficient to raise the end-plate potential
to threshold, and the muscle membrane is capable of
propagating a muscle action potential.
Attenuated Calcium Release
If a normal action potential is propagated over a
muscle membrane, but less calcium is released from the
lateral sacs, the contractile response will be attenuated.
The reduced amount of Ca2+ released would result in a
lower peak sarcoplasmic Ca^+ concentration and therefore
a reduced activation. Direct measurement of Ca release
in a fatigued muscle has not been reported. Despite this,
several authors have concluded that the mechanism
responsible for the fatigue they observed was reduced
Ca2+ release (15, 19). This conclusion is based on
results from one of two techniques: either a) all other
possibilities are eliminated or b) inference is obtained
from analysis of changes in the time to peak tension and
the peak rate of force development for a twitch. In the
former, evaluation of the functional state of the
neuromuscular junction and of the force generating
capacity of the muscle has revealed that these processes
are unaltered in the fatigued muscle. This leads one to


12
believe that the muscle has a reduced amount of Ca2+
released. In the latter, it is assumed that relaxation
occurs simultaneously with Ca2 + reuptake (4). Under these
circumstances, a reduction in contraction time would
result from a reduction in duration of activation (more
f
rapid reaccumulation or shorter duration of release). A
reduction in peak rate of force development without a
concommitant reduction in contraction time indicates
reduced activation, and this is interpreted as a reduced
amount of Ca2+ released. Brust has made observations
similar to these (reduced rate of force development in
fatigue with no change in contraction time) on mouse
soleus muscles in vitro, (10) and concluded that fatigue
was due to reduced Ca2+ release.
Similar observations would be expected if there was
an increase in the Ca2+ concentration at which binding to
troponin and subsequent contractile activity occurs. It
has been observed by Fuchs et al. (32) that the affinity
of troponin for Ca2+ can be altered by pH. This
possibility must be considered when dealing with
inferences from measurements of contraction time and
rate of force development. Fitts and Holloszy (29) have
presented data indicating that reduced pH may be
associated with fatigue. They support the theory that
reduced activation (and reduced rate of force development)
is due to a reduced affinity of troponin for Ca2+.


13
Another situation may occur in the muscle for
which the rate of force development declines with developed
tension while contraction time remains unchanged. A
reduction in contractile capacity would give the same
results. This possibility must be given consideration.
Some authors have tested for, and found, changes in the
contractile capacity of the muscle under study (30, 44).
These are discussed below.
Reduced Capacity of the Contractile Apparatus
Fatigue may be the result of a reduced ability of the
contractile proteins to generate tension. This could be
a result of either: i) damage to myofilaments (i.e.,
misalignment or inactivation) or ii) restricted availability
of energy. In either case, the effect would be a reduced
force generation under conditions of maximal activation.
This capacity to develop tension has been traditionally
tested with either a K+ contracture or a caffein
contracture. Both of these procedures result in maximal
activation (Ca2+ concentration high enough to saturate
the contractile apparatus). Tetanic stimulation has also
been used to evaluate the capacity of a muscle to generate
tension.
Fitts and Holloszy (30) observed that tetanic force
was reduced in the rat soleus muscle following a series
of tetanic contractions. They noted that recovery of the
force generating capacity occurred relatively quickly
(within minutes). No insight into the mechanism


14
responsible for the fatigue observed by these authors is
provided. Since recovery occurred quickly, it is obvious
that permanent damage to the myofilaments was not a
mechanism of the fatigue.
Spande and Schottelius (56) studied fatigue in the
mouse soleus muscle in vitro. They found that the
magnitude of the reduction in developed tension was
inversely proportional to the phosphoryl-creatine(PC)
concentration. PC serves as an immediate source of high
energy phosphate (~P), for rephosphorylation of ADP and
may also be involved in a transport capacity for ~P from
mitochondria to myofilaments (40, 55). The experiments
by Spande and Schottelius (56) involved contractions with
periods of anoxia and/or glucose deprivation, and this
must be kept in mind when comparing their results with
those of other authors. Under these circumstances
reduced energy availability appears to be related to the
fatigue. Fitts and Holloszy (29) have measured PC
changes during and following fatiguing contractions in
rat muscle. They found no relationship between PC and
the amount of fatigue or recovery from fatigue.
The final common mediator of energy availability is
the level of ATP in the muscle. Edwards reported that
ATP and PC concentrations were reduced in isolated
mouse soleus muscles during prolonged tetani under
anaerobic conditions. This was also the case when
muscles were fatigued in the presence of cyanide and


15
iodoacetic acid. In the former case, lactate accumulated
but in the latter case there was no accumulation of
lactate. It was noted that prolongation of relaxation
was associated with a reduction in ATP and PC levels.
This provides an indirect method of evaluating energy-
availability in the muscle. Relaxation would be expected
to be prolonged since it is dependent on reuptake of Ca^+.
Sequestering Ca^+ is an active transport process which
requires ATP (13). Reduced levels of ATP may also slow
the relaxation phase of individual cross-bridges. ATP
is required to permit dissociation of the actin and
myosin molecules (65). The extreme of this situation
occurs when rigor bonds form in the absence of ATP.
It can be concluded from the above discussion that
fatigue can be the result of any of several mechanisms.
The possibility exists that multiple mechanisms function
at once. For example, a reduced release of Ca^+ may be
accompanied by a limitation of energy availability. This
situation would complicate the elucidation of the
mechanism(s) responsible for the fatigue.
The following chapters present the details of
experiments conducted in an effort to gain an under
standing of fatigue in the gastrocnemius-plantaris
muscle group of the dog.


GENERAL METHODS
Mongrel dogs of either sex weighing 9-18 kg were
used in these studies. They were anesthetized with
intravenous sodium pentobarbitol, 30 mg/kg, with
additional 30 mg injections as needed. The animals
were intubated and maintained on a respirator throughout
the experiment. A Beckman LB-2 gas analyzer sampled gas
from the endotracheal tube continuously. Ventilation
was adjusted to maintain end-tidal CO2 at 4.5 .25 %.
Rectal temperature was monitored with a thermocouple,
and kept between 37.5 and 38C by appropriate adjustment
of a heating pad placed under the thorax of the supine
dog.
The left gastrocnemius-plantaris muscle was exposed
via an incision along the medial aspect of the left hind
limb. Muscles overlying the medial head of the
gastrocnemius-plantaris muscle group were tied twice
with butcher's cord and cut between the ties. These
muscles are: sartorius, gracilis, semitendinosis and
two heads of semimembranosis. All veins draining into
the popliteal vein were ligated except those branches
coming from the gastrocnemius-plantaris muscle (see
Figure 2). Any veins draining the muscle but not
entering the popliteal vein were ligated. These were
16


FIGURE 2.
The in situ dog gastrocnemius-plantaris
preparation (58).
G gastrocnemius-plantaris muscle
Gr- gracilis muscle
S sartorius muscle
SM- two heads of semimembranosis muscle
ST- semitendinosis muscle
muscle


VENOUS
SAMPLE
\
V
FIGURE 2
^///////////Z///////Z/ / /


19
only minor vessels which occur along the anterior or
lateral surfaces of the muscle. The popliteal vein was
cannulated. A cannulating type electromagnetic flow
probe (Narco Biosystems) (3mm I.D.) was placed in the
outflow tubing. The venous effluent was returned to the
dog via another cannula in the external jugular vein.
Heparin, 2000 U/kg (12 mg/kg) was administered I.V.
initially and 1000 U/kg was given half way through the
experiment, prevented coagulation of blood in the tubing.
A thin cannula passed through the wall of the outflow
tubing and threaded within it to the muscle provided a
sampling port for venous blood. A thermocouple was placed
alongside this thin tube. The tip of the probe was
within 1 cm of the muscle. The blood temperature here
was assumed to be an average temperature of all parts of
the muscle. A heat lamp focused on the abdomen and hind
limbs was used to maintain muscle temperature near 37C,
while the muscle was at rest. During contractions, the
lamp was turned off and the muscle temperature was
permitted to rise. The contralateral femoral artery was
cannulated and a Statham pressure transducer was connected
to the cannula. Output of the transducer was recorded
on a Grass polygraph model #5.
The Achilles tendon was severed close to the
calcaneous and securely fixed in an aluminum clamp.
The clamp was hooked to a slide bar which was fastened
to the cantilever beam of an isometric lever. Force


20
was measured with a displacement transducer detecting the
displacement of the free end of the cantilever beam. The
transducer output was linear for forces up to 20 kg. A
displacement at the transducer of 0.1 mm gave a full
scale deflection on the recorder. Output of the displace
ment transducer (tension) was amplified and recorded
directly. The amplified tension signal was also
differentiated with respect to time (Gould-Brush dif
ferentiator) The differentiated and direct signals
were recorded on a Gould-Brush Model 2400 recorder.
Blood flow and muscle temperature were also recorded
continuously. The maximal rate of change of the
amplified force signal never exceeded 80 v/sec. The
differentiator was calibrated with ramp signals and was
found to be linear through 120 v/sec.
The sciatic nerve was dissected free from
surrounding tissue. All branches of the nerve not
innervating the gastrocnemius-plantaris muscle were
severed. The nerve trunk was double ligated about 4
cm proximal to the muscle and cut between the ties. A
tubular stimulating electrode was placed on the distal
stump of the nerve. The nerve was stimulated with a
Grass Model SD9 stimulator with square pulses 0.2 msec
in duration and of 2-4 volts. This voltage was double
that necessary to produce a maximal contraction.
Contractions were isometric. The lever-arm of the
myograph was bolted to a cast iron base which was


21
clamped to the table. Bone nails were placed in the
tibia and femur (one each). These nails were firmly
attached to the base of the myograph. A turnbuckle
strut, placed between the lever-arm and one of the
bone nails prevented flexing of the lever-arm. The
muscle length was set 1-2 mm shorter than the length at
which developed tension was greatest (optimal length).
Optimal length was determined by measuring the developed
tension (total tension minus resting tension) of
contractions at (0.2/sec) at various lengths.


02 UPTAKE AND DEVELOPED TENSION
Introduction
Oxygen uptake (V02) of muscle can increase more than
40 times resting levels during repetitive stimulation (57).
At low frequencies of stimulation, V02 is proportional
to the isometric developed tension (AT) (total tension
minus rest tension) (66). This relationship was
observed for contractions following a period of fatiguing
contractions at 10-14 per sec for 30 minutes (66). By
stimulating the motor nerve with twin impulses, AT can
be increased. It is not known whether the proportionality
between V02 and At persists for twin impulses stimulation
before or after fatiguing contractions. It is of interest
to determine whether or not the muscle is capable of
increasing its V02 following fatigue, and if so, to see
if AT is still proportional to V02.
The purpose of this study is to investigate the
effect of fatigue and twin impulse stimulation on the
ratio between V02 and isometric developed tension in
the in situ dog gastrocnemius muscle. Further experiments
have also been conducted to determine the VO2:AT relation
ship for muscle "fatigued" by curare infusion or ischemia
during repetitive stimulation.
22


23
Methods
The preparation described in the General Methods
section was used in these experiments. Five series of
experiments were completed to determine the relationship
between muscle VO2 and AT.
Oxygen uptake by the muscle was calculated from the
venous outflow and the arteriovenous blood oxygen content
difference. Arterial samples were taken from the
contralateral femoral artery. Venous samples were taken
from the popliteal vein cannula via a thin catheter
threaded through the wall of the venous outflow tubing
to the end of the cannula close to the muscle group.
The blood samples, 0.8 ml each, were collected in glass
tuberculin syringes sealed with mercury-containing caps
and kept in ice until analyzed for O2 content with a
Lex O2 Con analyzer.
Series 1 and 2
Contractions began at the rate of 1/sec, and O2
uptake and developed tension were measured after a steady
level had been attained. Next, the muscle was fatigued
by stimulating it at a rate of 10-20 impulses / sec for
30-40 minutes. This reduced the developed tension in a
single twitch to about one-third to one-half of the pre
fatigue level. The muscle was allowed to recover for 30-
40 minutes so that the resting VO2 approached the pre
fatigue level. Three pairs of blood samples were taken
five minutes apart as the muscle continued to recover.


24
After this recovery period, the muscle was stimulated
at the same rate as before (1/sec) with twin impulses
(two impulses, 6.5 v in amplitude, 0.2 msec in duration
and separated by 10-20 msec), and O2 uptake and developed
tension were measured. The time between the twin impulses
was set by adjusting the delay between impulses until a
smooth contraction was obtained. Post fatigue stimulation
with twin impulses returned the developed tension
approximately to the level of single impulse stimulation
pre-fatigue. In the second series of experiments, both
single and twin impulse contractions were done before
and after the fatiguing contractions.
In each type of contraction, the muscle was allowed
to contract for at least four minutes before arterial and
venous samples were taken to ensure that developed tension
and blood flow had reached a steady level. After an
additional two to three minutes of contractions, a
second pair of arterial and venous samples was collected.
The O2 uptake rates calculated from the two pairs of
blood samples were averaged and the resting O2 uptake
rate was subtracted to give the net O2 uptake per minute.
This value was divided by the muscle weight and the
number of contractions per minute to give the O2 uptake
in microliters of O2 per gram of wet muscle per
contraction (yl O2 g--*- C_1) Developed tension was
expressed as grams of developed tension per gram of wet
muscle (g* g--*-) .


25
Series 3
Oxygen uptake and developed tension were measured
during the fatigue process. In separate experiments,
muscles were stimulated at rates of 3, 4, 5 and 6 impulses
per second. After the first five minutes of contractions,
blood samples were collected periodically as the muscle
fatigued during contractions for two hours. The decrease
in developed tension ranged from 34 to 45% over the two
hour period. Sixty to 80% of this decrease occurred in
the first 30 minutes. Although blood flow and developed
tension were sometimes changing rapidly, there was almost
no change in the arteriovenous blood oxygen content
differences. This allowed application of the Fick
equation for C>2 uptake calculation with confidence (64).
Series 4
Oxygen uptake and developed tension were measured in
muscles during different levels of reduced blood flow
produced by partially occluding the arterial inflow.
The muscles were stimulated to contract at one twitch
per second throughout these experiments.
Series 5
It is possible that a portion of the fatigue
observed in the experiments of Series 1-3 might be due
to presynaptic neural failure or neuromuscular junction
failure, particularly in the first series of experiments
in which the nerve-muscle preparation was stimulated at
rates of 10-20 impulses / sec for 30-40 minutes. To


26
investigate this possibility, two experiments were done,
in which neuromuscular transmission was completely
blocked by repeated injections of either curare or
succinylcholine. After the drug was given, the nerve
was stimulated at the rate of 20 impulses / sec for
30 minutes. Muscle contraction did not occur during this
30 minute period because of the presence of the blocking
drug. Developed tenion (at a stimulation rate of 1/sec)
was measured before the drug was injected and after the
effects of the drug had worn off. Therefore, any
difference in developed tension before and after the
period of high frequency stimulation with curare block
would be due to either nerve or neuromuscular junction
failure since the muscle did not contract during the 30
minutes of stimulation.
Neuromuscular fatigue was mimicked in a fresh muscle
by infusing curare into the animal at different rates to
block muscle contraction to varying degrees. 02 uptake
and developed tension were measured at the different
levels of neuromuscular blockade. The stimulation rate
was 1/sec.
Results
Resting O2 uptake for the gastrocnemius-plantaris
muscle averaged 7.7 y 1 O2 g-^ min--*-. This is somewhat
higher than average values previously reported (27, 57)
but well within the usual range. Mean arterial blood
pressure remained above 100 mmHg throughout all of the
experiments.


27
In the first series of experiments, analysis of
variance for repeated measures (8) on the ratios
between O2 uptake and developed tension observed before
and after fatigue revealed no significant difference
(p>.25). Table I shows the O2 uptake and developed
tension for each of the muscles both before and after
fatigue.
The results of the O2 uptake and developed tension
measurements in series 2-5 are summarized in Table II
and illustrated in Figures 3 and 4. Table II shows the
linear regression equations relating O2 uptake and
developed tension for each experiment. These equations
were calculated from data which included values from
the fatigued muscle as well as the fresh muscle. The
slopes of all but one (Experiment 8, p=.09) of the lines
are significantly different from zero (p<.05), despite
the small number of points used to determine each
regression equation. It is obvious from Figure 3 and
Table II that there was considerable variability between
animals. This has always been observed in this preparation
(27, 57, 66). However, despite differences in absolute
values between different animals, the same pattern of
response was observed in all cases. VO2 per contraction
and AT were directly related.
Results of four sample experiments from series 2-5
are shown in Figure 3. Figure 4 shows that all of the


UPTAKE (Ml 02 g-I C-l)
28
FIGURE 3. Results of four sample experiments.
Numbers refer to individual experiments.
Thirteen is from Series 2 (circled
numbers = post fatigue). Sixteen is
from Series 3. Nineteen is from
Series 4. Twenty-two is from Series
5.


29
FIGURE
X
it
Data from Series 2-5 normalized to the same
scale. Developed tension in percent of the
greatest developed tension in each experiment.
02 uptake in percent of the 02 uptake at the
greatest developed tension. Numbers refer to
individual experiments. Seven to fourteen are
Series 2 (circled numbers = post fatigue). Fifteen
to eighteen are Series 3. Nineteen to twenty-one
are Series 4. Twenty-two to twenty-five are
Series 5. The asterisk denotes (100% 100%)
which is common to all of the experiments. The
line in this figure is the line of identity (X=Y).


TABLE I
02 UPTAKE AND DEVELOPED TENSION BEFORE AND AFTER FATIGUE IN SERIES 1
Pre-Fatigue (Single Impulses) Post-Fatigue (Twin Impulses)
Developed Developed
Experiment
Tens ion
(a-g 1)
O2 Uptake
(ul 09-g~1-C1)
Tension
(q*q-1)
O2 Uptake
(yl 0?q-1-C-
1
148
.688
125
. 361
2
196
. 314
177
.356
3
254
.579
250
.446
4
134
. 396
174
.435
5
207
.452
178
.258
6
219
. 382
197
.476
Mean SEM
193 18
.468 + .057
184 16
.389 .033
Units are as given for Figure 3
u>
o


TABLE II
RELATIONSHIP BETWEEN 02 UPTAKE AND DEVELOPED TENSION IN SERIES 2-5
*N equals the number of data points in each experiment
+ Units for O2 uptake are microliters of O2 per gram of wet
muscle per contraction
Units for tension are grams per gram of wet muscle.


Series #
Expt. #
N*
Type of Fatigue
2
7
4
10/sec for
30
min
8
4
10/sec for
30
min
9
4
10/sec for
30
min
10
4
10/sec for
30
min
11
4
14/sec for
40
min
12
4
20/sec for
30
min
13
4
20/sec for
30
min
14
4
15/sec for
30
min
3
15
7
Continuous
at
3/sec
16
8
Continuous
at
4/sec
17
10
Continuous
at
5/sec
18
10
Continuous
at
6/sec
4
19
5
Ischemia
20
6
Ischemia
21
8
Ischemia
5
22
7
Partial Curare Block
23
8
Partial Curare Block
24
8
Partial Curare Block
25
7
Partial Curare Block
% Variance
Regression Equation* Explained
VO 2
=
3-3-10-3
T
-
9-3.10-2
99.8
VO 2
=
4 2103
T
+
90102
83.4
VO 2
=
3-6-10-3
T
-
2-6-10-1
89.7
VO 2
=
3-9-10-3
T
-
4-8-10"1
89.5
VO 2
-
2-8-10-3
T
-
2 010-1
98.0
VO 2
=
5-1-10-3
T
-
1-9-10-1
99.6
vo2
=
2-7-10-3
T
-
1-2-10"1
98.4
vo2
=
2-2-10-3
T
-
4 -5-10-2
98.6
VO 2
=
4-0-10-3
T
-
2-8-10-1
85.0
vo2
=
5-0-10"3
T
-
8-2-10-3
93.7
VO 2
=
3 4"103
T
+
8-1-10-2
95.2
VO 2
=
3-0-10-3
T
-
9-1-10-2
90.2
VO 2
=
1-3-10"3
T
-
1-3*10-2
91.6
VO 2
=
4 6 -10-3
T
-
7-6-10-2
88.9
vo2
=
5 1 103
T
-
2-2-10-1
91.6
VO 2
=
46103
T
+
1-1-10-1
97.2
vo2
=
1-5-10-3
T
-
7-2-10-2
97.7
VO 2
=
5-5-10-3
T
-
1*9*10-1
97.7
VO 2
=
52.103
T
-
2.6-10-1
95.9
oj
to


33
data follow the same pattern when normalized to the same
scale. In this figure, developed tension is plotted as
the percent of the highest tension developed in each
individual experiment, and O2 uptake is plotted as the
percent of the O2 uptake at the highest developed tension.
Most importantly, Figures 3 and 4, and Tables I and II
show that the relationship between O2 uptake and developed
tension was unchanged by the various treatments.
In two experiments, muscle contraction was completely
blocked by repeated injections of curare or succinylcholine
while the nerve was stimulated 20 times per second for
30 minutes. Injection of the blocker was discontinued
after the stimulation period and the effects of the
blocker were mostly dissipated within 10 minutes.
Developed tension was still at least 90% of the control
value. The observed reduction in contraction strength
may have been due to incomplete recovery from the
neuromuscular block. This 10% reduction in developed
tension can be compared with the 50-70% reduction
observed in the other experiments in which muscle
contraction was not blocked. It appears that most if
not all of the reduced contractile response was due to
alterations beyond the neuromuscular junction.
As pointed out in the Methods, the fatigued muscles
in the first and second series of experiments were allowed
to recover for 30-50 minutes. After this time, the
resting O2 uptake approached the pre-fatigue level.


34
However, developed tension recovered very little during
this time and was still only one-third to one-half of
the pre-fatigue value.
Discussion
Isometric developed tension at constant muscle length
was varied in this study by four methods: 1) twin impulses
stimulation, 2) fatigue produced by 30 minutes of
contractions at 20/sec, 3) ischemia caused by partial
occlusion of arterial inflow to the muscle, and 4) partial
block of neuromuscular transmission with curare. Figures
3 and 4, and Tables I and II show that none of these
treatments changed the relationship between 02 uptake
and developed tension. Stimulating the fatigued muscle
with twin impulses restored developed tension to pre
fatigue values. Fatigue did not increase the O2
requirement per unit of force developed, even when the
tension developed by the fatigued muscle was returned
to the pre-fatigue level by twin impulses stimulation.
The fatigued gastrocnemius-plantaris muscle is therefore
capable of increased developed tension and increased
V02. In addition, the 02 requirement per unit of force
developed was not altered during the development of
fatigue.
The dog gastrocnemius-plantaris muscle group has
certain advantages in studies of fatigue. Based on
histochemical staining properties, the dog gastrocnemius
contains only two motor unit types (43). These two


35
correspond to types FR and S (SR), (fast, fatigue
resistant and slow, fatigue resistant respectively),
described by Burke and colleagues (11) for hindlimb
muscles of the cat. Even though the dog gastrocnemius-
plantaris muscle group contains both FR and S units,
and the cat soleus muscle contains only type S units,
homogenates of cat soleus muscle have less than one-
third of the succinate oxidase activity of homogenates
of the dog gastrocnemius-plantaris muscle group (43).
From this, one might expect all of the dog gastrocnemius-
plantaris muscle units to be more resistant to fatigue
than any of the units of cat soleus muscles. However,
Burke and colleagues (11) have warned against extra
polation of histochemical and biochemical properties
to physiological properties.
There are several possible causes of the fatigue
observed in these experiments. In Series 1-3, fatigue
could have resulted from a failure in excitation-
contraction coupling, substrate depletion, accumulation
of metabolites, or a combination of these factors.
Since there are both FR and S fiber types in the
gastrocnemius, the fatigue might have been predominantly
in one of the fiber types.
It seems unlikely that neuromuscular junction failure
was a significant component of the fatigue observed in
Series 1-3. Testing for neuromuscular transmission
failure by direct stimulation of the dog gastronemius-
plantaris muscle group is not easy since its large size


36
makes constant field stimulation difficult. However,
several studies on other mammalian muscles (3, 41, 52)
have indicated that the possibility of neuromuscular
transmission failure at stimulation rates of less than
10/sec is minimal. In two experiments, nerve stimulation
at 20 impulses / sec for 30 minutes when muscle
contraction was blocked by curare or succinylcholine
caused less than a 10% decrease in developed tension.
Decreases in developed tension of 50-70% occurred under
the same stimulation conditions when muscle contraction
was not blocked. These findings indicate that presynaptic
failure of impulse propagation and inadequate release of
acetylcholine probably did not cause the fatigue observed
in our experiments. Desensitization of the endplate is
not ruled out by these results. However, neuromuscular
depression is presently believed to result from a
reduced number of released transmitter quanta and a
reduction of quantal size (42, 48).
In Series 4, the cause of fatigue might have been
muscular, neuromuscular, or a combination of the two
since ischemia can affect both the muscle and the
neuromuscular junction (16, 49). In Series 5, developed
tension was decreased by partial curare block which
presumably simulates neuromuscular junction failure.
These experiments do not allow identification of the
specific cause of fatigue. However, our results do
indicate that the oxygen uptake per unit of isometric


37
force production is unchanged by either muscle fatigue
or neuromuscular fatigue. This suggests that fatigue,
whether muscular, ischemic, neuromuscular, or a
combination of these three, does not cause any change in
the efficiency of energy transduction from ATP to
external tension development by the muscle, without
concommitant changes in the opposite direction for
energy transduction from foodstuffs to ATP. This is
not likely the case.
These results differ from those of Bronk (6),
Feng (28), Edwards and Hill (20) and Edwards, Hill
and Jones (21) in that they found that the energy
expenditure per unit of force production (or of tension
time) decreased during fatigue. Unlike our experiments,
however, these earlier studies used stimulus parameters
which caused partially to completely fused tetanic
contractions of relatively long duration. The present
study is of twitch or very brief tetanic contractions,
for which no plateau in developed tension occurs (see
Figure 5). There is little if any tension maintenance
involved.
The data presented in this study along with those
of Wilson and Stainsby (66) demonstrate a constant
coupling between C>2 uptake and developed tension in
isometric twitch contractions. In these two studies,
developed tension has been changed by stimulation
frequency, potassium ion infusions, twin impulse


FIGURE 5. Tracing of a recording of tension and
differential of tension for single impulse
and twin impulse contractions before and
after fatigue. Contractions are superimpos
for ease of comparison. There is no
maintained plateau in this type of
contraction.


TWITCH and TWIN
TENSION
dP/dt
FIGURE 5
CONTRACT ION
Post fatigue
OJ


40
stimulation, normal muscle fatigue, ischemic fatigue and
partial neuromuscular transmission block with curare.
During all of these treatments, the relationship between
O2 uptake and developed tension has been unaltered.
The data also show that although resting metabolic
rate following fatigue approaches pre-fatigue levels after
30 minutes, developed tension is still quite low.
Phosphorylcreatine and ATP levels should be fully
recovered following 30 minutes of rest (38, 51). Edwards
and coworkers (23) have also identified a long lasting
element of fatigue in humans that is not due to
depletion of high-energy phosphates. Further study
is warranted to determine whether or not there really
is a causative relationship between phosphorylcreatine
depletion and fatigue, as suggested by Spande and
Schottelius (56).


EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE
Introduction
Skeletal muscle has an attenuated response to a
single impulse following a prolonged period of twitch
contractions due to repetitive single impulse
stimulation (34, 66). This response is not necessarily
indicative of a reduced capacity of the muscle to develop
tension (23, 30). It is,however, a type of muscular
fatigue and warrants further investigation concerned
with determination of mechanisms responsible for this
"twitch fatigue."
Wilson and Stainsby (66) reported that twitch
fatigue occurs in the gastrocnemius-plantaris muscle
of the dog following 30-40 minutes of isometric
contractions (10-14/sec). They monitored recovery with
periods of low frequency stimulation over the course of
3-4 hours. An attenuation of developed tension was still
present following this recovery period. Little is known
of the mechanism responsible for this fatigue.
The purpose of the present investigation was to
study alterations in twitch contractions caused by
repetitive stimulation at three frequencies; 2.5, 5
and 10/sec. A twitch contraction can be characterized
41


42
by measurements of the magnitude and time-course of
tension development seen in the isometric myogram (3, 9).
These measurements are: developed tension (AT),
contraction time (Ct), half relaxation time (Rt 1/2),
peak rate of force development (dP/dt), and peak rate
of relaxation (-dP/dt) (see Figure 6). Sandow and Brust
(54) have named the changes in these measurements that
occur with repetitive stimulation the "fatigue patterns."
Fatigue patterns have been determined for single muscle
cells and whole mammalian and amphibian skeletal muscles
in vitro (9, 10, 35) Measurements on iii situ muscle
where direct determination of muscle force during
repetitive stimulation can be made while the muscle
maintains a normal circulation have not yet been
reported.
Methods
Twenty mongrel dogs of either sex weighing 9-18 kg
were used in this study. The gastrocnemius-plantaris
muscle group was prepared as described in the General
Methods chapter.
Fatigue as a result of 30 minutes of stimulation at
three frequencies 2.5, 5 or 10/sec was studied. Five
animals were used at each frequency. To study the fatigue
patterns of muscle, it is necessary to obtain fast traces
of contractions, before, during and after the fatiguing
contractions. To evaluate twitch contractions before
fatigue, the muscles were stimulated at either 1/sec or


43
Figure 6. Tracings of: Tension and dP/dt are presented to
demonstrate the manner by which the measurements
were made. See text for verbal description of
these terms.


44
2.5/sec for 2 minutes. After a fast trace (100 or 200
mm/sec paper speed) was obtained, contraction frequency
was either left at 2.5/sec or increased from 1/sec to 5
or 10/sec. Relaxation was not complete between
contractions when stimulation was 5/sec or 10/sec. To
facilitate measurement of the characteristics of a twitch,
the frequency of stimulation was reduced briefly, while
fast traces were obtained, then the fatiguing frequency
was restored (see Figure 7). During contractions at
2.5/sec complete relaxation occurs between contractions,
so fast traces were obtained without altering the
frequency of stimulation. Besides the contractions at
2 minutes, fast traces were obtained after 10 and 30
minutes of fatiguing contractions and after 10 and 40
minutes of recovery (see Figure 7). To get fast traces
during the recovery period which followed contractions
at 5/sec or 10/sec, the stimulator was turned on briefly
at 1/sec. Following the 30 minute period of contractions
at 2.5/sec, contractions were continued at a frequency
of 0.2/sec. Fast traces were obtained without altering
the frequency of stimulation. It has been reported that
contractions at this low frequency do not alter the
recovery process (66). In one experiment, a tetanic
contraction (200 msec duration, 100 impulses / sec) was
obtained, before and after the 10/sec fatiguing contractions.
This was done to permit evaluation of the contractile
capacity of the muscle. All fast traces were evaluated


45
for the characteristics of a twitch. These characteristics
are illustrated in Figure 6.
Arterial and venous blood samples (0.6 ml) were
obtained at regular intervals throughout the experiment.
Samples were drawn into glass tuberculin syringes, sealed
with mercury-containing caps, and placed in ice until
they were analyzed. These samples were analyzed for pH,
PCC>2 and PO2 at 37C with a radiometer (Copenhagen) blood
gas machine. These measurements permit evaluation of
viability of the animal and provide descriptive data
concerning metabolic status of the muscle.
At the end of each experiment, the fatigued muscle
was excised, trimmed of visible fat and connective tissue,
blotted and weighed. The force transducer was calibrated
after each experiment by hanging pre-weighed lead weights
on the lever.
In a few additional experiments, twitch contractions
were evaluated when AT was reduced by: ischemia, dantrolene
sodium or reduced stimulation voltage. Comparison of
these contractions with those obtained during and/or
following fatiguing contractions may provide some insight
into the mechanism of fatigue. To study the effects of
ischemia, the femoral artery was occluded while contractions
continued at 1/sec. Fatigue would not occur at this
frequency with an intact blood flow, but does occur with
ischemia. The occlusion was removed after AT fell to
about 50% of the pre-occlusion value (10-20 minutes)


46
Tension developed versus time. Contractions
in this case were 10/sec except as indicated.
Fast traces (not illustrated) were obtained
during the 1/sec stimulation.
FIGURE 7.


47
and recovery was observed. Dantrolene sodium, dissolved
in propylene glycol (25 mg/ml) was injected I.V. during
contractions at 0.2/sec. Dantrolene impairs release
of Ca from the lateral sacs (24). This is accomplished
without changes in the action potential and is apparently
a direct effect on the lateral sacs. Sufficient drug
was given to reduce AT at least 50% (2-5 mg/kg). To
study the effects of reducing the number of motor units
contracting, the stimulation voltage was reduced while
the muscle contracted 0.2/sec. This results in excitation
of fewer motor neurons and their motor units. Consequently
less tension is developed. Comparing the twitch
characteristics of a normal versus a fatigued muscle may
provide information leading to an understanding of the
mechanism(s) of fatigue.
Also, in a few experiments, samples of muscles were
obtained immediately following the 30 minute stimulation
period, and/or after 40 minutes of recovery. Samples
were frozen in situ with metal clamps pre-cooled in
liquid nitrogen. Small samples (30-80 mg) were then
homogenized (Vertis homogenizer) in perchloric acid
(.8% in 40% ethanol) and analyzed for phosphorylcreatine
by the method of Ennor and Stocken (25) (see Appendix).
Statistical analysis was by the two way analysis of
variance for repeated measures. Differences between means
were determined by Duncan's multiple range test (2).


48
Results
Blood samples were obtained before the contractions
began and at t = 10, 30, 40 and 70 minutes. Arterial PO2
was 87 2.3 mm Hg (mean SEM) before contractions and
did not change significantly throughout the experiments
(see Figure 8). Before contractions began, PvC>2 was
50.2 2.0 mm Hg. During the contraction period PvC>2
was lower, but none of the blood samples measured had
a P02 less than 12 mm Hg. Except for the experiments
where fatigue was caused by 2.5/sec contractions, PvC>2
was back to pre-fatigue values early in the recovery
period. Contractions were continued, 0.2/sec, during the
recovery period of these (2.5/sec) experiments; therefore,
it might be expected that PvC>2 would not be at rest levels.
Arterial PCO2 began at 31.6 0.8 mm Hg and fell
slowly during the experiments. The decrease in PaCC>2 was
statistically significant but probably is of minimal
physiological significance. Venous PCO2 was high during
the contraction period when PvC>2 was low, and returned
to pre-contraction levels early in the recovery period.
Arterial pH was 7.40 0.01 before contractions began,
and did not change significantly throughout the experiments.
Venous pH decreased from 7.37 at t = 0 minutes to 7.32
(for 2.5/sec) or 7.28 (for 5 or 10/sec) at t = 10 minutes.
By 10 minutes of recovery, venous pH had returned to pre
fatigue values (see Figure 8).


FIGURE 8. Blood flow and the blood gas measurements
versus time. The horizontal bar
indicates where fatiguing contractions
occurred. When means at a given time
(for different frequencies) were not
significantly different, means were
combined. Numbers refer to the
fatiguing frequency. Asterisks
indicate where measurements are
significantly different from the
original value (time = 0 minutes).
Vertical bars are SEM.


50
FIGURE 8


51
Although blood flow was measured continuously, only
those measurements corresponding to times when blood
samples were obtained are presented (see Figure 8).
Blood flow was higher during the contractions, but was
back to pre-fatigue values by 10 minutes of recovery.
There were no significant differences between frequencies
for blood flow response.
The first 2 minutes of contractions were at 1/sec
or 2.5/sec. There were no significant differences for
AT between these frequencies at t = 2 minutes. Mean AT
for all experiments was 2.3 15 g/g (wet wt) at this
time. The muscles weighed 48.5 t 3.3 g (wet wt).
Developed tension fell more rapidly during 10/sec
contractions than during 2.5/sec or 5/sec contractions
(see Figure 9). By 30 minutes all frequencies of
stimulation resulted in significant reductions in AT.
There was no significant recovery of AT during the 40
minutes following the fatiguing contractions.
Contraction time decreased during the fatiguing
contractions at 10/sec, but not during the contractions
at 2.5 or 5/sec. There was no significant difference
for Ct between recovery and pre-fatigue measurements at
any fatiguing frequency (see Figure 9).
Half relaxation time did not change during the
contractions or during the recovery except for recovery
of 2.5/sec fatigue. The Rt 1/2 was longer for contractions
at 0.2/sec than for 1/sec. If contractions during recovery


FIGURE 9. Developed tension, contraction time
and half relaxation time versus time
The horizontal bar indicates when
fatiguing contractions occurred.
Where there was no significant
difference between means, all
frequencies are combined. Numbers
refer to the fatiguing frequency.
Asterisks indicate where measure
ments are significantly different
from the original value (at time =
2 minutes). Vertical bars are SEM


FIGURE 9


54
for this frequency of fatiguing contractions had been 1/sec
(at t = 40 and 70 minutes only) then no difference from
pre-fatigue contractions would be expected.
Figure 10 illustrates the changes in dP/dt and -dP/dt
seen in these experiments. The changes seen for dP/dt
closely parallel those observed for AT. A positive and
significant correlation exists between dP/dt and AT
(r2 = 0.93) and between -dP/dt and AT (r^ = 0.82).
Muscle temperature rose during the fatiguing contrac
tions. The increase in temperature was only 1-2C. A
similar or smaller rise was seen during the 5/sec and
2.5/sec contractions. Muscle temperature fell slowly to
37C during recovery, but was not permitted to go below
37C.
Muscle samples obtained during a few of the experi
ments were analyzed for phosphorylcreatine (PC). Analysis
revealed that PC is low at t = 30 minutes (during
contractions), but is back to resting levels by t = 70
minutes (see Table III). The values of PC given in the
table are left:right ratios. PC was determined relative
to total creatine in the muscle sample. Harris (38) has
shown that total muscle creatine content does not change
during exercise and therefore can be used as an index of
muscle weight.
In one experiment, a tetanic contraction was obtained
before and after fatiguing contractions at 10/sec. Figure
11 illustrates the lack of change seen for this contraction.


TABLE III
PHOSPHORYLCREATINE ANALYSIS DURING CONTRACTIONS AND FOLLOWING THE RECOVERY PERIOD
Animal #
L: R ratio:
a) during contractions
b) following recovery
0.36 0.63 0.40 0.46
0.83 0.96 1.0 1.11 1.08 1.02 1.26 1.09 0.83 1.02
Phosphorylcreatine is presented above as a ratio:
PC Creatine--'- (left): PC Creatine--1- (right)
(See text for discussion of creatine as an index of muscle weight.)
U1
Ln


FIGURE 10. Peak rate of force development and peak rate
of relaxation. See Figure 5 for significance
of asterisks and numbers. Note similarity
between dP/dt versus time and AT versus time
(Figure 5).


100
DP/DT
(/o)
60
20
TIME (min)
i i i
10 25 40
5,10
FIGURE 10
i i
55 70
T
Ln


58
i
I
I
TETANIC CONTRACTIONS
dP/ dt
FIGURE 11. Tracings of recordings of tension and
differential of tension for tetanic
contractions (100/sec for 200 msec).
Developed tension following 30 minutes
of fatiguing contractions (10/sec) was
only slightly lower than that before
the 10/sec contractions. The differential
tracer were virtually superimposable.


59
The tetanic contraction is recovered at a time when
twitch AT is still reduced.
Contractions observed during ischemia demonstrated
a reduced AT and a prolonged Rt 1/2. Following restoration
of blood flow, recovery of both AT and Rt 1/2 was 50%
complete in 30 minutes. Figure 12 shows recordings from
one muscle for contractions pre-ischemia, during ischemia
and post-ischemia. These recordings are typical for
what was seen.
Comparing the effects of dantrolene sodium, ischemia
and reduced stimulation voltage on Rt 1/2 vs AT (see
Figure 13), indicates that ischemia and reduced voltage
cause large alterations in Rt 1/2 with concomitant
reductions in AT. With administration of dantrolene
sodium, the attenuation of AT is not accompanied by a
substantial change in Rt 1/2. This pattern seen with
dantrolene is similar to the changes seen during the
fatiguing contractions.
Discussion
In these experiments, twitch fatigue has resulted
from 30 minutes of contractions at 2.5, 5 or 10/sec.
Forty minutes after the fatiguing contractions were
ended, no significant recovery had occurred. Recovery
would eventually have occurred following several hours
of relative inactivity (66). Twitch fatigue, then,
results from a relatively persistent alteration in the muscle
which affects the contractile response to a single impulse.


60
FIGURE
ischemia
2. Tension and dP/dt (upper and lower curves
of each pair respectively) are shown.
1) a control contraction before occlusion
of blood flow
2) contraction during ischemia, 5 minutes
after occlusion of blood flow
3) a post-ischemia contraction, 50 minutes
after release of occlusion, contraction
frequency 1/sec.


61
FIGURE 13. Half relaxation time versus AT is presented
to illustrate the relative changes in Rt 1/2
when AT is reduced by ischemia, dantrolene
or reduced stimulation voltage. Each line
represents one dog. Lines were determined
by the least squares method common to all
three lines.


62
The response to tetanic stimulation is not altered.
Analysis of the fatigue patterns for this muscle group
may provide some insight into the mechanisms responsible
for this long-lasting fatigue.
The extent of comparisons between frequencies for
the fatigue patterns observed in these experiments is
limited. In the experiments where fatigue was caused
by contractions at 5/sec or 10/sec, the frequency of
stimulation was reduced to 1/sec to obtain fast traces.
This procedure was implemented because full relaxation
does not occur between contractions at 10/sec and 5/sec.
The measurements which have been made on these contractions
are affected by the resting tension. It was hoped that
by reducing the frequency to 1/sec for these contractions,
a true representation of the characteristics of a twitch
could be obtained. Although a common frequency is used,
it is evident that the preceding contractions did have
an effect on the measurements (see Figure 14). The
measurements made for the 2.5/sec series were made on
contractions at 2.5/sec during the 30 minute fatigue
period and at 0.2/sec during recovery. Due to the effect
of frequency and preceding contractions on the time
course of a twitch, caution must be exercised when
comparing values between frequencies. Comparing the trend
within one frequency with the trend within another
frequency is valid.


63
TENSION
DIFFERENTIAL
l/sec after
10/sec
FIGURE 14. Following fatiguing contractions at 10/sec,
switching the stimulator to l/sec results
in a negative staircase. The top tracing
is tension, and the lower one is dP/dt.
This demonstrates the inotropic effects
of previous contractions, and illustrates
the mechanisms responsible for the decrease
in developed tension seen following the 30
minute period of contractions. The single
contraction on the left immediately follows
10/sec contractions; the others (at a reduced
paper speed) were continued at l/sec.


64
If contractions of the fatigued muscle are compared
with contractions before the muscle was fatigued, the
following characteristics are noted. Developed tension
is reduced. This reduction appears to be greater when
a higher frequency of stimulation occurred during the
fatigue period. Contraction time and Rt 1/2 are not
different in the fatigued muscle in comparison with the
rested muscle for muscles fatigued at 10/sec and 5/sec.
The prolongation of contraction time in recovery from
fatiguing contractions at 2.5/sec is not due to fatigue,
but due to the stimulation frequency during recovery.
Contraction time gets shorter as frequency of stimulation
is increased. For the same reason, Ct is shorter during
the fatiguing contractions at 10/sec. This is an effect
of previous contractions (10/sec) altering the time-
course cf contractions at 1/sec. A decrease was seen for
dP/dt which was proportional to the decrease in AT.
Brust (10) reported similar fatigue patterns for the
mouse soleus muscle in vitro. The same author (9)
reported different fatigue patterns for frog semitend
inosis muscles. The major difference was that Rt 1/2 was
prolonged in the fatigued frog muscle but not in the
mammalian muscles. This difference is apparently not
species specific. Edwards et al. (22) reported that
there is a slowing of relaxation in mouse muscle
following a fatiguing effort. This slowing of relaxation
was correlated with low levels of ATP (22). This can


65
explain the increase in Rt 1/2 seen in ischemic fatigue
(see Figure 13). Since Rt 1/2 was not prolonged following
a period of fatiguing contractions with an intact blood
supply, it would appear that ATP was available within the
muscle.
The evidence presented above suggests that the fatigue
observed in these experiments did not occur as a result
of reduced ATP levels. Other evidence supports this
suggestion. Although PC levels were low during the
contraction period, (see Table III), resynthesis had
occurred before significant recovery of AT. Rapid
resynthesis of PC during recovery from a period of
contractions was also observed by Piiper and Spiller (51).
There seems to be no direct relationship between PC levels
and AT. This is contrary to a report by Spande and
Schottelius (56), but supports the observations of Fitts
and Holloszy (29). It should also be pointed out that
glycogen was still available after 30 minutes of stimulation
at 10/sec (14) and lactate production had declined (or
even reversed lactate uptake) (60). It would seem that
energy was available, but demand for energy was reduced.
In support of this conclusion, PvC>2 was not below minimum
critical PO2 (59) at any time that PvC>2 was measured.
The muscle is capable of developing more force than
that seen in a twitch. Twin impulse stimulation causes
developed tension to be double that seen with single


66
impulse stimulation (34). This relationship is maintained
for rested as well as fatigued muscles. It would appear
that the reduced AT of the fatigued muscle was due to
reduced activation.
A reduced activation of skeletal muscle could,
theoretically be the result of: a) reduced neuromuscular
transmission, b) reduced Ca2 + release, or c) reduced
responsiveness of the contractile elements to Ca2+.
Neuromuscular transmission appears to be intact (34).
This agrees with others (3, 35, 37) who have found only
minimal changes in muscle action potentials or electro
myographic response following comparable stimulation
O i
periods. There is a possibility that Ca release has
been attenuated. This could cause a reduced AT and
dP/dt without altering Ct or Rt 1/2 (15). Brust (10),
who observed similar fatigue patterns with the mouse
soleus muscle, suggests that the fatigue he observed was
a result of reduced Ca2+ release (see also (63) for the
converse). The factor(s) responsible for the reduced
Ca2+ release is/are not known. Bonner et al. (5) have
reported that muscle mitochondria accumulate Ca2+ during
exercise. This would reduce the pool of Ca2+ available
for recycling in the sarcoplasmic reticulum (67) and
consequently limit the amount available for release.
Dantrolene sodium apparently reduces the amount of
Ca2+ released per impulse (24). This drug was used in
these experiments to determine the effects of reduced


67
Ca2+ release on a twitch. A dantrolene treated muscle
contracts with a time-course similar to the fatigued
muscle. Ischemia or reduced stimulation voltage caused
a reduction in AT, but this was accompanied by changes
in Rt 1/2.
An alternative hypothesis involves a reduced binding
94-
of Ca to the regulatory proteins. During contractions,
muscle pH probably falls (33). Fuchs et al. (32) has
shown that binding of Ca2+ to troponin is inhibited by
lower pH. Fitts and Holloszy (30) has suggested this
may be a mechanism responsible for the fatigue seen in
their experiments.
The experiments reported herein do not permit
discrimination between these two potential mechanisms
of fatigue. The fatigue observed following 30 minutes
of stimulation at 2.5, 5 or 10/sec appears to be a
result of reduced activation. Further experiments will
need to be conducted to determine which of the theories
described above applied to these muscles.


EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH CONTRACTION
Introduction
Twitch developed tension is attenuated following a
period of repetitive stimulation. This attenuation is
apparently the result of a reduced activation, due either
to a reduced release of Ca2+ from the lateral sacs or
from a reduced responsiveness of the contractile proteins.
It is not due to a reduced availability of energy (ATP,
PC). Fitts and Holloszy (30) have suggested that the
reduced twitch response is a result of inhibition of the
contractile process due to a reduced intracellular pH.
Steinhagen et al. reported evidence supporting this
hypothesis (61). He reported that dog gastrocnemius
muscle fatigues more quickly during respiratory acidosis
than during normal pH balance.
Specific mechanisms which may contribute to this
acidosis-induced fatigue have been proposed. Nakamura
and Schwartz (46) reported that uptake of Ca2+ by
sarcoplasmic reticulum is accelerated in low pH medium.
This could reduce the duration of activation for a twitch
by reducing the Ca2 + concentration more quickly. Fuchs
et al. (32) have reported that Ca2+ binding to troponin
is inhibited by H+. These molecular mechanisms, if
effective under physiological conditions would cause a
reduction in AT, fatigue.
68


69
The purpose of this study was to observe the effects
of acidosis on the developed tension and the time-course
of a twitch contraction of the in situ dog gastrocnemius-
plantaris muscle. Intracellular pH can be reduced more
easily via respiratory acidosis than by metabolic acidosis
(12). For this reason, acidosis was induced by reducing
the ventilatory rate. The results of this study indicate
that acidosis is unlikely a direct cause of twitch fatigue.
Methods
The gastrocnemius-plantaris muscle preparation as
described in the General Methods section was used in this
series of experiments.
In each experiment, the nerve was stimulated at a
frequency of 0.2/sec. In three experiments, ventilation
was controlled to maintain arterial pH near 7.4 for the
duration of the experiment (2 hours). In four experiments,
after the muscle had been contracting (0.2/sec) for
20 minutes, ventilation was reduced to 3-4 breaths/minute.
The mixture of inspired gas was adjusted (with 9 5% C>2
and 5% CO2) to maintain normal arterial PO2 during the
hypoventilation. The period of hypoventilation was
continued until arterial pH was less than 7.1 (60-90
minutes). This period of hypoventilation was followed
by a period of hyperventilation (20 breaths/minute). The
hyperventilation was continued for 40-60 minutes (see
Figure 15).


70
With an additional four dogs, the sequence of
ventilatory adjustment was reversed (control, hyper
ventilation, hypoventilation) to permit evaluation of
a possible order effect.
At ten minute intervals throughout each experiment,
arterial and venous blood samples were obtained (0.8 ml)
in glass tuberculin syringes (1 ml capacity). The samples
were sealed with mercury containing caps and kept in ice
until they were analyzed for PO2, PCO2 and pH (Radiometer,
Copenhagen).
Fast traces of contractions were also obtained at 10
minute intervals (200 mm/sec on Gould-Brush Model 2400
recorder). The fast traces were measured for developed
tension (AT), half relaxation time (Rt 1/2), contraction
time (Ct), peak rate of force development (dP/dt) and
peak rate of relaxation (-dP/dt) (see Figure 6).
Statistical analysis was by a two way analysis of
variance for repeated measures (2). For statistical
analysis, the last two measurements (fast traces or blood
gases) before alteration of the ventilation were utilized
to represent the state in which they occurred (see
Figure 15).
Results
After 20 minutes of contractions, ventilation was
reduced to 3-4 breaths per minute (Group A) or increased
to 20 breaths per minute (Group B). This adjustment in
ventilation resulted in a reduction in arterial pH from


FIGURE 15. Developed tension, PO2, ventilation and
arterial pH for one dog, from Group A.
Blood samples and fast traces obtained
at were used for statistical analysis


72
200
DEV ELOPED
TIME (min)
FIGURE 15


73
7.37 t .01 (mean SEM) during the control period to
7.08 .03 in Group A and an increase in Group B from
7.36 .02 to 7.52 .02. After 60-90 minutes, ventilation
was increased to 20 breaths per minute in Group A and
reduced to 3-4 breaths per minute in Group B. This
second alteration in ventilatory frequency resulted in
an increase in pH to 7.4 in Group A and a reduction in
Group B to 7.2 (see Figure 16). In Group C, ventilation
was not altered, and arterial pH did not vary during the
experiments. The reduction in ventilation did not
significantly decrease arterial PC>2
When arterial pH remained constant for 2 hours
(Group C), AT increased with time. By the end of 2 hours,
AT was greater than it had been at the first 20 minute
period. As illustrated in Figure 17, AT increased with
time in Group A also but not in Group B. The only
significant difference in Groups A and B was that AT
during hyperventilation was greater than AT at any other
time period in Group A and greater than control in Group
B (see Table IV).
Despite the apparent effect of hyperventilation on AT,
there was no significant correlation of AT with arterial
or venous H+ concentration (see Table V). Furthermore,
AT was not reduced during hypoventilation. When arterial
pH fell to 7.09 .03, AT did not change significantly.
In Group C, dP/dt did not change significantly. In
Group A and Group B, however, dP/dt was higher during


74
TABLE IV
STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP
Group A Group B Group C
Rank
Rank
Rank
AT
.006
3 2 1
.05
3
2 1
.04
3 2 1
Rt 1/2
.18
NS
.003
1
2 3
.99
NS
Ct
.86
NS
.005
1
3 2
.98
NS
dP/dt
.001
3 2 1
.001
3
2 1
.122
NS
-dP/dt
. 002
3 12
.02
3
2 1
.91
NS
The p values are
given
for AT,
Rt
1/2,
Ct, dP/dt
and
-dP/dt for Group A (control, hypoventilation, hyper
ventilation), Group B (control, hyperventilation,
hypoventilation) and Group C (control). Means are
listed under the rank order. Rank indicates the
order from largest to smallest for Control, 1,
Acidosis, 2, and Alkalosis, 3. For Group C, rank
1, 2 and 3 indicate time periods; first 20 minutes,
1, next 60 minutes, 2, and last 40 minutes, 3.
Horizontal bars across ranks indicate means which
are not significantly different. NS indicates that
means are not significantly different.


FIGURE 16. Arterial and venous H+ concentration during
ventilatory states. The three columns on
the left are from Group A. The three columns
on the right are from Group B. Open columns
are arterial [H+]. Vertical bars are one
standard error of the mean. Upper bar is
for venous, lower bar for arterial SEM.


VENTILATION ( b re a t h s / mi nu te )
FIGURE 16


FIGURE 17. Mean arterial and venous PO2 during the
different ventilatory states. The three
columns on the left represent Group A.
The three on the right represent Group B.
Open bars represent venous PO2
and combined (open and closed) bars
represent arterial PO2. Vertical bars
represent one SEM.


100
ART ERIAL
and
VENOUS 50
po2
(mm Hq)
10 4 20 10 20 4
VENTILATION (breaths/minute)
FIGURE 17


79
hyperventilation than during the initial control period
(see Table I). There was no significant correlation
between arterial pH and dP/dt. This suggests, as seen
for AT, that the alterations in dP/dt associated with
ventilation patterns are not directly related to pH
(see Figure 18).
The peak rate of relaxation was greatest during
hyperventilation. In Group A, this was significantly
greater than both the control period and the period of
hypoventilation. In Group B, however, where arterial
pH increased to 7.52 during hyperventilation (as opposed
to 7.4 in Group A), -dP/dt during hyperventilation was
greater than that during normal ventilation, but not
significantly different from that during hypoventilation.
There was no significant correlation between -dP/dt and
arterial [H+].
In Groups A and C there were no significant changes
in Ct or Rt 1/2. In Group B, however, Ct was shorter
during hypoventilation than at other times. Rt 1/2 was
shorter during hyperventilation than at other times in
Group B (see Figure 19) .
Discussion
It has been suggested that intracellular acidification
is the cause of skeletal muscle fatigue (30). If there
is a direct influence of pH on the force of contraction,
this phenomenon would be independent of the manner in
which the pH change was obtained. It is apparent in


FIGURE 18. Mean developed tension, dP/dt and -dP/dt
during different ventilatory states.
Developed tension is % of highest in each
experiment. dP/dt and -dP/dt are % of
highest dP/dt in each experiment. Vertical
bars represent one standard error of the
mean.


aT mm dP/dt UZ3 -dP/dt CZJ
10 4 20 10 20
VENTILATION ( bre a t h s / m i nu te )
FIGURE 18


FIGURE 19. Mean contraction time and Half relaxation
time during different ventilatory states.
Vertical bars represent one SEM.


Rt 1/2 O Ct
I 0
20
VENTILATION ( brea t h s/m i nute )
FIGURE 19


84
TABLE V
STATISTICS FOR TWITCH CHARACTERISTICS VERSUS BLOOD GASES
Group
T
Rt
1/2
dP/dt
-dP/dt
Ct
p= r2=
P=
r2=
p= r2=
P=
r2=
P= r2=
Art [H+]
A
. 4
.001
00
00

. 8
.09
.17
B
.25
.06
. 26
.07
.1
C
. 33
.29
.4
.15
.22
ven [H+]
A
. 78
.002
.85
. 72
.2
.31
B
. 77
.13
. 75
.23
.03 .56
C
. 59
.26
.11
.24
.26
PaC02
A
. 25
.001
. 89
.57
.05
.52
.13
B
.04 .56
.04
.44
.04 .4
.01
.75
.25
C
.2
.13
. 15
.04
. 89
.06
Significance values are given for correlations between
twitch characteristics and blood gases. R2 values indicating
the percent of variability explained by the variable are
given for significant correlations (p<.05). Groups are
as defined in the legend for Table IV.


85
these experiments that the changes in Ct, Rt 1/2 and
-dP/dt are not directly associated with arterial pH.
This complication will be discussed further below.
However, to give consideration to the possibility that
acidosis causes fatigue, the results from Group A will
be discussed with respect to the likelihood that the
hypoventilation in these experiments resulted in alterations
in intracellular pH comparable to what might be expected
from contractions at 10/sec for 30 minutes.
In these experiments, arterial pH was reduced from
7.37 to 7.08 by hypoventilation. The acidosis accompanying
the hypercapnia was not associated with any change in aT,
Rt 1/2 or Ct. It would appear that the fatigue described
earlier cannot be a result of acidosis, unless intra
cellular pH during the fatiguing contractions changes
more than it did during hypoventilation.
The magnitude of the intracellular pH change occurring
in these experiments can be estimated from the results of
Burnell (12) Hypercapnia in dogs (PaCC>2 = 55 mm Hg)
resulted in a reduction of intracellular pH of neck
muscles from 6.85 to 6.57. Intracellular pH was
determined by the DMO method (12). Burnell (12)
observed that the maximal response had occurred within
15 minutes. In the experiments reported herein, arterial
PCO2 increased to 57 3.7 mm Hg. The measurements
presented were taken 60-90 minutes after the alteration
in ventilation. It is reasonable to assume that a very


86
similar change in intracellular pH occurred in these
experiments as was observed by Burnell, under almost
identical circumstances. It can therefore be concluded
that a change in intracellular pH values from normal
resting values (approximatley 6.85) to 6.57 does not
result in a change in developed tension.
The important point, however, is whether or not
intracellular pH fell to this level or lower during the
fatiguing contractions reported in earlier chapters.
Estimates of intracellular pH changes under these
circumstances can only be tentative. Venous PCO2 during
the fatiguing contractions was never greater than 57 mm Hg
(mean = 49 mm Hg at t = 10 minutes for 10/sec). This
hypercapnia would not cause an acidosis sufficient to reduce
AT (if reduced pH will cause reduced AT!). However, there
is lactic acid production during the first 5-20 minutes of
this type of stimulation (60). This is likely to contribute
to an intracellular acidification. Sahlin et al. (53)
report that in humans, performing maximal exercise to
exhaustion, intracellular pH drops from 7.08 to 6.6. This
is comparable to that seen by Hermanson and Osnes (39).
This decrease in pH was associated with an increase in
lactic acid production. Following the bout of exercise,
intracellular pH recovered to 7.0 within twenty minutes.
It is unlikely that intracellular pH changed as much in
the 10/sec fatigue as it did during the exhausting exercise
reported by Sahlin et al. (53). Furthermore, it seems


87
likely that intracellular pH would have returned to resting
levels after 40 minutes of recovery. Developed tension
at this time is still very much reduced (i.e., no significant
recovery has occurred). It seems reasonable to conclude
that the persistent fatigue caused by contractions at
10/sec for 30 minutes does not result directly from
acidosis. There may, however, be indirect ways in which
intracellular pH may affect the contractile process,
resulting in a change within the muscle which persists
beyond the time when pH has returned to control (26).
It is evident that changes in ventilatory pattern
do affect muscle contraction. During hyperventilation, AT
was increased. This was the case whether hypoventilation
preceded the period of hyperventilation or if normal
ventilation preceded it. In the former case (hypo
ventilation preceding) arterial pH returned to 7.4, so
there was no absolute arterial alkalinization. The
increase in AT under these circumstances was greater than
the increase seen in the latter case (hyperventilation
preceded by normal ventilation), despite the fact that
this procedure resulted in an increase in arterial pH
to 7.52. It seems that hyperventilation increases AT,
but hypoventilation only reduces AT when it is preceded
by hyperventilation.
The high p values reported in Table V reflect the
lack of relationship between AT and [H+]. The fact that
significant differences were observed between ventilation


88
periods is due not to absolute pH changes, but relative
changes possibly in conjunction with some other change
(ion distribution? i.e., see 26, 31) associated with
changes in ventilation.
The changes seen in Ct, dP/dt, -dP/dt and Rt 1/2
also suggest that alterations in ventilatory pattern
can affect contraction. The mechanisms responsible for
these changes are not clear.
In this study, it is likely that hypercapnia resulted
in intracellular acidosis. This acidification was not
accompanied by a reduction in AT. It can be concluded
that fatigue is not caused by acidosis if fatiguing
contractions do not cause any greater acidification than
that which occurred in these experiements.


SUMMARY
O2 Uptake and Developed Tension
The amount of oxygen used by a muscle was proportional
to the amount of tension developed. This occurred over a
wide range of forces when AT was altered by any of the
following:
a) Fatigue, after 30 minutes of stimulation at 14-20/sec
b) Fatigue, during fatiguing contractions at 3-6/sec
c) Twin impulse stimulation, before and after fatigue
at 14-20/sec
d) Fatigue, during contractions at 1/sec without blood
flow (ischemic fatigue)
e) Attenuated contraction, caused by administration of
curare in sufficient doses to reduce the force of
contraction by as much as 70%.
These results suggest that the major determinant of
energy utilization during an isometric contraction is the
magnitude of the developed tension. It should be emphasized
that these were twitches or very brief tetanic contractions
in which developed tension rose then fell, but did not
maintain a plateau of tension and can therefore be considered
to have a minimal "tension maintenance" component to the
determinants of energy utilization. The possibility that
neuromuscular junction failure may have contributed to
the observed fatigue was tested. It was demonstrated that
transmitter release was normal after 30 minutes of
stimulation at 20/sec.
89


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EHCQKHKWY_GSY6ZJ INGEST_TIME 2012-02-29T16:11:43Z PACKAGE AA00009126_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


FATIGUE IN SKELETAL MUSCLE
BY
BRIAN ROBERT MACINTOSH
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1979

ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to
Dr. W. N. Stainsby, Chairman of my Supervisory Committee,
for his valuable assistance and counsel over the past
four years. Acknowledgement is also due the other
members of my Committee: Dr. M. Fried, Dr. A. B. Otis,
Dr. P. Posner and Dr. C. W. Zauner. Each has unselfishly
contributed time and effort to provide me with the
guidance I needed to complete the requirements for this
degree.
Special thanks are expressed to Donna T. Dolbier, who
provided technical assistance and to Dr. L. Bruce Gladden
who collaborated with me on several research projects
during his Post-doctoral tenure with Dr. Stainsby.
Financial support for me during the pursuit of the
Ph.D degree has been provided by the following agencies
and departments:
NIH, grants to Drs. Stainsby, Otis and Cassin;
Department of Physiology (Teaching Assistantship);
College of Nursing (Teaching Assistantship).
The research reported in this dissertation has been
supported by The American Heart Association, Florida
Affiliate Grant # AG 7 and Sponsored Research Seed Grant
awarded to Dr. Stainsby.
I would like to thank Wendy Auerbach for doing an
excellent job of typing this manuscript.
li

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF FIGURES V
LIST OF TABLES vii
ABSTRACT viii
INTRODUCTION 1
EVENTS LEADING TO SKELETAL MUSCLE CONTRACTION .... 5
POSSIBLE FATIGUE MECHANISMS AND CURRENT THEORIES
OF FATIGUE 9
Central Nervous System 9
Neuromuscular Junction Failure 10
Attenuated Calcium Release 11
Reduced Capacity of the Contractile Apparatus . . 13
GENERAL METHODS 16
02 UPTAKE AND DEVELOPED TENSION 22
Introduction 22
Methods 23
Results 26
Discussion 34
EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE 41
Introduction 41
Methods 4 2
Results 48
Discussion 59
iii

Page
EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH
CONTRACTION 6 8
Introduction 68
Methods 69
Results 70
Discussion 79
SUMMARY 89
O2 Uptake and Developed Tension 89
Time-Course of the Twitch Contraction in
Fatigue 90
Acidosis and the Twitch Contraction 91
PROPOSED HYPOTHESES FOR SKELETAL MUSCLE FATIGUE . . 92
Depletion of Calcium at Lateral Sacs 92
Compartmentalization of Calcium Within the
Lateral Sacs 93
Attenuated Trigger for Release of Calcium ... 93
Reduced Binding Sensitivity for Calcium .... 94
CONCLUSIONS 9 5
APPENDIX 9 6
BIBLIOGRAPHY 100
BIOGRAPHICAL SKETCH 106
IV

LIST OF FIGURES
Page
1. Excitation-Contraction-Coupling 7
2. Gastrocnemius-Plantaris Muscle Preparation . . 18
3. Sample Experiments; VOg versus Developed
Tension 28
4. VOg versus Developed Tension; all Data,
Normalized 29
5. Twitch and Twin Contractions: Developed
Tension and dP/dt 39
6. Twitch Characteristics from Fast Traces of
Developed Tension and dP/dt 43
7. Procedure for Fatiguing Contractions with
Periodic Fast Traces 46
8. Q, POg, PCOg and pH During and Following
Fatiguing Contractions 50
9. Developed Tension, Half Relaxation Time and
Contraction Time 53
10. Peak Rate of Force Development and Peak Rate
of Relaxation 57
11. Tetanic Contraction Before and After Fatiguing
Contractions 58
12. Twitch Developed Tension and dP/dt Before,
During and After Ischemic Fatigue 60
13. Half Relaxation Time versus Developed Tension
for Dantrolene, Reduced Stimulation Voltage
and Ischemic Fatigue 61
14. The Effects of Preceding Contractions on
Developed Tension 63
15. Procedure for Ventilation and Sampling Pattern. 72
v

Page
16. Arterial and Venous [H+] 76
17. PC>2 During Different Ventilatory States .... 78
18. Developed Tension, dP/dt and -dP/dt During
Different Ventilatory States 81
19. Contraction Time and Half Relaxation Time
During Different Ventilatory States 83
vi

LIST OF TABLES
Page
I 02 UPTAKE AND DEVELOPED TENSION BEFORE AND
AFTER FATIGUE IN SERIES 1 30
II RELATIONSHIP BETWEEN 02 UPTAKE AND
DEVELOPED TENSION IN SERIES 2-5 32
III PHOSPHORYLCREATINE ANALYSIS DURING
CONTRACTIONS AND FOLLOWING THE RECOVERY
PERIOD 55
IV STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP 74
V STATISTICS FOR TWITCH CHARACTERISTICS
VERSUS BLOOD GASES 84
Vll

Abstract of Dissertation Presented to the Graduate
Council of the University of Florida
In Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
FATIGUE IN SKELETAL MUSCLE
By
Brian Robert Macintosh
June 1979
Chairman: Wendell N. Stainsby, D.Sc.
Major Department: Physiology
The in situ dog gastrocnemius-plantaris muscle
preparation has been used to study fatigue. Skeletal
muscle fatigue (reduced force output for a given stimulus)
results from a thirty minute period of isometric
contractions at 2.5 to 20/sec. This fatigue is not a
result of failure of motor nerve propagation or transmitter
release. The ratio of oxygen uptake to developed tension
(total tension minus resting tension) is unaltered during
or following fatiguing contractions. The economy of force
production is unaltered by twin impulse stimulation,
relative ischemia or administration of moderate doses of
curare or succinylcholine.
vii i

When developed tension is reduced due to repetitive
stimulation for thirty minutes at 2.5, 5 or 10/sec
contractions, the time to peak tension and half relaxation
times are unaltered. The peak rates of force development
and of relaxation are reduced proportionally to the
reduction in developed tension.
Following a forty minute period of recovery, the
twitch developed tension remains greatly attenuated, but
tetanic (200 msec of 100/sec stimulation) developed tension
is virtually the same as it was before the stimulation
period. Phosphorylcreatine is restored to resting levels
within forty minutes of recovery. Also, at this time,
blood flow and oxygen uptake have returned to pre-fatigue
values and venous PC>2, PCC>2 and pH are at resting levels.
The fatigue observed in these experiments appears to
be due to a reduction in the intensity of activation
obtained with a single impulse. Energy sources are
available and with maximal activation the contractile
mechanism is capable of the same force output it had
before the fatiguing contractions.
Further experiments were conducted to determine if
intracellular acidosis could have been the cause of the
reduced intensity of activation. A sixty to ninety
minute period of hypoventilation with an air mixture high
in O2 (arterial PO2 was maintained at 75-100 mm Hg) resulted
in a reduction in arterial pH to 7.08. There was no
reduction in twitch developed tension associated with this
IX

acidosis. It is likely that intracellular pH fell as
much during the respiratory acidosis as it did during
fatiguing contractions at 10/sec. The fatigue observed
during contractions at 10/sec could not be a result of
intracellular acidosis.
It can be concluded from these experiments that
twitch fatigue is not a result of energy deficiency,
reduced capacity of the contractile elements, intra¬
cellular acidosis (induced by reduced ventilation for
sixty to ninety minutes) or neuromuscular junction
failure. By the process of elimination it appears
that twitch fatigue results from a reduced activation
of the myofilaments during a twitch contraction. This
may be due to either a reduced sarcoplasmic Ca^+
concentration during contraction or a reduced response
of the myofilaments at a given Ca^+ concentration.
x

INTRODUCTION
The word fatigue has been used in the past with
several various definitions. Some authors (1, 52) equate
fatigue with exhaustion. Others (47, 50) use the word
fatigue to represent an inability to maintain a particular
work output. More recently (23, 30) skeletal muscle
fatigue has been defined as a reduced capacity of the
muscle to develop tension. Edwards et al. (23) and Fitts
and Holloszy (30) have observed that a twitch contraction
can still be attenuated when the force generating capacity
of the muscle is fully recovered.
A single impulse does not maximally activate the
contractile apparatus (17) and therefore does not permit
the full contractile response of which the muscle is
capable. The capacity to develop tension must be
evaluated under conditions of maximal activation. This
can be accomplished with a caffein or K+ induced
contracture or a maximal tetanic contraction.
If fatigue is defined as a reduced capacity of the
muscle to generate force, then what is it called if there
is an attenuated response to a single impulse? It appears
to be a separate phenomenon and probably occurs by a
separate mechanism (since a muscle recovers full capacity
to develop tension before twitch tension recovers to
pre-fatigue values). Edwards et al. point out (23) that
1

2
this "twitch fatigue" may be associated with perception
of increased effort necessary to maintain a given workload
or force output- For the purposes of this dissertation,
fatigue will be used as a generalization referring to a
reduced response of the muscle to a given stimulation.
Twitch fatigue will refer to an attenuated response to a
single impulse.
Wilson and Stainsby (66) have reported that twitch
developed tension of the in situ dog gastrocnemius-
plantaris muscle remains attenuated for hours following
a series of contractions at 10-14 per second. Fitts and
Holloszy (30) have reported that tetanic developed tension
recovers quickly following a period of repetitive
stimulation. This may also be the case for the in situ
dog gastrocnemius-plantaris muscle. If this is so, then
the fatigue observed by Wilson and Stainsby is only twitch
fatigue. This preparation, then, would provide a model
for studying twitch fatigue independent of tetanic fatigue.
Very little is known of the fatigability of the canine
gastrocnemius-plantaris muscle or of the mechanisms
responsible for that fatigue. The purpose of the present
study was to provide further information regarding the
fatiguing effects of repetitive stimulation on the
gastrocnemius-plantaris muscle.
To facilitate the reader's understanding of skeletal
muscle fatigue, a brief description of pertinent muscle
physiology and current theories of fatigue precedes the
sections describing the experiments which have been done.

3
The dog in situ gastrocnemius-plantaris muscle group
has been used throughout the series of studies reported
herein. To avoid repetition, the general procedures and
description of the preparation appear as a separate
chapter before any of the studies. The specific
procedures used in each study are described separately
in a Methods section for that study.
In the first study, the relationship between oxygen
uptake and developed tension has been determined for
skeletal muscle before, during and after fatiguing
contractions. These experiments were done to determine
whether or not the energy used by a muscle relative to the
amount of tension developed is altered by fatigue. There
are reports that indicate a change in either direction can
be expected (7, 21, 28).
Further studies were conducted to determine whether
or not changes occur in the time-course of the twitch as
a result of repetitive stimulation. Changes in the rate
of force development and in the time-course of a twitch
contraction have previously been interpreted as indications
of changes in the duration and intensity of activation of
the muscle (17, 36). These measurements may facilitate
an understanding of the mechanism(s) responsible for the
fatigue.
A third series of experiments has been conducted to
study the effects of respiratory acidosis on the twitch
contraction. Acidosis has been claimed to be one of

4
the major causes of fatigue (29). If this is the case,
respiratory acidosis should reduce the developed tension
of a twitch contraction.
In the final chapter of this dissertation, a brief
discussion of the possible theories for the mechanism of
fatigue observed in these experiments is presented. It
is evident that further research will be necessary to
permit evaluation of these theories with respect to
fatigue of the canine gastrocnemius-plantaris muscle
group. However, considerable evidence has accumulated
as a result of my studies, which disputes several of the
current theories of fatigue.

EVENTS LEADING TO
SKELETAL MUSCLE CONTRACTION
Muscular contraction is the result of a sequence of
chemical and physical events beginning with activity in
the central nervous system (CNS)(or sensory input to the
CNS). Failure or impairment at any site in this process
will result in a reduced contractile response of the
muscle. Fatigue and twitch fatigue, then, are results
of such failure. The identification of the site(s) of
failure in fatigue would provide a better understanding
of the mechanism(s) effecting the fatigue. Below, a
brief discussion of the normal sequence of events leading
to contraction is presented. CNS control of motor nerve
activity is complex and will not be described. For
simplicity, this discussion is based at the cellular level.
This sequence of events is described in a number of text¬
books (45, 62) and is illustrated in Figure 1. Following
the presentation of events leading to contraction, each
step in the sequence is considered as a potential site
for a mechanism of fatigue.
The sequence of events occurring at the nerve
terminal may be susceptible to failure. The arrival of
an action potential at the nerve terminal triggers the
release of acetylcholine from the terminal bouton.
Synaptic vesicles fuse to the terminal membrane and
5

6
release their contents into the synaptic cleft.
Acetylcholine diffuses the short distance across the
cleft (500 A).
Binding of acetylcholine to specific receptors
causes a transient increase in permeability of the muscle
membrane to Na+ and K+. This results in depolarization
of the end plate. The resulting change in membrane
potential is called the end-plate potential. Destruction
of the acetylcholine is accomplished by acetylcholines¬
terase which is located among the receptors on the post-
synaptic muscle membrane. Reconstitution of synaptic
vesicles is accomplished by reuptake of choline and
subsequent acetylation in the Golgi apparatus (enzyme:
choline-acetyl-transferase). Portions of the Golgi
complex, containing acetylcholine, are pinched off,
forming new synaptic vesicles.
A single action potential on a motor neuron usually
generates an end plate potential large enough to bring
the adjacent membrane area to threshold. Propagation of
an action potential over the membrane ensues. Transverse
tubules, located at regular intervals along the length of
the muscle fiber permit rapid communication with deep
portions of the muscle. Depolarization of transverse
tubules triggers release of calcium from the lateral sacs.
Lateral sacs are the terminal portions of the sarcoplasmic
reticulum lying adjacent to the transverse tubules. The
Ca^+ released from the lateral sacs raises the sarcoplasmic

7
FIGURE 1. The sequence of events occurring in
excitation-contraction coupling are
listed below. The numbers refer to
numbered events shown in the diaqram
above.
1. An action potential travels along a
motor nerve.
2. Acetylcholine which has been released
from the nerve terminal binds to
receptors on the muscle membrane,
causing depolarization - an end-plate
potential.
3. When the end-plate potential reaches
a threshold value an action potential
is fired. This action potential is
propagated over the entire muscle
membrane, and causes depolarization
of the transverse tubules.
4. Depolarization of the transverse tubules
triqgers release of Ca2 + from the lateral sacs.
5. Ca2 + which has been released, binds to troponin
which is associated with the thin myofilaments.
Contraction results.
6. Ca2+ is reaccumulated by an active transport
mechanism located in the longitudinal
tubules. Relaxation occurs.

8
free Ca2+ concentration. The subsequent binding of Ca2+
to troponin results in activation of the contractile
proteins in the muscle. The amount of Ca2+ released in
response to one action potential propagated over the
muscle membrane is not sufficient to saturate the troponin
molecules and therefore, incomplete activation occurs (17).
For complete activation and therefore maximal force
production, a period of nerve activity at a high frequency
is necessary. Relaxation occurs as Ca2+ is sequestered
(active transport) by the longitudinal sarcoplasmic
reticulum. Following reuptake, calcium is translocated
along the longitudinal reticulum to the lateral sacs,
completing the Ca2+ cycle (67) . The mechanism of this
translocation is unclear.

POSSIBLE FATIGUE MECHANISMS AND
CURRENT THEORIES OF FATIGUE
Central Nervous System Fatigue
Events initiating muscular contraction originate
from sensory input or directly in the central nervous
system. Any study of fatigue during exercise of the
whole animal must consider the possibilities of central
inhibition resulting in reduced muscular performance.
There are conflicting reports concerning the potential
for a central component in muscular fatigue. For example,
Merton (44) found that maximal voluntary effort was not
different from the response of the muscle to maximal
tetanic stimulation of the motor nerve. He was studying
brief contractions of the adductor pollicis of humans.
Conversely, Asmussen and Mazin (1) have reported that
"diverting activity" (visual stimulation) permits greater
muscular performance than that which is accomplished when
the eyes are closed. Further experiments demonstrated
that immediate recovery from exhausting exercise (with
eyes closed) occurred if the eyes were subsequently
opened.
It is apparent from the work of Asmussen and Mazin
(1) that central effects can alter muscular performance.
It is important to keep in mind though, that under some
9

10
circumstances (i.e., brief maximal effort) fatigue appears
to be due entirely to peripheral mechanisms (44).
Neuromuscular Junction Failure
In the normal sequence of events preceding a muscular
contraction, an action potential is propagated over the
muscle membrane. The occurrence of a normal muscle action
potential is dependent on transmitter release and muscle
membrane properties. Repetitive stimulation may alter
these properties, and this could result in alterations in
the contractile response. Merton (44) found no change
in fatigue in the electromyogram resulting from maximal
stimulation despite an attenuation of force output.
Bergmans (3) studying human extensor digitorum brevis
observed no change in the surface electromyogram during
fatiguing contractions. Electromyography is not the
most sensitive technique for measuring the membrane
response, but any large alteration in muscle action
potential generation and propagation would probably
have been detected.
Using small muscle bundles, and measuring intracellular
potentials, Hanson (37) noted only minor changes in the
rat soleus muscle resting potential and action potential
following repetitive stimulation. The amplitude of the
action potential was reduced in fatigue, but was restored
within a few minutes of recovery. Grabowski (35) noted
a reduced amplitude of the muscle action potential of
fatigued frog muscle fibers. A reduced amplitude could

11
also be produced in a rested muscle by reducing extra¬
cellular Na+ concentration. Under these conditions,
twitch height is not altered. It would appear from the
results of these experiments that following a period of
fatiguing contractions, the amount of neurotransmitter
released is sufficient to raise the end-plate potential
to threshold, and the muscle membrane is capable of
propagating a muscle action potential.
Attenuated Calcium Release
If a normal action potential is propagated over a
muscle membrane, but less calcium is released from the
lateral sacs, the contractile response will be attenuated.
The reduced amount of Ca2+ released would result in a
lower peak sarcoplasmic Ca^+ concentration and therefore
a reduced activation. Direct measurement of Ca release
in a fatigued muscle has not been reported. Despite this,
several authors have concluded that the mechanism
responsible for the fatigue they observed was reduced
Ca2+ release (15, 19). This conclusion is based on
results from one of two techniques: either a) all other
possibilities are eliminated or b) inference is obtained
from analysis of changes in the time to peak tension and
the peak rate of force development for a twitch. In the
former, evaluation of the functional state of the
neuromuscular junction and of the force generating
capacity of the muscle has revealed that these processes
are unaltered in the fatigued muscle. This leads one to

12
believe that the muscle has a reduced amount of Ca2+
released. In the latter, it is assumed that relaxation
occurs simultaneously with Ca2 + reuptake (4). Under these
circumstances, a reduction in contraction time would
result from a reduction in duration of activation (more
f
rapid reaccumulation or shorter duration of release). A
reduction in peak rate of force development without a
concommitant reduction in contraction time indicates
reduced activation, and this is interpreted as a reduced
amount of Ca2+ released. Brust has made observations
similar to these (reduced rate of force development in
fatigue with no change in contraction time) on mouse
soleus muscles in vitro, (10) and concluded that fatigue
was due to reduced Ca2+ release.
Similar observations would be expected if there was
an increase in the Ca2+ concentration at which binding to
troponin and subsequent contractile activity occurs. It
has been observed by Fuchs et al. (32) that the affinity
of troponin for Ca2+ can be altered by pH. This
possibility must be considered when dealing with
inferences from measurements of contraction time and
rate of force development. Fitts and Holloszy (29) have
presented data indicating that reduced pH may be
associated with fatigue. They support the theory that
reduced activation (and reduced rate of force development)
is due to a reduced affinity of troponin for Ca2+.

13
Another situation may occur in the muscle for
which the rate of force development declines with developed
tension while contraction time remains unchanged. A
reduction in contractile capacity would give the same
results. This possibility must be given consideration.
Some authors have tested for, and found, changes in the
contractile capacity of the muscle under study (30, 44).
These are discussed below.
Reduced Capacity of the Contractile Apparatus
Fatigue may be the result of a reduced ability of the
contractile proteins to generate tension. This could be
a result of either: i) damage to myofilaments (i.e.,
misalignment or inactivation) or ii) restricted availability
of energy. In either case, the effect would be a reduced
force generation under conditions of maximal activation.
This capacity to develop tension has been traditionally
tested with either a K+ contracture or a caffein
contracture. Both of these procedures result in maximal
activation (Ca2+ concentration high enough to saturate
the contractile apparatus). Tetanic stimulation has also
been used to evaluate the capacity of a muscle to generate
tension.
Fitts and Holloszy (30) observed that tetanic force
was reduced in the rat soleus muscle following a series
of tetanic contractions. They noted that recovery of the
force generating capacity occurred relatively quickly
(within minutes). No insight into the mechanism

14
responsible for the fatigue observed by these authors is
provided. Since recovery occurred quickly, it is obvious
that permanent damage to the myofilaments was not a
mechanism of the fatigue.
Spande and Schottelius (56) studied fatigue in the
mouse soleus muscle in vitro. They found that the
magnitude of the reduction in developed tension was
inversely proportional to the phosphoryl-creatine (PC)
concentration. PC serves as an immediate source of high
energy phosphate (~P), for rephosphorylation of ADP and
may also be involved in a transport capacity for ~P from
mitochondria to myofilaments (40, 55). The experiments
by Spande and Schottelius (56) involved contractions with
periods of anoxia and/or glucose deprivation, and this
must be kept in mind when comparing their results with
those of other authors. Under these circumstances
reduced energy availability appears to be related to the
fatigue. Fitts and Holloszy (29) have measured PC
changes during and following fatiguing contractions in
rat muscle. They found no relationship between PC and
the amount of fatigue or recovery from fatigue.
The final common mediator of energy availability is
the level of ATP in the muscle. Edwards reported that
ATP and PC concentrations were reduced in isolated
mouse soleus muscles during prolonged tetani under
anaerobic conditions. This was also the case when
muscles were fatigued in the presence of cyanide and

15
iodoacetic acid. In the former case, lactate accumulated
but in the latter case there was no accumulation of
lactate. It was noted that prolongation of relaxation
was associated with a reduction in ATP and PC levels.
This provides an indirect method of evaluating energy
availability in the muscle. Relaxation would be expected
to be prolonged since it is dependent on reuptake of Ca^+.
Sequestering Ca^+ is an active transport process which
requires ATP (13). Reduced levels of ATP may also slow
the relaxation phase of individual cross-bridges. ATP
is required to permit dissociation of the actin and
myosin molecules (65). The extreme of this situation
occurs when rigor bonds form in the absence of ATP.
It can be concluded from the above discussion that
fatigue can be the result of any of several mechanisms.
The possibility exists that multiple mechanisms function
at once. For example, a reduced release of Ca^+ may be
accompanied by a limitation of energy availability. This
situation would complicate the elucidation of the
mechanism(s) responsible for the fatigue.
The following chapters present the details of
experiments conducted in an effort to gain an under¬
standing of fatigue in the gastrocnemius-plantaris
muscle group of the dog.

GENERAL METHODS
Mongrel dogs of either sex weighing 9-18 kg were
used in these studies. They were anesthetized with
intravenous sodium pentobarbitol, 30 mg/kg, with
additional 30 mg injections as needed. The animals
were intubated and maintained on a respirator throughout
the experiment. A Beckman LB-2 gas analyzer sampled gas
from the endotracheal tube continuously. Ventilation
was adjusted to maintain end-tidal CO2 at 4.5 - .25 %.
Rectal temperature was monitored with a thermocouple,
and kept between 37.5 and 38°C by appropriate adjustment
of a heating pad placed under the thorax of the supine
dog.
The left gastrocnemius-plantaris muscle was exposed
via an incision along the medial aspect of the left hind
limb. Muscles overlying the medial head of the
gastrocnemius-plantaris muscle group were tied twice
with butcher's cord and cut between the ties. These
muscles are: sartorius, gracilis, semitendinosis and
two heads of semimembranosis. All veins draining into
the popliteal vein were ligated except those branches
coming from the gastrocnemius-plantaris muscle (see
Figure 2). Any veins draining the muscle but not
entering the popliteal vein were ligated. These were
16

FIGURE 2.
The in situ dog gastrocnemius-plantaris
preparation (58).
G - gastrocnemius-plantaris muscle
Gr- gracilis muscle
S - sartorius muscle
SM- two heads of semimembranosis muscle
ST- semitendinosis muscle
muscle

VENOUS
SAMPLE
FIGURE 2
6fY//////////////////// / /

19
only minor vessels which occur along the anterior or
lateral surfaces of the muscle. The popliteal vein was
cannulated. A cannulating type electromagnetic flow
probe (Narco Biosystems) (3mm I.D.) was placed in the
outflow tubing. The venous effluent was returned to the
dog via another cannula in the external jugular vein.
Heparin, 2000 U/kg (12 mg/kg) was administered I.V.
initially and 1000 U/kg was given half way through the
experiment, prevented coagulation of blood in the tubing.
A thin cannula passed through the wall of the outflow
tubing and threaded within it to the muscle provided a
sampling port for venous blood. A thermocouple was placed
alongside this thin tube. The tip of the probe was
within 1 cm of the muscle. The blood temperature here
was assumed to be an average temperature of all parts of
the muscle. A heat lamp focused on the abdomen and hind
limbs was used to maintain muscle temperature near 37°C,
while the muscle was at rest. During contractions, the
lamp was turned off and the muscle temperature was
permitted to rise. The contralateral femoral artery was
cannulated and a Statham pressure transducer was connected
to the cannula. Output of the transducer was recorded
on a Grass polygraph model #5.
The Achilles tendon was severed close to the
calcaneous and securely fixed in an aluminum clamp.
The clamp was hooked to a slide bar which was fastened
to the cantilever beam of an isometric lever. Force

20
was measured with a displacement transducer detecting the
displacement of the free end of the cantilever beam. The
transducer output was linear for forces up to 20 kg. A
displacement at the transducer of 0.1 mm gave a full
scale deflection on the recorder. Output of the displace¬
ment transducer (tension) was amplified and recorded
directly. The amplified tension signal was also
differentiated with respect to time (Gould-Brush dif¬
ferentiator) . The differentiated and direct signals
were recorded on a Gould-Brush Model 2400 recorder.
Blood flow and muscle temperature were also recorded
continuously. The maximal rate of change of the
amplified force signal never exceeded 80 v/sec. The
differentiator was calibrated with ramp signals and was
found to be linear through 120 v/sec.
The sciatic nerve was dissected free from
surrounding tissue. All branches of the nerve not
innervating the gastrocnemius-plantaris muscle were
severed. The nerve trunk was double ligated about 4
cm proximal to the muscle and cut between the ties. A
tubular stimulating electrode was placed on the distal
stump of the nerve. The nerve was stimulated with a
Grass Model SD9 stimulator with square pulses 0.2 msec
in duration and of 2-4 volts. This voltage was double
that necessary to produce a maximal contraction.
Contractions were isometric. The lever-arm of the
myograph was bolted to a cast iron base which was

21
clamped to the table. Bone nails were placed in the
tibia and femur (one each). These nails were firmly
attached to the base of the myograph. A turnbuckle
strut, placed between the lever-arm and one of the
bone nails prevented flexing of the lever-arm. The
muscle length was set 1-2 mm shorter than the length at
which developed tension was greatest (optimal length).
Optimal length was determined by measuring the developed
tension (total tension minus resting tension) of
contractions at (0.2/sec) at various lengths.

02 UPTAKE AND DEVELOPED TENSION
Introduction
Oxygen uptake (V02) of muscle can increase more than
40 times resting levels during repetitive stimulation (57).
At low frequencies of stimulation, V02 is proportional
to the isometric developed tension (AT) (total tension
minus rest tension) (66). This relationship was
observed for contractions following a period of fatiguing
contractions at 10-14 per sec for 30 minutes (66). By
stimulating the motor nerve with twin impulses, AT can
be increased. It is not known whether the proportionality
between V02 and At persists for twin impulses stimulation
before or after fatiguing contractions. It is of interest
to determine whether or not the muscle is capable of
increasing its V02 following fatigue, and if so, to see
if AT is still proportional to V02.
The purpose of this study is to investigate the
effect of fatigue and twin impulse stimulation on the
ratio between V02 and isometric developed tension in
the in situ dog gastrocnemius muscle. Further experiments
have also been conducted to determine the VO2:AT relation¬
ship for muscle "fatigued" by curare infusion or ischemia
during repetitive stimulation.
22

23
Methods
The preparation described in the General Methods
section was used in these experiments. Five series of
experiments were completed to determine the relationship
between muscle VO2 and AT.
Oxygen uptake by the muscle was calculated from the
venous outflow and the arteriovenous blood oxygen content
difference. Arterial samples were taken from the
contralateral femoral artery. Venous samples were taken
from the popliteal vein cannula via a thin catheter
threaded through the wall of the venous outflow tubing
to the end of the cannula close to the muscle group.
The blood samples, 0.8 ml each, were collected in glass
tuberculin syringes sealed with mercury-containing caps
and kept in ice until analyzed for O2 content with a
Lex O2 Con analyzer.
Series 1 and 2
Contractions began at the rate of 1/sec, and O2
uptake and developed tension were measured after a steady
level had been attained. Next, the muscle was fatigued
by stimulating it at a rate of 10-20 impulses / sec for
30-40 minutes. This reduced the developed tension in a
single twitch to about one-third to one-half of the pre¬
fatigue level. The muscle was allowed to recover for 30-
40 minutes so that the resting VO2 approached the pre¬
fatigue level. Three pairs of blood samples were taken
five minutes apart as the muscle continued to recover.

24
After this recovery period, the muscle was stimulated
at the same rate as before (1/sec) with twin impulses
(two impulses, 6.5 v in amplitude, 0.2 msec in duration
and separated by 10-20 msec), and O2 uptake and developed
tension were measured. The time between the twin impulses
was set by adjusting the delay between impulses until a
smooth contraction was obtained. Post fatigue stimulation
with twin impulses returned the developed tension
approximately to the level of single impulse stimulation
pre-fatigue. In the second series of experiments, both
single and twin impulse contractions were done before
and after the fatiguing contractions.
In each type of contraction, the muscle was allowed
to contract for at least four minutes before arterial and
venous samples were taken to ensure that developed tension
and blood flow had reached a steady level. After an
additional two to three minutes of contractions, a
second pair of arterial and venous samples was collected.
The O2 uptake rates calculated from the two pairs of
blood samples were averaged and the resting O2 uptake
rate was subtracted to give the net O2 uptake per minute.
This value was divided by the muscle weight and the
number of contractions per minute to give the O2 uptake
in microliters of O2 per gram of wet muscle per
contraction (pi O2 * g“l * C_1) . Developed tension was
expressed as grams of developed tension per gram of wet
muscle (g* g“l) .

25
Series 3
Oxygen uptake and developed tension were measured
during the fatigue process. In separate experiments,
muscles were stimulated at rates of 3, 4, 5 and 6 impulses
per second. After the first five minutes of contractions,
blood samples were collected periodically as the muscle
fatigued during contractions for two hours. The decrease
in developed tension ranged from 34 to 45% over the two
hour period. Sixty to 80% of this decrease occurred in
the first 30 minutes. Although blood flow and developed
tension were sometimes changing rapidly, there was almost
no change in the arteriovenous blood oxygen content
differences. This allowed application of the Fick
equation for C>2 uptake calculation with confidence (64).
Series 4
Oxygen uptake and developed tension were measured in
muscles during different levels of reduced blood flow
produced by partially occluding the arterial inflow.
The muscles were stimulated to contract at one twitch
per second throughout these experiments.
Series 5
It is possible that a portion of the fatigue
observed in the experiments of Series 1-3 might be due
to presynaptic neural failure or neuromuscular junction
failure, particularly in the first series of experiments
in which the nerve-muscle preparation was stimulated at
rates of 10-20 impulses / sec for 30-40 minutes. To

26
investigate this possibility, two experiments were done,
in which neuromuscular transmission was completely
blocked by repeated injections of either curare or
succinylcholine. After the drug was given, the nerve
was stimulated at the rate of 20 impulses / sec for
30 minutes. Muscle contraction did not occur during this
30 minute period because of the presence of the blocking
drug. Developed tenion (at a stimulation rate of 1/sec)
was measured before the drug was injected and after the
effects of the drug had worn off. Therefore, any
difference in developed tension before and after the
period of high frequency stimulation with curare block
would be due to either nerve or neuromuscular junction
failure since the muscle did not contract during the 30
minutes of stimulation.
Neuromuscular fatigue was mimicked in a fresh muscle
by infusing curare into the animal at different rates to
block muscle contraction to varying degrees. 02 uptake
and developed tension were measured at the different
levels of neuromuscular blockade. The stimulation rate
was 1/sec.
Results
Resting O2 uptake for the gastrocnemius-plantaris
muscle averaged 7.7 y 1 O2 ' g-^ * min--*-. This is somewhat
higher than average values previously reported (27, 57)
but well within the usual range. Mean arterial blood
pressure remained above 100 mmHg throughout all of the
experiments.

27
In the first series of experiments, analysis of
variance for repeated measures (8) on the ratios
between O2 uptake and developed tension observed before
and after fatigue revealed no significant difference
(p>.25). Table I shows the O2 uptake and developed
tension for each of the muscles both before and after
fatigue.
The results of the O2 uptake and developed tension
measurements in series 2-5 are summarized in Table II
and illustrated in Figures 3 and 4. Table II shows the
linear regression equations relating O2 uptake and
developed tension for each experiment. These equations
were calculated from data which included values from
the fatigued muscle as well as the fresh muscle. The
slopes of all but one (Experiment 8, p=.09) of the lines
are significantly different from zero (p<.05), despite
the small number of points used to determine each
regression equation. It is obvious from Figure 3 and
Table II that there was considerable variability between
animals. This has always been observed in this preparation
(27, 57, 66). However, despite differences in absolute
values between different animals, the same pattern of
response was observed in all cases. VO2 per contraction
and AT were directly related.
Results of four sample experiments from series 2-5
are shown in Figure 3. Figure 4 shows that all of the

UPTAKE (Ml 02 g-I C-l)
28
FIGURE 3. Results of four sample experiments.
Numbers refer to individual experiments.
Thirteen is from Series 2 (circled
numbers = post fatigue). Sixteen is
from Series 3. Nineteen is from
Series 4. Twenty-two is from Series
5.

29
FIGURE
X
it
Data from Series 2-5 normalized to the same
scale. Developed tension in percent of the
greatest developed tension in each experiment.
02 uptake in percent of the 02 uptake at the
greatest developed tension. Numbers refer to
individual experiments. Seven to fourteen are
Series 2 (circled numbers = post fatigue). Fifteen
to eighteen are Series 3. Nineteen to twenty-one
are Series 4. Twenty-two to twenty-five are
Series 5. The asterisk denotes (100%, 100%)
which is common to all of the experiments. The
line in this figure is the line of identity (X=Y).

TABLE I
02 UPTAKE AND DEVELOPED TENSION BEFORE AND AFTER FATIGUE IN SERIES 1
Pre-Fatigue (Single Impulses) Post-Fatigue (Twin Impulses)
Developed Developed
Experiment
Tension
(a-g-1)
O2 Uptake
(ul 09-g-1-C-1)
Tension
(g*q-1)
O2 Uptake
(y1 0?•g-1•C~
1
148
.688
125
. 361
2
196
. 314
177
.356
3
254
.579
250
.446
4
134
. 396
174
.435
5
207
.452
178
.258
6
219
. 382
197
.476
Mean ± SEM
193 ± 18
.468 + .057
184 ± 16
.389 ± .033
Units are as given for Figure 3
u>
o

TABLE II
RELATIONSHIP BETWEEN 02 UPTAKE AND DEVELOPED TENSION IN SERIES 2-5
*N equals the number of data points in each experiment
+ Units for O2 uptake are microliters of O2 per gram of wet
muscle per contraction
Units for tension are grams per gram of wet muscle.

Series #
Expt. #
N*
Type of Fatigue
2
7
4
10/sec for
30
min
8
4
10/sec for
30
min
9
4
10/sec for
30
min
10
4
10/sec for
30
min
11
4
14/sec for
40
min
12
4
20/sec for
30
min
13
4
20/sec for
30
min
14
4
15/sec for
30
min
3
15
7
Continuous
at
3/sec
16
8
Continuous
at
4/sec
17
10
Continuous
at
5/sec
18
10
Continuous
at
6/sec
4
19
5
Ischemia
20
6
Ischemia
21
8
Ischemia
5
22
7
Partial Curare Block
23
8
Partial Curare Block
24
8
Partial Curare Block
25
7
Partial Curare Block
% Variance
Regression Equation* Explained
VO 2
=
3-3-10-3
T
-
9-3.10-2
99.8
VO 2
=
4 - 2•10“3
T
+
9•0•10“2
83.4
VO 2
=
3-6-10-3
T
-
2-6-10-1
89.7
VO 2
=
3-9-10-3
T
-
4-8-10"1
89.5
VO 2
-
2-8-10-3
T
-
2-0-10"1
98.0
VO 2
=
5-1-10-3
T
-
1-9-10-1
99.6
vo2
=
2-7-10-3
T
-
1-2-10"1
98.4
vo2
=
2-2-10-3
T
-
4 -5-10-2
98.6
VO 2
=
4-0-10-3
T
-
2-8-10-1
85.0
vo2
=
5-0-10"3
T
-
8-2-10-3
93.7
VO 2
=
3 * 4"10“3
T
+
8-1-10-2
95.2
VO 2
=
3-0-10-3
T
-
9-1-10-2
90.2
vo2
=
1-3-10"3
T
-
1-3*10-2
91.6
VO 2
=
4 - 6 -10-3
T
-
7-6-10-2
88.9
vo2
=
5 * 1 • 10“3
T
-
2-2-10-1
91.6
VO 2
=
4•6•10“3
T
+
1-1-10"1
97.2
vo2
=
1-5-10-3
T
-
7-2-10-2
97.7
VO 2
=
5-5-10-3
T
-
1*9*10-1
97.7
VO 2
=
5•2.10“3
T
-
2-6-10-1
95.9
oj
to

33
data follow the same pattern when normalized to the same
scale. In this figure, developed tension is plotted as
the percent of the highest tension developed in each
individual experiment, and O2 uptake is plotted as the
percent of the O2 uptake at the highest developed tension.
Most importantly, Figures 3 and 4, and Tables I and II
show that the relationship between O2 uptake and developed
tension was unchanged by the various treatments.
In two experiments, muscle contraction was completely
blocked by repeated injections of curare or succinylcholine
while the nerve was stimulated 20 times per second for
30 minutes. Injection of the blocker was discontinued
after the stimulation period and the effects of the
blocker were mostly dissipated within 10 minutes.
Developed tension was still at least 90% of the control
value. The observed reduction in contraction strength
may have been due to incomplete recovery from the
neuromuscular block. This 10% reduction in developed
tension can be compared with the 50-70% reduction
observed in the other experiments in which muscle
contraction was not blocked. It appears that most if
not all of the reduced contractile response was due to
alterations beyond the neuromuscular junction.
As pointed out in the Methods, the fatigued muscles
in the first and second series of experiments were allowed
to recover for 30-50 minutes. After this time, the
resting O2 uptake approached the pre-fatigue level.

34
However, developed tension recovered very little during
this time and was still only one-third to one-half of
the pre-fatigue value.
Discussion
Isometric developed tension at constant muscle length
was varied in this study by four methods: 1) twin impulses
stimulation, 2) fatigue produced by 30 minutes of
contractions at 20/sec, 3) ischemia caused by partial
occlusion of arterial inflow to the muscle, and 4) partial
block of neuromuscular transmission with curare. Figures
3 and 4, and Tables I and II show that none of these
treatments changed the relationship between 02 uptake
and developed tension. Stimulating the fatigued muscle
with twin impulses restored developed tension to pre¬
fatigue values. Fatigue did not increase the O2
requirement per unit of force developed, even when the
tension developed by the fatigued muscle was returned
to the pre-fatigue level by twin impulses stimulation.
The fatigued gastrocnemius-plantaris muscle is therefore
capable of increased developed tension and increased
V02. In addition, the 02 requirement per unit of force
developed was not altered during the development of
fatigue.
The dog gastrocnemius-plantaris muscle group has
certain advantages in studies of fatigue. Based on
histochemical staining properties, the dog gastrocnemius
contains only two motor unit types (43). These two

35
correspond to types FR and S (SR), (fast, fatigue
resistant and slow, fatigue resistant respectively),
described by Burke and colleagues (11) for hindlimb
muscles of the cat. Even though the dog gastrocnemius-
plantaris muscle group contains both FR and S units,
and the cat soleus muscle contains only type S units,
homogenates of cat soleus muscle have less than one-
third of the succinate oxidase activity of homogenates
of the dog gastrocnemius-plantaris muscle group (43).
From this, one might expect all of the dog gastrocnemius-
plantaris muscle units to be more resistant to fatigue
than any of the units of cat soleus muscles. However,
Burke and colleagues (11) have warned against extra¬
polation of histochemical and biochemical properties
to physiological properties.
There are several possible causes of the fatigue
observed in these experiments. In Series 1-3, fatigue
could have resulted from a failure in excitation-
contraction coupling, substrate depletion, accumulation
of metabolites, or a combination of these factors.
Since there are both FR and S fiber types in the
gastrocnemius, the fatigue might have been predominantly
in one of the fiber types.
It seems unlikely that neuromuscular junction failure
was a significant component of the fatigue observed in
Series 1-3. Testing for neuromuscular transmission
failure by direct stimulation of the dog gastronemius-
plantaris muscle group is not easy since its large size

36
makes constant field stimulation difficult. However,
several studies on other mammalian muscles (3, 41, 52)
have indicated that the possibility of neuromuscular
transmission failure at stimulation rates of less than
10/sec is minimal. In two experiments, nerve stimulation
at 20 impulses / sec for 30 minutes when muscle
contraction was blocked by curare or succinylcholine
caused less than a 10% decrease in developed tension.
Decreases in developed tension of 50-70% occurred under
the same stimulation conditions when muscle contraction
was not blocked. These findings indicate that presynaptic
failure of impulse propagation and inadequate release of
acetylcholine probably did not cause the fatigue observed
in our experiments. Desensitization of the endplate is
not ruled out by these results. However, neuromuscular
depression is presently believed to result from a
reduced number of released transmitter quanta and a
reduction of quantal size (42, 48).
In Series 4, the cause of fatigue might have been
muscular, neuromuscular, or a combination of the two
since ischemia can affect both the muscle and the
neuromuscular junction (16, 49). In Series 5, developed
tension was decreased by partial curare block which
presumably simulates neuromuscular junction failure.
These experiments do not allow identification of the
specific cause of fatigue. However, our results do
indicate that the oxygen uptake per unit of isometric

37
force production is unchanged by either muscle fatigue
or neuromuscular fatigue. This suggests that fatigue,
whether muscular, ischemic, neuromuscular, or a
combination of these three, does not cause any change in
the efficiency of energy transduction from ATP to
external tension development by the muscle, without
concommitant changes in the opposite direction for
energy transduction from foodstuffs to ATP. This is
not likely the case.
These results differ from those of Bronk (6),
Feng (28), Edwards and Hill (20) and Edwards, Hill
and Jones (21) in that they found that the energy
expenditure per unit of force production (or of tension¬
time) decreased during fatigue. Unlike our experiments,
however, these earlier studies used stimulus parameters
which caused partially to completely fused tetanic
contractions of relatively long duration. The present
study is of twitch or very brief tetanic contractions,
for which no plateau in developed tension occurs (see
Figure 5). There is little if any tension maintenance
involved.
The data presented in this study along with those
of Wilson and Stainsby (66) demonstrate a constant
coupling between C>2 uptake and developed tension in
isometric twitch contractions. In these two studies,
developed tension has been changed by stimulation
frequency, potassium ion infusions, twin impulse

FIGURE 5. Tracing of a recording of tension and
differential of tension for single impulse
and twin impulse contractions before and
after fatigue. Contractions are superimpos
for ease of comparison. There is no
maintained plateau in this type of
contraction.

TWITCH and TWIN
TENSION
dP/dt
FIGURE 5
CONTRACT ION
Post - fatigue
OJ

40
stimulation, normal muscle fatigue, ischemic fatigue and
partial neuromuscular transmission block with curare.
During all of these treatments, the relationship between
O2 uptake and developed tension has been unaltered.
The data also show that although resting metabolic
rate following fatigue approaches pre-fatigue levels after
30 minutes, developed tension is still quite low.
Phosphorylcreatine and ATP levels should be fully
recovered following 30 minutes of rest (38, 51). Edwards
and coworkers (23) have also identified a long lasting
element of fatigue in humans that is not due to
depletion of high-energy phosphates. Further study
is warranted to determine whether or not there really
is a causative relationship between phosphorylcreatine
depletion and fatigue, as suggested by Spande and
Schottelius (56).

EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE
Introduction
Skeletal muscle has an attenuated response to a
single impulse following a prolonged period of twitch
contractions due to repetitive single impulse
stimulation (34, 66). This response is not necessarily
indicative of a reduced capacity of the muscle to develop
tension (23, 30). It is,however, a type of muscular
fatigue and warrants further investigation concerned
with determination of mechanisms responsible for this
"twitch fatigue."
Wilson and Stainsby (66) reported that twitch
fatigue occurs in the gastrocnemius-plantaris muscle
of the dog following 30-40 minutes of isometric
contractions (10-14/sec). They monitored recovery with
periods of low frequency stimulation over the course of
3-4 hours. An attenuation of developed tension was still
present following this recovery period. Little is known
of the mechanism responsible for this fatigue.
The purpose of the present investigation was to
study alterations in twitch contractions caused by
repetitive stimulation at three frequencies; 2.5, 5
and 10/sec. A twitch contraction can be characterized
41

42
by measurements of the magnitude and time-course of
tension development seen in the isometric myogram (3, 9).
These measurements are: developed tension (AT),
contraction time (Ct), half relaxation time (Rt 1/2),
peak rate of force development (dP/dt), and peak rate
of relaxation (-dP/dt) (see Figure 6). Sandow and Brust
(54) have named the changes in these measurements that
occur with repetitive stimulation the "fatigue patterns."
Fatigue patterns have been determined for single muscle
cells and whole mammalian and amphibian skeletal muscles
in vitro (9, 10, 35) . Measurements on iri situ muscle
where direct determination of muscle force during
repetitive stimulation can be made while the muscle
maintains a normal circulation have not yet been
reported.
Methods
Twenty mongrel dogs of either sex weighing 9-18 kg
were used in this study. The gastrocnemius-plantaris
muscle group was prepared as described in the General
Methods chapter.
Fatigue as a result of 30 minutes of stimulation at
three frequencies 2.5, 5 or 10/sec was studied. Five
animals were used at each frequency. To study the fatigue
patterns of muscle, it is necessary to obtain fast traces
of contractions, before, during and after the fatiguing
contractions. To evaluate twitch contractions before
fatigue, the muscles were stimulated at either 1/sec or

43
Figure 6. Tracings of: Tension and dP/dt are presented to
demonstrate the manner by which the measurements
were made. See text for verbal description of
these terms.

44
2.5/sec for 2 minutes. After a fast trace (100 or 200
mm/sec paper speed) was obtained, contraction frequency
was either left at 2.5/sec or increased from 1/sec to 5
or 10/sec. Relaxation was not complete between
contractions when stimulation was 5/sec or 10/sec. To
facilitate measurement of the characteristics of a twitch,
the frequency of stimulation was reduced briefly, while
fast traces were obtained, then the fatiguing frequency
was restored (see Figure 7). During contractions at
2.5/sec complete relaxation occurs between contractions,
so fast traces were obtained without altering the
frequency of stimulation. Besides the contractions at
2 minutes, fast traces were obtained after 10 and 30
minutes of fatiguing contractions and after 10 and 40
minutes of recovery (see Figure 7). To get fast traces
during the recovery period which followed contractions
at 5/sec or 10/sec, the stimulator was turned on briefly
at 1/sec. Following the 30 minute period of contractions
at 2.5/sec, contractions were continued at a frequency
of 0.2/sec. Fast traces were obtained without altering
the frequency of stimulation. It has been reported that
contractions at this low frequency do not alter the
recovery process (66). In one experiment, a tetanic
contraction (200 msec duration, 100 impulses / sec) was
obtained, before and after the 10/sec fatiguing contractions.
This was done to permit evaluation of the contractile
capacity of the muscle. All fast traces were evaluated

45
for the characteristics of a twitch. These characteristics
are illustrated in Figure 6.
Arterial and venous blood samples (0.6 ml) were
obtained at regular intervals throughout the experiment.
Samples were drawn into glass tuberculin syringes, sealed
with mercury-containing caps, and placed in ice until
they were analyzed. These samples were analyzed for pH,
PCC>2 and PO2 at 37°C with a radiometer (Copenhagen) blood
gas machine. These measurements permit evaluation of
viability of the animal and provide descriptive data
concerning metabolic status of the muscle.
At the end of each experiment, the fatigued muscle
was excised, trimmed of visible fat and connective tissue,
blotted and weighed. The force transducer was calibrated
after each experiment by hanging pre-weighed lead weights
on the lever.
In a few additional experiments, twitch contractions
were evaluated when AT was reduced by: ischemia, dantrolene
sodium or reduced stimulation voltage. Comparison of
these contractions with those obtained during and/or
following fatiguing contractions may provide some insight
into the mechanism of fatigue. To study the effects of
ischemia, the femoral artery was occluded while contractions
continued at 1/sec. Fatigue would not occur at this
frequency with an intact blood flow, but does occur with
ischemia. The occlusion was removed after AT fell to
about 50% of the pre-occlusion value (10-20 minutes)

46
Tension developed versus time. Contractions
in this case were 10/sec except as indicated.
Fast traces (not illustrated) were obtained
during the 1/sec stimulation.
FIGURE 7.

47
and recovery was observed. Dantrolene sodium, dissolved
in propylene glycol (25 mg/ml) was injected I.V. during
contractions at 0.2/sec. Dantrolene impairs release
of Ca from the lateral sacs (24). This is accomplished
without changes in the action potential and is apparently
a direct effect on the lateral sacs. Sufficient drug
was given to reduce AT at least 50% (2-5 mg/kg). To
study the effects of reducing the number of motor units
contracting, the stimulation voltage was reduced while
the muscle contracted 0.2/sec. This results in excitation
of fewer motor neurons and their motor units. Consequently
less tension is developed. Comparing the twitch
characteristics of a normal versus a fatigued muscle may
provide information leading to an understanding of the
mechanism(s) of fatigue.
Also, in a few experiments, samples of muscles were
obtained immediately following the 30 minute stimulation
period, and/or after 40 minutes of recovery. Samples
were frozen in situ with metal clamps pre-cooled in
liquid nitrogen. Small samples (30-80 mg) were then
homogenized (Vertis homogenizer) in perchloric acid
(.8% in 40% ethanol) and analyzed for phosphorylcreatine
by the method of Ennor and Stocken (25) (see Appendix).
Statistical analysis was by the two way analysis of
variance for repeated measures. Differences between means
were determined by Duncan's multiple range test (2).

48
Results
Blood samples were obtained before the contractions
began and at t = 10, 30, 40 and 70 minutes. Arterial PO2
was 87 ± 2.3 mm Hg (mean ± SEM) before contractions and
did not change significantly throughout the experiments
(see Figure 8). Before contractions began, PvC>2 was
50.2 ± 2.0 mm Hg. During the contraction period PvC>2
was lower, but none of the blood samples measured had
a PO2 less than 12 mm Hg. Except for the experiments
where fatigue was caused by 2.5/sec contractions, PvC>2
was back to pre-fatigue values early in the recovery
period. Contractions were continued, 0.2/sec, during the
recovery period of these (2.5/sec) experiments; therefore,
it might be expected that PvC>2 would not be at rest levels.
Arterial PCO2 began at 31.6 ± 0.8 mm Hg and fell
slowly during the experiments. The decrease in PaCC>2 was
statistically significant but probably is of minimal
physiological significance. Venous PCO2 was high during
the contraction period when PvC>2 was low, and returned
to pre-contraction levels early in the recovery period.
Arterial pH was 7.40 - 0.01 before contractions began,
and did not change significantly throughout the experiments.
Venous pH decreased from 7.37 at t = 0 minutes to 7.32
(for 2.5/sec) or 7.28 (for 5 or 10/sec) at t = 10 minutes.
By 10 minutes of recovery, venous pH had returned to pre¬
fatigue values (see Figure 8).

FIGURE 8. Blood flow and the blood gas measurements
versus time. The horizontal bar
indicates where fatiguing contractions
occurred. When means at a given time
(for different frequencies) were not
significantly different, means were
combined. Numbers refer to the
fatiguing frequency. Asterisks
indicate where measurements are
significantly different from the
original value (time = 0 minutes).
Vertical bars are ± SEM.

50
FIGURE 8

51
Although blood flow was measured continuously, only
those measurements corresponding to times when blood
samples were obtained are presented (see Figure 8).
Blood flow was higher during the contractions, but was
back to pre-fatigue values by 10 minutes of recovery.
There were no significant differences between frequencies
for blood flow response.
The first 2 minutes of contractions were at 1/sec
or 2.5/sec. There were no significant differences for
AT between these frequencies at t = 2 minutes. Mean AT
for all experiments was 2.3 - 15 g/g (wet wt) at this
time. The muscles weighed 48.5 t 3.3 g (wet wt).
Developed tension fell more rapidly during 10/sec
contractions than during 2.5/sec or 5/sec contractions
(see Figure 9). By 30 minutes all frequencies of
stimulation resulted in significant reductions in AT.
There was no significant recovery of AT during the 40
minutes following the fatiguing contractions.
Contraction time decreased during the fatiguing
contractions at 10/sec, but not during the contractions
at 2.5 or 5/sec. There was no significant difference
for Ct between recovery and pre-fatigue measurements at
any fatiguing frequency (see Figure 9).
Half relaxation time did not change during the
contractions or during the recovery except for recovery
of 2.5/sec fatigue. The Rt 1/2 was longer for contractions
at 0.2/sec than for 1/sec. If contractions during recovery

FIGURE 9. Developed tension, contraction time
and half relaxation time versus time
The horizontal bar indicates when
fatiguing contractions occurred.
Where there was no significant
difference between means, all
frequencies are combined. Numbers
refer to the fatiguing frequency.
Asterisks indicate where measure¬
ments are significantly different
from the original value (at time =
2 minutes). Vertical bars are ± SEM

FIGURE 9

54
for this frequency of fatiguing contractions had been 1/sec
(at t = 40 and 70 minutes only) then no difference from
pre-fatigue contractions would be expected.
Figure 10 illustrates the changes in dP/dt and -dP/dt
seen in these experiments. The changes seen for dP/dt
closely parallel those observed for AT. A positive and
significant correlation exists between dP/dt and AT
(r2 = 0.93) and between -dP/dt and AT (r^ = 0.82).
Muscle temperature rose during the fatiguing contrac¬
tions. The increase in temperature was only 1-2°C. A
similar or smaller rise was seen during the 5/sec and
2.5/sec contractions. Muscle temperature fell slowly to
37°C during recovery, but was not permitted to go below
37°C.
Muscle samples obtained during a few of the experi¬
ments were analyzed for phosphorylcreatine (PC). Analysis
revealed that PC is low at t = 30 minutes (during
contractions), but is back to resting levels by t = 70
minutes (see Table III). The values of PC given in the
table are left:right ratios. PC was determined relative
to total creatine in the muscle sample. Harris (38) has
shown that total muscle creatine content does not change
during exercise and therefore can be used as an index of
muscle weight.
In one experiment, a tetanic contraction was obtained
before and after fatiguing contractions at 10/sec. Figure
11 illustrates the lack of change seen for this contraction.

TABLE III
PHOSPHORYLCREATINE ANALYSIS DURING CONTRACTIONS AND FOLLOWING THE RECOVERY PERIOD
Animal #
L: R ratio:
a) during contractions
b) following recovery
0.36 0.63 0.40 0.46
0.83 0.96 1.0 1.11 1.08 1.02 1.26 1.09 0.83 1.02
Phosphorylcreatine is presented above as a ratio:
PC • Creatine--'- (left): PC • Creatine--1- (right)
(See text for discussion of creatine as an index of muscle weight.)
U1
Ln

FIGURE 10. Peak rate of force development and peak rate
of relaxation. See Figure 5 for significance
of asterisks and numbers. Note similarity
between dP/dt versus time and AT versus time
(Figure 5).

100
DP/DT
(°/o)
60
20
TIME (min)
10 25 40
5,10
FIGURE 10
i i
55 70
T
Ln

58
i
I
I
TETANIC CONTRACTIONS
dP/ dt
FIGURE 11. Tracings of recordings of tension and
differential of tension for tetanic
contractions (100/sec for 200 msec).
Developed tension following 30 minutes
of fatiguing contractions (10/sec) was
only slightly lower than that before
the 10/sec contractions. The differential
tracer were virtually superimposable.

59
The tetanic contraction is recovered at a time when
twitch AT is still reduced.
Contractions observed during ischemia demonstrated
a reduced AT and a prolonged Rt 1/2. Following restoration
of blood flow, recovery of both AT and Rt 1/2 was 50%
complete in 30 minutes. Figure 12 shows recordings from
one muscle for contractions pre-ischemia, during ischemia
and post-ischemia. These recordings are typical for
what was seen.
Comparing the effects of dantrolene sodium, ischemia
and reduced stimulation voltage on Rt 1/2 vs AT (see
Figure 13), indicates that ischemia and reduced voltage
cause large alterations in Rt 1/2 with concomitant
reductions in AT. With administration of dantrolene
sodium, the attenuation of AT is not accompanied by a
substantial change in Rt 1/2. This pattern seen with
dantrolene is similar to the changes seen during the
fatiguing contractions.
Discussion
In these experiments, twitch fatigue has resulted
from 30 minutes of contractions at 2.5, 5 or 10/sec.
Forty minutes after the fatiguing contractions were
ended, no significant recovery had occurred. Recovery
would eventually have occurred following several hours
of relative inactivity (66). Twitch fatigue, then,
results from a relatively persistent alteration in the muscle
which affects the contractile response to a single impulse.

60
FIGURE
ischemia
2. Tension and dP/dt (upper and lower curves
of each pair respectively) are shown.
1) a control contraction before occlusion
of blood flow
2) contraction during ischemia, 5 minutes
after occlusion of blood flow
3) a post-ischemia contraction, 50 minutes
after release of occlusion, contraction
frequency 1/sec.

61
FIGURE 13. Half relaxation time versus AT is presented
to illustrate the relative changes in Rt 1/2
when AT is reduced by ischemia, dantrolene
or reduced stimulation voltage. Each line
represents one dog. Lines were determined
by the least squares method common to all
three lines.

62
The response to tetanic stimulation is not altered.
Analysis of the fatigue patterns for this muscle group
may provide some insight into the mechanisms responsible
for this long-lasting fatigue.
The extent of comparisons between frequencies for
the fatigue patterns observed in these experiments is
limited. In the experiments where fatigue was caused
by contractions at 5/sec or 10/sec, the frequency of
stimulation was reduced to 1/sec to obtain fast traces.
This procedure was implemented because full relaxation
does not occur between contractions at 10/sec and 5/sec.
The measurements which have been made on these contractions
are affected by the resting tension. It was hoped that
by reducing the frequency to 1/sec for these contractions,
a true representation of the characteristics of a twitch
could be obtained. Although a common frequency is used,
it is evident that the preceding contractions did have
an effect on the measurements (see Figure 14). The
measurements made for the 2.5/sec series were made on
contractions at 2.5/sec during the 30 minute fatigue
period and at 0.2/sec during recovery. Due to the effect
of frequency and preceding contractions on the time
course of a twitch, caution must be exercised when
comparing values between frequencies. Comparing the trend
within one frequency with the trend within another
frequency is valid.

63
TENSION
DIFFERENTIAL
l/sec after
10/sec
FIGURE 14. Following fatiguing contractions at 10/sec,
switching the stimulator to l/sec results
in a negative staircase. The top tracing
is tension, and the lower one is dP/dt.
This demonstrates the inotropic effects
of previous contractions, and illustrates
the mechanisms responsible for the decrease
in developed tension seen following the 30
minute period of contractions. The single
contraction on the left immediately follows
10/sec contractions; the others (at a reduced
paper speed) were continued at l/sec.

64
If contractions of the fatigued muscle are compared
with contractions before the muscle was fatigued, the
following characteristics are noted. Developed tension
is reduced. This reduction appears to be greater when
a higher frequency of stimulation occurred during the
fatigue period. Contraction time and Rt 1/2 are not
different in the fatigued muscle in comparison with the
rested muscle for muscles fatigued at 10/sec and 5/sec.
The prolongation of contraction time in recovery from
fatiguing contractions at 2.5/sec is not due to fatigue,
but due to the stimulation frequency during recovery.
Contraction time gets shorter as frequency of stimulation
is increased. For the same reason, Ct is shorter during
the fatiguing contractions at 10/sec. This is an effect
of previous contractions (10/sec) altering the time-
course cf contractions at 1/sec. A decrease was seen for
dP/dt which was proportional to the decrease in AT.
Brust (10) reported similar fatigue patterns for the
mouse soleus muscle in vitro. The same author (9)
reported different fatigue patterns for frog semitend¬
inosis muscles. The major difference was that Rt 1/2 was
prolonged in the fatigued frog muscle but not in the
mammalian muscles. This difference is apparently not
species specific. Edwards et al. (22) reported that
there is a slowing of relaxation in mouse muscle
following a fatiguing effort. This slowing of relaxation
was correlated with low levels of ATP (22). This can

65
explain the increase in Rt 1/2 seen in ischemic fatigue
(see Figure 13). Since Rt 1/2 was not prolonged following
a period of fatiguing contractions with an intact blood
supply, it would appear that ATP was available within the
muscle.
The evidence presented above suggests that the fatigue
observed in these experiments did not occur as a result
of reduced ATP levels. Other evidence supports this
suggestion. Although PC levels were low during the
contraction period, (see Table III), resynthesis had
occurred before significant recovery of AT. Rapid
resynthesis of PC during recovery from a period of
contractions was also observed by Piiper and Spiller (51).
There seems to be no direct relationship between PC levels
and AT. This is contrary to a report by Spande and
Schottelius (56), but supports the observations of Fitts
and Holloszy (29). It should also be pointed out that
glycogen was still available after 30 minutes of stimulation
at 10/sec (14) and lactate production had declined (or
even reversed - lactate uptake) (60). It would seem that
energy was available, but demand for energy was reduced.
In support of this conclusion, PvC>2 was not below minimum
critical PO2 (59) at any time that PvC>2 was measured.
The muscle is capable of developing more force than
that seen in a twitch. Twin impulse stimulation causes
developed tension to be double that seen with single

66
impulse stimulation (34). This relationship is maintained
for rested as well as fatigued muscles. It would appear
that the reduced AT of the fatigued muscle was due to
reduced activation.
A reduced activation of skeletal muscle could,
theoretically be the result of: a) reduced neuromuscular
transmission, b) reduced Ca2 + release, or c) reduced
responsiveness of the contractile elements to Ca2+.
Neuromuscular transmission appears to be intact (34).
This agrees with others (3, 35, 37) who have found only
minimal changes in muscle action potentials or electro¬
myographic response following comparable stimulation
O i
periods. There is a possibility that Ca release has
been attenuated. This could cause a reduced AT and
dP/dt without altering Ct or Rt 1/2 (15). Brust (10),
who observed similar fatigue patterns with the mouse
soleus muscle, suggests that the fatigue he observed was
a result of reduced Ca2+ release (see also (63) for the
converse). The factor(s) responsible for the reduced
Ca2+ release is/are not known. Bonner et al. (5) have
reported that muscle mitochondria accumulate Ca2+ during
exercise. This would reduce the pool of Ca2+ available
for recycling in the sarcoplasmic reticulum (67) and
consequently limit the amount available for release.
Dantrolene sodium apparently reduces the amount of
Ca2+ released per impulse (24). This drug was used in
these experiments to determine the effects of reduced

67
Ca2+ release on a twitch. A dantrolene treated muscle
contracts with a time-course similar to the fatigued
muscle. Ischemia or reduced stimulation voltage caused
a reduction in AT, but this was accompanied by changes
in Rt 1/2.
An alternative hypothesis involves a reduced binding
94-
of Ca to the regulatory proteins. During contractions,
muscle pH probably falls (33). Fuchs et al. (32) has
shown that binding of Ca2+ to troponin is inhibited by
lower pH. Fitts and Holloszy (30) has suggested this
may be a mechanism responsible for the fatigue seen in
their experiments.
The experiments reported herein do not permit
discrimination between these two potential mechanisms
of fatigue. The fatigue observed following 30 minutes
of stimulation at 2.5, 5 or 10/sec appears to be a
result of reduced activation. Further experiments will
need to be conducted to determine which of the theories
described above applied to these muscles.

EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH CONTRACTION
Introduction
Twitch developed tension is attenuated following a
period of repetitive stimulation. This attenuation is
apparently the result of a reduced activation, due either
to a reduced release of Ca2+ from the lateral sacs or
from a reduced responsiveness of the contractile proteins.
It is not due to a reduced availability of energy (ATP,
PC). Fitts and Holloszy (30) have suggested that the
reduced twitch response is a result of inhibition of the
contractile process due to a reduced intracellular pH.
Steinhagen et al. reported evidence supporting this
hypothesis (61). He reported that dog gastrocnemius
muscle fatigues more quickly during respiratory acidosis
than during normal pH balance.
Specific mechanisms which may contribute to this
acidosis-induced fatigue have been proposed. Nakamura
and Schwartz (46) reported that uptake of Ca2+ by
sarcoplasmic reticulum is accelerated in low pH medium.
This could reduce the duration of activation for a twitch
by reducing the Ca2 + concentration more quickly. Fuchs
et al. (32) have reported that Ca2+ binding to troponin
is inhibited by H+. These molecular mechanisms, if
effective under physiological conditions would cause a
reduction in AT, fatigue.
68

69
The purpose of this study was to observe the effects
of acidosis on the developed tension and the time-course
of a twitch contraction of the in situ dog gastrocnemius-
plantaris muscle. Intracellular pH can be reduced more
easily via respiratory acidosis than by metabolic acidosis
(12). For this reason, acidosis was induced by reducing
the ventilatory rate. The results of this study indicate
that acidosis is unlikely a direct cause of twitch fatigue.
Methods
The gastrocnemius-plantaris muscle preparation as
described in the General Methods section was used in this
series of experiments.
In each experiment, the nerve was stimulated at a
frequency of 0.2/sec. In three experiments, ventilation
was controlled to maintain arterial pH near 7.4 for the
duration of the experiment (2 hours). In four experiments,
after the muscle had been contracting (0.2/sec) for
20 minutes, ventilation was reduced to 3-4 breaths/minute.
The mixture of inspired gas was adjusted (with 9 5% C>2
and 5% CO2) to maintain normal arterial PO2 during the
hypoventilation. The period of hypoventilation was
continued until arterial pH was less than 7.1 (60-90
minutes). This period of hypoventilation was followed
by a period of hyperventilation (20 breaths/minute). The
hyperventilation was continued for 40-60 minutes (see
Figure 15).

70
With an additional four dogs, the sequence of
ventilatory adjustment was reversed (control, hyper¬
ventilation, hypoventilation) to permit evaluation of
a possible order effect.
At ten minute intervals throughout each experiment,
arterial and venous blood samples were obtained (0.8 ml)
in glass tuberculin syringes (1 ml capacity). The samples
were sealed with mercury containing caps and kept in ice
until they were analyzed for PO2, PCO2 and pH (Radiometer,
Copenhagen).
Fast traces of contractions were also obtained at 10
minute intervals (200 mm/sec on Gould-Brush Model 2400
recorder). The fast traces were measured for developed
tension (AT), half relaxation time (Rt 1/2), contraction
time (Ct), peak rate of force development (dP/dt) and
peak rate of relaxation (-dP/dt) (see Figure 6).
Statistical analysis was by a two way analysis of
variance for repeated measures (2). For statistical
analysis, the last two measurements (fast traces or blood
gases) before alteration of the ventilation were utilized
to represent the state in which they occurred (see
Figure 15).
Results
After 20 minutes of contractions, ventilation was
reduced to 3-4 breaths per minute (Group A) or increased
to 20 breaths per minute (Group B). This adjustment in
ventilation resulted in a reduction in arterial pH from

FIGURE 15. Developed tension, PO2, ventilation and
arterial pH for one dog, from Group A.
Blood samples and fast traces obtained
at * were used for statistical analysis

72
200
DEV ELOPED
FIGURE 15

73
7.37 - .01 (mean ± SEM) during the control period to
7.08 ± .03 in Group A and an increase in Group B from
7.36 ± .02 to 7.52 Í .02. After 60-90 minutes, ventilation
was increased to 20 breaths per minute in Group A and
reduced to 3-4 breaths per minute in Group B. This
second alteration in ventilatory frequency resulted in
an increase in pH to 7.4 in Group A and a reduction in
Group B to 7.2 (see Figure 16). In Group C, ventilation
was not altered, and arterial pH did not vary during the
experiments. The reduction in ventilation did not
significantly decrease arterial PC>2 •
When arterial pH remained constant for 2 hours
(Group C), AT increased with time. By the end of 2 hours,
AT was greater than it had been at the first 20 minute
period. As illustrated in Figure 17, AT increased with
time in Group A also but not in Group B. The only
significant difference in Groups A and B was that AT
during hyperventilation was greater than AT at any other
time period in Group A and greater than control in Group
B (see Table IV).
Despite the apparent effect of hyperventilation on AT,
there was no significant correlation of AT with arterial
or venous H+ concentration (see Table V). Furthermore,
AT was not reduced during hypoventilation. When arterial
pH fell to 7.09 ± .03, AT did not change significantly.
In Group C, dP/dt did not change significantly. In
Group A and Group B, however, dP/dt was higher during

74
TABLE IV
STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP
Group A Group B Group C
Rank
Rank
Rank
AT
.006
3 2 1
.05
3
2 1
.04
3 2 1
Rt 1/2
.18
NS
.003
1
2 3
.99
NS
Ct
.86
NS
.005
1
3 2
.98
NS
dP/dt
.001
3 2 1
.001
3
2 1
.122
NS
-dP/dt
. 002
3 12
.02
3
2 1
.91
NS
The p values are
given
for AT,
Rt
1/2,
Ct, dP/dt
and
-dP/dt for Group A (control, hypoventilation, hyper¬
ventilation), Group B (control, hyperventilation,
hypoventilation) and Group C (control). Means are
listed under the rank order. Rank indicates the
order from largest to smallest for Control, 1,
Acidosis, 2, and Alkalosis, 3. For Group C, rank
1, 2 and 3 indicate time periods; first 20 minutes,
1, next 60 minutes, 2, and last 40 minutes, 3.
Horizontal bars across ranks indicate means which
are not significantly different. NS indicates that
means are not significantly different.

FIGURE 16. Arterial and venous H+ concentration during
ventilatory states. The three columns on
the left are from Group A. The three columns
on the right are from Group B. Open columns
are arterial [H+]. Vertical bars are one
standard error of the mean. Upper bar is
for venous, lower bar for arterial SEM.

VENTILATION ( b re a t h s / mi nu te )
FIGURE 16

FIGURE 17. Mean arterial and venous PO2 during the
different ventilatory states. The three
columns on the left represent Group A.
The three on the right represent Group B.
Open bars represent venous PO2
and combined (open and closed) bars
represent arterial PO2. Vertical bars
represent one SEM.

100
ART ERIAL
and
VENOUS 50
po2
(mm Hq)
10 4 20 10 20 4
VENTILATION (breaths/minute)
FIGURE 17

79
hyperventilation than during the initial control period
(see Table I). There was no significant correlation
between arterial pH and dP/dt. This suggests, as seen
for AT, that the alterations in dP/dt associated with
ventilation patterns are not directly related to pH
(see Figure 18).
The peak rate of relaxation was greatest during
hyperventilation. In Group A, this was significantly
greater than both the control period and the period of
hypoventilation. In Group B, however, where arterial
pH increased to 7.52 during hyperventilation (as opposed
to 7.4 in Group A), -dP/dt during hyperventilation was
greater than that during normal ventilation, but not
significantly different from that during hypoventilation.
There was no significant correlation between -dP/dt and
arterial [H+].
In Groups A and C there were no significant changes
in Ct or Rt 1/2. In Group B, however, Ct was shorter
during hypoventilation than at other times. Rt 1/2 was
shorter during hyperventilation than at other times in
Group B (see Figure 19) .
Discussion
It has been suggested that intracellular acidification
is the cause of skeletal muscle fatigue (30). If there
is a direct influence of pH on the force of contraction,
this phenomenon would be independent of the manner in
which the pH change was obtained. It is apparent in

FIGURE 18. Mean developed tension, dP/dt and -dP/dt
during different ventilatory states.
Developed tension is % of highest in each
experiment. dP/dt and -dP/dt are % of
highest dP/dt in each experiment. Vertical
bars represent one standard error of the
mean.

A T â– â–  dP/dt d -dp/dt Ed
10 4 20 10 20
VENTILATION ( bre a t h s / m i nu te )
FIGURE 18

FIGURE 19. Mean contraction time and Half relaxation
time during different ventilatory states.
Vertical bars represent one SEM.

Rt 1/2 O Ct
I 0
20
VENTILATION ( brea t h s/m i nute )
FIGURE 19

84
TABLE V
STATISTICS FOR TWITCH CHARACTERISTICS VERSUS BLOOD GASES
Group
T
Rt
1/2
dP/dt
-dP/dt
Ct
p= r2=
P=
r2=
p= r2=
P=
r2=
P= r2=
Art [H+]
A
. 4
.001
00
00
•
. 8
.09
.17
B
.25
.06
. 26
.07
.1
C
. 33
.29
.4
.15
.22
ven [H+]
A
. 78
.002
.85
. 72
.2
.31
B
. 77
.13
. 75
.23
.03 .56
C
. 59
.26
.11
.24
.26
PaC02
A
.25
.001
. 89
.57
.05
.52
.13
B
.04 .56
.04
.44
.04 .4
.01
.75
.25
C
.2
.13
. 15
.04
. 89
.06
Significance values are given for correlations between
twitch characteristics and blood gases. R2 values indicating
the percent of variability explained by the variable are
given for significant correlations (p<.05). Groups are
as defined in the legend for Table IV.

85
these experiments that the changes in Ct, Rt 1/2 and
-dP/dt are not directly associated with arterial pH.
This complication will be discussed further below.
However, to give consideration to the possibility that
acidosis causes fatigue, the results from Group A will
be discussed with respect to the likelihood that the
hypoventilation in these experiments resulted in alterations
in intracellular pH comparable to what might be expected
from contractions at 10/sec for 30 minutes.
In these experiments, arterial pH was reduced from
7.37 to 7.08 by hypoventilation. The acidosis accompanying
the hypercapnia was not associated with any change in aT,
Rt 1/2 or Ct. It would appear that the fatigue described
earlier cannot be a result of acidosis, unless intra¬
cellular pH during the fatiguing contractions changes
more than it did during hypoventilation.
The magnitude of the intracellular pH change occurring
in these experiments can be estimated from the results of
Burnell (12) . Hypercapnia in dogs (PaCC>2 = 55 mm Hg)
resulted in a reduction of intracellular pH of neck
muscles from 6.85 to 6.57. Intracellular pH was
determined by the DMO method (12). Burnell (12)
observed that the maximal response had occurred within
15 minutes. In the experiments reported herein, arterial
PCO2 increased to 57 ± 3.7 mm Hg. The measurements
presented were taken 60-90 minutes after the alteration
in ventilation. It is reasonable to assume that a very

86
similar change in intracellular pH occurred in these
experiments as was observed by Burnell, under almost
identical circumstances. It can therefore be concluded
that a change in intracellular pH values from normal
resting values (approximatley 6.85) to 6.57 does not
result in a change in developed tension.
The important point, however, is whether or not
intracellular pH fell to this level or lower during the
fatiguing contractions reported in earlier chapters.
Estimates of intracellular pH changes under these
circumstances can only be tentative. Venous PCO2 during
the fatiguing contractions was never greater than 57 mm Hg
(mean = 49 mm Hg at t = 10 minutes for 10/sec). This
hypercapnia would not cause an acidosis sufficient to reduce
AT (if reduced pH will cause reduced AT!). However, there
is lactic acid production during the first 5-20 minutes of
this type of stimulation (60). This is likely to contribute
to an intracellular acidification. Sahlin et al. (53)
report that in humans, performing maximal exercise to
exhaustion, intracellular pH drops from 7.08 to 6.6. This
is comparable to that seen by Hermanson and Osnes (39).
This decrease in pH was associated with an increase in
lactic acid production. Following the bout of exercise,
intracellular pH recovered to 7.0 within twenty minutes.
It is unlikely that intracellular pH changed as much in
the 10/sec fatigue as it did during the exhausting exercise
reported by Sahlin et al. (53). Furthermore, it seems

87
likely that intracellular pH would have returned to resting
levels after 40 minutes of recovery. Developed tension
at this time is still very much reduced (i.e., no significant
recovery has occurred). It seems reasonable to conclude
that the persistent fatigue caused by contractions at
10/sec for 30 minutes does not result directly from
acidosis. There may, however, be indirect ways in which
intracellular pH may affect the contractile process,
resulting in a change within the muscle which persists
beyond the time when pH has returned to control (26).
It is evident that changes in ventilatory pattern
do affect muscle contraction. During hyperventilation, AT
was increased. This was the case whether hypoventilation
preceded the period of hyperventilation or if normal
ventilation preceded it. In the former case (hypo¬
ventilation preceding) arterial pH returned to 7.4, so
there was no absolute arterial alkalinization. The
increase in AT under these circumstances was greater than
the increase seen in the latter case (hyperventilation
preceded by normal ventilation), despite the fact that
this procedure resulted in an increase in arterial pH
to 7.52. It seems that hyperventilation increases AT,
but hypoventilation only reduces AT when it is preceded
by hyperventilation.
The high p values reported in Table V reflect the
lack of relationship between AT and [H+]. The fact that
significant differences were observed between ventilation

88
periods is due not to absolute pH changes, but relative
changes possibly in conjunction with some other change
(ion distribution? i.e., see 26, 31) associated with
changes in ventilation.
The changes seen in Ct, dP/dt, -dP/dt and Rt 1/2
also suggest that alterations in ventilatory pattern
can affect contraction. The mechanisms responsible for
these changes are not clear.
In this study, it is likely that hypercapnia resulted
in intracellular acidosis. This acidification was not
accompanied by a reduction in AT. It can be concluded
that fatigue is not caused by acidosis if fatiguing
contractions do not cause any greater acidification than
that which occurred in these experiements.

SUMMARY
O2 Uptake and Developed Tension
The amount of oxygen used by a muscle was proportional
to the amount of tension developed. This occurred over a
wide range of forces when AT was altered by any of the
following:
a) Fatigue, after 30 minutes of stimulation at 14-20/sec
b) Fatigue, during fatiguing contractions at 3-6/sec
c) Twin impulse stimulation, before and after fatigue
at 14-20/sec
d) Fatigue, during contractions at 1/sec without blood
flow (ischemic fatigue)
e) Attenuated contraction, caused by administration of
curare in sufficient doses to reduce the force of
contraction by as much as 70%.
These results suggest that the major determinant of
energy utilization during an isometric contraction is the
magnitude of the developed tension. It should be emphasized
that these were twitches or very brief tetanic contractions
in which developed tension rose then fell, but did not
maintain a plateau of tension and can therefore be considered
to have a minimal "tension maintenance" component to the
determinants of energy utilization. The possibility that
neuromuscular junction failure may have contributed to
the observed fatigue was tested. It was demonstrated that
transmitter release was normal after 30 minutes of
stimulation at 20/sec.
89

90
Time-course of the Twitch Contraction in Fatigue
The time-course (Ct and Rt 1/2) and the rate of change
of force (dP/dt and -dP/dt) were observed when muscles were
fatigued with contractions at 2.5, 5 and 10/sec for 30
minutes. The pertinent observations are listed below.
a) Developed tension fell more rapidly during 10/sec
stimulation than 2.5 or 5/sec.
b) In the 40 minutes following the fatiguing
contractions, no significant recovery of
AT occurred.
c) The Ct and Rt 1/2 of a fatigued muscle are no
different from those of a rested muscle.
d) The dP/dt is greatly reduced in fatigue and is
significantly correlated with AT. A reduction
in -dP/dt is also seen in the fatigued muscle.
e) Despite the persistence of fatigue, the following
parameters have returned to pre-contraction values
after 40 minutes of recovery: venous pH, PO2, Q
and muscle phosphorylcreatine concentration.
It has been concluded from the above observations
that fatigue is not caused by a lack of availability of
energy. Fatigue appears to be the result of a reduced
activation of the muscle cells. This may be due to either
a reduced amount of Ca2 + released or a reduced sensitivity
of the contractile proteins. Since AT of a tetanic
contraction of the fatigued muscle was not different
from that of a rested muscle, it appears that the capacity
of the muscle to generate force is not reduced. Fatigue,
then must be a result of lower Ca2 + release or a change in
the binding relationship between Ca2 + and troponin (less
Ca2+ bound at a given Ca2+ concentration).

91
Acidosis and the Twitch Contraction
When acidosis was induced by hypoventilation, developed
tension did not decrease. It is likely that the intra¬
cellular pH reached a level in these experiments comparable
with that which was reached in the experiments summarized
above. This suggests that the reduced intensity of
activation observed as a result of fatiguing contractions
was not a result of intracellular acidosis.

PROPOSED HYPOTHESES FOR SKELETAL MUSCLE FATIGUE
Several hypotheses can be invoked to explain how there
may be less Ca2 + released from the lateral sacs. Following
is a discussion of a few hypothetical mechanisms to explain
the fatigue. These mechanisms are presented, not as an
explanation for the observed fatigue, but as hypotheses
which need to be tested to prove or disprove their
involvement in this fatigue. A detailed hypothesis of
the fatigue seen in these experiments is not justified
by the data available.
Depletion of Calcium at Lateral Sacs
Less Ca2+ would be released from the lateral sacs if
less Ca2+ were located at the lateral sacs. This situation
would occur if Ca2+ were lost from the cell or sequestered
by some other organelle within the cell. It is unlikely
that significant Ca2+ was lost from the cell during this
period of stimulation. The Ca concentration of the
interstitial fluid is high compared with the intracellular
concentration at rest or during contractions (18).
The amount of Ca within each muscle cell may be
unaltered in fatigue. Under these circumstances, the
amount of Ca^t available to the lateral sacs may be
limited if some organelle sequesters Ca2+. It has been
reported that mitochondria accumulate Ca during exercise
92

93
(5). It is conceivable that calcium accumulation by
mitochondria may reduce the amount of Ca2+ available
for recycling in the sarcoplasmic reticulum. This could
reduce the amount of Ca^ released with each impulse.
Other organelles may also sequester Ca , limiting the
amount of Ca2 + available to the sarcoplasmic reticulum.
The longitudinal tubules are known to accumulate
Ca2 + (67). It is unlikely that this accumulation would
permit the observed persistence of fatigue. Apparently
the time necessary for translocation of the Ca2+ from
the longitudinal reticulum to the lateral sacs is much
shorter (67) than the observed 40 minutes of recovery
during which no significant increase in AT occurred.
Compartmentalization Within the Lateral Sacs
A reduced Ca2+ release from lateral sacs would occur
if Ca were compartmentalized within the lateral sacs in a
manner which made it unavailable for release (i.e.f bound
vs free). There is no specific evidence available to
support this hypothesis. It is mentioned here only
because it is a hypothetical explanation of the data.
Attenuated Trigger Mechanism for Release of Calcium
A third potential mechanism which would result in
reduced Ca release would be an attenuation of the
2+
trigger mechanism for release of Ca . This mechanism
is poorly understood and therefore a discussion of how
this might be affected in fatigue is unwarranted. One
possibility, however, would be that conduction of the

muscle membrane depolarization into the transverse
tubular system is altered in a manner which affects the
trigger mechanism and therefore reduces Ca^ release.
Reduced Binding Sensitivity for Calcium
It is possible that the amount of Ca2 + released from
the lateral sacs is unaltered, but the degree of activation
accomplished at that concentration is reduced. It has been
observed that acidosis was not likely the cause of this
alteration, but other metabolic factors may be capable
of altering the binding relationship between Ca2+ and
troponin, thereby reducing the intensity of activation
• 9 +
at a given free Ca
concentration.

CONCLUSIONS
It is apparent that further research will be necessary
to elucidate the mechanism causing the fatigue observed
in the experiments reported herein. This research,
however, has eliminated several proposed mechanisms of
fatigue. A few hypotheses are presented which may
contribute to the fatigue observed in these experiments.
Each of these hypotheses is based on a central theme,
a reduced activation of the contractile proteins. It
remains to be seen which of these mechanisms (if any) is
the actual cause of the twitch fatigue observed in these
experiments.
95

APPENDIX
Method for Phosphorylcreatine Analysis
The method of Ennor and Stocken (25) was used to
determine phosphorylcreatine content of muscle samples.
This method determines creatine content before and after
a period of acid hydrolysis of phosphorylcreatine. The
key reaction for this determination is creatine with
diacetyl which yields a pink colored compound. The
intensity of the pink color is proportional to creatine
concentration when diacetyl is available in excess. The
optical density is determined with a spectrophotometer
at 525 nm.
Chemicals and Reagents for Deproteinization and Determination
I Perchloric Acid, stock 70-72% (Baker)
a) .8 M in 40% ethanol
b) .6 M (no ethanol)
II Potassium Carbonate, (Baker) 3 M containing
.5 M triethanolamine (Baker)
III Sodium Hydroxide (Malinkrodt)
a) 1 N
b) . 4 N
IV Hydrochloric acid (Fisher)
a) IN
b) . 4 N
V p Chloromercuric acid (Sigma) (also called p
Hydroxymercuric acid) .05 M purchased as
solution, or as powder then dissolved in
NaOH (1 N) then made to volume with H2O.
VI Naphthol (Sigma) 1% in stock alkali, mix new
daily
96

97
VII Diacetyl (Sigma)
a) stock solution 1%
b) mix 1:20 daily for use in assay
VIII Stock Alkali
for 1 liter: 160 g Na2CC>3 (Baker) and 60 g NaOH
(Malinkrodt)
IX Creatine (Sigma) (for standards)
16 yg/ml
Procedure for Deproteinization
Muscle samples weighing 30-80 mg were frozen in
situ with metal clamps pre-cooled in liquid N2* The
muscle samples were kept frozen until they were
homogenized in Perchloric acid. The samples were
homogenized (Vertis) for 60 seconds at high speed in
6 ml of ice cold .8 M Perchloric acid. The homogenate
was poured into a centrifuge tube (15 ml) and the blades
and bown were rinsed with 3 ml of .6 M Perchloric acid.
The .6 M Perchloric acid was retained in a separate
centrifuge tube.
The homogenate was spun down at 1200 g for 15 minutes
at 0°C. The supernatant was saved, and the pellet was
resuspended in the 3 ml rinse (.6 M Perchloric acid) .
This suspension was recentrifuged at 1200 g for 15
minutes and the supernatants were combined.
The combined supernatant was neutralized to pH
5.5-6.0 with addition of 3 M Na2CC>3. This was added
dropwise with constant mixing to avoid bubbling over.
Samples were subsequently certrifuged at room temperature
in a desk-top centrifuge at maximal rpm for 10 minutes.
The volume of the supernatant was determined and the

98
supernatant was stored in a freezer until further analysis
was done (usually same day, but a delay of 2-3 days made
no difference).
Determination of Free and Total Creatine
Duplicate analysis for free and total creatine were
made on each sample (4 aliquots per supernatant). Standards
also in duplicate were run with each determination.
Following, is a list of the procedures for free and total
determination:
1. Neutralized samples to pH = 7.0-7.3 with IN
NaOH (and IN HC1 if needed).
2. Measure 4 aliquots (.5-1 ml) of each supernatant
into graduated tubes.
3. Add H2O to 3 ml mark.
4. Place 2 (of 4) tubes in hot water bath (65°C)
to equilibrate.
5. Add 1 ml of .4 N HC1 to tubes which have
equilibrated to 65°C.
6. Replace in hot water bath for 9 minutes. These
are tubes for total creatine determination.
7. Remove the tubes from the bath and add 1 ml of
.4 N NaOH. Place in ice bath to cool quickly
to room temperature.
8. To each of the four tubes (2 free and 2 total),
add sequentially: 1 ml of p hydroxymercuribenzoate,
2 ml of Naphthol and 1 ml diacetyl.
9. Make volume to 10 ml, agitate and place in dark
for 20 minutes.
10.Read optical density at 525 nm.
Standards receive the same treatment as the samples
for free creatine (blank does not get 1 ml p hydroxy¬
mercuribenzoate. Creatine content of samples is

99
determined from the regression line obtained from the
standards. Phosphorylcreatine content is (total
creatine - free creatine)/total creatine.

BIBLIOGRAPHY
1. Asmussen, E. and B. Mazin. A central nervous
component in local muscular fatigue. Eur. J.
Appl, Physiol. 38: 9-15, 1978.
2. Barr, A. J., J. H. Goodnight, J. P. Sail and J. T. Helwig.
A Users Guide to SAS 76. Raleigh, North Carolina,
SAS Institute, 1976.
3. Bergmans, J., P. Geerinckx and N. Rosselle. The
kinetics of muscular fatigue in man. Electrical
Myography and Clinical Neurophysiology. 16: 25-46,
1976.
4. Blinks, J. R., R. Rudel and S. R. Taylor. Calcium
transients in isolated amphibian skeletal muscle
fibers: detection with aequorin. J. Physiol.
277: 291-323, 1978.
5. Bonner, H. G., S. W. Leslie, A. B. Combs and C. A. Tate.
Effects of exercise training and exhaustion on 45ca
uptake by rat skeletal muscle mitochondria and
sarcoplasmic reticulum. Research Comm, in Chemical
Path, and Pharm. 14: 767-770, 1976.
6. Bronk, D. W. The energy expended in maintaining a
muscular contraction. J, Physiol. 69: 306-315, 1930.
7. Brooks, G. A., K. J. Hittelman, J. A. Faulkner and
R. E. Beyer. Temperature, skeletal muscle mitochondrial
functions, and oxygen debt. Am. J. Physiol. 220: 1053-
1059, 1971.
8. Bruning, J. L. and B. L. Kintz. Computational Handbook
of Statistics. Atlanta, Scott, Foresman and Company,
1968, pp. 43-47.
9. Brust, M. Changes in contractility of frog muscle
due to fatigue and inhibitors. Am. J. Physiol. 206:
1043-1948, 1964.
10.Brust, M. Fatigue and caffeine effects in fast-twitch
and slow-twitch muscles of the mouse. Pflugers Arch.
367: 189-200, 1976.
100

101
11. Burke, R. E., D. N. Levine, P. Tsairis and
F. E. Zajac, III. Physiological types and
histochemical profiles in motor units of the
cat gastrocnemius. J. Physiol. 234: 723-
748, 1973.
12. Burnell, J. M. In vivo response of muscle to
changes in CO2 tension or extracellular bicarbonate.
Am. J. Physiol. 215: 1376-1383, 1968.
13. Chance, B. The energy-linked reaction of calcium
with mitochondria. J. Biol. Chem. 240: 2729-2748,
1965.
14. Chapler, C. K. and W. N. Stainsby. Carbohydrate
metabolism in contracting dog skeletal muscle in
situ. Am. J, Physiol. 215: 995-1004, 1968.
15. Connally, R., W. Gaugh and S. Winegrad. Characteristics
of isometric twitch of skeletal muscle immediately
after a tetanus. J. Gen. Physiol. 57: 697-709,
1971.
16. Dahlback, L. O., J. Ekstedt and E. Stalberg.
Ischemic effects on impulse transmission to
muscle fibers in man. Electroenceph. Clin.
Neurophysiol. 27: 540-543, 1969.
17. Desmedt, J. E. and K. Ilainaut. Kinetics of myofilament
activation in potentiated contraction: staircase
phenomenon in human skeletal muscle. Nature. 217:
529-532, 1958.
18. Ebashi, S. and M. Endo. Calcium ion and muscle
contraction. Progess in Biophysics and Molecular
Biology. 18: 125-183, 1968.
19. Eberstein, A. and A. Sandow. Fatigue mechanism in
muscle fibers. In: Effects of Use and Disuse on
Neuromuscular Functions. E. Gutmann (ed). pp 515-
526. Prague, Publication House Czecholsovak
Acad. Sci., 1963.
20. Edwards, R. H. T. and D. K. Hill. "Economy" of
force maintenance during electrically stimulated,
isometric contractions of human muscle. J. Physiol.
250: 13P-14P, 1975.
21. Edwards, R. H. T., D. K. Hill and D. A. Jones. Heat
production and chemical changes during isometric
contractions of the human quadriceps muscle.
J. Physiol. 251: 303-315, 1975.

102
22. Edwards, R. H. T., D. K. Hill and D. A. Jones.
Metabolic changes associated with the slowing of
relaxation in fatigued mouse muscle. J. Physiol.
251: 287-301, 1975.
23. Edwards, R. H. T., D. K. Hill, D. A. Jones and
P. A. Merton. Fatigue of long duration in human
skeletal muscle after exercise. J. Physiol. 272:
769-778, 1977.
24. Ellis, K. 0. and J. F. Carpenter. Studies on the
mechanism of action of dantrolene sodium. A
skeletal muscle relaxant. Naunyn-schmedeberg's
Arch. Pharmacol. 275: 83-94, 1972.
25. Ennor, A. H. and L. A. Stocken. Estimation of
creatine. Biochem. J. 42: 557-563, 1948.
26. Fabiato, A. and F. Fabiato. Effects of pH on the
myofilaments and the sarcoplasmic reticulum of
skinned cells from cardiac and skeletal muscles.
J. Physiol. 276: 233-255, 1978.
27. Fales, J. T., S. R. Heisey and K. L. Zierler.
Dependency of oxygen consumption of skeletal muscle
on number of stimuli during work in the dog.
Am. J. Physiol. 198: 1333-1342, 1960.
28. Feng, T. P. The heat-tension ratio in prolonged
tetanic contractions. Proc. Roy. Soc. (London) B.
108: 522-537, 1931.
29. Fitts, R. H. and J. 0. Holloszy. Lactate and
contractile force in frog muscle during development
of fatigue and recovery. Am. J. Physiol. 231:
430-431, 1976.
30. Fitts, R. H. and J. 0. Holloszy. Contractile
properties of rat soleus muscle: effects of
training and fatigue. Am. J. Physiol. Cell
233: C86-C91, 1977.
31. Fretthold, D. W. and L. C. Garg. The effect of
acid-base changes on skeletal muscle twitch tension.
Can. J. Physiol. Pharmacol. 56: 543-549, 1977.
32. Fuchs, F., V. Reddy and F. N. Briggs. The inter¬
action of cations with the calcium binding site
of troponin. Biochim. Biophys. Acta. 221: 407, 1970.
33. Furusawa, K. and R. M. T. Kerridge. The hydrogen
ion concentration of the muscles of the cat.
J. Physiol. 63: 33-41, 1927.

103
34. Gladden, L. B., B. R. Macintosh and W. N. Stainsby.
C>2 uptake and developed tension during and after
fatigue, curare block and ischemia. J. Appl. Physiol.
45: 751-755, 1978.
35. Grabowski, W., E. A. Lobsiger and H. C. Luttgau.
The effect of repetitive stimulation at low
frequencies upon the electrical and mechanical
activity of single muscle fibers. Pflugers Arch.
334: 222-239, 1972.
36. Hainaut, K. and M. Golde. Effect of ischemia on
contractile processes in human skeletal muscle.
Electromyog. Clin. Neurophysiol. 16: 67-74, 1976.
37. Hanson, J. The effects of repetitive stimulation on
the action potential and the twitch of rat muscle.
Acta. Physiol. Scand. 90: 387-400, 1974.
38. Harris, R. C., R. H. T. Edwards, E. Hultman,
L. O. Nordesjo, B. Nylind and K. Sahlin. The time
course of phosphorylcreatine resynthesis during
recovery of the quadriceps muscle in man. Pflugers
Archiv. 367: 137-142, 1976.
39. Hermanson, L. and J. B. Osnes. Blood and muscle
pH after maximal exercise in man. J. Appl. Physiol.
32: 304-308, 1972.
40. Jacobus, W. E. and A. Lehninger. Creatine kinase
of rat heart mitochondria. J. Biol. Chem. 248:
4803-4810, 1973.
41. Krnjevic, K. and R. Miledi. Failure of neuromuscular
propagation in rats. J. Physiol. 140: 440-461, 1958.
42. Kurihara, T. and J. E. Brooks. The mechanism of
neuromuscular fatigue: A study of mammalian muscle
using excitation-contraction uncoupling. Arch. Neurol.
32: 168-174, 1975.
43. Maxwell, L. C., J. K. Barclay, D. E. Mohrman and
J. A. Faulkner. Physiological characteristics of
skeletal muscle of dogs and cats. Am. J. Physiol.
233: C14-C18, 1977.
44. Merton, P. A. Voluntary strength and fatigue.
J. Physiol. 123: 553-564, 1954
45. Mountcastle, V. B. (ed). Medical Physiology. 13th
Edition. St. Louis, C. V. Mosby, 1974.

104
46. Nakamaru, Y. and A. Schwartz. The influence of
hydrogen ion concentration on calcium binding and
release by skeletal muscle sarcoplasmic reticulum.
J. Gen. Physiol. 59: 22-32, 1972.
47. Ochs, R. M. , J. C. Smith and V. R. Edgerton. Fatigue
characteristics of human gastrocnemius and soleus
muscles. Electromyography and Clin. Neurophysiol.
17: 297-306, 1977.
48. Otsuka, M., M. Endo and Y. Nonomura. Presynaptic
nature of neuromuscular depression. Jap. J. Physiol.
12: 573-583, 1962.
49. Paul, D. H. The effects of anoxia on the isolated
rat phrenic-nerve-diaphragm preparation. J. Physiol.
155: 358-371, 1961.
50. Petrofsky, J. S. Control of the recruitment and
firing frequencies of motor units in electrically
stimulated muscles in the cat. Medical and Biol.
Eng, and Ecmput. 16: 302-308, 1978.
51. Piiper, J. and P. Spiller. Repayment of 02 debt and
resynthesis of high-energy phosphates in gastrocnemius
muscle of the dog. J. Appl. Physiol. 28: 657-662,
1970 .
52. del Pozo, E. C. Transmission fatigue and contraction
fatigue. Am. J. Physiol. 135: 763-771, 1952.
53. Sahlin, K., R. C. Harris. B. Nylund and E. Hultman.
Lactate content and pH in muscle samples obtained
after dynamic exercise. Pflugers Archiv. 367:
143-149, 1976.
54. Sandow, A. and M. Brust. Effects of activity on
contractions of normal and dystrophic mouse muscles.
Am. J. Physiol. 202: 815-820, 1962.
55. Seraydarian, M. W., L. Artaza and B. C. Abbott.
Creatine and the control of energy metabolism in
cardiac and skeletal muscle cells in culture.
J. Molec. and Cellular Card. 6: 405-413, 1974.
56. Spande, J. I. and B. A. Schottelius. Chemical basis
of fatigue in isolated mouse soleus muscle.
Am. J. Physiol. 219: 1490-1495, 1970.
57. Stainsby, W. N. Oxygen uptake for isotonic and
isometric twitch contractions of dog skeletal muscle
in situ. Am. J. Physiol. 219: 435-439, 1970

105
58. Stainsby, W. N., J. T. Fales and J. L. Lilienthal, Jr.
Effect of passive stretch on oxygen consumption of
doq skeletal muscle in situ. Bull. Johns Hopkins Hosp.
99: 249-261, 1956.
59. Stainsby, W. N. and A. B. Otis. Blood flow, oxygen
tension, oxygen uptake and oxygen transport in
skeletal muscle. Am. J. Physiol. 206: 858-866,
1964 .
60. Stainsby, W. N. and H. G. Welch. Lactate metabolism
of contracting dog skeletal muscle in situ.
Am. J. Physiol. 211: 177-183, 1966.
61. Steinhagen, C., H. J. Hirche, H. W. Nestle,
U. Bovenkamp and I. Hosselman. The interstitial
pH of the working gastrocnemius muscle of the dog.
Pflugers Archiv. 367: 151-156, 1976.
62. Vander, A. J., J. H. Sherman and D. S. Luciano.
Human Physiology. The Mechanism of Body Function.
2nd Edition, New York, McGraw-Hill, 1975.
63. Vergara, J. L., S. I. Rapoport and V. Nassar-Centina.
Fatigue and post-tetanic potentiation in single
muscle fibers of the frog. Am. J. Physiol. Cell,
1: C185-C190, 1977.
64. Visscher, M. V. and J. A. Johnson. The Fick
principle: Analysis of potential errors in its
conventional application. J. Appl. Physiol. 5:
635-638, 1953.
65. Weber, A. and L. Winicur. The role of Ca++ in the
superprecipitation of actomyosin. J. Biol. Chem.
236: 3198-3202, 1961.
66. Wilson, B. A. and W. N. Stainsby. Relation between
oxygen uptake and developed tension in dog skeletal
muscle. J. Appl. Physiol.
67. Winegrad, S. Intracellular calcium movements of
frog skeletal muscle during recovery from tetanus.
J. Gen. Physiol. 51: 65-83, 1968.

BIOGRAPHICAL SKETCH
Brian Robert Macintosh was born in Owen Sound,
Ontario, March 26, 1952. He graduated from the
Owen Sound Collegiate and Vocational Institute in
1971. He received his Bachelor of Science degree
in Human Kinetics in 1975 at the University of
Guelph.
Brian was accepted into the Ph.D. program in
the Department of Physiology at the University of
Florida in 1975, and was admitted to candidacy for
the Doctor of Philosophy degree in 1977.
Brian is married to the former Patricia Dale
Wilkie. They have two children. Jennifer Louise
and Robert John David.
106

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.
tt
(
JL
Irina
W. N. Stainsby, Chairman
Professor of Physiology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Physiology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.
C '•'l
P. Posner
Associate Professor
of Physiology

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Biochemistry
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.
C~! w7 2fauner
Professor of Professional
Physical Education
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the require¬
ments for the degree of Doctor of Philosophy.
June, 1979
Dean, College of Medicine
Deá^V

m / saf
/?




PAGE 1

)$7,*8( ,1 6.(/(7$/ 086&/( %< %5,$1 52%(57 0$&,1726+ $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( &281&,/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

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f &ROOHJH RI 1XUVLQJ 7HDFKLQJ $VVLVWDQWVKLSf 7KH UHVHDUFK UHSRUWHG LQ WKLV GLVVHUWDWLRQ KDV EHHQ VXSSRUWHG E\ 7KH $PHULFDQ +HDUW $VVRFLDWLRQ )ORULGD $IILOLDWH *UDQW $* DQG 6SRQVRUHG 5HVHDUFK 6HHG *UDQW DZDUGHG WR 'U 6WDLQVE\ ZRXOG OLNH WR WKDQN :HQG\ $XHUEDFK IRU GRLQJ DQ H[FHOOHQW MRE RI W\SLQJ WKLV PDQXVFULSW OL

PAGE 3

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

PAGE 4

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

PAGE 5

/,67 2) ),*85(6 3DJH ([FLWDWLRQ&RQWUDFWLRQ&RXSOLQJ *DVWURFQHPLXV3ODQWDULV 0XVFOH 3UHSDUDWLRQ 6DPSOH ([SHULPHQWV 92J YHUVXV 'HYHORSHG 7HQVLRQ 92J YHUVXV 'HYHORSHG 7HQVLRQ DOO 'DWD 1RUPDOL]HG 7ZLWFK DQG 7ZLQ &RQWUDFWLRQV 'HYHORSHG 7HQVLRQ DQG G3GW 7ZLWFK &KDUDFWHULVWLFV IURP )DVW 7UDFHV RI 'HYHORSHG 7HQVLRQ DQG G3GW 3URFHGXUH IRU )DWLJXLQJ &RQWUDFWLRQV ZLWK 3HULRGLF )DVW 7UDFHV 4 32J 3&2J DQG S+ 'XULQJ DQG )ROORZLQJ )DWLJXLQJ &RQWUDFWLRQV 'HYHORSHG 7HQVLRQ +DOI 5HOD[DWLRQ 7LPH DQG &RQWUDFWLRQ 7LPH 3HDN 5DWH RI )RUFH 'HYHORSPHQW DQG 3HDN 5DWH RI 5HOD[DWLRQ 7HWDQLF &RQWUDFWLRQ %HIRUH DQG $IWHU )DWLJXLQJ &RQWUDFWLRQV 7ZLWFK 'HYHORSHG 7HQVLRQ DQG G3GW %HIRUH 'XULQJ DQG $IWHU ,VFKHPLF )DWLJXH +DOI 5HOD[DWLRQ 7LPH YHUVXV 'HYHORSHG 7HQVLRQ IRU 'DQWUROHQH 5HGXFHG 6WLPXODWLRQ 9ROWDJH DQG ,VFKHPLF )DWLJXH 7KH (IIHFWV RI 3UHFHGLQJ &RQWUDFWLRQV RQ 'HYHORSHG 7HQVLRQ 3URFHGXUH IRU 9HQWLODWLRQ DQG 6DPSOLQJ 3DWWHUQ Y

PAGE 6

3DJH $UWHULDO DQG 9HQRXV >+@ 3&! 'XULQJ 'LIIHUHQW 9HQWLODWRU\ 6WDWHV 'HYHORSHG 7HQVLRQ G3GW DQG G3GW 'XULQJ 'LIIHUHQW 9HQWLODWRU\ 6WDWHV &RQWUDFWLRQ 7LPH DQG +DOI 5HOD[DWLRQ 7LPH 'XULQJ 'LIIHUHQW 9HQWLODWRU\ 6WDWHV YL

PAGE 7

/,67 2) 7$%/(6 3DJH 837$.( $1' '(9(/23(' 7(16,21 %()25( $1' $)7(5 )$7,*8( ,1 6(5,(6 ,, 5(/$7,216+,3 %(7:((1 837$.( $1' '(9(/23(' 7(16,21 ,1 6(5,(6 ,,, 3+263+25
PAGE 8

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH &RXQFLO RI WKH 8QLYHUVLW\ RI )ORULGD ,Q 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ )$7,*8( ,1 6.(/(7$/ 086&/( %\ %ULDQ 5REHUW 0DFLQWRVK -XQH &KDLUPDQ :HQGHOO 1 6WDLQVE\ '6F 0DMRU 'HSDUWPHQW 3K\VLRORJ\ 7KH LQ VLWX GRJ JDVWURFQHPLXVSODQWDULV PXVFOH SUHSDUDWLRQ KDV EHHQ XVHG WR VWXG\ IDWLJXH 6NHOHWDO PXVFOH IDWLJXH UHGXFHG IRUFH RXWSXW IRU D JLYHQ VWLPXOXVf UHVXOWV IURP D WKLUW\ PLQXWH SHULRG RI LVRPHWULF FRQWUDFWLRQV DW WR VHF 7KLV IDWLJXH LV QRW D UHVXOW RI IDLOXUH RI PRWRU QHUYH SURSDJDWLRQ RU WUDQVPLWWHU UHOHDVH 7KH UDWLR RI R[\JHQ XSWDNH WR GHYHORSHG WHQVLRQ WRWDO WHQVLRQ PLQXV UHVWLQJ WHQVLRQf LV XQDOWHUHG GXULQJ RU IROORZLQJ IDWLJXLQJ FRQWUDFWLRQV 7KH HFRQRP\ RI IRUFH SURGXFWLRQ LV XQDOWHUHG E\ WZLQ LPSXOVH VWLPXODWLRQ UHODWLYH LVFKHPLD RU DGPLQLVWUDWLRQ RI PRGHUDWH GRVHV RI FXUDUH RU VXFFLQ\OFKROLQH YLL L

PAGE 9

:KHQ GHYHORSHG WHQVLRQ LV UHGXFHG GXH WR UHSHWLWLYH VWLPXODWLRQ IRU WKLUW\ PLQXWHV DW RU VHF FRQWUDFWLRQV WKH WLPH WR SHDN WHQVLRQ DQG KDOI UHOD[DWLRQ WLPHV DUH XQDOWHUHG 7KH SHDN UDWHV RI IRUFH GHYHORSPHQW DQG RI UHOD[DWLRQ DUH UHGXFHG SURSRUWLRQDOO\ WR WKH UHGXFWLRQ LQ GHYHORSHG WHQVLRQ )ROORZLQJ D IRUW\ PLQXWH SHULRG RI UHFRYHU\ WKH WZLWFK GHYHORSHG WHQVLRQ UHPDLQV JUHDWO\ DWWHQXDWHG EXW WHWDQLF PVHF RI VHF VWLPXODWLRQf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f UHVXOWHG LQ D UHGXFWLRQ LQ DUWHULDO S+ WR 7KHUH ZDV QR UHGXFWLRQ LQ WZLWFK GHYHORSHG WHQVLRQ DVVRFLDWHG ZLWK WKLV ,;

PAGE 10

DFLGRVLV ,W LV OLNHO\ WKDW LQWUDFHOOXODU S+ IHOO DV PXFK GXULQJ WKH UHVSLUDWRU\ DFLGRVLV DV LW GLG GXULQJ IDWLJXLQJ FRQWUDFWLRQV DW VHF 7KH IDWLJXH REVHUYHG GXULQJ FRQWUDFWLRQV DW VHF FRXOG QRW EH D UHVXOW RI LQWUDFHOOXODU DFLGRVLV ,W FDQ EH FRQFOXGHG IURP WKHVH H[SHULPHQWV WKDW WZLWFK IDWLJXH LV QRW D UHVXOW RI HQHUJ\ GHILFLHQF\ UHGXFHG FDSDFLW\ RI WKH FRQWUDFWLOH HOHPHQWV LQWUDn FHOOXODU DFLGRVLV LQGXFHG E\ UHGXFHG YHQWLODWLRQ IRU VL[W\ WR QLQHW\ PLQXWHVf RU QHXURPXVFXODU MXQFWLRQ IDLOXUH %\ WKH SURFHVV RI HOLPLQDWLRQ LW DSSHDUV WKDW WZLWFK IDWLJXH UHVXOWV IURP D UHGXFHG DFWLYDWLRQ RI WKH P\RILODPHQWV GXULQJ D WZLWFK FRQWUDFWLRQ 7KLV PD\ EH GXH WR HLWKHU D UHGXFHG VDUFRSODVPLF &DA FRQFHQWUDWLRQ GXULQJ FRQWUDFWLRQ RU D UHGXFHG UHVSRQVH RI WKH P\RILODPHQWV DW D JLYHQ &DA FRQFHQWUDWLRQ [

PAGE 11

,1752'8&7,21 7KH ZRUG IDWLJXH KDV EHHQ XVHG LQ WKH SDVW ZLWK VHYHUDO YDULRXV GHILQLWLRQV 6RPH DXWKRUV f HTXDWH IDWLJXH ZLWK H[KDXVWLRQ 2WKHUV f XVH WKH ZRUG IDWLJXH WR UHSUHVHQW DQ LQDELOLW\ WR PDLQWDLQ D SDUWLFXODU ZRUN RXWSXW 0RUH UHFHQWO\ f VNHOHWDO PXVFOH IDWLJXH KDV EHHQ GHILQHG DV D UHGXFHG FDSDFLW\ RI WKH PXVFOH WR GHYHORS WHQVLRQ (GZDUGV HW DO f DQG )LWWV DQG +ROORV]\ f KDYH REVHUYHG WKDW D WZLWFK FRQWUDFWLRQ FDQ VWLOO EH DWWHQXDWHG ZKHQ WKH IRUFH JHQHUDWLQJ FDSDFLW\ RI WKH PXVFOH LV IXOO\ UHFRYHUHG $ VLQJOH LPSXOVH GRHV QRW PD[LPDOO\ DFWLYDWH WKH FRQWUDFWLOH DSSDUDWXV f DQG WKHUHIRUH GRHV QRW SHUPLW WKH IXOO FRQWUDFWLOH UHVSRQVH RI ZKLFK WKH PXVFOH LV FDSDEOH 7KH FDSDFLW\ WR GHYHORS WHQVLRQ PXVW EH HYDOXDWHG XQGHU FRQGLWLRQV RI PD[LPDO DFWLYDWLRQ 7KLV FDQ EH DFFRPSOLVKHG ZLWK D FDIIHLQ RU LQGXFHG FRQWUDFWXUH RU D PD[LPDO WHWDQLF FRQWUDFWLRQ ,I IDWLJXH LV GHILQHG DV D UHGXFHG FDSDFLW\ RI WKH PXVFOH WR JHQHUDWH IRUFH WKHQ ZKDW LV LW FDOOHG LI WKHUH LV DQ DWWHQXDWHG UHVSRQVH WR D VLQJOH LPSXOVH" ,W DSSHDUV WR EH D VHSDUDWH SKHQRPHQRQ DQG SUREDEO\ RFFXUV E\ D VHSDUDWH PHFKDQLVP VLQFH D PXVFOH UHFRYHUV IXOO FDSDFLW\ WR GHYHORS WHQVLRQ EHIRUH WZLWFK WHQVLRQ UHFRYHUV WR SUHIDWLJXH YDOXHVf (GZDUGV HW DO SRLQW RXW f WKDW

PAGE 12

WKLV WZLWFK IDWLJXH PD\ EH DVVRFLDWHG ZLWK SHUFHSWLRQ RI LQFUHDVHG HIIRUW QHFHVVDU\ WR PDLQWDLQ D JLYHQ ZRUNORDG RU IRUFH RXWSXW )RU WKH SXUSRVHV RI WKLV GLVVHUWDWLRQ IDWLJXH ZLOO EH XVHG DV D JHQHUDOL]DWLRQ UHIHUULQJ WR D UHGXFHG UHVSRQVH RI WKH PXVFOH WR D JLYHQ VWLPXODWLRQ 7ZLWFK IDWLJXH ZLOO UHIHU WR DQ DWWHQXDWHG UHVSRQVH WR D VLQJOH LPSXOVH :LOVRQ DQG 6WDLQVE\ f KDYH UHSRUWHG WKDW WZLWFK GHYHORSHG WHQVLRQ RI WKH LQ VLWX GRJ JDVWURFQHPLXV SODQWDULV PXVFOH UHPDLQV DWWHQXDWHG IRU KRXUV IROORZLQJ D VHULHV RI FRQWUDFWLRQV DW SHU VHFRQG )LWWV DQG +ROORV]\ f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nV XQGHUVWDQGLQJ RI VNHOHWDO PXVFOH IDWLJXH D EULHI GHVFULSWLRQ RI SHUWLQHQW PXVFOH SK\VLRORJ\ DQG FXUUHQW WKHRULHV RI IDWLJXH SUHFHGHV WKH VHFWLRQV GHVFULELQJ WKH H[SHULPHQWV ZKLFK KDYH EHHQ GRQH

PAGE 13

7KH GRJ LQ VLWX JDVWURFQHPLXVSODQWDULV PXVFOH JURXS KDV EHHQ XVHG WKURXJKRXW WKH VHULHV RI VWXGLHV UHSRUWHG KHUHLQ 7R DYRLG UHSHWLWLRQ WKH JHQHUDO SURFHGXUHV DQG GHVFULSWLRQ RI WKH SUHSDUDWLRQ DSSHDU DV D VHSDUDWH FKDSWHU EHIRUH DQ\ RI WKH VWXGLHV 7KH VSHFLILF SURFHGXUHV XVHG LQ HDFK VWXG\ DUH GHVFULEHG VHSDUDWHO\ LQ D 0HWKRGV VHFWLRQ IRU WKDW VWXG\ ,Q WKH ILUVW VWXG\ WKH UHODWLRQVKLS EHWZHHQ R[\JHQ XSWDNH DQG GHYHORSHG WHQVLRQ KDV EHHQ GHWHUPLQHG IRU VNHOHWDO PXVFOH EHIRUH GXULQJ DQG DIWHU IDWLJXLQJ FRQWUDFWLRQV 7KHVH H[SHULPHQWV ZHUH GRQH WR GHWHUPLQH ZKHWKHU RU QRW WKH HQHUJ\ XVHG E\ D PXVFOH UHODWLYH WR WKH DPRXQW RI WHQVLRQ GHYHORSHG LV DOWHUHG E\ IDWLJXH 7KHUH DUH UHSRUWV WKDW LQGLFDWH D FKDQJH LQ HLWKHU GLUHFWLRQ FDQ EH H[SHFWHG f )XUWKHU VWXGLHV ZHUH FRQGXFWHG WR GHWHUPLQH ZKHWKHU RU QRW FKDQJHV RFFXU LQ WKH WLPHFRXUVH RI WKH WZLWFK DV D UHVXOW RI UHSHWLWLYH VWLPXODWLRQ &KDQJHV LQ WKH UDWH RI IRUFH GHYHORSPHQW DQG LQ WKH WLPHFRXUVH RI D WZLWFK FRQWUDFWLRQ KDYH SUHYLRXVO\ EHHQ LQWHUSUHWHG DV LQGLFDWLRQV RI FKDQJHV LQ WKH GXUDWLRQ DQG LQWHQVLW\ RI DFWLYDWLRQ RI WKH PXVFOH f 7KHVH PHDVXUHPHQWV PD\ IDFLOLWDWH DQ XQGHUVWDQGLQJ RI WKH PHFKDQLVPVf UHVSRQVLEOH IRU WKH IDWLJXH $ WKLUG VHULHV RI H[SHULPHQWV KDV EHHQ FRQGXFWHG WR VWXG\ WKH HIIHFWV RI UHVSLUDWRU\ DFLGRVLV RQ WKH WZLWFK FRQWUDFWLRQ $FLGRVLV KDV EHHQ FODLPHG WR EH RQH RI

PAGE 14

WKH PDMRU FDXVHV RI IDWLJXH f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

PAGE 15

(9(176 /($',1* 72 6.(/(7$/ 086&/( &2175$&7,21 0XVFXODU FRQWUDFWLRQ LV WKH UHVXOW RI D VHTXHQFH RI FKHPLFDO DQG SK\VLFDO HYHQWV EHJLQQLQJ ZLWK DFWLYLW\ LQ WKH FHQWUDO QHUYRXV V\VWHP &16fRU VHQVRU\ LQSXW WR WKH &16f )DLOXUH RU LPSDLUPHQW DW DQ\ VLWH LQ WKLV SURFHVV ZLOO UHVXOW LQ D UHGXFHG FRQWUDFWLOH UHVSRQVH RI WKH PXVFOH )DWLJXH DQG WZLWFK IDWLJXH WKHQ DUH UHVXOWV RI VXFK IDLOXUH 7KH LGHQWLILFDWLRQ RI WKH VLWHVf RI IDLOXUH LQ IDWLJXH ZRXOG SURYLGH D EHWWHU XQGHUVWDQGLQJ RI WKH PHFKDQLVPVf HIIHFWLQJ WKH IDWLJXH %HORZ D EULHI GLVFXVVLRQ RI WKH QRUPDO VHTXHQFH RI HYHQWV OHDGLQJ WR FRQWUDFWLRQ LV SUHVHQWHG &16 FRQWURO RI PRWRU QHUYH DFWLYLW\ LV FRPSOH[ DQG ZLOO QRW EH GHVFULEHG )RU VLPSOLFLW\ WKLV GLVFXVVLRQ LV EDVHG DW WKH FHOOXODU OHYHO 7KLV VHTXHQFH RI HYHQWV LV GHVFULEHG LQ D QXPEHU RI WH[Wn ERRNV f DQG LV LOOXVWUDWHG LQ )LJXUH )ROORZLQJ WKH SUHVHQWDWLRQ RI HYHQWV OHDGLQJ WR FRQWUDFWLRQ HDFK VWHS LQ WKH VHTXHQFH LV FRQVLGHUHG DV D SRWHQWLDO VLWH IRU D PHFKDQLVP RI IDWLJXH 7KH VHTXHQFH RI HYHQWV RFFXUULQJ DW WKH QHUYH WHUPLQDO PD\ EH VXVFHSWLEOH WR IDLOXUH 7KH DUULYDO RI DQ DFWLRQ SRWHQWLDO DW WKH QHUYH WHUPLQDO WULJJHUV WKH UHOHDVH RI DFHW\OFKROLQH IURP WKH WHUPLQDO ERXWRQ 6\QDSWLF YHVLFOHV IXVH WR WKH WHUPLQDO PHPEUDQH DQG

PAGE 16

UHOHDVH WKHLU FRQWHQWV LQWR WKH V\QDSWLF FOHIW $FHW\OFKROLQH GLIIXVHV WKH VKRUW GLVWDQFH DFURVV WKH FOHIW $f %LQGLQJ RI DFHW\OFKROLQH WR VSHFLILF UHFHSWRUV FDXVHV D WUDQVLHQW LQFUHDVH LQ SHUPHDELOLW\ RI WKH PXVFOH PHPEUDQH WR 1D DQG 7KLV UHVXOWV LQ GHSRODUL]DWLRQ RI WKH HQG SODWH 7KH UHVXOWLQJ FKDQJH LQ PHPEUDQH SRWHQWLDO LV FDOOHG WKH HQGSODWH SRWHQWLDO 'HVWUXFWLRQ RI WKH DFHW\OFKROLQH LV DFFRPSOLVKHG E\ DFHW\OFKROLQHVn WHUDVH ZKLFK LV ORFDWHG DPRQJ WKH UHFHSWRUV RQ WKH SRVW V\QDSWLF PXVFOH PHPEUDQH 5HFRQVWLWXWLRQ RI V\QDSWLF YHVLFOHV LV DFFRPSOLVKHG E\ UHXSWDNH RI FKROLQH DQG VXEVHTXHQW DFHW\ODWLRQ LQ WKH *ROJL DSSDUDWXV HQ]\PH FKROLQHDFHW\OWUDQVIHUDVHf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

PAGE 17

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

PAGE 18

IUHH &D FRQFHQWUDWLRQ 7KH VXEVHTXHQW ELQGLQJ RI &D WR WURSRQLQ UHVXOWV LQ DFWLYDWLRQ RI WKH FRQWUDFWLOH SURWHLQV LQ WKH PXVFOH 7KH DPRXQW RI &D UHOHDVHG LQ UHVSRQVH WR RQH DFWLRQ SRWHQWLDO SURSDJDWHG RYHU WKH PXVFOH PHPEUDQH LV QRW VXIILFLHQW WR VDWXUDWH WKH WURSRQLQ PROHFXOHV DQG WKHUHIRUH LQFRPSOHWH DFWLYDWLRQ RFFXUV f )RU FRPSOHWH DFWLYDWLRQ DQG WKHUHIRUH PD[LPDO IRUFH SURGXFWLRQ D SHULRG RI QHUYH DFWLYLW\ DW D KLJK IUHTXHQF\ LV QHFHVVDU\ 5HOD[DWLRQ RFFXUV DV &D LV VHTXHVWHUHG DFWLYH WUDQVSRUWf E\ WKH ORQJLWXGLQDO VDUFRSODVPLF UHWLFXOXP )ROORZLQJ UHXSWDNH FDOFLXP LV WUDQVORFDWHG DORQJ WKH ORQJLWXGLQDO UHWLFXOXP WR WKH ODWHUDO VDFV FRPSOHWLQJ WKH &D F\FOH f 7KH PHFKDQLVP RI WKLV WUDQVORFDWLRQ LV XQFOHDU

PAGE 19

3266,%/( )$7,*8( 0(&+$1,606 $1' &855(17 7+(25,(6 2) )$7,*8( &HQWUDO 1HUYRXV 6\VWHP )DWLJXH (YHQWV LQLWLDWLQJ PXVFXODU FRQWUDFWLRQ RULJLQDWH IURP VHQVRU\ LQSXW RU GLUHFWO\ LQ WKH FHQWUDO QHUYRXV V\VWHP $Q\ VWXG\ RI IDWLJXH GXULQJ H[HUFLVH RI WKH ZKROH DQLPDO PXVW FRQVLGHU WKH SRVVLELOLWLHV RI FHQWUDO LQKLELWLRQ UHVXOWLQJ LQ UHGXFHG PXVFXODU SHUIRUPDQFH 7KHUH DUH FRQIOLFWLQJ UHSRUWV FRQFHUQLQJ WKH SRWHQWLDO IRU D FHQWUDO FRPSRQHQW LQ PXVFXODU IDWLJXH )RU H[DPSOH 0HUWRQ f IRXQG WKDW PD[LPDO YROXQWDU\ HIIRUW ZDV QRW GLIIHUHQW IURP WKH UHVSRQVH RI WKH PXVFOH WR PD[LPDO WHWDQLF VWLPXODWLRQ RI WKH PRWRU QHUYH +H ZDV VWXG\LQJ EULHI FRQWUDFWLRQV RI WKH DGGXFWRU SROOLFLV RI KXPDQV &RQYHUVHO\ $VPXVVHQ DQG 0D]LQ f KDYH UHSRUWHG WKDW GLYHUWLQJ DFWLYLW\ YLVXDO VWLPXODWLRQf SHUPLWV JUHDWHU PXVFXODU SHUIRUPDQFH WKDQ WKDW ZKLFK LV DFFRPSOLVKHG ZKHQ WKH H\HV DUH FORVHG )XUWKHU H[SHULPHQWV GHPRQVWUDWHG WKDW LPPHGLDWH UHFRYHU\ IURP H[KDXVWLQJ H[HUFLVH ZLWK H\HV FORVHGf RFFXUUHG LI WKH H\HV ZHUH VXEVHTXHQWO\ RSHQHG ,W LV DSSDUHQW IURP WKH ZRUN RI $VPXVVHQ DQG 0D]LQ f WKDW FHQWUDO HIIHFWV FDQ DOWHU PXVFXODU SHUIRUPDQFH ,W LV LPSRUWDQW WR NHHS LQ PLQG WKRXJK WKDW XQGHU VRPH

PAGE 20

FLUFXPVWDQFHV LH EULHI PD[LPDO HIIRUWf IDWLJXH DSSHDUV WR EH GXH HQWLUHO\ WR SHULSKHUDO PHFKDQLVPV f 1HXURPXVFXODU -XQFWLRQ )DLOXUH ,Q WKH QRUPDO VHTXHQFH RI HYHQWV SUHFHGLQJ D PXVFXODU FRQWUDFWLRQ DQ DFWLRQ SRWHQWLDO LV SURSDJDWHG RYHU WKH PXVFOH PHPEUDQH 7KH RFFXUUHQFH RI D QRUPDO PXVFOH DFWLRQ SRWHQWLDO LV GHSHQGHQW RQ WUDQVPLWWHU UHOHDVH DQG PXVFOH PHPEUDQH SURSHUWLHV 5HSHWLWLYH VWLPXODWLRQ PD\ DOWHU WKHVH SURSHUWLHV DQG WKLV FRXOG UHVXOW LQ DOWHUDWLRQV LQ WKH FRQWUDFWLOH UHVSRQVH 0HUWRQ f IRXQG QR FKDQJH LQ IDWLJXH LQ WKH HOHFWURP\RJUDP UHVXOWLQJ IURP PD[LPDO VWLPXODWLRQ GHVSLWH DQ DWWHQXDWLRQ RI IRUFH RXWSXW %HUJPDQV f VWXG\LQJ KXPDQ H[WHQVRU GLJLWRUXP EUHYLV REVHUYHG QR FKDQJH LQ WKH VXUIDFH HOHFWURP\RJUDP GXULQJ IDWLJXLQJ FRQWUDFWLRQV (OHFWURP\RJUDSK\ LV QRW WKH PRVW VHQVLWLYH WHFKQLTXH IRU PHDVXULQJ WKH PHPEUDQH UHVSRQVH EXW DQ\ ODUJH DOWHUDWLRQ LQ PXVFOH DFWLRQ SRWHQWLDO JHQHUDWLRQ DQG SURSDJDWLRQ ZRXOG SUREDEO\ KDYH EHHQ GHWHFWHG 8VLQJ VPDOO PXVFOH EXQGOHV DQG PHDVXULQJ LQWUDFHOOXODU SRWHQWLDOV +DQVRQ f QRWHG RQO\ PLQRU FKDQJHV LQ WKH UDW VROHXV PXVFOH UHVWLQJ SRWHQWLDO DQG DFWLRQ SRWHQWLDO IROORZLQJ UHSHWLWLYH VWLPXODWLRQ 7KH DPSOLWXGH RI WKH DFWLRQ SRWHQWLDO ZDV UHGXFHG LQ IDWLJXH EXW ZDV UHVWRUHG ZLWKLQ D IHZ PLQXWHV RI UHFRYHU\ *UDERZVNL f QRWHG D UHGXFHG DPSOLWXGH RI WKH PXVFOH DFWLRQ SRWHQWLDO RI IDWLJXHG IURJ PXVFOH ILEHUV $ UHGXFHG DPSOLWXGH FRXOG

PAGE 21

DOVR EH SURGXFHG LQ D UHVWHG PXVFOH E\ UHGXFLQJ H[WUDn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f 7KLV FRQFOXVLRQ LV EDVHG RQ UHVXOWV IURP RQH RI WZR WHFKQLTXHV HLWKHU Df DOO RWKHU SRVVLELOLWLHV DUH HOLPLQDWHG RU Ef LQIHUHQFH LV REWDLQHG IURP DQDO\VLV RI FKDQJHV LQ WKH WLPH WR SHDN WHQVLRQ DQG WKH SHDN UDWH RI IRUFH GHYHORSPHQW IRU D WZLWFK ,Q WKH IRUPHU HYDOXDWLRQ RI WKH IXQFWLRQDO VWDWH RI WKH QHXURPXVFXODU MXQFWLRQ DQG RI WKH IRUFH JHQHUDWLQJ FDSDFLW\ RI WKH PXVFOH KDV UHYHDOHG WKDW WKHVH SURFHVVHV DUH XQDOWHUHG LQ WKH IDWLJXHG PXVFOH 7KLV OHDGV RQH WR

PAGE 22

EHOLHYH WKDW WKH PXVFOH KDV D UHGXFHG DPRXQW RI &D UHOHDVHG ,Q WKH ODWWHU LW LV DVVXPHG WKDW UHOD[DWLRQ RFFXUV VLPXOWDQHRXVO\ ZLWK &D UHXSWDNH f 8QGHU WKHVH FLUFXPVWDQFHV D UHGXFWLRQ LQ FRQWUDFWLRQ WLPH ZRXOG UHVXOW IURP D UHGXFWLRQ LQ GXUDWLRQ RI DFWLYDWLRQ PRUH I UDSLG UHDFFXPXODWLRQ RU VKRUWHU GXUDWLRQ RI UHOHDVHf $ UHGXFWLRQ LQ SHDN UDWH RI IRUFH GHYHORSPHQW ZLWKRXW D FRQFRPPLWDQW UHGXFWLRQ LQ FRQWUDFWLRQ WLPH LQGLFDWHV UHGXFHG DFWLYDWLRQ DQG WKLV LV LQWHUSUHWHG DV D UHGXFHG DPRXQW RI &D UHOHDVHG %UXVW KDV PDGH REVHUYDWLRQV VLPLODU WR WKHVH UHGXFHG UDWH RI IRUFH GHYHORSPHQW LQ IDWLJXH ZLWK QR FKDQJH LQ FRQWUDFWLRQ WLPHf RQ PRXVH VROHXV PXVFOHV LQ YLWUR f DQG FRQFOXGHG WKDW IDWLJXH ZDV GXH WR UHGXFHG &D UHOHDVH 6LPLODU REVHUYDWLRQV ZRXOG EH H[SHFWHG LI WKHUH ZDV DQ LQFUHDVH LQ WKH &D FRQFHQWUDWLRQ DW ZKLFK ELQGLQJ WR WURSRQLQ DQG VXEVHTXHQW FRQWUDFWLOH DFWLYLW\ RFFXUV ,W KDV EHHQ REVHUYHG E\ )XFKV HW DO f WKDW WKH DIILQLW\ RI WURSRQLQ IRU &D FDQ EH DOWHUHG E\ S+ 7KLV SRVVLELOLW\ PXVW EH FRQVLGHUHG ZKHQ GHDOLQJ ZLWK LQIHUHQFHV IURP PHDVXUHPHQWV RI FRQWUDFWLRQ WLPH DQG UDWH RI IRUFH GHYHORSPHQW )LWWV DQG +ROORV]\ f KDYH SUHVHQWHG GDWD LQGLFDWLQJ WKDW UHGXFHG S+ PD\ EH DVVRFLDWHG ZLWK IDWLJXH 7KH\ VXSSRUW WKH WKHRU\ WKDW UHGXFHG DFWLYDWLRQ DQG UHGXFHG UDWH RI IRUFH GHYHORSPHQWf LV GXH WR D UHGXFHG DIILQLW\ RI WURSRQLQ IRU &D

PAGE 23

$QRWKHU VLWXDWLRQ PD\ RFFXU LQ WKH PXVFOH IRU ZKLFK WKH UDWH RI IRUFH GHYHORSPHQW GHFOLQHV ZLWK GHYHORSHG WHQVLRQ ZKLOH FRQWUDFWLRQ WLPH UHPDLQV XQFKDQJHG $ UHGXFWLRQ LQ FRQWUDFWLOH FDSDFLW\ ZRXOG JLYH WKH VDPH UHVXOWV 7KLV SRVVLELOLW\ PXVW EH JLYHQ FRQVLGHUDWLRQ 6RPH DXWKRUV KDYH WHVWHG IRU DQG IRXQG FKDQJHV LQ WKH FRQWUDFWLOH FDSDFLW\ RI WKH PXVFOH XQGHU VWXG\ f 7KHVH DUH GLVFXVVHG EHORZ 5HGXFHG &DSDFLW\ RI WKH &RQWUDFWLOH $SSDUDWXV )DWLJXH PD\ EH WKH UHVXOW RI D UHGXFHG DELOLW\ RI WKH FRQWUDFWLOH SURWHLQV WR JHQHUDWH WHQVLRQ 7KLV FRXOG EH D UHVXOW RI HLWKHU Lf GDPDJH WR P\RILODPHQWV LH PLVDOLJQPHQW RU LQDFWLYDWLRQf RU LLf UHVWULFWHG DYDLODELOLW\ RI HQHUJ\ ,Q HLWKHU FDVH WKH HIIHFW ZRXOG EH D UHGXFHG IRUFH JHQHUDWLRQ XQGHU FRQGLWLRQV RI PD[LPDO DFWLYDWLRQ 7KLV FDSDFLW\ WR GHYHORS WHQVLRQ KDV EHHQ WUDGLWLRQDOO\ WHVWHG ZLWK HLWKHU D FRQWUDFWXUH RU D FDIIHLQ FRQWUDFWXUH %RWK RI WKHVH SURFHGXUHV UHVXOW LQ PD[LPDO DFWLYDWLRQ &D FRQFHQWUDWLRQ KLJK HQRXJK WR VDWXUDWH WKH FRQWUDFWLOH DSSDUDWXVf 7HWDQLF VWLPXODWLRQ KDV DOVR EHHQ XVHG WR HYDOXDWH WKH FDSDFLW\ RI D PXVFOH WR JHQHUDWH WHQVLRQ )LWWV DQG +ROORV]\ f REVHUYHG WKDW WHWDQLF IRUFH ZDV UHGXFHG LQ WKH UDW VROHXV PXVFOH IROORZLQJ D VHULHV RI WHWDQLF FRQWUDFWLRQV 7KH\ QRWHG WKDW UHFRYHU\ RI WKH IRUFH JHQHUDWLQJ FDSDFLW\ RFFXUUHG UHODWLYHO\ TXLFNO\ ZLWKLQ PLQXWHVf 1R LQVLJKW LQWR WKH PHFKDQLVP

PAGE 24

UHVSRQVLEOH IRU WKH IDWLJXH REVHUYHG E\ WKHVH DXWKRUV LV SURYLGHG 6LQFH UHFRYHU\ RFFXUUHG TXLFNO\ LW LV REYLRXV WKDW SHUPDQHQW GDPDJH WR WKH P\RILODPHQWV ZDV QRW D PHFKDQLVP RI WKH IDWLJXH 6SDQGH DQG 6FKRWWHOLXV f VWXGLHG IDWLJXH LQ WKH PRXVH VROHXV PXVFOH LQ YLWUR 7KH\ IRXQG WKDW WKH PDJQLWXGH RI WKH UHGXFWLRQ LQ GHYHORSHG WHQVLRQ ZDV LQYHUVHO\ SURSRUWLRQDO WR WKH SKRVSKRU\OFUHDWLQH3&f FRQFHQWUDWLRQ 3& VHUYHV DV DQ LPPHGLDWH VRXUFH RI KLJK HQHUJ\ SKRVSKDWH a3f IRU UHSKRVSKRU\ODWLRQ RI $'3 DQG PD\ DOVR EH LQYROYHG LQ D WUDQVSRUW FDSDFLW\ IRU a3 IURP PLWRFKRQGULD WR P\RILODPHQWV f 7KH H[SHULPHQWV E\ 6SDQGH DQG 6FKRWWHOLXV f LQYROYHG FRQWUDFWLRQV ZLWK SHULRGV RI DQR[LD DQGRU JOXFRVH GHSULYDWLRQ DQG WKLV PXVW EH NHSW LQ PLQG ZKHQ FRPSDULQJ WKHLU UHVXOWV ZLWK WKRVH RI RWKHU DXWKRUV 8QGHU WKHVH FLUFXPVWDQFHV UHGXFHG HQHUJ\ DYDLODELOLW\ DSSHDUV WR EH UHODWHG WR WKH IDWLJXH )LWWV DQG +ROORV]\ f KDYH PHDVXUHG 3& FKDQJHV GXULQJ DQG IROORZLQJ IDWLJXLQJ FRQWUDFWLRQV LQ UDW PXVFOH 7KH\ IRXQG QR UHODWLRQVKLS EHWZHHQ 3& DQG WKH DPRXQW RI IDWLJXH RU UHFRYHU\ IURP IDWLJXH 7KH ILQDO FRPPRQ PHGLDWRU RI HQHUJ\ DYDLODELOLW\ LV WKH OHYHO RI $73 LQ WKH PXVFOH (GZDUGV UHSRUWHG WKDW $73 DQG 3& FRQFHQWUDWLRQV ZHUH UHGXFHG LQ LVRODWHG PRXVH VROHXV PXVFOHV GXULQJ SURORQJHG WHWDQL XQGHU DQDHURELF FRQGLWLRQV 7KLV ZDV DOVR WKH FDVH ZKHQ PXVFOHV ZHUH IDWLJXHG LQ WKH SUHVHQFH RI F\DQLGH DQG

PAGE 25

LRGRDFHWLF DFLG ,Q WKH IRUPHU FDVH ODFWDWH DFFXPXODWHG EXW LQ WKH ODWWHU FDVH WKHUH ZDV QR DFFXPXODWLRQ RI ODFWDWH ,W ZDV QRWHG WKDW SURORQJDWLRQ RI UHOD[DWLRQ ZDV DVVRFLDWHG ZLWK D UHGXFWLRQ LQ $73 DQG 3& OHYHOV 7KLV SURYLGHV DQ LQGLUHFW PHWKRG RI HYDOXDWLQJ HQHUJ\ DYDLODELOLW\ LQ WKH PXVFOH 5HOD[DWLRQ ZRXOG EH H[SHFWHG WR EH SURORQJHG VLQFH LW LV GHSHQGHQW RQ UHXSWDNH RI &DA 6HTXHVWHULQJ &DA LV DQ DFWLYH WUDQVSRUW SURFHVV ZKLFK UHTXLUHV $73 f 5HGXFHG OHYHOV RI $73 PD\ DOVR VORZ WKH UHOD[DWLRQ SKDVH RI LQGLYLGXDO FURVVEULGJHV $73 LV UHTXLUHG WR SHUPLW GLVVRFLDWLRQ RI WKH DFWLQ DQG P\RVLQ PROHFXOHV f 7KH H[WUHPH RI WKLV VLWXDWLRQ RFFXUV ZKHQ ULJRU ERQGV IRUP LQ WKH DEVHQFH RI $73 ,W FDQ EH FRQFOXGHG IURP WKH DERYH GLVFXVVLRQ WKDW IDWLJXH FDQ EH WKH UHVXOW RI DQ\ RI VHYHUDO PHFKDQLVPV 7KH SRVVLELOLW\ H[LVWV WKDW PXOWLSOH PHFKDQLVPV IXQFWLRQ DW RQFH )RU H[DPSOH D UHGXFHG UHOHDVH RI &DA PD\ EH DFFRPSDQLHG E\ D OLPLWDWLRQ RI HQHUJ\ DYDLODELOLW\ 7KLV VLWXDWLRQ ZRXOG FRPSOLFDWH WKH HOXFLGDWLRQ RI WKH PHFKDQLVPVf UHVSRQVLEOH IRU WKH IDWLJXH 7KH IROORZLQJ FKDSWHUV SUHVHQW WKH GHWDLOV RI H[SHULPHQWV FRQGXFWHG LQ DQ HIIRUW WR JDLQ DQ XQGHUn VWDQGLQJ RI IDWLJXH LQ WKH JDVWURFQHPLXVSODQWDULV PXVFOH JURXS RI WKH GRJ

PAGE 26

*(1(5$/ 0(7+2'6 0RQJUHO GRJV RI HLWKHU VH[ ZHLJKLQJ NJ ZHUH XVHG LQ WKHVH VWXGLHV 7KH\ ZHUH DQHVWKHWL]HG ZLWK LQWUDYHQRXV VRGLXP SHQWREDUELWRO PJNJ ZLWK DGGLWLRQDO PJ LQMHFWLRQV DV QHHGHG 7KH DQLPDOV ZHUH LQWXEDWHG DQG PDLQWDLQHG RQ D UHVSLUDWRU WKURXJKRXW WKH H[SHULPHQW $ %HFNPDQ /% JDV DQDO\]HU VDPSOHG JDV IURP WKH HQGRWUDFKHDO WXEH FRQWLQXRXVO\ 9HQWLODWLRQ ZDV DGMXVWHG WR PDLQWDLQ HQGWLGDO &2 DW b 5HFWDO WHPSHUDWXUH ZDV PRQLWRUHG ZLWK D WKHUPRFRXSOH DQG NHSW EHWZHHQ DQG r& E\ DSSURSULDWH DGMXVWPHQW RI D KHDWLQJ SDG SODFHG XQGHU WKH WKRUD[ RI WKH VXSLQH GRJ 7KH OHIW JDVWURFQHPLXVSODQWDULV PXVFOH ZDV H[SRVHG YLD DQ LQFLVLRQ DORQJ WKH PHGLDO DVSHFW RI WKH OHIW KLQG OLPE 0XVFOHV RYHUO\LQJ WKH PHGLDO KHDG RI WKH JDVWURFQHPLXVSODQWDULV PXVFOH JURXS ZHUH WLHG WZLFH ZLWK EXWFKHUnV FRUG DQG FXW EHWZHHQ WKH WLHV 7KHVH PXVFOHV DUH VDUWRULXV JUDFLOLV VHPLWHQGLQRVLV DQG WZR KHDGV RI VHPLPHPEUDQRVLV $OO YHLQV GUDLQLQJ LQWR WKH SRSOLWHDO YHLQ ZHUH OLJDWHG H[FHSW WKRVH EUDQFKHV FRPLQJ IURP WKH JDVWURFQHPLXVSODQWDULV PXVFOH VHH )LJXUH f $Q\ YHLQV GUDLQLQJ WKH PXVFOH EXW QRW HQWHULQJ WKH SRSOLWHDO YHLQ ZHUH OLJDWHG 7KHVH ZHUH

PAGE 27

),*85( 7KH LQ VLWX GRJ JDVWURFQHPLXVSODQWDULV SUHSDUDWLRQ f JDVWURFQHPLXVSODQWDULV PXVFOH *U JUDFLOLV PXVFOH 6 VDUWRULXV PXVFOH 60 WZR KHDGV RI VHPLPHPEUDQRVLV PXVFOH 67 VHPLWHQGLQRVLV PXVFOH PXVFOH

PAGE 28

9(1286 6$03/( ? 9 ),*85( A==

PAGE 29

RQO\ PLQRU YHVVHOV ZKLFK RFFXU DORQJ WKH DQWHULRU RU ODWHUDO VXUIDFHV RI WKH PXVFOH 7KH SRSOLWHDO YHLQ ZDV FDQQXODWHG $ FDQQXODWLQJ W\SH HOHFWURPDJQHWLF IORZ SUREH 1DUFR %LRV\VWHPVf PP ,'f ZDV SODFHG LQ WKH RXWIORZ WXELQJ 7KH YHQRXV HIIOXHQW ZDV UHWXUQHG WR WKH GRJ YLD DQRWKHU FDQQXOD LQ WKH H[WHUQDO MXJXODU YHLQ +HSDULQ 8NJ PJNJf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r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

PAGE 30

ZDV PHDVXUHG ZLWK D GLVSODFHPHQW WUDQVGXFHU GHWHFWLQJ WKH GLVSODFHPHQW RI WKH IUHH HQG RI WKH FDQWLOHYHU EHDP 7KH WUDQVGXFHU RXWSXW ZDV OLQHDU IRU IRUFHV XS WR NJ $ GLVSODFHPHQW DW WKH WUDQVGXFHU RI PP JDYH D IXOO VFDOH GHIOHFWLRQ RQ WKH UHFRUGHU 2XWSXW RI WKH GLVSODFHn PHQW WUDQVGXFHU WHQVLRQf ZDV DPSOLILHG DQG UHFRUGHG GLUHFWO\ 7KH DPSOLILHG WHQVLRQ VLJQDO ZDV DOVR GLIIHUHQWLDWHG ZLWK UHVSHFW WR WLPH *RXOG%UXVK GLIn IHUHQWLDWRUf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

PAGE 31

FODPSHG WR WKH WDEOH %RQH QDLOV ZHUH SODFHG LQ WKH WLELD DQG IHPXU RQH HDFKf 7KHVH QDLOV ZHUH ILUPO\ DWWDFKHG WR WKH EDVH RI WKH P\RJUDSK $ WXUQEXFNOH VWUXW SODFHG EHWZHHQ WKH OHYHUDUP DQG RQH RI WKH ERQH QDLOV SUHYHQWHG IOH[LQJ RI WKH OHYHUDUP 7KH PXVFOH OHQJWK ZDV VHW PP VKRUWHU WKDQ WKH OHQJWK DW ZKLFK GHYHORSHG WHQVLRQ ZDV JUHDWHVW RSWLPDO OHQJWKf 2SWLPDO OHQJWK ZDV GHWHUPLQHG E\ PHDVXULQJ WKH GHYHORSHG WHQVLRQ WRWDO WHQVLRQ PLQXV UHVWLQJ WHQVLRQf RI FRQWUDFWLRQV DW VHFf DW YDULRXV OHQJWKV

PAGE 32

837$.( $1' '(9(/23(' 7(16,21 ,QWURGXFWLRQ 2[\JHQ XSWDNH 9f RI PXVFOH FDQ LQFUHDVH PRUH WKDQ WLPHV UHVWLQJ OHYHOV GXULQJ UHSHWLWLYH VWLPXODWLRQ f $W ORZ IUHTXHQFLHV RI VWLPXODWLRQ 9 LV SURSRUWLRQDO WR WKH LVRPHWULF GHYHORSHG WHQVLRQ $7f WRWDO WHQVLRQ PLQXV UHVW WHQVLRQf f 7KLV UHODWLRQVKLS ZDV REVHUYHG IRU FRQWUDFWLRQV IROORZLQJ D SHULRG RI IDWLJXLQJ FRQWUDFWLRQV DW SHU VHF IRU PLQXWHV f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n VKLS IRU PXVFOH IDWLJXHG E\ FXUDUH LQIXVLRQ RU LVFKHPLD GXULQJ UHSHWLWLYH VWLPXODWLRQ

PAGE 33

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n IDWLJXH OHYHO 7KH PXVFOH ZDV DOORZHG WR UHFRYHU IRU PLQXWHV VR WKDW WKH UHVWLQJ 92 DSSURDFKHG WKH SUHn IDWLJXH OHYHO 7KUHH SDLUV RI EORRG VDPSOHV ZHUH WDNHQ ILYH PLQXWHV DSDUW DV WKH PXVFOH FRQWLQXHG WR UHFRYHU

PAGE 34

$IWHU WKLV UHFRYHU\ SHULRG WKH PXVFOH ZDV VWLPXODWHG DW WKH VDPH UDWH DV EHIRUH VHFf ZLWK WZLQ LPSXOVHV WZR LPSXOVHV Y LQ DPSOLWXGH PVHF LQ GXUDWLRQ DQG VHSDUDWHG E\ PVHFf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r Jr r &Bf 'HYHORSHG WHQVLRQ ZDV H[SUHVVHG DV JUDPV RI GHYHORSHG WHQVLRQ SHU JUDP RI ZHW PXVFOH Jr Jrf

PAGE 35

6HULHV 2[\JHQ XSWDNH DQG GHYHORSHG WHQVLRQ ZHUH PHDVXUHG GXULQJ WKH IDWLJXH SURFHVV ,Q VHSDUDWH H[SHULPHQWV PXVFOHV ZHUH VWLPXODWHG DW UDWHV RI DQG LPSXOVHV SHU VHFRQG $IWHU WKH ILUVW ILYH PLQXWHV RI FRQWUDFWLRQV EORRG VDPSOHV ZHUH FROOHFWHG SHULRGLFDOO\ DV WKH PXVFOH IDWLJXHG GXULQJ FRQWUDFWLRQV IRU WZR KRXUV 7KH GHFUHDVH LQ GHYHORSHG WHQVLRQ UDQJHG IURP WR b RYHU WKH WZR KRXU SHULRG 6L[W\ WR b RI WKLV GHFUHDVH RFFXUUHG LQ WKH ILUVW PLQXWHV $OWKRXJK EORRG IORZ DQG GHYHORSHG WHQVLRQ ZHUH VRPHWLPHV FKDQJLQJ UDSLGO\ WKHUH ZDV DOPRVW QR FKDQJH LQ WKH DUWHULRYHQRXV EORRG R[\JHQ FRQWHQW GLIIHUHQFHV 7KLV DOORZHG DSSOLFDWLRQ RI WKH )LFN HTXDWLRQ IRU &! XSWDNH FDOFXODWLRQ ZLWK FRQILGHQFH f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

PAGE 36

LQYHVWLJDWH WKLV SRVVLELOLW\ WZR H[SHULPHQWV ZHUH GRQH LQ ZKLFK QHXURPXVFXODU WUDQVPLVVLRQ ZDV FRPSOHWHO\ EORFNHG E\ UHSHDWHG LQMHFWLRQV RI HLWKHU FXUDUH RU VXFFLQ\OFKROLQH $IWHU WKH GUXJ ZDV JLYHQ WKH QHUYH ZDV VWLPXODWHG DW WKH UDWH RI LPSXOVHV VHF IRU PLQXWHV 0XVFOH FRQWUDFWLRQ GLG QRW RFFXU GXULQJ WKLV PLQXWH SHULRG EHFDXVH RI WKH SUHVHQFH RI WKH EORFNLQJ GUXJ 'HYHORSHG WHQLRQ DW D VWLPXODWLRQ UDWH RI VHFf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n JA r PLQr 7KLV LV VRPHZKDW KLJKHU WKDQ DYHUDJH YDOXHV SUHYLRXVO\ UHSRUWHG f EXW ZHOO ZLWKLQ WKH XVXDO UDQJH 0HDQ DUWHULDO EORRG SUHVVXUH UHPDLQHG DERYH PP+J WKURXJKRXW DOO RI WKH H[SHULPHQWV

PAGE 37

,Q WKH ILUVW VHULHV RI H[SHULPHQWV DQDO\VLV RI YDULDQFH IRU UHSHDWHG PHDVXUHV f RQ WKH UDWLRV EHWZHHQ 2 XSWDNH DQG GHYHORSHG WHQVLRQ REVHUYHG EHIRUH DQG DIWHU IDWLJXH UHYHDOHG QR VLJQLILFDQW GLIIHUHQFH S!f 7DEOH VKRZV WKH 2 XSWDNH DQG GHYHORSHG WHQVLRQ IRU HDFK RI WKH PXVFOHV ERWK EHIRUH DQG DIWHU IDWLJXH 7KH UHVXOWV RI WKH 2 XSWDNH DQG GHYHORSHG WHQVLRQ PHDVXUHPHQWV LQ VHULHV DUH VXPPDUL]HG LQ 7DEOH ,, DQG LOOXVWUDWHG LQ )LJXUHV DQG 7DEOH ,, VKRZV WKH OLQHDU UHJUHVVLRQ HTXDWLRQV UHODWLQJ 2 XSWDNH DQG GHYHORSHG WHQVLRQ IRU HDFK H[SHULPHQW 7KHVH HTXDWLRQV ZHUH FDOFXODWHG IURP GDWD ZKLFK LQFOXGHG YDOXHV IURP WKH IDWLJXHG PXVFOH DV ZHOO DV WKH IUHVK PXVFOH 7KH VORSHV RI DOO EXW RQH ([SHULPHQW S f RI WKH OLQHV DUH VLJQLILFDQWO\ GLIIHUHQW IURP ]HUR Sf GHVSLWH WKH VPDOO QXPEHU RI SRLQWV XVHG WR GHWHUPLQH HDFK UHJUHVVLRQ HTXDWLRQ ,W LV REYLRXV IURP )LJXUH DQG 7DEOH ,, WKDW WKHUH ZDV FRQVLGHUDEOH YDULDELOLW\ EHWZHHQ DQLPDOV 7KLV KDV DOZD\V EHHQ REVHUYHG LQ WKLV SUHSDUDWLRQ f +RZHYHU GHVSLWH GLIIHUHQFHV LQ DEVROXWH YDOXHV EHWZHHQ GLIIHUHQW DQLPDOV WKH VDPH SDWWHUQ RI UHVSRQVH ZDV REVHUYHG LQ DOO FDVHV 92 SHU FRQWUDFWLRQ DQG $7 ZHUH GLUHFWO\ UHODWHG 5HVXOWV RI IRXU VDPSOH H[SHULPHQWV IURP VHULHV DUH VKRZQ LQ )LJXUH )LJXUH VKRZV WKDW DOO RI WKH

PAGE 38

837$.( 0O J, &Of ),*85( 5HVXOWV RI IRXU VDPSOH H[SHULPHQWV 1XPEHUV UHIHU WR LQGLYLGXDO H[SHULPHQWV 7KLUWHHQ LV IURP 6HULHV FLUFOHG QXPEHUV SRVW IDWLJXHf 6L[WHHQ LV IURP 6HULHV 1LQHWHHQ LV IURP 6HULHV 7ZHQW\WZR LV IURP 6HULHV

PAGE 39

),*85( ; LW 'DWD IURP 6HULHV QRUPDOL]HG WR WKH VDPH VFDOH 'HYHORSHG WHQVLRQ LQ SHUFHQW RI WKH JUHDWHVW GHYHORSHG WHQVLRQ LQ HDFK H[SHULPHQW XSWDNH LQ SHUFHQW RI WKH XSWDNH DW WKH JUHDWHVW GHYHORSHG WHQVLRQ 1XPEHUV UHIHU WR LQGLYLGXDO H[SHULPHQWV 6HYHQ WR IRXUWHHQ DUH 6HULHV FLUFOHG QXPEHUV SRVW IDWLJXHf )LIWHHQ WR HLJKWHHQ DUH 6HULHV 1LQHWHHQ WR WZHQW\RQH DUH 6HULHV 7ZHQW\WZR WR WZHQW\ILYH DUH 6HULHV 7KH DVWHULVN GHQRWHV b bf ZKLFK LV FRPPRQ WR DOO RI WKH H[SHULPHQWV 7KH OLQH LQ WKLV ILJXUH LV WKH OLQH RI LGHQWLW\ ;
PAGE 40

7$%/( 837$.( $1' '(9(/23(' 7(16,21 %()25( $1' $)7(5 )$7,*8( ,1 6(5,(6 3UH)DWLJXH 6LQJOH ,PSXOVHVf 3RVW)DWLJXH 7ZLQ ,PSXOVHVf 'HYHORSHG 'HYHORSHG ([SHULPHQW 7HQV LRQ DJ f 2 8SWDNH XO Ja&ff 7HQVLRQ TrTf 2 8SWDNH \O "fT& 0HDQ 6(0 s s s 8QLWV DUH DV JLYHQ IRU )LJXUH X! R

PAGE 41

7$%/( ,, 5(/$7,216+,3 %(7:((1 837$.( $1' '(9(/23(' 7(16,21 ,1 6(5,(6 r1 HTXDOV WKH QXPEHU RI GDWD SRLQWV LQ HDFK H[SHULPHQW 8QLWV IRU 2 XSWDNH DUH PLFUROLWHUV RI 2 SHU JUDP RI ZHW PXVFOH SHU FRQWUDFWLRQ 8QLWV IRU WHQVLRQ DUH JUDPV SHU JUDP RI ZHW PXVFOH

PAGE 42

6HULHV ([SW 1r 7\SH RI )DWLJXH VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ VHF IRU PLQ &RQWLQXRXV DW VHF &RQWLQXRXV DW VHF &RQWLQXRXV DW VHF &RQWLQXRXV DW VHF ,VFKHPLD ,VFKHPLD ,VFKHPLD 3DUWLDO &XUDUH %ORFN 3DUWLDO &XUDUH %ORFN 3DUWLDO &XUDUH %ORFN 3DUWLDO &XUDUH %ORFN b 9DULDQFH 5HJUHVVLRQ (TXDWLRQr ([SODLQHG 92 7 92 ff 7 fff 92 7 92 7 92 7 f f 92 7 YR 7 YR 7 92 7 YR 7 92 r f 7 92 7 92 7 r 92 7 YR r f f 7 92 fff 7 YR 7 92 7 rr 92 ff 7 RM WR

PAGE 43

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b RI WKH FRQWURO YDOXH 7KH REVHUYHG UHGXFWLRQ LQ FRQWUDFWLRQ VWUHQJWK PD\ KDYH EHHQ GXH WR LQFRPSOHWH UHFRYHU\ IURP WKH QHXURPXVFXODU EORFN 7KLV b UHGXFWLRQ LQ GHYHORSHG WHQVLRQ FDQ EH FRPSDUHG ZLWK WKH b UHGXFWLRQ REVHUYHG LQ WKH RWKHU H[SHULPHQWV LQ ZKLFK PXVFOH FRQWUDFWLRQ ZDV QRW EORFNHG ,W DSSHDUV WKDW PRVW LI QRW DOO RI WKH UHGXFHG FRQWUDFWLOH UHVSRQVH ZDV GXH WR DOWHUDWLRQV EH\RQG WKH QHXURPXVFXODU MXQFWLRQ $V SRLQWHG RXW LQ WKH 0HWKRGV WKH IDWLJXHG PXVFOHV LQ WKH ILUVW DQG VHFRQG VHULHV RI H[SHULPHQWV ZHUH DOORZHG WR UHFRYHU IRU PLQXWHV $IWHU WKLV WLPH WKH UHVWLQJ 2 XSWDNH DSSURDFKHG WKH SUHIDWLJXH OHYHO

PAGE 44

+RZHYHU GHYHORSHG WHQVLRQ UHFRYHUHG YHU\ OLWWOH GXULQJ WKLV WLPH DQG ZDV VWLOO RQO\ RQHWKLUG WR RQHKDOI RI WKH SUHIDWLJXH YDOXH 'LVFXVVLRQ ,VRPHWULF GHYHORSHG WHQVLRQ DW FRQVWDQW PXVFOH OHQJWK ZDV YDULHG LQ WKLV VWXG\ E\ IRXU PHWKRGV f WZLQ LPSXOVHV VWLPXODWLRQ f IDWLJXH SURGXFHG E\ PLQXWHV RI FRQWUDFWLRQV DW VHF f LVFKHPLD FDXVHG E\ SDUWLDO RFFOXVLRQ RI DUWHULDO LQIORZ WR WKH PXVFOH DQG f SDUWLDO EORFN RI QHXURPXVFXODU WUDQVPLVVLRQ ZLWK FXUDUH )LJXUHV DQG DQG 7DEOHV DQG ,, VKRZ WKDW QRQH RI WKHVH WUHDWPHQWV FKDQJHG WKH UHODWLRQVKLS EHWZHHQ XSWDNH DQG GHYHORSHG WHQVLRQ 6WLPXODWLQJ WKH IDWLJXHG PXVFOH ZLWK WZLQ LPSXOVHV UHVWRUHG GHYHORSHG WHQVLRQ WR SUHn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f 7KHVH WZR

PAGE 45

FRUUHVSRQG WR W\SHV )5 DQG 6 65f IDVW IDWLJXH UHVLVWDQW DQG VORZ IDWLJXH UHVLVWDQW UHVSHFWLYHO\f GHVFULEHG E\ %XUNH DQG FROOHDJXHV f IRU KLQGOLPE PXVFOHV RI WKH FDW (YHQ WKRXJK WKH GRJ JDVWURFQHPLXV SODQWDULV PXVFOH JURXS FRQWDLQV ERWK )5 DQG 6 XQLWV DQG WKH FDW VROHXV PXVFOH FRQWDLQV RQO\ W\SH 6 XQLWV KRPRJHQDWHV RI FDW VROHXV PXVFOH KDYH OHVV WKDQ RQH WKLUG RI WKH VXFFLQDWH R[LGDVH DFWLYLW\ RI KRPRJHQDWHV RI WKH GRJ JDVWURFQHPLXVSODQWDULV PXVFOH JURXS f )URP WKLV RQH PLJKW H[SHFW DOO RI WKH GRJ JDVWURFQHPLXV SODQWDULV PXVFOH XQLWV WR EH PRUH UHVLVWDQW WR IDWLJXH WKDQ DQ\ RI WKH XQLWV RI FDW VROHXV PXVFOHV +RZHYHU %XUNH DQG FROOHDJXHV f KDYH ZDUQHG DJDLQVW H[WUDn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

PAGE 46

PDNHV FRQVWDQW ILHOG VWLPXODWLRQ GLIILFXOW +RZHYHU VHYHUDO VWXGLHV RQ RWKHU PDPPDOLDQ PXVFOHV f KDYH LQGLFDWHG WKDW WKH SRVVLELOLW\ RI QHXURPXVFXODU WUDQVPLVVLRQ IDLOXUH DW VWLPXODWLRQ UDWHV RI OHVV WKDQ VHF LV PLQLPDO ,Q WZR H[SHULPHQWV QHUYH VWLPXODWLRQ DW LPSXOVHV VHF IRU PLQXWHV ZKHQ PXVFOH FRQWUDFWLRQ ZDV EORFNHG E\ FXUDUH RU VXFFLQ\OFKROLQH FDXVHG OHVV WKDQ D b GHFUHDVH LQ GHYHORSHG WHQVLRQ 'HFUHDVHV LQ GHYHORSHG WHQVLRQ RI b RFFXUUHG XQGHU WKH VDPH VWLPXODWLRQ FRQGLWLRQV ZKHQ PXVFOH FRQWUDFWLRQ ZDV QRW EORFNHG 7KHVH ILQGLQJV LQGLFDWH WKDW SUHV\QDSWLF IDLOXUH RI LPSXOVH SURSDJDWLRQ DQG LQDGHTXDWH UHOHDVH RI DFHW\OFKROLQH SUREDEO\ GLG QRW FDXVH WKH IDWLJXH REVHUYHG LQ RXU H[SHULPHQWV 'HVHQVLWL]DWLRQ RI WKH HQGSODWH LV QRW UXOHG RXW E\ WKHVH UHVXOWV +RZHYHU QHXURPXVFXODU GHSUHVVLRQ LV SUHVHQWO\ EHOLHYHG WR UHVXOW IURP D UHGXFHG QXPEHU RI UHOHDVHG WUDQVPLWWHU TXDQWD DQG D UHGXFWLRQ RI TXDQWDO VL]H f ,Q 6HULHV WKH FDXVH RI IDWLJXH PLJKW KDYH EHHQ PXVFXODU QHXURPXVFXODU RU D FRPELQDWLRQ RI WKH WZR VLQFH LVFKHPLD FDQ DIIHFW ERWK WKH PXVFOH DQG WKH QHXURPXVFXODU MXQFWLRQ f ,Q 6HULHV GHYHORSHG WHQVLRQ ZDV GHFUHDVHG E\ SDUWLDO FXUDUH EORFN ZKLFK SUHVXPDEO\ VLPXODWHV QHXURPXVFXODU MXQFWLRQ IDLOXUH 7KHVH H[SHULPHQWV GR QRW DOORZ LGHQWLILFDWLRQ RI WKH VSHFLILF FDXVH RI IDWLJXH +RZHYHU RXU UHVXOWV GR LQGLFDWH WKDW WKH R[\JHQ XSWDNH SHU XQLW RI LVRPHWULF

PAGE 47

IRUFH SURGXFWLRQ LV XQFKDQJHG E\ HLWKHU PXVFOH IDWLJXH RU QHXURPXVFXODU IDWLJXH 7KLV VXJJHVWV WKDW IDWLJXH ZKHWKHU PXVFXODU LVFKHPLF QHXURPXVFXODU RU D FRPELQDWLRQ RI WKHVH WKUHH GRHV QRW FDXVH DQ\ FKDQJH LQ WKH HIILFLHQF\ RI HQHUJ\ WUDQVGXFWLRQ IURP $73 WR H[WHUQDO WHQVLRQ GHYHORSPHQW E\ WKH PXVFOH ZLWKRXW FRQFRPPLWDQW FKDQJHV LQ WKH RSSRVLWH GLUHFWLRQ IRU HQHUJ\ WUDQVGXFWLRQ IURP IRRGVWXIIV WR $73 7KLV LV QRW OLNHO\ WKH FDVH 7KHVH UHVXOWV GLIIHU IURP WKRVH RI %URQN f )HQJ f (GZDUGV DQG +LOO f DQG (GZDUGV +LOO DQG -RQHV f LQ WKDW WKH\ IRXQG WKDW WKH HQHUJ\ H[SHQGLWXUH SHU XQLW RI IRUFH SURGXFWLRQ RU RI WHQVLRQn WLPHf GHFUHDVHG GXULQJ IDWLJXH 8QOLNH RXU H[SHULPHQWV KRZHYHU WKHVH HDUOLHU VWXGLHV XVHG VWLPXOXV SDUDPHWHUV ZKLFK FDXVHG SDUWLDOO\ WR FRPSOHWHO\ IXVHG WHWDQLF FRQWUDFWLRQV RI UHODWLYHO\ ORQJ GXUDWLRQ 7KH SUHVHQW VWXG\ LV RI WZLWFK RU YHU\ EULHI WHWDQLF FRQWUDFWLRQV IRU ZKLFK QR SODWHDX LQ GHYHORSHG WHQVLRQ RFFXUV VHH )LJXUH f 7KHUH LV OLWWOH LI DQ\ WHQVLRQ PDLQWHQDQFH LQYROYHG 7KH GDWD SUHVHQWHG LQ WKLV VWXG\ DORQJ ZLWK WKRVH RI :LOVRQ DQG 6WDLQVE\ f GHPRQVWUDWH D FRQVWDQW FRXSOLQJ EHWZHHQ &! XSWDNH DQG GHYHORSHG WHQVLRQ LQ LVRPHWULF WZLWFK FRQWUDFWLRQV ,Q WKHVH WZR VWXGLHV GHYHORSHG WHQVLRQ KDV EHHQ FKDQJHG E\ VWLPXODWLRQ IUHTXHQF\ SRWDVVLXP LRQ LQIXVLRQV WZLQ LPSXOVH

PAGE 48

),*85( 7UDFLQJ RI D UHFRUGLQJ RI WHQVLRQ DQG GLIIHUHQWLDO RI WHQVLRQ IRU VLQJOH LPSXOVH DQG WZLQ LPSXOVH FRQWUDFWLRQV EHIRUH DQG DIWHU IDWLJXH &RQWUDFWLRQV DUH VXSHULPSRV IRU HDVH RI FRPSDULVRQ 7KHUH LV QR PDLQWDLQHG SODWHDX LQ WKLV W\SH RI FRQWUDFWLRQ

PAGE 49

7:,7&+ DQG 7:,1 7(16,21 G3GW ),*85( &2175$&7 ,21 3RVW IDWLJXH 2-

PAGE 50

VWLPXODWLRQ QRUPDO PXVFOH IDWLJXH LVFKHPLF IDWLJXH DQG SDUWLDO QHXURPXVFXODU WUDQVPLVVLRQ EORFN ZLWK FXUDUH 'XULQJ DOO RI WKHVH WUHDWPHQWV WKH UHODWLRQVKLS EHWZHHQ 2 XSWDNH DQG GHYHORSHG WHQVLRQ KDV EHHQ XQDOWHUHG 7KH GDWD DOVR VKRZ WKDW DOWKRXJK UHVWLQJ PHWDEROLF UDWH IROORZLQJ IDWLJXH DSSURDFKHV SUHIDWLJXH OHYHOV DIWHU PLQXWHV GHYHORSHG WHQVLRQ LV VWLOO TXLWH ORZ 3KRVSKRU\OFUHDWLQH DQG $73 OHYHOV VKRXOG EH IXOO\ UHFRYHUHG IROORZLQJ PLQXWHV RI UHVW f (GZDUGV DQG FRZRUNHUV f KDYH DOVR LGHQWLILHG D ORQJ ODVWLQJ HOHPHQW RI IDWLJXH LQ KXPDQV WKDW LV QRW GXH WR GHSOHWLRQ RI KLJKHQHUJ\ SKRVSKDWHV )XUWKHU VWXG\ LV ZDUUDQWHG WR GHWHUPLQH ZKHWKHU RU QRW WKHUH UHDOO\ LV D FDXVDWLYH UHODWLRQVKLS EHWZHHQ SKRVSKRU\OFUHDWLQH GHSOHWLRQ DQG IDWLJXH DV VXJJHVWHG E\ 6SDQGH DQG 6FKRWWHOLXV f

PAGE 51

(9$/8$7,21 2) &+$1*(6 ,1 7+( 7:,7&+ &2175$&7,21 $662&,$7(' :,7+ )$7,*8( ,QWURGXFWLRQ 6NHOHWDO PXVFOH KDV DQ DWWHQXDWHG UHVSRQVH WR D VLQJOH LPSXOVH IROORZLQJ D SURORQJHG SHULRG RI WZLWFK FRQWUDFWLRQV GXH WR UHSHWLWLYH VLQJOH LPSXOVH VWLPXODWLRQ f 7KLV UHVSRQVH LV QRW QHFHVVDULO\ LQGLFDWLYH RI D UHGXFHG FDSDFLW\ RI WKH PXVFOH WR GHYHORS WHQVLRQ f ,W LVKRZHYHU D W\SH RI PXVFXODU IDWLJXH DQG ZDUUDQWV IXUWKHU LQYHVWLJDWLRQ FRQFHUQHG ZLWK GHWHUPLQDWLRQ RI PHFKDQLVPV UHVSRQVLEOH IRU WKLV WZLWFK IDWLJXH :LOVRQ DQG 6WDLQVE\ f UHSRUWHG WKDW WZLWFK IDWLJXH RFFXUV LQ WKH JDVWURFQHPLXVSODQWDULV PXVFOH RI WKH GRJ IROORZLQJ PLQXWHV RI LVRPHWULF FRQWUDFWLRQV VHFf 7KH\ PRQLWRUHG UHFRYHU\ ZLWK SHULRGV RI ORZ IUHTXHQF\ VWLPXODWLRQ RYHU WKH FRXUVH RI KRXUV $Q DWWHQXDWLRQ RI GHYHORSHG WHQVLRQ ZDV VWLOO SUHVHQW IROORZLQJ WKLV UHFRYHU\ SHULRG /LWWOH LV NQRZQ RI WKH PHFKDQLVP UHVSRQVLEOH IRU WKLV IDWLJXH 7KH SXUSRVH RI WKH SUHVHQW LQYHVWLJDWLRQ ZDV WR VWXG\ DOWHUDWLRQV LQ WZLWFK FRQWUDFWLRQV FDXVHG E\ UHSHWLWLYH VWLPXODWLRQ DW WKUHH IUHTXHQFLHV DQG VHF $ WZLWFK FRQWUDFWLRQ FDQ EH FKDUDFWHUL]HG

PAGE 52

E\ PHDVXUHPHQWV RI WKH PDJQLWXGH DQG WLPHFRXUVH RI WHQVLRQ GHYHORSPHQW VHHQ LQ WKH LVRPHWULF P\RJUDP f 7KHVH PHDVXUHPHQWV DUH GHYHORSHG WHQVLRQ $7f FRQWUDFWLRQ WLPH &Wf KDOI UHOD[DWLRQ WLPH 5W f SHDN UDWH RI IRUFH GHYHORSPHQW G3GWf DQG SHDN UDWH RI UHOD[DWLRQ G3GWf VHH )LJXUH f 6DQGRZ DQG %UXVW f KDYH QDPHG WKH FKDQJHV LQ WKHVH PHDVXUHPHQWV WKDW RFFXU ZLWK UHSHWLWLYH VWLPXODWLRQ WKH IDWLJXH SDWWHUQV )DWLJXH SDWWHUQV KDYH EHHQ GHWHUPLQHG IRU VLQJOH PXVFOH FHOOV DQG ZKROH PDPPDOLDQ DQG DPSKLELDQ VNHOHWDO PXVFOHV LQ YLWUR f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

PAGE 53

)LJXUH 7UDFLQJV RI 7HQVLRQ DQG G3GW DUH SUHVHQWHG WR GHPRQVWUDWH WKH PDQQHU E\ ZKLFK WKH PHDVXUHPHQWV ZHUH PDGH 6HH WH[W IRU YHUEDO GHVFULSWLRQ RI WKHVH WHUPV

PAGE 54

VHF IRU PLQXWHV $IWHU D IDVW WUDFH RU PPVHF SDSHU VSHHGf ZDV REWDLQHG FRQWUDFWLRQ IUHTXHQF\ ZDV HLWKHU OHIW DW VHF RU LQFUHDVHG IURP VHF WR RU VHF 5HOD[DWLRQ ZDV QRW FRPSOHWH EHWZHHQ FRQWUDFWLRQV ZKHQ VWLPXODWLRQ ZDV VHF RU VHF 7R IDFLOLWDWH PHDVXUHPHQW RI WKH FKDUDFWHULVWLFV RI D WZLWFK WKH IUHTXHQF\ RI VWLPXODWLRQ ZDV UHGXFHG EULHIO\ ZKLOH IDVW WUDFHV ZHUH REWDLQHG WKHQ WKH IDWLJXLQJ IUHTXHQF\ ZDV UHVWRUHG VHH )LJXUH f 'XULQJ FRQWUDFWLRQV DW VHF FRPSOHWH UHOD[DWLRQ RFFXUV EHWZHHQ FRQWUDFWLRQV VR IDVW WUDFHV ZHUH REWDLQHG ZLWKRXW DOWHULQJ WKH IUHTXHQF\ RI VWLPXODWLRQ %HVLGHV WKH FRQWUDFWLRQV DW PLQXWHV IDVW WUDFHV ZHUH REWDLQHG DIWHU DQG PLQXWHV RI IDWLJXLQJ FRQWUDFWLRQV DQG DIWHU DQG PLQXWHV RI UHFRYHU\ VHH )LJXUH f 7R JHW IDVW WUDFHV GXULQJ WKH UHFRYHU\ SHULRG ZKLFK IROORZHG FRQWUDFWLRQV DW VHF RU VHF WKH VWLPXODWRU ZDV WXUQHG RQ EULHIO\ DW VHF )ROORZLQJ WKH PLQXWH SHULRG RI FRQWUDFWLRQV DW VHF FRQWUDFWLRQV ZHUH FRQWLQXHG DW D IUHTXHQF\ RI VHF )DVW WUDFHV ZHUH REWDLQHG ZLWKRXW DOWHULQJ WKH IUHTXHQF\ RI VWLPXODWLRQ ,W KDV EHHQ UHSRUWHG WKDW FRQWUDFWLRQV DW WKLV ORZ IUHTXHQF\ GR QRW DOWHU WKH UHFRYHU\ SURFHVV f ,Q RQH H[SHULPHQW D WHWDQLF FRQWUDFWLRQ PVHF GXUDWLRQ LPSXOVHV VHFf ZDV REWDLQHG EHIRUH DQG DIWHU WKH VHF IDWLJXLQJ FRQWUDFWLRQV 7KLV ZDV GRQH WR SHUPLW HYDOXDWLRQ RI WKH FRQWUDFWLOH FDSDFLW\ RI WKH PXVFOH $OO IDVW WUDFHV ZHUH HYDOXDWHG

PAGE 55

IRU WKH FKDUDFWHULVWLFV RI D WZLWFK 7KHVH FKDUDFWHULVWLFV DUH LOOXVWUDWHG LQ )LJXUH $UWHULDO DQG YHQRXV EORRG VDPSOHV POf ZHUH REWDLQHG DW UHJXODU LQWHUYDOV WKURXJKRXW WKH H[SHULPHQW 6DPSOHV ZHUH GUDZQ LQWR JODVV WXEHUFXOLQ V\ULQJHV VHDOHG ZLWK PHUFXU\FRQWDLQLQJ FDSV DQG SODFHG LQ LFH XQWLO WKH\ ZHUH DQDO\]HG 7KHVH VDPSOHV ZHUH DQDO\]HG IRU S+ 3&&! DQG 32 DW r& ZLWK D UDGLRPHWHU &RSHQKDJHQf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b RI WKH SUHRFFOXVLRQ YDOXH PLQXWHVf

PAGE 56

7HQVLRQ GHYHORSHG YHUVXV WLPH &RQWUDFWLRQV LQ WKLV FDVH ZHUH VHF H[FHSW DV LQGLFDWHG )DVW WUDFHV QRW LOOXVWUDWHGf ZHUH REWDLQHG GXULQJ WKH VHF VWLPXODWLRQ ),*85(

PAGE 57

DQG UHFRYHU\ ZDV REVHUYHG 'DQWUROHQH VRGLXP GLVVROYHG LQ SURS\OHQH JO\FRO PJPOf ZDV LQMHFWHG ,9 GXULQJ FRQWUDFWLRQV DW VHF 'DQWUROHQH LPSDLUV UHOHDVH RI &D IURP WKH ODWHUDO VDFV f 7KLV LV DFFRPSOLVKHG ZLWKRXW FKDQJHV LQ WKH DFWLRQ SRWHQWLDO DQG LV DSSDUHQWO\ D GLUHFW HIIHFW RQ WKH ODWHUDO VDFV 6XIILFLHQW GUXJ ZDV JLYHQ WR UHGXFH $7 DW OHDVW b PJNJf 7R VWXG\ WKH HIIHFWV RI UHGXFLQJ WKH QXPEHU RI PRWRU XQLWV FRQWUDFWLQJ WKH VWLPXODWLRQ YROWDJH ZDV UHGXFHG ZKLOH WKH PXVFOH FRQWUDFWHG VHF 7KLV UHVXOWV LQ H[FLWDWLRQ RI IHZHU PRWRU QHXURQV DQG WKHLU PRWRU XQLWV &RQVHTXHQWO\ OHVV WHQVLRQ LV GHYHORSHG &RPSDULQJ WKH WZLWFK FKDUDFWHULVWLFV RI D QRUPDO YHUVXV D IDWLJXHG PXVFOH PD\ SURYLGH LQIRUPDWLRQ OHDGLQJ WR DQ XQGHUVWDQGLQJ RI WKH PHFKDQLVPVf RI IDWLJXH $OVR LQ D IHZ H[SHULPHQWV VDPSOHV RI PXVFOHV ZHUH REWDLQHG LPPHGLDWHO\ IROORZLQJ WKH PLQXWH VWLPXODWLRQ SHULRG DQGRU DIWHU PLQXWHV RI UHFRYHU\ 6DPSOHV ZHUH IUR]HQ LQ VLWX ZLWK PHWDO FODPSV SUHFRROHG LQ OLTXLG QLWURJHQ 6PDOO VDPSOHV PJf ZHUH WKHQ KRPRJHQL]HG 9HUWLV KRPRJHQL]HUf LQ SHUFKORULF DFLG b LQ b HWKDQROf DQG DQDO\]HG IRU SKRVSKRU\OFUHDWLQH E\ WKH PHWKRG RI (QQRU DQG 6WRFNHQ f VHH $SSHQGL[f 6WDWLVWLFDO DQDO\VLV ZDV E\ WKH WZR ZD\ DQDO\VLV RI YDULDQFH IRU UHSHDWHG PHDVXUHV 'LIIHUHQFHV EHWZHHQ PHDQV ZHUH GHWHUPLQHG E\ 'XQFDQnV PXOWLSOH UDQJH WHVW f

PAGE 58

5HVXOWV %ORRG VDPSOHV ZHUH REWDLQHG EHIRUH WKH FRQWUDFWLRQV EHJDQ DQG DW W DQG PLQXWHV $UWHULDO 32 ZDV s PP +J PHDQ s 6(0f EHIRUH FRQWUDFWLRQV DQG GLG QRW FKDQJH VLJQLILFDQWO\ WKURXJKRXW WKH H[SHULPHQWV VHH )LJXUH f %HIRUH FRQWUDFWLRQV EHJDQ 3Y&! ZDV s PP +J 'XULQJ WKH FRQWUDFWLRQ SHULRG 3Y&! ZDV ORZHU EXW QRQH RI WKH EORRG VDPSOHV PHDVXUHG KDG D 3 OHVV WKDQ PP +J ([FHSW IRU WKH H[SHULPHQWV ZKHUH IDWLJXH ZDV FDXVHG E\ VHF FRQWUDFWLRQV 3Y&! ZDV EDFN WR SUHIDWLJXH YDOXHV HDUO\ LQ WKH UHFRYHU\ SHULRG &RQWUDFWLRQV ZHUH FRQWLQXHG VHF GXULQJ WKH UHFRYHU\ SHULRG RI WKHVH VHFf H[SHULPHQWV WKHUHIRUH LW PLJKW EH H[SHFWHG WKDW 3Y&! ZRXOG QRW EH DW UHVW OHYHOV $UWHULDO 3&2 EHJDQ DW s PP +J DQG IHOO VORZO\ GXULQJ WKH H[SHULPHQWV 7KH GHFUHDVH LQ 3D&&! ZDV VWDWLVWLFDOO\ VLJQLILFDQW EXW SUREDEO\ LV RI PLQLPDO SK\VLRORJLFDO VLJQLILFDQFH 9HQRXV 3&2 ZDV KLJK GXULQJ WKH FRQWUDFWLRQ SHULRG ZKHQ 3Y&! ZDV ORZ DQG UHWXUQHG WR SUHFRQWUDFWLRQ OHYHOV HDUO\ LQ WKH UHFRYHU\ SHULRG $UWHULDO S+ ZDV EHIRUH FRQWUDFWLRQV EHJDQ DQG GLG QRW FKDQJH VLJQLILFDQWO\ WKURXJKRXW WKH H[SHULPHQWV 9HQRXV S+ GHFUHDVHG IURP DW W PLQXWHV WR IRU VHFf RU IRU RU VHFf DW W PLQXWHV %\ PLQXWHV RI UHFRYHU\ YHQRXV S+ KDG UHWXUQHG WR SUHn IDWLJXH YDOXHV VHH )LJXUH f

PAGE 59

),*85( %ORRG IORZ DQG WKH EORRG JDV PHDVXUHPHQWV YHUVXV WLPH 7KH KRUL]RQWDO EDU LQGLFDWHV ZKHUH IDWLJXLQJ FRQWUDFWLRQV RFFXUUHG :KHQ PHDQV DW D JLYHQ WLPH IRU GLIIHUHQW IUHTXHQFLHVf ZHUH QRW VLJQLILFDQWO\ GLIIHUHQW PHDQV ZHUH FRPELQHG 1XPEHUV UHIHU WR WKH IDWLJXLQJ IUHTXHQF\ $VWHULVNV LQGLFDWH ZKHUH PHDVXUHPHQWV DUH VLJQLILFDQWO\ GLIIHUHQW IURP WKH RULJLQDO YDOXH WLPH PLQXWHVf 9HUWLFDO EDUV DUH s 6(0

PAGE 60

),*85(

PAGE 61

$OWKRXJK EORRG IORZ ZDV PHDVXUHG FRQWLQXRXVO\ RQO\ WKRVH PHDVXUHPHQWV FRUUHVSRQGLQJ WR WLPHV ZKHQ EORRG VDPSOHV ZHUH REWDLQHG DUH SUHVHQWHG VHH )LJXUH f %ORRG IORZ ZDV KLJKHU GXULQJ WKH FRQWUDFWLRQV EXW ZDV EDFN WR SUHIDWLJXH YDOXHV E\ PLQXWHV RI UHFRYHU\ 7KHUH ZHUH QR VLJQLILFDQW GLIIHUHQFHV EHWZHHQ IUHTXHQFLHV IRU EORRG IORZ UHVSRQVH 7KH ILUVW PLQXWHV RI FRQWUDFWLRQV ZHUH DW VHF RU VHF 7KHUH ZHUH QR VLJQLILFDQW GLIIHUHQFHV IRU $7 EHWZHHQ WKHVH IUHTXHQFLHV DW W PLQXWHV 0HDQ $7 IRU DOO H[SHULPHQWV ZDV JJ ZHW ZWf DW WKLV WLPH 7KH PXVFOHV ZHLJKHG W J ZHW ZWf 'HYHORSHG WHQVLRQ IHOO PRUH UDSLGO\ GXULQJ VHF FRQWUDFWLRQV WKDQ GXULQJ VHF RU VHF FRQWUDFWLRQV VHH )LJXUH f %\ PLQXWHV DOO IUHTXHQFLHV RI VWLPXODWLRQ UHVXOWHG LQ VLJQLILFDQW UHGXFWLRQV LQ $7 7KHUH ZDV QR VLJQLILFDQW UHFRYHU\ RI $7 GXULQJ WKH PLQXWHV IROORZLQJ WKH IDWLJXLQJ FRQWUDFWLRQV &RQWUDFWLRQ WLPH GHFUHDVHG GXULQJ WKH IDWLJXLQJ FRQWUDFWLRQV DW VHF EXW QRW GXULQJ WKH FRQWUDFWLRQV DW RU VHF 7KHUH ZDV QR VLJQLILFDQW GLIIHUHQFH IRU &W EHWZHHQ UHFRYHU\ DQG SUHIDWLJXH PHDVXUHPHQWV DW DQ\ IDWLJXLQJ IUHTXHQF\ VHH )LJXUH f +DOI UHOD[DWLRQ WLPH GLG QRW FKDQJH GXULQJ WKH FRQWUDFWLRQV RU GXULQJ WKH UHFRYHU\ H[FHSW IRU UHFRYHU\ RI VHF IDWLJXH 7KH 5W ZDV ORQJHU IRU FRQWUDFWLRQV DW VHF WKDQ IRU VHF ,I FRQWUDFWLRQV GXULQJ UHFRYHU\

PAGE 62

),*85( 'HYHORSHG WHQVLRQ FRQWUDFWLRQ WLPH DQG KDOI UHOD[DWLRQ WLPH YHUVXV WLPH 7KH KRUL]RQWDO EDU LQGLFDWHV ZKHQ IDWLJXLQJ FRQWUDFWLRQV RFFXUUHG :KHUH WKHUH ZDV QR VLJQLILFDQW GLIIHUHQFH EHWZHHQ PHDQV DOO IUHTXHQFLHV DUH FRPELQHG 1XPEHUV UHIHU WR WKH IDWLJXLQJ IUHTXHQF\ $VWHULVNV LQGLFDWH ZKHUH PHDVXUHn PHQWV DUH VLJQLILFDQWO\ GLIIHUHQW IURP WKH RULJLQDO YDOXH DW WLPH PLQXWHVf 9HUWLFDO EDUV DUH s 6(0

PAGE 63

),*85(

PAGE 64

IRU WKLV IUHTXHQF\ RI IDWLJXLQJ FRQWUDFWLRQV KDG EHHQ VHF DW W DQG PLQXWHV RQO\f WKHQ QR GLIIHUHQFH IURP SUHIDWLJXH FRQWUDFWLRQV ZRXOG EH H[SHFWHG )LJXUH LOOXVWUDWHV WKH FKDQJHV LQ G3GW DQG G3GW VHHQ LQ WKHVH H[SHULPHQWV 7KH FKDQJHV VHHQ IRU G3GW FORVHO\ SDUDOOHO WKRVH REVHUYHG IRU $7 $ SRVLWLYH DQG VLJQLILFDQW FRUUHODWLRQ H[LVWV EHWZHHQ G3GW DQG $7 U f DQG EHWZHHQ G3GW DQG $7 UA f 0XVFOH WHPSHUDWXUH URVH GXULQJ WKH IDWLJXLQJ FRQWUDFn WLRQV 7KH LQFUHDVH LQ WHPSHUDWXUH ZDV RQO\ r& $ VLPLODU RU VPDOOHU ULVH ZDV VHHQ GXULQJ WKH VHF DQG VHF FRQWUDFWLRQV 0XVFOH WHPSHUDWXUH IHOO VORZO\ WR r& GXULQJ UHFRYHU\ EXW ZDV QRW SHUPLWWHG WR JR EHORZ r& 0XVFOH VDPSOHV REWDLQHG GXULQJ D IHZ RI WKH H[SHULn PHQWV ZHUH DQDO\]HG IRU SKRVSKRU\OFUHDWLQH 3&f $QDO\VLV UHYHDOHG WKDW 3& LV ORZ DW W PLQXWHV GXULQJ FRQWUDFWLRQVf EXW LV EDFN WR UHVWLQJ OHYHOV E\ W PLQXWHV VHH 7DEOH ,,,f 7KH YDOXHV RI 3& JLYHQ LQ WKH WDEOH DUH OHIWULJKW UDWLRV 3& ZDV GHWHUPLQHG UHODWLYH WR WRWDO FUHDWLQH LQ WKH PXVFOH VDPSOH +DUULV f KDV VKRZQ WKDW WRWDO PXVFOH FUHDWLQH FRQWHQW GRHV QRW FKDQJH GXULQJ H[HUFLVH DQG WKHUHIRUH FDQ EH XVHG DV DQ LQGH[ RI PXVFOH ZHLJKW ,Q RQH H[SHULPHQW D WHWDQLF FRQWUDFWLRQ ZDV REWDLQHG EHIRUH DQG DIWHU IDWLJXLQJ FRQWUDFWLRQV DW VHF )LJXUH LOOXVWUDWHV WKH ODFN RI FKDQJH VHHQ IRU WKLV FRQWUDFWLRQ

PAGE 65

7$%/( ,,, 3+263+25
PAGE 66

),*85( 3HDN UDWH RI IRUFH GHYHORSPHQW DQG SHDN UDWH RI UHOD[DWLRQ 6HH )LJXUH IRU VLJQLILFDQFH RI DVWHULVNV DQG QXPEHUV 1RWH VLPLODULW\ EHWZHHQ G3GW YHUVXV WLPH DQG $7 YHUVXV WLPH )LJXUH f

PAGE 67

'3'7 rRf 7,0( PLQf L L L ),*85( L L 7 /Q

PAGE 68

L , 7(7$1,& &2175$&7,216 G3 GW ),*85( 7UDFLQJV RI UHFRUGLQJV RI WHQVLRQ DQG GLIIHUHQWLDO RI WHQVLRQ IRU WHWDQLF FRQWUDFWLRQV VHF IRU PVHFf 'HYHORSHG WHQVLRQ IROORZLQJ PLQXWHV RI IDWLJXLQJ FRQWUDFWLRQV VHFf ZDV RQO\ VOLJKWO\ ORZHU WKDQ WKDW EHIRUH WKH VHF FRQWUDFWLRQV 7KH GLIIHUHQWLDO WUDFHU ZHUH YLUWXDOO\ VXSHULPSRVDEOH

PAGE 69

7KH WHWDQLF FRQWUDFWLRQ LV UHFRYHUHG DW D WLPH ZKHQ WZLWFK $7 LV VWLOO UHGXFHG &RQWUDFWLRQV REVHUYHG GXULQJ LVFKHPLD GHPRQVWUDWHG D UHGXFHG $7 DQG D SURORQJHG 5W )ROORZLQJ UHVWRUDWLRQ RI EORRG IORZ UHFRYHU\ RI ERWK $7 DQG 5W ZDV b FRPSOHWH LQ PLQXWHV )LJXUH VKRZV UHFRUGLQJV IURP RQH PXVFOH IRU FRQWUDFWLRQV SUHLVFKHPLD GXULQJ LVFKHPLD DQG SRVWLVFKHPLD 7KHVH UHFRUGLQJV DUH W\SLFDO IRU ZKDW ZDV VHHQ &RPSDULQJ WKH HIIHFWV RI GDQWUROHQH VRGLXP LVFKHPLD DQG UHGXFHG VWLPXODWLRQ YROWDJH RQ 5W YV $7 VHH )LJXUH f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f 7ZLWFK IDWLJXH WKHQ UHVXOWV IURP D UHODWLYHO\ SHUVLVWHQW DOWHUDWLRQ LQ WKH PXVFOH ZKLFK DIIHFWV WKH FRQWUDFWLOH UHVSRQVH WR D VLQJOH LPSXOVH

PAGE 70

),*85( LVFKHPLD 7HQVLRQ DQG G3GW XSSHU DQG ORZHU FXUYHV RI HDFK SDLU UHVSHFWLYHO\f DUH VKRZQ f D FRQWURO FRQWUDFWLRQ EHIRUH RFFOXVLRQ RI EORRG IORZ f FRQWUDFWLRQ GXULQJ LVFKHPLD PLQXWHV DIWHU RFFOXVLRQ RI EORRG IORZ f D SRVWLVFKHPLD FRQWUDFWLRQ PLQXWHV DIWHU UHOHDVH RI RFFOXVLRQ FRQWUDFWLRQ IUHTXHQF\ VHF

PAGE 71

),*85( +DOI UHOD[DWLRQ WLPH YHUVXV $7 LV SUHVHQWHG WR LOOXVWUDWH WKH UHODWLYH FKDQJHV LQ 5W ZKHQ $7 LV UHGXFHG E\ LVFKHPLD GDQWUROHQH RU UHGXFHG VWLPXODWLRQ YROWDJH (DFK OLQH UHSUHVHQWV RQH GRJ /LQHV ZHUH GHWHUPLQHG E\ WKH OHDVW VTXDUHV PHWKRG FRPPRQ WR DOO WKUHH OLQHV

PAGE 72

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f 7KH PHDVXUHPHQWV PDGH IRU WKH VHF VHULHV ZHUH PDGH RQ FRQWUDFWLRQV DW VHF GXULQJ WKH PLQXWH IDWLJXH SHULRG DQG DW VHF GXULQJ UHFRYHU\ 'XH WR WKH HIIHFW RI IUHTXHQF\ DQG SUHFHGLQJ FRQWUDFWLRQV RQ WKH WLPH FRXUVH RI D WZLWFK FDXWLRQ PXVW EH H[HUFLVHG ZKHQ FRPSDULQJ YDOXHV EHWZHHQ IUHTXHQFLHV &RPSDULQJ WKH WUHQG ZLWKLQ RQH IUHTXHQF\ ZLWK WKH WUHQG ZLWKLQ DQRWKHU IUHTXHQF\ LV YDOLG

PAGE 73

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f ZHUH FRQWLQXHG DW OVHF

PAGE 74

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f DOWHULQJ WKH WLPH FRXUVH FI FRQWUDFWLRQV DW VHF $ GHFUHDVH ZDV VHHQ IRU G3GW ZKLFK ZDV SURSRUWLRQDO WR WKH GHFUHDVH LQ $7 %UXVW f UHSRUWHG VLPLODU IDWLJXH SDWWHUQV IRU WKH PRXVH VROHXV PXVFOH LQ YLWUR 7KH VDPH DXWKRU f UHSRUWHG GLIIHUHQW IDWLJXH SDWWHUQV IRU IURJ VHPLWHQGn LQRVLV PXVFOHV 7KH PDMRU GLIIHUHQFH ZDV WKDW 5W ZDV SURORQJHG LQ WKH IDWLJXHG IURJ PXVFOH EXW QRW LQ WKH PDPPDOLDQ PXVFOHV 7KLV GLIIHUHQFH LV DSSDUHQWO\ QRW VSHFLHV VSHFLILF (GZDUGV HW DO f UHSRUWHG WKDW WKHUH LV D VORZLQJ RI UHOD[DWLRQ LQ PRXVH PXVFOH IROORZLQJ D IDWLJXLQJ HIIRUW 7KLV VORZLQJ RI UHOD[DWLRQ ZDV FRUUHODWHG ZLWK ORZ OHYHOV RI $73 f 7KLV FDQ

PAGE 75

H[SODLQ WKH LQFUHDVH LQ 5W VHHQ LQ LVFKHPLF IDWLJXH VHH )LJXUH f 6LQFH 5W ZDV QRW SURORQJHG IROORZLQJ D SHULRG RI IDWLJXLQJ FRQWUDFWLRQV ZLWK DQ LQWDFW EORRG VXSSO\ LW ZRXOG DSSHDU WKDW $73 ZDV DYDLODEOH ZLWKLQ WKH PXVFOH 7KH HYLGHQFH SUHVHQWHG DERYH VXJJHVWV WKDW WKH IDWLJXH REVHUYHG LQ WKHVH H[SHULPHQWV GLG QRW RFFXU DV D UHVXOW RI UHGXFHG $73 OHYHOV 2WKHU HYLGHQFH VXSSRUWV WKLV VXJJHVWLRQ $OWKRXJK 3& OHYHOV ZHUH ORZ GXULQJ WKH FRQWUDFWLRQ SHULRG VHH 7DEOH ,,,f UHV\QWKHVLV KDG RFFXUUHG EHIRUH VLJQLILFDQW UHFRYHU\ RI $7 5DSLG UHV\QWKHVLV RI 3& GXULQJ UHFRYHU\ IURP D SHULRG RI FRQWUDFWLRQV ZDV DOVR REVHUYHG E\ 3LLSHU DQG 6SLOOHU f 7KHUH VHHPV WR EH QR GLUHFW UHODWLRQVKLS EHWZHHQ 3& OHYHOV DQG $7 7KLV LV FRQWUDU\ WR D UHSRUW E\ 6SDQGH DQG 6FKRWWHOLXV f EXW VXSSRUWV WKH REVHUYDWLRQV RI )LWWV DQG +ROORV]\ f ,W VKRXOG DOVR EH SRLQWHG RXW WKDW JO\FRJHQ ZDV VWLOO DYDLODEOH DIWHU PLQXWHV RI VWLPXODWLRQ DW VHF f DQG ODFWDWH SURGXFWLRQ KDG GHFOLQHG RU HYHQ UHYHUVHG ODFWDWH XSWDNHf f ,W ZRXOG VHHP WKDW HQHUJ\ ZDV DYDLODEOH EXW GHPDQG IRU HQHUJ\ ZDV UHGXFHG ,Q VXSSRUW RI WKLV FRQFOXVLRQ 3Y&! ZDV QRW EHORZ PLQLPXP FULWLFDO 32 f DW DQ\ WLPH WKDW 3Y&! ZDV PHDVXUHG 7KH PXVFOH LV FDSDEOH RI GHYHORSLQJ PRUH IRUFH WKDQ WKDW VHHQ LQ D WZLWFK 7ZLQ LPSXOVH VWLPXODWLRQ FDXVHV GHYHORSHG WHQVLRQ WR EH GRXEOH WKDW VHHQ ZLWK VLQJOH

PAGE 76

LPSXOVH VWLPXODWLRQ f 7KLV UHODWLRQVKLS LV PDLQWDLQHG IRU UHVWHG DV ZHOO DV IDWLJXHG PXVFOHV ,W ZRXOG DSSHDU WKDW WKH UHGXFHG $7 RI WKH IDWLJXHG PXVFOH ZDV GXH WR UHGXFHG DFWLYDWLRQ $ UHGXFHG DFWLYDWLRQ RI VNHOHWDO PXVFOH FRXOG WKHRUHWLFDOO\ EH WKH UHVXOW RI Df UHGXFHG QHXURPXVFXODU WUDQVPLVVLRQ Ef UHGXFHG &D UHOHDVH RU Ff UHGXFHG UHVSRQVLYHQHVV RI WKH FRQWUDFWLOH HOHPHQWV WR &D 1HXURPXVFXODU WUDQVPLVVLRQ DSSHDUV WR EH LQWDFW f 7KLV DJUHHV ZLWK RWKHUV f ZKR KDYH IRXQG RQO\ PLQLPDO FKDQJHV LQ PXVFOH DFWLRQ SRWHQWLDOV RU HOHFWURn P\RJUDSKLF UHVSRQVH IROORZLQJ FRPSDUDEOH VWLPXODWLRQ 2 L SHULRGV 7KHUH LV D SRVVLELOLW\ WKDW &D UHOHDVH KDV EHHQ DWWHQXDWHG 7KLV FRXOG FDXVH D UHGXFHG $7 DQG G3GW ZLWKRXW DOWHULQJ &W RU 5W f %UXVW f ZKR REVHUYHG VLPLODU IDWLJXH SDWWHUQV ZLWK WKH PRXVH VROHXV PXVFOH VXJJHVWV WKDW WKH IDWLJXH KH REVHUYHG ZDV D UHVXOW RI UHGXFHG &D UHOHDVH VHH DOVR f IRU WKH FRQYHUVHf 7KH IDFWRUVf UHVSRQVLEOH IRU WKH UHGXFHG &D UHOHDVH LVDUH QRW NQRZQ %RQQHU HW DO f KDYH UHSRUWHG WKDW PXVFOH PLWRFKRQGULD DFFXPXODWH &D GXULQJ H[HUFLVH 7KLV ZRXOG UHGXFH WKH SRRO RI &D DYDLODEOH IRU UHF\FOLQJ LQ WKH VDUFRSODVPLF UHWLFXOXP f DQG FRQVHTXHQWO\ OLPLW WKH DPRXQW DYDLODEOH IRU UHOHDVH 'DQWUROHQH VRGLXP DSSDUHQWO\ UHGXFHV WKH DPRXQW RI &D UHOHDVHG SHU LPSXOVH f 7KLV GUXJ ZDV XVHG LQ WKHVH H[SHULPHQWV WR GHWHUPLQH WKH HIIHFWV RI UHGXFHG

PAGE 77

&D UHOHDVH RQ D WZLWFK $ GDQWUROHQH WUHDWHG PXVFOH FRQWUDFWV ZLWK D WLPHFRXUVH VLPLODU WR WKH IDWLJXHG PXVFOH ,VFKHPLD RU UHGXFHG VWLPXODWLRQ YROWDJH FDXVHG D UHGXFWLRQ LQ $7 EXW WKLV ZDV DFFRPSDQLHG E\ FKDQJHV LQ 5W $Q DOWHUQDWLYH K\SRWKHVLV LQYROYHV D UHGXFHG ELQGLQJ RI &D WR WKH UHJXODWRU\ SURWHLQV 'XULQJ FRQWUDFWLRQV PXVFOH S+ SUREDEO\ IDOOV f )XFKV HW DO f KDV VKRZQ WKDW ELQGLQJ RI &D WR WURSRQLQ LV LQKLELWHG E\ ORZHU S+ )LWWV DQG +ROORV]\ f KDV VXJJHVWHG WKLV PD\ EH D PHFKDQLVP UHVSRQVLEOH IRU WKH IDWLJXH VHHQ LQ WKHLU H[SHULPHQWV 7KH H[SHULPHQWV UHSRUWHG KHUHLQ GR QRW SHUPLW GLVFULPLQDWLRQ EHWZHHQ WKHVH WZR SRWHQWLDO PHFKDQLVPV RI IDWLJXH 7KH IDWLJXH REVHUYHG IROORZLQJ PLQXWHV RI VWLPXODWLRQ DW RU VHF DSSHDUV WR EH D UHVXOW RI UHGXFHG DFWLYDWLRQ )XUWKHU H[SHULPHQWV ZLOO QHHG WR EH FRQGXFWHG WR GHWHUPLQH ZKLFK RI WKH WKHRULHV GHVFULEHG DERYH DSSOLHG WR WKHVH PXVFOHV

PAGE 78

())(&76 2) 5(63,5$725< $&,'26,6 21 7+( 7:,7&+ &2175$&7,21 ,QWURGXFWLRQ 7ZLWFK GHYHORSHG WHQVLRQ LV DWWHQXDWHG IROORZLQJ D SHULRG RI UHSHWLWLYH VWLPXODWLRQ 7KLV DWWHQXDWLRQ LV DSSDUHQWO\ WKH UHVXOW RI D UHGXFHG DFWLYDWLRQ GXH HLWKHU WR D UHGXFHG UHOHDVH RI &D IURP WKH ODWHUDO VDFV RU IURP D UHGXFHG UHVSRQVLYHQHVV RI WKH FRQWUDFWLOH SURWHLQV ,W LV QRW GXH WR D UHGXFHG DYDLODELOLW\ RI HQHUJ\ $73 3&f )LWWV DQG +ROORV]\ f KDYH VXJJHVWHG WKDW WKH UHGXFHG WZLWFK UHVSRQVH LV D UHVXOW RI LQKLELWLRQ RI WKH FRQWUDFWLOH SURFHVV GXH WR D UHGXFHG LQWUDFHOOXODU S+ 6WHLQKDJHQ HW DO UHSRUWHG HYLGHQFH VXSSRUWLQJ WKLV K\SRWKHVLV f +H UHSRUWHG WKDW GRJ JDVWURFQHPLXV PXVFOH IDWLJXHV PRUH TXLFNO\ GXULQJ UHVSLUDWRU\ DFLGRVLV WKDQ GXULQJ QRUPDO S+ EDODQFH 6SHFLILF PHFKDQLVPV ZKLFK PD\ FRQWULEXWH WR WKLV DFLGRVLVLQGXFHG IDWLJXH KDYH EHHQ SURSRVHG 1DNDPXUD DQG 6FKZDUW] f UHSRUWHG WKDW XSWDNH RI &D E\ VDUFRSODVPLF UHWLFXOXP LV DFFHOHUDWHG LQ ORZ S+ PHGLXP 7KLV FRXOG UHGXFH WKH GXUDWLRQ RI DFWLYDWLRQ IRU D WZLWFK E\ UHGXFLQJ WKH &D FRQFHQWUDWLRQ PRUH TXLFNO\ )XFKV HW DO f KDYH UHSRUWHG WKDW &D ELQGLQJ WR WURSRQLQ LV LQKLELWHG E\ + 7KHVH PROHFXODU PHFKDQLVPV LI HIIHFWLYH XQGHU SK\VLRORJLFDO FRQGLWLRQV ZRXOG FDXVH D UHGXFWLRQ LQ $7 IDWLJXH

PAGE 79

7KH SXUSRVH RI WKLV VWXG\ ZDV WR REVHUYH WKH HIIHFWV RI DFLGRVLV RQ WKH GHYHORSHG WHQVLRQ DQG WKH WLPHFRXUVH RI D WZLWFK FRQWUDFWLRQ RI WKH LQ VLWX GRJ JDVWURFQHPLXV SODQWDULV PXVFOH ,QWUDFHOOXODU S+ FDQ EH UHGXFHG PRUH HDVLO\ YLD UHVSLUDWRU\ DFLGRVLV WKDQ E\ PHWDEROLF DFLGRVLV f )RU WKLV UHDVRQ DFLGRVLV ZDV LQGXFHG E\ UHGXFLQJ WKH YHQWLODWRU\ UDWH 7KH UHVXOWV RI WKLV VWXG\ LQGLFDWH WKDW DFLGRVLV LV XQOLNHO\ D GLUHFW FDXVH RI WZLWFK IDWLJXH 0HWKRGV 7KH JDVWURFQHPLXVSODQWDULV PXVFOH SUHSDUDWLRQ DV GHVFULEHG LQ WKH *HQHUDO 0HWKRGV VHFWLRQ ZDV XVHG LQ WKLV VHULHV RI H[SHULPHQWV ,Q HDFK H[SHULPHQW WKH QHUYH ZDV VWLPXODWHG DW D IUHTXHQF\ RI VHF ,Q WKUHH H[SHULPHQWV YHQWLODWLRQ ZDV FRQWUROOHG WR PDLQWDLQ DUWHULDO S+ QHDU IRU WKH GXUDWLRQ RI WKH H[SHULPHQW KRXUVf ,Q IRXU H[SHULPHQWV DIWHU WKH PXVFOH KDG EHHQ FRQWUDFWLQJ VHFf IRU PLQXWHV YHQWLODWLRQ ZDV UHGXFHG WR EUHDWKVPLQXWH 7KH PL[WXUH RI LQVSLUHG JDV ZDV DGMXVWHG ZLWK b &! DQG b &2f WR PDLQWDLQ QRUPDO DUWHULDO 32 GXULQJ WKH K\SRYHQWLODWLRQ 7KH SHULRG RI K\SRYHQWLODWLRQ ZDV FRQWLQXHG XQWLO DUWHULDO S+ ZDV OHVV WKDQ PLQXWHVf 7KLV SHULRG RI K\SRYHQWLODWLRQ ZDV IROORZHG E\ D SHULRG RI K\SHUYHQWLODWLRQ EUHDWKVPLQXWHf 7KH K\SHUYHQWLODWLRQ ZDV FRQWLQXHG IRU PLQXWHV VHH )LJXUH f

PAGE 80

:LWK DQ DGGLWLRQDO IRXU GRJV WKH VHTXHQFH RI YHQWLODWRU\ DGMXVWPHQW ZDV UHYHUVHG FRQWURO K\SHUn YHQWLODWLRQ K\SRYHQWLODWLRQf WR SHUPLW HYDOXDWLRQ RI D SRVVLEOH RUGHU HIIHFW $W WHQ PLQXWH LQWHUYDOV WKURXJKRXW HDFK H[SHULPHQW DUWHULDO DQG YHQRXV EORRG VDPSOHV ZHUH REWDLQHG POf LQ JODVV WXEHUFXOLQ V\ULQJHV PO FDSDFLW\f 7KH VDPSOHV ZHUH VHDOHG ZLWK PHUFXU\ FRQWDLQLQJ FDSV DQG NHSW LQ LFH XQWLO WKH\ ZHUH DQDO\]HG IRU 32 3&2 DQG S+ 5DGLRPHWHU &RSHQKDJHQf )DVW WUDFHV RI FRQWUDFWLRQV ZHUH DOVR REWDLQHG DW PLQXWH LQWHUYDOV PPVHF RQ *RXOG%UXVK 0RGHO UHFRUGHUf 7KH IDVW WUDFHV ZHUH PHDVXUHG IRU GHYHORSHG WHQVLRQ $7f KDOI UHOD[DWLRQ WLPH 5W f FRQWUDFWLRQ WLPH &Wf SHDN UDWH RI IRUFH GHYHORSPHQW G3GWf DQG SHDN UDWH RI UHOD[DWLRQ G3GWf VHH )LJXUH f 6WDWLVWLFDO DQDO\VLV ZDV E\ D WZR ZD\ DQDO\VLV RI YDULDQFH IRU UHSHDWHG PHDVXUHV f )RU VWDWLVWLFDO DQDO\VLV WKH ODVW WZR PHDVXUHPHQWV IDVW WUDFHV RU EORRG JDVHVf EHIRUH DOWHUDWLRQ RI WKH YHQWLODWLRQ ZHUH XWLOL]HG WR UHSUHVHQW WKH VWDWH LQ ZKLFK WKH\ RFFXUUHG VHH )LJXUH f 5HVXOWV $IWHU PLQXWHV RI FRQWUDFWLRQV YHQWLODWLRQ ZDV UHGXFHG WR EUHDWKV SHU PLQXWH *URXS $f RU LQFUHDVHG WR EUHDWKV SHU PLQXWH *URXS %f 7KLV DGMXVWPHQW LQ YHQWLODWLRQ UHVXOWHG LQ D UHGXFWLRQ LQ DUWHULDO S+ IURP

PAGE 81

),*85( 'HYHORSHG WHQVLRQ 32 YHQWLODWLRQ DQG DUWHULDO S+ IRU RQH GRJ IURP *URXS $ %ORRG VDPSOHV DQG IDVW WUDFHV REWDLQHG DW r ZHUH XVHG IRU VWDWLVWLFDO DQDO\VLV

PAGE 82

'(9 (/23(' 7,0( PLQf ),*85(

PAGE 83

W PHDQ s 6(0f GXULQJ WKH FRQWURO SHULRG WR s LQ *URXS $ DQG DQ LQFUHDVH LQ *URXS % IURP s WR  $IWHU PLQXWHV YHQWLODWLRQ ZDV LQFUHDVHG WR EUHDWKV SHU PLQXWH LQ *URXS $ DQG UHGXFHG WR EUHDWKV SHU PLQXWH LQ *URXS % 7KLV VHFRQG DOWHUDWLRQ LQ YHQWLODWRU\ IUHTXHQF\ UHVXOWHG LQ DQ LQFUHDVH LQ S+ WR LQ *URXS $ DQG D UHGXFWLRQ LQ *URXS % WR VHH )LJXUH f ,Q *URXS & YHQWLODWLRQ ZDV QRW DOWHUHG DQG DUWHULDO S+ GLG QRW YDU\ GXULQJ WKH H[SHULPHQWV 7KH UHGXFWLRQ LQ YHQWLODWLRQ GLG QRW VLJQLILFDQWO\ GHFUHDVH DUWHULDO 3&! f :KHQ DUWHULDO S+ UHPDLQHG FRQVWDQW IRU KRXUV *URXS &f $7 LQFUHDVHG ZLWK WLPH %\ WKH HQG RI KRXUV $7 ZDV JUHDWHU WKDQ LW KDG EHHQ DW WKH ILUVW PLQXWH SHULRG $V LOOXVWUDWHG LQ )LJXUH $7 LQFUHDVHG ZLWK WLPH LQ *URXS $ DOVR EXW QRW LQ *URXS % 7KH RQO\ VLJQLILFDQW GLIIHUHQFH LQ *URXSV $ DQG % ZDV WKDW $7 GXULQJ K\SHUYHQWLODWLRQ ZDV JUHDWHU WKDQ $7 DW DQ\ RWKHU WLPH SHULRG LQ *URXS $ DQG JUHDWHU WKDQ FRQWURO LQ *URXS % VHH 7DEOH ,9f 'HVSLWH WKH DSSDUHQW HIIHFW RI K\SHUYHQWLODWLRQ RQ $7 WKHUH ZDV QR VLJQLILFDQW FRUUHODWLRQ RI $7 ZLWK DUWHULDO RU YHQRXV + FRQFHQWUDWLRQ VHH 7DEOH 9f )XUWKHUPRUH $7 ZDV QRW UHGXFHG GXULQJ K\SRYHQWLODWLRQ :KHQ DUWHULDO S+ IHOO WR s $7 GLG QRW FKDQJH VLJQLILFDQWO\ ,Q *URXS & G3GW GLG QRW FKDQJH VLJQLILFDQWO\ ,Q *URXS $ DQG *URXS % KRZHYHU G3GW ZDV KLJKHU GXULQJ

PAGE 84

7$%/( ,9 67$7,67,&6 )25 ',))(5(1&(6 %(7:((1 9(17,/$7,21 67$7(6 )25 ($&+ *5283 *URXS $ *URXS % *URXS & 5DQN 5DQN 5DQN $7 5W 16 16 &W 16 16 G3GW 16 G3GW 16 7KH S YDOXHV DUH JLYHQ IRU $7 5W &W G3GW DQG G3GW IRU *URXS $ FRQWURO K\SRYHQWLODWLRQ K\SHUn YHQWLODWLRQf *URXS % FRQWURO K\SHUYHQWLODWLRQ K\SRYHQWLODWLRQf DQG *URXS & FRQWUROf 0HDQV DUH OLVWHG XQGHU WKH UDQN RUGHU 5DQN LQGLFDWHV WKH RUGHU IURP ODUJHVW WR VPDOOHVW IRU &RQWURO $FLGRVLV DQG $ONDORVLV )RU *URXS & UDQN DQG LQGLFDWH WLPH SHULRGV ILUVW PLQXWHV QH[W PLQXWHV DQG ODVW PLQXWHV +RUL]RQWDO EDUV DFURVV UDQNV LQGLFDWH PHDQV ZKLFK DUH QRW VLJQLILFDQWO\ GLIIHUHQW 16 LQGLFDWHV WKDW PHDQV DUH QRW VLJQLILFDQWO\ GLIIHUHQW

PAGE 85

),*85( $UWHULDO DQG YHQRXV + FRQFHQWUDWLRQ GXULQJ YHQWLODWRU\ VWDWHV 7KH WKUHH FROXPQV RQ WKH OHIW DUH IURP *URXS $ 7KH WKUHH FROXPQV RQ WKH ULJKW DUH IURP *URXS % 2SHQ FROXPQV DUH DUWHULDO >+@ 9HUWLFDO EDUV DUH RQH VWDQGDUG HUURU RI WKH PHDQ 8SSHU EDU LV IRU YHQRXV ORZHU EDU IRU DUWHULDO 6(0

PAGE 86

9(17,/$7,21 E UH D W K V PL QX WH f ),*85(

PAGE 87

),*85( 0HDQ DUWHULDO DQG YHQRXV 32 GXULQJ WKH GLIIHUHQW YHQWLODWRU\ VWDWHV 7KH WKUHH FROXPQV RQ WKH OHIW UHSUHVHQW *URXS $ 7KH WKUHH RQ WKH ULJKW UHSUHVHQW *URXS % 2SHQ EDUV UHSUHVHQW YHQRXV 32 DQG FRPELQHG RSHQ DQG FORVHGf EDUV UHSUHVHQW DUWHULDO 32 9HUWLFDO EDUV UHSUHVHQW RQH 6(0

PAGE 88

$57 (5,$/ DQG 9(1286 SR PP +Tf 9(17,/$7,21 EUHDWKVPLQXWHf ),*85(

PAGE 89

K\SHUYHQWLODWLRQ WKDQ GXULQJ WKH LQLWLDO FRQWURO SHULRG VHH 7DEOH ,f 7KHUH ZDV QR VLJQLILFDQW FRUUHODWLRQ EHWZHHQ DUWHULDO S+ DQG G3GW 7KLV VXJJHVWV DV VHHQ IRU $7 WKDW WKH DOWHUDWLRQV LQ G3GW DVVRFLDWHG ZLWK YHQWLODWLRQ SDWWHUQV DUH QRW GLUHFWO\ UHODWHG WR S+ VHH )LJXUH f 7KH SHDN UDWH RI UHOD[DWLRQ ZDV JUHDWHVW GXULQJ K\SHUYHQWLODWLRQ ,Q *URXS $ WKLV ZDV VLJQLILFDQWO\ JUHDWHU WKDQ ERWK WKH FRQWURO SHULRG DQG WKH SHULRG RI K\SRYHQWLODWLRQ ,Q *URXS % KRZHYHU ZKHUH DUWHULDO S+ LQFUHDVHG WR GXULQJ K\SHUYHQWLODWLRQ DV RSSRVHG WR LQ *URXS $f G3GW GXULQJ K\SHUYHQWLODWLRQ ZDV JUHDWHU WKDQ WKDW GXULQJ QRUPDO YHQWLODWLRQ EXW QRW VLJQLILFDQWO\ GLIIHUHQW IURP WKDW GXULQJ K\SRYHQWLODWLRQ 7KHUH ZDV QR VLJQLILFDQW FRUUHODWLRQ EHWZHHQ G3GW DQG DUWHULDO >+@ ,Q *URXSV $ DQG & WKHUH ZHUH QR VLJQLILFDQW FKDQJHV LQ &W RU 5W ,Q *URXS % KRZHYHU &W ZDV VKRUWHU GXULQJ K\SRYHQWLODWLRQ WKDQ DW RWKHU WLPHV 5W ZDV VKRUWHU GXULQJ K\SHUYHQWLODWLRQ WKDQ DW RWKHU WLPHV LQ *URXS % VHH )LJXUH f 'LVFXVVLRQ ,W KDV EHHQ VXJJHVWHG WKDW LQWUDFHOOXODU DFLGLILFDWLRQ LV WKH FDXVH RI VNHOHWDO PXVFOH IDWLJXH f ,I WKHUH LV D GLUHFW LQIOXHQFH RI S+ RQ WKH IRUFH RI FRQWUDFWLRQ WKLV SKHQRPHQRQ ZRXOG EH LQGHSHQGHQW RI WKH PDQQHU LQ ZKLFK WKH S+ FKDQJH ZDV REWDLQHG ,W LV DSSDUHQW LQ

PAGE 90

),*85( 0HDQ GHYHORSHG WHQVLRQ G3GW DQG G3GW GXULQJ GLIIHUHQW YHQWLODWRU\ VWDWHV 'HYHORSHG WHQVLRQ LV b RI KLJKHVW LQ HDFK H[SHULPHQW G3GW DQG G3GW DUH b RI KLJKHVW G3GW LQ HDFK H[SHULPHQW 9HUWLFDO EDUV UHSUHVHQW RQH VWDQGDUG HUURU RI WKH PHDQ

PAGE 91

D7 PP G3GW 8= G3GW &=9(17,/$7,21 EUH D W K V P L QX WH f ),*85(

PAGE 92

),*85( 0HDQ FRQWUDFWLRQ WLPH DQG +DOI UHOD[DWLRQ WLPH GXULQJ GLIIHUHQW YHQWLODWRU\ VWDWHV 9HUWLFDO EDUV UHSUHVHQW RQH 6(0

PAGE 93

5W 2 &W 9(17,/$7,21 EUHD W K VP L QXWH f ),*85(

PAGE 94

7$%/( 9 67$7,67,&6 )25 7:,7&+ &+$5$&7(5,67,&6 9(5686 %/22' *$6(6 *URXS 7 5W G3GW G3GW &W S U 3 U S U 3 U 3 U $UW >+@ $ f % & YHQ >+@ $ % & 3D& $ % & 6LJQLILFDQFH YDOXHV DUH JLYHQ IRU FRUUHODWLRQV EHWZHHQ WZLWFK FKDUDFWHULVWLFV DQG EORRG JDVHV 5 YDOXHV LQGLFDWLQJ WKH SHUFHQW RI YDULDELOLW\ H[SODLQHG E\ WKH YDULDEOH DUH JLYHQ IRU VLJQLILFDQW FRUUHODWLRQV Sf *URXSV DUH DV GHILQHG LQ WKH OHJHQG IRU 7DEOH ,9

PAGE 95

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n FHOOXODU S+ GXULQJ WKH IDWLJXLQJ FRQWUDFWLRQV FKDQJHV PRUH WKDQ LW GLG GXULQJ K\SRYHQWLODWLRQ 7KH PDJQLWXGH RI WKH LQWUDFHOOXODU S+ FKDQJH RFFXUULQJ LQ WKHVH H[SHULPHQWV FDQ EH HVWLPDWHG IURP WKH UHVXOWV RI %XUQHOO f +\SHUFDSQLD LQ GRJV 3D&&! PP +Jf UHVXOWHG LQ D UHGXFWLRQ RI LQWUDFHOOXODU S+ RI QHFN PXVFOHV IURP WR ,QWUDFHOOXODU S+ ZDV GHWHUPLQHG E\ WKH '02 PHWKRG f %XUQHOO f REVHUYHG WKDW WKH PD[LPDO UHVSRQVH KDG RFFXUUHG ZLWKLQ PLQXWHV ,Q WKH H[SHULPHQWV UHSRUWHG KHUHLQ DUWHULDO 3&2 LQFUHDVHG WR s PP +J 7KH PHDVXUHPHQWV SUHVHQWHG ZHUH WDNHQ PLQXWHV DIWHU WKH DOWHUDWLRQ LQ YHQWLODWLRQ ,W LV UHDVRQDEOH WR DVVXPH WKDW D YHU\

PAGE 96

VLPLODU FKDQJH LQ LQWUDFHOOXODU S+ RFFXUUHG LQ WKHVH H[SHULPHQWV DV ZDV REVHUYHG E\ %XUQHOO XQGHU DOPRVW LGHQWLFDO FLUFXPVWDQFHV ,W FDQ WKHUHIRUH EH FRQFOXGHG WKDW D FKDQJH LQ LQWUDFHOOXODU S+ YDOXHV IURP QRUPDO UHVWLQJ YDOXHV DSSUR[LPDWOH\ f WR GRHV QRW UHVXOW LQ D FKDQJH LQ GHYHORSHG WHQVLRQ 7KH LPSRUWDQW SRLQW KRZHYHU LV ZKHWKHU RU QRW LQWUDFHOOXODU S+ IHOO WR WKLV OHYHO RU ORZHU GXULQJ WKH IDWLJXLQJ FRQWUDFWLRQV UHSRUWHG LQ HDUOLHU FKDSWHUV (VWLPDWHV RI LQWUDFHOOXODU S+ FKDQJHV XQGHU WKHVH FLUFXPVWDQFHV FDQ RQO\ EH WHQWDWLYH 9HQRXV 3&2 GXULQJ WKH IDWLJXLQJ FRQWUDFWLRQV ZDV QHYHU JUHDWHU WKDQ PP +J PHDQ PP +J DW W PLQXWHV IRU VHFf 7KLV K\SHUFDSQLD ZRXOG QRW FDXVH DQ DFLGRVLV VXIILFLHQW WR UHGXFH $7 LI UHGXFHG S+ ZLOO FDXVH UHGXFHG $7f +RZHYHU WKHUH LV ODFWLF DFLG SURGXFWLRQ GXULQJ WKH ILUVW PLQXWHV RI WKLV W\SH RI VWLPXODWLRQ f 7KLV LV OLNHO\ WR FRQWULEXWH WR DQ LQWUDFHOOXODU DFLGLILFDWLRQ 6DKOLQ HW DO f UHSRUW WKDW LQ KXPDQV SHUIRUPLQJ PD[LPDO H[HUFLVH WR H[KDXVWLRQ LQWUDFHOOXODU S+ GURSV IURP WR 7KLV LV FRPSDUDEOH WR WKDW VHHQ E\ +HUPDQVRQ DQG 2VQHV f 7KLV GHFUHDVH LQ S+ ZDV DVVRFLDWHG ZLWK DQ LQFUHDVH LQ ODFWLF DFLG SURGXFWLRQ )ROORZLQJ WKH ERXW RI H[HUFLVH LQWUDFHOOXODU S+ UHFRYHUHG WR ZLWKLQ WZHQW\ PLQXWHV ,W LV XQOLNHO\ WKDW LQWUDFHOOXODU S+ FKDQJHG DV PXFK LQ WKH VHF IDWLJXH DV LW GLG GXULQJ WKH H[KDXVWLQJ H[HUFLVH UHSRUWHG E\ 6DKOLQ HW DO f )XUWKHUPRUH LW VHHPV

PAGE 97

OLNHO\ WKDW LQWUDFHOOXODU S+ ZRXOG KDYH UHWXUQHG WR UHVWLQJ OHYHOV DIWHU PLQXWHV RI UHFRYHU\ 'HYHORSHG WHQVLRQ DW WKLV WLPH LV VWLOO YHU\ PXFK UHGXFHG LH QR VLJQLILFDQW UHFRYHU\ KDV RFFXUUHGf ,W VHHPV UHDVRQDEOH WR FRQFOXGH WKDW WKH SHUVLVWHQW IDWLJXH FDXVHG E\ FRQWUDFWLRQV DW VHF IRU PLQXWHV GRHV QRW UHVXOW GLUHFWO\ IURP DFLGRVLV 7KHUH PD\ KRZHYHU EH LQGLUHFW ZD\V LQ ZKLFK LQWUDFHOOXODU S+ PD\ DIIHFW WKH FRQWUDFWLOH SURFHVV UHVXOWLQJ LQ D FKDQJH ZLWKLQ WKH PXVFOH ZKLFK SHUVLVWV EH\RQG WKH WLPH ZKHQ S+ KDV UHWXUQHG WR FRQWURO f ,W LV HYLGHQW WKDW FKDQJHV LQ YHQWLODWRU\ SDWWHUQ GR DIIHFW PXVFOH FRQWUDFWLRQ 'XULQJ K\SHUYHQWLODWLRQ $7 ZDV LQFUHDVHG 7KLV ZDV WKH FDVH ZKHWKHU K\SRYHQWLODWLRQ SUHFHGHG WKH SHULRG RI K\SHUYHQWLODWLRQ RU LI QRUPDO YHQWLODWLRQ SUHFHGHG LW ,Q WKH IRUPHU FDVH K\SRn YHQWLODWLRQ SUHFHGLQJf DUWHULDO S+ UHWXUQHG WR VR WKHUH ZDV QR DEVROXWH DUWHULDO DONDOLQL]DWLRQ 7KH LQFUHDVH LQ $7 XQGHU WKHVH FLUFXPVWDQFHV ZDV JUHDWHU WKDQ WKH LQFUHDVH VHHQ LQ WKH ODWWHU FDVH K\SHUYHQWLODWLRQ SUHFHGHG E\ QRUPDO YHQWLODWLRQf GHVSLWH WKH IDFW WKDW WKLV SURFHGXUH UHVXOWHG LQ DQ LQFUHDVH LQ DUWHULDO S+ WR ,W VHHPV WKDW K\SHUYHQWLODWLRQ LQFUHDVHV $7 EXW K\SRYHQWLODWLRQ RQO\ UHGXFHV $7 ZKHQ LW LV SUHFHGHG E\ K\SHUYHQWLODWLRQ 7KH KLJK S YDOXHV UHSRUWHG LQ 7DEOH 9 UHIOHFW WKH ODFN RI UHODWLRQVKLS EHWZHHQ $7 DQG >+@ 7KH IDFW WKDW VLJQLILFDQW GLIIHUHQFHV ZHUH REVHUYHG EHWZHHQ YHQWLODWLRQ

PAGE 98

SHULRGV LV GXH QRW WR DEVROXWH S+ FKDQJHV EXW UHODWLYH FKDQJHV SRVVLEO\ LQ FRQMXQFWLRQ ZLWK VRPH RWKHU FKDQJH LRQ GLVWULEXWLRQ" LH VHH f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

PAGE 99

6800$5< 2 8SWDNH DQG 'HYHORSHG 7HQVLRQ 7KH DPRXQW RI R[\JHQ XVHG E\ D PXVFOH ZDV SURSRUWLRQDO WR WKH DPRXQW RI WHQVLRQ GHYHORSHG 7KLV RFFXUUHG RYHU D ZLGH UDQJH RI IRUFHV ZKHQ $7 ZDV DOWHUHG E\ DQ\ RI WKH IROORZLQJ Df )DWLJXH DIWHU PLQXWHV RI VWLPXODWLRQ DW VHF Ef )DWLJXH GXULQJ IDWLJXLQJ FRQWUDFWLRQV DW VHF Ff 7ZLQ LPSXOVH VWLPXODWLRQ EHIRUH DQG DIWHU IDWLJXH DW VHF Gf )DWLJXH GXULQJ FRQWUDFWLRQV DW VHF ZLWKRXW EORRG IORZ LVFKHPLF IDWLJXHf Hf $WWHQXDWHG FRQWUDFWLRQ FDXVHG E\ DGPLQLVWUDWLRQ RI FXUDUH LQ VXIILFLHQW GRVHV WR UHGXFH WKH IRUFH RI FRQWUDFWLRQ E\ DV PXFK DV b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

PAGE 100

7LPHFRXUVH RI WKH 7ZLWFK &RQWUDFWLRQ LQ )DWLJXH 7KH WLPHFRXUVH &W DQG 5W f DQG WKH UDWH RI FKDQJH RI IRUFH G3GW DQG G3GWf ZHUH REVHUYHG ZKHQ PXVFOHV ZHUH IDWLJXHG ZLWK FRQWUDFWLRQV DW DQG VHF IRU PLQXWHV 7KH SHUWLQHQW REVHUYDWLRQV DUH OLVWHG EHORZ Df 'HYHORSHG WHQVLRQ IHOO PRUH UDSLGO\ GXULQJ VHF VWLPXODWLRQ WKDQ RU VHF Ef ,Q WKH PLQXWHV IROORZLQJ WKH IDWLJXLQJ FRQWUDFWLRQV QR VLJQLILFDQW UHFRYHU\ RI $7 RFFXUUHG Ff 7KH &W DQG 5W RI D IDWLJXHG PXVFOH DUH QR GLIIHUHQW IURP WKRVH RI D UHVWHG PXVFOH Gf 7KH G3GW LV JUHDWO\ UHGXFHG LQ IDWLJXH DQG LV VLJQLILFDQWO\ FRUUHODWHG ZLWK $7 $ UHGXFWLRQ LQ G3GW LV DOVR VHHQ LQ WKH IDWLJXHG PXVFOH Hf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f

PAGE 101

$FLGRVLV DQG WKH 7ZLWFK &RQWUDFWLRQ :KHQ DFLGRVLV ZDV LQGXFHG E\ K\SRYHQWLODWLRQ GHYHORSHG WHQVLRQ GLG QRW GHFUHDVH ,W LV OLNHO\ WKDW WKH LQWUDn FHOOXODU S+ UHDFKHG D OHYHO LQ WKHVH H[SHULPHQWV FRPSDUDEOH ZLWK WKDW ZKLFK ZDV UHDFKHG LQ WKH H[SHULPHQWV VXPPDUL]HG DERYH 7KLV VXJJHVWV WKDW WKH UHGXFHG LQWHQVLW\ RI DFWLYDWLRQ REVHUYHG DV D UHVXOW RI IDWLJXLQJ FRQWUDFWLRQV ZDV QRW D UHVXOW RI LQWUDFHOOXODU DFLGRVLV

PAGE 102

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f 7KH DPRXQW RI &D ZLWKLQ HDFK PXVFOH FHOO PD\ EH XQDOWHUHG LQ IDWLJXH 8QGHU WKHVH FLUFXPVWDQFHV WKH DPRXQW RI &DAW DYDLODEOH WR WKH ODWHUDO VDFV PD\ EH OLPLWHG LI VRPH RUJDQHOOH VHTXHVWHUV &D ,W KDV EHHQ UHSRUWHG WKDW PLWRFKRQGULD DFFXPXODWH &D GXULQJ H[HUFLVH

PAGE 103

f ,W LV FRQFHLYDEOH WKDW FDOFLXP DFFXPXODWLRQ E\ PLWRFKRQGULD PD\ UHGXFH WKH DPRXQW RI &D DYDLODEOH IRU UHF\FOLQJ LQ WKH VDUFRSODVPLF UHWLFXOXP 7KLV FRXOG UHGXFH WKH DPRXQW RI &DA UHOHDVHG ZLWK HDFK LPSXOVH 2WKHU RUJDQHOOHV PD\ DOVR VHTXHVWHU &D OLPLWLQJ WKH DPRXQW RI &D DYDLODEOH WR WKH VDUFRSODVPLF UHWLFXOXP 7KH ORQJLWXGLQDO WXEXOHV DUH NQRZQ WR DFFXPXODWH &D f ,W LV XQOLNHO\ WKDW WKLV DFFXPXODWLRQ ZRXOG SHUPLW WKH REVHUYHG SHUVLVWHQFH RI IDWLJXH $SSDUHQWO\ WKH WLPH QHFHVVDU\ IRU WUDQVORFDWLRQ RI WKH &D IURP WKH ORQJLWXGLQDO UHWLFXOXP WR WKH ODWHUDO VDFV LV PXFK VKRUWHU f WKDQ WKH REVHUYHG PLQXWHV RI UHFRYHU\ GXULQJ ZKLFK QR VLJQLILFDQW LQFUHDVH LQ $7 RFFXUUHG &RPSDUWPHQWDOL]DWLRQ :LWKLQ WKH /DWHUDO 6DFV $ UHGXFHG &D UHOHDVH IURP ODWHUDO VDFV ZRXOG RFFXU LI &D ZHUH FRPSDUWPHQWDOL]HG ZLWKLQ WKH ODWHUDO VDFV LQ D PDQQHU ZKLFK PDGH LW XQDYDLODEOH IRU UHOHDVH LHI ERXQG YV IUHHf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

PAGE 104

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f DW D JLYHQ IUHH &D FRQFHQWUDWLRQ

PAGE 105

&21&/86,216 ,W LV DSSDUHQW WKDW IXUWKHU UHVHDUFK ZLOO EH QHFHVVDU\ WR HOXFLGDWH WKH PHFKDQLVP FDXVLQJ WKH IDWLJXH REVHUYHG LQ WKH H[SHULPHQWV UHSRUWHG KHUHLQ 7KLV UHVHDUFK KRZHYHU KDV HOLPLQDWHG VHYHUDO SURSRVHG PHFKDQLVPV RI IDWLJXH $ IHZ K\SRWKHVHV DUH SUHVHQWHG ZKLFK PD\ FRQWULEXWH WR WKH IDWLJXH REVHUYHG LQ WKHVH H[SHULPHQWV (DFK RI WKHVH K\SRWKHVHV LV EDVHG RQ D FHQWUDO WKHPH D UHGXFHG DFWLYDWLRQ RI WKH FRQWUDFWLOH SURWHLQV ,W UHPDLQV WR EH VHHQ ZKLFK RI WKHVH PHFKDQLVPV LI DQ\f LV WKH DFWXDO FDXVH RI WKH WZLWFK IDWLJXH REVHUYHG LQ WKHVH H[SHULPHQWV

PAGE 106

$33(1',; 0HWKRG IRU 3KRVSKRU\OFUHDWLQH $QDO\VLV 7KH PHWKRG RI (QQRU DQG 6WRFNHQ f ZDV XVHG WR GHWHUPLQH SKRVSKRU\OFUHDWLQH FRQWHQW RI PXVFOH VDPSOHV 7KLV PHWKRG GHWHUPLQHV FUHDWLQH FRQWHQW EHIRUH DQG DIWHU D SHULRG RI DFLG K\GURO\VLV RI SKRVSKRU\OFUHDWLQH 7KH NH\ UHDFWLRQ IRU WKLV GHWHUPLQDWLRQ LV FUHDWLQH ZLWK GLDFHW\O ZKLFK \LHOGV D SLQN FRORUHG FRPSRXQG 7KH LQWHQVLW\ RI WKH SLQN FRORU LV SURSRUWLRQDO WR FUHDWLQH FRQFHQWUDWLRQ ZKHQ GLDFHW\O LV DYDLODEOH LQ H[FHVV 7KH RSWLFDO GHQVLW\ LV GHWHUPLQHG ZLWK D VSHFWURSKRWRPHWHU DW QP &KHPLFDOV DQG 5HDJHQWV IRU 'HSURWHLQL]DWLRQ DQG 'HWHUPLQDWLRQ 3HUFKORULF $FLG VWRFN b %DNHUf Df 0 LQ b HWKDQRO Ef 0 QR HWKDQROf ,, 3RWDVVLXP &DUERQDWH %DNHUf 0 FRQWDLQLQJ 0 WULHWKDQRODPLQH %DNHUf ,,, 6RGLXP +\GUR[LGH 0DOLQNURGWf Df 1 Ef 1 ,9 +\GURFKORULF DFLG )LVKHUf Df ,1 Ef 1 9 S &KORURPHUFXULF DFLG 6LJPDf DOVR FDOOHG S +\GUR[\PHUFXULF DFLGf 0 SXUFKDVHG DV VROXWLRQ RU DV SRZGHU WKHQ GLVVROYHG LQ 1D2+ 1f WKHQ PDGH WR YROXPH ZLWK +2 9, 1DSKWKRO 6LJPDf b LQ VWRFN DONDOL PL[ QHZ GDLO\

PAGE 107

9,, 'LDFHW\O 6LJPDf Df VWRFN VROXWLRQ b Ef PL[ GDLO\ IRU XVH LQ DVVD\ 9,,, 6WRFN $ONDOL IRU OLWHU J 1D&&! %DNHUf DQG J 1D2+ 0DOLQNURGWf ,; &UHDWLQH 6LJPDf IRU VWDQGDUGVf \JPO 3URFHGXUH IRU 'HSURWHLQL]DWLRQ 0XVFOH VDPSOHV ZHLJKLQJ PJ ZHUH IUR]HQ LQ VLWX ZLWK PHWDO FODPSV SUHFRROHG LQ OLTXLG 1r 7KH PXVFOH VDPSOHV ZHUH NHSW IUR]HQ XQWLO WKH\ ZHUH KRPRJHQL]HG LQ 3HUFKORULF DFLG 7KH VDPSOHV ZHUH KRPRJHQL]HG 9HUWLVf IRU VHFRQGV DW KLJK VSHHG LQ PO RI LFH FROG 0 3HUFKORULF DFLG 7KH KRPRJHQDWH ZDV SRXUHG LQWR D FHQWULIXJH WXEH POf DQG WKH EODGHV DQG ERZQ ZHUH ULQVHG ZLWK PO RI 0 3HUFKORULF DFLG 7KH 0 3HUFKORULF DFLG ZDV UHWDLQHG LQ D VHSDUDWH FHQWULIXJH WXEH 7KH KRPRJHQDWH ZDV VSXQ GRZQ DW J IRU PLQXWHV DW r& 7KH VXSHUQDWDQW ZDV VDYHG DQG WKH SHOOHW ZDV UHVXVSHQGHG LQ WKH PO ULQVH 0 3HUFKORULF DFLGf 7KLV VXVSHQVLRQ ZDV UHFHQWULIXJHG DW J IRU PLQXWHV DQG WKH VXSHUQDWDQWV ZHUH FRPELQHG 7KH FRPELQHG VXSHUQDWDQW ZDV QHXWUDOL]HG WR S+ ZLWK DGGLWLRQ RI 0 1D&&! 7KLV ZDV DGGHG GURSZLVH ZLWK FRQVWDQW PL[LQJ WR DYRLG EXEEOLQJ RYHU 6DPSOHV ZHUH VXEVHTXHQWO\ FHUWULIXJHG DW URRP WHPSHUDWXUH LQ D GHVNWRS FHQWULIXJH DW PD[LPDO USP IRU PLQXWHV 7KH YROXPH RI WKH VXSHUQDWDQW ZDV GHWHUPLQHG DQG WKH

PAGE 108

VXSHUQDWDQW ZDV VWRUHG LQ D IUHH]HU XQWLO IXUWKHU DQDO\VLV ZDV GRQH XVXDOO\ VDPH GD\ EXW D GHOD\ RI GD\V PDGH QR GLIIHUHQFHf 'HWHUPLQDWLRQ RI )UHH DQG 7RWDO &UHDWLQH 'XSOLFDWH DQDO\VLV IRU IUHH DQG WRWDO FUHDWLQH ZHUH PDGH RQ HDFK VDPSOH DOLTXRWV SHU VXSHUQDWDQWf 6WDQGDUGV DOVR LQ GXSOLFDWH ZHUH UXQ ZLWK HDFK GHWHUPLQDWLRQ )ROORZLQJ LV D OLVW RI WKH SURFHGXUHV IRU IUHH DQG WRWDO GHWHUPLQDWLRQ 1HXWUDOL]HG VDPSOHV WR S+ ZLWK ,1 1D2+ DQG ,1 +& LI QHHGHGf 0HDVXUH DOLTXRWV POf RI HDFK VXSHUQDWDQW LQWR JUDGXDWHG WXEHV $GG +2 WR PO PDUN 3ODFH RI f WXEHV LQ KRW ZDWHU EDWK r&f WR HTXLOLEUDWH $GG PO RI 1 +& WR WXEHV ZKLFK KDYH HTXLOLEUDWHG WR r& 5HSODFH LQ KRW ZDWHU EDWK IRU PLQXWHV 7KHVH DUH WXEHV IRU WRWDO FUHDWLQH GHWHUPLQDWLRQ 5HPRYH WKH WXEHV IURP WKH EDWK DQG DGG PO RI 1 1D2+ 3ODFH LQ LFH EDWK WR FRRO TXLFNO\ WR URRP WHPSHUDWXUH 7R HDFK RI WKH IRXU WXEHV IUHH DQG WRWDOf DGG VHTXHQWLDOO\ PO RI S K\GUR[\PHUFXULEHQ]RDWH PO RI 1DSKWKRO DQG PO GLDFHW\O 0DNH YROXPH WR PO DJLWDWH DQG SODFH LQ GDUN IRU PLQXWHV 5HDG RSWLFDO GHQVLW\ DW QP 6WDQGDUGV UHFHLYH WKH VDPH WUHDWPHQW DV WKH VDPSOHV IRU IUHH FUHDWLQH EODQN GRHV QRW JHW PO S K\GUR[\n PHUFXULEHQ]RDWH &UHDWLQH FRQWHQW RI VDPSOHV LV

PAGE 109

GHWHUPLQHG IURP WKH UHJUHVVLRQ OLQH REWDLQHG IURP WKH VWDQGDUGV 3KRVSKRU\OFUHDWLQH FRQWHQW LV WRWDO FUHDWLQH IUHH FUHDWLQHfWRWDO FUHDWLQH

PAGE 110

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

PAGE 111

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f SS 3UDJXH 3XEOLFDWLRQ +RXVH &]HFKROVRYDN $FDG 6FL (GZDUGV 5 + 7 DQG +LOO (FRQRP\ RI IRUFH PDLQWHQDQFH GXULQJ HOHFWULFDOO\ VWLPXODWHG LVRPHWULF FRQWUDFWLRQV RI KXPDQ PXVFOH 3K\VLRO 33 (GZDUGV 5 + 7 +LOO DQG $ -RQHV +HDW SURGXFWLRQ DQG FKHPLFDO FKDQJHV GXULQJ LVRPHWULF FRQWUDFWLRQV RI WKH KXPDQ TXDGULFHSV PXVFOH 3K\VLRO

PAGE 112

(GZDUGV 5 + 7 +LOO DQG $ -RQHV 0HWDEROLF FKDQJHV DVVRFLDWHG ZLWK WKH VORZLQJ RI UHOD[DWLRQ LQ IDWLJXHG PRXVH PXVFOH 3K\VLRO (GZDUGV 5 + 7 +LOO $ -RQHV DQG 3 $ 0HUWRQ )DWLJXH RI ORQJ GXUDWLRQ LQ KXPDQ VNHOHWDO PXVFOH DIWHU H[HUFLVH 3K\VLRO (OOLV DQG ) &DUSHQWHU 6WXGLHV RQ WKH PHFKDQLVP RI DFWLRQ RI GDQWUROHQH VRGLXP $ VNHOHWDO PXVFOH UHOD[DQW 1DXQ\QVFKPHGHEHUJnV $UFK 3KDUPDFRO (QQRU $ + DQG / $ 6WRFNHQ (VWLPDWLRQ RI FUHDWLQH %LRFKHP )DELDWR $ DQG ) )DELDWR (IIHFWV RI S+ RQ WKH P\RILODPHQWV DQG WKH VDUFRSODVPLF UHWLFXOXP RI VNLQQHG FHOOV IURP FDUGLDF DQG VNHOHWDO PXVFOHV 3K\VLRO )DOHV 7 6 5 +HLVH\ DQG / =LHUOHU 'HSHQGHQF\ RI R[\JHQ FRQVXPSWLRQ RI VNHOHWDO PXVFOH RQ QXPEHU RI VWLPXOL GXULQJ ZRUN LQ WKH GRJ $P 3K\VLRO )HQJ 7 3 7KH KHDWWHQVLRQ UDWLR LQ SURORQJHG WHWDQLF FRQWUDFWLRQV 3URF 5R\ 6RF /RQGRQf % )LWWV 5 + DQG +ROORV]\ /DFWDWH DQG FRQWUDFWLOH IRUFH LQ IURJ PXVFOH GXULQJ GHYHORSPHQW RI IDWLJXH DQG UHFRYHU\ $P 3K\VLRO )LWWV 5 + DQG +ROORV]\ &RQWUDFWLOH SURSHUWLHV RI UDW VROHXV PXVFOH HIIHFWV RI WUDLQLQJ DQG IDWLJXH $P 3K\VLRO &HOO && )UHWWKROG : DQG / & *DUJ 7KH HIIHFW RI DFLGEDVH FKDQJHV RQ VNHOHWDO PXVFOH WZLWFK WHQVLRQ &DQ 3K\VLRO 3KDUPDFRO )XFKV ) 9 5HGG\ DQG ) 1 %ULJJV 7KH LQWHUn DFWLRQ RI FDWLRQV ZLWK WKH FDOFLXP ELQGLQJ VLWH RI WURSRQLQ %LRFKLP %LRSK\V $FWD )XUXVDZD DQG 5 0 7 .HUULGJH 7KH K\GURJHQ LRQ FRQFHQWUDWLRQ RI WKH PXVFOHV RI WKH FDW 3K\VLRO

PAGE 113

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f 0HGLFDO 3K\VLRORJ\ WK (GLWLRQ 6W /RXLV & 9 0RVE\

PAGE 114

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

PAGE 115

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
PAGE 116

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

PAGE 117

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nfnO 3 3RVQHU $VVRFLDWH 3URIHVVRU RI 3K\VLRORJ\

PAGE 118

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 3URIHVVRU RI %LRFKHPLVWU\ FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ &a :7 ]IDXQHU 3URIHVVRU RI 3URIHVVLRQDO 3K\VLFDO (GXFDWLRQ 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI 0HGLFLQH DQG WR WKH *UDGXDWH &RXQFLO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHn PHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ -XQH 'HDQ &ROOHJH RI 0HGLFLQH 'H£A9

PAGE 119

P I ""


m / 52 f
/?7?