A dissertation presented. Jennifer A. Carr. To The Department of Biology. in the field of. Biology

Transcription

1 Muscle Function During Swimming and Running in Aquatic, Semi-Aquatic and Cursorial Birds A dissertation presented by Jennifer A. Carr To The Department of Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Biology Northeastern University Boston, Massachusetts January 2008

2 Muscle Function During Swimming and Running in Aquatic, Semi-Aquatic and Cursorial Birds ii A dissertation presented by Jennifer A. Carr ABSTRACT OF DISSERTATION Submitted n partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology in the Graduate School of Arts and Sciences of Northeastern University, January 2008

3 Abstract iii The function of large hindlimb muscles with long fascicles and complicated architecture has been studied in many organisms specialized for terrestrial locomotion. Studies of muscle function, energetics and blood flow have allowed a to more accurate determination of which muscles play an important roles during running. One muscle that has been hypothesized to play such a role in birds is the Iliotibialis Lateralis pars Post Acetabularis (ILPO). The ILPO is a large muscle in the hindlimb of cursorial birds, receives a large proportion of the blood flow during running at high speeds, and tends to be reduced or lost in birds that do not employ walking or running as their primary form of locomotion. One of the goals of this study was to comprehensively characterize the function of the ILPO, taking into account the ILPO s long fascicles that vary in length, the muscle s complex origin and insertion, and its anatomical variations seen across different bird orders. In order to characterize the ILPO s function during running, length changes and electrical activity were measured along the posterior fascicle of the ILPO and between the anterior and posterior fascicle of the ILPO in the Helmeted Guinea Fowl (Numida meleagris). These results were compared to those predicted by a moment arm and kinematics model developed in the guinea fowl. To determine how the ILPO functions during other types of locomotion in species that are not specialized for cursorial locomotion, the function of the ILPO was measured during swimming and running in the Common Moorhen (Gallinula chloropus), a bird that employs both swimming and terrestrial forms of locomotion, and the Mallard (Anas platyrhynchus), a bird that while capable of terrestrial locomotion locomotes primarily via surface swimming. Finally,

4 the function of another muscle was measured in the mallard that, based on blood flow iv and anatomical studies, appears to play an important role during swimming, the Flexor Cruris Lateralis (FCL). The FCL, like the ILPO, is a large muscle with a complex origin and insertion in cursorial locomotors. However unlike the ILPO this muscle does not appear to be reduced or absent in birds that locomote via swimming: instead it appears to have been significantly modified in birds that employ swimming as a primary form of locomotion. Our results demonstrate that, when active, the ILPO in the guinea fowl experiences similar amounts of length change and has the same average velocity during active lengthening and active shortening between fascicles. It is hypothesized that the varying moment arm at the hip as well as the tendinous aponeurosis play a role in maintaining a uniform amount of active strain among fascicles of different lengths. Along a fascicle, the strain in the ILPO was uniform in the different segments of the posterior ILPO during the active shortening part of the length change cycle and differential strain was found to occur between proximal, central and distal segments during active lengthening, passive lengthening and passive shortening. These proximal to distal differences may be caused by different amounts of length change when the ILPO is being passively shortened followed by compensatory length-tension effects during the period when the ILPO is being actively lengthened. In the common moorhen and mallard, the ILPO s function during terrestrial locomotion appears to be relatively conserved and a similar strain pattern during cursorial locomotion is observed in all three species. Although the ILPO is active during swimming in both the mallard and the common moorhen, our results suggest that the

5 ILPO does not play a large functional role during swimming in either species. Our v results show that although the ILPO is active in the common moorhen during swimming, it experiences less strain than it does during running. Our measurements of ILPO function in a swimming mallard show that there is a significant decrease in both strain and electrical activity. Finally, there is no significant effect of speed on emg activity of the ILPO during swimming in either the common moorhen or mallard, indicating that although the limb as a whole must produce more work to go faster, it is not producing the extra work needed using the ILPO in either species of swimming birds. Based on our results, the FCL of the mallard appears to function in both terrestrial locomotion and during swimming. During running, the FCL actively shortens during the stance phase, producing positive work which likely counteracts cocontracting knee extensors and driving knee flexion. During swimming the FCL is actively lengthened, and later isometric, for a large portion of the swim cycle. When the FCL is isometric it is possible that the FCL is holding the knee and hip in a flexed position and decreasing the amount of drag on the mallard by increasing the streamlined appearance of the body. This research demonstrates how muscles can function in different types of locomotion and in different species during the same type of locomotion. The variations observed within the ILPO and the FCL in terms of both structure and function demonstrates the importance of muscles functioning to produce positive work and demonstrates how muscles play an important role in stability during both swimming and running either by remaining isometric or by being lengthened while active.

6 Acknowledgements vi I would like to begin by acknowledging my advisor Dr. Richard Marsh, without his support and knowledge these experiments would not have been possible, through his guidance I have become a better scientist and a better teacher. I would like to thank my committee members Gwilym Jones, Fred Davis, Rebeca Rosengaus and Thomas Roberts who have always been available for assistance, for moral support and encouragement. This dissertation would not have been possible without the support of my coworkers in the lab both past and present who assisted with surgeries, bird care and experiments including David Ellerby, Jonas Rubenson, Havalee Henry, Thomas Hoogendyk, Gwenn Catterfeld, Rosemary Truong, Amanda Flynn, Jade McPherson and Matthew Propert. I would like to thank my family and friends who have been there supporting me throughout my thesis project especially my mom who made me go back to school, Kris Severi, Jessica Gonyor and Nathan McDaniel. Finally I would like to thank the person who was an invaluable source of love and support my husband Francis (Igor) Carr, who helped me deal with both the joys and frustrations associated with this project and kept me focused on what was important in life.

7 Table of Contents vii Table of Contents Abstract Acknowledgements Table of Contents List of Abbreviations List of Figures iii vi vii xiii xiv Chapter 1 Differential Strain in an Active Muscle During Locomotion I. Introduction 1 II. Materials and Methods A. Animals and Training 4 B. Muscle Architecture 4 C. Surgery 5 D. Recordings 6 E. Videography 6 F. Myofilament Length and Length-Tension Curve 7 G. Sarcomere Measurements 8 H. Length and Velocity Changes 9 I. Statistical Analysis 11 III. Results

8 A. Length Changes 13 viii B. Velocity Changes 14 C. Electromyography 14 D. Predicted Sarcomere Operating Lengths 15 IV. Discussion A. Length Changes and Electrical Activity in the ILPO 16 B. Alternative Hypothesis for Differential Strain 17 C. Length Tension Effects 19 D. Summary 20 V. Literature Cited 22 Chapter 2 Compensatory Mechanisms in a Muscle with Varying Moment Arms I. Introduction 33 II. Materials and Methods A. Animals and Training 39 B. Moment Arm Measurements 39 C. Statistical Analysis 41 III. Results

9 A. Length and Velocity Changes 43 ix B. Moment Arm Measurements 43 C. Calculated vs. Experimental Length Changes 44 IV. Discussion 46 VI. Literature Cited 51 Chapter 3 Iliotibialis Lateralis pars Post Acetabularis (ILPO) Function in Helmeted Guinea Fowl (Numida meleagris) I. Introduction 62 II. Materials and Methods A. Animals and Training 66 B. Sonomicrometry and Electromyography 66 C. Recordings 67 D. Videography 68 E. Sarcomere Measurements 69 F. Length and Velocity Changes 70 G. Blood Flow versus Emg Measurements 72 H. Statistical Analysis 73 III. Results

10 A: Muscle Length Change 74 x B. Velocity Changes as a Function of Speed and Incline 75 C. Sarcomere Measurements 75 D. Electrical Activity 76 E. Blood Flow versus Emg Comparison 76 IV. Discussion A. Function of Active Lengthening in the ILPO 79 B. Function of the ILPO During Active Shortening 82 C. Length Tension Effects 82 D. Emg Analysis versus Blood Flow 84 E. Summary 84 V. Literature Cited 86 Chapter 4: Iliotibialis Lateralis pars Post Acetabularis (ILPO) Function During Swimming and Running in an Aquatic and Semi-Aquatic Bird Species I. Introduction 105 II. Materials and Methods A. Animals and Training 110 B. Surgery 111

11 C. Recordings 112 xi D. Videography 112 E. Sarcomere Measurements 113 F. Length Changes 115 G. Statistical Analysis 119 III. Results A. ILPO Length Changes During Running & Swimming 120 B. ILPO Electrical Activity During Running & Swimming 122 IV. Discussion A. ILPO Function During Running 125 B. Length and Electrical Activity Changes During Swimming 127 C. Conclusion 129 V. Literature Cited 131 Chapter 5: A Swimming Muscle with a Novel Function I. Introduction 146 II. Materials and Methods A. Animals and Training 151 B. Surgery 151

12 C. Recordings 152 xii D. Videography 153 E. Sarcomere Measurements 154 F. Length Changes 155 G. Statistical Analysis 159 III. Results A. General Pattern of Length Change and Electrical Activity 160 B. Length Change in the FCL 160 C. Electrical Activity in the FCL 161 D. Swimming and Running Kinematics 161 IV. Discussion A. Function of the FCL During Running 163 B. FCL Function During Swimming 164 C. Summary 166 V. Literature Cited 167

13 List of Abbreviations xiii EMG FCL FCLP FCLA FCM FT IC IF ILPO L 0 L L t V Electromyography Flexor Cruris Lateralis Flexor Cruris Lateralis pars Pelvica Flexor Cruris Lateralis pars Accessoria Flexor Cruris Medialis Femerotibialis Iliotibialis cranialis Iliofibularis Iliotibialis Lateralis pars Post Acetabularis Optimal length Length Change in length Time Velocity

14 List of Figures xiv Figure 1 Posterior ILPO Sonomicrometry Crystal Placement 25 Figure 2 Proximal, Central, Distal Length Changes 26 Figure 3 Length Change versus Speed in the Posterior ILPO 27 Figure 4 Average Velocity in the Posterior ILPO 28 Figure 5 Emg Start and Stop Times in the Posterior ILPO 29 Figure 6 Average and Integrated Emg s in the Posterior ILPO 30 Figure 7 Length-Tension Curve in the Posterior ILPO 31 Figure 8 Sarcomere Length versus Speed in the Posterior ILPO 32 Figure 9 Anterior and Posterior ILPO Crystal Placement 53 Figure 10 Moment Arm Measurement Setup 54 Figure 11 Anterior and Posterior Length Changes 55 Figure 12 Strain versus Speed in the Anterior and Posterior ILPO 56 Figure 13 Average Velocity in the Anterior and Posterior ILPO 57 Figure 14 Moment Arm at the Hip of the ILPO 58 Figure 15 Moment Arm at the Knee of the ILPO 59 Figure 16 Joint Angles in the Helmeted Guinea Fowl 60 Figure 17 Calculated versus Actual Strain in the ILPO 61 Figure 18 Superficial Muscles of the Helmeted Guinea Fowl 93 Figure 19 ILPO Reduction or Elimination 94 Figure 20 Length Change in the Anterior and Posterior ILPO 95 Figure 21 Active Strain in the ILPO on the Level and on an Incline 96 Figure 22 Passive Strain in the ILPO on the Level and on an Incline 97

15 Figure 23 Average Velocity on the Level and on an Incline 98 xv Figure 24 Sarcomere Length versus Speed on the Level and Incline 99 Figure 25 Average emg and integrated emg values 100 Figure 26 Emg Duration, Start and Stop Times 101 Figure 27 ILPO Oxygen Consumption and Emg Activity 102 Figure 28 Joint Kinematics during Walking and Running 103 Figure 29 Length-Tension Curve on the Level and Incline 104 Figure 30 Crystal Placement in Moorhen & Mallard 137 Figure 31 ILPO Presence, Absence of Reduction 138 Figure 32 Average Length Change in Guinea Fowl, Moorhen & Mallard 139 Figure 33 Average Length Change During Swimming 140 Figure 34 Active Strain During Running and Swimming in Moorhen 141 Figure 35 Active Strain During Running and Swimming in Mallard 142 Figure 36 Average emg and Integrated emg in Common Moorhen 143 Figure 37 Average emg and Integrated emg in Mallard 144 Figure 38 Joint Kinematics in Common Moorhen and Mallard 145 Figure 39 FCLA, FCLP Presence or Absence 173 Figure 40 FCL Anatomy in the Mallard & Helmeted Guinea Fowl 174 Figure 41 FCL in Mallard with Sonomicrometry and Emg Placement 175 Figure 42 FCL Length Change During Running and Swimming 176 Figure 43 Active Strain in the FCL During Running and Swimming 177 Figure 44 Average emg and Integrated emg in the FCL 178 Figure 45 Emg duration, start time and stop time in the FCL 179

16 xvi Figure 46 Joint Kinematics During Running and Swimming in the Mallard 180 Figure 47 Average Maximum Continuous Angular Changes 181

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18 Chapter 1: Differential Strain in an Active Muscle During Locomotion 1 Introduction When conducting in vitro and in vivo studies, many researchers assume that all portions of the muscle experience a uniform strain pattern (Huijing, 1999; McGowan et al., 2006; Zajac, 1989). However differential strain among fascicles in parallel, and along fascicles in individual muscles, has been reported. Differential strain along a fascicle has been previously reported in the semitendinosus muscle of the American Toad (Bufo americanus) where individual segments along the fascicle showed different amounts of strain (Ahn et al., 2003). Differential strain among fascicles in parallel has been measured in the iliofibularis in the Helmeted Guinea Fowl (Numida meleagris) during running, and in the biceps brachii in humans during elbow flexion (Hoogendyk et al., 2005; Pappas et al., 2002). Differential strain both along a single fascicle and among fascicles in parallel has the potential to affect many hypotheses of muscle function. If differential strain within a muscle is not accounted for then it can affect calculations of the amount of force produced by the muscle due to length-tension or force-velocity affects. Differential strain, if not accounted for, can also result in an overestimation or underestimation of the amount of work being produced or absorbed by a muscle depending on whether the actual length change across the whole muscle is greater or less than the amount occurring in the particular segment being measured. In studies where differential strain has been demonstrated, many hypotheses have been put forward as to why this phenomenon occurs. Differences in strain amplitude seen along a fascicle of an individual muscle have been hypothesized to occur due to

19 differences in individual muscle properties. Differential strain along fascicles in the 2 toad semitendinosus are hypothesized to occur because individual segments of the muscle are operating at different regions of their force-length and force-velocity relationships (Ahn et al., 2003). Differential strain among fascicles in parallel has in some cases been attributed to differences in muscle architecture and differences in the moment arm of individual fibers within a single muscle. For example differential strain seen between the anterior and posterior fascicles of the iliofibularis is hypothesized to be influenced by differences in the moment arm at the hip. In the biceps brachii, differential strain is thought to be caused by fascicle interactions with an internal aponeurosis during shortening (Hoogendyk et al., 2005; Pappas et al., 2002). The goals of my study were to determine whether differential strain occurs along a single fascicle and eliminating effects due to differences in muscle architecture architecture between fascicles, and (if differential strain is observed) to determine why it occurs along the fascicle. To eliminate differences in the moment arms or architectural differences in the point of origin or insertion, I measured strain in proximal, central and distal positions along a single fascicle in the Iliotibialis Lateralis pars Post Actabularis (ILPO) in Helmeted Guinea Fowl (Numida meleagris). I chose to study the ILPO in guinea fowl for several reasons. Guinea fowl are a commonly-used experimental species for locomotion studies due to their size and their running ability. The ILPO has long ( mm) posterior fascicles that can be implanted with multiple sonomicrometry crystals and emg electrodes to measure length changes and electrical activity. The ILPO is the largest single muscle in the hindlimb of the guinea fowl, accounting for 12.88±0.64% of the total muscle mass in the hindlimb, and receiving more than 12.27% of the total

20 hindlimb blood flow during running at 2.5m*s -1 (Ellerby et al., 2005; Marsh et al., ). The function of the ILPO during level, uphill and downhill running has been examined in two studies in guinea fowl (Buchanan, 1999; McGowan et al., 2006) and one study in turkeys (Roberts et al., 2007), however these studies measured the function of the ILPO in only a portion of the fascicle, and assumed that the observed length changes were uniform across the fascicle. The goal of my study was to test this assumption. I hypothesized that strain would be uniform along the posterior fascicles of the guinea fowl. Along the posterior fascicle of the ILPO in-series fibers have the same moment arm at the hip and knee and there is no internal aponeurosis to affect function of different portions of the fascicle. I tested my hypothesis by implanting the posterior ILPO with four sonomicrometry crystals to measure length change (see Fig. 1). The sonomicrometry crystals were implanted so that they spanned the majority of the fascicle and divided the fascicle into proximal, central and distal segments. I implanted emg s to measure electrical activity. Using post-mortem measurements of sarcomere length compared to sonomicrometer segment length, I normalized my length measurements (taken via sonomicrometry) to sarcomere lengths.

21 4 Materials and Methods Animals and Training Five guinea fowl averaging 1.543±0.08 kg body mass were used for this experiment. The guinea fowl were housed in individual cages at the Northeastern University Animal Care Facility, with food and water provided ad libitum on a 10:14 light-dark cycle. Prior to surgery and experimental recordings, the birds were trained to run inside a three-sided box on a motorized treadmill. The birds were trained 3-5 days per week for a minimum of five weeks. Each training session lasted for 30 minutes. After training the birds could, in an individual session, maintain level speeds of 2.5 ms -1 for 15 minutes, 2.78 ms -1 for 3 minutes, and 3.0 ms -1 for 3 minutes. Once a bird was sufficiently trained, surgery was performed to insert sonomicrometry and emg sensors as described below. After surgery, birds were allowed to recover in individual cages for at least 40 hours prior to any experimental recordings. After recovery, birds were run on the treadmill and experimental recordings were taken as described below. Birds were run at speeds of 0.5, 1.0, 1.5, 2.0, 2.5, 2.78 and 3.0 ms -1 on a level incline for at least 30 seconds per speed. Each bird was sacrificed after experimental recordings were taken. Muscle Architecture The ILPO originates on the dorsolateral iliac crest, the terminal iliac process and the posterior ischium; it inserts onto the patella, its tendon and the knee joint capsule (Gatesy, 1999). The ILPO covers the lateral caudal surface of the thigh. I chose to implant the long posterior fascicles because they insert close to the knee joint and had much less in-series elastic tissue than did the anterior fascicles. The posterior fascicles in

22 which crystals were implanted were 82.62±3.37 mm long. The crystals implanted into 5 the posterior fascicles spanned 73.19±3.12% of the total fascicle length. Surgery All transducers were sterilized (12 hour ethylene oxide treatment) and implanted using sterile surgical techniques under general anesthesia (isofluorane). Four custommade 1-mm sonomicrometry transducers made from PZT discs (Boston Piezo-Optics, Inc.) and attached to stainless steel holders (Gatesy, 1999; Olson and Marsh, 1998) were implanted along posterior fascicles of the ILPO, thus dividing the fascicles into proximal, central and distal segments (Fig. 1). Small punctures, approximately 1 mm in length, were created in the connective tissue overlying the muscle to a depth of approximately 3mm. The crystals were then placed between the fascicles. The holders were secured to the overlying connective tissue using 6-0 silk sutures. Care was taken to ensure that the four crystals were always implanted between the same fascicles along the length of the muscle. Two fine-wire bipolar emg electrodes were implanted into each segment of the fascicles to measure muscle activity. The emg electrodes were constructed from 3T stainless steel Teflon-coated wire (Medwire) that was twisted along its length. The tips of the electrodes were bared 1.5 mm at each end and the preactive ends spaced 3 mm apart. The emg electrodes were implanted in the ILPO with a chamfered 25 gauge needle at positions that were immediately anterior and posterior to each segment. Post mortem examination of the implanted limb verified the placement of the sonomicrometry crystals and emg electrodes.

23 Recordings 6 Sonomicrometry measurements were acquired digitally with a sampling frequency of 613 Hz using Sonoview software and a Sonometrics TRX Series 8 interface. The emg signals were amplified by WPI model DAM-50 preamplifiers with highand low-pass filters set at 10 and 3000 Hz, respectively. The amplified and filtered signals were collected at a frequency of 10kHz using an ADI Instruments Power Lab/16SP and a Macintosh PowerMac. The emg data were subsequently analyzed using the application Igor Pro (version 5.0). The signals were first filtered using a finite impulse response filter with a band pass of 90 Hz to 1000 Hz. Emg signals were rectified and the start time, stop time, and emg duration were all measured. The burst amplitudes were expressed in three ways: the integrated value of the burst duration; the average emg per burst (equal to the integrated value divided by the burst duration); and the average emg per stride (equal to the integrated value divided by the stride duration). Videography Video was recorded to VHS tape with a NAC HV-1000 high-speed video camera operating at 500 fields per second. I synchronized the emg and sonomicrometry traces with the high-speed video recordings using a square-wave generated by my emg recording system which was recorded in the video fields with a NAC wave inserter. Visual markers were made using either white paint or reflective tape. These markers were placed above the caudal and cranial pelvis; on the hip, ankle, tarsometatarsalphalangeal and interphalangeal joints; and on the toe of the bird. The knee was not marked because skin movement makes surface markers unreliable for locating this joint. The knee position was calculated from the measured hip and ankle positions during

24 7 running and the distance from the hip to the knee and knee to the ankle measured postmortem. Data were collected for ten steady speed strides at each speed for each bird. The strides were analyzed for changes in muscle strain, muscle velocity and electrical activity. Myofilament Length and Length-Tension Curve An idealized sarcomere length-tension curve for the guinea fowl ILPO was estimated by measuring the myofilament lengths of the sarcomeres and applying assumptions based on previous single-fiber length-tension curves. The thin filament length has been estimated to be 2.26 µm [corrected from data in Buchanan (1999)]. Assuming a bare zone length of 0.2 µm on the thick filament, the plateau region of the length-tension curve was calculated to span µm. These measurements of thin filament lengths are similar to those measured in the domestic fowl (Littlefield and Fowler, 2002). Thick filament lengths in the ILPO of guinea fowl are 1.6µm (Buchanan, 1999), a value consistent with most measurements of vertebrate skeletal muscle thick filaments (Gordon et al., 1966b; Littlefield and Fowler, 2002). Based on these measurements I predict that the upper end of the descending limb would occur at a length of 3.86 µm where there is no longer any overlap of the thick and thin filaments. In addition, it is assumed that the shallow ascending limb transitions to the steep ascending limb at a length of 1.6 µm, the point at which the thick filaments would contact the z-line. Note that in the literature, the point at which the steep ascending limb falls to zero does not seem to correspond to any of the landmarks of the shortening process (Gordon et al., 1966a; Gordon et al., 1966b). However Gordon et al. found that the steep ascending limb

25 fell to zero at a value that was approximately 60% of L 0 (Gordon et al., 1966b). Based 8 on this finding, I assume that my steep ascending limb reaches zero at 1.39 µm. Sarcomere Measurements After each animal s experimental recordings were completed, the animal was sacrificed and the instrumented leg was bolted into place with the hip and knee in a flexed position. The limb was allowed to sit in a bolted position for six hours until the leg muscles were in full rigor. Once the fixed limb was in rigor, a reference sonomicrometry measurement (denoted L so, ref ) was measured. In addition, a muscle temperature measurement was taken to correct for temperature effects on the speed of sound. Two samples of the ILPO tissue, each measuring ~1 cm 3, were removed from between each crystal pair. These muscle samples were frozen using isopentane chilled in liquid nitrogen, and then sectioned using a cryostat. For each muscle sample, at least 10 sections were placed on slides and stained using Weingert Iron Hematoxylin (Humason, 1979). From each slide (containing one stained section), at least three fibers were photographed at 1000X magnification and a Nikon camera with a resolution of pixels per µm. Sarcomere lengths were measured using a compound microscope at 1000X magnification. Sarcomere measurements were performed using a digital imaging program (NIH Image J version 1.38). In Image J, a series of at least 10 sarcomeres was measured from each fiber, and the average individual sarcomere length (denoted L sc, ref ) was calculated by dividing the total length of the in-series sarcomeres by the number of sarcomeres in series. Using the reference sonomicrometry length and measured sarcomere length, in vivo sonomicrometry lengths (L so ) were converted to estimated in vivo sarcomere lengths (L sc ) using the equation:

26 9 L sc = L sc,ref L so L so,ref (1) Length measurements in this study are either reported as µm, or are converted to %L 0 using the following equation: %L 0 = 100 L sc 2.36 (2) where L sc is the calculated sarcomere length from equation 1, and 2.36 µm is the estimated sarcomere length in the middle of the plateau of maximum force of the calculated length-tension curve. Length and Velocity Changes Muscle segment lengths were measured from the sonomicrometry traces after the lengths in mm had been converted to mm. The length values measured were: L fd = minimum length of the ILPO, which occurred near the time of foot down L max,st = maximum length of the ILPO occurring during stance L min = minimum length of the ILPO, which occurred near the time of toe off L max,sw = maximum length of the ILPO occurring during swing L emg,on = length of the ILPO 25ms after the start of the emg activity L emg,off = length of the ILPO 25ms after the stop of the emg activity These measured lengths were used to calculate active lengthening, active shortening, passive lengthening and passive shortening. When calculating active lengthening and active shortening, I assumed an electromechanical delay of 25ms. I are not aware of studies done on the ILPO to confirm this assumption, but this time period falls within values measured for other muscles (Cavanagh and Komi, 1979; Gabriel and

27 10 Boucher, 1998; Moritani et al., 1987; Muraoka et al., 2004; Winter and Brookes, 1991; Zhou et al., 1995). The equation used to calculate a given length change was dependent on the timing of L emg,on and L emg,off relative to other lengths measured during a stride. Active lengthening ( L al ) was calculated using one of two equations. If L emg,on occurred before L fd then: If L emg,on occurred after L fd then: L al = L max,st - L fd (3) L al = L max,st L emg,on (4) Active shortening ( L as ) was calculated using one of two equations. If L emg,off occurred after L min then: If L emg,off occurred before L min then: L as = L max,st L min (5) L as = L max,st L emg,off (6) Passive lengthening ( L pl ) was calculated using one of two equations. If L emg,off occurred before L min then: If L emg,off occurred after L min then: L pl = L max,sw L min (7) L pl = L max,sw L emg,off (8) Passive shortening ( L ps ) was calculated using one of two equations. If L emg,on occurred after L fd then: If L emg,on occurred before L fd then: L ps = L min,sw L fd (9) L ps = L min,sw L emg,on (10)

28 All length changes are reported as %L 0 where L 0 is the middle of the plateau region of 11 the length tension curve. Average velocities were calculated during active lengthening and shortening and in each compartment and at each speed. Average velocities in Ls -1 were calculated by dividing the amount of active lengthening or active shortening (expressed as a fraction of L 0 ) that occurred during a stride by the duration of lengthening or shortening. Thus the velocity during active lengthening (V al ) is: V al = L al (2.36t al ) (11) where L al is the active lengthening from equation 3 or 4, 2.36µm is the length at the center of the plateau region of the length-tension curve, and t al is equal to the duration of active lengthening in seconds. Similarly, the velocity during active shortening (V as ) is: V as = L as (2.36t as ) (12) where L as is the active shortening from equation 5 or 6, 2.36µm is the length at the center of the plateau region of the length-tension curve, and t as is the duration of active shortening in seconds. Statistical Analysis Multivariate and single-variate analyses were conducted using the general linear model in the application SPSS (version 15.0 for Microsoft Windows) to test for significant differences between proximal, central and distal length changes, velocity changes and electrical activity variables. An animal identifier was entered as a factor in

29 the model and speed was entered as a covariate in the model: this removed the 12 variation among the individual animals and allowed analysis of the effect of speed on length and electrical activity changes in the different segments. As part of the analysis, multiple comparisons analyses were performed using a posthoc Bonferroni correction to determine whether there was a significant difference in length change, velocity changes, and electrical activity between the proximal, central and distal segments. All measurements are reported as a mean value plus or minus one standard error.

30 Results 13 Length Changes The proximal (Fig. 2A), central (Fig. 2B) and distal (Fig. 2C) segments of the posterior ILPO show the same general pattern of lengthening and activation during running. Electrical activity in the posterior ILPO starts in late swing and proceeds through most of the stance phase. During the stance phase, the posterior ILPO is initially lengthened while active, and then shortens in the latter half of the stance. During the swing phase, when the muscle is inactive, it is passively lengthened and then passively shortened. While the general pattern of length change was similar in all of the segments of the posterior ILPO, the amount of length change that occurred during some portions of the stride demonstrated significant differences as a function of position. Active lengthening (Fig. 3A) was significantly different between the proximal and distal segments (p < 0.001), and between the central and distal segments (p < 0.001) of the posterior ILPO. No significant difference in active lengthening occurred between the proximal and central segments (p = 1.00) of the posterior ILPO. Active shortening (Fig. 3B) demonstrated no significant effect of position (p = 0.621). Significant differences in passive shortening (Fig. 3D) were measured between the proximal and distal segments (p < 0.001), the distal and central segments (p < 0.001), and the proximal and central segments (p = 0.036). Significant differences in passive lengthening (Fig. 3C) were measured between the proximal and distal segments (p < 0.001) and between the central and distal segments (p < 0.001); no significant difference was found between the central and proximal segments (p = 0.354). Speed was found to have a significant effect on

31 active lengthening (p = 0.001) (Fig. 3A), active shortening (p < 0.001) (Fig. 3B), 14 passive lengthening (p < 0.001) (Fig. 3C), and passive shortening (p < 0.001) (Fig. 3D). Velocity Changes I found a significant effect on the average sarcomere velocity during active lengthening (Fig. 4A) of both speed (p < 0.001) and position (p < 0.001) in the proximal, central and distal portions of the posterior ILPO. Average velocity during active lengthening in the proximal section of the posterior ILPO was significantly different from the central (p = 0.003) and the distal (p < 0.001). I also found that the average velocity during active lengthening in the central segment was significantly different from the distal (p < 0.001). Average velocity during active shortening (Fig. 4B) showed a significant effect of speed (p < 0.001) but no effect of position (p = 0.282). Electromyography As a function of position, I found no significant differences in the emg start time (p = 0.23) and stop time (p = 0.957) relative to foot down (Fig. 5); emg duration (p = 0.549) (Fig. 5); average emg per burst (p = 0.765) (Fig. 6A); average emg per stride (p = 0.057) (Fig. 6B); or integrated emg (p = 0.062) (Fig. 6C). There was a significant effect of speed on emg start time (p < 0.001) and emg stop time (p < 0.001) relative to foot down. There was also a significant effect of speed (p < 0.001) on average emg per burst and average emg per stride. There was no significant difference as a function of speed in the integrated emg values (p = 0.444). I found that even though the average emg values per burst did increase with speed, the emg duration decreased so that the integrated emg values did not significantly change as a function of speed. I did find a significant effect

32 of speed in my measurements of start time (p < 0.001) and stop time (p < 0.001) 15 relative to foot down, average emg per burst (p < 0.001), and average emg per stride (p < 0.001). Predicted Sarcomere Operating Lengths Based on my calculated length-tension curves, active lengthening of all of the segments of the ILPO, regardless of speed, occurred on the shallow ascending limb of the curve (Fig. 7). My results showed that the sarcomere length at the start of active lengthening (Fig. 8B) showed a significant effect of position (p = 0.002) but not of speed (p = 0.580). The sarcomere length at which active lengthening stopped (Fig. 8A) showed a significant effect of speed (p = 0.004), but position was not significant (p = 0.290).

33 Discussion 16 Length Changes and Electrical Activity in the ILPO My results demonstrate that significant differences in strain occur among portions of the posterior ILPO during the active lengthening, passive shortening and passive lengthening portions of the stride. The amount of strain that occurs during these portions of the stride is always significantly greater in the distal portion of the fascicle than it is in the proximal and central portions. This data refutes my hypothesis, since strain in the posterior ILPO is not uniform across the fascicle except during the period of active shortening. During the period of time when the ILPO is being lengthened while active, the distal portion lengthens significantly more than either the proximal or central portions of the muscle. Differential strain among fascicles in parallel in other studies has been demonstrated in other muscles to be caused by differential activation (Chanaud et al., 1991; Hoogendyk et al., 2005). However in the studies where there is differential activation among fascicles, there has usually been found to be a difference in moment arm among fascicles, which allows parts of muscles with a greater moment arm to impart more angular change at a particular joint for a given amount of shortening. For example in the study done in the cat by Chanaud et al. (1991), the anterior portion of the biceps femoris has greater moment arm at the hip and is active when hip extensor torque is being produced. The medial portion of the biceps femoris is active during periods of hip extension even though it is capable of acting as a knee flexor and hip extensor. The posterior fibers of the biceps femoris have a greater moment arm at the knee than the rest of the muscle, and its activity pattern correlates with knee flexion. In my experiment

34 17 with the guinea fowl, differential activation in the posterior ILPO would have to occur in in-series fibers along a single fascicle with the same moment arm at both the hip and knee, rather than among fascicles in a muscle where different portions of the muscle have different moment arms. In order for differential activation to explain differential strain in the posterior ILPO, the proximal portion of the posterior ILPO would have to be activated prior to (earlier onset times) or more intensely (greater average emg per burst, greater average emg per stride or greater integrated emg s) than the distal portion in order to recruit enough motor units to begin shortening sooner against the force produced by knee flexion. However, differential strain in the posterior ILPO is not accompanied by any significant changes in average emg per stride, average emg per burst, integrated emg or emg start and stop times between segments. Alternative Hypothesis for Differential Strain I hypothesize that the differential strain that occurs in the posterior ILPO can be explained by the differences in length change that occur in the ILPO during the passive portion of its length-shortening cycle followed by compensatory length-tension effects. The ILPO inserts onto the patella, the patellar tendon and the knee joint capsule (Gatesy, 1999). Several other muscles that are active during the swing phase of running also insert on the patellar tendon including the Iliotibialis Lateralis pars Preacetabularis (ILPR), the Iliotibialis cranialis (IC), parts of the Femerotibialis (FT) and part of the Medial Gastrocnemius (MG) (Gatesy, 1999; Marsh et al., 2004). Differential strain in the posterior ILPO, while passive during swing could be caused by interactions of the passive ILPO with active swing phase muscles. Subsequent differences in active lengthening would then be due to compensatory length tension effects in the ILPO when the muscle is

35 18 subsequently activated during stance. The ILPO during walking and running exhibits a passive stretch shortening cycle during swing followed by an active stretch shortening cycle during stance. My data demonstrates that the ILPO experiences differential strain during both passive lengthening and passive shortening. The distal segment is passively lengthened and shortened a greater amount than either the central or proximal segments and the central segment is passively shortened a greater amount than the distal segment. The resultant effect is that the proximal, central and distal segments are shortened to different points along the sarcomere length-tension curve, with the distal segment beginning activity at the shortest sarcomere length and the proximal segment beginning activity at the longest sarcomere length at the onset of active lengthening. I hypothesize that differential strain occurs while the muscle is being actively lengthened in order to prevent muscle damage via sarcomere popping (Butterfield et al., 2005; Morgan, 1990; Proske and Morgan, 2001). In order to prevent muscle damage the shorter distal sarcomeres should be actively lengthened (before shortening begins) to the same sarcomere length as the central and proximal portions of the posterior ILPO. If all of the sarcomeres along the entire fascicle were not operating on the same portion of the length-tension curve, then the longer (hence stronger) sarcomeres in the proximal and central segments would, when actively shortening, lengthen the shorter (hence weaker) sarcomeres in the distal compartment, until all of the sarcomeres were homogenous and capable of producing the same amount of force (Woledge et al., 1985). Other studies have hypothesized that sarcomere inhomogeneity occurs during active shortening in other muscles (Blemker et al., 2005; Ettema and Huijing, 1994; Huijing, 1985; Huijing, 1996; Pappas et al., 2002; van Eijden and Raadsheer, 1992). This does not seem to occur in the

36 posterior fascicle of the ILPO: there was no significant difference in the amount of 19 active shortening that occurred in the proximal, central or distal segments. Most of the muscles studied that have demonstrated nonhomogenous shortening or nonhomogenous sarcomere length have internal aponeuroses incorporated as part of their architectural design. The ILPO does not have an internal aponeurosis, and in terms of the muscle s internal architecture, the ILPO is a relatively simple parallel-fibered muscle. Length Tension Effects During level running, all of the segments of the posterior ILPO are lengthening and shortening on the shallow ascending limb of the length tension curve. If the ILPO is maximizing force and work during level running, then it should be doing some of its active shortening on the plateau region of the curve, where it is capable of producing the greatest amount of force and work. However during level running, the ILPO may not have to produce the maximum amount of force and work. Other studies have shown that, in response to a 12% incline, there are increased amounts of active shortening in the ILPO (Roberts et al., 2007). Because more work is required on the incline, it is possible that the ILPO shifts its operating range farther up onto the plateau region of the lengthtension curve in order to produce the needed increases in power and work output (Carr, 2008, Chapter 3). Studies done on the ILPO during downhill running in turkeys demonstrate that the ILPO experiences greater amounts of active lengthening than when running on a level incline (Roberts et al., 2007). If the ILPO ended its period of active lengthening on the level of the plateau region of the length-tension curve, then an increase in active lengthening has the potential to cause the ILPO s length at the cessation of active lengthening to occur on the steep descending limb of the length-tension curve

37 prior to the onset of active shortening. Previous studies have shown that it is 20 disadvantageous for a muscle to operate on this portion of the length-tension curve because the muscle is more likely to be damaged: sarcomeres in this region are more likely to experience sarcomere popping (Butterfield et al., 2005; Cutts, 1989; Morgan, 1990; Proske and Morgan, 2001). Summary I found that the long posterior fascicles of the ILPO undergo differential strain during both active and passive portions of the biphasic length-shortening cycle. Differential strain is not caused by any changes in electrical activity across the muscle. I hypothesize that differential strain in the posterior ILPO is caused by the differential strain that occurs during the swing phase when the ILPO is passive. Differential strain during the passive portion of the cycle may be caused by the ILPO interacting via a complex insertion point with other active muscles during the swing phase. The differential strain that occurs in the passive ILPO is followed by a compensatory lengthtension effect that occurs when the muscle is actively lengthened and leads to sarcomere homogeneity during active shortening. Because differential strain has been demonstrated in the ILPO, it would benefit researchers to attempt to sample along the entire muscle fascicle when possible, particularly in muscles with long parallel fibers, that experience active lengthening and have a shared origin or insertion point with other muscles. Sampling across the majority of the fascicle will ensure that the strains measured in one segment of the muscle are homogenous along the entire length of the muscle. In addition, differential strain should be taken into consideration when modeling muscle

38 function because different amounts of force are produced at different points of the 21 fascicle.

39 Literature Cited 22 Ahn, A. N., Monti, R. J. and Biewener, A. A. (2003). In vivo and in vitro heterogeneity of segment length changes in the semimembranosus muscle of the toad. J. Physiol. 549, Blemker, S. S., Pinsky, P. M. and Delp, S. L. (2005). A 3D model of muscle reveals the causes of nonuniform strains in the biceps brachii. J. Biomech. 38, Buchanan, C. I. (1999). Muscle Function and Tendon Adaptation in Guinea Fowl (Numida meleagris). In Biology, vol. Ph D., pp Boston: Northeastern University. Butterfield, T. A., Leonard, T. R. and Herzog, W. (2005). Differential serial sarcomere number adaptations in knee extensor muscles of rats is contraction type dependent. J. Appl. Physiol. 99, Cavanagh, P. R. and Komi, P. V. (1979). Electromechanical delay in human skeletal muscle under concentric and eccentric contractions. Eur. J. Appl. Physiol. Occup. Physiol. 42, Chanaud, C. M., Pratt, C. A. and Loeb, G. E. (1991). Functionally complex muscles of the cat hindlimb. II. Mechanical and architectural heterogenity within the biceps femoris. Exp. Brain Res. 85, Cutts, A. (1989). Sarcomere length changes in muscles of the human thigh during walking. J. Anat. 166, Ellerby, D. J., Henry, H. T., Carr, J. A., Buchanan, C. I. and Marsh, R. L. (2005). Blood flow in guinea fowl Numida meleagris as an indicator of energy expenditure by individual muscles during walking and running. J. Physiol. 564, Ettema, G. J. and Huijing, P. A. (1994). Effects of distribution of muscle fiber length on active length-force characteristics of rat gastrocnemius medialis. Anat. Rec. 239, Gabriel, D. A. and Boucher, J. P. (1998). Effects of repetitive dynamic contraction upon electromechanical delay. Eur. J. Appl. Physiol. Occup. Physiol. 79, Gatesy, S. M. (1999). Guineafowl hindlimb function II: electromyographic analysis and motor pattern evolution. J. Morphol. 240, Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966a). Tension development in highly stretched vertebrate muscle fibres. J. Physiol. 184,

40 23 Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966b). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, Hoogendyk, T., Carr, J. A., Henry, H. T., Rubenson, J. and Marsh, R. L. (2005). Mechanical and neural determinants of differences in fascicle strain between functionally distinct compartments in M. Iliofibularis during terrestrial locomotion in guinea fowl (Numida meleagris). Integr. Comp. Biol. 45. Huijing, P. A. (1985). Architecture of the human gastrocnemius muscle and some functional consequences. Acta Anat (Basel) 123, Huijing, P. A. (1996). Important experimental factors for skeletal muscle modelling: non-linear changes of muscle length force characteristics as a function of degree of activity. Eur. J. Morphol. 34, Huijing, P. A. (1999). Muscle as a collagen fiber reinforced composite: a review of force transmission in muscle and whole limb. J. Biomech. 32, Humason, G. L. (1979). Animal Tissue Techniques. San Francisco: W. H. Freeman and Company. Littlefield, R. and Fowler, V. M. (2002). Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images. Biophys. J. 82, Marsh, R. L., Ellerby, D. J., Carr, J. A., Henry, H. T. and Buchanan, C. I. (2004). Partitioning the energetics of walking and running: swinging the limbs is expensive. Science 303, McGowan, C. P., Duarte, H. A., Main, J. B. and Biewener, A. A. (2006). Effects of load carrying on metabolic cost and hindlimb muscle dynamics in guinea fowl (Numida meleagris). J. Appl. Physiol. 101, Morgan, D. L. (1990). New insights into the behavior of muscle during active lengthening. Biophys. J. 57, Moritani, T., Berry, M. J., Bacharach, D. W. and Nakamura, E. (1987). Gas exhange parameters, muscle blood flow and electromechanical properties of the plantar flexors. Eur. J. Appl. Physiol. Occup. Physiol. 56, Muraoka, T., Muramatsu, T., Fukunaga, T. and Kanehisa, H. (2004). Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo. J. Appl. Physiol. 96, Olson, J. M. and Marsh, R. L. (1998). Activation patterns and length changes in hindlimb muscles of the bullfrog Rana catesbeiana during jumping. J. Exp. Biol. 201,

41 Pappas, G. P., Asakawa, D. S., Delp, S. L., Zajac, F. E. and Drace, J. E. (2002). Nonuniform shortening in the biceps brachii during elbow flexion. J. Appl. Physiol. 92, Proske, U. and Morgan, D. L. (2001). Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J. Physiol. 537, Roberts, T. J., Higginson, B. K., Nelson, F. E. and Gabaldon, A. M. (2007). Muscle strain is modulated more with running slope than speed in wild turkey knee and hip extensors. J. Exp. Biol. 210, van Eijden, T. M. and Raadsheer, M. C. (1992). Heterogeneity of fiber and sarcomere length in the human masseter muscle. Anat. Rec. 232, Winter, E. M. and Brookes, F. B. (1991). Electromechanical response times and muscle elasticity in men and women. Eur. J. Appl. Physiol. Occup. Physiol. 63, Woledge, R. C., Curtin, N. A. and Homsher, E. (1985). Energetic Aspects of Muscle Contraction. London: Academic Press Incorporated. Zajac, F. E. (1989). Muscle and tendon: properties, models, scaling, and application to biomechanics and motor control. Crit. Rev. Biomed. Eng. 17, Zhou, S., Lawson, D. L., Morrison, W. E. and Fairweather, I. (1995). Electromechanical delay in isometric muscle contractions evoked by voluntary, reflex and electrical stimulation. Eur. J. Appl. Physiol. Occup. Physiol. 70,

42 25 Figure 1: Superficial muscles in the Helmeted Guinea Fowl hindlimb. Red circles indicate sonomicrometry crystal placement. At least one emg electrode was placed along the fascicle per pair of crystals. (Drawn by Dr. David Ellerby)

43 Length (µm) Millivolts Proximal ILPO Central ILPO Distal ILPO % Stride Duration Figure 2: The proximal (green), central (red) and distal (blue) length changes expressed as sarcomere length and electrical activity of the posterior fascicles of the guinea fowl during one stride at 2.5 ms -1. The black arrow indicates the point where the toe comes off the ground. The shaded areas represent ± one standard error from the mean length change (solid line).

44 27 12 A Active Lengthening *, ** 8 4 *** *** B Active Shortening ns % L C Passive Lengthening *, ** *** *** D Passive Shortening *, ** **, *** *, *** Speed (ms -1 ) Figure 3: Active lengthening (A), active shortening (B), passive lengthening (C) and passive shortening (D) in the proximal ( ), central ( ), and distal ( ) portions of the posterior ILPO. If the paired comparisons demonstrated a significant difference due to position, the results are displayed as * significantly different from proximal, ** significantly different from central, *** significantly different from distal or ns not significant. Proximal and distal values are offset slightly from actual speed.

45 Velocity During Active Lengthening A *, ** *, *** 1.0 **, *** 0.5 Velocity (Ls -1 ) Velocity During Active Shortening B 3.0 ns Speed (ms -1 ) Figure 4: Average velocity during active lengthening (A) and average velocity during active shortening (B) in the proximal ( ), central ( ), and distal ( ) portions of the posterior ILPO. If the paired comparisons demonstrated a significant difference due to position, the results are displayed as * significantly different from proximal, ** significantly different from central, *** significantly different from distal or ns not significant. Proximal and distal results are offset slightly from actual speeds.

46 ms ms ms ms -1 Proximal ILPO Central ILPO Distal ILPO 1.5 ms ms ms Time (ms) 0.3 Figure 5: Emg start and stop times relative to foot down in the proximal (green), central (red), and distal (blue) portions of the posterior ILPO at speeds between 0.5 and 3.0 m*s -1. All timing variables are presented relative to foot down (0.0). Error bars represent ± one standard error.

47 Average Emg per Burst (mv) A 30 Average Emg per Stride (mv) B Integrated Emg (mv * s) 35x C Proximal ILPO Central ILPO Distal ILPO m/s 1.0 m/s 1.5 m/s 2.0 m/s 2.5 m/s 2.78 m/s 3.0 m/s Speed (ms -1 ) Figure 6: Average emg per burst (A), average emg per stride (B) and integrated emg (C) as a function of speed for the proximal (green), central (red) and distal (blue) portions of the posterior ILPO.

48 ms ms -1 Tension (% of Maximum) ms Sarcomere Length (µm) Figure 7: The lengths on the Helmeted Guinea Fowl length tension curve that the proximal (green), central (red) and distal (blue) portions of the posterior ILPO are operating at while walking and running at 0.5, 1.5 and 2.5 ms -1 during active lengthening.

49 A Sarcomere Length (µm) B Speed (ms -1 ) Figure 8: Maximum (A) and minimum (B) sarcomere lengths as a function of speed that occur during active lengthening in the proximal ( ), central ( ), and distal ( ) portions of the posterior ILPO.

50 Chapter 2: Compensatory Mechanisms in a Muscle with Varying Moment Arms 33 Introduction An understanding of the relationship between muscle architecture and muscle function can help us better understand how a muscle functions during legged locomotion. However, during legged locomotion it is difficult to gain this understanding because muscles can function in many different ways, while experimental methods to resolve these differences may be constrained by the anatomy of a particular muscle, for example the presence or absence of a free tendon to which a strain gauge could be attached. It is especially challenging to measure muscle function in humans in vivo, so many researchers choose to make in vivo measurements in other species where more invasive techniques can be employed. For example, in animals other than humans I can directly measure some aspects of muscle function such as length change and electrical activity by inserting sonomicrometry crystals (to measure length changes) and emg electrodes (to measure electrical activity) directly into the belly of the muscle. In vivo measurements of muscle function are particularly valuable in muscles with complex anatomical connections because it allows us to accurately determine what is happening in the muscle. A large muscle with complex origins and insertions may have varying architectural characteristics across the muscle that have the potential to cause differential strain between fascicles within the single muscle (Chanaud et al., 1991; Pappas et al., 2002). Some of the architectural characteristics known to vary across a muscle include the moment arm, fascicle length and physiological cross sectional area (Chanaud et al., 1991; Lieber and Friden, 2000). The moment arm experienced by a given fascicle can

51 vary depending on how far away the origin or insertion of the fascicle is from the 34 center of rotation. Hence a muscle with a broad origin may have a fascicle that is located closer to the joint and will experience a smaller moment arm than another fascicle in the same muscle that originates further away from the joint. The fascicle with the larger moment arm will experience a greater change in length for a given angular change at the joint. Large muscles also can experience the problem that they are made up of fibers with different fascicle lengths that have the same moment arm. Fascicles with the same moment arm should experience the same length changes for a given change in angle; however in shorter fascicles the same length change will lead to a greater change in percent strain and velocity when normalized to the fascicle length. Variations in velocity could lead to variations in force output across a muscle because different fascicles would be operating at different parts of the force-velocity curve (Lieber and Friden, 2000; Woledge et al., 1985). Complexities associated with muscle architecture have consequences when doing complex modeling of muscle function, for example during studies of animal or human locomotion or in a clinical setting (Herzog and ter Keurs, 1988; Huijing, 1996; Koh and Herzog, 1998; Lieber and Friden, 2000; Woittiez et al., 1984). Many models of muscle function, particularly those done on humans, depend on external measures of kinematics and measurements of muscle moment arm to describe the length changes experienced by a muscle during movement. In these models, muscles are often represented as having a single line of action so that all of the fascicles in the muscle experience the same moment arm at a given angle (Delp et al., 1990; Lloyd and Besier, 2003; Macfadden and Brown, 2007). This methodology, known as the lump parameter model, has also been used in

52 muscles that have variable moment arms due to having either broad origins or 35 insertions. Recent studies have demonstrated that using this methodology limits the ability of models to accurately represent the paths of muscles with complex geometry (Blemker and Delp, 2005). Finally, even when using more complex three dimensional modeling techniques that account for variable moment arms within a muscle, many researchers are still commonly making the assumption that the moment arm of the muscle and the external kinematics are accurate ways of predicting the muscle s fascicle length changes. This assumption may be incorrect in muscles that share either an origin or insertion point with other muscles. The complex origin or insertion may allow for interactions between muscles and may cause length changes during the movement cycle that are different than those predicted by external kinematics and muscle moment arm. In this study I set out to determine how differences in muscle architecture, such as variations in moment arm and fascicle length among fascicles, affect the active strain pattern observed in vivo between fascicles within a single muscle. Another goal was to determine whether or not I could predict the strain pattern that occurred in different fascicles in a leg muscle that has both a broad origin (and hence a variable moment arm at the hip joint) and a common tendon of insertion (so all the fascicles experience the same moment arm at the knee) using joint kinematics and in vitro measurements of a muscles moment arms. The muscle chosen for this study was the Iliotibialis Lateralis pars Postacetabularis (ILPO) (Fig. 9) in the Helmeted Guinea Fowl (Numida meleagris). The ILPO in guinea fowl has a broad origin spanning the posterior portion of the pelvis, and its fascicles experience different moment arms depending on the fascicle location relative

53 to the hip joint center. The ILPO has fascicles that vary in length from 30 to over 36 80mm and it has a common insertion point on the patella and its associated tendon so fascicles of different lengths experience a similar moment arm at the knee. Other studies have demonstrated that during running, the ILPO of the guinea fowl and in the turkey undergoes both eccentric and concentric contraction. During cursorial locomotion the ILPO is initially actively lengthened and thus absorbs work during the first part of stance, and subsequently actively shortens producing work during the latter half of stance (Buchanan, 1999; Marsh, 1999; McGowan et al., 2006; Roberts et al., 2007). The ILPO is thought to be an important muscle during running. The ILPO is the largest muscle in the hindlimb of the guinea fowl, accounting for 12.88±0.64% of the total muscle mass in the hindlimb, and receiving more than 12.27% of the total hindlimb blood flow during running at 2.5ms -1 (Ellerby et al., 2005; Marsh et al., 2004). Finally, guinea fowl are a commonly-used experimental species for locomotion studies due to their size and their running ability. In order to determine length changes that occur in the ILPO during running, I implanted both the anterior and posterior fascicles of the ILPO with sonomicrometry crystals to measure length change, and electromyography electrodes to measure electrical activity parameters. The shorter fascicles of the anterior ILPO were implanted with two sonomicrometry crystals. The longer posterior fascicles could not be spanned by a single set of crystals so in order to get an accurate representation of the length change occurring along the fascicle, the posterior ILPO was implanted with four sonomicrometry crystals (Fig. 9). The proximal, central and distal segment length changes were summed to estimate the total length change that occurred in the posterior ILPO. I implanted emg s to

54 measure electrical activity. Using a technique described in a previous study (Carr, , Chapter 1), I normalized my length measurements (taken via sonomicrometry) into sarcomere measurements so that I could directly compare the anterior and posterior fascicles length changes. In addition to measuring length changes and electrical activity in the ILPO, I performed post-mortem measurements of muscle moment arms at both the hip and the knee for the anterior and posterior fascicles. Expected length change was calculated at both positions in the muscle using measurements of the muscle moment arms and joint angles that occurred during the stride. The calculated measurements were normalized to the same length as the actual measurements using the maximum length at midstance, which has been shown to remain fairly constant among and within a fascicle at a particular speed (Carr, 2008, Chapters 1 and 3). The calculated length changes were then compared to the actual measured length changes that occurred during running in the anterior and posterior ILPO. Due to the similar moment arm at the knee, one would expect the anterior fascicles to experience similar absolute length changes but greater relative length changes than the posterior fascicles during knee movement. However if the ILPO functioned in this manner, then the anterior fascicles would be at a mechanical disadvantage due to the large relative strain values, and this could lead to muscle damage (particularly during active lengthening) via mechanisms such as sarcomere popping (Butterfield et al., 2005; Proske and Morgan, 2001). I hypothesize that the anterior fascicles experience a similar relative amount of strain as the posterior fascicles. This would allow the anterior fascicles to maintain a similar mechanical function by operating at the same velocity and at the same point along the length tension curve. In addition, by

55 avoiding large strains, the anterior ILPO could prevent muscle damage especially 38 during eccentric activity.

56 39 Materials and Methods A detailed description of sonomicrometry and electromyography surgery and technique, videography, sarcomere measurements and length and velocity calculations, is presented by Carr (2008, Chapter 1). Animals and Training Four guinea fowl averaging 1.59±0.1 kg body mass were used for this experiment. The guinea fowl were housed in individual cages at the Northeastern University Animal Care Facility, with food and water provided ad libitum on a 10:14 L:D cycle. Prior to surgery and experimental recordings, the birds were trained to run inside a three-sided box on a motorized treadmill. The birds were trained 3-5 days/week for a minimum of five weeks. Each training session lasted for 30 minutes. After training the birds could, in an individual session, maintain level speeds of 2.5 ms -1 for 15 minutes, 2.78 ms -1 for 3 minutes, and 3.0 ms -1 for 3 minutes. Once a bird was sufficiently trained, surgery was performed to insert sonomicrometry and emg sensors as described below. After surgery, birds were allowed to recover in individual cages for at least 40 hours prior to any experimental recordings. After recovery, birds were run on the treadmill and experimental recordings were taken as described below. Birds were run at speeds of 0.5, 1.0, 1.5, 2.0, 2.5, 2.78 and 3.0ms -1 on a level incline for at least 30 seconds per speed. Each bird was sacrificed after its experimental recordings were taken. Moment Arm Measurements Post mortem measurements of moment arm were made using the tendon excursion method (Spoor and van Leeuwen, 1992) using simultaneous measurements of

57 the change in angle at the joint and the tendon excursion. In order to measure angular 40 change, the limb was videotaped at a field rate of 60Hz using a JVC Digital Video Camera model #GR-DVL9800. The limb was moved through angular excursions at either the hip or knee joint while video and length measurements were recorded. The video was synchronized with length measurements using a synchronized voltage pulse that turned on an LED in the camera s field of view. To measure the moment arms of the anterior and posterior fascicles at the hip, the femur was fixed in place using a bone clamp, and the pelvis was the movable bone in the system (Fig. 10, bottom). Cranial and caudal markers were placed on the pelvis, and the femur was marked at its centers of rotation at the hip and knee. The anterior and posterior origins on the pelvis were marked. All musculature was removed from the pelvis. Holes were drilled through the pelvis at the anterior and posterior fascicle origins, and silk thread was attached to the pelvis at the origin point and threaded through a guide placed to keep the thread in the same orientation relative to the femur as the fascicle. The end of the thread was connected to a counterweighted Harvard Bioscience isotonic length transducer (# ) such that the thread remained perpendicular to the lever arm of the length transducer. To measure moment arm at the knee, the femur was fixed in place using a bone clamp and the tibiotarsus was the movable bone of the system (Fig. 10, top). All of the muscle was removed from the femur but the ILPO s tendon of insertion at the knee was left intact. Because the insertion for the ILPO is a fairly broad piece of connective tissue, stainless steel insect pins were threaded perpendicularly across the tendon to maintain the tendon shape across the knee. The tendon (via the insect pin) was then attached to a silk thread that was in turn attached to the isotonic length transducer so that

58 the thread was perpendicular to the arm of the length transducer. Three reflective 41 markers were placed on the limb: one at the center of rotation of the hip, one at the center of rotation for the knee and one for the center of rotation for the ankle. These markers were tracked using video to measure changes in joint angle. Length change signals were collected at a frequency of 1000 Hz using an ADI Instruments Power Lab/16SP and the application Chart running on a Macintosh PowerMac. Length signals were exported from Chart into the application Igor Pro (version 5.0) where they were downsampled to the same frequency as the video and the relevant length changes that corresponded to the video were identified. After the experiment, the video of the limbs was digitized, deinterlaced and imported into NIH Image J, the reflective markers were autotracked in Image J to get the (x, y) coordinates used to calculate the angles. Graphs were made of length change versus angle in radians; these curves were fitted to a polynomial using a stepwise least squares regression analysis (significance p < 0.05). The joints were moved through an angular excursion that was greater than what the joint experienced during running to minimize end effects of the curve fitting process. The resulting polynomial equation was differentiated to calculate the instantaneous moment arm as a function of angle. Moment arms at the hip and knee were used along with kinematics measured from the guinea fowl running at 0.5, 1.5 and 2.5 ms -1 to calculate the length change predicted from moment arm and angle measurements. Statistical Analysis Multivariate and single-variable analyses were conducted using the general linear model in the application SPSS (versions 14.0 and 15.0 for Windows) to test for significant effects of speed and position on length changes, velocity changes and

59 electrical activity variables. Speed and incline were used as covariates in the model. 42 The results of incline are discussed by Carr (2008, Chapter 3). An animal identifier and the position across the fasicle were added as factors in the model. To compare my predicted length changes versus my actual length change measurements in the anterior and posterior ILPO, paired t-tests were performed. To compare predicted length changes between the anterior and posterior fascicles, unpaired t-tests were done. Results of all statistical tests were considered significant if the p-values were less than All measurements are reported as a mean value plus or minus one standard error.

60 Results 43 Length and Velocity Changes Electrical activity in the anterior and posterior ILPO starts near the end of the swing or the first part of the stance phase and lasts throughout the majority of stance (Fig. 11). During stance the ILPO is initially lengthened while active and then actively shortens in the latter part of stance (Fig. 11). Strain during active lengthening (Fig. 12A) and active shortening (Fig. 12B) in the anterior and posterior ILPO both demonstrate significant effects of speed (p < 0.05). There was no significant difference in the strain during active lengthening (p = 0.275) or active shortening (p = 0.377) between the anterior and posterior fascicles. Average velocity during active shortening (Fig. 13A) showed significant effects of speed (p < 0.001) but was not significantly different between the anterior and posterior fascicle of the ILPO (p = 0.480). Average velocity during active lengthening (Fig. 13B) also showed a significant effect of speed (p < 0.001) but again there was no significant difference between the anterior and posterior fascicles (p = 0.693). Average velocities during active shortening and during active lengthening, in both the anterior and posterior ILPO, increased as a function of speed. Moment Arm Measurements The average moment arm for fascicles of the anterior ILPO (Fig. 14A) as a function of joint angle increases with angle until it reaches a maximum and then decreases. The maximum moment arm at the hip of the anterior fascicles occurs at 95 degrees and is ± 0.66 mm. The average moment arm at the hip for posterior fascicles of the ILPO as a function of joint angle (Fig. 14B) also increases with angle.

61 44 The moment arm at the hip of the posterior fascicles at 95 degrees is ± 1.86mm - almost four times as large as the moment in the anterior fascicles at the same angle. Because all of the fascicles of the ILPO insert onto the same tendon on the knee, they have the same moment arms as a function of angle. The moment arm of the ILPO at the knee as a function of angle (Fig. 15) increases as a function of angle, until it reaches a maximum at 95 degrees of ± 0.383mm, and then begins to decrease as angle increases further. Calculated versus Experimental Length Changes Figure 16 shows the average angular excursions of the hip, knee and ankle joints at 0.5 ms -1 (A), 1.5 ms -1 (B) and 2.5 ms -1 (C). Individual bird average kinematics at the knee and hip, along with the each bird s individual moment arm measurements, were used to calculate the expected length change at 0.5 (Fig. 17A), 1.5 (Fig. 17B), and 2.5 ms - 1 (Fig. 17C). There were no significant differences found in between the calculated and measured length changes that occur in the anterior ILPO during active shortening at 0.5 (p = 0.15), 1.5 (p = 0.28), or 2.5 ms -1 (p = 0.26), nor were any significant differences found between the calculated and measured length changes that occur in the anterior ILPO during the period of active lengthening at 0.5 (p = 0.78) or 1.5 (p = 0.11) ms -1. Significant differences were found between the calculated and measured length changes that occur during active lengthening (p = 0.006) in the anterior ILPO at 2.5 ms -1. No significant differences were found between calculated and measured lengths in the posterior ILPO during active lengthening or active shortening (p > 0.05) at any speed. My moment arm kinematic model predicted that active lengthening at 2.5 ms -1 would be

62 significantly greater in the posterior ILPO than it would be in the anterior ILPO (p < ).

63 Discussion 46 I were not able to measure any significant differences in active lengthening, active shortening, average lengthening velocity or average shortening velocity between the anterior and posterior fascicles of the ILPO despite differences in moment arm at the hip and fascicle length. The moment arm and kinematics model was, in most cases, able to predict the amount of active shortening and active lengthening that occurred in the anterior and posterior ILPO and the predicted and measured values were not significantly different. However my model was not able to predict the amount of active lengthening that occurred in the anterior ILPO at 2.5 ms -1. Even though differences in muscle architecture do not yield differences in muscle strain I still need to explain why the moment arm and kinematics model was not able to accurately predict the length change that occurred in the anterior fascicles at 2.5 ms -1, and why there is a uniform strain pattern between the anterior and posterior fascicles of the ILPO. Despite differences in fascicle length between anterior and posterior fascicles no significant differences in strain were measured during active shortening at these locations and similar strains were also predicted to occur based on the moment arm and kinematics model. During active shortening, which occurs in the latter half of stance, the hip and knee are both going through angular changes but the knee is going through a smaller angular change than the hip. Because the majority of angle change during late stance and early swing is occurring at the hip, the length change in these fascicles is dominated by the moment arms experienced by the different fascicles at the hip. The difference in moment arm at the hip compensates for the differences in fascicle length such that the relative strains experienced by the anterior and posterior fascicles of the ILPO is similar

64 during active shortening. The anterior fascicles of the ILPO have a smaller moment 47 arm at the hip and experience a decreased absolute change in length as a function of angle when compared to the posterior ILPO with its larger moment arm. Even though the anterior fascicles go through less absolute length change, as a function of angle they are shorter so the relative length change is similar in both the anterior and posterior ILPO during this period of the stride. This allows the anterior and posterior fascicles to experience strains during active shortening and passive lengthening of similar amplitudes and at similar velocities, a condition of particular importance during active shortening because it allows the anterior and posterior fascicles to have very similar mechanical functions and to produce the same force and power. Similar compensatory mechanisms have been modeled in the vastus lateralis and rectus femoris in humans using threedimensional muscle models and have been hypothesized to occur in the biceps femoris of cats (Blemker and Delp, 2005; Chanaud et al., 1991): the length change that was predicted to occur in various fascicles was not constant but varied depending on the fascicle s position in three dimensional space. In contrast to the predictions for active shortening, the moment arm and kinematics model predicted significant differences in strain between the anterior and posterior ILPO during active lengthening at 2.5 ms -1. The model predicted that during active lengthening, a greater amount of strain would occur in the anterior ILPO than the posterior ILPO. Active lengthening in the ILPO occurs during the first half of stance and the predicted strain is primarily being driven by the large amounts of knee flexion occurring during this part of stance. Because the anterior and posterior ILPO share the same moment arm about the knee joint, my model predicted that they would experience

65 the same absolute amount of length change, which would translate to a larger strain in 48 the shorter anterior fascicles. The amount of active lengthening in the anterior ILPO was predicted to be much larger than the actual length change that occurs, while the amount of lengthening predicted to occur in the posterior ILPO was not significantly different than what was actually measured in this part of the muscle. During active lengthening at 2.5 ms -1 the sarcomeres of the anterior fascicle are lengthened to 2.24 µm. If the anterior fascicles at 2.5 ms -1 were lengthened the amount predicted by the moment arm measurements, the sarcomeres within the anterior fascicle would be lengthened to a length of 2.37µm, which is located quite a bit further up on the length tension curve (plateau region µm). Other studies have shown that the sarcomere length increases both with speed and with incline (Carr, 2008, Chapter 3). If the anterior fascicles were lengthened to the point predicted by the moment arm and kinematics, and then subsequently lengthened to even greater lengths at higher speeds and greater inclines, then it is possible that they would be lengthened beyond the plateau region of the length tension curve and onto the inherently unstable descending limb of the curve. Muscles should not operate on the descending limb, particularly during eccentric activity, because there is a greater chance of sarcomere popping occurring in the weakened sarcomeres leading to muscle damage (Butterfield et al., 2005; Gordon et al., 1966; Morgan, 1990; Proske and Morgan, 2001). The question I am left with is why am I not seeing the length change that is predicted by the moment arm and kinematics at 2.5 ms -1 when my model is very good at predicting length changes in the posterior fascicle and in the anterior fascicle at slower speeds?

66 Closer examination of the anatomy of the ILPO shows that the anterior 49 fascicles insert on a broad tendinous sheath that attaches to the patella at the knee. I hypothesize that during active lengthening this tendinous sheath is stretched while the muscle is active, and some of the length change imposed by the moment arm at the knee actually goes into lengthening the anterior fascicle s tendon of insertion rather than acting directly upon the anterior fascicle and lengthening it. I calculated that the anterior fascicle s tendon of insertion at 2.5 ms -1 has to stretch 3.11±0.34% of its total length to account for the difference I observed between the predicted fascicle length and the actual fascicle length. This tendon strain is well below the 8-10% strain at which tendon failure occurs (Biewener, 2003) and it allows room for even greater tendon stretch as the amount of active lengthening in the anterior ILPO increases with increased speed and incline (Carr, 2008, Chapter 3). At speeds lower than 2.5 ms -1, less force would be acting on the tendon, less length change would occur in the tendon and I get a closer march between my actual and predicted values in the anterior ILPO during active lengthening. The posterior fascicles insert close to the end of the long aponeurotic tendon, therefore there is very little tendon available for stretch. This may explain why I am able to accurately predict length changes in the posterior fascicle based on moment arm and joint kinematics at all speeds. Despites its architectural complexity, the ILPO shows similar amounts of length changes during most of its stretch-shortening cycle. The variable moments that occur for different fascicles at the hip play a compensatory role during active shortening in late stance so that the strain values are homogenous across the muscle. During early stance the ILPO is actively lengthened and these length changes in the anterior and posterior

67 fascicles are driven primarily by the large amount of knee flexion that is occurring. 50 Even though the moment arm at the knee is constant, the anterior fascicles at 2.5 ms -1 experience the same relative amount of strain as the posterior fascicles. I hypothesize that this is due to stretching that occurs in the long, tendinous aponeurosis that the anterior fascicles insert on, which absorbs some of the length change being transmitted from the hip, so that the anterior fascicles are not lengthened the same absolute amount as the posterior fascicles. In conclusion, while models based on moment arms and kinematics may be useful to predict length changes in muscles that have relatively simple and small origins and insertions, these models are not always useful in accurately predicting length changes in the ILPO a muscle with a larger and more complex origin and insertion. My ILPO model based on moment arm and external kinematics was capable of predicting some of the length change patterns seen in the ILPO, however it overestimated other length changes, particularly at high speeds. Without actual measurements made in vivo, erroneous conclusions about muscle function - particularly during active lengthening - could have been made, leading researchers to incorrect conclusions about the velocity of active lengthening, the amount of force and work produced by the muscle, and the lengthtension operating range of the muscle. By doing studies that combine experimental verification of models as well as modeling, I should be able to better refine existing models so that they include more variables and give a more accurate representation of what is occurring during locomotion in complex muscles (Blemker and Delp, 2005; Blemker et al., 2005; Delp et al., 1990; Pappas et al., 2002).

68 Literature Cited 51 Biewener, A. A. (2003). Animal Locomotion. Oxford: Oxford University Press. Blemker, S. S. and Delp, S. L. (2005). Three-dimensional representation of complex muscle architectures and geometries. Ann. Biomed. Eng. 33, Blemker, S. S., Pinsky, P. M. and Delp, S. L. (2005). A 3D model of muscle reveals the causes of nonuniform strains in the biceps brachii. J. Biomech. 38, Buchanan, C. I. (1999). Muscle Function and Tendon Adaptation in Guinea Fowl (Numida meleagris). In Biology, vol. Ph D., pp Boston: Northeastern University. Butterfield, T. A., Leonard, T. R. and Herzog, W. (2005). Differential serial sarcomere number adaptations in knee extensor muscles of rats is contraction type dependent. J. Appl. Physiol. 99, Chanaud, C. M., Pratt, C. A. and Loeb, G. E. (1991). Functionally complex muscles of the cat hindlimb. II. Mechanical and architectural heterogenity within the biceps femoris. Exp. Brain Res. 85, Delp, S. L., Loan, J. P., Hoy, M. G., Zajac, F. E., Topp, E. L. and Rosen, J. M. (1990). An interactive graphics-based model of the lower extremity to study orthopaedic surgical procedures. IEEE Trans. Biomed. Eng. 37, Ellerby, D. J., Henry, H. T., Carr, J. A., Buchanan, C. I. and Marsh, R. L. (2005). Blood flow in guinea fowl Numida meleagris as an indicator of energy expenditure by individual muscles during walking and running. J. Physiol. 564, Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, Herzog, W. and ter Keurs, H. E. (1988). A method for the determination of the force-length relation of selected in-vivo human skeletal muscles. Pflugers Arch. 411, Huijing, P. A. (1996). Important experimental factors for skeletal muscle modelling: non-linear changes of muscle length force characteristics as a function of degree of activity. Eur. J. Morphol. 34, Koh, T. J. and Herzog, W. (1998). Increasing the moment arm of the tibialis anterior induces structural and functional adaptation: implications for tendon transfer. J. Biomech. 31,

69 Lieber, R. L. and Friden, J. (2000). Functional and clinical significance of skeletal muscle architecture. Muscle Nerve 23, Lloyd, D. G. and Besier, T. F. (2003). An EMG-driven musculoskeletal model to estimate muscle forces and knee joint moments in vivo. J. Biomech. 36, Macfadden, L. N. and Brown, N. A. (2007). Biarticular hip extensor and knee flexor muscle moment arms of the feline hindlimb. J. Biomech. Marsh, R. L. (1999). How muscles deal with real-world loads: the influence of length trajectory on muscle performance. J. Exp. Biol. 202, Marsh, R. L., Ellerby, D. J., Carr, J. A., Henry, H. T. and Buchanan, C. I. (2004). Partitioning the energetics of walking and running: swinging the limbs is expensive. Science 303, McGowan, C. P., Duarte, H. A., Main, J. B. and Biewener, A. A. (2006). Effects of load carrying on metabolic cost and hindlimb muscle dynamics in guinea fowl (Numida meleagris). J. Appl. Physiol. 101, Morgan, D. L. (1990). New insights into the behavior of muscle during active lengthening. Biophys. J. 57, Pappas, G. P., Asakawa, D. S., Delp, S. L., Zajac, F. E. and Drace, J. E. (2002). Nonuniform shortening in the biceps brachii during elbow flexion. J. Appl. Physiol. 92, Proske, U. and Morgan, D. L. (2001). Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J. Physiol. 537, Roberts, T. J., Higginson, B. K., Nelson, F. E. and Gabaldon, A. M. (2007). Muscle strain is modulated more with running slope than speed in wild turkey knee and hip extensors. J. Exp. Biol. 210, Spoor, C. W. and van Leeuwen, J. L. (1992). Knee muscle moment arms from MRI and from tendon travel. J. Biomech. 25, Woittiez, R. D., Huijing, P. A., Boom, H. B. and Rozendal, R. H. (1984). A three-dimensional muscle model: a quantified relation between form and function of skeletal muscles. J. Morphol. 182, Woledge, R. C., Curtin, N. A. and Homsher, E. (1985). Energetic Aspects of Muscle Contraction. London: Academic Press Incorporated.

70 53 Figure 9: Superficial muscles in the guinea fowl hindlimb. The ILPO is pictured in white. Red circles indicate sonomicrometry crystal placement in the anterior and posterior fascicles of the ILPO. At least one emg electrode was placed along the fascicle per pair of crystals. Drawn by Dr. David Ellerby.

71 Figure 10: Moment arm measurement set up for the knee (top) and for the hip (bottom). 54

72 110 A %L B Millivolts % Stride Cycle Figure 11: Anterior (red) and posterior (blue) emg activity and length as a function of L 0 at 0.5 ms -1, a walk (A), and 2.5 ms -1 a run (B) during a single stride. Black arrow represents toe-up and grey bands are ± one standard error.

73 A %L B 4 2 Posterior ILPO Anterior ILPO Speed (ms -1 ) Figure 12: Length change expressed as a %L 0 during active lengthening (A, circles) and active shortening (B, squares) during running in the anterior (red) and posterior (blue) ILPO. Anterior markers are offset slightly from speed.

74 A Velocity (Ls -1 ) B Speed (ms -1 ) Figure 13: Average active shortening velocity (A, squares) and active lengthening velocity (B, circles) during running in the anterior (red) and posterior (blue) ILPO. Anterior markers are offset slightly from speed.

75 A Moment Arm (mm) Angle (degrees) B Moment Arm (mm) Angle (degrees) Figure 14: Average moment arm of the anterior (A) and posterior (B) fascicles at the hip vs. hip joint angle. Grey bands are ± one standard error.

76 59 ILPO Moment Arm at Knee 10 8 Moment Arm (mm) Angle (degrees) Figure 15: Average moment arm of ILPO at the knee vs. knee joint angle. Grey bands are ± one standard error.

77 A B Joint Angle (degrees) C % Stride Cycle Figure 16: Knee (blue) and hip (red) joint angles in the guinea fowl during running on the level at 0.5 (A), 1.5 (B) and 2.5 ms -1 (C). Black arrow indicates point of toe off. Grey bands are ± one standard error.

78 A 110 B C D %L E F % Stride Cycle Figure 17: Calculated (dashed line) vs. actual strain (solid line) in the anterior (red) and posterior (blue) ILPO during running at 0.5 ms -1 (A&B), 1.5 ms -1 (C&D) and 2.5 ms -1 (E&F) in bird B334. Significant difference between calculated and actual values occurred in the anterior ILPO while running at 2.5 ms -1 during the period of active lengthening.

79 Chapter 3: Iliotibialis Lateralis pars Post Acetabularis (ILPO) Function in Helmeted Guinea Fowl (Numida meleagris) 62 Introduction Research studies have demonstrated that during running, leg muscles can function in many different ways. Some muscles can even have two functions during locomotion. An example of this would be a muscle that is initially actively lengthened and subsequently actively shortens: this occurs in the vastus lateralis in dogs during galloping (Carrier et al., 1998) and in rats during running (Gillis and Biewener, 2001). Another muscle that has more than one function during running the muscle I examined for this study is the Iliotibialis Lateralis pars Post Acetabularis in the Helmeted Guinea Fowl (Numida meleagris). The ILPO in guinea fowl is illustrated in Figure 18. In guinea fowl, the ILPO is the largest single muscle in the hindlimb, and it has important functions during running. The ILPO accounts for 12.88±0.64% of the total muscle mass in the hindlimb, and uses a large amount of energy during running at high speeds but comparatively little during walking (Ellerby et al., 2005; Marsh et al., 2004). The morphology of the ILPO across different bird species appears to be associated with terrestrial running. In extant cursorial birds, such as the guinea fowl, the ILPO is a large muscle. However the ILPO shows notable variation in other orders of birds that employ different forms of locomotion (Fig. 19). The ILPO has been lost or reduced in species spanning 17 orders of birds. The ILPO has been lost in several orders and families of birds that are excellent flyers including Procellariiformes, Falconiformes, Psittaciformes, Coliiformes,

80 Caprimulgiformes, Strigiformes, and Apodiformes as well as some taxa in 63 Passeriformes including Tyrannidae (tyrant flycatchers) and Hirundinidae (swallows and martins). Swallows and Flycatchers feed primarily on insects, employing aerial hawking, where they seize the insects directly in their bills. They spend little time on the ground and their terrestrial locomotion is usually restrained to one or two hops covering a short distance (Sibley, 2001). The ILPO has also been reduced or lost in birds that spend a large amount of time swimming, including members of the orders Anseriformes, Pelecaniformes, Sphenisciformes, Ciconiiformes, and Charadriiformes. The New World Vultures (Ciconiiformes) has a well-developed ILPO muscle. It is possible that the ILPO was not lost in this group of birds that fly well because they are both also known for spending large amounts of time on the ground. The anatomical data demonstrates that the ILPO is reduced or absent in many birds that locomote primarily by swimming or by flying, but is present in birds that are considered good cursorial locomotors such as the ratites (many of which are incapable of flight) and Galliformes. Two notable exceptions to this general observation are the Grebes (Podicipediformes) and Loons (Gaviiformes). Both of these orders have well-developed ILPO s. However, they have, switched the orientation of their legs to an abducted posture during swimming, allowing them to swim in a manner similar to that of frogs and toads. In birds, muscles are needed to hold the limb in an abducted position (overcoming the weight of the limb) and Raikow (1970) hypothesized based on anatomy that the Iliotibialis is acting to overcome the weight of the limb and maintain the abducted position of the hip and knee. This leg position maximizes the amount of thrust that can be produced in a two-propulsor system when the feet are swept backward until they meet

81 behind the animal (Daniel, 1984). In addition, their legs are shifted farther back on 64 their bodies, making walking on land difficult at best. Both of these adaptations should increase the economy of aquatic locomotion (Raikow, 1970). Because the ILPO is so prevalent in birds that are good cursorial locomotors and because it uses so much energy during rapid running, knowing the function of the ILPO could tell us about the mechanical functions important during rapid running. Other studies have demonstrated that during running, the ILPO in the guinea fowl and turkey actively lengthens and absorbs work during the first part of stance, and actively shortens to produce work during the latter half of stance (Buchanan, 1999; Marsh, 1999; McGowan et al., 2006; Roberts et al., 2007). However even though the ILPO has been studied extensively, more recent studies done in my lab have demonstrated the need to employ relatively complex models in order to accurately represent and predict the muscle s overall function: the ILPO shows proximal to distal differences in strain; it can show differences in passive strain between the anterior and posterior fascicles due to effects of muscle architecture; and it is able to adjust its operating range on the length tension curve in response to changes in speed (Carr, 2008, Chapters 1 & 2). My study will be the first survey of ILPO function that accounts for these effects therefore giving a more accurate representation of the muscle s overall function. I chose to use guinea fowl to study the function of the ILPO for several reasons. Guinea fowl are commonly used for experimental locomotion studies due to their size and their running ability; two other studies have been done in guinea fowl so there is no interspecific variation to obfuscate my comparisons (Buchanan, 1999; Marsh, 1999; McGowan et al., 2006). I hypothesized that there will be significant effects of both speed and incline on

82 the length operating range of the ILPO, on the amount of strain that occurs in the 65 muscle while active and passive, and on the electrical activity that occurs in the ILPO. Because I have both blood flow measurements and electrical activity measurements, I will be able to determine how well the blood flow (which acts as a measure of total muscle energy use) correlates with electrical activity (which has been used in other studies as a de facto measure of muscle energetics). I tested these hypotheses by implanting the anterior ILPO with two sonomicrometry crystals that spanned the majority of the fascicle, and implanting the posterior ILPO with four sonomicrometry crystals to measure length change (see Fig. 18). The posterior sonomicrometry crystals were implanted so that they spanned the majority of the fascicle and divided the fascicle into proximal, central and distal segments. The proximal, central and distal segment length changes were summed to get the total length change that occurred in the posterior ILPO. I implanted emg s to measure electrical activity, and using a technique described in a previous study (Carr, 2008, Chapter 1), I normalized my length measurements (taken via sonomicrometry) into sarcomere measurements.

83 Materials and Methods 66 Animals and Training Four Helmeted Guinea Fowl averaging 1.59±0.1 kg body mass were used for this experiment. The guinea fowl were housed in individual cages at the Northeastern University Animal Care Facility, with food and water provided ad libitum. Prior to surgery and experimental recordings, the birds were trained to run inside a three-sided box on a motorized treadmill. The birds were trained 3-5 days/week for a minimum of five weeks. Each training session lasted for 30 minutes. After training the birds could, in an individual session, maintain level speeds of 2.5 m*s -1 for 15 minutes, 2.78 ms -1 for 3 minutes, and 3.0 ms -1 for 3 minutes. Once a bird was sufficiently trained, surgery was performed to insert sonomicrometry and emg sensors as described below. After surgery, birds were allowed to recover in individual cages for at least 40 hours prior to any experimental recordings. After recovery, birds were run on the treadmill and experimental recordings were taken as described below. Birds were run at speeds of 0.5, 1.0, 1.5, 2.0, 2.5, 2.78 and 3.0 ms -1 on a level incline for at least 30 seconds per speed. Each bird was sacrificed after its experimental recordings were taken. Sonomicrometry and Electromyography All transducers were sterilized (12 hour ethylene oxide treatment) and implanted using sterile surgical techniques under general anesthesia (isofluorane). Six custommade 1-mm sonomicrometry transducers were made from PZT discs (Boston Piezo- Optics, Inc.) and attached to stainless steel holders (Olson and Marsh, 1998). Four crystals were implanted along posterior fascicles of the ILPO, thus dividing the fascicles into proximal, central and distal segments (Fig. 18). Crystals were placed in the posterior

84 ILPO so that they spanned a large portion of the total fascicle length, allowing us to 67 account for differential strain and get an accurate measure of total fascicle strain (Carr, 2008, Chapter 1). These length changes in the proximal, central and distal compartments were summed to obtain the total length change that occurs in the posterior ILPO. Two crystals were also placed in the shorter anterior fascicles, spanning the center of the fascicle to get a measure of length change in the anterior portion of the ILPO. Small punctures approximately 1 mm in length were created in the connective tissue overlying the muscle to a depth of approximately 3mm. The crystals were then placed between the fascicles. The holders were secured to the overlying connective tissue using 6-0 silk sutures. Care was taken to ensure that the anterior and posterior crystals were always implanted between the same fascicles along the length of the muscle. Fine-wire bipolar emg electrodes were implanted into the ILPO to measure muscle activity, including 5 electrodes in the posterior ILPO and 2 in the anterior ILPO. The emg electrodes were constructed from 3T stainless steel Teflon-coated wire (Medwire) that was twisted along its length. The tips of the electrodes were bared 1.5 mm at each end and preactive ends spaced 3 mm apart. Emg electrodes were implanted in the ILPO with a chamfered 25 gauge needle at positions that were immediately anterior and posterior to each segment. Post mortem examinations of verified the placement of sonomicrometry crystals and emg electrodes. Recordings Sonomicrometry measurements were acquired digitally with a sampling frequency of 613 Hz using Sonoview software and a Sonometrics TRX Series 8 interface.

85 68 Emg signals were amplified by WPI model DAM-50 preamplifiers with highand low-pass filters set at 10 and 3000 Hz, respectively. Emg signals were collected at a frequency of 10kHz using an ADI Instruments Power Lab/16SP and a Macintosh PowerMac. The emg data were subsequently analyzed using the application Igor Pro (version 5.0). The signals were first filtered using a finite impulse response filter with a band pass of 90Hz to 1000Hz. Emg signals were rectified and the start time, stop time, and emg duration were all measured. The burst amplitudes were expressed in three ways: the integrated value of the burst duration; the average emg per burst (equal to the integrated value divided by the burst duration); and the average emg per stride (equal to the integrated value divided by the stride duration). Videography Video was recorded to VHS tape with a NAC HV-1000 high-speed video camera operating at 500 fields per second. I synchronized the emg and sonomicrometry traces with the high-speed video recordings using a square-wave generated by my emg recording system which was recorded in the video fields with a NAC wave inserter. Visual markers were made using either white paint or reflective tape. These markers were placed above the caudal and cranial pelvis; on the hip, ankle, tarsometatarsalphalangeal and interphalangeal joints; and on the toe of the bird. The knee angle was calculated using the law of cosines and several distances, including the distance between the hip and the ankle markers based on post mortem measures of the distance between the center of rotation of the hip and center of rotation of the knee, and the distance between the center of rotation of the knee and the center of rotation of the ankle. Data were

86 collected for ten steady speed strides at each speed for each bird. The strides were 69 analyzed for changes in muscle strain, muscle velocity and electrical activity. Sarcomere Measurements After each animal s experimental recordings had been completed, the animal was sacrificed and its instrumented leg was bolted into place with the hip and knee in a flexed position. The limb was allowed to sit in a bolted position for six hours until the leg muscles were in full rigor. Once the fixed limb was in rigor, a reference sonomicrometry measurement (denoted L so, r ) was measured. In addition, a body temperature measurement was taken to correct for temperature effects on the speed of sound. Two samples of the ILPO tissue, each measuring ~1cm 3, were removed from between each crystal pair. These muscle samples were frozen using liquid nitrogen and isopentane, and then sectioned using a cryostat. For each muscle sample, at least ten sections were placed on slides and stained using Weingert Iron Hematoxylin (Humason, 1979). From each slide (containing one stained section), at least three fibers were captured. Sarcomere lengths were measured using a compound microscope at 1000X magnification. Sarcomere measurements were performed using a digital imaging program (NIH Image J version 1.38). In Image J, a series of at least 10 sarcomeres was measured from each fiber, and the average individual sarcomere length (denoted L sc, ref ) was calculated by dividing the total length of the in-series sarcomeres by the number of sarcomeres in series. Using the reference sonomicrometry length and the measured sarcomere length, in vivo sonomicrometry lengths (L so ) were converted to estimated in vivo sarcomere lengths (L sc ) using the equation:

87 70 L sc,ref L so L so,ref = L sc (1) Length measurements in this paper are either reported as µm, or are converted to %L 0 using the following equation: %L 0 = L sc 100 (2) 2.36µm where L sc is the calculated sarcomere length from equation 1, and 2.36µm is the estimated sarcomere length for maximum force production based on the length in the middle of the plateau region of the calculated length-tension curve described in chapter 1. Length and Velocity Changes Muscle segment lengths were measured from the sonomicrometry traces after the lengths in mm had been converted to µm. The length values measured were: L fd = minimum length of the ILPO which occurred near the time of foot down L max,st = maximum length of the ILPO occurring during stance L min = minimum length of the ILPO, which occurred near the time of toe off L max,sw = maximum length of the ILPO occurring during swing L emg,on = length of the ILPO 25ms after the start of the emg activity L emg,off = length of the ILPO 25ms after the stop of the emg activity These measured lengths were used to calculate active lengthening, active shortening, passive lengthening and passive shortening. When calculating active lengthening and active shortening I assumed an electromechanical delay of 25ms. I are not aware of studies done on the ILPO to confirm this assumption, but this time period falls within

88 values measured for other muscles (Cavanagh and Komi, 1979; Gabriel and Boucher, ; Moritani et al., 1987; Muraoka et al., 2004; Winter and Brookes, 1991; Zhou et al., 1995). The equation used to calculate a given length change was dependent on the timing of L emg,on and L emg,off relative to other lengths measured during a stride. Active lengthening ( L al ) was calculated using one of two equations. If L emg,on occurred before L fd then: L al = L max,st - L fd (3) If L emg,on occurred after L fd then: L al = L max,st L emg,on (4) Active shortening ( L as ) was also calculated using one of two equations. If L emg,off occurred after L min then: L as = L max,st L min (5) If L emg,off occurred before L min then: L as = L max,st L emg,off (6) Passive lengthening ( L pl ) was calculated using one of two equations. If L emg,off occurred before L min then: L pl = L max,sw L min (7) If L emg,off occurred after L min then: L pl = L max,sw L emg,off (8) Passive shortening ( L ps ) was calculated using one of two equations. If L emg,on occurred after L fd then: L ps = L min,sw L fd (9) If L emg,on occurred before L fd then:

89 L ps = L min,sw L emg,on (10) 72 All length changes are reported as %L 0 where L 0 is the middle of the plateau region of the length tension curve. Average velocities were calculated during active lengthening and shortening and in each compartment and at each speed. Average velocities in L*s -1 were calculated by dividing the amount of active lengthening or active shortening (expressed as a fraction of L 0 ) that occurred during a stride by the duration of lengthening or shortening. Thus velocity during active lengthening (V al ) is: V al = L al (2.36µm t al ) (11) where L al is the active lengthening from equation 3 or 4, 2.36µm is the length at the center of the plateau region of the length-tension curve, and t al is equal to the duration of active lengthening in seconds. Similarly, the velocity during active shortening (V as ) is: V as = L as (2.36µm t as ) (12) where L as is the active shortening from equation 5 or 6, 2.36µm is the length at the center of the plateau region of the length-tension curve, and t as is the duration of active shortening in seconds. Blood Flow vs. Emg Measurements Using blood flow and oxygen consumption measurements from Rubenson et al. (2006) and Ellerby et al. (2005), and assuming that blood flow is directly proportional to oxygen consumption, I were able to predict the oxygen consumption above rest of the ILPO during locomotion at 0.5, 1.5 and 2.78 ms -1 during level running and at 1.5 ms -1

90 73 during running on a 15% incline. The oxygen consumption values and the emg values measured in this experiment were then converted to a percentage by dividing by the value measured for each variable at 2.78 ms -1. The final percentage emg values were individually compared to the oxygen consumption percentages to determine if there was a correlation. Statistical Analysis Multivariate and single-variate analyses were conducted using the general linear model in the application SPSS (versions 15.0 for Windows) to test for significant effects of speed and incline on length changes, velocity changes and electrical activity variables. An animal identifier was added as a factor in the model: this removed the variation among the individual animals. Speed and incline were both used as covariates in the model. To test for differences between blood flow and emg values, Pearson s correlation coefficients were calculated to determine if blood flow could be correlated with measurements of muscle activity. Results were considered statistically significant if the p-value was less than All measurements are reported as a mean value plus or minus one standard error.

91 Results 74 Muscle Length Change The length change that I measured in the anterior and posterior ILPO during a walk (Fig. 20A) and during a run (Fig. 20B) was similar to measurements taken in other studies (Buchanan, 1999; McGowan et al., 2006; Roberts and Scales, 2004). The ILPO is active throughout stance: it is initially actively lengthened and in the latter half of stance it actively shortens. During swing, the ILPO shows periods of both when it is passively lengthened and passively shortened. Active lengthening (Fig. 21A) demonstrated a significant effect of both incline (p<0.022) and speed (p=0.017) however there was no significant effect of anterior-posterior position (p=0.275). The amount the ILPO is actively lengthened during the early part of stance tends to increase as speed increases and decrease as incline increases. Active shortening (Fig. 21B) demonstrated a significant effect of incline (p<0.001) and speed (p<0.001) but not anterior-posterior position (p=0.377). The amount of active shortening (Fig. 21B) experienced by the anterior and posterior ILPO tends to increase with increasing speed and with increasing incline. These results are similar to what has been observed in the ILPO of turkeys (Roberts and Scales, 2004). Passive lengthening (Fig. 22A) demonstrated a significant effect of anteriorposterior position (p<0.025), incline (p<0.025) and speed (p<0.05). The amount of passive lengthening experienced by the anterior and posterior ILPO during running tends to increase with both speed and incline and is greater in the anterior ILPO than it is in the posterior ILPO. Passive shortening (Fig. 22B) demonstrated a significant effect of speed (p<0.001) but not of anterior or posterior position (p=0.051) or incline (p=0.659). The

92 amount of passive shortening experienced by both the anterior and posterior ILPO 75 tended to increase with speed. Velocity Changes as a Function of Speed and Incline Average active shortening velocity (Fig. 23A) demonstrated a significant effect of incline (p<0.001) and speed (p<0.001) but not anterior-posterior position (p=0.480). During running, active shortening velocity in the anterior ILPO and posterior ILPO increased with both speed and incline. Average active lengthening velocity (Fig. 23B) demonstrated a significant effect of speed (p<0.001) but not incline (p=0.683) or anterior posterior position (p=0.693). During running, the average active lengthening velocity increased with speed. Sarcomere Measurements My L emg,on values demonstrated a significant effect of incline (p<0.001) but there was no significant effect of either speed (p=0.864) or anterior-posterior position (p=0.063). The L start values showed a significant effect of incline (p<0.001) but were not significantly affected by anterior-posterior position (p=0.063 or speed (p=0.864). The average L start values at each incline were 2.07±0.01µm during level running, 2.16±0.01µm during running on the 10% incline, and 2.29±0.02µm during running on the 20% incline. The L max values (Fig. 24A) demonstrated a significant effect of incline (p<0.001) and speed (p=0.001) and tended to increase as both speed and incline increased, however there was no significant effect of anterior-posterior position (p=0.752) on L max. The L min values (Fig. 24B) demonstrated a significant effect of speed (p<0.001) and incline (p=0.028) but not position (p=0.065).

93 Electrical Activity 76 The electrical activity variables that I measured are average emg per burst (Fig 25A), average emg per stride (Fig. 25B), integrated emg (Fig. 25C), emg duration, and emg start and stop time relative to foot down (Fig. 26). All of these variables measured, except for stop time relative to foot down (p=0.174), demonstrated a significant effect of incline (p<0.05) but showed variable results of significance with regard to speed and anterior-posterior position. Measured values of average emg per burst (Fig. 25A) showed a significant effect of speed (p<0.001) but not anterior-posterior position (p=0.067). Integrated emg values demonstrated no significant effects of position (p=0.214) or speed (p=0.773). My integrated emg values (Fig. 25C) showed no significant effect of speed because, even though the average emg increased with speed, the duration of the emg decreased with speed and the two changes cancelled themselves out. This cancellation is the reason why I used integrated values to calculate average emg per stride (Fig. 25B). Average emg per stride demonstrated a significant effect of speed (p<0.001), however there was no significant effect of anterior-posterior position (p=0.092). Average emg per stride generally tended to increase with speed. The emg timing variables I measured, including emg duration and emg start and stop time relative to foot down (Fig. 26), all demonstrated a significant effect of speed (p<0.001) but there was no significant effect of anterior-posterior position on any of these variables (p>0.05). Blood Flow vs. Emg Comparison Figure 27 shows normalized emg measurements and ILPO oxygen consumption measurements at specific speeds and inclines. By calculating a Pearson s correlation coefficient between ILPO oxygen consumption and emg intensity values, I are able to

94 show that the oxygen consumption values correlate with the average emg per burst 77 values (p=0.011), average emg per stride values (p=0.04) and integrated emg values (p=0.047).

95 Discussion 78 The anterior and posterior ILPO demonstrate quite similar patterns of lengthening and activation, being actively lengthened during the first part of stance followed by a period of active shortening. The only significant difference between the anterior and posterior ILPO occurs during the period of passive lengthening in early stance. Passive lengthening occurs during the first part of swing, when the knee is extending (Fig 28). The fascicles of the ILPO have the same insertion and therefore the same moment arm at the knee. However the fascicles of the anterior ILPO are much shorter, so for a given angular excursion the anterior ILPO will undergo the same absolute amount of length change as the posterior ILPO but a larger relative length change. I hypothesize that this is what causes the difference in passive lengthening seen between the anterior and posterior ILPO. Because the muscle is passive, the increase in active lengthening experienced by the anterior ILPO will not cause sarcomere damage via popping (Butterfield et al., 2005; Cutts, 1989; Gordon et al., 1966; Huijing, 1996; Koh and Herzog, 1998; Proske and Morgan, 2001) so compensatory mechanisms to prevent muscle damage during this phase of the lengthen-shorten cycle are not necessary. Another chapter (Carr, 2008, Chapter 2) discusses the compensatory mechanisms between the anterior and posterior fascicles of the ILPO, including the aponeurotic tendon and the variable moment arm at the hip, that keep strain uniform between fascicles during the active part of the cycle. Because those compensatory effects have already been discussed elsewhere, this chapter will focus more on the general pattern of function in the ILPO, including the function of the ILPO when it is being actively

96 lengthened, its function during active shortening, and why I believe the ILPO shifts in 79 length-tension operating range as incline increases. Function of Active Lengthening in the ILPO It has been hypothesized that active lengthening in the ILPO increases the amount of force it can produce during shortening via stretch-induced activation (McGowan et al., 2006). While stretch-induced activation probably occurs in the posterior ILPO, I do not think that this is the primary function of the active lengthening in this muscle. Active lengthening does increase the amount of work produced when shortening occurs (Edman et al., 1982), however the amount of work that goes into the muscle to actively lengthen it to such a large extent (4 to 10% at the highest speeds) is greater than the extra amount of work that come out of the muscle during subsequent active shortening (Josephson and Ellington, 1997; Takarada et al., 1997). I hypothesize that the primary function of the ILPO during active lengthening is to stabilize the knee joint, both by helping to maintain the stiffness of the limb at the level of the knee joint and also by absorbing work by increasing the amount of active lengthening that may occur during a perturbation at the joint when the foot hits the ground (Buchanan, 1999; Marsh, 1999). The ILPO early in stance is coactivated (Marsh Lab, unpublished results) and actively lengthened, producing a force to oppose the action of the knee flexors including the flexor cruris medialis and the posterior iliofibularis (Gatesy, 1999; Hoogendyk et al., 2005) that are active during this period. During active lengthening, a muscle has active fibers that are operating on the negative velocity portion of the force-velocity curve. By operating on the negative velocity portion of the force velocity curve, the ILPO is able to produce a large and stable force opposing muscles to

97 contract against and is capable of absorbing a large amount of work (Josephson and 80 Stokes, 1999). Coactivation and eccentric contractions appear to be common in many animals during cursorial locomotion. These have been hypothesized as a method of regulating joint stiffness in order to keep the movement of the joint steady and may play a role in response to perturbation (Lamontagne et al., 2000; Loeb, 1995). Coactivation and eccentric contractions occur in major leg muscles of the cockroach during steady state running (Full and Stokes, 1998). In human running, the gluteus maximus shows increased emg activity during foot descent as speed increases (Mann et al., 1986). This muscle probably acts to decelerate the thigh prior to foot contact. Also, the gluteus medius and tensor fascia undergo eccentric contraction during the beginning of the support phase. Another set of human muscles that undergo coactivation during walking and running are the quadriceps and hamstrings (Osternig et al., 1995). Coactivation of the quadriceps and the hamstrings appears to be particularly important during the first part of stance when the knee is flexing and the foot is hitting the ground. Researchers have hypothesized that hamstring coactivation provides a mode of dynamic joint stiffness during knee extension and aids the anterior cruciate ligament in maintaining joint stability by exerting a torque opposite that produced by the quadriceps (Osternig et al., 1995). Incorporating joint stiffness into models of locomotion in various organisms has been one method used to obtain stable locomotion. One such model by Wagner and Blickhan (2003) stabilized the knee in the absence of neuronal feedback by incorporating increased cocontraction of an antagonistic-flexor extensor system around a knee joint with a

98 moving center of rotation. Wagner and Blickhan (2003) found that by increasing 81 cocontraction around the joint, the joint stabilized itself independent of any neuronal feedback. Coactivation does decrease the economy of walking and running, by increasing the amount of negative work done at a joint, however coactivation may be an adaptive, pre-flexive response to perturbations (Dietz et al., 1990; Loeb, 1995) that may compensate for an abrupt change in foot placement and thus possibly prevent damage to other structures. Measured quadriceps emg signals in human walkers have been shown to increase during the stance phase of walking (Lamontagne et al., 2000). It has been hypothesized that this increase in emg signals during stance may automatically and rapidly contribute to and compensate for an unexpected hole or obstacle (Dietz et al., 1990). Studies have shown that if landing of the swing limb is perturbed, an enlarged emg signal is seen in the biceps femoris and the gastrocnemius of the stance leg, and there is increased cocontraction in the rectus femoris and tibialis anterior (Dietz et al., 1986). Coactivation has also been hypothesized to play a role in preventing ligament damage. In a study by Pope et al. (Pope et al., 1979), muscle activation times and the amount of torque required for the knee joint to feel pain were measured. A skiing injury that produced enough force to rupture the medial collateral ligament was simulated to determine if inactive muscles could become active and produce a sustained contraction fast enough to prevent rupture of this ligament. The results were that 39ms after a perturbation, ligament loading began, while pain was estimated to be perceived at 51.9ms and ligament rupture occurred at 73ms. The average reflex response was not observed until 128ms after the perturbation and a forceful sustained contraction did not occur until

99 215ms after the perturbation. If the antagonistic muscle was not activated prior to the 82 perturbation, then the ligaments at the knee would be overloaded before a muscle could counter the extra force at the knee joint. In essence a coactivating muscle may be acting as an additional safety factor to prevent tendon damage by absorbing excess force at the joint. Function of the ILPO During Active Shortening The ILPO is actively shortening in the latter half of stance while both the hip and the knee are extending. Previous studies in my lab have demonstrated the ILPO has a significant moment at the knee and at the hip (Carr, 2008, Chapter 2). I hypothesize that the ILPO is producing positive work during active shortening that contributes to both knee and hip extension during that portion of its cycle. V max for the lateralis gastrocnemius in the guinea fowl has been estimated at 15.3 L*s -1 based on allometry and studies done in turkeys (Askew et al., 2001; Henry, 2003; Nelson et al., 2004). The ILPO during active shortening experiences an average velocity no greater than 2.24±0.20 L*s -1 at 3.0 ms -1 during level running well below the V max estimated in the lateral gastrocnemius allowing the muscle to produce large amounts of force and work at this speed. Separating out how much of the positive work done by the ILPO is acting about the knee and how much is acting about the hip is beyond the scope of this experiment. Length Tension Effects Roberts et al (2007) found that the ILPO in the turkey demonstrated a shift in muscle function from net energy absorption during downhill running to net energy production during uphill running as evinced by the decrease in active lengthening and the increase in active shortening as incline increased. My birds were not run on a downhill

100 slope, however I also have shown a decrease in active lengthening and an increase in 83 active shortening as a function of incline. In addition to changes in overall strain, the ILPO appears to demonstrate a functional shift on the length tension curve that allows it to produce more work per active fiber during active shortening by shifting the length at which active shortening begins to points that are further up along the length tension curve (Fig. 29): this allows the muscle to actively shorten for a longer period of time along the plateau region of the length tension curve. Even though the muscle is capable of producing more force due to this shift along the length tension curve, it is shortening at a greater velocity (Fig. 23A), therefore the shift to greater forces along the length tension curve will be at least partially cancelled out by the increase in velocity because force is going to decrease with the increase in velocity. These velocity effects may be partially compensated for by the increases in activation seen as a function of incline, as demonstrated by the increase in both average emg per burst and average emg per stride as a function of incline (Fig. 25A & B). While my results also demonstrate that the amount of active lengthening decreases as a function of incline, the muscle is shifted to a higher operating range on the length tension curve (Fig. 29) and at lower velocities (Fig. 23B): both conditions allow the muscle to produce more force and hence more work despite the smaller length change. My results support the Roberts et al. (2007) hypothesis that there appears to be a shift from force absorption to force production in response to incline, but because I have no direct force measurements from the muscle, I cannot be sure how much the amount of force and hence work is modulated by the muscle as a function of increased speed or incline.

101 Emg Analysis versus Blood Flow 84 Many studies have used emg measurements as a relative measure of how much energy a muscle is using (Dufour et al., 2007; Mian et al., 2006; Smith and Newham, 2007). My study found that there was a significant correlation between the emg activation levels measured and the oxygen consumption of the ILPO above rest. Because the emg and ILPO oxygen consumption measurements were done on different animals, it is not possible to directly compare birds to one another, and all I can say is that there is a significant correlation between the two sets of values. However based on this study, the emg parameters measured (average emg per stride, average emg per burst and integrated emg) do significantly correlate with muscle oxygen consumption by the ILPO as a function of speed and incline. Summary I believe that during active lengthening in running the ILPO plays an important role in stabilizing the limb against external destabilizing forces and possibly preventing tendon damage. Even though active lengthening and work absorption decrease the economy of locomotion, they may act to stabilize the joint and provide a protective mechanism preventing the ligaments around joints from being damaged in the case of an unexpected perturbation such as a rock or other obstacle encountered during locomotion. The ILPO may help stabilize the knee joint by coactivation during early stance when it is actively lengthened acting as an agonist to active knee flexors. I also hypothesize that the ILPO acts as both a hip and knee extensor during active shortening although I are unable at this time to separate out how much work the muscle does at each joint. The ILPO appears to be capable of modulating its function in response to both incline and speed

102 although how much the actual amount of work produced by the muscle changes as a 85 function of either of these variables is obfuscated by corresponding changes in lengthtension, velocity and muscle activation. Finally, common electrical activity variables that have been used as de facto measurements of muscle energetics do appear to be correlated with muscle energy use as measured by blood flow (hence oxygen consumption) of the ILPO.

103 86 Literature Cited Askew, G. N., Marsh, R. L. and Ellington, C. P. (2001). The mechanical power output of the flight muscles of blue-breasted quail (Coturnix chinensis) during take-off. J. Exp. Biol. 204, Berger, A. J. (1952). The comparative functional morphology of the pelvic appendage in three genera of Cuculidae. Am. Midl. Nat. 47, Berger, A. J. (1956). Myology of Sandhill Crane. Wilson Bull. 68, Berman, S. L. (1984). The hindlimb musculature of the White-Fronted Amazon (Amazona albicans, Psittaciformes). Auk 101, Berman, S. L. and Raikow, R. J. (1982). The hindlimb musculature of mousebirds (Coliiformes). Auk 99, Buchanan, C. I. (1999). Muscle Function and Tendon Adaptation in Guinea Fowl (Numida meleagris). In Biology, vol. Ph D., pp Boston: Northeastern University. Butterfield, T. A., Leonard, T. R. and Herzog, W. (2005). Differential serial sarcomere number adaptations in knee extensor muscles of rats is contraction type dependent. J. Appl. Physiol. 99, Carrier, D. R., Gregersen, C. S. and Silverton, N. A. (1998). Dynamic gearing in running dogs. J. Exp. Biol. 201, Cavanagh, P. R. and Komi, P. V. (1979). Electromechanical delay in human skeletal muscle under concentric and eccentric contractions. Eur. J. Appl. Physiol. Occup. Physiol. 42, Cracraft, J. (1971). The functional morphology of the hind limb of the domestic pigeon, Columba livia. Bull. Am. Mus. Nat. Hist. 14, Cracraft, J. (1988). The major clades of birds. Oxford: Clarendon Press. Cutts, A. (1989). Sarcomere length changes in muscles of the human thigh during walking. J. Anat. 166, Daniel, T. L. (1984). Unsteady aspects of aquatic locomotion. Am. Zool. 24, 121-

104 87 Dietz, V., Faist, M. and Pierrot-Deseilligny, E. (1990). Amplitude modulation of the quadriceps H-reflex in the human during the early stance phase of gait. Exp. Brain Res. 79, Dietz, V., Quintern, J., Boos, G. and Berger, W. (1986). Obstruction of the swing phase during gait: phase-dependent bilateral leg muscle coordination. Brain Res. 384, Dufour, S. P., Doutreleau, S., Lonsdorfer-Wolf, E., Lampert, E., Hirth, C., Piquard, F., Lonsdorfer, J., Geny, B., Mettauer, B. and Richard, R. (2007). Deciphering the metabolic and mechanical contributions to the exercise-induced circulatory response: insights from eccentric cycling. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R Edman, K. A., Elzinga, G. and Noble, M. I. (1982). Residual force enhancement after stretch of contracting frog single muscle fibers. J. Gen. Physiol. 80, Ellerby, D. J., Henry, H. T., Carr, J. A., Buchanan, C. I. and Marsh, R. L. (2005). Blood flow in guinea fowl Numida meleagris as an indicator of energy expenditure by individual muscles during walking and running. J. Physiol. 564, Fisher, H. I. (1946). Adaptations an comparative anatomy of the locomotor apparatus of new world vultures. Am. Midl. Nat. 35, Full, R. J. and Stokes, D. (1998). Energy absorption during running by leg muscles in a cockroach. J. Exp. Biol Gabriel, D. A. and Boucher, J. P. (1998). Effects of repetitive dynamic contraction upon electromechanical delay. Eur. J. Appl. Physiol. Occup. Physiol. 79, Gangl, D., Weissengruber, G. E., Egerbacher, M. and Forstenpointer, G. (2004). Anatomical description of the muscles of the pelvic limb in the ostrich (Struthio camelus). Anat. Histol. Embryol. 33, Garrod, A. H. (1873). On certain muscles of the thigh of birds and their value in classification. J. Zool. (Lond.), Gatesy, S. M. (1999). Guineafowl hindlimb function II: electromyographic analysis and motor pattern evolution. J. Morphol. 240, George, J. C. and Berger, A. J. (1966). Avian Myology. New York and London: Academic Press.

105 Gillis, G. B. and Biewener, A. A. (2001). Hindlimb muscle function in relation to speed and gait: in vivo patterns of strain and activation in a hip and knee extensor of the rat (Rattus norvegicus). J. Exp. Biol. 204, Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, Henry, H. T. (2003). Jumping Performance in Guineafowl (Numida meleagris). In Biology, vol. Master of Science, pp. 73. Boston: Northeastern University. Hoogendyk, T., Carr, J. A., Henry, H. T., Rubenson, J. and Marsh, R. L. (2005). Mechanical and neural determinants of differences in fascicle strain between functionally distinct compartments in M. Iliofibularis during terrestrial locomotion in guinea fowl (Numida meleagris). Integr. Comp. Biol. 45. Hudson, G. E. (1937). Studies on the muscles of the pelvic appendages of birds. Am. Midl. Nat. 18, Hudson, G. E., Hoff, K. M., Vanden Berge, J. and Trivette, E. C. (1969). A numerical study of the wing and leg muscle of Lari and Alcae. Ibis (Lond. 1859) 111, Hudson, G. E., Lanzillotti, P. J. and Edwards, G. E. (1959). Muscles of the pelvic limb in galliform birds. Am. Midl. Nat. 61, Hudson, G. E., Schreiweis, D. O., Wang, S. Y. and Lancaster, D. A. (1972). A numerical study of the wing and leg muscle of tinamous. Northwest Sci. 46, Huijing, P. A. (1996). Important experimental factors for skeletal muscle modelling: non-linear changes of muscle length force characteristics as a function of degree of activity. Eur. J. Morphol. 34, Humason, G. L. (1979). Animal Tissue Techniques. San Francisco: W. H. Freeman and Company. Josephson, R. and Ellington, C. (1997). Power output from a flight muscle of the bumblebee Bombus terrestris. I. Some features of the dorso-ventral flight muscle. J. Exp. Biol. 200, Josephson, R. K. and Stokes, D. R. (1999). The force-velocity properties of a crustacean muscle during lengthening. J. Exp. Biol. 202 (Pt 5), Klemm, R. D. (1969). Comparative myology of the hindlimb of procellariiform birds. Southern Illinois University Monographs,

106 89 Koh, T. J. and Herzog, W. (1998). Increasing the moment arm of the tibialis anterior induces structural and functional adaptation: implications for tendon transfer. J. Biomech. 31, Lamontagne, A., Richards, C. L. and Malouin, F. (2000). Coactivation during gait as an adaptive behavior after stroke. J. Electromyogr. Kinesiol. 10, Loeb, G. E. (1995). Control implications of musculosketelal mechanics. Conf. Proc. IEEE Eng. Med. Biol. Soc. 17, Mann, R. A., Moran, G. T. and Dougherty, S. E. (1986). Comparative electromyography of the lower extremity in jogging, running, and sprinting. Am. J. Sports Med. 14, Marsh, R. L. (1999). How muscles deal with real-world loads: the influence of length trajectory on muscle performance. J. Exp. Biol. 202, Marsh, R. L., Ellerby, D. J., Carr, J. A., Henry, H. T. and Buchanan, C. I. (2004). Partitioning the energetics of walking and running: swinging the limbs is expensive. Science 303, Maurer, D. R. and Raikow, R. J. (1981). Appendicular myology, phylogeny and classification of the avian order Coraciiformes (Including Trogoniformes). Annals of Carnegie Museum 50, McGowan, C. (1979). The hind limb musculature of brown kiwi, Apteryx australis mantelli. J. Morphol. 160, McGowan, C. P., Duarte, H. A., Main, J. B. and Biewener, A. A. (2006). Effects of load carrying on metabolic cost and hindlimb muscle dynamics in guinea fowl (Numida meleagris). J. Appl. Physiol. 101, McKitrick, M. C. (1991). Phylogentic analysis of avian hindlimb musculature. Miscellaneous Publication Museum of Zoology, University of Michigan, Mellett, F. D. (1994). A note on the musculature of the proximal part of the pelvic limb of the ostrich (Struthis camelus). J. S. Afr. Vet. Assoc. 65, 5-9. Mian, O. S., Thom, J. M., Ardigo, L. P., Narici, M. V. and Minetti, A. E. (2006). Metabolic cost, mechanical work, and efficiency during walking in young and older men. Acta Physiol. (Oxf.) 186, Miller, A. H. Structural Modifications in the Hawaiian goose (Nesochen sandvicensis). University of California Museum of Vertebrate Zoology 42, Mindell, D. P., Sorenson, M. D., Huddleston, C., Miranda, H., Knight, A., Sawchuk, S. J. and Yuri, T. (1997). Phylogenetic relationships among and within select

107 avian orders based on mitochondrial DNA. In Avian Molecular Evolution and Systematics, (ed. D. P. Mindell). San Diego: Academic Press. 90 Mitchell, P. C. (1915). Anatomical notes on the gruiform birds Aramus giganteus and Rhinocetes kagu. J. Zool. (Lond.), Moritani, T., Berry, M. J., Bacharach, D. W. and Nakamura, E. (1987). Gas exhange parameters, muscle blood flow and electromechanical properties of the plantar flexors. Eur. J. Appl. Physiol. Occup. Physiol. 56, Muraoka, T., Muramatsu, T., Fukunaga, T. and Kanehisa, H. (2004). Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo. J. Appl. Physiol. 96, Nelson, F. E., Gabaldon, A. M. and Roberts, T. J. (2004). Force-velocity properties of two avian hindlimb muscles. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 137, Olson, J. M. and Marsh, R. L. (1998). Activation patterns and length changes in hindlimb muscles of the bullfrog Rana catesbeiana during jumping. J. Exp. Biol. 201, Osternig, L. R., Caster, B. L. and James, C. R. (1995). Contralateral hamstring (biceps femoris) coactivation patterns and anterior cruciate ligament dysfunction. Med. Sci. Sports Exerc. 27, Owre, O. T. (1967). Adaptations for locomotion and feeding in the anhinga and the double-crested cormorant. In Ornithological Mongraphs #6, (ed. R. L. Zusi): The American Ornithologists Union. Patak, A. E. and Baldwin, J. (1998). Pelvic Limb Musculature in the Emu Dromaius novhollandiae (Aves: Struthioniformes: Dromaiidae): Adaptations to High- Speed Running. J. Morphol. 238, Podulka, S., Rohrbaugh, R. W. and Bonney, R. E. (2004). Handbook of Bird Biology. Ithaca New York: Cornell Lab of Ornithology. Pope, M. H., Johnson, R. J., Brown, D. W. and Tighe, C. (1979). The role of the musculature in injuries to the medial collateral ligament. J. Bone Joint Surg. Am. 61, Proske, U. and Morgan, D. L. (2001). Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J. Physiol. 537, Raikow, R. J. (1970). Evolution of Diving Adaptations in the Stifftail Duck. University of California Publications in Zoology 94.

108 Raikow, R. J. (1976). Pelvic myology of the hawaiian honeycreepers (Drepanidae). Auk 93, Raikow, R. J. (1978). Appendicular myology and relationships to the New World Nine Primaries Oscines (Aves: Passeriformes). Bulletin of Carnegie Museum of Natural History 7, Raikow, R. J. (1980). Appendicular myology and relationships of the shrikes (Aves: Passeriformes: Laniidae). Annals of Carnegie Museum 49, Raikow, R. J. (1985). Avian Myology. In Form and Function in Birds, eds. A. S. King and J. McLelland), pp : Academic Press Inc. Roberts, T. J., Higginson, B. K., Nelson, F. E. and Gabaldon, A. M. (2007). Muscle strain is modulated more with running slope than speed in wild turkey knee and hip extensors. J. Exp. Biol. 210, Roberts, T. J. and Scales, J. A. (2004). Adjusting muscle function to demand: joint work during acceleration in wild turkeys. J. Exp. Biol. 207, Rosser, B. W., Secoy, D. M. and Riegert, P. W. (1982). The leg muscles of the American coot (Fulica atra gmelin). Can. J. Zool. 60, Ruck, P. R. (1949). Studies on the Muscles of the Pelvic Appendage in Certain Anseriform Birds. In Biology, pp Pullman: State College of Washington. Schreiweis, D. O. (1982). A comparative study of the appendicular musculature of penguins (Aves: Sphenisciformes). Smithsonian Contribution to Zoology 341, Sibley, C. G. and Alquist, J. E. (1990). Phylogeny and Classification of Birds: A Study in Molecular Evolution. New Haven, CT: Yale University Press. Sibley, D. A. (2001). The Sibley Guide to Bird Life and Behavior. New York: Alfred A. Knopf. Smith, I. C. and Newham, D. J. (2007). Fatigue and functional performance of human biceps muscle following concentric or eccentric contractions. J. Appl. Physiol. 102, Swierczewki, E. V. and Raikow, R. J. (1981). Hind limb morphology, phylogeny, and classification of the piciformes. Auk 98, Takarada, Y., Iwamoto, H., Sugi, H., Hirano, Y. and Ishii, N. (1997). Stretchinduced enhancement of mechanical work production in frog single fibers and human muscle. J. Appl. Physiol. 83,

109 Vanden Berge, J. (1982). Notes of the myology of the pelvic limb in kiwi (Apteryx) and in other birds. Auk 99, Verstappen, M., Aerts, P. and De Vree, F. (1998). Functional morphology of the hindlimb musculature of the black-billed magpie, Pica pica (Aves, Corvidae). Zoomorphology 118, Wilcox, H. H. (1952). The pelvic musculature of the loon, Gavia immer. Am. Midl. Nat. 48, Winter, E. M. and Brookes, F. B. (1991). Electromechanical response times and muscle elasticity in men and women. Eur. J. Appl. Physiol. Occup. Physiol. 63, Zhou, S., Lawson, D. L., Morrison, W. E. and Fairweather, I. (1995). Electromechanical delay in isometric muscle contractions evoked by voluntary, reflex and electrical stimulation. Eur. J. Appl. Physiol. Occup. Physiol. 70, Zusi, R. L. and Bentz, G. D. (1982). Variation of a muscle in hummingbirds and swifts and its systematic implications. Proceedings of the Biological Society of Washington 95, Zusi, R. L. and Bentz, G. D. (1984). Myology of the Purple-throated Carib (Eulampis jugularis) and Other Hummingbirds (Aves: Throchilidae). Smithsonian Contribution to Zoology 385, 1-70.

110 93 Figure 18: Superficial muscles in the guinea fowl hindlimb. The ILPO is pictured in white. Red circles indicate sonomicrometry crystal placement. At least one emg electrode was placed along the fascicle per pair of crystals. Drawn by Dr. David Ellerby

111 94 ILPO is Missing or Reduced ILPO is Present Pelacaniiformes ** ( Pelicans, Boobies, Tinamiformes (Tinamous) Cormorants, Anhingas & Frigate birds) Dinornithiformes (Kiwis) Procellariformes (Albatrosses, Shearwaters and Rheiformes (Rheas) Petrels) Struthioniformes (Ostriches) Sphenisciformes (Penguins) Casuariiformes (Cassowaries & Emus) Falconiformes (Old World Vultures & Diurnal Galliformes (Turkeys, Pheasants and Guinea Fowl) Birds of Prey) Podicipediformes (Grebes) Chadradriiformes (Shorebirds, Gulls and Auks) Gaviiformes (Loons) Psittaciformes (Parrots, Parakeets, Lories & Ciconiiformes (Herons, Ibis s, Storks & New World Macaws) Vultures) *** Coliiformes (Mousebirds) Gruiformes (Cranes, Rails and Allies) Musophagiformes (Turaco) Columbiformes (Pigeons and Doves) Strigiiformes (Owls) Cuculiformes (Cuckoos, Roadrunners & Anis) Caprimulgiformes (Frogmouth, Nightjar & Opisthocomiformes (Hoatzin) Goatsucker) Phoenicopteriformes (Flamingos) Apodiformes (Swifts and Hummingbirds) Trogoniiformes (Trogons) Upupiformes (Hoopoes & Woodhoopoes) Coraciiformes (Kingfishers, Hornbills and Allies) Piciformes (Woodpeckers and Allies) Passeriformes (Perching Birds) * Anseriformes (Ducks, Geese & Swans) Figure 19: Bird Orders in which the ILPO is missing or reduced. * ILPO is reduced in some families of Passeriformes including the Tyrannidae, Hirundinidae and Cotingidae. ** It is unknown whether the ILPO is reduced in the pelicans. *** The ILPO is reduced in the family Sulidae and the Marabou Stork. It is unknown whether the ILPO is present or absent in this order. Anatomy taken from (Berger, 1952; Berger, 1956; Berman, 1984; Berman and Raikow, 1982; Cracraft, 1971; Fisher, 1946; Gangl et al., 2004; Garrod, 1873; George and Berger, 1966; Hudson, 1937; Hudson et al., 1969; Hudson et al., 1959; Hudson et al., 1972; Klemm, 1969; Maurer and Raikow, 1981; McGowan, 1979; McKitrick, 1991; Mellett, 1994; Miller, ; Mitchell, 1915; Owre, 1967; Patak and Baldwin, 1998; Raikow, 1970; Raikow, 1976; Raikow, 1978; Raikow, 1980; Raikow, 1985; Rosser et al., 1982; Ruck, 1949; Schreiweis, 1982; Swierczewki and Raikow, 1981; Vanden Berge, 1982; Verstappen et al., 1998; Wilcox, 1952; Zusi and Bentz, 1982; Zusi and Bentz, 1984). Phylogeny compiled from (Cracraft, 1988; Mindell et al., 1997; Podulka et al., 2004; Sibley and Alquist, 1990)

112 110 A %L B Millivolts % Stride Cycle Figure 20: Length change normalized to %L 0 and emg activity across a stride cycle from foot down to foot down in the anterior (red) and posterior (blue) at 0.5 ms -1 and 2.5 ms -1 in the guinea fowl. Grey bands represent ± one standard error of the mean. Black arrow represents toe off.

113 96 12 A %L 0 25 B Speed (ms -1 ) Figure 21: Active lengthening (A) and active shortening (B) strain in the ILPO on the level (black), 10% incline (blue) and 20% incline (red) as a function of speed in the anterior ( ) and posterior ( ) ILPO. A significant effect of speed and incline was demonstrated for active lengthening and active shortening. Anterior values are slightly offset from actual speeds.

114 97 30 A %L B Speed (ms -1 ) Figure 22: Passive lengthening (A) and passive shortening (B) strain in the ILPO on the level (black), 10% incline (blue) and 20% incline (red) as a function of speed in the anterior ( ) and posterior ( ) ILPO. A significant effect of speed, incline and position was demonstrated for passive lengthening. Anterior values are slightly offset from actual speeds.

115 A Velocity (Ls -1 ) B Speed (ms -1 ) Figure 23: Active shortening velocity (A) and active lengthening velocity (B) in the in the anterior ( ) and posterior ( ) ILPO on the level (black), 10% incline (blue) and 20% incline (red) as a function of speed. There was a significant effect of speed and incline on average velocity during active shortening. Anterior values are slightly offset speed.

116 A Length (µm) B Speed (ms -1 ) Figure 24: L max values (A) and L smin values (B) in the anterior ( ) and posterior ( ) ILPO on the level (black), 10% incline (blue) and 20% incline (red) as a function of speed. Anterior values are slightly offset speed.

117 A Average Emg per Burst (mv) Average Emg per Stride (mv) B C Integrated Emg (mv * s) Speed (ms -1 ) Figure 25: Average emg values per burst (A), average emg values per stride (B) and integrated emg (C) in the anterior ( ) and posterior ( ) ILPO on the level (black), 10% incline (blue) and 20% incline (red) as a function of speed. Anterior values are slightly offset from speed.

118 Speed (ms -1 ) Time Relative to Foot Down (s) 0.4 Figure 26: Emg duration, start and stop time relative to foot down (0) in the anterior ILPO on the level (red), 10% incline (green), 20% incline (black) and in the posterior ILPO on the level (blue), 10% incline (yellow) and 20% incline (grey) vs. speed. Bars represent ± one standard error.

119 Percentage of Values at 2.78ms Level 1.5 Level (*) 1.5 Level (**) % Incline 2.78 Level Speed (ms -1 ) & Incline Figure 27: Predicted ILPO oxygen consumption over rest (red), average emg per burst (blue), average emg per stride (green) and integrated emg (black) all normalized to a percentage of the value measured at 2.78 ms -1 during level running. * value at 1.5 ms -1 is from Ellerby et al. (2005), ** value at 1.5 ms -1 is from Rubenson et al. (2006).

120 A B Joint Angle (degrees) C % Stride Cycle Figure 28: Knee (blue) and hip (red) joint angles in the guinea fowl during running on the level at 0.5 (A), 1.0 (B) and 2.5 (C) ms -1.

121 100 Active Lengthening Range Tension Active Shortening Range Level 10% Incline 20% Incline Length (µm) Figure 29: Active lengthening range (top) and active shortening range (bottom) during running at 2.0 ms -1 on the level (red), 10% incline (blue) and 20% incline (green).

122 105 Chapter 4: Iliotibialis Lateralis pars Post Acetabularis (ILPO) Function During Swimming and Running in an Aquatic and a Semi-Aquatic Bird Species Introduction Differences in habitat utilization and natural selection can lead to morphological variations, and these variations are often indicative of functional differences (Lowell, 1965). One of the ways animals utilize different habitats is by employing different forms of locomotion; this has led to adaptations that improve locomotion for a specific environment. Muscle structure and function is one morphological character that can be variable. In the hindlimb of birds I have studied a muscle, the Iliotibialis Lateralis pars Post Acetabularis (ILPO). The ILPO demonstrates adaptations that indicate it plays an important in terrestrial locomotion. The ILPO in the Helmeted Guinea Fowl (Numida melaeagris) (Fig. 30), a bird well known for its running ability, is the single largest muscle of the hindlimb of that species. The ILPO comprises 12.88±0.64% of the total muscle mass in the hindlimb, and receives more than 12.27% of the total hindlimb blood flow during running at 2.5ms -1 (Ellerby et al., 2005; Henry, 2003). It has been hypothesized that the ILPO plays an important role in running but the ILPO has a relatively complex strain cycle that has made it difficult to definitively determine its function. There have been several studies that looked at the function of the ILPO during running on the level, uphill and downhill, and the ILPO has been shown to perform two different functions (Buchanan, 1999; McGowan et al., 2006; Roberts et al., 2007).

123 106 During early stance the ILPO is initially lengthened while active and then actively shortens in the latter half of stance. Previous chapters of this dissertation have hypothesized that the ILPO, when it is being actively lengthened, stabilizes the knee joint by maintaining stiffness around the joint and by preventing damage to structures around the knee during external perturbations. During active shortening I have hypothesized that the ILPO acts as both a hip and a knee extensor (Carr, 2008, Chapter 3). Many comparative anatomical studies offer support to the hypotheses I have developed about the ILPO s important functions during terrestrial locomotion (Fig. 31). Unfortunately most of the studies do not offer precise quantitative information about muscle mass and dimensions and are more qualitative in nature, discussing muscles in terms of their presence, absence or reduction. However these studies do reveal that the ILPO demonstrates differences in muscle morphology that are correlated with the primary form of locomotion employed. The ILPO shows notable variation in orders of birds that employ different forms of locomotion. The ILPO has been lost or reduced in species in 17 orders of birds (Fig. 31). The ILPO has been lost in several orders and families of birds that are excellent flyers including the orders Procellariiformes, Falconiformes, Psittaciformes, Coliiformes, Caprimulgiformes, Strigiformes, and Apodiformes as well as representatives of the Passeriformes including Tyrannidae (tyrant flycatchers) and Hirundinidae (swallows and martins). Swallows and Flycatchers feed primarily on insects, employing aerial hawking, where they seize the insects directly in their bills. They spend little time on the ground and their terrestrial locomotion is usually restrained to one or two hops covering a short distance (Sibley, 2001). The ILPO has

124 107 also been reduced or lost in birds that spend a large amount of time swimming at the surface of the water, including members of Anseriformes, Pelecaniformes, Sphenisciformes, Ciconiiformes, and Charadriiformes. The New World Vultures (Ciconiiformes) have a well developed ILPO. It is possible that the ILPO was not lost in this group of birds that fly well because they are known to spend large amounts of time on the ground. The anatomical data demonstrate that the ILPO is reduced or absent in many birds that locomote primarily by foot-propelled surface swimming or by flying. In contrast to the foot-propelled surface swimmers grebes and loons (Podicipediformes and Gaviiformes), which are highly specialized foot-propelled divers, are reported to have a well-developed ILPO. However, substantial changes in limb morphology in these groups of birds may allow the ILPO to play a role in underwater swimming (Daniel, 1984; Raikow, 1970) since these birds do not spend large amounts of time locomoting terrestrially. In addition the ILPO is present in birds that are considered good cursorial locomotors such as the ratites (which are incapable of flight) and the Galliformes. Because the ILPO has been reduced or is absent in birds that do not locomote extensively on land, I hypothesize that in animals using both surface and terrestrial locomotion, the ILPO will be active during legged locomotion on land but not during foot-propelled surface swimming. If the ILPO plays a role in stabilizing the limb during running, then it is reasonable to observe that this muscle is reduced in birds that swim. Swimming birds with few exceptions are neutrally buoyant and do not encounter large destabilizing forces, therefore do not have need of muscles that produce stability against external perturbing forces. In addition the primary function of muscles during swimming

125 108 is to produce work and power to overcome drag, so a muscle that absorbed work while active would be an energetic liability and most likely would be selected for elimination or reduction via natural selection. In order to test my hypothesis I chose two species of bird and implanted the ILPO in both species with sonomicrometry crystals to measure length change and emg electrodes to measure muscle activity during both swimming and running. The goal was to choose species that routinely use both surface swimming and terrestrial locomotion but it is difficult to find species equally adapted to both locomotor modes. One species that I chose for these experiments, the Common Moorhen (Gallinula chloropus), has a body form more similar to terrestrial locomotors, and while it has no webbing between its toes, it is a competent swimmer (Taylor, 1998). The other species I chose the Mallard (Anas platyrhynchus) is considered to be more adapted to surface swimming but does routinely use walking and low speed running on land (Raikow, 1970). Both the common moorhen and the mallard have an ILPO that, while smaller, is similar in morphology to the guinea fowl. In addition to measuring length change and electrical activity during swimming and running, high speed video was recorded so that the activity of the ILPO could be related to the portion of the stride/swim cycle (stance versus swing or power stroke versus recovery stroke). Kinematic measurements taken from high-speed video included hip, knee and ankle angles during both swimming and running. In this chapter, measurements from the common moorhen and the mallard are compared within each species during both swimming and running. In addition, measurements are compared between the mallard, the common moorhen and the guinea

126 109 fowl during running and walking at speeds with similar duty factors. Similar duty factors were chosen instead of similar Froude numbers because Froude numbers assume isometric scaling (Alexander, 2005) which does not occur between these species of birds since they are adapted for quite different primary form of locomotion.

127 110 Materials and Methods Animals and Training Four male mallards and five common moorhens averaging 1.113±0.03 kg and 0.274±0.02 kg body mass were used for this experiment. Both species of birds were housed in communal cages at the Northeastern University Animal Care Facility with access to open water. Food and water was provided for both species ad libitum and kept of a 12:12 L:D cycle. Prior to surgery and experimental recordings, the birds were trained to run inside a three-sided box on a motorized treadmill. The birds were trained 3 days per week for a minimum of fourteen weeks. Each training session lasted for 20 minutes. After training the mallards and common moorhens could, in an individual session, maintain level speeds of 0.5 ms -1 for 5 minutes, 0.75 ms -1 for 5 minutes and 1.0 ms -1 for 2 minutes. Birds were also trained to swim inside a flow tank at a constant speed. Common moorhens at the end of training were able to maintain speeds of 0.5 ms -1 for 3 minutes, and mallards could maintain 0.7 ms -1 for 3 minutes; both speeds are at or very close to the animals hull speeds (Prange and Schmidt-Nielsen, 1970). Once a bird was sufficiently trained, a surgery was performed to insert sonomicrometry and emg sensors as described below. After surgery, birds were allowed to recover in individual cages for at least 40 hours prior to any experimental recordings. After recovery, birds were run on the treadmill and experimental recordings were taken as described below. Birds were run at speeds of 0.22, 0.33, 0.5, 0.67, 0.78, 0.89 and 1.0 ms -1 (moorhens were also run at speeds of 1.22 and 1.39 ms -1 ) on a level incline for at least 30 seconds per speed. Mallards were swam in the flow tank at speeds of 0.3, 0.35, 0.4, 0.45, 0.5, 0.55,

128 , 0.65 and 0.7 ms -1. Common moorhens swam in the flow tank at speeds of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5 ms -1. All of the mallards and two of the moorhens were sacrificed after their experimental recordings were taken, and the rest of the moorhens were sacrificed at a later date. Surgery All transducers were sterilized (12 hour ethylene oxide treatment) and implanted using sterile surgical techniques under general anesthesia (isofluorane). Two custommade 1-mm sonomicrometry transducers made from PZT discs (Boston Piezo-Optics, Inc.) and attached to stainless steel holders (Olson and Marsh, 1998) were implanted along fascicles of the ILPO (Fig. 30). Small punctures approximately 1mm in length were created in the connective tissue overlying the muscle to a depth of approximately 3mm. The crystals were then placed between the fascicles. The holders were secured to the overlying connective tissue using 6-0 silk sutures. Care was taken to ensure that the two crystals were always implanted between the same fascicles along the length of the muscle. Two fine-wire bipolar emg electrodes were implanted into each segment of the fascicles to measure muscle activity. The emg electrodes were constructed from 3T stainless steel Teflon-coated wire (Medwire) that was twisted along its length. The tips of the electrodes were bared 1.5 mm at each end and preactive ends spaced 3 mm apart. Emg electrodes were implanted in the ILPO with a chamfered 25 gauge needle at positions that were immediately anterior and posterior to each pair of crystals. Post

129 112 mortem examinations verified the placement of the sonomicrometry crystals and emg electrodes. Recordings Sonomicrometry measurements were acquired digitally with a sampling frequency of 613 Hz using Sonoview software and a Sonometrics TRX Series 8 interface. Emg signals were amplified by WPI model DAM-50 preamplifiers with high- and low-pass filters set at 10 and 3000 Hz respectively. Emg signals were collected at a frequency of 10kHz using an ADI Instruments Power Lab/16SP and a Macintosh PowerMac. The emg data was subsequently analyzed using the application Igor Pro (version 5.0). The signals were first filtered using a finite impulse response filter with a band pass of 90Hz to 1000Hz. Emg signals were rectified and the start time, stop time, and emg duration were all measured. For comparative measurements between species, all timing values were normalized to percent stride duration. The burst amplitudes were expressed in three ways: the integrated value of the burst duration; the average emg per burst (equal to the integrated value divided by the burst duration); and the average emg per stride (equal to the integrated value divided by the stride duration). Videography Video was recorded for the running mallards to VHS tape with a NAC HV-1000 high-speed video camera operating at 500 fields per second. I synchronized the emg and sonomicrometry traces during mallard running with the high-speed video recordings using a square-wave generated by the emg recording system which was recorded in the video fields with a NAC wave inserter. Mallard swimming, common moorhen running

130 113 and common moorhen swimming were recorded to VHS tape at 240Hz using a JVC Digital Video Camera model #GR-DVL9800. The JVC recordings were synchronized with the emg and sonomicrometry traces using a light pulse generated by the emg recording system which was placed in the visual field of the JVC. Visual markers were made using reflective tape. These markers were placed above the caudal and cranial pelvis; on the ankle, tarsometatarsal-phalangeal and interphalangeal joints; and on the toe of the bird. The hip position was calculated using a circle-circle intersection formula (Weisstein) and several distance measurements, including between the cranial pelvic marker and the hip, the caudal pelvic marker and the hip, and the cranial and caudal pelvic marker. Knee angle was calculated using the law of cosines and measured distances including the distance between the hip and the ankle markers based on post mortem measures of the distance between the center of rotation of the hip and center of rotation of the knee, and the distance between the center of rotation of the knee and the center of rotation of the ankle. Data were collected for at least ten steady speed strides at each speed for each bird. Sarcomere Measurements For two moorhens and four mallards, after each animal s experimental recordings had been completed, the animal was sacrificed and the instrumented leg was bolted into place with the hip and knee in a flexed position. The limb was allowed to sit in a bolted position for six hours until the leg muscles were in full rigor. Once the fixed limb was in rigor, a reference sonomicrometry measurement (denoted L so, ref ) was measured. In addition, a muscle temperature measurement was taken to correct for temperature effects

131 114 on the speed of sound. Two samples of the ILPO tissue, each measuring ~1cm 3, were removed from between each crystal pair. These muscle samples were frozen using liquid nitrogen and isopentane, and then sectioned using a cryostat. From each muscle sample, at least ten sections were placed on slides and stained using Weingert Iron Hematoxylin (Humason, 1979). From each slide (containing one stained section), pictures of at least three fibers were captured. Sarcomere lengths were measured using a compound microscope at 1000X magnification. Sarcomere measurements were performed using a digital imaging program (NIH Image J version 1.38). In Image J, a series of at least 20 sarcomeres was measured from each fiber, and the average individual sarcomere length (denoted L sc, ref ) was calculated by dividing the total length of the in-series sarcomeres by the number of sarcomeres in series. Using the reference sonomicrometry length and the measured sarcomere length, in vivo sonomicrometry lengths (L so ) were converted to estimated in vivo sarcomere lengths (L sc ) using the equation: L sc,ref L so L so,ref = L sc (1) Length measurements for the mallard are either reported as mm, or are converted to %L 0 using the following equation: %L 0 = L sc 100 (2) 2.36µm where L sc is the calculated sarcomere length from equation 1, and 2.36mm is the estimated sarcomere length for maximum force production based on the length in the middle of the plateau region of the calculated length-tension curve. Since I did not have sarcomere measurements for all of the common moorhens, their length measurements are

132 115 reported at %L 0 where L 0 is the maximum length of the ILPO measured during the stance phase of the stride cycle (L max, st ). Based on the two common moorhens with sarcomere measurements, I think this is probably a slight underestimate of the actual L 0 value. Length Changes Muscle segment lengths were measured from the sonomicrometry traces after the lengths in mm had been converted to µm in the case of the mallards or were measured in mm for the common moorhens. The length values measured during running were: L fd = minimum length of the ILPO, which occurred at or near the time of foot down L max,st = maximum length of the ILPO, which occurred during stance L min = minimum length of the ILPO, which occurred near the time of toe off L max,sw = maximum length of the ILPO, which occurred during swing L emg,on = length of the ILPO 25ms after the start of the emg activity L emg,off = length of the ILPO 25ms after the stop of the emg activity These measured lengths were used to calculate active shortening during swing, active lengthening during stance, active shortening during swing, passive lengthening and passive shortening. When calculating active lengthening and active shortening I assumed an electromechanical delay of 25 ms. I am not aware of studies done on the ILPO to confirm this assumption, but this time period falls within values measured for other muscles (Cavanagh and Komi, 1979; Gabriel and Boucher, 1998; Moritani et al., 1987; Muraoka et al., 2004; Winter and Brookes, 1991; Zhou et al., 1995). The equation used

133 116 to calculate a given length change was dependent on the timing of L emg,on and L emg,off relative to other lengths measured during a stride. Active shortening during swing ( L as,sw ) was calculated using one of three equations. If L emg,on occurred before L fd but after L max,sw then: L as,sw = L emg,on - L fd (3) If L emg,on occurred before L max,sw then: L as,sw = L max,sw L fd (4) If L emg,on occurred after L fd then: L as,sw = 0 (5) Active lengthening ( L al ) was calculated using one of two equations. If L emg,on occurred before L fd then: L al = L max,st - L fd (6) If L emg,on occurred after L fd then: L al = L max,st L emg,on (7) Active shortening during stance ( L as,st ) was also calculated using one of two equations. If L emg,stop occurred after L min then: L as,st = L max,st L min (8) If L emg,off occurred before L min then: L as,st = L max,st L emg,off (9) Passive lengthening ( L pl ) was calculated using one of four equations. If L emg,off occurred before L min and L emg,on for the next stride occurred after L max,sw then: L pl = L max,sw L min (10)

134 117 If L emg,off occurred after L min and L emg,on for the next stride occurred after L max,sw then: L pl = L max,sw L emg,off (11) If L emg,off occurred before L min and L emg,on for the next stride occurred before L max,sw then: L pl = L emg,on L min (12) If L emg,off occurred after L min and L emg,on for the next stride occurred before L max,sw then: L pl = L emg,on L emg,off (13) Passive shortening ( L ps ) was calculated using one of three equations. If L emg,on occurred before L max,sw then: L ps = 0 (14) If L emg,on occurred after L max,sw but before L fd then: L ps = L max,sw L emg,on (15) If L emg,on occurred after L max,sw and after L fd then: L ps = L max,sw L fd (16) The length values measured were during swimming in the common moorhen and mallard were: L max,ps = maximum length of the ILPO, which occurred at or near the time of the start of the power stroke L min,rs = minimum length of the ILPO, which occurred at or near the time of the start of the return stroke L emg,on = length of the ILPO 25ms after the start of the emg activity

135 118 L emg,off = length of the ILPO 25ms after the stop of the emg activity These measured lengths were used to calculate active shortening, active lengthening, and passive shortening. Active shortening ( L as,swim ) was calculated using one of four formulas. If L emg,on occurred before L max,ps and L emg,off occurred after L min,rs then: L as,swim = L max,ps - L min,rs (17) If L emg,on occurred before L max,ps and L emg,off occurred before L min,rs then: L as,swim = L max,ps L emg,off (18) If L emg,on occurred after L max,ps and L emg,off occurred after L min,rs then: L as,swim = L emg,on L min,rs (19) If L emg,on occurred after L max,ps and L emg,off occurred before L min,rs then: L as,swim = L emg,on L emg,off (20) Active lengthening ( L al,swim ) was calculated using one of two formulas. If L emg,off occurred after L min,rs then: L al,swim = L emg,off L min,rs (21) If L emg,off occurred before L min,rs then: L al,swim = 0 (22) Passive lengthening ( L pl,swim ) was calculated using one of four formulas. If L emg,off occurred after L min,rs and L emg,on for the next stride occurred after L max,ps then: L pl,swim = L max,ps - L emg,off (23) If L emg,off occurred before L min,rs and L emg,on for the next stride occurred after L max,ps then: L pl,swim = L max,ps - L min,rs (24)

136 119 If L emg,off occurred after L min,rs and L emg,on for the next stride occurred before L max,ps then: L pl,swim = L emg,on - L emg,off (25) If L emg,off occurred before L min,rs and L emg,on for the next stride occurred before L max,ps then: L pl,swim = L emg,on - L min,rs (26) All length changes are reported as %L 0, where L 0 for the mallards is the middle of the plateau region of the length tension curve, and L 0 for the common moorhens is L max,st. Statistical Analysis To test for significant differences in length change and electrical activity as a function of speed during swimming and running within a species, multivariate analyses were conducted using the general linear model in the application SPSS (version 15.0 for Microsoft Windows). Speed was used as a covariate in the model and an individual bird identifier was added to factor out variation between birds of the same species. Within a species, multivariate analyses were performed to determine whether the differences in strain were significant between running and swimming; swimming versus terrestrial locomotion was used as a factor in this model but specific speeds were not used. To determine if there was a significant difference in the emg timing between the FCL and the ILPO during swimming, a paired t-test was performed between the start and stop times of emg activity for the two muscles. Results were considered statistically significant if p All measurements are reported as a mean value plus or minus one standard error.

137 120 Results ILPO Length Changes During Running and Swimming Figure 32 shows the length change and duration of electrical activity in the ILPO during running on the level at 1.5 ms -1 in the guinea fowl (Fig. 32A), and at 1.0 ms -1 in the common moorhen (Fig. 32B) and the mallard (Fig. 32C). These speeds were picked for comparison because they had a similar duty factor. The length changes that occur in the ILPO in the guinea fowl during running have been previously described in another manuscript (Carr, 2008, Chapters 1, 2 and 3) so will not be dealt with in detail here. The ILPO in the common moorhen (Fig. 32B) has electrical activity that begins midway through the swing phase and continues through the stance phase of locomotion. This muscle in the moorhen, when active, demonstrates a period of shortening during swing, followed by a period of lengthening during stance and again shortens during the latter half of stance. The ILPO in the common moorhen also demonstrates a period of passive lengthening during the early part of stance and passive shortening during the latter half of stance. The mallard (Fig. 32C) shows a pattern of activation and length change during running that is similar to the common moorhen. The ILPO begins its activity in late swing, demonstrating a small amount of active shortening during swing, then is actively lengthened during early stance and actively shortens during the latter part of stance. The ILPO in the mallard also demonstrates a period of passive lengthening during the first part of swing and a period of passive shortening during the latter half of swing. Figure 33 shows ILPO length change and emg duration during a swim cycle, from the start of the power stroke to the end of the return stroke, in the mallard (Fig. 33A) at

138 ms -1 and the common moorhen at 0.35 ms -1 (Fig. 33B). During swimming in the mallard (Fig. 33A) the ILPO begins its activity towards the end of the return stroke and is active through the entirety of the power stroke. Very little active or passive length change occurs in the ILPO during this time although there are small amounts of active lengthening and active shortening that I attempted to quantify. In the common moorhen (Fig. 33B) the ILPO begins its activity at the very end of the return stroke and is actively shortening through the majority of the power stroke it goes through a small amount of active lengthening towards the end of the power stroke and then is passively lengthened through the majority of the return stroke. When comparing length change between terrestrial locomotion and swimming, active lengthening during the stance phase of terrestrial locomotion was compared to active lengthening during swimming and active shortening during the stance phase of terrestrial locomotion was compared with active shortening during swimming. In the common moorhen, active shortening (Fig. 34A) demonstrated a significant effect of speed during both terrestrial locomotion (p<0.001) and swimming (p=0.006). Active shortening was also found to be significantly different (p<0.001) between terrestrial locomotion and swimming. Active lengthening during terrestrial locomotion (p=0.011) and during swimming (p=0.018) both demonstrated a significant effect of speed and there was found to be a significant difference between active lengthening (Fig. 34B) during terrestrial locomotion and swimming (p<0.001). The length changes that occurred during active shortening and active lengthening were found to be greater during terrestrial

139 122 locomotion than during swimming and showed a general trend to increase as speed increased until the leveled out at the highest running speeds. During terrestrial locomotion in the mallard there was no significant effect of speed on the amount of active lengthening that occured (p=0.223), however there was a significant effect of speed on the amount of active lengthening that occured during swimming (p=0.014) (Fig. 35A). There was a significant effect of speed on active shortening during terrestrial locomotion (p=0.002) and during swimming (p=0.025) (Fig. 35B). The amount of active lengthening and active shortening that occurred in the ILPO in the mallard during swimming was significantly less than what occurred during terrestrial locomotion (Fig. 35A&B) (p<0.001). ILPO Electrical Activity During Running and Swimming Significant effects of speed were seen in electrical activity variables measured in the common moorhen during terrestrial locomotion. Speed had a significant effect on average emg per burst (p<0.001) (Fig. 36A), integrated emg (p=0.037) (Fig. 36B) and average emg per stride values (p<0.001) (Fig. 36C) in the common moorhen: there was an increase in all three variables as a function of speed up to 1.0 ms -1. However speed had no significant effect on any of the emg variables measured during swimming in the common moorhen (p>0.05) (Fig. 36A, B & C). When I looked at measurements of emg intensity between swimming and terrestrial locomotion in the common moorhen, there was no significant difference between the two forms of locomotion for the average emg per burst (Fig. 36A) (p=0.051) or the integrated emg values (Fig. 36B) (p=0.437). There was a significant difference in the average emg per stride values (Fig. 36C) (p=0.026).

140 123 This trend is probably due to the fact that stride times at the same speed in the same bird are approximately 33% shorter during swimming than during terrestrial locomotion. Significant effects of speed were demonstrated in the mallard for average emg per burst (p=0.001) (Fig. 37A), integrated emg (p<0.001) (Fig. 37B) and average emg per stride (p=0.002) (Fig. 37C) and all three variables increased with speed up to 0.89 ms -1. Speed did not have a significant effect on any of the emg variables measured during swimming in the mallard (p>0.05) (Fig. 37 A, B and C). In the mallard the differences between terrestrial locomotion and swimming were more pronounced than in the common moorhen. The emg intensity variables also showed a similar trend with the average emg per stride (Fig. 37A), the integrated emg (Fig. 37B) and the average emg per burst (Fig. 37C) always being significantly less (p 0.001) during swimming than in terrestrial locomotion. Because the emg values during swimming in the mallard were so small, and with the ILPO being relatively small compared to underlying larger muscles, one possibility is that the emg electrodes in the ILPO might have been picking up crosstalk from one of the other larger deep muscles in the thigh. A likely candidate for this would be the FCL which is a very large muscle located in the posterior thigh deep to the ILPO. To eliminate this as a possibility, I compared start and stop times relative to the start of power stroke measured in the FCL (unpublished results) and the ILPO to each other using paired t-tests. Significant differences were found between the FCL and the ILPO for start time relative to the start of power stroke (p=0.002) and for stop time relative to the start

141 124 of power stroke (p<0.001) demonstrating that the emg signals measured in the ILPO are not actually cross-talk measured from the FCL.

142 125 Discussion ILPO Function During Running My hypothesis that the ILPO is active during legged locomotion is supported by my results. Despite differences in outward appearance and primary locomotory modes the general function of the ILPO during terrestrial locomotion appears to be mostly conserved among the three species in which I did measurements. In all three species the ILPO experiences during early stance a period of active lengthening followed by active shortening. Even though the general pattern of activity and length change appear conserved, there are some important differences including differences in active lengthening and the period of active shortening that occurs during swing. Guinea fowl, common moorhens and mallards all have periods of active lengthening during stance. In the guinea fowl and the common moorhen, active lengthening during stance increases as a function of speed (Carr, 2008, Chapter 3), however in the mallard active lengthening does not increase with speed. If the function of active lengthening is to counteract knee flexion and cancel out forces produced by external perturbations (Carr, 2008; Buchanan, 1999; Marsh, 1999), then it would be necessary to produce a relatively constant force to oppose knee flexion and cancel out any perturbing force during early stance thus maintaining stiffness around the joint. However, mallards locomote primarily by waddling and other studies have demonstrated that waddling birds do not experience as great of a fluctuation in fore-aft ground reaction when compared with other animals that use terrestrial locomotion (Griffin and Kram, 2000). Therefore it is possible that the ILPO does not have to be actively lengthened as much in the mallard because the forces

143 126 they experience in the fore-aft direction may be more constant than either the common moorhen or the guinea fowl which do not use a waddling gait. In addition the mallard is the only bird I used whose toes are webbed. Webbing between the toes increases both the surface area of an animal s base of support and its step width, and thus provides increased stability (Donelan et al., 2004). The increase in stability due to the increase of the size of the base of support may allow the mallard to locomote terrestrially without increasing the amount of active lengthening with speed. The common moorhen experiences a period of active shortening during swing that is not seen in either the mallard or the guinea fowl. I believe that this is explained by the leg morphology of the common moorhen. The common moorhen is a wading bird with long legs relative to its body size (Taylor, 1998) and particularly long toes that it uses to grab onto and walk on floating reeds and grasses in its environment. In order for the common moorhen s long legs to clear the ground during swing, the moorhen experiences a large amount of extension at the knee and the hip in the latter part of swing (Fig. 38), and this may have to be actively powered by muscles. If hip and knee extension needs to be actively powered by muscles then this would explain the active shortening that the ILPO in the common moorhen experiences during swing. In order to test this hypothesis, studies would have to be performed to measure the joint moments and forces acting on the joints. Both the common moorhen and the mallard experience significant effects of speed on the magnitude of the average emg per burst and the average emg per stride. This is similar to what occurs in the guinea fowl (Carr, 2008, Chapter 3). However both

144 127 the common moorhen and the mallard duck experience increases in their integrated emg values as a function of speed. In the guinea fowl the increase in average emg with speed is offset by a decrease in duration so that the integrated emg remains constant (Carr, 2008, Chapter 3). It may be that either the average emg in the common moorhen or the mallard does not increase at the same rate as in the guinea fowl or the emg duration does not decrease at the same rate as the guinea fowl, leading to an increase in integrated emg as a function of speed in these two species. Length and Electrical Activity Changes During Swimming My hypothesis that the ILPO is not active during surface swimming in either the common moorhen or the mallard is not supported by my results. The ILPO in the common moorhen and the mallard are active during swimming, however the pattern of activation as a function of speed and the amount of the length change that occurs during swimming both suggest that the ILPO does not play as large a role during swimming as it does during running, particularly in the more aquatic mallard. Wave drag (the primary force acting on animals swimming at the water s surface) shows a general trend to increase with speed, the work to overcome that drag also needs to increase with speed (Fish, 2000; Fish and Baudinette, 1999). However, our results demonstrate that active shortening, the period where work would be being produced, is significantly less during swimming than it is during terrestrial locomotion in both the mallard and the common moorhen. In the mallard, active shortening is very small (less than 2%) except at the two highest swimming speeds (0.65 and 0.75 ms -1 ). The electrical activity variables I measured demonstrated no significant effects of speed for either the

145 128 common moorhen or for the mallard. This indicates that even though muscle strain increases with speed, the amount of muscle activation does not. If muscle strains are increasing but muscle activation is not changing than the muscle is shortening and lengthening faster but not recruiting a greater cross-sectional area (Woledge et al., 1985). If a muscle is operating at a higher velocity then the amount of force it can produce will decrease. Work is equal to force times distance, hence by not increasing the amount of force being produced by the muscle during length changes, the muscle is actually producing less force than it is capable of if a greater cross sectional area of the muscle was recruited. By not activating a greater cross-sectional area of the ILPO these species are producing less force in the muscle than they would be if the muscle activation increased, because force is proportional to cross sectional area (Biewener, 2003; Woledge et al., 1985). The argument that the ILPO does not play a very important role during swimming is also supported by blood flow data from other species of ducks. If the ILPO plays a role in swimming then its metabolic rate and the rate of blood flow to the muscle should increase during swimming. However in a Butler et al. study, per gram blood flow during swimming to the ILPO did not significantly increase from the values measured at rest (Butler et al., 1988). This lends support to the argument that the ILPO does not play a very important role during swimming, at least in terms of what is hypothesized to be the primary function of muscles during swimming, to shorten and produce work (Schmidt- Nielsen, 1972). This may explain why the ILPO has been lost or reduced in many orders of birds that use swimming as their primary form of locomotion.

146 129 Conclusion One of the current hypotheses for the function of the ILPO during active lengthening is that the ILPO is actively lengthened during the first part of stance to produce a relatively large stable force around the knee to oppose knee flexion and oppose any perturbing external forces (Buchanan, 1999; Marsh, 1999). Stability is an important function of muscles during terrestrial locomotion since one of the primary objectives during locomotion is to not fall down (Garcia et al., 1998) and one of the mechanisms hypothesized for assisting with stability is the cocontraction of antagonistic muscles around joints (Wagner and Blickhan, 2003). My measurements of length change and electrical activity in the mallard and the moorhen appear to support the stability hypothesis. The ILPO is active in the mallard, the common moorhen and the guinea fowl during terrestrial locomotion when there is the potential for external destabilizing forces acting on the knee joint. In addition I observe that the mallard, a bird which may experience less variation in the fore-aft forces it experiences due to its waddling gait (Griffin and Kram, 2000), does not demonstrate a significant increase in the amount of active lengthening seen in the ILPO, unlike the common moorhens and the guinea fowl species that do not have waddling gaits during terrestrial locomotion. During swimming the knee does not experience large external destabilizing forces so there is no need to produce a large force around the knee to stabilize against external perturbations. Although the ILPO is active during swimming in both the mallard and the common moorhen, my results suggest that the ILPO plays a minor functional role during swimming in either species. My results show that although the ILPO is active in the

147 130 common moorhen during swimming, it experiences less strain than it does during running. My measurements of ILPO function during swimming in the mallard show that there is a significant decrease in both strain and emg activity. Finally, there is no significant effect of speed on emg activity of the ILPO during swimming in either the common moorhen or the mallard, indicating that even though the limb as a whole must produce more work to go faster (Fish, 2000; Fish and Baudinette, 1999), it is not producing the extra work needed using the ILPO in either species of swimming birds. In conclusion the measured function of the ILPO during swimming in multiple species of birds that commonly use swimming as a form of locomotion provides support for the ILPO stability hypothesis. In a bird that is highly adapted for swimming such as the mallard, the ILPO shows significantly less electrical activity and strain during swimming than in the common moorhen, a bird that does not demonstrate as many specializations for swimming.

148 131 Literature Cited Alexander, R. M. (2005). Models and the scaling of energy costs for locomotion. J. Exp. Biol. 208, Berger, A. J. (1952). The comparative functional morphology of the pelvic appendage in three genera of Cuculidae. Am. Midl. Nat. 47, Berger, A. J. (1956). Myology of Sandhill Crane. Wilson Bull. 68, Berman, S. L. (1984). The hindlimb musculature of the White-Fronted Amazon (Amazona albicans, Psittaciformes). Auk 101, Berman, S. L. and Raikow, R. J. (1982). The hindlimb musculature of mousebirds (Coliiformes). Auk 99, Biewener, A. A. (2003). Animal Locomotion. Oxford: Oxford University Press. Buchanan, C. I. (1999). Muscle Function and Tendon Adaptation in Guinea Fowl (Numida meleagris). In Biology, vol. Ph D., pp Boston: Northeastern University. Butler, P. J., Turner, D. L., Al-Wassia, A. and Bevan, R. M. (1988). Regional distribution of blood flow during swimming in the tufted duck (Aythya fuligula). J. Exp. Biol. 135, Cavanagh, P. R. and Komi, P. V. (1979). Electromechanical delay in human skeletal muscle under concentric and eccentric contractions. Eur. J. Appl. Physiol. Occup. Physiol. 42, Cracraft, J. (1971). The functional morphology of the hind limb of the domestic pigeon, Columba livia. Bull. Am. Mus. Nat. Hist. 14, Cracraft, J. (1988). The major clades of birds. Oxford: Clarendon Press Daniel, T. L. (1984). Unsteady aspects of aquatic locomotion. Am. Zool. 24, 121- Donelan, J. M., Shipman, D. W., Kram, R. and Kuo, A. D. (2004). Mechanical and metabolic requirements for active lateral stabilization in human walking. J. Biomech. 37, Ellerby, D. J., Henry, H. T., Carr, J. A., Buchanan, C. I. and Marsh, R. L. (2005). Blood flow in guinea fowl Numida meleagris as an indicator of energy expenditure by individual muscles during walking and running. J. Physiol. 564,

149 132 Fish, F. E. (2000). Biomechanics and energetics in aquatic and semiaquatic mammals: platypus to whale. Physiol. Biochem. Zool. 73, Fish, F. E. and Baudinette, R. V. (1999). Energetics of locomotion by the Australian water rat (Hydromys chrysogaster): Comparison of swimming and running in a semiaquatic mammal. J. Exp. Biol. 202, Fisher, H. I. (1946). Adaptations an comparative anatomy of the locomotor apparatus of new world vultures. Am. Midl. Nat. 35, Gabriel, D. A. and Boucher, J. P. (1998). Effects of repetitive dynamic contraction upon electromechanical delay. Eur. J. Appl. Physiol. Occup. Physiol. 79, Gangl, D., Weissengruber, G. E., Egerbacher, M. and Forstenpointer, G. (2004). Anatomical description of the muscles of the pelvic limb in the ostrich (Struthio camelus). Anat. Histol. Embryol. 33, Garcia, M., Chatterjee, A., Ruina, A. and Coleman, M. (1998). The simplest walking model: stability, complexity, and scaling. J. Biomech. Eng. 120, Garrod, A. H. (1873). On certain muscles of the thigh of birds and their value in classification. J. Zool. (Lond.), George, J. C. and Berger, A. J. (1966). Avian Myology. New York and London: Academic Press. Griffin, T. M. and Kram, R. (2000). Penguin waddling is not wasteful. Nature 408, 929. Henry, H. T. (2003). Jumping Performance in Guineafowl (Numida meleagris). In Biology, vol. Master of Science, pp. 73. Boston: Northeastern University. Hudson, G. E. (1937). Studies on the muscles of the pelvic appendages of birds. Am. Midl. Nat. 18, Hudson, G. E., Hoff, K. M., Vanden Berge, J. and Trivette, E. C. (1969). A numerical study of the wing and leg muscle of Lari and Alcae. Ibis (Lond. 1859) 111, Hudson, G. E., Lanzillotti, P. J. and Edwards, G. E. (1959). Muscles of the pelvic limb in galliform birds. Am. Midl. Nat. 61, Hudson, G. E., Schreiweis, D. O., Wang, S. Y. and Lancaster, D. A. (1972). A numerical study of the wing and leg muscle of tinamous. Northwest Sci. 46,

150 133 Humason, G. L. (1979). Animal Tissue Techniques. San Francisco: W. H. Freeman and Company. Klemm, R. D. (1969). Comparative myology of the hindlimb of procellariiform birds. Southern Illinois University Monographs, Lowell, S. W. (1965). Climbing and pecking adaptations in some north american woodpeckers. Condor 67, Marsh, R. L. (1999). How muscles deal with real-world loads: the influence of length trajectory on muscle performance. J. Exp. Biol. 202, Maurer, D. R. and Raikow, R. J. (1981). Appendicular myology, phylogeny and classification of the avian order Coraciiformes (Including Trogoniformes). Annals of Carnegie Museum 50, McGowan, C. (1979). The hind limb musculature of brown kiwi, Apteryx australis mantelli. J. Morphol. 160, McGowan, C. P., Duarte, H. A., Main, J. B. and Biewener, A. A. (2006). Effects of load carrying on metabolic cost and hindlimb muscle dynamics in guinea fowl (Numida meleagris). J. Appl. Physiol. 101, McKitrick, M. C. (1991). Phylogentic analysis of avian hindlimb musculature. Miscellaneous Publication Museum of Zoology, University of Michigan, Mellett, F. D. (1994). A note on the musculature of the proximal part of the pelvic limb of the ostrich (Struthis camelus). J. S. Afr. Vet. Assoc. 65, 5-9. Miller, A. H. Structural Modifications in the Hawaiian goose (Nesochen sandvicensis). University of California Museum of Vertebrate Zoology 42, Mindell, D. P., Sorenson, M. D., Huddleston, C., Miranda, H., Knight, A., Sawchuk, S. J. and Yuri, T. (1997). Phylogenetic relationships among and within select avian orders based on mitochondrial DNA. In Avian Molecular Evolution and Systematics, (ed. D. P. Mindell). San Diego: Academic Press. Mitchell, P. C. (1915). Anatomical notes on the gruiform birds Aramus giganteus and Rhinocetes kagu. J. Zool. (Lond.), Moritani, T., Berry, M. J., Bacharach, D. W. and Nakamura, E. (1987). Gas exhange parameters, muscle blood flow and electromechanical properties of the plantar flexors. Eur. J. Appl. Physiol. Occup. Physiol. 56,

151 Muraoka, T., Muramatsu, T., Fukunaga, T. and Kanehisa, H. (2004). Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo. J. Appl. Physiol. 96, Olson, J. M. and Marsh, R. L. (1998). Activation patterns and length changes in hindlimb muscles of the bullfrog Rana catesbeiana during jumping. J. Exp. Biol. 201, Owre, O. T. (1967). Adaptations for locomotion and feeding in the anhinga and the double-crested cormorant. In Ornithological Mongraphs #6, (ed. R. L. Zusi): The American Ornithologists Union. Patak, A. E. and Baldwin, J. (1998). Pelvic Limb Musculature in the Emu Dromaius novhollandiae (Aves: Struthioniformes: Dromaiidae): Adaptations to High- Speed Running. J. Morphol. 238, Podulka, S., Rohrbaugh, R. W. and Bonney, R. E. (2004). Handbook of Bird Biology. Ithaca New York: Cornell Lab of Ornithology. Prange, H. D. and Schmidt-Nielsen, K. (1970). The metabolic cost of swimming in ducks. J. Exp. Biol. 53, Raikow, R. J. (1970). Evolution of Diving Adaptations in the Stifftail Duck. University of California Publications in Zoology 94. Raikow, R. J. (1976). Pelvic myology of the hawaiian honeycreepers (Drepanidae). Auk 93, Raikow, R. J. (1978). Appendicular myology and relationships to the New World Nine Primaries Oscines (Aves: Passeriformes). Bulletin of Carnegie Museum of Natural History 7, Raikow, R. J. (1980). Appendicular myology and relationships of the shrikes (Aves: Passeriformes: Laniidae). Annals of Carnegie Museum 49, Raikow, R. J. (1985). Avian Myology. In Form and Function in Birds, eds. A. S. King and J. McLelland), pp : Academic Press Inc. Roberts, T. J., Higginson, B. K., Nelson, F. E. and Gabaldon, A. M. (2007). Muscle strain is modulated more with running slope than speed in wild turkey knee and hip extensors. J. Exp. Biol. 210, Rosser, B. W., Secoy, D. M. and Riegert, P. W. (1982). The leg muscles of the American coot (Fulica atra gmelin). Can. J. Zool. 60,

152 Ruck, P. R. (1949). Studies on the Muscles of the Pelvic Appendage in Certain Anseriform Birds. In Biology, pp Pullman: State College of Washington. Schmidt-Nielsen, K. (1972). Locomotion: energy cost of swimming, flying, and running. Science 177, Schreiweis, D. O. (1982). A comparative study of the appendicular musculature of penguins (Aves: Sphenisciformes). Smithsonian Contribution to Zoology 341, Sibley, C. G. and Alquist, J. E. (1990). Phylogeny and Classification of Birds: A Study in Molecular Evolution. New Haven, CT: Yale University Press. Sibley, D. A. (2001). The Sibley Guide to Bird Life and Behavior. New York: Alfred A. Knopf. Swierczewki, E. V. and Raikow, R. J. (1981). Hind limb morphology, phylogeny, and classification of the piciformes. Auk 98, Taylor, B. (1998). Rails. New Haven and London: Yale University Press. Vanden Berge, J. (1982). Notes of the myology of the pelvic limb in kiwi (Apteryx) and in other birds. Auk 99, Verstappen, M., Aerts, P. and De Vree, F. (1998). Functional morphology of the hindlimb musculature of the black-billed magpie, Pica pica (Aves, Corvidae). Zoomorphology 118, Wagner, H. and Blickhan, R. (2003). Stabilizing function of antagonistic neuromusculoskeletal systems: an analytical investigation. Biol. Cybern. 89, Weisstein, E. W. Circle-Circle Intersection. In Circle-Circle Intersection: Wolfram Web Resource. Wilcox, H. H. (1952). The pelvic musculature of the loon, Gavia immer. Am. Midl. Nat. 48, Winter, E. M. and Brookes, F. B. (1991). Electromechanical response times and muscle elasticity in men and women. Eur. J. Appl. Physiol. Occup. Physiol. 63, Woledge, R. C., Curtin, N. A. and Homsher, E. (1985). Energetic Aspects of Muscle Contraction. London: Academic Press Incorporated. Zhou, S., Lawson, D. L., Morrison, W. E. and Fairweather, I. (1995). Electromechanical delay in isometric muscle contractions evoked by voluntary, reflex and electrical stimulation. Eur. J. Appl. Physiol. Occup. Physiol. 70,

153 136 Zusi, R. L. and Bentz, G. D. (1982). Variation of a muscle in hummingbirds and swifts and its systematic implications. Proceedings of the Biological Society of Washington 95, Zusi, R. L. and Bentz, G. D. (1984). Myology of the Purple-throated Carib (Eulampis jugularis) and Other Hummingbirds (Aves: Throchilidae). Smithsonian Contribution to Zoology 385, 1-70.

154 137 Figure 30: Superficial muscles in the hindlimb. The ILPO is labeled with arrow. The red circles indicate sonomicrometry crystal placements. Blue cicles represent relative emg electrode placement. Note that in the mallard the ILPO does not cover as much of the lateral surface of the thigh. Drawn by Dr. David Ellerby.

155 138 ILPO is Missing or Reduced ILPO is Present Anseriformes (Ducks and Geese) Tinamiformes (Tinamous) Pelacaniiformes** (Pelicans, Boobies, Dinornithiformes (Kiwis) Cormorants, Anhingas & Frigate birds) Rheiformes (Rheas) Procellariformes (Albatrosses, Shearwaters and Struthioniformes (Ostriches) Petrels) Casuariiformes (Cassowaries & Emus) Sphenisciformes (Penguins) Galliformes (Turkeys, Pheasants and Guinea Falconiformes (Old World Vultures & Diurnal Fowl) Birds of Prey) Podicipediformes (Grebes) Chadradriiformes (Shorebirds, Gulls and Auks) Gaviiformes (Loons) Psittaciformes (Parrots, Parakeets, Lories & Ciconiiformes (Herons, Ibis s, Storks & New Macaws) World Vultures) *** Coliiformes (Mousebirds) Gruiformes (Cranes, Rails and Allies) Musophagiformes (Turaco) Columbiformes (Pigeons and Doves) Strigiiformes (Owls) Cuculiformes (Cuckoos, Roadrunners & Anis) Caprimulgiformes (Frogmouth, Nightjar & Opisthocomiformes (Hoatzin) j Goatsucker) Phoenicopteriformes (Flamingos) Apodiformes (Swifts and Hummingbirds) Trogoniiformes (Trogons) Upupiformes (Hoopoes & Woodhoopoes) Coraciiformes (Kingfishers, Hornbills and Allies) Piciformes (Woodpeckers and Allies) Passeriformes (Perching Birds) * Figure 31: Bird Orders in which the ILPO is missing or reduced. * ILPO is reduced in some families of Passeriformes including the Tyrannidae, Hirundinidae and Cotingidae. ** It is unknown whether the ILPO is reduced in the pelicans. *** The ILPO is reduced in the family Sulidae and the Marabou Stork. It is unknown whether the ILPO is present or absent in this order. Anatomy taken from (Berger, 1952; Berger, 1956; Berman, 1984; Berman and Raikow, 1982; Cracraft, 1971; Fisher, 1946; Gangl et al., 2004; Garrod, 1873; George and Berger, 1966; Hudson, 1937; Hudson et al., 1969; Hudson et al., 1959; Hudson et al., 1972; Klemm, 1969; Maurer and Raikow, 1981; McGowan, 1979; McKitrick, 1991; Mellett, 1994; Miller, ; Mitchell, 1915; Owre, 1967; Patak and Baldwin, 1998; Raikow, 1970; Raikow, 1976; Raikow, 1978; Raikow, 1980; Raikow, 1985; Rosser et al., 1982; Ruck, 1949; Schreiweis, 1982; Swierczewki and Raikow, 1981; Vanden Berge, 1982; Verstappen et al., 1998; Wilcox, 1952; Zusi and Bentz, 1982; Zusi and Bentz, 1984). Phylogeny compiled from (Cracraft, 1988; Mindell et al., 1997; Podulka et al., 2004; Sibley and Alquist, 1990)

156 A B %L Millivolts C % Stride Cycle Figure 32: Average length change expressed as a function of %L 0 and electrical activity during running for in the guinea fowl at 1.5 ms 1 (A, red), the common moorhen running at 1.0 ms 1 (B, blue) and the mallard running at 1.0 ms 1 (C, green). Averages were computed by interpolating at least 10 individual running strides to 100 points, then taking the average and standard errors of the interpolated strides. The grey bands represent ± one standard error. The black arrow represents the average point where the toe comes off the ground during the stride.

157 A %L B Millivolts % Swim Cycle Figure 33: This figure shows the length change and emg activity that occurs in the ILPO in the mallard at 0.5 ms -1 (A, green) and the commom moorhen at 0.35 ms -1 (B, blue) swimming speeds that correspond to the speed of the minimum cost of transport in both species (Carr, unpublished results). Averages for swimming strides were calculated in the same manner as previously described for running only swimming started at the start of the power stroke and went through to the start of the next power stroke. The black arrow represents the start of the return stroke and the grey bars represent ± one standard error.

158 A Swimming Terrestrial Locomotion %L B Speed (m*s -1 ) Figure 34: Active shortening (A) and active lengthening (B) in the common moorhen during terrestrial (red) locomotion and swimming (blue) as a function of speed. Significant differences between terrestrial locomotion and swimming values were demonstrated for both active shortening (p<0.001) and active lengthening (p<0.001). The error bars represent ± one standard error.

159 A %L B Terrestrial Locomotion Swimming Speed (m*s -1 ) Figure 35: Active shortening (A) and active lengthening (B) in the mallard during terrestrial locomotion (green) and swimming (blue). Significant differences were seen between terrestrial locomotion and swimming for both active lengthening (p<0.001) and active shortening (p<0.001). The error bars represent ± one standard error.

160 143 Average Emg per Stride (mv) A Integrated Emg (mv*s) B Terrestrial Locomotion Swimming Average Emg per Burst (mv) C Speed (m*s -1 ) Figure 36: Average emg per stride (A), integrated emg (B) and average emg per burst in the ILPO of the common moorhen during terrestrial locomotion (red) and swimming (blue). No significant difference was found between running and swimming average emg per burst values (p=0.051) or integrated emg values (p=0.437). However there was a significant difference between terrestrial locomotion and swimming for the average emg per stride values (p=0.026) with the swimming values usually being greater than the terrestrial values. The error bars represent ± one standard error.

161 144 Average Emg per Stride (mv) A B Terrestrial Locomotion Swimming Integrated Emg (mv*s) Average Emg per Burst (mv) C Speed (m*s -1 ) Figure 37: Average emg per stride (A), integrated emg (B) and average emg per burst (C) during terrestrial locomotion (green) and swimming (blue) in the mallard. Significant differences were found between terrestrial locomotion and swimming for the average emg values per stride (p<0.001) and per burst (p=0.001) as well as for the integrated emg values (p<0.001). The error bars represent ± one standard error.

162 145 A B Joint Angle (degrees) C D % of Swim Cycle Figure 38: Average hip (red), knee (blue) and ankle (green) joint angles across a single stride/swim cycle in the mallard during swimming at 0.5 ms -1 (A) and running at 1.0 ms -1 (B) and in the common moorhen during swimming at 0.35 ms -1 (C) and running at 1.0 ms 1 (D). Grey bands are ± one standard error.

163 Chapter 5: A Swimming Muscle with a Novel Function 146 Introduction Very few studies have been done that measure muscle function in animals swimming at the water s surface (Biewener and Corning, 2001; Gillis and Biewener, 2000). To my knowledge, only one study has looked at the proximal muscles of the thigh in swimming birds (Butler et al., 1988): this study examined flow to various muscles during swimming, which gives us a good idea of the amount of energy used by particular muscles during swimming, but does not tell us anything more about how they function in terms of length change and electrical activity. During locomotion, muscles can function as struts, remaining isometric when active and allowing for the storage and recovery of elastic energy in tendinous structures. Muscles that act as struts during level running include the lateral gastrocnemius in turkeys (Roberts et al., 1997), the digital flexor IV and the lateral gastrocnemius in guinea fowl (Daley and Biewener, 2003; Gabaldon et al., 2004), and the semimembranosus in dogs (Gregersen et al., 1998). Muscles can also act like motors, actively shortening and producing positive work during running. Muscles that actively shorten and produce positive work during level running include the lateral gastrocnemius of mallard duck (Biewener and Corning, 2001), the fibularis longus of turkey (Gabaldon et al., 2004) and the vastus lateralis of dogs (Carrier et al., 1998). Some muscles act as motors during uphill running in the lateral gastrocnemius of the guinea fowl and the turkey and digital flexor IV in the guinea fowl (Carrier et al., 1998; Daley and Biewener, 2003; Gabaldon et al., 2004). Muscles can be lengthened while active, thereby absorbing

164 work and acting as a stabilizer or brake. This work-absorbing mode occurs in the 147 trochanter-femoral extensor muscles in the cockroach (Blaberus discoidalis) (Full and Stokes, 1998), and in the vastus lateralis of the dog (Carrier et al., 1998; Full and Stokes, 1998). During swimming it has generally been assumed that muscles act as motors, actively shortening and producing positive work: this is because during swimming an animal must supply a force equal to the drag that is encounters in order to propel itself forward (Schmidt-Nielsen, 1972). During drag-based swimming, the main function for many muscles is to impart enough kinetic energy to the paddling appendage to accelerate the mass of the appendage, plus the fluid it entrains, in order to produce thrust to propel the animal forward (Blake, 1981; Vogel, 1994). Therefore, many of the muscles active during swimming should primarily operate to produce positive work and actively shorten. The lateral gastocnemius in swimming mallards (Biewener and Corning, 2001) and the semimembranosus, the plantaris, the gluteus and cruralis in the marine toad function in this manner, actively shortening and producing work (Gillis and Biewener, 2000). In birds that are adapted for swimming (e.g., ducks), fluid is accelerated by the foot primarily by extension and flexion of the tarsometatarsus (Raikow, 1970). According to Raikow s observations during slow swimming, the femur and tibiotarsus are held stationary, and the power stroke is caused by extension of the tarsometatarsus. However during rapid swimming, the extension of the tarsometatarsus is augmented by knee flexion and hip extension (Raikow, 1970). The toes are spread during the power stroke to maximize surface area and folded together during the recovery stroke to minimize surface area (Raikow, 1970).

165 `Despite the observation that the majority of fluid acceleration and hence 148 thrust is produced at the distal joints, there is a proximal muscle, the Flexor Cruris Lateralis (FCL), that receives a large amount of blood flow during swimming (Butler et al., 1988) and is the largest single muscle in the hindlimb of mallards (Carr, personal observations). The FCL is variable in its morphology within extant bird orders (Fig 39). The FCL is a two-joint muscle in mallards: it originates on the pelvis and crosses the knee joint, inserting on the tibiotarsus (Fig. 40A). The FCL in orders that are more cursorial, such as the ratites and the galliformes, is split into two different muscles, the Flexor Cruris Lateralis pars Pelvica (FCLP) and the Flexor Cruris Lateralis pars Accessoria (FCLA) (Fig. 40B). In addition the FCLA is connected to the intermediate gastrocnemius, a muscle which crosses the ankle, allowing this series of muscles to act at the hip, the knee and the ankle during running (Ellerby et al., 2003). The FCLA is missing in some orders that spend little time on the ground and are well-known for their flying ability, including the Orders Falconiiformes, Caprimulgiformes, Strigiiformes, Apodiformes, Piciformes, Coraciiformes and Trogoniiformes (Fig. 39). The FCLP and FCLA though are found in the more cursorial Secretary Bird, a member of the order Falconiformes. The FCLA is also missing in many orders of birds that are primarily aquatic locomotors, including the Orders Anseriformes, Pelecaniformes, Procellariformes, Podicipediformes, Gaviiformes, Sphenisciformes and Chadradriiformes (Fig. 39). Due to the FCL s origin and insertion in aquatic birds, it is capable of acting as both a hip extensor and knee flexor. However neither the knee nor the hip go through significant angular changes during swimming (Biewener and Corning, 2001; Raikow,

166 1970), leaving unanswered many questions about the FCL s function during 149 swimming. In order to determine the function of the FCL in swimming birds I chose a species that had an FCL but that was missing the accessory head, and also was capable of swimming in a flow tank and walking and running on a treadmill. The species of bird I chose for these experiments is the Mallard (Anas platyrhynchus). In addition to meeting all of the stated conditions, the mallard is a common North American species (and easy to obtain from commercial sources), and it has been the subject of previous studies looking at muscle function in surface swimming birds (Biewener and Corning, 2001). Because neither the hip nor the knee go through large angular changes during swimming, it is possible that the FCL does not actively shorten and produce work during swimming. I hypothesized that the FCL in swimming ducks is active and nearly isometric during the power stroke, acting to hold the knee in a flexed position. This action of the FCL would act against the propulsive force: the FCL would hold the paddling appendage close to the body, reducing interference drag and increasing the fineness ratio of the body (Fish, 1993; Vogel, 1994). During running in the duck, the knee experiences a large amount of flexion during stance (~100 degrees) (Biewener and Corning, 2001), however this flexion could be passively driven by the flexor moment at the knee created by the ground reaction force which could be driving the flexion of this joint. Therefore I hypothesized that the FCL in mallards is inactive during running and only functions during swimming. I tested these hypotheses by implanting the FCL with two sonomicrometry crystals that measured length change (see Fig. 41). I implanted emg s to measure electrical activity, and using a technique described in a previous study (Carr, 2008,

167 Chapter 1), I normalized my length measurements (taken via sonomicrometry) into 150 sarcomere measurements. I also measured kinematics using high-speed video during both swimming and terrestrial locomotion in the mallard.

168 Materials and Methods 151 Animals and Training Four mallards averaging 1.113±0.03 kg body mass were used for this experiment. The mallards were housed in communal cages at the Northeastern University Animal Care Facility with access to open water. Food and water was provided for ad libitum. Prior to surgery and experimental recordings, the birds were trained to run inside a threesided box on a motorized treadmill. The birds were trained 3 days per week for a minimum of fourteen weeks. Each training session lasted for 20 minutes. After training the mallards could, in an individual session, maintain level speeds of 0.5 ms -1 for 5 minutes, 0.75 ms -1 for 5 minutes and 1.0 ms -1 for 2 minutes. Birds were also trained to swim inside a flow tank at a constant speed; mallards could maintain 0.7 ms -1 for 3 minutes, a speed that is at or very close to the animal s hull speed (Prange and Schmidt- Nielsen, 1970). Once a bird was sufficiently trained, a surgery was performed to insert sonomicrometry and emg sensors as described below. After surgery, birds were allowed to recover in individual cages for at least 40 hours prior to any experimental recordings. After recovery, birds were run on the treadmill and experimental recordings were taken as described below. Birds were run at speeds of 0.22, 0.33, 0.5, 0.67, 0.78, 0.89 and 1.0 ms -1 on a level incline for at least 30 seconds per speed, and swam in the flow tank at speeds of 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65 and 0.7 ms -1. Each bird was sacrificed after its experimental recordings were taken, and electrode placement was verified. Surgery All transducers were sterilized (12 hour ethylene oxide treatment) and implanted using sterile surgical techniques under general anesthesia (isofluorane). Two custom-

169 152 made 1-mm sonomicrometry transducers made from PZT discs (Boston Piezo-Optics, Inc.) and attached to stainless steel holders (Olson and Marsh, 1998) were implanted along fascicles of the FCL, and these crystals spanned approximately 30% of the fascicle length (Fig. 41). Small punctures approximately 1mm in length were created in the connective tissue overlying the muscle to a depth of approximately 3mm. The crystals were then placed between the fascicles. The holders were secured to the overlying connective tissue using 6-0 silk sutures. Care was taken to ensure that the two crystals were always implanted between the same fascicles along the length of the muscle. Two fine-wire bipolar EMG electrodes were implanted into each segment of the fascicles to measure muscle activity. The emg electrodes were constructed from 3T stainless steel Teflon-coated wire (Medwire) that was twisted along its length. The tips of the electrodes were bared 1.5 mm at each end and preactive ends spaced 3 mm apart. Emg electrodes were implanted in the FCL with a chamfered 25 gauge needle at positions that were immediately anterior and posterior to each pair of crystals. Post mortem examinations verified verified the placement of the sonomicrometry crystals and emg electrodes. Recordings Sonomicrometry measurements were acquired digitally with a sampling frequency of 613 Hz using Sonoview software and a Sonometrics TRX Series 8 interface. Emg signals were amplified by WPI model DAM-50 preamplifiers with high- and low-pass filters set at 10 and 3000Hz, respectively. Emg signals were collected at a frequency of 10kHz using an ADI Instruments Power Lab/16SP and a Macintosh PowerMac. The Emg data was subsequently analyzed using the application Igor Pro (v.

170 ). The signals were first filtered using a finite impulse response filter with a band pass of 90Hz to 1000Hz. Emg signals were rectified and the start time, stop time, and emg duration were all measured. The burst amplitudes were expressed in three ways: the integrated value of the burst duration; the average emg per burst (equal to the integrated value divided by the burst duration); and the average emg per stride (equal to the integrated value divided by the stride duration). Videography Video was recorded for the running mallards to VHS tape with a NAC HV-1000 high-speed video camera operating at 500 fields per second. I synchronized the emg and sonomicrometry traces during mallard running with the high-speed video recordings using a square-wave generated by my emg recording system which was recorded in the video fields with a NAC wave inserter. Mallard swimming was recorded to VHS tape at 240Hz using a JVC Digital Video Camera model #GR-DVL9800. The JVC recordings were synchronized with the emg and sonomicrometry traces using a light pulse generated by my emg recording system which was placed in the visual field of the JVC. Visual markers were made using reflective tape. These markers were placed above the caudal and cranial pelvis; on the ankle, tarsometatarsal-phalangeal, and interphalangeal joints; and on the toe of the bird. The hip position was calculated using a circle-circle intersection formula (Weisstein) and several distances measured between the cranial pelvic marker and the hip, the caudal pelvic marker and the hip, and the cranial and caudal pelvic marker. The knee position was calculated using the law of cosines and several measured distances: the distance between the hip and the ankle markers based on post mortem measures of the distance between the center of rotation of the hip and center

171 154 of rotation of the knee, and the distance between the center of rotation of the knee and the center of rotation of the ankle. The hip and knee positions were calculated via these methods (instead of directly marked and measured from the video footage) so that I could minimize the amount of feathers that had to be removed from the bird, and thus minimize waterlogged feathers and thermoregulatory stresses during swimming. Data were collected for at least ten steady speed strides at each speed for each bird. Sarcomere Measurements After each animal s experimental recordings were completed, the animal was sacrificed, and the instrumented leg was bolted into place with the hip and knee in a flexed position. The limb was allowed to sit in a bolted position for six hours until the leg muscles were in full rigor. Once the fixed limb was in rigor, a reference sonomicrometry measurement (denoted L so, ref ) was measured. In addition, a body temperature measurement was taken to correct for temperature effects on the speed of sound. Two samples of the FCL tissue, each measuring ~1cm 3, were removed from between each crystal pair. These muscle samples were frozen using liquid nitrogen and isopentane, and then sectioned using a cryostat. For each muscle sample, at least ten sections were placed on slides and stained using Weingert Iron Hematoxylin (Humason, 1979). From each slide (containing one stained section), pictures of at least three fibers were captured. Sarcomere lengths were measured using a compound microscope at 1000X magnification. Sarcomere measurements were performed using a digital imaging program (NIH Image J version 1.38). In Image J, a series of at least 20 sarcomeres was measured from each fiber, and the average individual sarcomere length (denoted L sc, ref ) was calculated by dividing the total length of the in-series sarcomeres by the number of

172 sarcomeres in series. Using the reference sonomicrometry length and the measured 155 sarcomere length, in vivo sonomicrometry lengths (L so ) were converted to estimated in vivo sarcomere lengths (L sc ) using the equation: L sc,ref L so L so,ref = L sc (1) Length measurements for the mallard are either reported as µm, or are converted to %L 0 using the following equation: %L 0 = L sc 100 (2) 2.36µm where L sc is the calculated sarcomere length from equation 1, and 2.36µm is the estimated sarcomere length for maximum force production based on the length in the middle of the plateau region of the calculated length-tension curve from a previous study (Carr, 2008, Chapter 1). Length Changes Muscle segment lengths were measured from the sonomicrometry traces after the lengths in mm had been converted to µm. The length values measured were during running were: L max,run = maximum length of the FCL, which occurred at or near the start of stance L min,run = minimum length of the FCL, which occurred at of near the time of toe off L emg,on = length of the FCL 25ms after the start of the emg activity L emg,off = length of the FCL 25ms after the stop of the emg activity

173 These measured lengths were used to calculate active shortening, active lengthening, 156 and passive lengthening. When calculating active lengthening and active shortening, I assumed an electromechanical delay of 25 milliseconds. I am not aware of studies done on the FCL to confirm this assumption, but this time period falls within values measured for other muscles (Cavanagh and Komi, 1979; Gabriel and Boucher, 1998; Moritani et al., 1987; Muraoka et al., 2004; Winter and Brookes, 1991; Zhou et al., 1995). The equation used to calculate a given length change was dependent on the timing of L emg,on and L emg,off relative to other lengths measured during a stride. Active shortening during the stance ( L as,run ) was calculated using one of four equations. If L emg,on occurred before L max,run and L emg,off occurred after L min,run then: L as,run = L max,run L min,run (3) If L emg,on occurred before L max,run and L emg,off occurred before L min,run then: L as,run = L max,run L emg,off (4) If L emg,on occurred after L max,run and L emg,off occurred after L min,run then: L as,run = L emg,on L min,rum (5) If L emg,on occurred after L max,run and L emg,off occurred before L min,run then: L as,run = L emg,on L emg,off (6) Active lengthening ( L al,run ) was calculated using one of two equations. If L emg,off occurred before L min,run then: L al,run = 0.0 (7) If L emg,off occurred after L min then: L al,run = L emg,off L min,run (8)

174 Passive lengthening ( L p,runl ) was also calculated using one of four equations. If 157 L emg,off occurred after L min,run and L emg,on for the next stride occurred after L max,run for the next stride then: L pl,run = L max,run L emg,off (9) If L emg,off occurred before L min,run and L emg,on for the next stride occurred after L max,run then: L pl,run = L max,run L min,run (10) If L emg,off occurred before L min,run and L emg,on for the next stride occurred before L max,run then: L pl,run = L emg,on L min,run (11) If L emg,off occurred after L min,run and L emg,on for the next stride occurred before L max,run then: L pl,run = L emg,on L emg,off (12) The length values measured during swimming in the mallard were: L max,swim = maximum length of the FCL which occurred at or near the time of the start of the power stroke L min,swim = minimum length of the ILPO, which occurred during the return stroke L emg,on = length of the ILPO 25ms after the start of the emg activity L emg,off = length of the ILPO 25ms after the stop of the emg activity These measured lengths were used to calculate active lengthening, active shortening, and passive lengthening in the mallard. Active lengthening ( L al,swim ) was calculated using one of two formulas. If L emg,on occurred before L max,swim then: L al,swim = L max,swim L start (13) If L emg,on occurred after L max,swim then: L al,swim = 0.0 (14)

175 158 Active shortening L as,swim ) was calculated using one of four formulas. If L emg,on occurred before L max,swim and L emg,off occurred after L min,rs then: L as,swim = L max - L min (15) If L emg,on occurred before L max and L emg,off occurred before L min,swim then: L as,swim = L max,swim L emg,off (16) If L emg,on occurred after L max,swim and L emg,off occurred after L min,swim then: L as,swim = L emg,on L min,swim (17) If L emg,on occurred after L max,swim and L emg,off occurred before L min,swim then: L as,swim = L emg,on L emg,off (18) Passive lengthening ( L pl,swim ) was calculated using one of four formulas. If L emg,off occurred after L min,swim and L emg,on for the next stride occurred after L max,swim then: L pl,swim = L max,swim - L emg,off (19) If L emg,off occurred before L min,swim and L emg,on for the next stride occurred after L max,swim then: L pl,swim = L max,swim - L min,swim (20) If L emg,off occurred after L min,swim and L emg,on for the next stride occurred before L max,swim then: L pl,swim = L emg,on - L emg,off (21) If L emg,off occurred before L min,swim and L emg,on for the next stride occurred before L max,swim then: L pl,swim = L emg,on - L min,swim (22) All length changes are reported as %L 0 where L 0 for the mallards is the middle of the plateau region of the length tension curve (2.36µm).

176 159 Statistical Analysis To test for significant differences in length change, electrical activity and kinematic variables as a function of speed and type of locomotion, multivariate analyses were conducted using the general linear model in the application SPSS (version 15.0 for Microsoft Windows). For the analysis of running, a bird identifier was used as a factor and speed was used as a covariate. For the analysis of swimming, a bird identifier was used as a factor and speed was used as a covariate. Within each species, multivariate analyses were performed to determine whether the differences in strain or angular change were significant between running and swimming; swimming and terrestrial locomotion were used as factors in this model but specific speeds were not used. When comparing angular strains, the longest continuous period of flexion or extension during running in any one joint was compared to the longest continuous period of flexion or extension during swimming in any one joint. All measurements are reported as a mean value plus or minus one standard error.

177 Results 160 General Pattern of Length Change and Electrical Activity During Locomotion The general pattern of length change and electrical activity during swimming and running in the FCL is shown in Figure 42. Figures 42A and 42B shows the length change and electrical activity duration in the mallard during swimming at speeds of 0.5 ms -1 (the minimum cost of transport speed during swimming) and 0.7 ms -1 (the hull speed in mallards (Prange and Schmidt-Nielsen, 1970)). The FCL during swimming starts its electrical activity towards the end of the return stroke and then is active throughout the power stroke and into the next return stroke. During the power stroke, the FCL shows relatively little change in length, but during the return stroke the FCL actively shortens, and then is passively and then actively lengthened. Figures 42C and 42D demonstrate the length change and electrical activity duration in the FCL during walking at 0.67 ms -1 and during running at 1.0 ms -1. Electrical activity in the FCL during running and walking starts during the end of the swing phase and continues through most of the stance phase. The FCL during running and walking demonstrates periods of active shortening and is both actively and passively lengthened. Length Change in the FCL Active lengthening changes in the FCL during running and swimming are demonstrate in figure 43. Active lengthening during swimming (Fig. 43A) did not show a significant effect of speed (p=0.376) and was not significantly different from active lengthening during running (p=0.762) (Fig. 43A) which also did not demonstrate a significant effect of speed (p=0.887). During both running and swimming, the amount of active lengthening that was done to the FCL was relatively small (less than 4%). Active

178 shortening during swimming and running (Fig. 43B) did demonstrate a significant 161 effect of speed (p<0.001) and the strains during swimming were significantly less then the running strains (p<0.001). Electrical Activity in the FCL There was a significant effect of speed on average emg per stride (Fig. 44A) and average emg per burst (Fig. 44C) during both running and swimming (p<0.05). Both the per-burst and per-stride average emg values tended to increase with speed during both swimming and running. There was not a significant effect of speed on the integrated emg values (Fig. 44B) during running or swimming. There was no significant difference between running and swimming for any of the measured intensity values (p>0.05). Speed had no significant effect of the start time of emg activity relative to foot down during running (Fig. 45A), but speed did have a significant effect on stop time relative to foot down during running (p=0.006). Stop time relative to foot down tended to decrease as speed increased. Speed was also shown to not have a significant effect on the start or stop time relative to the start of power stroke during swimming (Fig. 45B). Swimming and Running Kinematics Figure 46 shows average hip, knee and ankle joint changes during a walk (A) and a run (B), and during swimming at 0.5 ms -1 (C) and 0.7 ms -1 (D). During running and walking, the hip experiences a period of initial flexion, followed by extension during stance and swing. The knee is flexed throughout the stance phase and then extends during the swing phase. The ankle during stance first flexes by a small amount then extends, and during swing first flexes by a large amount then extends by a large amount. During swimming, the hip and knee experience a small amount of flexion and extension

179 during the power and return stroke. The ankle during the power stroke is 162 continuously extending and then during the return stroke the foot folds up and the ankle flexes back to its original position for the next power stroke. During running there is no significant effect of speed on any of the angular excursions seen at any of the joints. During swimming the only significant increases seen with speed occurred during knee extension (p<0.001) and ankle extension (p=0.003): in both of these cases the excursion of the joint increased with speed. When I compared the maximum amount of continuous angular change during running and swimming at all the joints, the amount of angular change was significantly smaller during swimming than during running (p<0.05) except for the ankle joint (Fig. 47). At the ankle the amount of flexion that occurred during swimming was significantly less than what occurred during running (p=0.016) but the amount of ankle extension that occurred was not significantly different between running and swimming (p=0.355).

180 Discussion 163 Function of the FCL During Running The FCL in the mallard actively shortens during the stance phase of running and walking. During early stance the hip joint is flexing, and the knee is flexing throughout the stance phase while the FCL is active. The resultant electrical activity and length change measured in the FCL refutes my hypothesis about the FCL s function during running. I hypothesized that the FCL would be inactive during stance and that knee flexion would be passively driven by the joint moment caused by the ground reaction force at the knee. However based upon my angular data, at foot down, the moment produced by the ground reaction force and acting at the knee and hip may be an extensor moment if the center of pressure for the ground reaction force goes through the center of the foot. If both the hip and the knee were experiencing an extensor moment and the joints are flexing, this would require active shortening and work production of a muscle such as the FCL, which is capable of acting as both a hip and knee flexor, in order to produce the positive work needed to counteract the joint moment and cause the angular change in the early part of stance (Lloyd and Besier, 2003; Winter, 2004). It is also possible that cocontraction is occurring at the knee and hip joint and the FCL is actively shortening to counteract the negative work produced by active knee extensors. Other studies done on mallards have shown that the ILPO, a hip and knee extensor, is active during early stance (Carr, 2008, Chapter 4) and is actively lengthening, it is possible that the ILPO is not the only active knee extensor during early stance. If knee extensors are active during knee flexion then it is possible that the FCL is actively shortening and producing work to oppose the activity of the knee extensors so that knee flexion can

181 occur. In late stance when the ground reaction force should be behind the knee, 164 causing a flexor moment at the knee, it is possible that the flexor moment at the knee is not large enough to passively cause the angular change seen at the knee joint and has to be supplemented by active muscle work provided by the FCL. This would also explain the trends I saw in the FCL with speed. If the instantaneous ground reaction force acting at the joint increases with speed, then the amount of active shortening and the increase in muscle activation would increase the amount of work done by the muscle to counteract the increased moments at the hip and knee (Dutto et al., 2004; Roberts and Belliveau, 2005; Roberts et al., 1998). To my knowledge no studies have been done that measure joint forces and moments at the knee in the mallard so I cannot determine exactly what the moments of the joint are at any given time or how much of a force is needed around the knee and hip to produce the corresponding change in angle that occurs while the FCL is active. FCL Function During Swimming During swimming the FCL is doing three things when it is active. When active the FCL in swimming mallards is isometric, actively shortens and is actively lengthened. Active shortening during the first part of the return stroke occurs during a period of knee and hip flexion, and the small amount of active shortening occurring in the FCL may be causing this small amount of flexion at both the hip and knee. The amount of work that needs to be produced by the appendage during the power stroke has to increase to produce the drag required to produce the increased forward thrust (Fish, 1984; Fish, 1993; Fish, 2000; Howell, 1937; Tarasoff et al., 1972). The FCL during active shortening experiences the same amount of active shortening regardless of speed, but

182 increases the amount of activation of the FCL, indicating an increase in recruitment. 165 Because muscle force is proportional to cross-sectional area, if the amount of crosssectional area that is recruited increases while the amount of lengthening remains the same, the amount of work produced by the muscle will increase (Biewener, 2003; Marsh, 1999; Woledge et al., 1985). The active lengthening that occurs in the FCL occurs during a period when both the hip and the knee are transitioning between extension in late return stroke and flexion during early power stroke. It may be that while the FCL is being actively lengthened, it is absorbing work produced at the end of the return stroke (thus acting as a brake) and also assisting in transitioning the hip and knee joints between extension and flexion (Full and Stokes, 1998; Marsh, 1999). The third function of the FCL during swimming is to remain isometric during the first part of the power stroke, holding the knee in a flexed position. During this time period, the ankle is extending, moving the limb and the entrained water in the posterior direction to create forward thrust. By holding the muscle isometric and maintaining the knee in a flexed position, the FCL could serve two functions. The force produced by the moving entrained water has the potential to produce a reaction force oriented in front of the knee joint, thus creating an extensor moment at the knee. The FCL may be producing force isometrically to counteract this moment and maintain the knee in its flexed position. If small angular changes occur at the knee and hip during swimming, these changes would allow birds that are adapted for swimming to keep their hip and knee flexed towards the body, allowing the tibiotarsus and phallangeals (the paddle) to remain close to the body. If mallards extended the hip during swimming and moved the propulsor

183 farther away from the body, this would increase drag on the body due to interference 166 between the body and the propulsor, increasing the required amount of power (Fish, 1993; Pennycuick et al., 1996; Vogel, 1994). Extending the hip would also decrease the body s fineness ratio (the fineness ratio reflects the streamlined profile of the body: FR = maximum length divided by maximum thickness) (Fish, 1993) and increase the amount of drag on the animal. Summary To my knowledge this study is the first of its kind to demonstrate that muscles during swimming do more than actively shorten and produce work: I have demonstrated that the FCL is actively lengthened, and later isometric, for a large portion of the swim cycle. During swimming at the surface the mallard hindlimb must supply a force equal to the drag that it encounters in order to propel itself forward. One method of increasing the efficiency of surface swimming is to decrease the amount of drag that an animal encounters so that the forces that need to be produced to propel the animal forward do not have to be as large. It is possible that the FCL s anatomy and function in the mallard hindlimb evolved in response to selective pressure s to increase the efficiency of surface swimming locomotion which is the primary form of locomotion employed by the mallard.

184 Literature Cited 167 Berger, A. J. (1952). The comparative functional morphology of the pelvic appendage in three genera of Cuculidae. Am. Midl. Nat. 47, Berger, A. J. (1956). Myology of Sandhill Crane. Wilson Bull. 68, Berman, S. L. (1984). The hindlimb musculature of the White-Fronted Amazon (Amazona albicans, Psittaciformes). Auk 101, Berman, S. L. and Raikow, R. J. (1982). The hindlimb musculature of mousebirds (Coliiformes). Auk 99, Biewener, A. A. (2003). Animal Locomotion. Oxford: Oxford University Press. Biewener, A. A. and Corning, W. R. (2001). Dynamics of mallard (Anas platyrynchos) gastrocnemius function during swimming versus terrestrial locomotion. J. Exp. Biol. 204, Blake, R. W. (1981). Mechanics of Drag-based Mechanisms of Propulsion in Aquatic Vertebrates. Symp. Zool. Zoc. Lond. 48, Butler, P. J., Turner, D. L., Al-Wassia, A. and Bevan, R. M. (1988). Regional distribution of blood flow during swimming in the tufted duck (Aythya fuligula). J. Exp. Biol. 135, Carrier, D. R., Gregersen, C. S. and Silverton, N. A. (1998). Dynamic gearing in running dogs. J. Exp. Biol. 201, Cavanagh, P. R. and Komi, P. V. (1979). Electromechanical delay in human skeletal muscle under concentric and eccentric contractions. Eur. J. Appl. Physiol. Occup. Physiol. 42, Cracraft, J. (1971). The functional morphology of the hind limb of the domestic pigeon, Columba livia. Bull. Am. Mus. Nat. Hist. 14, Cracraft, J. (1988). The major clades of birds. Oxford: Clarendon Press. Daley, M. A. and Biewener, A. A. (2003). Muscle force-length dynamics during level versus incline locomotion: a comparison of in vivo performance of two guinea fowl ankle extensors. J. Exp. Biol. 206, Dutto, D. J., Hoyt, D. F., Cogger, E. A. and Wickler, S. J. (2004). Ground reaction forces in horses trotting up an incline and on the level over a range of speeds. J. Exp. Biol. 207,

185 Ellerby, D. J., Marsh, R. L., Carr, J. A. and Henry, H. T. (2003). Muscle Function in running guinea fowl: Linked muscles spanning multiple joints. In SICB Annual Meeting & Exhibition SICB. Anaheim, CA. 168 Fish, F. E. (1984). Mechanics, power output and efficiency of the swimming muskrat (Ondatra zibethicus). J. Exp. Biol. 110, Fish, F. E. (1993). Influence of hydrodynamic design and propulsive mode on mammalian swimming energetics. Aust. J. Zool. 42, Fish, F. E. (2000). Biomechanics and energetics in aquatic and semiaquatic mammals: platypus to whale. Physiol. Biochem. Zool. 73, Fisher, H. I. (1946). Adaptations an comparative anatomy of the locomotor apparatus of new world vultures. Am. Midl. Nat. 35, Full, R. J. and Stokes, D. (1998). Energy absorption during running by leg muscles in a cockroach. J. Exp. Biol Gabaldon, A. M., Nelson, F. E. and Roberts, T. J. (2004). Mechanical function of two ankle extensors in wild turkeys: shifts from energy production to energy absorption during incline versus decline running. J. Exp. Biol. 207, Gabriel, D. A. and Boucher, J. P. (1998). Effects of repetitive dynamic contraction upon electromechanical delay. Eur. J. Appl. Physiol. Occup. Physiol. 79, Gangl, D., Weissengruber, G. E., Egerbacher, M. and Forstenpointer, G. (2004). Anatomical description of the muscles of the pelvic limb in the ostrich (Struthio camelus). Anat. Histol. Embryol. 33, Garrod, A. H. (1873). On certain muscles of the thigh of birds and their value in classification. J. Zool. (Lond.), George, J. C. and Berger, A. J. (1966). Avian Myology. New York and London: Academic Press. Gillis, G. B. and Biewener, A. A. (2000). Hindlimb extensor muscle function during jumping and swimming in the toad (Bufo marinus). J. Exp. Biol. 203, Gregersen, C. S., Silverton, N. A. and Carrier, D. R. (1998). External work and potential for elastic storage at the limb joints of running dogs. J. Exp. Biol. 201, Haughton, A. (1867). Notes of Animal Mechanics No. X - Muscular anatomy of the Emu (Dromaeus navae hollandiae). Proc. R. Ir. Acad. 9,

186 169 Howell, A. B. (1937). The swimming mechanism of the platypus. J. Mammal. 18, Hudson, G. E. (1937). Studies on the muscles of the pelvic appendages of birds. Am. Midl. Nat. 18, Hudson, G. E., Hoff, K. M., Vanden Berge, J. and Trivette, E. C. (1969). A numerical study of the wing and leg muscle of Lari and Alcae. Ibis (Lond. 1859) 111, Hudson, G. E., Lanzillotti, P. J. and Edwards, G. E. (1959). Muscles of the pelvic limb in galliform birds. Am. Midl. Nat. 61, Hudson, G. E., Schreiweis, D. O., Wang, S. Y. and Lancaster, D. A. (1972). A numerical study of the wing and leg muscle of tinamous. Northwest Sci. 46, Humason, G. L. (1979). Animal Tissue Techniques. San Francisco: W. H. Freeman and Company. Klemm, R. D. (1969). Comparative myology of the hindlimb of procellariiform birds. Southern Illinois University Monographs, Lloyd, D. G. and Besier, T. F. (2003). An EMG-driven musculoskeletal model to estimate muscle forces and knee joint moments in vivo. J. Biomech. 36, Marsh, R. L. (1999). How muscles deal with real-world loads: the influence of length trajectory on muscle performance. J. Exp. Biol. 202, Maurer, D. R. and Raikow, R. J. (1981). Appendicular myology, phylogeny and classification of the avian order Coraciiformes (Including Trogoniformes). Annals of Carnegie Museum 50, McGowan, C. (1979). The hind limb musculature of brown kiwi, Apteryx australis mantelli. J. Morphol. 160, McKitrick, M. C. (1991). Phylogentic analysis of avian hindlimb musculature. Miscellaneous Publication Museum of Zoology, University of Michigan, Mellett, F. D. (1994). A note on the musculature of the proximal part of the pelvic limb of the ostrich (Struthis camelus). J. S. Afr. Vet. Assoc. 65, 5-9. Miller, A. H. Structural Modifications in the Hawaiian goose (Nesochen sandvicensis). University of California Museum of Vertebrate Zoology 42, Mindell, D. P., Sorenson, M. D., Huddleston, C., Miranda, H., Knight, A., Sawchuk, S. J. and Yuri, T. (1997). Phylogenetic relationships among and within select

187 avian orders based on mitochondrial DNA. In Avian Molecular Evolution and Systematics, (ed. D. P. Mindell). San Diego: Academic Press. 170 Mitchell, P. C. (1915). Anatomical notes on the gruiform birds Aramus giganteus and Rhinocetes kagu. J. Zool. (Lond.), Moritani, T., Berry, M. J., Bacharach, D. W. and Nakamura, E. (1987). Gas exhange parameters, muscle blood flow and electromechanical properties of the plantar flexors. Eur. J. Appl. Physiol. Occup. Physiol. 56, Muraoka, T., Muramatsu, T., Fukunaga, T. and Kanehisa, H. (2004). Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo. J. Appl. Physiol. 96, Olson, J. M. and Marsh, R. L. (1998). Activation patterns and length changes in hindlimb muscles of the bullfrog Rana catesbeiana during jumping. J. Exp. Biol. 201, Owre, O. T. (1967). Adaptations for locomotion and feeding in the anhinga and the double-crested cormorant. In Ornithological Mongraphs #6, (ed. R. L. Zusi): The American Ornithologists Union. Patak, A. E. and Baldwin, J. (1998). Pelvic Limb Musculature in the Emu Dromaius novhollandiae (Aves: Struthioniformes: Dromaiidae): Adaptations to High- Speed Running. J. Morphol. 238, Pennycuick, C. J., Klaassen, M., Kvist, A. and Lindstrom, A. (1996). Wingbeat Frequency and the Body Drag Anomaly: Wind-Tunnel Observations on a Thrush Nightingale (Luscinia luscinia) and a Teal (Anas crecca). J. Exp. Biol. 199, Podulka, S., Rohrbaugh, R. W. and Bonney, R. E. (2004). Handbook of Bird Biology. Ithaca New York: Cornell Lab of Ornithology. Prange, H. D. and Schmidt-Nielsen, K. (1970). The metabolic cost of swimming in ducks. J. Exp. Biol. 53, Raikow, R. J. (1970). Evolution of Diving Adaptations in the Stifftail Duck. University of California Publications in Zoology 94. Raikow, R. J. (1976). Pelvic myology of the hawaiian honeycreepers (Drepanidae). Auk 93, Raikow, R. J. (1978). Appendicular myology and relationships to the New World Nine Primaries Oscines (Aves: Passeriformes). Bulletin of Carnegie Museum of Natural History 7, 1-43.

188 171 Raikow, R. J. (1980). Appendicular myology and relationships of the shrikes (Aves: Passeriformes: Laniidae). Annals of Carnegie Museum 49, Raikow, R. J. (1985). Avian Myology. In Form and Function in Birds, eds. A. S. King and J. McLelland), pp : Academic Press Inc. Roberts, T. J. and Belliveau, R. A. (2005). Sources of mechanical power for uphill running in humans. J. Exp. Biol. 208, Roberts, T. J., Chen, M. S. and Taylor, C. R. (1998). Energetics of bipedal running. II. Limb design and running mechanics. J. Exp. Biol. 201, Roberts, T. J., Marsh, R. L., Weyand, P. G. and Taylor, C. R. (1997). Muscular force in running turkeys: the economy of minimizing work. Science 275, Rosser, B. W., Secoy, D. M. and Riegert, P. W. (1982). The leg muscles of the American coot (Fulica atra gmelin). Can. J. Zool. 60, Ruck, P. R. (1949). Studies on the Muscles of the Pelvic Appendage in Certain Anseriform Birds. In Biology, pp Pullman: State College of Washington. Schmidt-Nielsen, K. (1972). Locomotion: energy cost of swimming, flying, and running. Science 177, Schreiweis, D. O. (1982). A comparative study of the appendicular musculature of penguins (Aves: Sphenisciformes). Smithsonian Contribution to Zoology 341, Sibley, C. G. and Alquist, J. E. (1990). Phylogeny and Classification of Birds: A Study in Molecular Evolution. New Haven, CT: Yale University Press. Swierczewki, E. V. and Raikow, R. J. (1981). Hind limb morphology, phylogeny, and classification of the piciformes. Auk 98, Tarasoff, F. J., Bisaillon, A., Pierard, J. and Whitt, A. P. (1972). Locomotory patterns and external morphology of the river otter, sea otter, and harp seal (Mammalia). Can. J. Zool. 50, Vanden Berge, J. (1982). Notes of the myology of the pelvic limb in kiwi (Apteryx) and in other birds. Auk 99, Verstappen, M., Aerts, P. and De Vree, F. (1998). Functional morphology of the hindlimb musculature of the black-billed magpie, Pica pica (Aves, Corvidae). Zoomorphology 118, Press. Vogel, S. (1994). Life in Moving Fluids. Princeton, NJ: Princeton University

189 Weisstein, E. W. Circle-Circle Intersection. In Circle-Circle Intersection: Wolfram Web Resource. 172 Wilcox, H. H. (1952). The pelvic musculature of the loon, Gavia immer. Am. Midl. Nat. 48, Winter, D. A. (2004). Biomechanics and Motor Control of Human Movement: John Wiley & Sons. Winter, E. M. and Brookes, F. B. (1991). Electromechanical response times and muscle elasticity in men and women. Eur. J. Appl. Physiol. Occup. Physiol. 63, Woledge, R. C., Curtin, N. A. and Homsher, E. (1985). Energetic Aspects of Muscle Contraction. London: Academic Press Incorporated. Zhou, S., Lawson, D. L., Morrison, W. E. and Fairweather, I. (1995). Electromechanical delay in isometric muscle contractions evoked by voluntary, reflex and electrical stimulation. Eur. J. Appl. Physiol. Occup. Physiol. 70, Zusi, R. L. and Bentz, G. D. (1982). Variation of a muscle in hummingbirds and swifts and its systematic implications. Proceedings of the Biological Society of Washington 95, Zusi, R. L. and Bentz, G. D. (1984). Myology of the Purple-throated Carib (Eulampis jugularis) and Other Hummingbirds (Aves: Throchilidae). Smithsonian Contribution to Zoology 385, 1-70.

190 173 FCLP/FCLA Present FCLA Absent FCLP/FCLA Absent Tinamiformes (Tinamous Pelcaniformes (Pelicans, Tropicbirds, Boobies, Cormorants, Anhingas & Falconiformes (Old World Vultures & Diurnal Birds of Prey) Dinornithiformes (Kiwis) Frigatebirds) Rheiformes (Rheas) Struthioniformes (Ostriches) Casuariiformes (Cassowaries & Emus) Procellariformes (Albatrosees, Shearwaters and Petrels) Podicipediformes (Grebes) Gaviiformes (Loons) Strigiformes (Owls) Apodiformes (Swifts and Hummingbirds) Galliformes (Turkeys, Pheasants & Guinea Fowl) Ciconiiformes (Herons, Ibis s, Storks, New World Vultures) Gruiformes (Cranes and Rails) Columbiformes (Pigeons & Doves) Psittaciformes (parrots, Parakeets, Lories & Macaws) Coliiformes (Mousebirds) Cuculiformes (Cuckoos, Roadrunners and Anis) Sphenisciformes (Penguins) Chadradriiformes (Shorebirds, Gulls and Auks) Musophagiformes (Turacos) Caprimulgiformes (Frogmouth, Nightjar & Goatsuckers) Trogoniformes (Trogons) Coraciiformes (Kingfishers, Hornbills & Allies) Piciformes (Woodpeckers & Allies) Opisthocomiformes (Hoatzin) Upupiformes (Hoopoes & Woodhoopoes) Passeriformes (Perching Birds) Figure 39: This figure is designed to show the presence/absence of the FCLA and the presence/absense of the FCLP. Bird orders in the first column have both an FCLP and an FCLA, bird orders in the second column have lost the FCLA but still have an FCLP, and bird orders in the third column have lost the FCLP and FCLA in its entirety. FCLA missing in family Heliornithidae from this order Both the FCLP and FCLA are missing in the family Fregatidae in this order FCLA and FCLP present in the secretary bird It is unknown whether the FCLP or FCLA is present or absent in this order Anatomy taken from (Berger, 1952; Berger, 1956; Berman, 1984; Berman and Raikow, 1982; Cracraft, 1971; Fisher, 1946; Gangl et al., 2004; Garrod, 1873; George and Berger, 1966; Haughton, 1867; Hudson, 1937; Hudson et al., 1969; Hudson et al., 1959; Hudson et al., 1972; Klemm, 1969; Maurer and Raikow, 1981; McGowan, 1979; McKitrick, 1991; Mellett, 1994; Miller, ; Mitchell, 1915; Owre, 1967; Patak and Baldwin, 1998; Raikow, 1970; Raikow, 1976; Raikow, 1978; Raikow, 1980; Raikow, 1985; Rosser et al., 1982; Ruck, 1949; Schreiweis, 1982; Swierczewki and Raikow, 1981; Vanden Berge, 1982; Verstappen et al., 1998; Wilcox, 1952; Zusi and Bentz, 1982; Zusi and Bentz, 1984) Phylogeny compiled from ((Cracraft, 1988; Mindell et al., 1997; Podulka et al., 2004; Sibley and Alquist, 1990)

191 A 174 B FCLP FCLA IG Figure 40: Flexor Cruris Lateralis (FCL) as seen in the mallard (Anas platyrhynchus) (A). Flexor Cruris Lateralis pars Pelvica (FCLP), Flexor Cruris Lateralis pars Accessoria (FCLA) and the Intermediate Gastrocnemius (IG) as seen in the Guinea Fowl (Numida meleagris) (B). Drawn by Dr. David Ellerby.