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Evolution of respiratory physiology for extreme high-altitude flight in the bar-headed goose Scott, Graham 2009

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EVOLUTION OF RESPIRATORY PHYSIOLOGY FOR EXTREME HIGH-ALTITUDE FLIGHT IN THE BAR-HEADED GOOSE  by  Graham Scott B.Sc., McMaster University, 2002 M.Sc., University of British Columbia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2009  © Graham Scott, 2009  ii ABSTRACT  Bar-headed geese migrate over the Himalayas at up to 9000m elevation where they must sustain the high metabolic rates needed for flight despite being severely hypoxic. The present thesis studied the respiratory physiology of this species to better understand the basis for this impressive feat. Evolutionary changes important for high altitude flight were deduced by comparing bar-headed geese to several low altitude species (greylag geese, pink-footed geese, barnacle geese, mallard ducks, or pekin ducks). Theoretical modelling of the O2 transport pathway was first used to determine traits with the greatest influence over O2 transport in hypoxia. This suggested that a heightened capacity to increase ventilation, a high haemoglobin O2 affinity, and an enhanced O2 diffusing capacity in the muscle should be most influential for enhancing O2 supply. Therefore, the remainder of this thesis tested the general hypothesis that these traits have evolved in bar-headed geese. Total ventilation was higher in bar-headed geese than in low altitude waterfowl during severe environmental hypoxia. This was entirely due to larger tidal volumes, which should have further enhanced effective ventilation of the gas exchange surface and thus increased O2 loading and arterial O2 tension. Two primary mechanisms accounted for this difference, (i) a reduction in metabolic depression during hypoxia and (ii) a blunted chemosensitivity to respiratory hypocapnia. These studies also supported previous research showing that bar-headed geese have a higher haemoglobin O2 affinity. Oxygen diffusing capacity in the flight muscle was also enhanced in bar-headed  iii geese. This was due to an increased number of capillaries surrounding each muscle fiber and a redistribution of mitochondria within these fibers towards the cell membrane, closer to capillaries. There were also slight increases in aerobic capacity and alterations in the control of mitochondrial respiration, which could help sustain ATP turnover during prolonged flight in hypoxia. This thesis has shown that high altitude adaptation in bar-headed geese has involved a suite of evolutionary changes at multiple steps in the O2 transport pathway. This work provides important insights into how respiratory systems evolve, and helps explain the incredible ability of bar-headed geese to fly at extremely high altitudes.  iv TABLE OF CONTENTS  Abstract ......................................................................................................................... ii Table of Contents ......................................................................................................... iv List of Tables ................................................................................................................. x List of Figures............................................................................................................... xi List of Abbreviations................................................................................................... xv Acknowledgements...................................................................................................... xx Co-Authorship Statement ......................................................................................... xxii 1. Introduction............................................................................................................... 1 1.1. Evolution of Respiratory Systems ................................................................. 1 1.2. High Altitude Adaptation.............................................................................. 4 1.3. The Bar-Headed Goose................................................................................. 5 1.4. Thesis Objectives and Hypotheses ................................................................ 6 1.5. References .................................................................................................... 9 2. Flying high: a theoretical analysis of the factors limiting exercise performance in birds at altitude ............................................................................. 14 2.1. Introduction ................................................................................................ 14 2.1.1. The ventilatory and pulmonary systems and oxygen transport ..................................................................................... 16 2.1.2. The circulatory system and oxygen transport ................................ 18 2.1.3. Flight muscle and oxygen transport .............................................. 20 2.1.4. How do birds fly high? ................................................................. 22  v 2.2. Methods...................................................................................................... 22 2.2.1. Theoretical model of O2 transport................................................. 22 2.2.2. Solution of the model ................................................................... 25 2.2.3. Sensitivity analysis ....................................................................... 27 2.2.4. Assumptions................................................................................. 28 2.3. Results........................................................................................................ 28 2.3.1. Oxygen transport in the lungs and tissues ..................................... 28 2.3.2. Effects of P50 on oxygen consumption .......................................... 29 2.3.3. Sensitivity analysis in normoxia ................................................... 30 2.3.4. Sensitivity analysis in moderate hypoxia ...................................... 32 2.3.5. Sensitivity analysis in severe hypoxia........................................... 32 •  2.3.6. V and DTO 2 during flight at extreme altitude ................................ 34 2.4. Discussion .................................................................................................. 35 !  ! Assumptions and starting data of the model.................................. 36 2.4.1. 2.4.2. What limits exercise performance in birds? .................................. 37 2.4.3. Potential adaptations for high altitude flight in birds..................... 39 2.4.4. Conclusions.................................................................................. 42  2.5. Summary of Chapter................................................................................... 42 2.6. References .................................................................................................. 52 3. Control of breathing and adaptation to high altitude in the bar-headed goose..................................................................................................... 60 3.1. Introduction ................................................................................................ 60 3.2. Materials and Methods................................................................................ 63  vi 3.2.1. Animals........................................................................................ 63 3.2.2. Surgical procedures ...................................................................... 63 3.2.3. Measurements .............................................................................. 64 3.2.4. Experimental protocol .................................................................. 65 3.2.5. Data and statistical analyses.......................................................... 66 3.3. Results........................................................................................................ 67 3.3.1. Poikilocapnic hypoxic ventilatory responses................................. 67 3.3.2. Isocapnic hypoxic ventilatory responses ....................................... 69 3.3.3. Acid-base regulation during hypoxia ............................................ 70 3.3.4. Ventilatory response to CO2/pH.................................................... 71 3.3.5. Breathing mechanics during hypoxia ............................................ 72 3.3.6. Metabolic responses to hypoxia and elevated breathing ................ 73 3.4. Discussion .................................................................................................. 74 3.4.1. Oxygen chemosensitivity is not enhanced in bar-headed geese ..... 75 3.4.2. Has sensitivity to CO2 changed in bar-headed geese? ................... 76 3.4.3. Pulmonary mechanics may have evolved in bar-headed geese ...... 78 3.4.4. Haemoglobin has evolved in bar-headed geese ............................. 79 3.4.5. The metabolic response to hypoxia has evolved in bar-headed geese .......................................................................... 80 3.4.6. Effective ventilation is enhanced in bar-headed geese................... 82 3.4.7. Implications for high altitude flight............................................... 83 3.4.8. Can phylogenetic history explain the interspecific differences? ................................................................................. 85  vii 3.5. Summary of Chapter................................................................................... 86 3.6. References ................................................................................................ 100 4. Body temperature depression and peripheral heat loss during hypoxia are reduced in bar-headed geese........................................................................... 108 4.1. Introduction .............................................................................................. 108 4.2. Materials and Methods.............................................................................. 110 4.2.1. Animals...................................................................................... 110 4.2.2. Surgical procedures .................................................................... 110 4.2.3. Measurements ............................................................................ 111 4.2.4. Experimental protocols............................................................... 112 4.2.5. Data and statistical analyses........................................................ 113 4.3. Results...................................................................................................... 114 4.3.1. Thermoregulatory responses to stepwise hypoxia ....................... 114 4.3.2. Metabolic responses to stepwise hypoxia.................................... 115 4.3.3. Ventilatory responses to stepwise hypoxia.................................. 116 4.3.4. Responses to prolonged hypoxia................................................. 117 4.3.5. Interactions of O2 loading with thermoregulation and metabolism................................................................................. 118 4.3.6. Effects of vagotomy on the responses to stepwise hypoxia ......... 119 4.4. Discussion ................................................................................................ 120 4.4.1. Hypoxic responses of waterfowl................................................. 120 4.4.2. Interspecific differences in hypoxia responses ............................ 123 4.4.3. High altitude adaptation in bar-headed geese .............................. 126  viii 4.5. Summary of Chapter................................................................................. 126 4.6. References ................................................................................................ 138 5. Evolution of muscle phenotype for extreme high altitude flight in the bar-headed goose................................................................................................... 145 5.1. Introduction .............................................................................................. 145 5.2. Materials and Methods.............................................................................. 146 5.2.1. Animals...................................................................................... 146 5.2.2. Muscle histology ........................................................................ 147 5.2.3. Respiration of muscle mitochondria............................................ 149 5.2.4. Statistical analyses...................................................................... 150 5.3. Results and Discussion.............................................................................. 151 5.3.1. Flight muscle phenotype in geese ............................................... 151 5.3.2. Respiration of muscle mitochondria in geese .............................. 156 5.3.3. Evolution of O2 transport for flight at high altitude ..................... 157 5.3.4. Conclusions................................................................................ 160 5.4. Summary of Chapter................................................................................. 160 5.5. References ................................................................................................ 178 6. Control of respiration in flight muscle fibers from the high altitude bar-headed goose and low altitude birds.............................................................. 184 6.1. Introduction .............................................................................................. 184 6.2. Materials and Methods.............................................................................. 186 6.2.1. Animals...................................................................................... 186 6.2.2. Permeabilized muscle fiber experiments..................................... 187  ix 6.2.3. Enzyme activity assays on homogenized muscle......................... 188 6.2.4. Creatine kinase activity assays on isolated mitochondria ............ 190 6.2.5. Data and statistical analyses........................................................ 191 6.3. Results...................................................................................................... 192 6.3.1. Respiration of permeabilized muscle fibers................................. 192 6.3.2. Muscle enzyme activities............................................................ 194 6.4. Discussion ................................................................................................ 194 6.4.1. Aerobic capacity of bar-headed goose flight muscle ................... 195 6.4.2. Control of respiration in bar-headed goose flight muscle ............ 197 6.4.3. Evolution of hypoxia tolerance ................................................... 200 6.5. Summary of Chapter................................................................................. 202 6.6. References ................................................................................................ 212 7. General Discussion and Conclusions .................................................................... 219 7.1. Mechanisms Underlying Extreme High Altitude Flight in the Bar-Headed Goose.................................................................................... 219 7.1.1. Ventilatory and pulmonary systems ............................................ 220 7.1.2. Circulatory systems .................................................................... 221 7.1.3. Oxygen diffusing capacity in the muscle..................................... 222 7.1.4. Oxygen utilization in the muscle................................................. 223 7.1.5. Costs and tradeoffs associated with high altitude flight ability .... 224 7.2. Evolution of Respiratory Systems ............................................................. 224 7.3. Future Directions ...................................................................................... 226 7.4. References ................................................................................................ 229  x LIST OF TABLES  Table 2.1. Potential physiological adaptations for high altitude bird flight..................... 44 Table 2.2. Literature values used for the calculations in the model ................................ 45 Table 3.1. Respiratory variables during poikilocapnic hypoxia...................................... 87 Table 3.2. Blood gas variables during poikilocapnic hypoxia ........................................ 88 Table 3.3. Respiratory variables during isocapnic hypoxia ............................................ 89 Table 3.4. Blood gas variables during isocapnic hypoxia............................................... 90 Table 3.5. Blood gas variables during hypercapnia at moderate hypoxia........................ 91 Table 4.1. Respiratory variables during prolonged hypoxia ......................................... 128 Table 4.2. Respiratory variables during stepwise hypoxia in vagotomized ducks......... 129 Table 5.1. Histological measurements from the flight muscle of geese ........................ 162 Table 6.1. Aerobic capacity is enhanced in bar-headed geese ...................................... 203 Table 6.2. Statistical results from phylogenetically independent contrast analysis........ 204 Table 6.3. Activities of metabolic enzymes in flight muscle ........................................ 205  xi LIST OF FIGURES  Fig. 2.1. Schematic of the oxygen transport pathway in birds ........................................ 46 Fig. 2.2. Oxygen tensions in the lung and tissue capillaries during normoxia................. 47 Fig. 2.3. Effects of biochemical features of haemoglobin on O2 consumption................ 48 Fig. 2.4. Effects of different physiological variables on O2 consumption ....................... 49 Fig. 2.5. Effect of varying the temperature effect on haemoglobin on O2 consumption in severe hypoxia...................................................................................... 50 Fig. 2.6. Assessment of the ventilation rates and tissue diffusion capacities that could achieve the rate of oxygen consumption for flight ......................................... 51 Fig. 3.1. Total ventilation was higher in bar-headed geese during severe poikilocapnic hypoxia than in both greylag geese and pekin ducks................................ 92 Fig. 3.2. The ventilatory response of bar-headed geese to poikilocapnic hypoxia involved a different breathing pattern............................................................... 93 Fig. 3.3. Ventilation was not higher in bar-headed geese in isocapnic hypoxia............... 94 Fig. 3.4. Breathing pattern during isocapnic hypoxia was similar in bar-headed geese, greylag geese, and pekin ducks........................................................................... 95 Fig. 3.5. Acid-base regulation was similar during hypoxia in all species........................ 96 Fig. 3.6. Total ventilation, breathing frequency, and tidal volume as a function of arterial CO2 tension................................................................................................... 97 Fig. 3.7. Total ventilation relative to peak inspiratory flow and peak expiratory flow in bar-headed geese, greylag geese, and pekin ducks ............................................. 98 Fig. 3.8. The responses of oxygen consumption rate to hypoxia were different  xii between species............................................................................................................. 99 Fig. 4.1. Body temperature depression during stepwise hypoxia was less severe in bar-headed geese than in greylag geese or pekin ducks................................. 130 Fig. 4.2. Surface temperatures of the bill increased in bar-headed geese, greylag geese, and pekin ducks during stepwise hypoxia ............................................. 131 Fig. 4.3. Bill surface temperatures increased in less severe hypoxia in greylag geese and pekin ducks than in bar-headed geese ............................................. 132 Fig. 4.4. Oxygen consumption rate increased during stepwise hypoxia in bar-headed geese, greylag geese, and pekin ducks ................................................... 133 Fig. 4.5. Total ventilation, breathing frequency, tidal volume, and air convection requirements during stepwise hypoxia recovery......................................... 134 Fig. 4.6. Body temperature change, oxygen consumption rate, and total ventilation during sustained 9% inspired O2 ................................................................ 135 Fig. 4.7. Relationships between measured variables and arterial oxygen loading during stepwise hypoxia ................................................................................. 136 Fig. 4.8. Bilateral vagotomy did not eliminate body temperature depression in response to stepwise hypoxia................................................................................... 137 Fig. 5.1. Hypothesized phylogenetic relationships among the species examined in Chapter 5................................................................................................. 163 Fig. 5.2. Histochemical staining of goose pectoralis muscle ........................................ 164 Fig. 5.3. Succinate dehydrogenase staining of flight muscle from geese ...................... 165 Fig. 5.4. Myosin-ATPase staining of flight muscle from geese .................................... 166 Fig. 5.5. Muscle fiber composition is altered in the pectoralis muscle of  xiii bar-headed geese......................................................................................................... 167 Fig. 5.6. Capillarity is enhanced in the pectoralis muscle of bar-headed geese ............. 168 Fig. 5.7. Capillary staining of flight muscle from geese ............................................... 169 Fig. 5.8. Relationship between muscle fiber area and body mass in geese.................... 170 Fig. 5.9. Regressions between standardized independent contrasts of muscle traits and migration altitude strategy............................................................................ 171 Fig. 5.10. Representative transmission electron micrographs from bar-headed geese......................................................................................................... 172 Fig. 5.11. Relationship between mitochondrial abundance and capillary density across muscle depths in geese...................................................................................... 173 Fig. 5.12. Mitochondria are redistributed towards the cell membrane in the oxidative fibers of bar-headed goose muscle ............................................................... 174 Fig. 5.13. Mitochondria isolated from the flight muscle of different bird species had similar respiration rates and phosphorylation efficiencies...................................... 175 Fig. 5.14. Oxygen kinetics of mitochondria isolated from the flight muscle was similar in bar-headed geese to in other bird species .............................................. 176 Fig. 5.15. Relationships between mitochondrial respiration rate, O2 tension, and mitochondrial P50 .................................................................................................. 177 Fig. 6.1. Hypothesized phylogenetic tree for species in the present study..................... 206 Fig. 6.2. Representative experiment showing the respiration of permeabilized fibers from the flight muscle of a bar-headed goose..................................................... 207 Fig. 6.3. Respiration of permeabilized fibers from the flight muscle ............................ 208 Fig. 6.4. Effects of changing respiration state on the relative changes in  xiv O2 consumption rate.................................................................................................... 209 Fig. 6.5. ADP kinetics of permeabilized fibers from the flight muscle ......................... 210 Fig. 6.6. Regressions between standardized independent contrasts of muscle respiration traits and flight altitude strategy ..................................................... 211  xv LIST OF ABBREVIATIONS  AA (IIa,m)  Areal density of type IIa fibers in the muscle  AA (IIb,m)  Areal density of type IIb fibers in the muscle  !  ACR  Air convention requirement or Acceptor control ratio  !  C:F  Number of capillaries per muscle fiber  CBF  Cerebral blood flow  CD  Capillary density  CacapO 2  O2 content of blood leaving a lung capillary  CaO 2  Arterial O2 content  CcapO 2  Instantaneous O2 content in a capillary  !  CHb  Concentration of haemoglobin in the blood  !  CK  Creatine kinase  COX  Cytochrome oxidase  CPT  Carnitine palmitoyltransferase  Cr  Creatine  CS  Citrate synthase  CV  Coefficient of variation  CvO 2  Mixed venous O2 content  Cv O 2  Venous O2 content  !  ! !  dCcapO 2 dt  DL O 2 !  !  Rate of change of the instantaneous O2 content in a capillary O2 diffusing capacity in the lungs  xvi  DTO 2  O2 diffusing capacity in the tissues  FCCP  Carbonylcyanide-p-trifluoromethoxyphenylhydrazone  !  (used to uncouple mitochondrial respiration)  FCL CO 2  Fraction of CO2 in the clavicular air sac  FICO 2  Fraction of CO2 in the inspired gas  FI O 2  Fraction of O2 in the inspired gas  FO 2  Fraction of O2 in a gas phase  !  fR  Breathing frequency  !  Hb  Haemoglobin  [HCO3-]a  Arterial concentration of bicarbonate  [HCO3-]v  Venous concentration of bicarbonate  HOAD  3-Hydroxyacyl-coA dehydrogenase  HVR  Hypoxic ventilatory response  IPC  Intrapulmonary chemoreceptors  Km  ADP concentration at 50% of maximum state 3 respiration  LDH  Lactate dehydrogenase  mi-CK  Mitochondrial isoenzyme of creatine kinase  n  Hill coefficient describing the cooperativity of haemoglobin-O2 binding  N  Number of individuals  ns  Not significant  P/O ratio  Quotient of ADP consumed and atomic oxygen consumed  Pa CO 2  Arterial partial pressure of CO2  PaO 2  Arterial partial pressure of O2  ! !  ! !  xvii PB  Barometric pressure  PcapO 2  Instantaneous PO 2 in a capillary  PCO 2  Partial pressure of CO2  PCTO 2  ! Partial pressure of O2 in the caudal thoracic air sac  PE O 2  Partial pressure of O2 in the expired gas  !  pHa  pH of arterial blood  !  pHv  pH of venous blood  PIO 2  Partial pressure of O2 in the inspired gas  PiO 2  Partial pressure of O2 in the intracellular compartment  PmitO 2  Partial pressure of O2 at the mitochondria  PO 2  Partial pressure of O2  PparaO 2  Instantaneous PO 2 in a parabronchiole  Pv CO 2  Venous partial pressure of CO2  PvO 2  ! Mixed venous partial pressure of O2  Pv O 2  Venous partial pressure of O2  !  PK  Pyruvate kinase  !  P50  Partial pressure of O2 at either 50% haemoglobin saturation or 50% of the  ! !  ! ! ! ! ! !  normoxic rate of mitochondrial respiration •  Q •  ! !  Cardiac output or the rate of blood flow  Q cap  Rate of blood flow through a capillary  Q10  Coefficient for the fractional effect of a 10°C temperature change  xviii r  Pearson product-moment correlation coefficient  R  Ideal gas constant  RQ  Respiratory quotient (quotient of the rates of CO2 production and O2 consumption)  RCR  Respiratory control ratio  SDH  Succinate dehydrogenase  Sv(cristae,mito)  Mitochondrial cristae surface density  T  Temperature  Tb  Body temperature  Tbill  Surface temperature of the bill  tcap  Total transit time through a capillary  TE  Expiratory time  TI  Inspiratory time  TMPD  N,N,N',N'-tetramethyl-p-phenylenediamine (used to maximally stimulate cytochrome oxidase)  •  V •  VO 2  Ventilation rate Rate of O2 consumption  ! •  ! !  !  VO 2MAX  Maximum rate of O2 consumption  VT  Tidal volume  •  V Tot  Total ventilation rate  Vv(lipid,f)  Intracellular lipid volume density  Vv(lipid,m)  Intramuscular lipid volume density  xix Vv(mito,f)  Mitochondrial volume density within muscle fibers  VV (mito,IIa) Mitochondrial volume density in type IIa fibers VV (mito,IIb) Mitochondrial volume density in type IIb fibers !  VV (mito,m)  Average mitochondrial volume density at a location in the muscle  !  ∆pH(a-v)  pH difference between arterial and venous blood  !  φ  Bohr coefficient  xx ACKNOWLEDGEMENTS  I have been incredibly lucky to have completed my graduate training in such a stimulating intellectual environment, and there are several people that deserve my sincerest thanks. Firstly, and most importantly, I would like to thank my supervisor Bill Milsom. Although his wisdom extended well beyond the boundaries of science (to quality wines and food or great north shore treks), he taught me how to find simplicity in the complexity of biology. Bill is an excellent scientist, a champion of comparative physiology, and a great mentor. I would also like to express my sincerest gratitude to a few other individuals that have had an immense influence on my success and intellectual development: Jeff Richards, for wooing me into the realm of biochemistry and for many years of support and guidance; Trish Schulte, for impressing upon me the importance of thinking broadly; and Chris Wood, for bringing me into science, for many years of insightful and constructive feedback, and for opening so many doors. I have the utmost respect for these individuals and I would not be where I am now without them. I would also like to thank my committee members, Colin Brauner, Darren Irwin, Dave Jones, and Bill Sheel. They were all excellent resources and invested a lot of time into me and my thesis. They deserve my deepest gratitude for keeping me on my toes! The comparative physiology group at UBC is a great environment because of an amazing group of graduate students and postdocs. I would like to thank several people in the Milsom lab for their friendship and support, including both current (Cosima Ciuhandu, Angelina Fong, Stella Lee, Catalina Reyes) and past (Dennis Andrade, Andrea  xxi Corcoran, Barb Gajda, and Angie O’Neill) members. I have many good friends and colleagues from other labs as well, far more than I can mention here, but Dan Baker, Jason Bystrianski, Charles Darveau, Nann Fangue, Anne Dalziel, Rush Dhillon, Jeremy Goldbogen, Milica Mandic, Jodie Rummer, Ben Speers-Roesch, and Anne Todgham deserve special mention for helping and inspiring me along the way. There were a lot of other people that provided technical help that was invaluable to completing this thesis. Arthur Vanderhorst and Sam Gopaul provided exceptional animal care for my birds and I am indebted to them. Stuart Egginton and Glenn Tattersall were great collaborators and made important contributions to my research. Rowan Barrett, Bruce Bowen, Viviana Cadena, Ted Garland, Tamara Godbey, Gordon Gray, Jordon Guenette, Chris Harvey-Clark, Wayne Maddison, Fahima Syeda, and Sheila Thornton all provided important technical instruction. Derrick Horne was a brilliant help with the electron microscope. Thanks as well to the various agencies that funded my salary and travel during the last five years. The money I received from NSERC, the Killam Trusts, IODE Canada, the Company of Biologists, the Canadian Society of Zoologists, Novo Nordisk, EPCOR Water Limited, and the Faculty of Graduate Studies and the Department of Zoology at UBC is very much appreciated. Finally, I am thankful for all the support from my parents, Gary and Melonie, as well as my wonderful wife Angela. In particular, I thank Angela for tolerating me during the hard times, for accommodating my sometimes-extensive travel schedule, and for just being there always. I sincerely thank everyone that made my time at UBC so amazing.  xxii CO-AUTHORSHIP STATEMENT  The manuscripts presented in chapters 2 through 6 contain one to three authors in addition to the author of the present thesis. G.R. Scott devised the general outline and objectives of the thesis, and was the primary contributor to experimental design, data collection and analysis, and manuscript preparation. W.K. Milsom provided valuable supervision to all components of the thesis, helped design all experiments, and provided useful criticism to all manuscripts. Additional co-authors (V. Cadena, S. Egginton, J.G. Richards, and G.J. Tattersall) made important secondary contributions to various aspects of the research.  1 1. INTRODUCTION  The ecology, behaviour, and biogeographic distribution of organisms are critically dependent on their underlying physiology. Physiology facilitates homeostasis, both during periods of relative ease and during stressful environmental challenges, and thus allows organisms to survive, interact with others, and reproduce. For this reason, studies aimed at understanding how physiological systems evolve help explain the mechanistic basis of emergent traits such as performance and life history. Respiratory systems are particularly useful for studying the patterns and processes of physiological evolution. Oxygen is the terminal electron acceptor in the mitochondrial reactions that produce cellular energy in the form of ATP, and is thus essential for normal metabolism in most eukaryotic organisms. However, as organisms get larger or their metabolic requirements increase, diffusion of O2 from the environment can be insufficient to meet the O2 demands of the body; efficient systems for convective O2 supply then become essential (Burggren, 2004). Thus, the respiratory system of most vertebrates is crucial for transporting O2 from the environment, and has been shaped by evolution to meet the diverse gas exchange needs of organisms (e.g., Farmer, 1999; Hicks, 2002; Maina, 2002; Brainerd and Owerkowicz, 2006).  1.1. Evolution of Respiratory Systems Oxygen transport occurs along a pathway from environment to mitochondria that can be conceptually divided into a series of five interacting steps: (i) breathing, which brings O2 into contact with the respiratory surface; (ii) pulmonary (or branchial) diffusion  2 across the air-blood (or water-blood) interface; (iii) circulation via the blood, which delivers O2 throughout the body; (iv) tissue diffusion across the blood-mitochondria interface; and (v) mitochondrial O2 utilization, which creates ATP (Weibel, 1984). The intriguing question is how evolutionary changes in flux through the entire O2 transport pathway occur: must every step in this pathway evolve, or can pathway flux be altered by changing only some but not all of these steps? An early theory addressing this question suggested that the capacity of every step in the pathway must increase equally to enhance overall capacity (Weibel et al., 1981; Weibel et al., 1991). This theory was termed symmorphosis, and was based on the suppositions that (i) the maximum capacity for flux that is realized by the whole pathway in vivo is near the structural (morphological) limits of each step in the pathway, and (ii) there is strong selection against excess structural capacity beyond the attainable limits in vivo. The prediction of this theory is that all structural components of the O2 pathway are equally rate-limiting for gas exchange, and that this should apply across species and training states. This theory has good correlative support from interspecific comparisons of species with different body sizes or exercise capacities (see publications that follow from Taylor and Weibel, 1981; Taylor et al., 1996). The theory has been subject to strict criticism, however, based on theoretical arguments against optimality models and how evolutionary change occurs, as well as specific examples where the theory does not hold (Garland and Huey, 1987; Dudley and Gans, 1991; Jones, 1998). Symmorphosis is therefore considered a useful null hypothesis rather than a causal and unifying principle of pathway evolution (Diamond, 1992; Jones, 1998).  3 A more recent theory of respiratory pathway flux argues that overall control arises from the summed influence of each step in the pathway, and that different steps can have unequal contributions to control (analogous to metabolic control theory) (Darveau et al., 2002; Hochachka and Burelle, 2004). The theory implies by extension that changes in the overall capacity for O2 transport during evolution can involve changes of varying magnitudes at different steps. This theory is less at odds with the criticisms faced by the symmorphosis theory, and has persuasive support from examples of single-species adaptation (Jones, 1998) and artificial selection experiments that have monitored the evolution of aerobic capacity over short time-scales (Henderson et al., 2002; Gonzalez et al., 2006a; Gonzalez et al., 2006b). The most parsimonious explanation for how the O2 pathway evolves should reconcile the observations forming the basis for each of these two theories. Firstly, individual steps in the O2 pathway can influence pathway flux and may evolve without concurrent changes in other steps. Signatures of this mode of pathway evolution should be most apparent over short evolutionary time scales (e.g., intraspecific population comparisons or in artificial selection experiments). Secondly, natural selection against excess structural capacity (when it is of little selective advantage and not somehow constrained) may equalize the structural capacities of individual steps over longer time scales, resulting in apparent signatures of symmorphosis. The intriguing question that arises is which steps in the O2 transport pathway are most likely to change during evolution? The most satisfying answer may be that steps with more control, and potentially greater selective advantage, are more likely to evolve, but this idea lacks empirical exploration.  4 1.2. High Altitude Adaptation High altitude environments are extremely challenging. For every kilometer one ascends, the barometric pressure falls over 10%, so that at the peak of the world’s highest mountains the oxygen pressure is only 1/4 of what it is at sea level. Supplying enough O2 to sustain aerobic metabolism can be a challenge for low altitude species in hypoxia, and can even be debilitating; indeed, the very best human mountaineers can barely sustain basal metabolism at the summit of Mount Everest (West, 2000). However, wherever there is a food source, high altitude environments contain a variety of species that have adapted successfully. Paramount in these species are evolutionary changes that increase the capacity for O2 transport when O2 availability is low. Animals that are adapted (in an evolutionary sense) to high altitude thus provide an excellent opportunity for studying the patterns of respiratory system evolution. Several previous studies have explored the unique physiological characteristics of animals that are native to high altitude. The species studied span the vertebrates, and include mammals (mice, pika, camelids, human populations), birds (waterfowl, chicken, vultures, hummingbirds, passerines), amphibians (Andean frog), and even fish (scaleless carp). Alterations in the O2-binding properties of haemoglobin are by far the best-studied characteristic (Weber, 2007; Storz and Moriyama, 2008), but examples of putativelyadaptive differences in high altitude species have been reported for every step in the O2 cascade (e.g., Ge et al., 1998; Hammond et al., 2001; Mathieu-Costello, 2001; Sheafor, 2003; Brutsaert et al., 2005). Nevertheless, a clear picture of the full suite of evolutionary changes necessary to succeed at high altitude has yet to emerge, largely because the whole O2 pathway is rarely studied in a single species in its entirety (humans being a  5 possible exception). Doing so could provide significant insights into the physiological mechanisms of high altitude adaptation and into how respiratory systems evolve.  1.3. The Bar-Headed Goose One of the most celebrated high altitude performers is the bar-headed goose (Anser indicus), which flies over the Himalayas twice a year on its migration between southern and central Asia. This species is one of at least ten closely-related goose species within the genus Anser, and is thought to have arisen within the last 2.5 million years (Ruokonen et al., 2000; Donne-Goussé et al., 2002) concurrent with the rapid up-thrust of the Himalayas (Valdiya, 2002). This species has been reported to transcend the highest peaks in the range, at altitudes of up to 9000m, and does not seem to show preference for low altitude migration routes through riverine valleys (Swan, 1970; Javed et al., 2000). This contrasts the majority of other waterfowl species, whose migration routes are generally at low to moderate altitudes (Cramp and Simmons, 1977) and rarely involves venturing to greater heights (e.g., Manville, 1963), suggesting that high altitude migration is a uniquely derived characteristic of bar-headed geese. Incredibly, the metabolic costs of flight in bar-headed geese require 10- to 20-fold increases in O2 consumption rate (Ward et al., 2002), which must be sustained despite the severe hypoxia at high altitudes. Being impossible for low altitude birds, the impressive physiological feat of this species should require unique specializations for matching O2 supply and demand at high altitudes. Indeed, bar-headed geese have greater hypoxia tolerance than both closely- and distantly-related low altitude waterfowl (Black and Tenney, 1980). This species has also evolved a unique haemoglobin protein sequence,  6 which causes an inherently higher O2 affinity than that of low altitude waterfowl (Petschow et al., 1977; Jessen et al., 1991; Zhang et al., 1996). However, when the present thesis work began these were the only potential high altitude adaptations demonstrated in bar-headed geese. Otherwise, the physiological basis for the elevated O2 transport capacity of this species in hypoxia was poorly understood (Fedde et al., 1985; Faraci, 1991). Bar-headed geese are therefore a fascinating system for studying the evolution of performance at high altitude, and understanding the mechanisms underlying their extreme physiological feat was of interest.  1.4. Thesis Objectives and Hypotheses The general objective of this thesis was to determine how the O2 transport pathway of bar-headed geese has evolved to facilitate high altitude migration. This required an integrative approach employing a diversity of methods, to test the general hypothesis that multiple steps in the O2 transport pathway are enhanced in bar-headed geese to sustain performance in severe hypoxia. In subsequent chapters, five manuscripts will be presented that tested this general hypothesis for different steps in the O2 pathway. Other physiological systems that are relevant to exercise performance in hypoxia will also be explored in these chapters. Chapter 2 starts with a general review of hypoxia tolerance in birds, highlighting previous work in bar-headed geese and other species that provides a foundation for the remainder of the thesis. This is followed by a theoretical analysis of the factors limiting exercise performance in low altitude birds, which identified the steps in the O2 transport pathway with the greatest potential for increasing O2 transport during hypoxia. The  7 general hypothesis of the thesis thus became more specific: those steps in the O2 pathway with the greatest control over O2 transport will show the greatest improvements associated with high altitude flight in bar-headed geese. Subsequent experiments (with their own specific hypotheses) were designed to test this general hypothesis. The specific hypothesis tested in chapters 3 and 4 is that the hypoxic ventilatory response of bar-headed geese is enhanced compared with that of low altitude species. These chapters contain a series of experiments examining the control of breathing, as well as the influence of breathing on O2 loading into the blood. These chapters also examine other physiological changes that influence breathing and metabolism at high altitude, such as changes in acid-base status and body temperature. The specific hypothesis tested in chapter 5 is that the capacity for O2 diffusion from the blood to mitochondria is enhanced in the flight muscle of bar-headed geese compared with that of low altitude species. Multiple morphometric features related to muscle O2 transport are investigated, as are numerous traits related to muscle aerobic capacity and O2 utilization. Following from the results of chapter 5, chapter 6 further examines muscle aerobic capacity in bar-headed geese. This chapter also tests the hypothesis that the control of mitochondrial respiration is altered in this species to help match ATP supply and demand and promote metabolite stability. The general outcome of this thesis is an integrated understanding of how the O2 transport pathway has evolved in bar-headed geese to support high altitude flight, as well as how evolutionary changes in this pathway have influenced other important  8 physiological systems. In doing so, this thesis contributes appreciable insight into the general mechanisms of how physiological systems evolve.  9 1.5. References Black, C. P. and Tenney, S. M. (1980). Oxygen transport during progressive hypoxia in high altitude and sea level waterfowl. Respir. Physiol. 39, 217-239. Brainerd, E. L. and Owerkowicz, T. (2006). Functional morphology and evolution of aspiration breathing in tetrapods. Respir. Physiol. Neurobiol. 154, 73-88. Brutsaert, T. D., Parra, E. J., Shriver, M. D., Gamboa, A. and León-Velarde, F. (2005). Ancestry explains the blunted ventilatory response to sustained hypoxia and lower exercise ventilation of Quechua altitude natives. Am. J. Physiol. Reg. Integr. Comp. Physiol. 289, R225-R234. Burggren, W. W. (2004). What is the purpose of the embryonic heart beat? Or how facts can ultimately prevail over physiological dogma. Physiol. Biochem. Zool. 77, 333-345. Cramp, S. and Simmons, K. E. L. (1977). Handbook of the birds of Europe, the Middle East, and North Africa: the birds of the Western Palearctic. Oxford, UK: Oxford University Press. Darveau, C. A., Suarez, R. K., Andrews, R. D. and Hochachka, P. W. (2002). Allometric cascade as a unifying principle of body mass effects on metabolism. Nature 417, 166-170. Diamond, J. M. (1992). Evolutionary physiology. The red flag of optimality. Nature 355, 204-206. Donne-Goussé, C., Laudet, V. and Hänni, C. (2002). A molecular phylogeny of anseriformes based on mitochondrial DNA analysis. Mol. Phylogenet. Evol. 23, 339-356.  10 Dudley, R. and Gans, C. (1991). A critique of symmorphosis and optimality models in physiology. Physiol. Zool. 64, 627-637. Faraci, F. M. (1991). Adaptations to hypoxia in birds: how to fly high. Annu. Rev. Physiol. 53, 59-70. Farmer, C. G. (1999). Evolution of the vertebrate cardio-pulmonary system. Annu. Rev. Physiol. 61, 573-592. Fedde, M. R., Faraci, F. M., Kilgore, D. L., Cardinet, G. H. and Chatterjee, A. (1985). Cardiopulmonary adaptations in birds for exercise at high altitude. In Circulation, Respiration, and Metabolism (eds. R. Gilles), pp. 149-163. Berlin: Springer-Verlag. Garland, T. and Huey, R. B. (1987). Testing symmorphosis: does structure match functional requirements? Evolution 41, 1404-1409. Ge, R. L., Kubo, K., Kobayashi, T., Sekiguchi, M. and Honda, T. (1998). Blunted hypoxic pulmonary vasoconstrictive response in the rodent Ochotona curzoniae (pika) at high altitude. Am. J. Physiol. Heart Circ. Physiol. 274, H1792-H1799. Gonzalez, N. C., Howlett, R. A., Henderson, K. K., Koch, L. G., Britton, S. L., Wagner, H. E., Favret, F. and Wagner, P. D. (2006a). Systemic oxygen transport in rats artificially selected for running endurance. Respir. Physiol. Neurobiol. 151, 141-150. Gonzalez, N. C., Kirkton, S. D., Howlett, R. A., Britton, S. L., Koch, L. G., Wagner, H. E. and Wagner, P. D. (2006b). Continued divergence in VO2max of rats artificially selected for running endurance is mediated by greater convective blood O2 delivery. J. Appl. Physiol. 101, 1288-1296.  11 Hammond, K. A., Szewczak, J. and Krol, E. (2001). Effects of altitude and temperature on organ phenotypic plasticity along an altitudinal gradient. J. Exp. Biol. 204, 1991-2000. Henderson, K. K., Wagner, H., Favret, F., Britton, S. L., Koch, L. G., Wagner, P. D. and Gonzalez, N. C. (2002). Determinants of maximal O2 uptake in rats selectively bred for endurance running capacity. J. Appl. Physiol. 93, 1265-1274. Hicks, J. W. (2002). The physiological and evolutionary significance of cardiovascular shunting patterns in reptiles. News Physiol. Sci. 17, 241-245. Hochachka, P. W. and Burelle, Y. (2004). Control of maximum metabolic rate in humans: dependence on performance phenotypes. Mol. Cell. Biochem. 256, 95103. Javed, S., Takekawa, J. Y., Douglas, D. C., Rahmani, A. R., Kanai, Y., Nagendran, M., Choudhury, B. C. and Sharma, S. (2000). Tracking the spring migration of a bar-headed goose (Anser indicus) across the Himalaya with satellite telemetry. Global Environ. Res. 2, 195-205. Jessen, T.-H., Weber, R. E., Fermi, G., Tame, J. and Braunitzer, G. (1991). Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering. Proc. Natl. Acad. Sci. USA 88, 6519-6522. Jones, J. H. (1998). Optimization of the mammalian respiratory system: symmorphosis versus single species adaptation. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 120, 125-138. Maina, J. N. (2002). Structure, function and evolution of the gas exchangers: comparative perspectives. J. Anat. 201, 281-304.  12 Manville, R. H. (1963). Altitude record for mallard. Wilson Bull. 75, 92. Mathieu-Costello, O. (2001). Muscle adaptation to altitude: tissue capillarity and capacity for aerobic metabolism. High Alt. Med. Biol. 2, 413-425. Petschow, D., Würdinger, I., Baumann, R., Duhm, J., Braunitzer, G. and Bauer, C. (1977). Causes of high blood O2 affinity of animals living at high altitude. J. Appl. Physiol. 42, 139-143. Ruokonen, M., Kvist, L. and Lumme, J. (2000). Close relatedness between mitochondrial DNA from seven Anser goose species. J. Evol. Biol. 13, 532-540. Sheafor, B. A. (2003). Metabolic enzyme activities across an altitudinal gradient: an examination of pikas (genus Ochotona). J. Exp. Biol. 206, 1241-1249. Storz, J. F. and Moriyama, H. (2008). Mechanisms of hemoglobin adaptation to high altitude hypoxia. High Alt. Med. Biol. 9, 148-157. Swan, L. W. (1970). Goose of the Himalayas. Nat. Hist. 70, 68-75. Taylor, C. R. and Weibel, E. R. (1981). Design of the mammalian respiratory system. I. Problem and strategy. Respir. Physiol. 44, 1-10. Taylor, C. R., Weibel, E. R., Weber, J. M., Vock, R., Hoppeler, H., Roberts, T. J. and Brichon, G. (1996). Design of the oxygen and substrate pathways. I. Model and strategy to test symmorphosis in a network structure. J. Exp. Biol. 199, 16431649. Valdiya, K. S. (2002). Emergence and evolution of Himalaya: reconstructing history in the light of recent studies. Prog. Phys. Geog. 26, 360-399.  13 Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weber, R. E. (2007). High-altitude adaptations in vertebrate hemoglobins. Respir. Physiol. Neurobiol. 158, 132-142. Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA, USA: Harvard University Press. Weibel, E. R., Taylor, C. R., Gehr, P., Hoppeler, H., Mathieu, O. and Maloiy, G. M. O. (1981). Design of the mammalian respiratory system. IX. Functional and structural limits for oxygen flow. Respir. Physiol. 44, 151-164. Weibel, E. R., Taylor, C. R. and Hoppeler, H. (1991). The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Natl. Acad. Sci. USA 88, 10357-10361. West, J. B. (2000). Human limits for hypoxia: the physiological challenge of climbing Mt. Everest. Ann. N. Y. Acad. Sci. 899, 15-27. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493.  14 2. FLYING HIGH: A THEORETICAL ANALYSIS OF THE FACTORS LIMITING EXERCISE PERFORMANCE IN BIRDS AT ALTITUDE1  2.1. Introduction Oxidative metabolism provides the energy for cellular processes in the majority of living organisms, yet many animals can survive and reproduce in environments with very little oxygen. Living in these environments generally requires many physiological, cellular, and molecular specializations that match O2 supply to O2 demand. On the one hand, there are examples of species in all vertebrate classes that reduce O2 demand in hypoxia by suppressing total metabolism (Boutilier, 2001). On the other hand, some animals have a remarkable capacity to increase O2 supply in hypoxia, and generally do so by increasing the flux capacity of O2 transport pathways (Hochachka, 1985). This strategy is especially important in species that exercise in hypoxic environments, and there is perhaps no better example of animals that perform high intensity exercise during hypoxia than birds flying at altitude. Indeed, O2 consumption may increase 10-20 fold during flight (Butler et al., 1977; Ward et al., 2002; Peters et al., 2005), and some birds are known to fly at exceptionally high altitudes. Bar-headed geese (Anser indicus) are perhaps the best known in this regard: this species flies over the Himalayan mountains twice a year on its migration between Tibet and India and has been sighted flying above the highest peaks in the range (9000m above sea level) (Swan, 1970).  1  A version of this chapter has been published: Scott, G. R. and Milsom, W. K. (2006). Flying high: a theoretical analysis of the factors limiting exercise performance in birds at altitude. Respir. Physiol. and Neurobiol. 154, 284-301.  15 How species such as the bar-headed goose fly at altitudes that can render many mammals comatose (Tucker, 1968) remains a mystery. It seems reasonable, however, that an enhanced O2 flux capacity along the pathway from environment to mitochondria plays a substantial role. This oxygen cascade consists of many steps, including ventilation, diffusion of O2 into the blood, circulation of O2 throughout the body, and diffusion from the blood to the tissue mitochondria. Important specializations in high altitude fliers could exist at multiple steps in this cascade. In fact, if we consider symmorphosis as a null hypothesis (Weibel et al., 1991), evolutionary changes that enhance O2 flux could exist at all steps in the cascade. Substantial research has explored some of these steps in high altitude birds, as will be discussed in the following sections, but others remain largely unexplored. The present study was undertaken to address the question of how some birds are able to fly at extreme altitudes. The potential physiological specializations that support high altitude flight have attracted a great deal of attention in the past (Faraci, 1991) and this research will first be reviewed briefly (highlighted in Table 2.1). A theoretical sensitivity analysis will then be presented that explores the possible factors that could limit exercise performance, which must be overcome in birds flying at high altitude. In doing so, the primary objective of this analysis was to quantitatively assess the physiological variables that should have the greatest adaptive benefit for flight in high altitude hypoxia.  16 2.1.1. The ventilatory and pulmonary systems and oxygen transport The bird lung is unique among the lungs of air-breathing vertebrates, with a blood flow that is crosscurrent to gas flow, and a gas flow that occurs unidirectionally through rigid parabronchioles. As such, bird lungs are inherently more efficient than the lungs of other air-breathing vertebrates (Piiper and Scheid, 1972; Piiper and Scheid, 1975). While this may partially account for the greater hypoxia tolerance of birds in general when compared to mammals (cf. Scheid, 1990), its presence in all birds excludes the crosscurrent lung as a possible adaptation that is specific to high altitude fliers. Similarly, an extremely small diffusion distance across the blood-gas interface compared to other air breathers seems to be a characteristic of all bird lungs, and not just those of high fliers (Maina and King, 1982; Powell and Mazzone, 1983; Shams and Scheid, 1989; West et al., 2006). Partly because of this small diffusion distance, the inherent O2 diffusing capacity across the gas-blood interface ( DL O 2 ) is generally high in birds (Shams and Scheid, 1989), although other factors can conspire to decrease the efficiency of lung to  ! ventilation-perfusion heterogeneity may occur in some blood O2 transfer. For example, birds during hypoxia (Schmitt et al., 2002) and could limit O2 exchange, but it remains to be seen if this also occurs in high altitude birds. Birds generally increase ventilation during hypoxia, primarily (but not exclusively) due to increases in breathing frequency (Bouverot et al., 1976; Colacino et al., 1977; Brackenbury et al., 1982; Powell et al., 2000). A heightened capacity to increase ventilation during severe hypoxia might benefit high altitude birds, because it could reduce the difference between inspired PO 2 and arterial PO 2 (Tucker, 1968; Shams and Scheid, 1989). However, although it is known that bar-headed geese can increase  !  !  17 ventilation by nearly 10-fold during severe poikilocapnic hypoxia (3% O2, equivalent to 11580m elevation) (Black and Tenney, 1980) and during treadmill exercise in moderately severe poikilocapnic hypoxia (7% O2, 7620m) (Fedde et al., 1989), it is unclear to what extent they differ from low altitude birds (Black and Tenney, 1980; Kiley et al., 1985). Several other important issues are still unresolved. Previous studies of running and swimming birds elicited only 2-3-fold increases in O2 consumption (Kiley et al., 1985; Butler and Turner, 1988; Fedde et al., 1989), compared to the 15-20-fold increases seen during flight (Ward et al., 2002), and differences in the capacities of the respiratory systems in low and high altitude birds may only become apparent when ventilatory demands increase to those seen during flight. Furthermore, hyperventilation at altitude is associated with a respiratory hypocapnia/alkalosis, which tends to inhibit breathing. If high altitude birds are less sensitive to changes in CO2/pH they might be better able to sustain increases in ventilation during severe hypoxia coupled with exercise. The CO2/pH sensitivity of ventilation is commonly assessed by comparing the isocapnic and poikilocapnic hypoxic ventilatory responses; however, the isocapnic ventilatory responses to hypoxia of both low and high altitude birds have not been compared (but see Powell et al., 2000). In this regard, the ventilatory response in high altitude birds may also depend on their capacity to maintain intracellular pH during alkalosis (Weinstein et al., 1985), or to buffer changes in extracellular pH due to hyperventilation (Dodd and Milsom, 1987; Dodd et al., 2007). It therefore remains to be determined whether high altitude fliers have a greater capacity to increase ventilation during severe hypoxia.  18 2.1.2. The circulatory system and oxygen transport After diffusing into the blood in the lungs, O2 is circulated throughout the body primarily bound to haemoglobin. A high cardiac output is therefore important for exercise at high altitude to supply the working muscle with adequate amounts of O2. Indeed, animals selectively bred for exercise performance have higher maximum cardiac outputs (Hussain et al., 2001), as do species that have evolved for exercise performance (Hoppeler and Weibel, 1998). Whether cardiac output limits exercise performance per se, however, is less clear; other factors may limit intense exercise, and in more athletic species (or individuals) cardiac output may be higher simply out of necessity (Wagner, 1996b). Excessive cardiac output may even be detrimental if blood transit times in the lungs or tissues are substantially reduced. Unfortunately, very little is known about cardiac performance in high flying birds. Both the high altitude bar-headed goose and the low altitude pekin duck (Anas platyrhynchos) can increase cardiac output at least 5-fold during hypoxia at rest (Black and Tenney, 1980), but no comparison of maximum cardiac performance has been made between high and low altitude birds. Changes in blood flow distribution during hypoxia have received a great deal of attention in birds. In general, hypoxia-induced hyperventilation causes hypocapnia and respiratory alkalosis. Because cerebral blood flow (CBF) is primarily regulated by the  PCO 2 /pH of the cerebrospinal fluid, high altitude results in cerebral vasoconstriction and a severe reduction in O2 delivery to the brain in most mammals (Faraci and Fedde, 1986).  !  Interestingly, whereas the effects of hypercapnia/acidosis on CBF appears to be similar in birds and mammals, CBF in birds appears to be insensitive to the effects of hypocapnia/alkalosis, unlike in mammals (Grubb et al., 1978; Faraci and Fedde, 1986).  19 However, the ability to maintain CBF during hypocapnia/alkalosis is thought to be present in all birds, so differences in CBF regulation are unlikely to be a unique specialization for flying at extreme altitudes; indeed, cerebral O2 delivery is maintained and is equivalent in both bar-headed geese and pekin ducks during severe hypoxia (Faraci et al., 1984b). Not only must O2 delivery be maintained to the brain, it must also increase substantially to the heart and pectoral muscle during flight at extreme altitudes. A portion of this increase in O2 delivery arises from redistributing blood flow away from the splanchnic region, the kidneys, and the skin, towards the exercising muscle; however, the •  contribution of flow redistribution to the large increase in VO 2 during flight is probably small. This suggestion comes from research comparing the impact of redistributing blood  ! flow to working muscle between sedentary and trained individuals or species: for •  individuals/species with a high VO 2MAX , the impact of supplying proportionally more blood to the muscles is diminished (Hochachka, 1985). Instead, high altitude species have frequently adapted to ! hypoxia by increasing the affinity of their haemoglobin (Hb) for O2 (Snyder, 1985; Samaja et al., 2003). This is the case for many high-flying birds (Petschow et al., 1977) whose increased Hb-O2 affinity is frequently based on only one or a few amino acid substitutions (Braunitzer and Hiebl, 1988; Weber et al., 1988; Weber et al., 1993; Zhang et al., 1996; Weber and Fago, 2004). As a result, species such as the barheaded goose can sustain greater O2 delivery to muscles than low altitude birds during hypoxia, because at any given blood PO 2 their blood O2 content will be higher (Faraci et al., 1984b).  !  20 Even though haemoglobin with a lower P50 increases oxygen loading across the lungs, and can result in higher tissue O2 delivery, the absolute benefit of a low P50 on O2 •  consumption ( VO 2 ) is unclear (but see Chappell and Snyder, 1984). This is primarily because a low P50 inhibits oxygen unloading at the tissues, so it has been suggested that other!characteristics of Hb that facilitate oxygen unloading may be important in high altitude-adapted animals (Samaja et al., 2003). Previous suggestions include variations in the Bohr (CO2/pH) effect on Hb-O2 affinity and the nature of Hb-O2 cooperativity. It is notable in this regard that the Bohr effect in bar-headed geese is less than in other waterfowl (Liang et al., 2001), suggesting that traits other than Hb biochemistry may increase O2 unloading in high flying birds. The concentration of Hb in the blood could potentially influence O2 transport at altitude as well. Increased Hb content often accompanies altitude acclimatization in some mammals. Blood Hb concentration is generally the same in low and high altitude birds (Black and Tenney, 1980), however, and acclimation to hypoxia increases blood Hb in low altitude birds but not in high altitude birds. These results suggest that blood Hb concentration is not adaptive for high altitude flight.  2.1.3. Flight muscle and oxygen transport Once oxygenated blood is circulated to the tissues, O2 moves to the tissue mitochondria, the site of oxidative phosphorylation and O2 consumption. Transport of O2 from the blood to the mitochondria involves several steps. Oxygen must first dissociate from Hb and diffuse through the various compartments of the blood, but in both birds and mammals the conductances of these steps are high, and are unlikely to impose much of a  21 limitation to O2 transport (Phu et al., 1986). In contrast, diffusion across the vascular wall and through the extracellular spaces is thought to provide the greatest limitation to O2 transport (Wittenberg and Wittenberg, 2003). Consequently, the size of the capillarymuscle fiber interface is an extremely important determinant of a muscle’s aerobic capacity (Hepple, 2000; Mathieu-Costello, 2001). Finally, O2 diffuses across the muscle fiber membrane and moves through the cytoplasm to cytochrome oxidase, the terminal O2 acceptor in the mitochondrial electron transport chain. Myoglobin probably assists intracellular O2 transport and thus reduces the resistance to O2 flux (Gayeski and Honig, 1988; Wittenberg and Wittenberg, 2003). Physiological traits that increase the O2 diffusing capacity of muscle could be important for flight at extreme altitude in birds. Because the capillary to muscle fiber •  interface appears to provide the greatest resistance to O2 flux, maximum VO 2 could be increased in high altitude birds by increases in flight muscle and heart muscle  ! capillarization. This is indeed the case in the flight muscle of finches, as those living at moderate altitude have higher capillarization than those living at sea level (MathieuCostello et al., 1998). Similar differences appear to exist between the leg muscles of high altitude and sea level geese: bar-headed geese reared in normoxia had greater gastrocnemius muscle capillarity than Canada geese (Branta canadensis), and this difference persisted even after Canada geese were reared in moderate hypoxia (Snyder et al., 1984). In contrast, myoglobin contents of the pectoral, cardiac, and gastrocnemius muscles are equivalent between bar-headed geese and other low-flying waterfowl (Snyder et al., 1984; Saunders and Fedde, 1991). It remains to be seen if capillarity, and thus O2 diffusing capacity, is enhanced in the flight muscle of bar-headed geese.  22 In addition to the diffusive pathways for O2 flux in the muscle, O2 consumption is influenced by certain characteristics of mitochondrial metabolism in the muscle. Both the maximum aerobic capacity and the mitochondrial O2 kinetics (e.g., the ‘affinity’ of mitochondria for O2) can influence the O2 consumption rate, so adaptation to high altitude could increase tissue mitochondria content or mitochondrial O2 affinity (Hochachka, 1985; St-Pierre et al., 2000; Mathieu-Costello, 2001). Additionally, high altitude adaptation could increase the activity of enzymes involved in aerobic metabolism (Hochachka, 1985; Sheafor, 2003). Bird flight muscle already has a very high aerobic capacity (Turner and Butler, 1988; Suarez et al., 1991; Suarez, 1993; Mathieu-Costello et al., 1998), yet it is unclear if this or other biochemical aspects of O2 demand are altered in high-flying birds.  2.1.4. How do birds fly high? From the above discussion it is obvious that the mechanistic bases for the ability of some bird species to fly at extreme altitude is poorly understood. The remainder of this study will thus use theoretical sensitivity analysis to explore traits that are potentially adaptive for high altitude flight.  2.2. Methods 2.2.1. Theoretical model of O2 transport This model has a similar structure to previous theoretical analyses of O2 transport in mammals and reptiles (Wagner, 1996a; Wang and Hicks, 2002; Wang and Hicks, 2004), but is altered to accommodate the crosscurrent arrangement of the avian lung (Fig.  23 2.1). Oxygen transport at the lung is modelled using parabronchial ‘tubes’ containing perfectly mixed gas, which contact a finite number of blood capillaries along their length at right angles. When gas enters the parabronchi and encounters the first lung capillary, O2 diffuses from the parabronchi into the blood, as dictated by the following equation (analogous to Fick’s diffusion equation).  dCcapO 2 dt  where  dCcapO 2 dt  =  DL O 2 •  tcap Q cap  (2.1)  (PparaO 2 " PcapO 2 )  is the rate of change of the instantaneous O2 content in the capillary !  ( CcapO 2 ), DL O 2 is the diffusion conductance of the lung for O2, tcap is the total capillary !  !  •  transit time, Q cap is blood flow rate through the capillary, and PparaO 2 and PcapO 2 are the ! instantaneous PO 2 in the parabronchi and the capillary, respectively. The O2 tension of  ! ! ! the gas encountering the second capillary is reduced, and is calculated by mass ! conservation. •  V (PparaO 2 ,1 " PparaO 2 ,2 ) R Tb PB  •  = Q cap (CacapO 2 " CvO 2 )  (2.2)  •  where ventilation ( V ) is assumed constant along the parabronchiole (i.e., a respiratory ! quotient of 1), PparaO 2 ,1 and PparaO 2 ,2 are the PparaO 2 at the 1st and 2nd capillary, R is the gas ! constant, Tb is avian body temperature in degrees Kelvin (314K), PB is barometric  ! and Cacap ! and Cv (mixed ! pressure, venous CO 2 ) are the O2 contents leaving and O2 O2 entering the first capillary, respectively. Equations 2.1 and 2.2 are then repeated for the  ! capillary,!and all subsequent capillaries, ! second and the resulting arterial PO 2 ( PaO 2 ) is calculated from arterial O2 content ( CaO 2 , the average value from all the CacapO 2 leaving  ! !  !  !  24 the capillaries) using equation 2.4, below. The arterial PO 2 is independent of the arbitrary number of capillaries used in the model (above approximately 20 capillaries), so the • ! number of capillaries along the length of the parabronchi was set at 50. Q cap was •  therefore set as the total cardiac output ( Q ) divided by 50.  ! Oxygen transport at the tissues is modelled using capillaries containing perfectly ! capillary network has the Pa determined from the mixed blood. Blood entering the O2 lung calculations above, and loses O2 to the surrounding cells as determined by an  !  equation similar to equation 2.1 above.  dCcapO 2 dt  =  "DTO 2 •  tcap Q  (PcapO 2 " PmitO 2 )  (2.3)  where DTO 2 is diffusion conductance of the tissues for O2, and PmitO 2 is the instantaneous  ! PO 2 at the mitochondria (set at zero, see below). The mixed venous PO 2 ( PvO 2 ) is simply  ! !  ! the PO 2 of blood leaving the tissue capillaries. When integrated over the length of the ! ! capillaries, tcap cancels out of equations 2.1 and 2.3, so need not be known.  !  Three additional equations, in addition to the above 3 primary equations describing O2 transport, are important for the model. The first is the Hill equation, which describes the relationship between O2 content and O2 tension in the blood.  CO 2 = 4 CHb  PO 2 n PO 2 n + P50 n  (2.4)  where CHb is the concentration of haemoglobin (Hb) in the blood, n is the Hill coefficient  ! (describes the cooperativity of Hb-O2 binding), and P50 is the O2 tension at which Hb is 50% saturated with oxygen.  25 The second additional equation describes the Bohr effect on P50.  P50 = P50i "10#"$pH(a%v)  (2.5)  where P50i is the P50 before the Bohr effect, φ is the Bohr coefficient, and ∆pH(a-v) is the  ! arterio-venous pH difference. Using this equation, P50 increases at the tissues so that the effects of CO2/pH could be accounted for in the model. The third additional equation is an empirical relationship that accounts for the effect of ventilation on the PO 2 in the caudal thoracic air sac ( PCTO 2 ; approximates the •  ! !  PO 2 entering the parabronchi): as V increases, dead space gas contributes less to the ! ! PCTO 2 , and so the difference between PIO 2 and PCTO 2 decreases. ! •  PCTO 2 = PIO 2 " 33.2 e"0.43 V (2.6) ! ! This relationship was derived using data from the available studies on pekin ducks that ! various degrees of hypoxia (Colacino et al., 1977; Shams and included normoxia and  Scheid, 1989; Shams and Scheid, 1993), and described most of the variation in their data (R2=0.95). PCTO 2 is used in a somewhat similar way to alveolar PO 2 in previous •  theoretical models (Wagner, 1996a; Wang and Hicks, 2004), and allows the effect of V ! ! on the difference between PIO 2 and PCTO 2 to be accounted for in the sensitivity analysis !  (described below).  !  !  2.2.2. Solution of the model The model used data from the literature on pekin ducks when available to •  reproduce in vivo conditions near maximal O2 consumption ( VO 2MAX ) during normoxia  !  26 ( PIO 2 of 150 Torr), moderate hypoxia (84 Torr), and severe hypoxia (30 Torr) (Table •  •  2.2). Several measured parameters, namely PIO 2 , V , Q , CHb, P50, φ, ∆pH(a-v), and n,  ! were put directly into the model, after which DL O 2 and DTO 2 were calculated to ! ! ! •  reproduce the measured in vivo PaO 2 , PvO 2 , and oxygen consumption rate ( VO 2 ). Given ! ! the assumptions below, the model solves equations 2.1-2.3 for the 3 unknown outcome  ! ! ! • variables PaO 2 , PvO 2 , and PE O 2 ( PO 2 of expired gas), and then calculates VO 2 using 2 separate equations.  !  !  !  !  !  •  •  VO 2 = •  V (PIO 2 " PE O 2 ) R Tb PB •  VO 2 = Q (CaO 2 " CvO 2 )  (2.7)  (2.8)  ! The model was solved iteratively using a program written in Matlab (version 7) as ! estimate of Pv , values for Pa and PE were calculated by follows. From a starting O2 O2 O2  integrating equation 2.1 then solving equation 2.2 (‘lung equations’) for each capillary in  ! From the determined ! ! of Pa , equation 2.3 (‘tissue the lungs, as described above. value O2 equation’) was integrated to calculate a new value for PvO 2 . The lung and tissue  ! equations were then repeated iteratively, using the outcome from each calculation as ! inputs in the next calculation, until a stable solution was reached (defined as the point when 2 successive calculation of PaO 2 and PvO 2 were both within 0.1 Torr). Within the •  range of data reported in this study, VO 2 calculated by equations 2.7 and 2.8 always ! ! agreed to within 0.1 mmol/min. Stable solution of the model generally took less than 10  !  27 iterations, and the final outcome was independent of the starting estimate of PvO 2 (it did, however, influence the number of required iterations).  ! 2.2.3. Sensitivity analysis •  From the starting point at each PIO 2 , reproducing in vivo conditions near VO 2MAX in the pekin duck, physiological parameters were then treated as independent variables in  ! ! the model. Changing these independent variables over a wide range altered the values of •  the dependent outcome variables, PaO 2 , PvO 2 , and PE O 2 , and therefore changed VO 2 . As •  such, the effect on VO 2 of changing each physiological parameter individually was ! ! ! ! assessed. When some physiological parameters were changed drastically, stable solutions could not!always be reached, so for these cases data are not reported. Because high altitude is characterized by extremely low temperatures, the effect of temperature on P50 was also assessed in severe hypoxia. For this part of our analysis, cold inspired air was assumed to cool blood in the lungs by 10oC, so haemoglobin in the blood was at 41oC in the tissues and 31oC in the lungs. Temperature was modelled to have a direct effect on P50 (in Torr/oC temperature change), such that the P50 decreased in the lungs and returned to normal at the tissues. The temperature effect was assessed over a range of temperature sensitivities from 0 to 2 Torr/oC, at starting P50 values of both 40 and 25 Torr.  28 2.2.3. Assumptions The assumptions in our model are similar to those in other theoretical studies of O2 transport (Wagner, 1996a; Wang and Hicks, 2002; Wang and Hicks, 2004). Limitations on O2 transport due to ventilation/perfusion heterogeneity in the lungs, metabolism/perfusion heterogeneity in the tissues, shunting of blood flow, and imperfect mixing of gas or blood are all treated as diffusion limitations to O2 flux, and therefore contribute to the diffusion conductances in the lungs and tissues ( DL O 2 and DTO 2 ). The ‘tissues’ are treated as one compartment, and it is assumed that total blood volume is  ! flight!at altitude no doubt constant in both the lung and tissue compartments. Whereas involves some non-steady state changes in physiological variables, solution of our model only accounts for steady state conditions. Finally, mitochondrial PO 2 was set to be zero, rather than at a slightly higher and variable value as it exists in vivo (Gayeski and Honig,  ! for solving the model. The data 1988; Wagner, 1996a); this was an unfortunate necessity •  used in this study are from pekin ducks performing treadmill exercise near VO 2 MAX (Kiley et al., 1985; Butler and Turner, 1988) or for pekin ducks at their limit of hypoxia  ! • tolerance (Black and Tenney, 1980). Although VO 2 MAX was not rigorously determined, these studies include all the required measurements and provided the best available data sets we could find.  !  2.3. Results 2.3.1. Oxygen transport in the lungs and tissues In the crosscurrent arrangement of the avian lung, blood PO 2 varies in both the direction of blood flow and the direction of gas flow (Fig. 2.2A). In our theoretical  !  29 model, venous blood enters the lung capillaries and its PO 2 rises as it travels perpendicular to the parabronchi, until it leaves the capillaries and becomes part of the  ! into the blood and parabronchial O2 arterial blood supply. Oxygen therefore diffuses levels fall as gas travels along the length of the parabronchi. As a result, blood PO 2 rises more if it travels through capillaries at the start of the parabronchi than at the end. PaO 2 results from the mixing of blood leaving all the capillaries.  !  Oxygen transport in avian tissues is modelled in the same way as!in other vertebrates (Fig. 2.2B). Blood enters the tissue capillaries with the same PaO 2 that left the lungs. Oxygen then diffuses out of the blood continuously along the length of the capillaries. After leaving the tissues, blood has an O2 tension of!PvO 2 and is then circulated to the lungs. As described in the methods, our model reaches a stable solution  ! (Fig. 2.2A) and tissues (Fig. by iterating between gas transport calculations in the lungs 2.2B).  2.3.2. Effects of P50 on oxygen consumption Altering the affinity of haemoglobin for oxygen (P50) had different effects on O2 consumption in normoxia, moderate hypoxia, and severe hypoxia (Fig. 2.3A). In normoxia, increasing P50 from the model’s starting value (from literature values on pekin •  •  ducks near VO 2MAX ) of 40 Torr, and thus decreasing Hb-O2 affinity, increased VO 2 by up to 2% until approximately 60 Torr. Above 60 Torr, further increases in P50 decreased  ! •! VO 2 slightly. Decreasing P50 from the model’s starting value, however, decreased O2 •  consumption sharply, such that at a P50 of 25 Torr VO 2 was reduced by 20%. A similar  ! !  30 •  trend was observed in moderate hypoxia, except the highest VO 2 occurred at a P50 of 40 •  Torr, the starting point in the model. In contrast, VO 2 decreased sharply when P50 was ! •  increased in severe hypoxia, and VO 2 increased when P50 was decreased. The highest ! •  VO 2 in severe hypoxia occurred at a P50 of 10 Torr, at which point O2 consumption was ! •  nearly 20% above the starting value of the model, and beyond this value VO 2 decreased  !  sharply as P50 decreased further.  ! As discussed in the introduction, Hb-O2 affinity is frequently higher in birds at high altitude. Bar-headed geese have a P50 of approximately 25 Torr, whereas many closely related waterfowl have a P50 of approximately 40 Torr (including pekin ducks, the species from which starting data for this study were derived). Many high altitude birds are already known to have a low P50, so we performed the remaining sensitivity analyses at both 40 Torr and 25 Torr. In doing so, we tested whether there is an interaction between P50 and other physiological variables, and consequently, whether the low P50 in high altitude species changes the potential benefit of different physiological traits.  2.3.3. Sensitivity analysis in normoxia Altering biochemical properties of Hb other than P50 influenced O2 consumption in normoxia. Altering the Bohr coefficient (φ) had a nearly linear effect for a P50 of both •  40 and 25 Torr, such that increasing φ had a positive effect on VO 2 during exercise (Fig. 2.3B,C). The lower P50 enhanced the influence of φ, but in each case large alterations in φ  ! • resulted in no more than a 10% change in VO 2 . Changing the Hill coefficient (n) had  !  31 •  more variable effects on VO 2 (Fig. 2.3D,E). At a P50 of 40 Torr, increasing n from the •  •  starting value of 2.8 to about 3.7 enhanced VO 2 slightly (<1%), after which VO 2 declined. ! •  Below 2.8, lowering n decreased VO 2 more sharply. The influence of the Hill coefficient ! ! •  was very different at a P50 of 25 Torr: the peak VO 2 shifted from an n=3.3 at a P50 of 40 ! Torr, to n=1.7 at a P50 of 25 Torr. The lower P50 therefore favoured less Hb-O2  ! cooperativity in normoxia (i.e., a straighter Hb-O2 saturation curve). On either side of 1.7, •  increasing or decreasing n reduced VO 2 sharply. Data for other physiological variables are summarized for normoxia in Fig. 2.4A,  ! • which shows the effect of a 2-fold increase or decrease of each variable on VO 2 during exercise. At a P50 of 40 Torr, increasing both cardiovascular variables (Hb concentration,  ! • CHb, and cardiac output, Q ) and respiratory variables (lung O2 diffusing capacity, DL O 2 , •  •  and ventilation rate, V ) had a small influence on VO 2 (<5%). Only increasing tissue O2 ! ! •  diffusing capacity ( DTO 2 ) had a substantial positive effect on VO 2 (30%). At a P50 of 25 ! ! Torr, the effect of increasing cardiovascular variables was enhanced (to 20%) and the  ! influence ! of respiratory variables was reduced (<1%). Furthermore, the benefit of DTO 2 almost doubled (to 57%) at the lower P50. In general, for normoxia as well as moderate  ! hypoxia and severe hypoxia, decreasing physiological variables had a greater effect than •  increasing them, with the exception of CHb, Q , and DTO 2 at the lower P50. Furthermore, •  the effect of altering multiple physiological variables on VO 2 was additive rather than ! ! interactive (data not shown).  !  32 2.3.4. Sensitivity analysis in moderate hypoxia •  The effects of the biochemical properties of Hb on VO 2 during exercise in moderate hypoxia were very similar to their effects in normoxia. The Bohr coefficient  ! had a modest positive linear effect for both P50 values, and the lower P50 enhanced the influence of φ (Fig. 2.3B,C). Similar to normoxia, decreasing the P50 from 40 Torr to 25 Torr in moderate hypoxia reduced the most beneficial Hill coefficient, from a value greater than 5 to 2.6 (Fig. 2.3D,E). Compared to normoxia, moderate hypoxia favoured more Hb-O2 cooperativity (higher Hill coefficient) at both P50 values. Changing other physiological variables had similar effects in moderate hypoxia as in normoxia (Fig. 2.4B). The only substantial exception to this was the interaction •  •  between P50 on CHb, Q , and DTO 2 . The benefit of increasing CHb and Q was similar •  between normoxia and moderate hypoxia at a P50 of 40 Torr, but CHb and Q increased ! ! ! •  VO 2 by only 13% at a P50 of 25 Torr (compared to 20% in normoxia). The benefit of ! increasing DTO 2 at a P50 of 40 Torr was higher in moderate hypoxia (38%) than in !  normoxia (30%), but at a P50 of 25 Torr DTO 2 had nearly equivalent effects (56% and  ! 57%). ! 2.3.5. Sensitivity analysis in severe hypoxia The effects of Hb properties on O2 consumption during exercise were very different in severe hypoxia than in normoxia and moderate hypoxia. The lower P50 still enhanced the influence of the Bohr effect, but the difference between the two P50 values •  was much greater (Fig. 2.3B,C). Altering the Bohr coefficient had less influence on VO 2  !  33 at a P50 of 40 Torr (much shallower slope), and increasing φ beyond about 0.6 did not •  increase VO 2 . The interaction between P50 and the Hill coefficient was also different in severe hypoxia; lowering the P50 increased the most beneficial Hill coefficient from 1.5 to  !2.4 (Fig. 2.3D,E). In normoxia and moderate hypoxia, lowering P50 reduced the most beneficial value for the Hill coefficient. Furthermore, whereas moderate hypoxia favoured more Hb-O2 cooperativity than normoxia (higher Hill coefficient) at both P50 values, severe hypoxia favoured less cooperativity than normoxia. The effects of changing other physiological variables were also very different in severe hypoxia than in normoxia and moderate hypoxia (Fig. 2.4C). At a P50 of 40 Torr, •  •  the benefit of increasing CHb, Q , and V increased (to 10%, 10%, and 41%, respectively), and the benefit of increasing DTO 2 decreased (to only 2%). The effects of lowering P50 on  ! • ! the benefit of CHb and Q were reversed in severe hypoxia compared to normoxia and ! moderate hypoxia, because the benefit of increasing them was much lower (only 2%) at a ! • P50 of 25 Torr. Furthermore, the negative effect of the lower P50 on the influence of V •  •  was eliminated in severe hypoxia (increasing V enhanced VO 2 by 33%). The strong ! interaction between P50 and DTO 2 was still apparent in severe hypoxia, however: increasing DTO 2  ! ! only enhanced VO 2 at a P50 of 25 Torr (by 34%). Increasing DL O 2 still ! •  •  had a minor influence on VO 2 in severe hypoxia. ! ! ! Changing the effect of temperature on Hb P50 could have a large influence on  ! • VO 2 . This was true for starting P50 values of both 40 and 25 Torr, but its influence was greater at the lower P50 (Fig. 2.5). The significance of the temperature effect must be  !  34 taken with some caution, however, as the degree of cooling of pulmonary blood during flight in low ambient temperatures is unknown. This part of the model assumes that pulmonary blood is cooled 10oC. If this does occur, then at a physiologically realistic •  value for the temperature effect (~1.5 Torr/oC; Maginniss et al., 1997) VO 2 could increase by 40-60%.  ! •  2.3.6. V and DTO 2 during flight at extreme altitude The results of the sensitivity analysis suggest that ventilation rate and tissue O2 !  ! • diffusing capacity are likely to have the greatest influence on VO 2 in severe hypoxia. Using a simple set of equations, we sought to assess the possible combinations of  ! • physiological variables that could sustain the VO 2 required for flight at high altitude. Barheaded geese have been known to fly at altitudes of 9000m ( PIO 2 ≈ 38 Torr), and during  ! flight in a wind tunnel they consume oxygen at rates of 15 to 20 mmol/min (Ward et al.,  ! • • 2002). The possible combinations of V and PE O 2 that could sustain this VO 2 were •  calculated using equation 2.7 (Fig. 2.6A). The minimum ventilation rate for this VO 2 at a ! ! ! PIO 2 of 38 Torr is approximately 7 L/min, but this assumes complete removal of O2 from  ! • the inspired gas. Over a physiologically realistic range of PE O 2 , V would need to be  !  between 10 and 20 L/min (depending on the amount of O2 extracted from the inspired ! ! gas; see arrows in Fig 2.6A), which is approximately 2- to 4-fold higher than the starting •  VO 2MAX values from the in vivo pekin duck data used in the sensitivity analysis.  !  35 •  •  The possible combinations of DTO 2 and Q that could sustain VO 2 during flight were determined primarily using equations 2.3 and 2.8 (Fig. 2.6B). The calculations were  ! for Pa! (38 and 30 Torr), ! made with two different values so the effect of a low versus O2 high difference between inspired and arterial PO 2 could be determined. By doing so, it  ! was determined that when the inspired-arterial PO 2 difference was low ( PaO 2 ≈ 38 Torr), ! • concurrent 2- to 3-fold increases in DTO 2 (0.135 to ~0.4 mmol/Torr/min) and Q (1.8 to ! ! •  ~6.0 L/min) above the starting values from pekin ducks near VO 2MAX (middle arrow, Fig. ! ! •  •  2.6B) could achieve the required VO 2 for flight. Larger increases in either DTO 2 or Q ! would reduce the amount of change required in the other variable (see arrows, Fig. 2.6B).  ! ! ! greater Furthermore, if the inspired-arterial PO 2 difference was higher, much increases in •  DTO 2 and Q would be required (grey curve, Fig. 2.6B). Overall, the assessment in Fig. ! •  !  •  2.6 suggests that physiologically reasonable increases in V , DTO 2 , and Q (2- to 3-fold) ! •  from the starting values obtained from pekin ducks near VO 2MAX (Table 2.2) would allow ! ! ! species like the bar-headed goose to fly at extreme altitude.  ! 2.4. Discussion Unlike in vivo studies, theoretical sensitivity analyses allow individual physiological variables to be altered independently so their isolated effects on O2 consumption can be assessed. By applying this analysis to hypoxia in birds, we feel we can predict which factors most likely limit O2 consumption and exercise performance (Table 2.1). As a consequence, our analysis identifies the steps in the O2 cascade for  36 which evolutionary changes have the greatest potential for improving O2 supply and supporting flight at high altitude, namely ventilation and O2 diffusing capacity of the flight muscle.  2.4.1. Assumptions and starting data of the model As discussed in section 2.3, the assumptions in our model (as outlined in the methods) are similar to those in previous theoretical studies of oxygen transport and a thorough discussion of many of these assumptions are included in these earlier works (Wagner, 1996a; Wang and Hicks, 2002; Wang and Hicks, 2004). While the assumptions of the model may in some cases limit its predictive power, they eliminate a great deal of complexity, much of which cannot be modelled due to a lack of mechanistic knowledge. The model assumes that ventilation/perfusion heterogeneity in the lungs and metabolism/perfusion heterogeneity in the tissues can be treated as part of DL O 2 and  DTO 2 . The effects of changing these ‘apparent’ lung and tissue diffusion capacities should ! therefore be considered to represent the role of lung and tissue gas exchange in general. !  Otherwise, an exceedingly complex multi-compartment heterogeneous model would be necessary, and in vivo data addressing these potential heterogeneities are lacking, particularly at the tissues (but see Powell and Wagner, 1982; Schmitt et al., 2002). With regard to DTO 2 the model also treats the ‘tissues’ as a single compartment. Whereas O2 •  diffusing capacity of any one tissue is assumed to be constant at VO 2 MAX , regardless of ! the level of hypoxaemia, different tissues may have different tissue diffusion capacities  ! (Hogan et al., 1988). Indeed, the observation that the calculated starting values for DTO 2 in the sensitivity analysis increase progressively from normoxia to severe hypoxia  !  37 suggests that blood flow is being redistributed as O2 levels fall. Interestingly, calculated  DTO 2 declines with altitude in humans (Wagner, 1996a), suggesting that there are differences in muscle perfusion with altitude between birds and mammals, or differential  !  responses of the structural determinants of tissue O2 diffusing capacity. Unfortunately, as mentioned above, the assumptions in the model do not allow us to predict the potential benefits of these heterogeneities for high altitude performance. Since our interest was in the factors limiting exercise performance at altitude, the starting data for our model were obtained from previous studies on pekin ducks near maximal O2 consumption. These ducks were exercising on a treadmill, however, and were not flying. Unfortunately, to the best of our knowledge only one previous study has made all the required measurements for this analysis during flight, and this was only done in normoxia (in pigeons, Butler et al., 1977). Pekin ducks are the only species for which we could find all the required measurements for our analysis during exercise in both normoxia and hypoxia (Black and Tenney, 1980; Kiley et al., 1985). Only the lung and tissue diffusion capacities remained to be calculated in our analysis, but previous experimental determinations of DL O 2 in pekin ducks were similar to the values calculated in this study (Scheid et al., 1977). Similar values for DTO 2 are not available.  ! 2.4.2. What limits exercise performance in ! birds? Our analysis suggests that O2 diffusing capacity in the tissues poses the greatest limitation to exercise performance in birds during normoxia, similar to what may also be the case in mammals (Wagner, 1996a,b). Qualitatively, this appears to be true regardless of haemoglobin O2 affinity, although the benefit of increasing DTO 2 was greater at a low  !  38 P50. In normoxia, increasing DTO 2 may be more beneficial to birds with a lower P50 because their Hb does not desaturate until a lower PO 2 . Mean capillary PO 2 , the driving  ! force for diffusion into exercising muscle, would therefore be lower, so a higher DTO 2 ! ! • • should be required to maintain VO 2 during exercise. At this lower P50, Q and CHb also ! •  appear to limit exercise performance in birds, but changes in V have less influence, as in ! ! mammals and some reptiles (Wagner, 1996a; Frappell et al., 2002). This was not the case at the higher P50, where DTO 2  ! • was the only substantial limitation to VO 2 . Because this  model ignores the contribution of changes in cardiac output to changes in perfusion of  ! specific tissues!and tissue blood volume, however, the influence of cardiac output may be greater than determined by our analysis. DL O 2 does not appear to pose much of a •  limitation to VO 2 in normoxia, similar to the situation in humans and some but not all ! lizards (Wagner, 1996a; Frappell et al., 2002; Wang and Hicks, 2004).  !  The physiological variables limiting exercise performance in birds during  moderate hypoxia are similar to those limiting performance in normoxia. DTO 2 continues •  to pose the greatest limitation, and limitations imposed by the circulation ( Q and CHb) are ! •  still greater at a lower P50. Unlike normoxia, however, VO 2 in moderate hypoxia appears ! to be limited less by the circulation and more by respiratory variables, as is also the case in humans (Wagner, 1996a).  !  The most substantial difference between severe hypoxia and normoxia/moderate hypoxia is in the effects of altering ventilation. Ventilation appears to become a major •  limitation to exercise performance at extreme altitude. DTO 2 also appears to limit VO 2 in  !  !  39 severe hypoxia, but only at lower P50 values. This is not entirely unsurprising: in severe hypoxia the venous blood of pekin ducks (a species which has a higher P50) is almost completely deoxygenated in vivo (Black and Tenney, 1980), so there are no possible benefits of increasing DTO 2 . At the lower P50, there is a substantially higher arterial O2 content, so more O2 can be removed, and increasing DTO 2 can have a greater influence. In  ! • modelling of humans during severe hypoxia, DTO 2 , DL O 2 , and V have the greatest ! influence on exercise performance (Wagner, 1996a). That DL O 2 appears to pose less of a ! ! ! limitation to exercise performance at extreme altitude in birds may reflect the extremely small diffusion distances across the lungs of most!birds (Maina and King, 1982; Powell and Mazzone, 1983; Shams and Scheid, 1989).  2.4.3. Potential adaptations for high altitude flight in birds Hb-O2 affinity is known to be higher in many species adapted to high altitude, so it is perhaps not surprising that our modelling suggests that this trait is beneficial at high altitude. Historically, a high Hb-O2 affinity was believed to be important because of the effect of a low P50 on oxygen loading at the lungs (Samaja et al., 2003). The benefit of a low P50 alone on total O2 consumption is less certain, however, because Hb with a high affinity inhibits O2 unloading at the tissues. This is perhaps why decreasing P50 alone •  could only increase VO 2 during exercise by 10-20% in this study. It has been suggested that other factors influencing Hb-O2 affinity, such as the Bohr effect, could concurrently  ! facilitate oxygen unloading; however, our results suggest that a larger Bohr effect has  40 •  surprisingly little influence on VO 2MAX . Consistent with this, the Bohr effect is actually lower in the high altitude bar-headed goose than in other waterfowl (Liang et al., 2001).  ! capacity in the tissues should also be beneficial in high altitude The O2 diffusing birds with a high haemoglobin O2 affinity. In the present study, a simultaneous decrease •  in P50 (from 40 to 25 Torr) and increase in DTO 2 (2-fold) increased VO 2 by 51%. Thus, in high flying birds that are known to have a low P50, such as the bar-headed goose and  ! ! increases in the O2 diffusing Rüppell’s griffon (Gyps rueppellii), capacity of the flight muscle should be very beneficial. However, increased DTO 2 may not be a specific adaptation to high altitude, but could also be a consequence of other factors (e.g., lower mean capillary PO 2 in normoxia) as discussed!in Section 2.4.2. Regardless of the evolutionary forces producing the change, increased DTO 2 should be tremendously  ! beneficial to high altitude fliers with a low P50. Furthermore, our results suggest that there may be interesting interactions between P50!and DTO 2 in general, which should be considered when assessing the adaptive benefit of a high Hb-O2 affinity in high altitude animals.  !  Our analysis suggests that an enhanced capacity to increase ventilation should also benefit birds significantly in severe hypoxia, and could therefore be important for sustaining high altitude flight. This is likely true regardless of P50; although there is a •  small amount of interaction between P50 and ventilation, increasing V always had a substantial effect on O2 consumption rate. However, as discussed in section 2.1.1, it ! remains to be determined whether a greater capacity to increase ventilation is a  characterisitic of high altitude flyers.  41 Differences in the Bohr effect or the Hill coefficient did not appear to be beneficial for flight at high altitude. Neither variable had a major influence on O2 consumption in severe hypoxia, as the benefits of changing these variables did not generally exceed 10%. Previous studies in many bird species have shown that the Hill coefficient increases with PO 2 , possibly enhancing O2 transport (Maginniss et al., 1997), but this possibility was not addressed in the present analysis.  ! to the Bohr effect and Hill coefficient, the temperature effect on Hb-O2 In contrast binding affinity may have a substantial effect on O2 consumption, and may therefore be beneficial for high altitude flight (Maginniss et al., 1997). This will only be possible if the ventilation of cold air cools pulmonary blood. This would reduce the P50 of Hb in the lungs, and thus facilitate O2 uptake. When this blood enters the exercising muscles it would then be re-warmed to body temperature or higher, and O2 would be released from Hb. Our modelling suggests that a temperature effect on Hb could significantly enhance •  VO 2 . The greater the temperature difference between blood in the lungs and in the muscles, and the greater the temperature effect on Hb-O2 binding, the greater the increase  !  •  •  in VO 2 . At normal levels of temperature sensitivity, the increase in VO 2 was approximately 5% for every 1oC difference. Altering the magnitude of the temperature  !  effect on Hb while allowing lung temperature to fall could!therefore be beneficial at high altitude. At present, however, it is unknown whether the Hb of high altitude birds has a heightened sensitivity to temperature, or whether pulmonary blood is actually cooled during high altitude flight. Increasing other physiological variables that we studied, namely maximum cardiac output, Hb concentration, and lung O2 diffusing capacity, are likely of little  42 advantage at high altitude. As discussed above, these traits do not seem to limit exercise performance at extreme altitude, and theoretically increasing them had only small benefits for O2 transport. The effect of some other variables (e.g., blood flow distribution, pH regulation, tissue oxidative capacity, etc.; Table 2.1) could not be examined using our model, so we cannot exclude a potentially important role for these physiological traits.  2.4.4. Conclusions Using a theoretical sensitivity analysis that allows individual physiological variables to be independently altered, we have identified the factors most likely to limit O2 consumption and exercise performance in birds, and by extension, the physiological changes that are likely beneficial for high altitude flight. Some of these changes, in particular haemoglobin O2 affinity, are already thought to be adaptive for flying at high altitude. For other traits, such as an enhanced hypoxic ventilatory response or an enhanced O2 diffusing capacity of the flight muscle, evolutionary changes have not yet been conclusively demonstrated in vivo. The remaining chapters of this thesis will therefore determine if these traits are enhanced in birds that are adapted to high altitude.  2.5. Summary of Chapter A sensitivity analysis was performed using a theoretical model of O2 transport to help  •  determine the factors limiting exercise performance in birds. Haemoglobin (Hb) O2 affinity, total ventilation, and O2 diffusing capacity in the  •  •  tissues ( DTO 2 ) had the greatest influences on VO 2 in severe hypoxia.  !  !  43 •  There was a beneficial interaction between DTO 2 and the P50 of Hb, such that  •  increasing DTO 2 had a greater influence on VO 2 when P50 was low. ! Changes in the O2 diffusing capacity of the lung, cardiac output, blood Hb  •  ! ! • concentration, the Bohr coefficient, or the Hill coefficient had little influence on VO 2 in severe hypoxia.  !  44 Table 2.1. Potential physiological adaptations for flying at high altitude in birds Physiological Trait Respiratory System High maximum ventilation rates Reduced sensitivity of ventilation to hypocapnia/alkalosis Small diffusion barrier Reduced pulmonary vasoconstriction Circulatory System High cardiac output High Hb-O2 affinity Enhanced blood flow to brain and heart Effective extracellular pH regulation Tissues Greater capillary density Greater abundance of mitochondria Effective intracellular pH regulation  Reference Tucker, 1968 Scheid, 1990 Maina and King, 1982 Faraci et al., 1984a Black and Tenney, 1980 Petschow et al., 1977 Faraci et al., 1984b Dodd and Milsom, 1987 Fedde et al., 1985 Fedde et al., 1985 Weinstein et al., 1985  The above list represents previous suggestions of what traits may be uniquely beneficial in high altitude birds, but many have yet to be thoroughly explored in vivo. See text for details.  45 Table 2.2. Literature values used for the calculations in the model Variable  PIO 2 (Torr) •  V (BTPS, L/min) •  ! !  ! ! !  Q (L/min) PaO 2 (Torr) PvO 2 (Torr) CHb (mM) P50 (Torr) φ ∆pH(a-v) n TB (oC) •  VO 2 (mmol/min) DL O 2 (mmol/Torr/min)* DTO 2 (mmol/Torr/min) * ! ! !  ! ! !  Normoxia (sea level)  Moderate Hypoxia (3500m)  Severe Hypoxia (10000m)  150  84  30  3.0  5.0  4.5  1.2 98 43 2.3 40 0.4 0.1 2.8 41  1.4 60 33 2.3 40 0.4 0.1 2.8 41  1.8 26 5 2.3 40 0.4 0.1 2.8 41  4.9 0.075 0.080  5.5 0.250 0.120  2.0 0.500 0.135  •  •  PIO 2 , inspired O2 tension; V , total ventilation; Q , cardiac output; PaO 2 , arterial O2 tension; PvO 2 , mixed venous O2 tension; CHb, haemoglobin (Hb) concentration; P50, PO 2 at half-saturation of Hb with O2; φ, Bohr coefficient; ∆pH(a-v), arterio-venous pH • ! ! difference; n, Hill coefficient; TB, body temperature; V! O 2 , oxygen consumption rate; !DL O , O2 diffusing capacity in the lung; DTO , O2 diffusing capacity in the tissues. 2 2 Literature sources for most starting data: normoxia and moderate hypoxia, Kiley et al. (1985); severe hypoxia, Black and Tenney (1980). Other starting data came from Black ! and Tenney (1980) (P50 and φ) and Weber and Fago (2004) (n), and TB was set at the * ! DL O 2 and DTO 2 were calculated using all other commonly accepted value for birds. literature values. !  !  46  Fig. 2.1. Schematic of the oxygen transport pathway in birds. The crosscurrent parabronchial lung is unidirectionally ventilated by air sacs, and oxygen diffuses into blood capillaries from air capillaries (not shown) all along the length of the parabronchi. Oxygen is then circulated in the blood, and diffuses to mitochondria in the tissues. The rate of oxygen transport at both the lungs and tissues can be calculated using the Fick equation, and the amount of O2 transferred from the lungs into the blood can be calculated using an oxygen conservation equation. See the Methods for definitions of all variables, as well as other equations used in the model and additional details.  47  Fig. 2.2. Oxygen tensions in the lung (A) and tissue (B) capillaries during normoxia. In the crosscurrent avian lung, PO 2 increases along the path of blood flow through the lungs, but does not increase by as much at the end of the parabronchi as at the start (gas PO 2 decreases along the length of the parabronchi). In the tissues, blood PO 2 decreases continuously along!the capillary length as O2 diffuses to tissue mitochondria. The direction of blood and gas flows are depicted by arrows. Data were collected using the ! theoretical model of oxygen transport that is described in the methods. To reach a ! in the lungs (A) and solution, our model iterates between gas transport calculations tissues (B) until a stable result is reached.  48  Fig. 2.3. The effects of varying different biochemical features of haemoglobin (Hb) on oxygen consumption during exercise in normoxia ( PIO 2 of 150 Torr; black), moderate hypoxia (84 Torr; dark grey dashed), and severe hypoxia (30 Torr; light grey). P50 is the PO 2 at 50% Hb saturation (see methods for a mathematical description of variables). In (B) through (E), each variable was assessed ! at the P50 of pekin ducks (40 Torr; B,D) and of bar-headed geese (25 Torr; C,E).  !  49  Fig. 2.4. The effects of a 2-fold increase (dark bars) or decrease (light bars) in different physiological variables on oxygen consumption during exercise in (A) normoxia ( PIO 2 of 150 Torr), (B) moderate hypoxia (84 Torr), and (C) severe hypoxia (30 Torr). The effects were assessed at the P50 of pekin ducks (40 Torr, solid bars) as well as the P50 of bar•  headed geese (25 Torr, hatched bars). CHb, blood haemoglobin concentration; Q , cardiac ! •  output; DL O 2 , diffusion conductance of the lungs for O2; V , ventilation rate; DTO 2 , diffusion conductance of the tissues for O2. See Table 2.2 for starting values obtained • ! from pekin ducks near VO 2 MAX . ! ! !  !  50  Fig. 2.5. The effect of varying the magnitude of the effect of temperature on the P50 of haemoglobin (Hb) on oxygen consumption during exercise in severe hypoxia (30 Torr). Arterial blood was assumed to be cooled by 10oC in the lungs (see methods). The effects of each variable were assessed at the P50 of pekin ducks (40 Torr; solid) as well as the P50 of bar-headed geese (25 Torr; dashed).  51  Fig. 2.6. A theoretical assessment of the ventilation rates (A) and tissue diffusion capacities (B) that could achieve the rate of oxygen consumption typical of an adult bar•  headed goose during flight ( VO 2 =15 mmol/min). A PIO 2 of 38 Torr simulates an altitude •  of approximately 9000 m. In (A), the possible combinations of V and PE O 2 were •  calculated using equation 2.7. In (B), the possible combinations of DTO 2 and Q were ! ! determined with equations 2.3-2.5 and 2.8, for two possible values for PaO 2 . ♦ represents ! the condition for pekin ducks (see Table 2.2). As the arrows ! in (A) and (B) show, greater • ! ! increases in either V or DT reduce the required lung oxygen extraction or cardiac O2  • ! output, respectively, to sustain the VO 2 during flight. P50 was set at 25 Torr, and the values for all other parameters are shown in Table 2.2. ! !  !  52 2.6. References Black, C. P. and Tenney, S. M. (1980). Oxygen transport during progressive hypoxia in high altitude and sea level waterfowl. Respir. Physiol. 39, 217-239. Boutilier, R. G. (2001). Mechanisms of cell survival in hypoxia and hypothermia. J. Exp. Biol. 204, 3171-3181. Bouverot, P., Hildwein, G. and Oulhen, P. (1976). Ventilatory and circulatory O2 convection at 4000 m in pigeon at neutral or cold temperature. Respir. Physiol. 28, 371-385. Brackenbury, J. H., Gleeson, M. and Avery, P. (1982). Control of ventilation in running birds: effects of hypoxia, hyperoxia, and CO2. J. Appl. Physiol. 53, 13971404. Braunitzer, G. and Hiebl, I. (1988). Molekulare aspekte der höhenatmung von vögeln. Hämoglobine der streifengans (Anser indicus), der andengans (Chloephaga melanoptera) und des sperbergeiers (Gyps rueppellii). Naturwissenschaften 75, 280-287. Butler, P. J. and Turner, D. L. (1988). Effect of training on maximal oxygen uptake and aerobic capacity of locomotory muscles in tufted ducks, Aythya fuligula. J. Physiol. 401, 347-359. 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Hoppeler, H. and Weibel, E. R. (1998). Limits for oxygen and substrate transport in mammals. J. Exp. Biol. 201, 1051-1064.  55 Hussain, S. O., Barbato, J. C., Koch, L. G., Metting, P. J. and Britton, S. L. (2001). Cardiac function in rats selectively bred for low- and high-capacity running. Am. J. Physiol. Reg. Integr. Comp. Physiol. 281, R1787-R1791. Kiley, J. P., Faraci, F. M. and Fedde, M. R. (1985). Gas exchange during exercise in hypoxic ducks. Respir. Physiol. 59, 105-115. Liang, Y. H., Hua, Z. Q., Liang, X., Xu, Q. and Lu, G. Y. (2001). The crystal structure of bar-headed goose hemoglobin in deoxy form: The allosteric mechanism of a hemoglobin species with high oxygen affinity. J. Mol. Biol. 313, 123-137. Maginniss, L. A., Bernstein, M. H., Deitch, M. A. and Pinshow, B. (1997). Effects of chronic hypobaric hypoxia on blood oxygen binding in pigeons. J. Exp. Zool. 277, 293-300. Maina, J. N. and King, A. S. (1982). Morphometrics of the avian lung. 2. The wild mallard (Anas platyrhynchos) and greylag goose (Anser anser). Respir. Physiol. 50, 299-310. Mathieu-Costello, O. (2001). Muscle adaptation to altitude: tissue capillarity and capacity for aerobic metabolism. High Alt. Med. Biol. 2, 413-425. Mathieu-Costello, O., Agey, P. J., Wu, L., Szewczak, J. M. and MacMillen, R. E. (1998). Increased fiber capillarization in flight muscle of finch at altitude. Respir. Physiol. 111, 189-199. Peters, G. W., Steiner, D. A., Rigoni, J. A., Mascilli, A. D., Schnepp, R. W. and Thomas, S. P. (2005). Cardiorespiratory adjustments of homing pigeons to steady wind tunnel flight. J. Exp. Biol. 208, 3109-3120.  56 Petschow, D., Würdinger, I., Baumann, R., Duhm, J., Braunitzer, G. and Bauer, C. (1977). Causes of high blood O2 affinity of animals living at high altitude. J. Appl. Physiol. 42, 139-143. Phu, D. N., Yamaguchi, K., Scheid, P. and Piiper, J. (1986). Kinetics of oxygen uptake and release by red blood cells of chicken and duck. J. Exp. Biol. 125, 15-27. Piiper, J. and Scheid, P. (1972). Maximum gas transfer efficacy of models for fish gills, avian lungs and mammalian lungs. Respir. Physiol. 14, 115-124. Piiper, J. and Scheid, P. (1975). Gas transport efficacy of gills, lungs and skin: theory and experimental data. Respir. Physiol. 23, 209-221. Powell, F. L., Dwinell, M. R. and Aaron, E. A. (2000). Measuring ventilatory acclimatization to hypoxia: comparative aspects. Respir. Physiol. 122, 271-284. Powell, F. L. and Mazzone, R. W. (1983). Morphometrics of rapidly frozen goose lungs. Respir. Physiol. 51, 319-332. Powell, F. L. and Wagner, P. D. (1982). Ventilation-perfusion inequality in avian lungs. Respir. Physiol. 48, 233-241. Samaja, M., Crespi, T., Guazzi, M. and Vandegriff, K. D. (2003). Oxygen transport in blood at high altitude: role of the hemoglobin-oxygen affinity and impact of the phenomena related to hemoglobin allosterism and red cell function. Eur. J. Appl. Physiol. 90, 351-359. Saunders, D. K. and Fedde, M. R. (1991). Physical conditioning: effect on the myoglobin concentration in skeletal and cardiac muscle of bar-headed geese. Comp. Biochem. Physiol. A. Physiol. 100A, 349-352.  57 Scheid, P. (1990). Avian respiratory system and gas exchange. In Hypoxia: the Adaptations (eds. J. R. Sutton, G. Coates and J. E. Remmers), pp. 4-7. Toronto, Canada: B. C. Decker. Scheid, P., Worth, H., Holle, J. P. and Meyer, M. (1977). Effects of oscillating and intermittent ventilatory flow on efficacy of pulmonary O2 transfer in duck. Respir. Physiol. 31, 251-258. Schmitt, P. M., Powell, F. L. and Hopkins, S. R. (2002). Ventilation-perfusion inequality during normoxic and hypoxic exercise in the emu. J. Appl. Physiol. 93, 1980-1986. Shams, H. and Scheid, P. (1989). Efficiency of parabronchial gas exchange in deep hypoxia: measurements in the resting duck. Respir. Physiol. 77, 135-146. Shams, H. and Scheid, P. (1993). Effects of hypobaria on parabronchial gas exchange in normoxic and hypoxic ducks. Respir. Physiol. 91, 155-163. Sheafor, B. A. (2003). Metabolic enzyme activities across an altitudinal gradient: an examination of pikas (genus Ochotona). J. Exp. Biol. 206, 1241-1249. Snyder, G. K., Byers, R. L. and Kayar, S. R. (1984). Effects of hypoxia on tissue capillarity in geese. Respir. Physiol. 58, 151-160. Snyder, L. R. G. (1985). Low P50 in deer mice native to high altitude. J. Appl. Physiol. 58, 193-199. St-Pierre, J., Tattersall, G. J. and Boutilier, R. G. (2000). Metabolic depression and enhanced O2 affinity of mitochondria in hypoxic hypometabolism. Am. J. Physiol. Reg. Integr. Comp. Physiol. 279, R1205-R1214.  58 Suarez, R. K. (1993). Oxidative metabolism in hummingbird flight muscles. In The Vertebrate Gas Transport Cascade: Adaptations to Environment and Mode of Life (eds. J. E. P. W. Bicudo), pp. 279-285. Boca Raton, USA: CRC Press. Suarez, R. K., Lighton, J. R., Brown, G. S. and Mathieu-Costello, O. (1991). Mitochondrial respiration in hummingbird flight muscles. Proc. Natl. Acad. Sci. USA 88, 4870-4873. Swan, L. W. (1970). Goose of the Himalayas. Nat. Hist. 70, 68-75. Tucker, V. A. (1968). Respiratory physiology of house sparrows in relation to highaltitude flight. J. Exp. Biol. 48, 55-66. Turner, D. L. and Butler, P. J. (1988). The aerobic capacity of locomotory muscles in the tufted duck, Aythya fuligula. J. Exp. Biol. 135, 445-460. Wagner, P. D. (1996a). A theoretical analysis of factors determining Vo2max at sea level and altitude. Respir. Physiol. 106, 329-343. Wagner, P. D. (1996b). Determinants of maximal oxygen transport and utilization. Annu. Rev. Physiol. 58, 21-50. Wang, T. and Hicks, J. W. (2002). An integrative model to predict maximum O2 uptake in animals with central vascular shunts. Zoology 105, 45-53. Wang, T. and Hicks, J. W. (2004). Why savannah monitor lizards hyperventilate during activity: a comparison of model predictions and experimental data. Respir. Physiol. Neurobiol. 144, 251-261. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356.  59 Weber, R. E. and Fago, A. (2004). Functional adaptation and its molecular basis in vertebrate hemoglobins, neuroglobins, and cytoglobins. Respir. Physiol. Neurobiol. 144, 141-159. Weber, R. E., Hiebl, I. and Braunitzer, G. (1988). High altitude and hemoglobin function in the vultures Gyps rueppellii and Aegypius monachus. Biol. Chem. Hoppe Seyler 369, 233-240. Weber, R. E., Jessen, T. H., Malte, H. and Tame, J. (1993). Mutant hemoglobins (α119-Ala and β55-Ser): functions related to high-altitude respiration in geese. J. Appl. Physiol. 75, 2646-2655. Weibel, E. R., Taylor, C. R. and Hoppeler, H. (1991). The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Natl. Acad. Sci. USA 88, 10357-10361. Weinstein, Y., Bernstein, M. H., Bickler, P. E., Gonzales, D. V., Samaniego, F. C. and Escobedo, M. A. (1985). Blood respiratory properties in pigeons at high altitudes: effects of acclimation. Am. J. Physiol. 249, R765-R775. West, J. B., Watson, R. R. and Fu, Z. X. (2006). The honeycomb-like structure of the bird lung allows a uniquely thin blood-gas barrier. Respir. Physiol. Neurobiol. 152, 115-118. Wittenberg, J. B. and Wittenberg, B. A. (2003). Myoglobin function reassessed. J. Exp. Biol. 206, 2011-2020. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493.  60  3. CONTROL OF BREATHING AND ADAPTATION TO HIGH ALTITUDE IN THE BAR-HEADED GOOSE2  3.1. Introduction To sustain physiological function at high altitude cellular O2 supply and demand must remain matched, even though environmental O2 availability is reduced. Substantial reductions in O2 demand are not possible in animals that remain active in hypoxia, however, so the capacity to transport O2 must be increased in high altitude species. Oxygen transport occurs along the pathway from air to mitochondria, which can be conceptually separated into 4 steps: ventilation, pulmonary diffusion, circulation (perfusion), and tissue diffusion (Weibel, 1984; Chapter 2; Scott and Milsom, 2006). In animals that adapt (in an evolutionary sense) to high altitude, natural selection could act at any step of the O2 pathway. Physiological theories predict that the capacity for O2 transport in hypoxia could evolve either through equal changes in every step of the O2 pathway (the symmorphosis concept) (Weibel et al., 1996), or through disproportionate changes at only one or a few steps in the pathway (the control coefficients concept) (Hochachka and Burelle, 2004). As the first step in the O2 transport pathway, breathing is responsible for gas transfer between the lungs and the environment. Exposure to environmental hypoxia causes an immediate increase in breathing due to stimulation of arterial chemoreceptors, followed by many subsequent time dependent modifications that are thought to originate 2  A version of this chapter has been published: Scott, G. R. and Milsom, W. K. (2007). Control of breathing and adaptation to high altitude in the bar-headed goose. Am. J. Physiol. Reg. Integr. Comp. Physiol. 293, R379-R391. (used with permission)  61 from multiple sites in the reflex pathway (Powell et al., 1998). In addition to these modifications, changes in metabolic state and differences in pulmonary mechanics will also influence breathing. Evolutionary changes in ventilatory control in high altitude adapted species are poorly documented. Multiple human populations have adapted to different high altitude regions of the world, and although it is clear that evolutionary changes in respiratory control exist in these groups, different changes have arisen in each: high altitude Quechuans from the Andean plateau have an inherently reduced hypoxic ventilatory response (HVR) compared to low altitude residents (Brutsaert et al., 2005), whereas high altitude Tibetans have an enhanced HVR (Wu and Kayser, 2006). Little is known about how control of breathing has evolved in other vertebrates that are adapted to high altitude. Birds in general have excellent hypoxia tolerance and can maintain normal function during hypoxia too severe for most mammals to survive (Tucker, 1968). Some species of birds are exceptional in this regard. The bar-headed goose (Anser indicus) flies over the Himalayan mountains twice a year on its migration between its wintering grounds in southern Asia and its summering grounds on the Tibetan plateau. This species has been repeatedly observed flying above the highest peaks in the range (Swan, 1970; Javed et al., 2000), where oxygen levels can be only 25% of those at sea level. This feat is especially impressive considering that the bar-headed goose increases its rate of O2 consumption 10-20-fold above resting levels during flight (Ward et al., 2002). Several previous physiological studies have sought to determine how bar-headed geese can sustain the aerobic requirements of flight at such high altitudes (reviewed in  62 Faraci, 1991; Chapter 2; Scott and Milsom, 2006). Haemoglobin of this species has been shown to have an inherently higher O2 affinity, which is almost entirely due to a single amino acid substitution in the αA polypeptide chain (Petschow et al., 1977; Weber et al., 1993; Zhang et al., 1996). This substitution is probably adaptive in bar-headed geese by increasing O2 loading during hypoxia. However, although many other physiological attributes of birds in general may be beneficial for high altitude flight, no other unique specializations have yet been found in bar-headed geese (Black and Tenney, 1980a; Black and Tenney, 1980b; van Nice et al., 1980; Faraci et al., 1984; Faraci et al., 1985; Fedde et al., 1985; Faraci and Fedde, 1986; Fedde et al., 1989; Saunders and Fedde, 1991). In a recent theoretical study, we found that a heightened ability to increase ventilation should enhance O2 transport substantially during hypoxia in birds (Chapter 2; Scott and Milsom, 2006). Based on these findings, we hypothesized that high altitude adaptation in bar-headed geese would involve changes in ventilatory control such that this species would have an enhanced HVR (i.e., it would breathe more) and load more O2 into the blood during hypoxia. To test this hypothesis, we compared the control of breathing in bar-headed geese and two other waterfowl species: the greylag goose (Anser anser), which is a close relative of the bar-headed goose, and the pekin duck (Anas platyrhynchos), which is a more distant relative (pekin ducks are a domesticated subspecies of mallard ducks). These species are thought to migrate primarily at low to moderate altitudes (<4000m), even though there have been occasional sightings at higher elevations (Swan, 1961; Manville, 1963; Swan, 1970). We predicted that differences in control of breathing would exist between bar-headed geese and both low altitude species,  63 which due to the evolutionary relationship between the species selected would probably represent a uniquely derived specialization that had not existed in its ancestors.  3.2. Materials and Methods 3.2.1. Animals Experiments were performed on 8 bar-headed geese (1.9-2.6 kg), 6 greylag geese (3.2-4.9 kg), and 10 pekin ducks (2.3-3.7 kg). All animals were bred and raised at sea level, either at the Animal Care Facility of the University of British Columbia (UBC) or by local suppliers. Animals were housed outdoors at UBC, and had no prior exposure to high altitude. Birds were starved for one day before each experiment but continued to receive unlimited access to water. All animal care and experimentation was conducted according to UBC animal care protocol #A04-1013.  3.2.2. Surgical procedures All birds underwent surgery one to two days before experimentation. Birds were given an intramuscular injection of telazol (Wyeth; 20-25 mg/kg) to induce moderate anaesthesia and were lightly restrained in dorsal recumbency. Animals were maintained under this general anaesthesia with 1-2 additional injections of telazol (8-10 mg/kg) as needed. Flexible polyethylene cannulae (PE-90) filled with 1000 IU/ml heparinized saline were implanted under local anaesthesia (Lidocaine, Langford; administered subcutaneously) in the right brachial artery and vein (lateral to the humerus), and were slowly advanced approximately 8 cm. The clavicular air sac was intubated with a small tube (7 mm diameter and ~10 mm long) containing a rubber plug under local anaesthesia.  64 The tube was secured to the skin and underlying air sac membrane with suture. On the day of the experiment the rubber plug was removed and replaced with a similar plug that allowed the passage of a heat-flared PE-90 cannula, such that gas samples could be drawn continuously from the clavicular air sac.  3.2.3. Measurements Body plethysmography was used to measure ventilation, as previously described (Dodd and Milsom, 1987; Dodd et al., 2007). Briefly, the plethysmograph consisted of two parts, a body compartment and a head compartment, separated from each other by a flexible latex collar. The head compartment was used to administer specific gas mixtures for the animals to breathe. The composition of dry gas flowing into the head compartment was controlled by mixing N2, O2, and CO2 through a series of calibrated flow meters, and was monitored with oxygen (Raytech) and carbon dioxide (Beckman, LB-2; hypercapnic hypoxia exposure only) analyzers. Gas was humidified before entering the head compartment, and the flow rate was never less than 15 l/min. Changes in body volume (due to ventilatory movements) in the body compartment were detected with a pneumotachograph (Fleisch) connected to a differential pressure transducer (Validyne), which was calibrated to yield a measurement of ventilatory flow. Arterial blood pressure was continuously monitored using a physiological pressure transducer (Narco Scientific) connected to the brachial artery cannula. Gas was continuously drawn from the clavicular air sac (poikilocapnic and isocapnic hypoxia exposures only) at a rate of approximately 100 ml/min, and its fractional CO2 composition ( FCL CO 2 ) was measured with a CO2 analyzer (Beckman).  !  65 Ventilatory flow, fractional O2 composition of gas entering and leaving the head compartment, airflow through the head compartment, and FCL CO 2 were recorded to a computer at a 125 Hz sampling frequency per channel using Windaq data acquisition software (Dataq Instruments).  !  After being drawn, arterial and mixed venous blood samples (0.7 ml) were immediately used to measure blood gases, pH, and O2 content. Arterial and venous O2 ( PO 2 ) and CO2 ( PCO 2 ) tensions, and arterial and venous pH were determined using Radiometer blood gas/pH electrodes maintained at avian core body temperature (41oC).  !  ! The electrodes were calibrated before each sample using saturated gases and commercially prepared pH buffers (VWR). Arterial and venous O2 contents were determined at avian body temperature using the method of Tucker (1967). The bicarbonate ion concentrations of blood were calculated using the Henderson-Hasselbach equation, assuming a pK of 6.090 and a solubility coefficient of 0.0282 mmol/l/torr in plasma (Helbecka et al., 1964).  3.2.4. Experimental protocol Birds were placed in the experimental apparatus and allowed 60-90 minutes to adjust to their surroundings. The birds were then exposed to progressive step reductions in the fractional O2 composition of inspired gas (in order of declining FIO 2 , 0.209, 0.12, 0.09, 0.07, 0.05, and in bar-headed geese only, 0.04). This hypoxia exposure was  ! under isocapnic performed twice, once under poikilocapnic conditions and once conditions. The poikilocapnic protocol was performed first in half the experiments and the isocapnic protocol was performed first in the other half, such that the effects of time-  66 dependent processes between poikilocapnic and isocapnic data were minimized. For poikilocapnic hypoxia exposure, FIO 2 was reduced as described above and inspired CO2 fraction ( FICO 2 ) was zero. Because FICO 2 was not manipulated, blood CO2 levels (and thus  ! FCL CO 2 ) fell proportionately with the hypoxia-induced hyperventilation. During isocapnic ! ! hypoxia exposure, FICO 2 was increased slowly as necessary while monitoring FCL CO 2 to !  maintain blood CO2 levels constant, and FIO 2 was reduced in the same manner as for  ! ! poikilocapnic hypoxia. After 15 minutes of exposure to each FIO 2 , arterial and venous ! blood samples were withdrawn slowly and analyzed as described above. As much of the sampled blood as possible was returned to the bird.!Birds were exposed to each FIO 2 for 25 minutes. Each bird was allowed approximately 90 minutes of recovery between the  ! and FCL had first and second hypoxia exposures, at which time respiratory variables CO 2 returned to resting levels. After both hypoxia exposures were complete, birds recovered  ! for 15 minutes (at FIO 2 =0.12, FICO 2 =0) and were then exposed to hypercapnia ( FIO 2 =0.12, FICO 2 =0.05) for 25 minutes, with blood samples being withdrawn at 15 minutes as before. ! !  !  !  3.2.5. Data and statistical analyses All data acquired in Windaq were analyzed using a specially written Matlab (version 7, Mathworks) program. Average values were calculated for each variable during the interval between 10-20 minutes of exposure to each inspired gas level. Inspiratory tidal volume (VT) was determined by integrating positive periods of ventilatory flow. Total ventilation was calculated from the product of VT and breathing frequency. The rate of oxygen consumption was calculated from the product of airflow  67 through the head compartment and the FO 2 difference between the compartment’s inflow and outflow (i.e., the respiratory quotient, RQ, was assumed to be 1). While RQ values in  ! 0.7 to 1.0, and our calculations ignore this potential birds typically range from below source of variation, we used the same value in all cases and so this will not alter our results. Because water vapour was removed from this gas before analysis, the rates of O2 consumption were determined as described by Withers (Withers, 1977). Air convection requirement was calculated as the quotient of total ventilation and the rate of oxygen consumption. Average values for inspiratory time, expiratory time, peak inspiratory flow rate, peak expiratory flow rate, and FCL CO 2 were also determined for each minute analyzed.  ! means ± standard error. Within each species, all data in each Data are reported as experiment were analyzed using repeated measures analysis of variance, using HolmSidak post-hoc tests. Comparisons between species were made for respiratory and blood gas data using two-factor (species and FIO 2 ) repeated measures analysis of variance and Holm-Sidak post-hoc tests within each FIO 2 . Least squares linear regression was used to  ! assess the relationships between total ventilation and peak inspiratory/expiratory flow ! rates. Statistical tests were performed using Sigmastat software (version 3.0, Systat Software Inc.). A significance level of p<0.05 was used throughout.  3.3. Results 3.3.1. Poikilocapnic hypoxic ventilatory responses During the poikilocapnic hypoxia experiment, each progressive step reduction in FIO2 increased total ventilation and reduced arterial O2 tension ( Pa O 2 ) for all 3 species  !  68 (Fig. 3.1). The ventilatory response was significantly greater in bar-headed geese: this species increased total ventilation by 7.2-fold above normoxic levels at 5% FIO 2 , compared to only 4.2-fold in greylag geese and 4.9-fold in pekin ducks (Fig. 3.1A). Pa O 2  ! was generally higher (4-7 Torr) in bar-headed geese at all levels of hypoxia, and this was ! FI , which significant at 5% FIO 2 (by 6 Torr). Only bar-headed geese could tolerate 4% O2 resulted in a 8.6-fold increase in ventilation above normoxic levels. Interestingly, at both  ! FI , Pa in bar-headed geese was approximately equal!to inspired P (the 5 and 4% O2 O2 O2 theoretical maximum, grey dashed line in Fig. 3.1B); Pa O 2 never reached inspired levels  ! ! ! in either greylag geese or pekin ducks. The enhanced ventilatory response to ! apparent in the relationship between poikilocapnic hypoxia in bar-headed geese is ventilation and Pa O 2 (Fig. 3.1C). The higher total ventilation in bar-headed geese was caused by an enhanced tidal  ! response to poikilocapnic hypoxia (Fig. 3.2). Tidal volume was significantly volume higher in bar-headed geese than in both other species at 9%, 7%, and 5% FIO 2 , but breathing frequency was generally similar. As a result, for any level of total ventilation, bar-headed geese had a higher tidal volume and lower breathing!frequency (right-shifted curve in Fig. 3.2). The ventilatory response to poikilocapnic hypoxia involved reductions in inspiratory time (TI) and expiratory time (TE) in all three species, and TE tended to fall more such that the ratio of TI/TE increased (Table 3.1). In concert with progressive reductions in Pa O 2 during poikilocapnic hypoxia were declines in arterial O2 content ( Ca O 2 ), venous O2 tension, and venous O2 content (Table  ! 3.2). Bar-headed geese maintained a higher Ca O 2 than greylag geese and pekin ducks at ! !  69 5% FIO2, consistent with the observed Pa O 2 differences between species. When Ca O 2 was expressed relative to Pa O 2 , and the data were fitted with a sigmoidal Hill equation  ! ! (i.e., Ca O 2 = Pa O 2 n/(P50n + Pa O 2 n)), bar-headed geese, greylag geese, and pekin ducks had ! in vivo values of P50 (the PO 2 causing 50% saturation of blood O2 binding capacity) of !  ! ! 35.4 torr, 32.7 torr, and 35.8 torr, respectively. ! 3.3.2. Isocapnic hypoxic ventilatory responses As during poikilocapnic hypoxia (when CO2 levels in the blood were allowed to fall), during isocapnic hypoxia (when CO2 levels in the blood were maintained) each reduction in FIO2 normally increased total ventilation and reduced Pa O 2 (Fig. 3.3). The only exception was that total ventilation did not increase during exposure to the lowest  ! FIO 2 studied. In general, total ventilation and Pa O 2 were higher in isocapnic hypoxia than in poikilocapnic hypoxia in all 3 species. However, the ventilatory response of bar-  !  ! not enhanced compared to pekin ducks: both headed geese to isocapnic hypoxia was species increased total ventilation by ~7-fold above normoxic levels at 5% FIO 2 (Fig. 3.3A). Greylag geese had lower total ventilation than both other species at 7% and 5%  ! FIO 2 , and could only increase total ventilation 4-4.5-fold above normoxic levels. Pa O 2 was similar in all species at all levels of FIO 2 , and it approached inspired PO 2 at 7% FIO 2  !  and below (Fig. 3.3B).  !  ! ! ! 3.4) than Tidal volumes were generally higher during isocapnic hypoxia (Fig. during poikilocapnic hypoxia (Fig. 3.2). Furthermore, and in contrast to poikilocapnic hypoxia, there were few differences between species in tidal volume responses to  70 isocapnic hypoxia. Tidal volume was reduced in greylag geese at 9%, 7%, and 5% FIO 2 , but otherwise all species responses were similar for both tidal volume and breathing frequency.  !  The ventilatory response to isocapnic hypoxia involved reductions in inspiratory time (TI) and expiratory time (TE), and an increase in TI/TE (Table 3.3). Arterial O2 content ( Ca O 2 ), venous O2 tension, and venous O2 content (Table 3.4) all decreased progressively. For both poikilocapnic and isocapnic hypoxia in all species, venous O2  !content was almost always near zero for the lowest FI studied; however, this tended to O2 occur in conjunction with lower Pv O 2 levels in bar-headed geese. When Ca O 2 was  ! expressed relative to Pa O 2 and fitted with a Hill equation as for poikilocapnic hypoxia ! ! above, bar-headed geese, greylag geese, and pekin ducks had in vivo values of P50 of 38.2 ! and 48.1 torr, respectively. torr, 45.6 torr, 3.3.3. Acid-base regulation during hypoxia The increases in ventilation during poikilocapnic hypoxia caused progressive reductions in the CO2 tension of arterial blood ( Pa CO 2 ) (Fig. 3.5A), venous blood ( Pv CO 2 ) (Table 3.2), and the respiratory system (clavicular air sac, PCL CO 2 ) (Table 3.1) in all 3  ! ! species. This respiratory hypocapnia did not cause any significant arterial pH disturbance ! partly due to progressive reductions in in any species (Fig. 3.5A), and this may have been arterial bicarbonate concentrations (assuming calculated [HCO3-] is representative of actual [HCO3-]). There were statistically insignificant trends suggestive of a respiratory alkalosis during moderate hypoxia, followed by metabolic acidosis at more severe levels of hypoxia in all species.  71 Because CO2 was added to inspired gas during isocapnic hypoxia, Pa CO 2 remained constant in all species (Fig. 3.5B), and Pv CO 2 (Table 3.4) and PCL CO 2 (Table 3.3)  ! experienced only small inconsistent changes. Arterial pH homeostasis was therefore ! ! maintained during moderate hypoxia. During more severe hypoxia, all species experienced an isocapnic metabolic acidosis of arterial blood, in conjunction with reductions in arterial bicarbonate. For both poikilocapnic and isocapnic hypoxia experiments, venous pH and bicarbonate levels changed in a similar manner to those in arterial blood (Tables 3.2 and 3.4). Although there were small differences in absolute values between species, their pattern of acid-base regulation during both poikilocapnic and isocapnic hypoxia were the same: there did not appear to be any differences between species in the responses of pH,  PCO 2 , or calculated bicarbonate concentrations of arterial blood (Fig. 3.5).  !  3.3.4. Ventilatory response to CO2/pH In addition to both poikilocapnic and isocapnic conditions, all species were exposed to hypercapnia (5% inspired CO2) at 12% FIO 2 . As a result, the CO2/pH sensitivity of all three species could be assessed during moderate hypoxia using 3  ! pH (pHa). All 3 species increased ventilation different levels of arterial PCO 2 and arterial via increases in both breathing frequency and tidal volume in response to increased Pa CO 2  ! and decreased pHa (Fig. 3.6). There were no statistically significant differences between ! and 3.6B), species in the response of total ventilation to either Pa CO 2 or pHa (Figs. 3.6A but there was an insignificant trend of a reduced ventilatory sensitivity to reductions in  !  72 pHa in greylag geese (due to a reduced tidal volume response). Curiously, Pa CO 2 was lower in greylag geese than the other species during hypercapnia. Pekin ducks appeared  ! enhanced tidal volume to have a reduced breathing frequency response (Fig. 3.6C) and an response (Fig. 3.6E) to Pa CO 2 , but these differences were not significant. Most importantly, there were no clear differences between bar-headed geese and the two low  ! during hypercapnic exposure. altitude species Similar to the responses of arterial PCO 2 and pH to hypercapnia (Fig. 3.6), venous  PCO 2 increased and venous pH decreased compared to isocapnic levels (Table 3.5). ! Hypercapnia also had modest effects on blood oxygen levels. Pa O 2 was generally !  elevated by the enhancement of ventilation during hypercapnia compared to isocapnia, but this was not always accompanied by an increase!in O2 content (possibly due to effects of CO2/pH on blood O2 binding) (Table 3.5).  3.3.5. Breathing mechanics during hypoxia A preliminary assessment of how breathing mechanics might influence the hypoxic ventilatory responses of each species was performed by analyzing the relationships between total ventilation and inspiratory/expiratory flow rates (using data from both poikilocapnia and isocapnia). There was an extremely strong correlation (R2=0.97) between total ventilation and peak inspiratory flow using data from all three species (Fig. 3.7A). Notably, however, the maximum observed peak inspiratory flow in greylag geese was much lower than in the other species (dashed vertical line in Fig. 3.7A). The analogous correlation between total ventilation and peak expiratory flow was also strong (R2=0.96), but the isocapnic data points for greylag geese at 7% and 5% FIO 2  !  73 deviated from the regression (Fig. 3.7B). Therefore, while the maximum observed peak expiratory flows in this species were 67-75% of those in bar-headed geese and pekin ducks (dashed vertical lines in Fig. 3.7), the maximum peak inspiratory flows were only 46-56%. Therefore, an inability to increase peak inspiratory flow may have limited the hypoxic ventilatory response of greylag geese, especially during severe isocapnic hypoxia. In particular, it may explain why greylag geese could not concurrently generate large tidal volumes and high breathing frequencies. These results were supported by flow-volume loops constructed from representative breath sequences of all individuals (data not shown), suggesting that limitations in the generation of inspiratory flow may exist across the breathing cycle in greylag geese.  3.3.6. Metabolic responses to hypoxia and elevated breathing The rate of oxygen consumption increased in all species during both poikilocapnic and isocapnic hypoxia (Fig. 3.8), which likely reflects the metabolic costs of respiratory and cardiac muscle work. Furthermore, there was variability in the metabolic responses to hypoxia between species. During poikilocapnic hypoxia, barheaded geese had the greatest increase in oxygen consumption and greylag geese had the lowest (Fig. 3.8A). Similar trends were observed during isocapnic hypoxia, but the differences between bar-headed geese and the other two species were less pronounced (Fig. 3.8B). As a result, only small differences between species were observed in their air convection requirements (total ventilation expressed relative to oxygen consumption rate, also called the ventilatory equivalent) during poikilocapnic hypoxia (Fig. 3.8C). This suggests that the species differences in total ventilation during severe hypoxia were due  74 to differences in metabolic demands, and that the higher total ventilation per se did not cause the enhanced O2 loading in bar-headed geese during severe hypoxia (Fig. 3.1B). The enhanced tidal volume response (which suggests an increase in effective ventilation, see Discussion) may instead account for the enhanced O2 loading in this species (Fig. 3.2). Interestingly, air convection requirements were lower in bar-headed geese at 7%  FIO 2 than in both other species (Fig. 3.8D).  !  3.4. Discussion During their biannual migration over the Himalayan mountains, bar-headed geese fly at altitudes up to 9000m, where oxygen levels in the air can be exceedingly low. Sustained flight elevates metabolic rate 10-20-fold above resting levels in birds and must be accompanied by similar increases in O2 uptake. Our present findings suggest that amongst the respiratory specializations that have evolved in bar-headed geese to facilitate O2 loading during severe environmental hypoxia is an enhanced ventilatory response to poikilocapnic hypoxia (Fig. 3.1). Because this was primarily due to a larger tidal volume response (Fig. 3.2), there would have been a large enhancement of parabronchial (effective) ventilation. Arterial PO 2 and O2 content were therefore substantially higher in bar-headed geese during severe hypoxia. This improved ability of bar-headed geese to load O2 into the blood!may at least partially account for their incredible ability to fly high. We will consider several mechanisms that could account for the heightened breathing response to hypoxia in bar-headed geese. Chemoreceptors stimulate breathing in response to reductions in arterial PO 2 (sensed by arterial chemoreceptors) as well as  !  75 increases in arterial PCO 2 and decreases in pH (sensed by arterial and central chemoreceptors) (Jones and Purvis, 1970; Bouverot, 1978; Monge and León-Velarde,  ! 1991; Hempleman et al., 1992). During environmental hypoxia arterial PO 2 is reduced, stimulating increases in breathing. This augments CO2 loss, however, which causes  ! respiratory hypocapnia, partially offsetting the ventilatory response. The enhanced breathing response of bar-headed geese could therefore be due to an increased sensitivity to arterial hypoxia or a reduced sensitivity to hypocapnia (and any associated pH disturbance). Differences in ventilation between species could have also been caused by differences in the mechanical constraints on ventilatory flows or in the magnitude of the metabolic responses to hypoxia. All of these possibilities will be discussed in more detail below.  3.4.1. Oxygen chemosensitivity is not enhanced in bar-headed geese The enhanced ventilatory response of bar-headed geese to poikilocapnic hypoxia was probably not caused by an increased chemosensitivity to reductions in arterial PO 2 . Ventilatory responses to isocapnic hypoxia are not inhibited by respiratory hypocapnia as  ! they are during poikilocapnic hypoxia (because arterial PCO 2 is held constant), making the isocapnic HVR a reasonable indicator of O2 chemosensitivity. Because the isocapnic  ! ducks (Fig. 3.3), we believe their HVR was the same in bar-headed geese and pekin chemoreceptor sensitivities to O2 are similar. The isocapnic HVR in greylag geese was reduced compared to the other species but this was probably not due to reduced O2 chemosensitivity but to pulmonary mechanical constraints, as discussed below. This contrasts with reports in high altitude populations of humans, where evolutionary  76 alterations in O2 chemosensitivity of breathing (both increases and decreases) have been demonstrated (Brutsaert et al., 2005; Wu and Kayser, 2006) (see Introduction). The isocapnic HVR in our experiment may not represent a sole effect of changes in arterial hypoxia per se for 2 reasons. Firstly, because birds possess intrapulmonary CO2 chemoreceptors (IPC), adding CO2 to the inspired gas to maintain arterial isocapnia would have altered IPC discharge (Hempleman and Posner, 2004; Milsom et al., 2004). IPC discharge has slight inhibitory effects on breathing (but primarily alters breathing pattern), and discharge declines as CO2 increases (Milsom et al., 2004; Dodd et al., 2007). While the net effect might be a slight stimulation of breathing, at elevated inspired CO2, IPC discharge would be low in all species. We therefore anticipate that potential differences in IPC discharge had a minimal influence on our results in isocapnic hypoxia, bearing in mind that our understanding of the sensitivities and roles of IPCs in intact animals (particularly bar-headed geese) is restricted. Secondly, although Pa CO 2 was held constant during the isocapnic HVR experiment, all birds experienced a metabolic  ! acidosis. This could also have stimulated breathing, so the measured isocapnic HVR may have been greater than a response to arterial hypoxia alone. Since the measured acidosis was similar in all birds, this effect was probably equivalent in all species.  3.4.2. Has sensitivity to CO2 changed in bar-headed geese? The enhanced ventilatory response of bar-headed geese to poikilocapnic hypoxia was not caused by differences in arterial PCO 2 or pH, because changes in these variables during hypoxia were similar among species in both the current study (Fig. 3.5A) and  ! and Tenney, 1980a). Both extracellular and those previously conducted (Black  77 intracellular acid-base regulation are exceptional in waterfowl (Weinstein et al., 1985; Dodd and Milsom, 1987; Dodd et al., 2007), which could account for the tight regulation of arterial pH during respiratory hypocapnia. The responses of Pa CO 2 and pHa to isocapnic hypoxia were also similar between species (Fig. 3.5B). Unlike poikilocapnic  ! hypoxia, however, all species exhibited a metabolic acidosis. The cause of this acidosis is unclear, but it could have resulted from an increase in anaerobic metabolism, or a hypoxaemia-induced reduction in blood bicarbonate levels as occurs during poikilocapnic hypoxia. There was no difference between species in the hypercapnic ventilatory response measured during moderate hypoxia. In all species at 12% FIO 2 , breathing responded similarly to elevated arterial PCO 2 and reduced arterial pH (Fig. 3.6). Curiously, the  ! changes in breathing pattern in response to hypercapnia varied between species, but these ! statistically significant. While these results suggest that reduced differences were not CO2/pH chemosensitivity does not cause the enhanced poikilocapnic HVR in bar-headed geese, this suggestion is not unequivocal. There could still be reduced sensitivity to hypocapnia (reduced CO2) but not hypercapnia (elevated CO2). Furthermore, CO2/pH sensitivity may also change as a function of arterial PO 2 , such that bar-headed geese may only be less sensitive to hypocapnia when severely hypoxic. Indeed, total ventilation in  ! of Pa bar-headed geese appeared nearly independent CO 2 and pHa in severe hypoxia (compare Figs. 3.1 and 3.3). Interactions between O2 and CO2/pH sensitivity are well described in mammals (Day and Wilson,!2007), and it is likely that the nature of this interaction could change through evolution. However, such a low sensitivity to CO2/pH is surprising, and is not typical of other species of mammal or birds (Bouverot, 1978;  78 Powell et al., 2000). A reduced chemosensitivity to hypocapnia may also explain the increased tidal volume and parabronchial ventilation, and therefore the enhanced O2 loading, in bar-headed geese during severe hypoxia. We are unaware of any other examples of reduced hypocapnic chemosensitivity, but hypercapnic chemosensitivity is known to be lower in burrowing animals (who are chronically exposed to higher levels of CO2) (Kilgore et al., 1985), and can increase (not decrease) during ventilatory acclimatization to hypoxia in mammals (Engwall and Bisgard, 1990; Fatemian and Robbins, 1998). The effects of poikilocapnic hypoxia on ventilation have been studied previously in bar-headed geese, and produced results that are largely consistent with our present findings: bar-headed geese increase ventilation more than pekin ducks in severe hypoxia (Black et al., 1978; Black and Tenney, 1980a; van Nice et al., 1980). They also maintained higher rates of O2 consumption (Black and Tenney, 1980a), the importance of which is discussed below. In contrast to our present findings, however, previous studies found that pekin ducks started to increase ventilation at a higher FIO 2 than bar-headed geese. The reasons for this are not clear.  ! 3.4.3. Pulmonary mechanics may have evolved in bar-headed geese The ventilatory response of greylag geese appears to be limited by their capacity to generate high inspiratory flows (Fig. 3.7), possibly due to a reduced ability to generate high inspiratory pressure. This would reduce the capacity for greylag geese to increase tidal volume and breathing frequency concurrently, and may account for this species’ diminished tidal volume responses to hypoxia and hypercapnia (Figs. 3.2, 3.4, and 3.6).  79 Human endurance athletes are known to approach the mechanical limits of their respiratory system for generating inspiratory pressure during maximal exercise (Johnson et al., 1992). This and other mechanical limitations can reduce arterial oxygen loading, even in normoxia (Dempsey and Wagner, 1999). Bar-headed geese attained higher peak inspiratory flows than both other species in the current study. During high altitude flight both hypoxia and exercise would intensely stimulate breathing and the data suggest that bar-headed geese may have an enhanced mechanical capacity to generate high ventilatory flows, at least compared to their close relative, the greylag goose.  3.4.4. Haemoglobin has evolved in bar-headed geese Bar-headed geese are known to have a higher inherent haemoglobin O2 affinity than low altitude waterfowl, primarily due to a single amino acid substitution in the αA polypeptide chain (Petschow et al., 1977; Weber et al., 1993; Zhang et al., 1996). Barheaded goose whole blood has a P50 of 25 Torr at standard conditions in vitro, compared to 40 Torr in greylag geese (Petschow et al., 1977). Bar-headed goose haemoglobin also lacks a salt bridge important for the Bohr effect (Liang et al., 2001). Interestingly, studies of bar-headed goose haemoglobin were some of the best early examples of putatively adaptive changes at the molecular level (Golding and Dean, 1998). In vivo O2 equilibrium curves from data in the present study illustrate these findings. The in vivo O2 equilibrium curves for isocapnic hypoxia reveal a left-shifted P50 for the bar-headed goose (38 Torr, versus 46 and 48 Torr in the other 2 species), while those for poikilocapnic hypoxia reveal similar P50 values for all 3 species (35, 33, and 36 Torr). Given the parallel changes in PaCO2 and pHa in all 3 species, this simply reflects the reduced Bohr effect in  80 bar-headed geese. The potential benefit of a reduced Bohr effect in bar-headed geese is unclear. While it may have arisen to restrict increases in O2 affinity during hypocapnia/alkalosis (Chapter 2; Scott and Milsom, 2006), it could also reduce the beneficial effects of the Bohr effect on O2 unloading to the tissues that normally occurs during exercise-induced tissue acidosis.  3.4.5. The metabolic response to hypoxia has evolved in bar-headed geese During hypoxia at rest, metabolism can change as a result of 2 processes: elevated O2 demand by respiratory muscle (and possibly demand from cardiac muscle and tissue acid-base regulation), and reduced O2 demand due to metabolic depression in other tissues (Guppy and Withers, 1999). In the present study, the sum of these processes resulted in a net increase in metabolism during hypoxia in all three species (Fig. 3.8). Thus, while hypoxia depresses whole-body metabolism in many other vertebrates, elevated metabolism occurs in some (but not all) bird species during hypoxia (Tucker, 1968; Black and Tenney, 1980a), and this likely resulted from the augmented cardiorespiratory requirements for O2 transport. While no previous studies have directly assessed the cost of breathing during severe hypoxia in birds, some previous work suggests that it might be appreciable. In Canada geese (Branta canadensis), tripling breathing frequency at constant tidal volume increases the metabolic costs of breathing approximately 4-fold (Funk et al., 1997); tidal volume also increases substantially during hypoxia, which would further increase the cost of breathing (Scheid and Piiper, 1969). In elite human athletes at maximal exercise, both oxygen consumption and total ventilation increase 15- to 20-fold above resting levels (Johnson et al., 1992). At these high rates of  81 total ventilation the metabolic costs of breathing can increase substantially, and account for 15% or more of the total oxygen consumption rate (i.e., 15% of VO 2 MAX ) (Johnson et al., 1992). If that metabolic cost of breathing is compared to the total oxygen consumption rate at rest, just increasing ventilation would!result in a 2.5- to 3-fold increase in oxygen consumption rate. These previous studies, as well as observations that blood flow to respiratory muscles increases several fold during hypoxia in waterfowl (Faraci et al., 1985), suggest that the increases in metabolic rate (i.e., oxygen consumption rate) observed in the present study (Fig. 3.8) can be partly attributed to the increased cost of breathing. Furthermore, the data also suggest that some of the difference between bar-headed geese and the other two species in the rise of metabolic rate observed at 5% FIO 2 may be attributed to the differences in breathing. Total ventilation generally increased in all species as the air they breathed became  ! more hypoxic. Other than at 5% FI , total ventilation was similar between species, O2 raising the question of what accounts for the higher metabolic rates in bar-headed geese at less severe levels of ! hypoxia. Since increasing ventilation via tidal volume is thought to be more costly than increasing ventilation via breathing frequency (Milsom, 1991), the higher tidal volumes in bar-headed geese could partially explain their higher rates of metabolism. Breathing pattern aside, low altitude waterfowl are known to restrict blood flow to the most hypoxia-sensitive tissues during hypoxia (i.e., heart and brain) but this does not occur in bar-headed geese (Faraci et al., 1984; Faraci et al., 1985). Thus, despite net increases in total metabolism, low altitude waterfowl may have also experienced some degree of regional metabolic depression, which may not have occurred in barheaded geese. The ability of bar-headed geese to avoid metabolic depression in flight  82 muscle during severe hypoxia is undoubtedly essential for high altitude flight. Work in mammals suggests that hypoxic metabolic depression is controlled by hypothalamic sites that reduce metabolism, body temperature, and ventilation (Barros et al., 2006; Gargaglioni et al., 2006). A relative metabolic depression in low altitude species may therefore result from central inhibitory influences that also reduce the ventilatory response to hypoxia, which may not have occurred in bar-headed geese. Future studies will explore this possibility (Chapter 4).  3.4.6. Effective ventilation is enhanced in bar-headed geese The enhanced O2 loading in bar-headed geese is best attributed to a large increase in parabronchial (effective) ventilation. With each breath, only part of the inspired air ventilates the gas exchange surface, while the rest only ventilates dead space. Total ventilation is thus comprised of both effective ventilation and dead space ventilation. Deeper breaths (i.e., larger tidal volume breaths) reduce the contribution of total ventilation that ventilates dead space, and will therefore result in greater effective ventilation of the gas exchange surface (assuming there are no large differences in anatomical dead space volume between species). Total ventilation and metabolism were evenly matched in all species during poikilocapnic hypoxia, because air convection requirements (total ventilation normalized to oxygen consumption rate) were similar (Fig. 3.8). This suggests that differences in total ventilation per se did not enhance arterial PO 2 or O2 loading in bar-headed geese; total ventilation merely kept pace with the higher  ! ventilation metabolic rate sustained by this species in hypoxia. The difference in effective between bar-headed geese and low altitude waterfowl was probably even greater than the  83 difference in total ventilation, however, because bar-headed geese had higher tidal volumes and lower breathing frequencies at any given total ventilation during hypoxia (Fig. 3.2). This should increase the ratio of parabronchial ventilation to metabolism in bar-headed geese compared to low altitude waterfowl, and therefore cause the enhanced arterial PO 2 and O2 loading during severe hypoxia.  ! 3.4.7. Implications for high altitude flight During steady flight in normoxia, bar-headed geese and other birds increase their rate of oxygen consumption between 10- and 20-fold (Bernstein, 1987; Butler, 1991; Ward et al., 2002). This appears to be matched by a nearly equivalent increase in ventilation (Bernstein, 1987) such that air convection requirements are approximately constant between rest and flight exercise (at least in steady state). It is unknown whether this is also true of flight in hypoxia, as no previous studies have measured breathing or metabolism under these combined conditions. However, it raises the question of whether these animals can increase ventilation in hypoxia 10-fold to meet resting metabolic demands, and then another 10- to 20-fold to accommodate the metabolic costs of flight. In other words, can the hypoxic ventilatory response be fully sustained during the exercise of flight? The air convection requirement increases in a similar fashion going from rest to exercise during running in bar-headed geese and pekin ducks in both normoxia and hypoxia (Kiley et al., 1985; Fedde et al., 1989), suggesting that hypoxia and exercise normally have additive effects on ventilation. Bar-headed geese may therefore be capable of exceptionally high maximum ventilation rates, but the upper limits of this species’ ventilatory system have yet to be determined.  84 Another set of intriguing questions arises from the fact that breathing frequency is coordinated with wing beat frequency during flight (Boggs, 1997). This coordination is thought to reduce the energetic costs of breathing (Funk et al., 1997), and geese are known to exhibit wing beat/respiration ratios between 1:1 and 4:1 (Butler and Woakes, 1980; Funk et al., 1992; Funk et al., 1993). This raises the reciprocal questions of whether respiratory-locomotor coupling is disrupted by hypoxia or whether the hypoxic ventilatory response is disrupted by respiratory-locomotor coupling. Within this context is the question of how the differences we observed between the breathing pattern responses of bar-headed geese and the other species at rest apply during flight. The data do suggest that altering respiratory drive will alter the entrainment ratio (from 1:1 to 2:1 for instance) (Funk et al., 1993) suggesting that flying birds can adjust their breathing patterns to enhance O2 loading into the blood and still coordinate breathing with wing flapping. Finally, the partial pressure of O2 is reduced at high altitude due to declines in total barometric pressure (hypobaria) rather than reduced FIO 2 . An important, and as of yet unanswered, question is how hypobaria influences breathing and O2 loading in bar-  ! headed geese. Previous studies comparing the responses to normobaric and hypobaric hypoxia suggest that parabronchial ventilation, aerodynamic valving, and O2 loading are largely unaffected by reductions in barometric pressure alone (Shams et al., 1990; Shams and Scheid, 1993). Analogous experiments have not been conducted on bar-headed geese, so it remains to be seen whether pulmonary mechanics or O2 loading are affected by hypobaria in this species.  85 3.4.8. Can phylogenetic history explain the interspecific differences? Bar-headed geese, greylag geese, and ross geese (Anser rossii) form a monophyletic group within the genus Anser (subfamily Anserinae) so greylag geese are a close evolutionary relative of bar-headed geese (Donne-Goussé et al., 2002). Pekin ducks are more distantly related and belong to a neighbouring subfamily (Anatinae). There are undoubtedly many characteristics that have diverged between geese and ducks that can be explained by neutral evolutionary processes, such that greylag geese and bar-headed geese are more alike to one another than to pekin ducks (Garland and Adolph, 1994). With regards to the potential limitation for generating inspiratory airflow in greylag geese, this observation was probably not caused by neutral evolution but is inconsistent with hypoxia adaptation. However, pekin ducks and greylag geese were similar in the majority of their responses to hypoxia, whereas bar-headed geese were often different. This suggests that the enhanced effective ventilation and O2 loading of bar-headed geese in hypoxia were not a product of phylogenetic history, and that these uniquely derived phenotypes may be related to the exceptional hypoxia tolerance of this species. The Himalayan mountains are a formidable barrier to avian migration, and many species that migrate between the northern and southern sides of the range fly around or employ longer routes through riverine valleys (Swan, 1961; Swan, 1970; Javed et al., 2000; Irwin and Irwin, 2005). Bar-headed geese routinely migrate along direct routes over the highest peaks in the Himalayas, an exceptional feat as exemplified by its rarity amongst migrating Asian birds. The extreme nature of this migration, along with the known importance of breathing for O2 transport in hypoxia, strongly suggest that the enhanced effective ventilation and O2 loading of this species are adaptive.  86 3.5. Summary of Chapter •  Control of breathing in bar-headed geese was compared to that in low altitude waterfowl (greylag geese and pekin ducks) by exposing birds to step decreases in inspired O2 under both poikilocapnic (uncontrolled CO2) and isocapnic (with blood CO2 experimentally maintained) conditions.  •  Bar-headed geese breathed substantially more during severe poikilocapnic hypoxia. This was due primarily to an enhanced tidal volume response, which increased arterial PO2 and O2 loading into the blood.  •  There were no differences between species in ventilatory sensitivities to isocapnic hypoxia, the hypoxia-induced changes in blood CO2 tensions or pH, or hypercapnic ventilatory sensitivities.  •  Bar-headed geese maintain higher rates of metabolism (reflected by O2 consumption rate) during hypoxia.  •  Bar-headed geese may be capable of generating higher inspiratory airflows.  87 Table 3.1. Respiratory variables during poikilocapnic hypoxia  FI O 2 Bar-Headed Goose  !  21% O2 12% O2 9% O2 7% O2 5% O2 4% O2  Greylag Goose 21% O2 12% O2 9% O2 7% O2 5% O2  Pekin Duck 21% O2 12% O2 9% O2 7% O2 5% O2  TI  TE  TI/TE  PCL CO 2  2.32 ± 0.13a 2.21 ± 0.11a 1.99 ± 0.09b 1.61 ± 0.05c 0.84 ± 0.11d 0.73 ± 0.05d  3.73 ± 0.25a 3.26 ± 0.27a 2.72 ± 0.24a 1.83 ± 0.10b 0.93 ± 0.12c 0.79 ± 0.04c  0.64 ± 0.05a 0.70 ± 0.03a 0.76 ± 0.04b 0.89 ± 0.04c 0.90 ± 0.02c 0.92 ± 0.05c  41.5 ± 2.4a 31.3 ± 2.3b 23.1 ± 1.7c 16.0 ± 1.1d 7.9 ± 1.1e 6.1 ± 1.3f  2.02 ± 0.28a 1.82 ± 0.27a 1.56 ± 0.12a 1.13 ± 0.09b 0.88 ± 0.13b  2.43 ± 0.40a 2.61 ± 0.43a 2.04 ± 0.24ab 1.26 ± 0.17b 0.83 ± 0.04c  0.86 ± 0.07ab 0.72 ± 0.05a 0.79 ± 0.07ab 0.93 ± 0.07b 1.06 ± 0.11b  39.8 ± 3.2a 29.6 ± 2.4b 22.2 ± 1.5c 15.9 ± 0.6d 10.1 ± 0.8e  1.92 ± 0.22a 1.55 ± 0.12a 1.27 ± 0.10a 1.10 ± 0.10b 0.98 ± 0.10b  3.06 ± 0.26a 2.72 ± 0.26a 1.87 ± 0.24a 1.49 ± 0.20b 1.12 ± 0.15c  0.65 ± 0.10a 0.58 ± 0.04a 0.71 ± 0.05a 0.77 ± 0.04a 0.89 ± 0.03b  31.9 ± 2.4a 25.6 ± 1.7b 18.5 ± 1.3c 13.3 ± 1.0d 7.7 ± 1.1e  !  FIO 2 , inspired O2 fraction (%); TI, inspiratory time (s); TE, expiratory time (s); PCL CO 2 , clavicular air sac CO2 tension (torr). Statistics were performed within each species only. For each parameter treatments with different letters are significantly different (P<0.05). !  !  88 Table 3.2. Blood gas variables during poikilocapnic hypoxia  Pv O 2  Pv CO 2  pHv  [HCO3-]v  Cv O 2  40.9 ± 4.3a 31.0 ± 2.4ab ! 24.2 ± 1.6bc 22.3 ± 1.7c 16.5 ± 1.7c 16.6 ± 4.3c  32.6 ± 1.3a 28.3 ± 1.8ab 24.6 ± 1.5bc 19.4 ± 1.5cd 14.4 ± 1.4 de 11.0 ± 2.0e  7.36 ± 0.04a 7.33 ± 0.08a 7.37 ± 0.08a 7.36 ± 0.11a 7.23 ± 0.12a 7.26 ± 0.11a  17.0 ± 1.3a 14.3 ± 2.1b ! 13.7 ± 1.8b 11.4 ± 2.8c 6.4 ± 1.9d 5.3 ± 2.4d  2.60 ± 0.37a 2.27 ± 0.29a 1.15 ± 0.30b 1.10 ± 0.22b 0.69 ± 0.30bc 0.16 ± 0.07c  4.96 ± 0.15a 4.60 ± 0.16b 4.02 ± 0.14c 2.95 ± 0.29d 1.54 ± 0.22e  55.8 ± 3.8a 38.1 ± 1.4b 31.8 ± 0.9bc 25.8 ± 2.7c 21.6 ± 3.0c  31.3 ± 1.3a 27.2 ± 1.4ab 21.7 ± 0.8b 19.3 ± 1.2bc 15.7 ± 1.2c  7.37 ± 0.06a 7.35 ± 0.07a 7.32 ± 0.10a 7.36 ± 0.04a 7.19 ± 0.10a  17.6 ± 3.8a 14.4 ± 2.6b 11.0 ± 2.9c 10.1 ± 1.2d 5.0 ± 2.4e  3.23 ± 0.30a 2.85 ± 0.12a 1.86 ± 0.25b 1.28 ± 0.26b 0.38 ± 0.15c  4.83 ± 0.32a 3.83 ± 0.29b 2.94 ± 0.25c 2.28 ± 0.21d 1.97 ± 0.11e  60.8 ± 1.1a 36.8 ± 1.8ab 28.7 ± 1.2bc 24.4 ± 1.2c 21.5 ± 0.7c  34.2 ± 1.7a 27.5 ± 1.4b 22.4 ± 1.2c 18.7 ± 1.1c 20.8 ± 5.6c  7.41 ± 0.06a 7.44 ± 0.03a 7.34 ± 0.11a 7.32 ± 0.14a 7.23 ± 0.18a  20.3 ± 3.1a 17.1 ± 2.5b 11.8 ± 2.1c 10.1 ± 1.9d 6.7 ± 2.2e  3.40 ± 0.25a 2.22 ± 0.25b 1.43 ± 0.24c 1.23 ± 0.14c 0.89 ± 0.10d  FI O 2  Ca O 2  Bar-Headed Goose  !  21% O2 12% O2 ! 9% O2 7% O2 5% O2 4% O2  4.57 ± 0.20a 4.54 ± 0.30a ! 3.47 ± 0.36b 3.18 ± 0.31c 2.31 ± 0.20d 1.10 ± 0.13e  Greylag Goose 21% O2 12% O2 9% O2 7% O2 5% O2  Pekin Duck 21% O2 12% O2 9% O2 7% O2 5% O2  !  FIO 2 , inspired O2 fraction (%); Ca O 2 , arterial O2 content (mM); Pv O 2 , venous O2 tension (torr); Pv CO 2 , venous CO2 tension (torr); pHv, venous pH; [HCO3-]v, venous bicarbonate concentration (mM); Cv O 2 , venous O2 content (mM). Statistics were performed within each species only. For each letters are significantly different ! parameter treatments with different ! ! (P<0.05). !  89 Table 3.3. Respiratory variables during isocapnic hypoxia TI  TE  TI/TE  PCL CO 2  2.35 ± 0.16a 2.09 ± 0.11b 1.68 ± 0.10c 1.21 ± 0.11d 0.87 ± 0.06e 0.92 ± 0.08e  3.48 ± 0.25a 2.54 ± 0.18b 1.74 ± 0.11c 1.42 ± 0.12cd 1.25 ± 0.19cd 1.13 ± 0.05d  0.69 ± 0.06a 0.83 ± 0.05ab 0.97 ± 0.04b 0.86 ± 0.02 ab 0.73 ± 0.05 a 0.82 ± 0.07 ab  36.7 ± 4.4a 34.5 ± 4.2a 32.4 ± 3.2a 37.3 ± 2.0a 37.1 ± 1.1a 37.1 ± 2.4a  2.32 ± 0.16a 1.93 ± 0.21a 1.31 ± 0.23b 1.04 ± 0.09b 1.02 ± 0.12b  3.04 ± 0.22a 2.05 ± 0.24b 1.36 ± 0.22c 1.02 ± 0.09c 0.96 ± 0.09c  0.79 ± 0.07a 0.96 ± 0.05b 0.96 ± 0.03b 1.02 ± 0.05b 1.06 ± 0.08b  42.0 ± 1.9a 39.7 ± 1.5ab 38.4 ± 1.7ab 36.1 ± 1.2bc 34.4 ± 1.1c  2.07 ± 0.11a 1.83 ± 0.10a 1.29 ± 0.04b 0.81 ± 0.04c 0.87 ± 0.04c  3.24 ± 0.29a 2.11 ± 0.10b 1.50 ± 0.07c 0.86 ± 0.08d 0.94 ± 0.08d  0.67 ± 0.08a 0.73 ± 0.11ab 0.87 ± 0.03ab 0.96 ± 0.04b 0.94 ± 0.04b  31.1 ± 1.8a 28.7 ± 1.9b 28.8 ± 1.9b 29.3 ± 1.9b 30.7 ± 2.2b  FI O 2 Bar-Headed Goose  !  21% O2 12% O2 9% O2 7% O2 5% O2 4% O2  Greylag Goose 21% O2 12% O2 9% O2 7% O2 5% O2  Pekin Duck 21% O2 12% O2 9% O2 7% O2 5% O2  !  FIO 2 , inspired O2 fraction (%); TI, inspiratory time (s); TE, expiratory time (s); PCL CO 2 , clavicular air sac CO2 tension (torr). Statistics were performed within each species only. For each parameter treatments with different letters are significantly different (P<0.05). !  !  90 Table 3.4. Blood gas variables during isocapnic hypoxia  Pv O 2  Pv CO 2  pHv  [HCO3-]v  Cv O 2  38.9 ± 2.8a 42.6 ± 5.7a ! 37.9 ± 1.3a 32.4 ± 2.5a 17.6 ± 1.7b 14.2 ± 2.1b  31.8 ± 1.7a 35.9 ± 2.3a 38.3 ± 2.6a 36.9 ± 2.7a 42.0 ± 1.0a 36.4 ± 1.8a  7.37 ± 0.05a 7.36 ± 0.06a 7.24 ± 0.10ab 7.21 ± 0.07ab 6.99 ± 0.09b 6.93 ± 0.10b  16.9 ± 1.4a 18.7 ± 1.6a ! 16.0 ± 3.1a 13.4 ± 1.2ab 9.5 ± 1.5b 6.9 ± 0.8b  2.19 ± 0.28a 1.99 ± 0.25a 1.45 ± 0.47ab 1.95 ± 0.18a 0.47 ± 0.17b 0.12 ± 0.06b  5.62 ± 0.26a 5.13 ± 0.18a 4.05 ± 0.34b 3.07 ± 0.19c 1.61 ± 0.40d  57.7 ± 4.5a 47.8 ± 2.2b 40.2 ± 2.5b 24.2 ± 3.8c 19.6 ± 3.7c  31.5 ± 1.6a 29.4 ± 1.5a 33.9 ± 1.8a 37.0 ± 2.8a 31.7 ± 2.6a  7.39 ± 0.05a 7.38 ± 0.04a 7.24 ± 0.03ab 7.12 ± 0.02b 7.01 ± 0.01b  17.7 ± 2.7a 15.8 ± 0.7ab 13.2 ± 0.4ab 11.1 ± 1.2b 7.2 ± 0.7b  2.86 ± 0.20a 2.76 ± 0.16a 1.80 ± 0.38b 0.63 ± 0.16c 0.13 ± 0.08c  5.04 ± 0.18a 4.17 ± 0.15b 3.82 ± 0.13b 2.46 ± 0.12c 1.77 ± 0.22d  57.0 ± 2.6a 47.7 ± 2.9b 43.7 ± 1.9b 34.1 ± 3.6c 21.8 ± 3.3d  33.9 ± 1.1a 36.8 ± 2.0ab 38.3 ± 1.2ab 43.5 ± 2.3b 44.8 ± 1.0b  7.48 ± 0.01a 7.36 ± 0.07a 7.40 ± 0.05a 7.25 ± 0.03ab 7.06 ± 0.08b  22.8 ± 1.4a 29.0 ± 2.8a 22.1 ± 2.9a 17.0 ± 2.5ab 11.7 ± 1.8b  3.33 ± 0.25a 2.75 ± 0.43ab 2.23 ± 0.21b 1.35 ± 0.12c 0.31 ± 0.11d  FI O 2  Ca O 2  Bar-Headed Goose  !  21% O2 12% O2 ! 9% O2 7% O2 5% O2 4% O2  4.86 ± 0.15a 4.60 ± 0.28a ! 3.93 ± 0.36b 3.55 ± 0.36c 2.38 ± 0.26d 1.21 ± 0.14e  Greylag Goose 21% O2 12% O2 9% O2 7% O2 5% O2  Pekin Duck 21% O2 12% O2 9% O2 7% O2 5% O2  !  FIO 2 , inspired O2 fraction (%); Ca O 2 , arterial O2 content (mM); Pv O 2 , venous O2 tension (torr); Pv CO 2 , venous CO2 tension (torr); pHv, venous pH; [HCO3-]v, venous bicarbonate concentration (mM); Cv O 2 , venous O2 content (mM). Statistics were performed within each species only. For each!parameter treatments with different ! letters are significantly different ! (P<0.05). !  91 Table 3.5. Blood gas variables during hypercapnia (5% inspired CO2) at moderate hypoxia (12% inspired O2)  Pa O 2  [HCO3-]a  Ca O 2  Pv O 2  Pv CO 2  pHv  [HCO3-]v  Cv O 2  16.7 ± 1.8  4.00 ± 0.53  36.6 ± 3.8  42.7 ± 2.8  7.20 ± 0.09  15.7 ± 2.3  2.65 ± 0.58  !  !  !  10.5 ± 1.8  5.12 ± 0.23  55.4 ± 5.7  37.1 ± 2.0  7.12 ± 0.11  11.8 ± 3.0  2.62 ± 0.37  20.0 ± 1.4  4.57 ± 0.16  61.4 ± 3.2  46.8 ± 1.1  7.32 ± 0.06  22.7 ± 3.0  3.37 ± 0.15  Bar-Headed Goose 75.5 ± 2.2  !  Greylag Goose 68.1 ± 1.8  !  Pekin Duck 73.4 ± 3.5  !  Pa O 2 , arterial O2 tension (torr); [HCO3-]a, arterial bicarbonate concentration (mM); Ca O 2 , arterial O2 content (mM); Pv O 2 , venous O2 tension (torr); Pv CO 2 , venous CO2 tension (torr); pHv, venous pH; [HCO3-]v, venous bicarbonate concentration (mM); Cv O 2 , venous O2 content (mM). ! !  ! !  92  !  Fig. 3.1. Total ventilation (A) was higher in bar-headed geese during severe poikilocapnic (uncontrolled CO2) hypoxia than in both greylag geese and pekin ducks. This resulted in higher arterial O2 tensions ( PO 2 ) (B) during reduced inspired O2 fraction ( FO 2 ). The grey dashed line in (B) represents the PO 2 of inspired air. The response of total ventilation to reductions in arterial PO 2 (C) was greater in bar-headed geese. For each species, total ventilation increased!significantly and arterial PO2 decreased significantly after each step reduction of inspired FO! (P<0.05). * Significant difference between bar2 headed geese and both low ! altitude species (P<0.05).  !  93  Fig. 3.2. The ventilatory response of bar-headed geese to poikilocapnic (uncontrolled CO2) hypoxia involved a different breathing pattern than that of both greylag geese and pekin ducks: for any total ventilation, bar-headed geese had a higher tidal volume and lower breathing frequency (right-shifted curve). Grey lines represent breathing frequency (fR) isopleths. * Significant difference in tidal volume between bar-headed geese and both low altitude species.  Significant difference in tidal volume between low altitude species (P<0.05).  94  Fig. 3.3. Total ventilation (A) was not higher in bar-headed geese than in pekin ducks during isocapnic (constant arterial CO2) hypoxia, but greylag geese had a reduced ventilatory response to isocapnic hypoxia. There were no differences in arterial O2 tension ( PO 2 ) (B) at any level of inspired O2 fraction ( FO 2 ). The grey dashed line in (B) represents the PO 2 of inspired air. * Significant difference between bar-headed geese and both low altitude species.  Significant difference between low altitude species (P<0.05).  !  ! !  95  Fig. 3.4. The change in breathing pattern during isocapnic (constant arterial CO2) hypoxia was similar in bar-headed geese, greylag geese, and pekin ducks, except in greylag geese during severe hypoxia. Grey lines represent breathing frequency (fR) isopleths.  Significant difference in tidal volume between low altitude species (P<0.05).  96  Fig. 3.5. Acid-base regulation was similar during both poikilocapnic (uncontrolled CO2) and isocapnic (constant arterial CO2) hypoxia in all species. (A) During poikilocapnic hypoxia, no statistically significant pH imbalance occurred in arterial blood. (B) During isocapnic hypoxia, all species experienced a metabolic acidosis of arterial blood at severe levels of hypoxia.  97  Fig. 3.6. Total ventilation (A,B), breathing frequency (C,D), and tidal volume (E,F) as a function of arterial CO2 tension ( PCO 2 ) (A,C,E) or arterial pH (B,D,F) in bar-headed geese, greylag geese, and pekin ducks.  !  98  Fig. 3.7. Total ventilation relative to (A) peak inspiratory flow (PIF) and (B) peak expiratory flow (PEF) in bar-headed geese, greylag geese, and pekin ducks. (A) There was a tight correlation (R2=0.97) between ventilation and PIF in a regression including both poikilocapnic and isocapnic data from all species (symbols for isocapnic data are marked with a white or black +). (B) There was also a tight correlation (R2=0.96) between ventilation and PEF, but greylag geese fell off this regression line in severe isocapnic hypoxia (at 7% and 5% FIO 2 ). Dashed vertical lines represent the maximum observed peak inspiratory and expiratory flows in greylag geese and dashed horizontal lines represent the total ventilations predicted by the regression equations from these data. !  99  Fig. 3.8. The responses of oxygen consumption rate to (A) poikilocapnic (uncontrolled CO2) and (B) isocapnic (constant arterial CO2) hypoxia were different between species. (C) There were small differences between greylag geese and the other two species during poikilocapnic hypoxia when total ventilation was expressed relative to oxygen consumption rate (air convection requirement, also called the ventilatory equivalent). (D) During isocapnic hypoxia, air convection requirement was lower in bar-headed geese at 7% inspired O2. * Significant difference between bar-headed geese and both low altitude species.  Significant difference between low altitude species (P<0.05).  100 3.6. References Barros, R. C. H., Branco, L. G. S. and Cárnio, E. C. (2006). 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Y. (2001). The crystal structure of bar-headed goose hemoglobin in deoxy form: The allosteric mechanism of a hemoglobin species with high oxygen affinity. J. Mol. Biol. 313, 123-137. Manville, R. H. (1963). Altitude record for mallard. Wilson Bulletin 75, 92.  105 Milsom, W. K. (1991). Intermittent breathing in vertebrates. Annu. Rev. Physiol. 53, 87105. Milsom, W. K., Abe, A. S., Andrade, D. V. and Tattersall, G. J. (2004). Evolutionary trends in airway CO2/H+ chemoreception. Respir. Physiol. Neurobiol. 144, 191202. Monge, C. and León-Velarde, F. (1991). Physiological adaptation to high altitude: oxygen transport in mammals and birds. Physiol. Rev. 71, 1135-1172. Petschow, D., Würdinger, I., Baumann, R., Duhm, J., Braunitzer, G. and Bauer, C. (1977). Causes of high blood O2 affinity of animals living at high altitude. J. Appl. Physiol. 42, 139-143. Powell, F. L., Dwinell, M. R. and Aaron, E. A. (2000). Measuring ventilatory acclimatization to hypoxia: comparative aspects. Respir. 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Respir. Physiol. 91, 155-163. Swan, L. W. (1961). The ecology of the high Himalayas. Sci. Am. 205, 68-78. Swan, L. W. (1970). Goose of the Himalayas. Nat. Hist. 70, 68-75. Tucker, V. A. (1967). Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23, 410-414. Tucker, V. A. (1968). Respiratory physiology of house sparrows in relation to highaltitude flight. J. Exp. Biol. 48, 55-66. van Nice, P., Black, C. P. and Tenney, S. M. (1980). A comparative study of ventilatory responses to hypoxia with reference to hemoglobin O2-affinity in llama, cat, rat, duck and goose. Comp. Biochem. Physiol. A. Physiol. 66A, 347350. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weber, R. E., Jessen, T. H., Malte, H. and Tame, J. (1993). Mutant hemoglobins (α119-Ala and β55-Ser): functions related to high-altitude respiration in geese. J. Appl. Physiol. 75, 2646-2655. Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA, USA: Harvard University Press.  107 Weibel, E. R., Taylor, C. R., Weber, J. M., Vock, R., Roberts, T. J. and Hoppeler, H. (1996). Design of the oxygen and substrate pathways. VII. Different structural limits for oxygen and substrate supply to muscle mitochondria. J. Exp. Biol. 199, 1699-1709. Weinstein, Y., Bernstein, M. H., Bickler, P. E., Gonzales, D. V., Samaniego, F. C. and Escobedo, M. A. (1985). Blood respiratory properties in pigeons at high altitudes: effects of acclimation. Am. J. Physiol. 249, R765-R775. Withers, P. C. (1977). Measurement of Vo2, Vco2, and evaporative water loss with a flow-through mask. J. Appl. Physiol. 42, 120-123. Wu, T. and Kayser, B. (2006). High altitude adaptation in Tibetans. High Alt. Med. Biol. 7, 193-208. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493.  108  4. BODY TEMPERATURE DEPRESSION AND PERIPHERAL HEAT LOSS DURING HYPOXIA ARE REDUCED IN BAR-HEADED GEESE3  4.1. Introduction In order to survive in hypoxic environments, animals must continue to balance cellular oxygen supply and demand. In many hypoxia-adapted animals the capacity to maintain this balance is enhanced by a combination of physiological and biochemical responses that increase O2 supply and/or reduce O2 demand (Hochachka, 1985). Coordinated metabolic depression appears to be the most pervasive strategy for reducing O2 demand and surviving in severe hypoxia, and occurs through concerted responses by individual cells and whole physiological control systems (Hochachka et al., 1996; Guppy and Withers, 1999; Boutilier, 2001). Reductions in body temperature (Tb) should facilitate metabolic depression during hypoxia by reducing temperature-dependent O2 demands. Tb depression is believed to result from a decrease in Tb setpoint that is regulated by thermoregulatory control regions in the hypothalamus and/or spinal cord (Crawshaw et al., 1985; Simon et al., 1986; Wood and Gonzales, 1996; Bicego et al., 2007), presumably by altering the balance between metabolic heat generation and heat loss. Peripheral heat loss is regulated by controlling blood flow to specific regions of the body surface, which alters surface temperature and thus the temperature differential driving heat dissipation (Klir and Heath, 1994; Mauck et  3  A version of this chapter has been published: Scott, G. R., Cadena, V., Tattersall, G. J., and Milsom, W. K. (2007). Body temperature depression and peripheral heat loss accompany the metabolic and ventilatory responses to hypoxia in low and high altitude birds. J. Exp. Biol. 211, 1326-1335.  109 al., 2003). These ‘thermal windows’ are typically poorly insulated, and include the ears, feet, and nose of mammals (Klir and Heath, 1992), or the bill and feet of birds (Kilgore and Schmidt-Nielsen, 1975; Baudinette et al., 1976; Hagan and Heath, 1980). Despite its known importance for thermoregulation in general, the role and control of peripheral heat loss from thermal windows during hypoxic Tb depression has received very little attention (but see Tattersall and Milsom, 2003). In addition to reductions in O2 demand via metabolic depression, O2 supply during hypoxia can be improved. The O2 transport pathway from environment to mitochondria has several components, including ventilation, pulmonary diffusion, circulation, and tissue diffusion (Weibel, 1984). Control of this O2 supply pathway is well understood in vertebrates (Bouverot, 1978; Taylor et al., 1999), but the relative importance of alterations in O2 supply versus O2 demand during hypoxia is unclear. Hypoxia adaptation could enhance the capacity for either O2 transport or metabolic depression, depending on the selective pressure driving hypoxia tolerance. For example, the bar-headed goose (Anser indicus) flies over the Himalayas on its migratory route between South and Central Asia, at altitudes of up to 9000m where O2 pressures are 5fold lower than at sea level (Swan, 1970; Javed et al., 2000). O2 consumption must concurrently increase 10- to 20-fold above resting levels in this species to sustain flight (Ward et al., 2002). Metabolic depression is clearly not feasible in bar-headed geese while flying in hypoxia, and it is conceivable that this species should minimize heat loss and Tb depression during hypoxia. Little comparative data exist concerning the use of Tb depression as a strategy for matching O2 supply and demand. In the present study we investigate heat loss and Tb  110 depression during hypoxia in birds, and examine the relationship between these thermoregulatory variables and the metabolic and ventilatory responses to hypoxia. Barheaded geese are compared to two low altitude waterfowl species, the closely related greylag goose (Anser anser) and the more distantly related pekin duck (Anas platyrynchos). We hypothesized that thermal windows would be used to help depress Tb in birds during hypoxia, and that the degree of Tb depression would be inversely related to the capacity for maintaining O2 supply. We also hypothesized that bar-headed geese would minimize metabolic depression in hypoxia, and would therefore reduce heat loss and Tb depression compared to low altitude birds.  4.2. Materials and Methods 4.2.1. Animals Experiments were performed on seven bar-headed geese (2.1-3.1 kg), four greylag geese (3.8-4.8 kg), and 13 pekin ducks (2.7-3.9 kg). All animals were bred and raised at sea level, either at the Animal Care Facility of the University of British Columbia (UBC) or by local suppliers. Animals were housed outdoors at UBC, and were fasted for one day before each experiment but continued to receive free access to water. All animal care and experimentation was conducted according to UBC animal care protocol #A04-1013.  4.2.2. Surgical procedures Six of the ducks were bilaterally vagotomized to determine the responses of waterfowl to hypoxia in the absence of stimulation by peripheral chemoreceptor  111 afferents. A surgical plane of anaesthesia was maintained with isoflurane, and local analgesia (Lidocaine) was applied to the site of incision. Both vagi were isolated in the upper region of the neck and then cut, after which the skin was sutured closed.  4.2.3. Measurements Body plethysmography was used to measure breathing, as described previously (Dodd and Milsom, 1987; Dodd et al., 2007; Scott and Milsom, 2007; Chapter 3). The plethysmograph consisted of two parts, a body compartment and a head compartment, separated from each other by a flexible latex collar. The head compartment was used to administer specific gas mixtures, using calibrated N2 and O2 flowmeters, which were monitored with an oxygen analyzer (Raytech, Vancouver, BC, Canada). Changes in body volume (due to ventilatory movements) were detected with a pneumotachograph (Fleisch, Richmond, VA, USA) connected to a differential pressure transducer (Validyne, Northridge, CA, USA) to yield a measurement of ventilatory flow. Ventilatory flow, Tb (measured with a flexible rectal thermometer), fractional O2 composition of gas entering and leaving the head compartment, and airflow through the head compartment were recorded using Windaq data acquisition software (Dataq Instruments, Akron, OH, USA). Bill surface temperatures (Tbill) were measured using a portable infrared thermal imaging camera (Model 7515; Mikron Instruments, Oakland, NJ, USA). The camera was mounted directly above the head compartment of the plethysmograph, which was sealed with transparent polyvinylidene chloride (PVDC) film (Saran Wrap®, S.C. Johnson and Son, Brantford, ON, Canada) to provide a window with minimal absorption for infrared  112 radiation to pass. Commercial software (MikroSpec RT; Mikron Instruments) was used to determine average Tbill from thermal images. Data were corrected for the slight decrease in detected temperature (~0.2 oC) that was caused by heat absorption by the PVDC film.  4.2.4. Experimental protocols For all experiments on intact birds, the animals were placed in the water-jacketed plethysmograph (held between 11-13oC) and allowed 60-90 min to adjust to their surroundings. This temperature is well within the thermoneutral zone of all species (V. Cadena, G. R. Scott, W. K. Milsom, and G. J. Tattersall, unpublished), and birds can be held at this temperature for several hours in normoxia and exhibit no significant changes in Tb, metabolism, or breathing. In the first experiment (stepwise hypoxia), seven barheaded geese, seven pekin ducks, and four greylag geese were used. Birds were exposed to progressive step reductions in the fractional O2 composition of inspired gas (FI O 2 : 21%, 12%, 9%, 7%, and in bar-headed geese only, 5%) with each step lasting 15 min.  ! The most severe level of hypoxia was followed by a 20 min normoxic recovery period. In the second experiment (prolonged hypoxia), birds (five bar-headed geese and four greylag geese) were exposed to 9% O2 for 60 min, followed by a 30 min recovery in normoxia. At the end of each experiment the bird was returned to the Animal Care Facility. The first experiment was also performed on vagotomized ducks. However, many bird species do not tolerate chronic bilateral vagotomy (Fedde and Burger, 1963), so these ducks were placed directly into the experimental apparatus after surgery to  113 minimize the chronic effects of vagotomy. Birds were kept in normoxia for 60-90 min to adjust to their surroundings and allow time for the residual effects of isoflurane anaesthesia to diminish. Three vagotomized ducks were then exposed to the stepwise hypoxia protocol described above. Three other vagotomized ducks were exposed to normoxia for the same duration to control for the effects of vagotomy and anaesthesia alone (in absence of hypoxia). Birds were sacrificed with an intramuscular overdose of sodium pentobarbital after completing the protocol.  4.2.5. Data and statistical analyses All data acquired in Windaq were analyzed using a specially written Matlab (version 7, Mathworks) program. Inspiratory tidal volume (VT), breathing frequency (fR), •  total ventilation (product of VT and fR), oxygen consumption rate ( VO 2 ), and air •  convection requirement (quotient of total ventilation and VO 2 ) were determined as ! previously described (Chapter 3). Due to the effect of temperature on metabolism, we  ! • also calculated what mean VO 2 would have been in absence of hypoxic Tb depression •  ( VO 2 ,corrected ).  !  •  •  VO 2 ,corrected = VO 2 " Q10 #Tb !  10  (4.1)  In equation 4.1, ∆Tb is the magnitude of body temperature depression (using values measured after 5, 10,!or 15 min at each FI O 2 , as appropriate), and we made these calculations assuming Q10 values of both 2 and 3.  ! ± standard error. Two factor (species and time) Data are reported as means repeated measures analysis of variance and Holm-Sidak post-hoc tests were used to  114 determine statistical significance within and between species (using a significance level of p<0.05). Least squares linear regression was used to assess the relationships between •  arterial O2 content (using previously collected data from Chapter 3) and Tb, Tbill, or VO 2 . Statistical tests were performed using Sigmastat software (version 4, Systat Software Inc., San Jose, CA, USA).  !  4.3. Results 4.3.1. Thermoregulatory responses to stepwise hypoxia All three species depressed body temperature in response to step reductions in inspired O2, and the extent of Tb depression increased with the severity of hypoxia (Fig. 4.1A). Initial body temperatures were not significantly different between species (barheaded geese, 41.3 ± 0.2 oC; greylag geese, 41.6 ± 0.2 oC; and pekin ducks, 41.9 ± 0.3 o  C; p>0.05). Unlike the low altitude species however, whose initial reductions in Tb  occurred after 9 min (pekin ducks) and 14 min (greylag geese) of 12% inspired O2 (FI O 2 ), bar-headed geese did not significantly reduce Tb until 10 min into 9% FI O 2 . Furthermore,  ! bar-headed geese experienced less body temperature depression than both low altitude species during 7% FI O 2 (Fig. 4.1A). Both low altitude species ! had reduced Tb by more than 1oC during 7% FI O 2 , but the same degree of Tb depression did not occur in bar-  ! headed geese until the later stages of 5% FI O 2 . We did not expose greylag geese or pekin ! ducks to this lowest level of hypoxia since it is not well tolerated by these species, unlike ! much deeper levels of hypoxia (Black and Tenney, bar-headed geese which can survive 1980; Scott and Milsom, 2007; Chapter 3).  115 All species increased bill surface temperature (Tbill) in response to stepwise hypoxia (Figs. 4.2 and 4.3A). Bill warming tended to begin at the end or along the midline of the bill, then spread over the rest of the bill surface. The statistically significant onset of bill warming occurred after 4 min and 3 min of 9% FI O 2 in greylag geese and pekin ducks, respectively, but not until 4 min of 7% FI O 2 in bar-headed geese.  ! As a result, bar-headed geese demonstrated significantly less bill warming during the majority of exposure to 9% FI O 2 (Fig. 4.3A). There was!a trend towards bill warming in greylag geese and pekin ducks at 12% FI O 2 , and after 6-7 min of 12% FI O 2 Tbill was  ! higher in greylag geese than in bar-headed geese. Initial Tbill values were similar between species (bar-headed geese, 25.3!± 0.5 oC; greylag geese, 27.0 ±!1.5 oC; and pekin ducks, 26.8 ± 0.4 oC; p>0.05). Normoxic recovery of Tb after severe hypoxia was slightly different in pekin ducks than in either goose species (Fig. 4.1B). Both geese started recovering Tb within 20 min, but no significant recovery occurred in ducks. Consistent with this difference, barheaded geese and greylag geese immediately reduced Tbill with the onset of normoxia (thus favouring heat retention), but in pekin ducks there was not an immediate reduction (Fig. 4.3B). After this initial reduction, Tbill generally continued to decline throughout recovery. Greylag geese exhibited the greatest Tb recovery during the 20 min recovery, possibly due to their slightly larger size (which should favour heat retention).  4.3.2. Metabolic responses to stepwise hypoxia Oxygen consumption rates were similar between species in normoxia, and generally increased with each successive decrease in FI O 2 (Fig. 4.4A). The duration of  !  116 exposure to each level of hypoxia (assessed at 5, 10, and 15 min) did not alter the hypoxic metabolic responses. Bar-headed geese generally maintained higher metabolic rates during hypoxia: metabolism in this species was higher than greylag geese for all levels of hypoxia, and higher than both low altitude species during 7% FI O 2 . However, the differences in metabolism could not be entirely explained by differences in Tb. After  ! correcting oxygen consumption rates for the differences in Tb depression between species, using Q10 values of either 2 or 3 (short or long dashed lines, respectively, in Fig. 4.4A), the mean rates were still elevated in bar-headed geese compared to the low altitude species. Oxygen consumption rates recovered to pre-hypoxia levels rapidly in all species after they were returned from hypoxia to normoxia (Fig. 4.4B) (the first 2 min of recovery could not be measured due to methodological issues). Even though metabolism during hypoxia was different between species, these differences were abolished after 4 min of recovery. Small changes in metabolism also appeared to occur in all species throughout recovery.  4.3.3. Ventilatory responses to stepwise hypoxia Breathing increased in all species in response to stepwise poikilocapnic (uncontrolled CO2) hypoxia, due to increases in both tidal volume and breathing frequency (Fig. 4.5A). As the severity of hypoxia increased, time-dependent changes in the ventilatory responses became apparent in the low altitude species. In greylag geese breathing frequency declined over time at each FI O 2 , whereas in pekin ducks tidal volume decreased over time at 7% FI O 2 . These changes were partially offset by changes in tidal  ! !  117 volume or breathing frequency, respectively, so total ventilation changed only slightly with duration. Unlike the low altitude species however, the ventilatory response of barheaded geese to poikilocapnic hypoxia was extremely stable. Bar-headed geese also exhibited different breathing patterns than the low altitude species, generally having higher tidal volumes and lower breathing frequencies. Total ventilation was generally matched to metabolism in all species, because air convection requirements (quotient of total ventilation and oxygen consumption rate) changed very little during exposure to moderate levels of hypoxia (Fig. 4.5A). At more severe levels of hypoxia air convection requirements increased in bar-headed geese and greylag geese (5% and 7% FI O 2 , respectively). All breathing variables generally returned to pre-hypoxic levels within 20 min of normoxic recovery (Fig. 4.5B).  ! 4.3.4. Responses to prolonged hypoxia During prolonged hypoxia (9% FI O 2 ), the greatest changes in body temperature, metabolism, and breathing occurred during the first 15-20 min of exposure (Fig. 4.6).  ! throughout 60 min of hypoxia in both bar-headed geese Slight reductions in Tb continued and greylag geese, but appeared to approach a stable value (at least in bar-headed geese). Species differed in the overall Tb response to hypoxia (p<0.05 for species×time interaction), and greylag geese tended to exhibit more profound reductions in Tb; however, both species recovered Tb at similar rates after being returned to normoxia. Oxygen consumption increased immediately in bar-headed geese, was sustained for the duration of hypoxia, and then returned rapidly to control levels during recovery; curiously, this increase in metabolism was less than the increase during stepwise hypoxia  118 (Fig. 4.4 versus Fig. 4.6). O2 consumption appeared to increase immediately in greylag geese as well (although this was not significant until 20 min), but the recovery occurred more slowly. Breathing increased immediately in response to 9% O2 in bar-headed geese, and was very stable throughout prolonged hypoxia. Bar-headed geese breathed substantially more than greylag geese, particularly early in hypoxia exposure, as the initial (measured after 5 min) increase in breathing was larger in bar-headed geese (1.6fold) than in greylag geese (1.3-fold) (Fig. 4.6). However, breathing increased progressively over time in greylag geese due to increases in tidal volume (Table 4.1). The higher total ventilation in bar-headed geese was due to both an overall higher tidal volume and a more pronounced hypoxic breathing frequency response (Table 4.1). Ventilation was well matched to metabolism: there were only small insignificant changes in air convection requirements (Table 4.1).  4.3.5. Interactions of O2 loading with thermoregulation and metabolism When body temperature and bill temperature during stepwise hypoxia were expressed as a function of the O2 content in arterial blood (from Chapter 3; Scott and Milsom, 2007), instead of time (which allows comparison of different species at each inspired O2), differences between bar-headed geese and the low altitude species were much less prominent (Fig. 4.7). Body temperature fell by approximately 0.5 oC for every 1 mM fall in arterial O2 content in all three species (see Fig. 4.7 legend) (Fig. 4.7A). Bill temperature increased by 2-2.5 oC for every 1 mM fall in O2 content in bar-headed geese and pekin ducks (Fig. 4.7B). Although Tbill in greylag geese increased less as a function of O2 content overall (~1 oC/mM), this species was similar to the other two if the deepest  119 level of hypoxia is excluded (~2 oC/mM). In contrast to Tb and Tbill, the relationships between O2 consumption rates and arterial O2 content were different between species (Fig. 4.7C). Metabolism increased the most in bar-headed geese as O2 content fell, followed by pekin ducks, then greylag geese.  4.3.6. Effects of vagotomy on the responses to stepwise hypoxia Removing vagal chemoafferent input to the central nervous system did not abolish body temperature depression during stepwise hypoxia in pekin ducks. Ducks that were bilaterally vagotomized reduced Tb more than intact ducks during hypoxia, such that by the end of hypoxia exposure Tb fell by greater than 2 oC in these birds (Fig. 4.8A). This contrasted with the effect of vagotomy on bill surface temperatures during hypoxia. Vagotomized ducks did not change Tbill during hypoxia, unlike the increase in intact ducks, though there appeared to be a slight decrease in Tbill during the most severe hypoxia (Fig. 4.8B). These results were not non-specific effects of vagotomy, because Tb and Tbill were constant in time-matched vagotomized normoxic controls (Fig. 4.8). Vagotomy attenuated the ventilatory responses to hypoxia (Table 4.2). Although ventilatory responses appeared to be greater in hypoxic vagotomized ducks, total ventilation tended to increase in both hypoxic ducks and normoxic control ducks over time, and these groups were statistically indistinguishable. Interestingly, vagotomized ducks exposed to the deepest level of hypoxia reduced oxygen consumption rates slightly compared to pre-hypoxia controls (Table 4.2).  120 4.4. Discussion Body temperature depression occurs across all vertebrate classes during hypoxia, and is believed to reduce O2 demands and thus help balance O2 supply and demand (Wood and Gonzales, 1996; Bicego et al., 2007). In the present study we show that Tb depression (Figs. 4.1 and 4.6) during hypoxia occurs in concert with increases in the surface temperature of thermal windows in the bill (Figs. 4.2 and 4.3), supporting the idea that hypoxia leads to specific physiological adjustments that reduce the Tb setpoint. Hypoxic Tb depression also appeared to relate inversely to O2 supply: Tb depression and bill warming occurred at lower inspired O2 in bar-headed geese than in low altitude waterfowl, but this could be explained by higher blood O2 loading in this species (Fig. 4.7). This ability of bar-headed geese to minimize the depressive effects of hypoxia on Tb and metabolism is undoubtedly essential for maintaining the high metabolic rates necessary for flight at high altitude, and suggests that evolutionary changes that enhance O2 supply are important for some species in hypoxia.  4.4.1. Hypoxic responses of waterfowl The generality of hypoxic Tb depression across bird species (e.g., Novoa et al., 1991; Kilgore et al., 2007) and other vertebrate classes suggests that the mechanisms responsible for Tb depression are widespread; however, responses of the thermoregulatory control system to hypoxia are still poorly understood (Bicego et al., 2007). Previous studies in mammals indicate that hypothalamic O2 sensors may initiate Tb depression during hypoxia, and can operate in absence of peripheral chemoreceptor inputs (Iriki and Kozawa, 1976; Fewell et al., 1997; Barros et al., 2006; Gargaglioni et  121 al., 2006). Our findings suggest that this could also be the case in birds: vagotomy, which in birds eliminates afferent input from all arterial and pulmonary chemoreceptors, did not eliminate Tb depression during hypoxia in ducks (Fig. 4.8A). Consistent with this hypothesis, metabolic O2 limitation in the brain of ducks (Bryan and Jones, 1980) occurs at similar levels of hypoxia to that initiating Tb depression in the present study. Vagotomy did eliminate bill warming during hypoxia (Fig. 4.8B) however, so at least part of the hypoxic thermoregulatory response relies on information from peripheral chemoreceptors or thermoafferents carried by the vagus. Regardless of the sensors involved, our conclusions agree with work in pigeons showing that the magnitude of Tb depression is strongly influenced by changes in O2 loading (Barnas and Rautenberg, 1990). Enhancement of O2 supply to neural sensors can therefore alleviate the occurrence of Tb depression during hypoxia. The increases in bill surface temperature observed during hypoxia (Figs. 4.2 and 4.3) strongly suggest that perfusion of thermal windows is specifically controlled to facilitate heat loss during hypoxia, similar to what occurs during heat stress (Bech et al., 1982). Local regulation of blood flow to the bill in response to cellular O2 limitation probably cannot explain our results; preferential perfusion of this tissue for supplying O2 per se is unlikely, because blood flow is redistributed to the heart and brain during hypoxia, and away from less hypoxia-sensitive tissues (Faraci et al., 1984; Faraci et al., 1985). Increasing heat loss from the bill was probably not the only means of Tb depression during hypoxia in our study. Tb depression occurred without any change in bill temperature in vagotomized ducks (Fig. 4.8); hypoxic Tb depression in these animals  122 may have therefore occurred by reducing heat generation or by increasing heat loss from routes not dependent on vagal feedback. Biochemical adjustments that decrease proton leak across the mitochondrial inner membrane could reduce thermogenesis in hypoxia, which would have the added effect of reducing temperature-independent rates of metabolism and O2 demand (Gnaiger et al., 2000; St-Pierre et al., 2000). Evaporative heat loss from respiratory surfaces could increase during hypoxia (Tattersall and Gerlach, 2005; Hoffman et al., 2007), particularly when total ventilation increases (although ventilatory responses are attenuated by vagotomy). Heat loss from the feet probably occurs in hypoxia as well, and it is unclear whether this route of heat loss requires intact vagi. The relative contributions of these mechanisms remain unclear. A regulated decline in Tb during hypoxia undoubtedly reduces O2 demands, and probably facilitates metabolic depression in tissues that need not remain active. However, higher workloads of respiratory and cardiac muscles (and possibly tissues involved in acid-base regulation) should increase their metabolic requirements during hypoxia. The response of a whole animal is therefore the sum of factors that either increase or decrease global metabolism. In the current study and previous studies of birds during hypoxia (Tucker, 1968; Bouverot and Hildwein, 1978; Black and Tenney, 1980; Novoa et al., 1991; Scott and Milsom, 2007; Chapter 3), this sum caused a net increase in whole animal metabolism. This is unlike the situation in mammals (e.g., Barros et al., 2001; Tattersall et al., 2002), which may relate to the exceptional ability of birds to increase ventilation (with its associated metabolic costs) (Scheid, 1990). Birds primarily experience hypoxia when flying, so it is conceivable that metabolic depression was selected against during the origins of flight (but see Bucher and Chappell, 1997).  123 Nevertheless, depression of Tb during hypoxia will lead to reductions in metabolism that may not decrease whole-animal O2 consumption, but certainly diminish the global metabolic demands that would exist without this response.  4.4.2. Interspecific differences in hypoxia responses Many previous studies of bar-headed geese suggest that this species is exceptional at maintaining O2 supply to mitochondria during hypoxia. Bar-headed geese have an enhanced poikilocapnic hypoxic ventilatory response (HVR), particularly during severe hypoxia (Scott and Milsom, 2007; Chapter 3). Poikilocapnic hypoxia is environmentally realistic, but the decrease in blood CO2 that occurs because of the initial ventilatory response reflexly inhibits breathing. Because the isocapnic HVR (when CO2 is experimentally maintained) of bar-headed geese is the same as that of other species, the enhanced poikilocapnic HVR may be partly caused by a ventilatory insensitivity to hypocapnia (Chapter 3; Scott and Milsom, 2007). Bar-headed geese also have an increased haemoglobin-O2 affinity (Petschow et al., 1977; Weber et al., 1993), and both of these evolutionary changes should enhance O2 loading during hypoxia. Based on our previous theoretical calculations, these specializations in the O2 transport pathway of barheaded geese should impart considerable benefit for maintaining high metabolic rates during flight at high altitude (Chapter 2; Scott and Milsom, 2006). In the current study we confirm our previous findings on the hypoxic ventilatory response of bar-headed geese (Chapter 3; Scott and Milsom, 2007). This species breathes with much larger tidal volumes than low altitude species (Fig. 4.5; Table 4.1), which should reduce dead space ventilation, increase effective ventilation of the gas exchange  124 surface, and enhance O2 loading (Chapter 3; Scott and Milsom, 2007). In addition, total ventilation in bar-headed geese was higher than in greylag geese during prolonged hypoxia at 9% inspired O2 (Fig. 4.6). This is inconsistent with the results at 9% O2 of acute stepwise hypoxia experiments (Fig. 4.5; Chapter 3; Scott and Milsom, 2007), but consistent with previous observations that bar-headed geese breathe substantially more than low altitude species at 5% inspired O2 (Chapter 3; Scott and Milsom, 2007). Time domains of the ventilatory response to poikilocapnic hypoxia appear to differ between bar-headed geese and low altitude waterfowl. Total ventilation, as well as its components, breathing frequency and tidal volume, increased rapidly and then changed very little throughout the duration of hypoxia in bar-headed geese, in both the stepwise and prolonged protocols (Figs. 4.5 and 4.6; Table 4.1). In contrast, greylag geese and pekin ducks exhibited time-dependent changes in breathing pattern. During the stepwise protocol, the acute response to hypoxia was followed by decreases in either breathing frequency (greylag geese) or tidal volume (pekin ducks at 7% O2), and in greylag geese this appeared to be offset by increases in tidal volume (Fig. 4.5). Tidal volume also increased progressively during the prolonged hypoxia protocol in greylag geese (Table 4.1), but was not offset by declines in breathing frequency in this experiment, such that a gradual increase in total ventilation occurred (Fig. 4.6). Unfortunately, we do not at present have sufficient information to define these changes in terms of established time domains of the hypoxic ventilatory response (Powell et al., 1998; Mitchell et al., 2001). The onset of Tb depression and bill warming did not occur until more severe levels of hypoxia in bar-headed geese, but the relationships between body or bill  125 temperature change and arterial O2 content were similar between species. Previous research supports this finding, having shown that bar-headed geese have higher arterial O2 content and reduce Tb less than pekin ducks during hypoxia (Black and Tenney, 1980; Faraci et al., 1984; Scott and Milsom, 2007; Chapter 3). Rather than being because of differences in how thermoregulatory control centres respond to changes in arterial O2, the reduced Tb depression of bar-headed geese may result from a lower magnitude of hypoxaemia at any given inspired O2. However, the extent of Tb depression may decrease at higher ambient temperatures (Faraci et al., 1984), suggesting that the relationship between O2 loading and Tb depression depends on the thermal environment. The higher rates of metabolism in bar-headed geese during hypoxia could not be explained by differences in O2 loading or the Q10 effects of changes in Tb (Figs. 4.4 and 4.7), but could reflect a higher metabolic cost of O2 transport or a reduction in the temperature-independent means of metabolic depression. Assuming the latter, evolutionary changes at multiple steps in the pathway of O2 transport and utilization could help sustain higher metabolic rates in bar-headed geese. For example, this species has a higher capillarity within leg muscle, which should enhance the capacity for O2 diffusion from the blood (Snyder et al., 1984). Mitochondria of bar-headed geese could also be better at maintaining rates of ATP supply when intracellular O2 is low. These possibilities could be especially important for maintaining high rates of metabolism for flight during hypoxia, and will be further explored in Chapters 5 and 6.  126 4.4.3. High altitude adaptation in bar-headed geese Bar-headed geese and greylag geese have a close phylogenetic relationship within the genus Anser, while ducks are more distantly related (Donne-Goussé et al., 2002). Therefore, if the differences between species were caused by neutral evolutionary processes, greylag geese and bar-headed geese should have been more alike to one another than to pekin ducks. This was generally not the case: bar-headed geese often responded differently to hypoxia than both low altitude species, suggesting that these unique phenotypes are related to high altitude adaptation. Because bar-headed geese must increase metabolic rates substantially during flight at high altitude, any suppressive effect of hypoxia on their metabolism will be detrimental to performance. The ability of bar-headed geese to minimize body temperature and metabolic depression during hypoxia could therefore be essential to this species’ extraordinary migration. We believe this results from the enhanced capacity of bar-headed geese to load O2 into the blood, which likely has immense value for high altitude flight.  4.5. Summary of Chapter •  The thermoregulatory, metabolic, and ventilatory responses to hypoxia of bar-headed geese were compared to those of low altitude waterfowl (greylag geese and pekin ducks).  •  All birds reduced body temperature (Tb) during hypoxia, by up to 1-1.5 oC.  127 •  Reductions in Tb were due in part to regulated increases in heat loss, reflected by increases in bill surface temperatures (up to 5oC). Vagotomy showed that bill warming required vagal afferent inputs (probably peripheral chemoreceptors).  •  Bar-headed geese required more severe hypoxia to initiate Tb depression and heat loss from the bill.  •  Differences in Tb could not entirely account for the higher rates of metabolism (reflected by O2 consumption rate) during hypoxia in bar-headed geese.  •  There was less time-dependent variation in the hypoxic ventilatory response (i.e., fewer time domains) in bar-headed geese than in low altitude waterfowl.  128 Table 4.1. Respiratory variables during prolonged hypoxia (9% inspired O2) Time (min) Bar-Headed Geese 21% O2 0 9% O2 5 10 15 20 30 40 50 60 21% O2 65 70 75 80 85 90 Greylag Geese 21% O2 0 9% O2 5 10 15 20 30 40 50 60 21% O2 65 70 75 80 85 90 †  Breathing Frequency (min-1)  Tidal Volume (ml kg-1)  Air Convection Requirement (ml mmol-1)  11.9 ± 0.6  47.8 ± 1.9*  690 ± 110  16.7 ± 1.7 16.4 ± 1.5 16.5 ± 1.9 17.2 ± 2.6† 16.2 ± 1.4 16.5 ± 2.0 17.4 ± 1.4† 16.8 ± 1.7†  54.1 ± 4.0* 58.5 ± 4.7*,† 54.5 ± 3.6* 54.1 ± 4.8* 56.8 ± 4.1* 52.6 ± 5.2* 53.9 ± 4.4* 54.9 ± 3.3*  789 ± 116 777 ± 96 744 ± 104 715 ± 92 740 ± 107 737 ± 132 735 ± 95 731 ± 99  12.1 ± 0.9* 12.1 ± 0.6* 12.9 ± 0.9* 12.7 ± 0.8 13.0 ± 0.6 12.8 ± 0.5  44.2 ± 3.8* 44.7 ± 3.5* 45.5 ± 4.0* 44.5 ± 3.6* 45.9 ± 2.3* 44.3 ± 3.6*  566 ± 89 617 ± 105 680 ± 131 620 ± 91 606 ± 91 655 ± 104  16.2 ± 2.4  29.5 ± 4.7  560 ± 126  16.4 ± 0.8 15.3 ± 1.1 16.0 ± 0.9 16.1 ± 0.9 16.4 ± 1.1 17.8 ± 2.0 16.1 ± 1.1 15.9 ± 1.2  36.8 ± 1.2 39.7 ± 0.8 40.4 ± 0.5 40.7 ± 0.5† 42.0 ± 1.1† 39.0 ± 1.0 42.9 ± 1.2† 42.8 ± 1.2†  579 ± 84 595 ± 102 624 ± 121 541 ± 36 582 ± 76 592 ± 68 643 ± 124 577 ± 85  17.8 ± 1.1 17.3 ± 1.7 17.5 ± 2.4 17.1 ± 2.0 17.0 ± 2.1 16.3 ± 1.2  30.8 ± 2.7 29.5 ± 2.6 29.0 ± 3.0 29.3 ± 2.6 28.1 ± 2.4 30.2 ± 1.6  425 ± 45 458 ± 79 466 ± 98 524 ± 149 551 ± 196 533 ± 159  Significant difference from pre-hypoxic control (within species). * Significant difference from greylag geese (p<0.05).  129 Table 4.2. Respiratory variables during stepwise hypoxia in vagotomized ducks Time FI O 2 (min) Hypoxia 0 21% ! !15 12% 30 9% 45 7% Control 0 15 30 45  •  •  •  •  V Tot (ml kg-1 min-1)  fR (min-1)  VT (ml kg-1)  VO 2 (mmol kg-1 min-1)  V Tot VO 2 (ml mmol-1)  591 ± 105 700 ± 148 914 ± 179 1504 ± 411†  7.6 ± 1.0 8.6 ± 1.9 12.2 ± 4.2 16.5 ± 6.0  78.1 ± 11.1 81.9 ± 3.0 ! 86.3 ± 16.1 104.2 ± 17.9  1.64 ± 0.07 ! 1.70 ± 0.09 1.65 ± 0.10 1.46 ± 0.08†  363 ± 72 409 ± 82 564 ± 119 1080 ± 333†*  473 ± 137 637 ± 193 743 ± 240 857 ± 312  5.5 ± 1.8 6.9 ± 3.1 8.1 ± 3.5 10.9 ± 4.9  89.5 ± 8.6 103.8 ± 14.9 100.8 ± 12.9 84.8 ± 8.7  1.50 ± 0.30 1.62 ± 0.33 1.52 ± 0.34 1.54 ± 0.32  301 ± 44 388 ± 73 468 ± 72 524 ± 102  21% 21% 21% 21%  •  •  FI O 2 , inspired O2 fraction; V Tot , total ventilation; fR, breathing frequency; VT, tidal volume; VO 2 , •  !  •  oxygen consumption rate; V Tot VO 2 , air convection requirement. Data shown are the average values after 10-15 minutes at each FI O 2 . † Significant difference from time 0 (within species). ! * ! Significant difference between hypoxia and normoxia-control treatments (p<0.05).  !  !  130  Fig. 4.1. (A) Body temperature (Tb) depression during stepwise hypoxia was less severe in bar-headed geese (light grey triangles) than in greylag geese (black squares) or pekin ducks (dark grey circles). Significant Tb depression began earlier in greylag geese (black arrow) and pekin ducks (dark grey arrow) than in bar-headed geese (light grey dashed arrow), and bar-headed geese reduced Tb less during 7% inspired O2 (asterisk). (B) Tb increased significantly during 20 min of recovery in normoxia in bar-headed geese (light grey dashed arrow) and greylag geese (black arrow) but not in pekin ducks. The body temperature change in each individual was determined by subtracting its pre-hypoxia Tb value (see Results).  131  Fig. 4.2. Surface temperatures of the bill increased in bar-headed geese (BG, top row), greylag geese (GG, middle row) and pekin ducks (PD, bottom row) during stepwise hypoxia. Inspired O2 was reduced from 21% to 12%, 9%, 7%, and then in bar-headed geese only 5%. Each level of hypoxia was sustained for 15 min. Scale bar represents 10 cm.  132  Fig. 4.3. (A) Bill surface temperatures increased during stepwise hypoxia in bar-headed geese (light grey triangles), greylag geese (black squares), and pekin ducks (dark grey circles). This increase became significant much earlier in greylag geese (black arrow) and pekin ducks (dark grey arrow) than in bar-headed geese (light grey dashed arrow), and was lower in bar-headed geese during 9% inspired O2 (asterisk). (B) Bill temperature decreased after the first minute of normoxic recovery in bar-headed geese (light grey dashed arrow) and greylag geese (black arrow) and after the second minute in pekin ducks (dark grey arrow). The bill temperature change in each individual was determined by subtracting its pre-hypoxia value (see Results). Representative thermal images are shown in Fig. 4.2.  133  Fig. 4.4. (A) Oxygen consumption rate increased during stepwise hypoxia in bar-headed geese (light grey triangles), greylag geese (black squares), and pekin ducks (dark grey circles). Bar-headed geese had higher oxygen consumption rates than both other species during 7% inspired O2 (asterisks). Differences between species persisted after mean oxygen consumption rates were corrected for differences in body temperature depression (see Materials and Methods), using Q10 values of either 2 (short dashed lines) or 3 (long dashed lines). (B) Oxygen consumption rates decreased rapidly during normoxic recovery in all species.  134  Fig. 4.5. Total ventilation, breathing frequency, tidal volume, and air convection requirements (total ventilation divided by oxygen consumption rate) during stepwise hypoxia (A) and subsequent normoxic recovery (B) in bar-headed geese (light grey triangles), greylag geese (black squares), and pekin ducks (dark grey circles). * Significant difference between bar-headed geese and both greylag geese and pekin ducks.  135  Fig. 4.6. Body temperature change, oxygen consumption rate, and total ventilation during 9% inspired O2 that was sustained for 60 min, followed by 30 min normoxic recovery, in bar-headed geese (light grey triangles) and greylag geese (squares). * Significant difference between bar-headed geese and greylag geese.  Significant difference from 5 min and 10 min time points (white squares) within greylag geese.  136  Fig. 4.7. Relationships between measured variables and arterial O2 content (Chapter 3) during stepwise hypoxia. Data are after 15 min at each level of hypoxia. Slopes of regressions in (A) were similar between species (bar-headed geese, 0.46 ± 0.08; greylag geese, 0.55 ± 0.03; pekin ducks, 0.56 ± 0.07). Slopes in (B) were steeper in bar-headed geese (-2.5 ± 0.6) and pekin ducks (-2.0 ± 0.4) than in greylag geese (-0.80 ± 0.25). In (C) the slope was steepest in bar-headed geese (-0.68 ± 0.21), followed by pekin ducks (-0.42 ± 0.12), and greylag geese (-0.26 ± 0.04). All regressions were statistically significant.  137  Fig. 4.8. (A) Bilateral vagotomy did not eliminate body temperature (Tb) depression in response to stepwise hypoxia. Vagotomized ducks exposed to hypoxia (black and white circles) began reducing Tb during 9% inspired O2 (black arrow), and reached lower mean Tb than intact ducks (dark grey circles). (B) Bilatereral vagotomy abolished the increase in bill surface temperature that occurs in intact ducks during hypoxia. 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L. and Schmidt-Nielsen, K. (1975). Heat loss from ducks feet immersed in cold water. Condor 77, 475-478. Klir, J. J. and Heath, J. E. (1992). An infrared thermographic study of surface temperature in relation to external thermal stress in three species of foxes: the red  142 fox (Vulpes vulpes), Arctic fox (Alopex lagopus), and kit fox (Vulpes macrotis). Physiol. Zool. 65, 1011-1021. Klir, J. J. and Heath, J. E. (1994). Thermoregulatory responses to thermal stimulation of the preoptic anterior hypothalamus in the red fox (Vulpes vulpes). Comp. Biochem. Physiol. A. Physiol. 109, 557-566. Mauck, B., Bilgmann, K., Jones, D. D., Eysel, U. and Dehnhardt, G. (2003). Thermal windows on the trunk of hauled-out seals: hot spots for thermoregulatory evaporation? J. Exp. Biol. 206, 1727-1738. Mitchell, G. S., Powell, F. L., Hopkins, S. R. and Milsom, W. K. (2001). Time domains of the hypoxic ventilatory response in awake ducks: episodic and continuous hypoxia. Respir. Physiol. 124, 117-128. 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K. (2003). Transient peripheral warming accompanies the hypoxic metabolic response in the golden-mantled ground squirrel. J. Exp. Biol. 206, 33-42. Taylor, E. W., Jordan, D. and Coote, J. H. (1999). Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiol. Rev. 79, 855-916. Tucker, V. A. (1968). Respiratory physiology of house sparrows in relation to highaltitude flight. J. Exp. Biol. 48, 55-66. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weber, R. E., Jessen, T. H., Malte, H. and Tame, J. (1993). Mutant hemoglobins (α119-Ala and β55-Ser): functions related to high-altitude respiration in geese. J. Appl. Physiol. 75, 2646-2655. Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA, USA: Harvard University Press. Wood, S. C. and Gonzales, R. (1996). Hypothermia in hypoxic animals: mechanisms, mediators, and functional significance. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 113, 37-43.  145 5. EVOLUTION OF MUSCLE PHENOTYPE FOR EXTREME HIGH ALTITUDE FLIGHT IN THE BAR-HEADED GOOSE4  5.1. Introduction High altitude environments are particularly challenging to animal life, due to their characteristically low atmospheric temperatures and O2 levels. The hypoxia at high altitude can be particularly debilitating for lowland species, and for even the best human mountaineers, O2 levels at the world’s highest peaks can barely sustain basal metabolism (West, 2000). However, numerous species have acquired genetically based physiological adaptations that minimize performance decrements at high altitude (Chappell and Snyder, 1984; Beall, 2007; Storz and Moriyama, 2008). These species provide an exceptional opportunity for studying how complex physiological systems evolve, since the mechanisms of O2 transport and utilization are well understood. Because the O2 transport pathway is composed of a series of physiological steps (breathing, pulmonary O2 diffusion, circulation via the blood, tissue O2 diffusion, and mitochondrial O2 utilization) (Weibel, 1984), the complex nature of high altitude adaptation is best understood by considering all steps in the O2 pathway. One of the most celebrated high altitude performers is the bar-headed goose (Anser indicus). This species crosses the Himalayas on its biannual migration between southern and central Asia, flying over the highest mountains in the world and reaching altitudes of up to 9000m (Swan, 1970). Incredibly, bar-headed geese not only tolerate the 4  A version of this chapter has been published: Scott, G. R., Egginton, S., Richards, J. G., and Milsom, W. K. (2009). Evolution of muscle phenotype for extreme high altitude flight in the bar-headed goose. Proc. R. Soc. B. 276, 3645-3653. (http://rspb.royalsocietypublishing.org/)  146 severe hypoxia at these elevations, they also sustain the 10- to 20-fold increase in O2 consumption rate that is necessary to fuel flapping flight (Ward et al., 2002). The physiological basis for this elevated O2 transport capacity is not completely understood, but it results in part from evolutionary changes in the cardiorespiratory system that improve O2 uptake and circulation during hypoxia (Petschow et al., 1977; Jessen et al., 1991; Scott and Milsom, 2007; Chapter 3). Much less is known about the flight muscle of this species, but theoretical modeling suggests that an enhanced capacity for O2 diffusion from blood into muscle, which can be realized with increased muscle capillarity, should also improve O2 transport in hypoxia (Scott and Milsom, 2006; Chapter 2). Here we demonstrate, using a comparative phylogenetic approach, a striking difference in flight muscle phenotype related to high altitude flight in bar-headed geese. This difference was unique compared to multiple low altitude migratory species, and was not due to physiological plasticity or interspecific variation in body mass. The unique flight muscle phenotype in bar-headed geese is therefore inherent and probably serves to enhance muscle O2 transport during flight at extremely high altitudes.  5.2. Materials and Methods 5.2.1. Animals Histological measurements were made on 6 bar-headed geese (1.9-2.7 kg), 8 barnacle geese (Branta leucopsis) (1.6-2.2 kg), and 8 pink-footed geese (Anser brachyrhynchus) (2.3-3.5 kg). Mitochondrial respiration was performed on the same barheaded geese, 3 different barnacle geese (1.9-2.0 kg), 4 greylag geese (Anser anser) (3.95.1 kg), and 9 mallard ducks (Anas platyrhynchos) (1.1-1.6 kg). All birds were young  147 adults of similar age (3-5 years old) that had been bred and raised in captivity at sea level by registered breeders. Measurements were made on both male and female individuals for each species, none of which had ever flown. All animal care and experimentation was conducted according to UBC animal care protocol #A04-1013. These species were chosen due to their close phylogenetic relationships to barheaded geese (Fig. 5.1). Barnacle geese, pink-footed geese, greylag geese, and mallard ducks all live at low altitudes, and although fine-scale migration patterns are not known for all of these species, they generally follow low to moderate altitude migration routes (Cramp and Simmons, 1977). This suggests that high altitude migration is a derived characteristic of bar-headed geese.  5.2.2. Muscle histology Birds were terminally anaesthetized with an overdose of intravenously injected sodium pentobarbital and the pectoralis major muscle was dissected. Samples were taken half way along the length of the sternum, 3-5 cm lateral to the carina, at surface (immediately subcutaneous), intermediate (50% depth), and deep (adjacent to the sternum) muscle depths. Samples were coated in mounting medium on cork disks and then rapidly frozen in 2-methylbutane (cooled in liquid N2). Muscle was sectioned (10 µm) transverse to fiber length in a -20°C cryostat. Additional muscle from an intermediate depth was fixed at resting length using 2.5% glutaraldehyde in cacodylate buffer (0.1M, pH 7.4), then post-fixed in buffered OsO4 (1%) for 1h, dehydrated in ethanol, and embedded in epoxy resin. Transverse semi-thin sections (0.5 µm) were  148 stained with toluidine blue, and ultrathin sections (~80 nm) were stained with uranyl acetate and lead citrate. Cryostat sections were stained for succinate dehydrogenase, myosin-ATPase (preincubated at pH 4.6), and alkaline phosphatase activities (Deveci et al., 2001), and these sections as well as the semithin sections (used to identify intramuscular lipid) were imaged using light microscopy. Ultrathin sections were imaged using transmission electron microscopy. Unbiased measurements for determining all histological variables were collected and analyzed as previously described (Weibel, 1979; Egginton, 1990). Sufficient images (≥5) were analyzed for each sample to account for heterogeneity, determined by the number of replicates necessary to yield a stable mean value. Mitochondria were classified as subsarcolemmal if they were located between the cell membrane and the outer edges of peripheral myofibrils. Representative alkaline phosphatase-stained images (four individuals from each species, surface muscle depth) were digitized to determine capillary domain area (the area around each capillary whose boundary is equidistant from each adjacent capillary) and an estimate of the heterogeneity of capillary spacing (the coefficient of variation of all domain diameters in an image), both as previously described (Degens et al., 1992; Degens et al., 2006). A representative image showing capillary domain areas and the more traditional Krogh cylinder areas is shown in Fig. 5.2. Average mitochondrial volume density at each muscle depth ( VV (mito,m) ) was calculated by equation 5.1:  !  VV (mito,m) = VV (mito,IIa) " AA (IIa,m) + VV (mito,IIb) " AA (IIb,m)  !  (5.1)  149 where VV (mito,IIa) and VV (mito,IIb) are the mitochondrial volume densities in type IIa and IIb fibers, and AA (IIa,m) and AA (IIb,m) are the areal densities of type IIa and IIb !  !  fibers. !  !  5.2.3. Respiration of muscle mitochondria A mixed population of subsarcolemmal and intermyofibrillar mitochondria were isolated from various muscle depths. Briefly, birds were terminally anaesthetized and a large sample (approximately 6g) of pectoralis major muscle was removed, immediately transferred to 15 ml of ice-cold isolation buffer (in mM: 100 sucrose, 50 tris base, 5 MgCl2, 5 EGTA, 100 KCl, 1 ATP; pH 7.4), and minced. Muscle was digested for 5 min by adding 10 ml more buffer containing nagarse proteinase (1 mg/g muscle), and homogenized gently with 5 passes of a Potter-Elvehjem homogenizer (100 rpm). Homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatant was filtered through cheesecloth and then centrifuged at 8700g for 10 min at 4°C (same for all further centrifugation). The pellet was re-suspended in 20 ml of isolation buffer and centrifuged, re-suspended in 10 ml of storage buffer (in mM: 0.5 EGTA, 3 MgCl2, 60 Kmethanesulfonate, 20 taurine, 10 KH2PO4, 20 hepes, 110 sucrose, 1 MgCl2, 0.02 vitamin E succinate, 2 pyruvate, 2 malate; pH 7.1), centrifuged, and finally re-suspended in 3 ml of storage buffer. Mitochondria were stored on ice until experimentation. Respiration of mitochondria (0.04 mg mitochondrial protein) was measured by high-resolution respirometry (Oxygraph-2k, Oroboros) in 2 ml of respiration buffer (in mM: 0.5 EGTA, 3 MgCl2, 60 K-MES, 20 taurine, 10 KH2PO4, 20 hepes, 110 sucrose, 1 MgCl2, 1 g/l fatty acid-free bovine serum albumin; pH 7.1; O2 solubility factor of 0.92) at  150 avian body temperature (41°C). Two separate experiments were performed using two different combinations of substrates. In experiment 1, state 2 was first stimulated with malate (2 mM) and pyruvate (5 mM). P/O ratios were determined twice by the conventional method (Gnaiger et al., 2000) by adding 125 µM of ADP (the average P/O is reported). After state 4 respiration was reached, state 3 was induced with maximal ADP (0.7-1.0 mM) and mitochondria were allowed to deplete all the O2. After a period of anoxia (5 min), the O2 tension was raised slightly, the remaining ADP was consumed, and all O2 was consumed in state 4. After anoxia, the O2 tension was raised, cytochrome oxidase was maximally stimulated with ADP (1.25 mM), TMPD (N,N,N',N'-tetramethylp-phenylenediamine; 0.5 mM) and ascorbate (0.5 mM), and all O2 was consumed. After anoxia, the O2 tension was raised and mitochondria were uncoupled with FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone; 0.5 µM). Experiment 2 was the same as experiment 1, except that succinate (10 mM) was also present to spark state 2. The O2 tension that reduced mitochondrial respiration to 50% of the normoxic rate (P50) was determined for states 3 and 4, and for TMPD-stimulated respiration, as previously described (Gnaiger, 2001). Respiration rates were corrected for background O2 flux and are expressed relative to mitochondrial protein concentration.  5.2.4. Statistical analyses Data are generally reported as means ± standard error. One- or two-factor ANOVA and Student-Newman-Keuls post-hoc tests were used, as appropriate, and abnormally distributed data were log-transformed before testing. Ordinary least squares (OLS) and reduced major axis (RMA) linear regressions were used for correlations.  151 RMA regressions were calculated with the program RMA (Bohonak and van der Linde, 2004). All other traditional statistical tests were performed using Sigmastat (Systat). We also performed phylogenetically independent contrast analysis (Midford et al., 2005; Maddison and Maddison, 2009) for traits that were uniquely different in barheaded geese. We used our previously published phylogenetic tree (Lee et al., 2008) pruned to include only the species in question (Fig. 5.1). Low and high altitude migration strategies were coded as 0 or 1 dummy variable (Garland et al., 1992), respectively. We tested for significant positive relationships between the standardized independent contrasts of muscle traits and flight altitude by determining the one-tailed p-values for correlations computed through the origin (degrees of freedom, df=1 for all). A significance level of P<0.05 was used throughout.  5.3. Results and Discussion 5.3.1. Flight muscle phenotype in geese The flight muscle of geese contained fast oxidative (type IIa) and fast glycolytic (type IIb) fiber types, which exhibit stereotypical differences in succinate dehydrogenase (a mitochondrial enzyme) and myosin-ATPase (an index of contractility) activities (Fig. 5.2). Consistent with what is known for other bird species (Rosser et al., 1987; Torrella et al., 1998), type IIa fibers were most abundant across the muscle, and slow oxidative (type I) fibers were not observed. There was a striking difference in the muscle fiber composition of bar-headed geese compared to the low altitude birds (Figs. 5.3-5.5; Table 5.1). The flight muscle of barnacle geese and pink-footed geese had a significant proportion of fast glycolytic fibers  152 near the muscle surface, but as muscle depth increased so too did the proportion of fast oxidative fibers; this appears to be a general characteristic of birds (Mathieu-Costello et al., 1998a). In contrast, the flight muscle of bar-headed geese had a much higher proportion of fast oxidative fibers. This was apparent for average values across the whole muscle (bar-headed geese, 82.5 ±0.9%; barnacle geese, 76.8 ± 1.0%; pink-footed geese, 71.2 ± 2.3%) and was exaggerated near the muscle surface, where the potential for increasing oxidative fiber density was greatest. Consistent with the differences in fiber composition, the flight muscle of barheaded geese had more capillaries than that of low altitude geese (Figs. 5.6-5.7). Both the number of capillaries per fiber (C:F) and the capillary density (CD) increased with muscle depth in barnacle geese and pink-footed geese, appropriately increasing O2 supply capacity in more oxidative regions of the muscle. C:F was much higher in bar-headed geese at surface and intermediate muscle depths, as well as for the global average (barheaded geese, 2.13 ± 0.09; barnacle geese, 1.72 ± 0.08; pink-footed geese, 1.69 ± 0.07), such that it was as high in the superficial regions as in the deep regions. CD was also higher in bar-headed geese than in both low altitude species near the muscle surface, and the overall average was higher than in pink-footed geese (bar-headed geese, 2287 ± 112 mm-2; barnacle geese, 2270 ± 118 mm-2; pink-footed geese, 1802 ± 107 mm-2). The less pronounced interspecific differences in CD than in C:F probably resulted from a confounding effect of allometry on the muscle fiber size of barnacle geese (Fig. 5.8; Table 5.1). Nevertheless, consistent with the differences in CD near the muscle surface, the volume of muscle supplied by each capillary (as indicated by the capillary domain areas) was smaller in bar-headed geese than in both low altitude species (Table 5.1).  153 Muscle capillaries also appeared to be more homogeneously spaced in bar-headed geese, because the coefficient of variation of capillary spacing in this species was significantly less than in barnacle geese and lower on average than in pink-footed geese (Table 5.1). We also performed phylogenetically independent contrast analysis on muscle traits that were uniquely different in bar-headed geese, by testing for positive relationships between the standardized contrasts of these traits and flight altitude (Fig. 5.9; see Materials and Methods). There were positive relationships between flight altitude and oxidative fiber areal density (Pearson product-moment correlation coefficient, r=0.996), myosin-ATPase type IIa areal density (r=0.999), and CD (r=0.994), all at the muscle surface, as well as C:F at surface (r=0.998), intermediate (r=0.989), and deep (r=0.999) muscle depths. Therefore, the associations between the unique characteristics of bar-headed geese and their high altitude migration strategy are independent of phylogeny. Because differences in fiber composition per se will influence capillarity (due to concomitant changes in O2 demands), we next sought to determine if the increased proportion of oxidative fibers could account for the increased capillarity in bar-headed goose muscle. To do so we first assessed the mitochondrial abundance in oxidative and glycolytic fibers in all species (Fig. 5.10). There were no statistically significant differences between species in the mitochondrial volume densities within oxidative or glycolytic fibers, or in the densities of intramuscular or intracellular lipid droplets (Table 5.1). Mitochondrial cristae surface densities were very high (~60 µm2/µm3), similar to those in the highly aerobic flight muscle of hummingbirds (Suarez et al., 1991), but were also not different between species. With these data we then calculated the average  154 mitochondrial volume density at each muscle depth for each species. C:F was more strongly related to average mitochondrial volume density (R2=0.94) (Fig. 5.6) than CD was when only low altitude species were considered (compare Figs. 5.6b and 5.11), and was thus a better index for comparing capillarity between species. In doing so, we found that bar-headed geese had more capillaries per muscle fiber for a given mitochondrial volume density at surface and intermediate muscle depths. Therefore, capillarity and O2 diffusing capacity are higher in bar-headed goose muscle than can be accounted for by the increased proportion of oxidative fibers. This result has clear benefits for sustaining muscle O2 flux when capillary O2 tensions fall during hypoxia. Although C:F is higher in birds living at high altitude (León-Velarde et al., 1993), in some cases this has been entirely attributed to concurrent increases in mitochondrial abundance (Mathieu-Costello et al., 1998b). Furthermore, exercise and hypoxia would have been potential stimulants of angiogenesis in these studies, in contrast to the inherently higher capillarity that exists in bar-headed geese before ever flying or experiencing high altitude. Therefore, despite some disagreement about its role during high altitude acclimatization (Mathieu-Costello, 2001), increases in the O2 diffusing capacity of the muscle have evolved in some high altitude species, which probably improves aerobic performance in hypoxia. Although mitochondrial abundance within each fiber type is similar between species, the proportion of all mitochondria that are subsarcolemmal (rather than intermyofibrillar) was much higher in bar-headed geese (Fig. 5.12). Furthermore, the positive relationship between subsarcolemmal mitochondria proportion and flight altitude persisted after correcting for phylogeny (r=0.999 for correlation shown in Fig. 5.9). This  155 redistribution of mitochondria towards the cell membrane and closer to capillaries should reduce intracellular diffusion distances, which like our results for capillarity is an inherent difference that is not dependent on exercise or prior hypoxia exposure. Its importance for enhancing O2 transport is emphasized by previous human studies showing that increases in aerobic performance after exercise training are associated with a preferential proliferation of subsarcolemmal mitochondria (Hoppeler et al., 1985). In fact, the proportion of subsarcolemmal mitochondria in bar-headed goose muscle is even higher than in the highly aerobic flight muscle of hummingbirds (Suarez et al., 1991). Although this mitochondrial distribution should increase O2 transport (Mainwood and Rakusan, 1982), it could hinder the intracellular movement of ATP equivalents and other metabolites (Kinsey et al., 2007). This suggests that bar-headed goose flight muscle may rely on an effective system for intracellular shuttling of ATP equivalents, such as the creatine kinase shuttle (Andrienko et al., 2003). This possibility will be explored in Chapter 6. Although the mechanisms accounting for the altered muscle phenotype in barheaded geese are unclear, they may involve inherent differences in muscle development (Bassel-Duby and Olson, 2006; Biressi et al., 2007). Activity-dependent regulators of muscle phenotype, such as neural stimulation related to muscle contraction, did not cause the differences in this study because no species had been allowed to fly. The differences probably instead reflect evolutionary changes in the gene networks regulating skeletal muscle. Flight muscle aerobic capacity develops rapidly after birth (Bishop et al., 1995), and it is probable that bar-headed geese acquire their altered phenotype in the short time before their first migration.  156 5.3.2. Respiration of muscle mitochondria in geese The maximum capacities for respiration of mitochondria isolated from the flight muscle were extremely similar between bar-headed geese and low altitude species (Fig. 5.13). This was assessed for the entire electron transport chain (state 3, maximal ADP stimulation), for cytochrome oxidase (treatment with TMPD, an exogenous electron donor), and for ATP synthase (treatment with FCCP, a mitochondrial uncoupler). These results suggest that the alteration in fiber composition (Fig. 5.3), rather than any change in the respiration or density of mitochondria within muscle cells, is the primary factor increasing aerobic capacity in bar-headed goose muscle. Bar-headed geese were also no more effective at making ATP, as indicated by an equivalent phosphorylation efficiency (indicated by the P/O ratio) to that of other species (Fig. 5.13). The effects of low oxygen on mitochondrial respiration were largely similar between species (Fig. 5.14). This was particularly true at high respiration rates (mitochondrial P50 during state 3 or cytochrome oxidase stimulation), as would occur during flight exercise, although bar-headed geese had the highest P50 at low respiration rates (state 4). Overall, the state-dependent differences in O2 kinetics resulted in a strong positive relationship between P50 and respiration rate for all species (Fig. 5.15). The P50 values we observed were slightly higher than those for mammalian mitochondria (Gnaiger, 2001), potentially because we used a higher temperature (avian body temperature of 41°C). There is a well-known hyperbolic relationship between O2 tension and mitochondrial respiration rate (Gnaiger, 2001) (shown in Fig. 5.15). Mild O2 limitation (e.g., inhibition by 5-10%) will therefore occur at significantly higher O2 tensions than  157 the P50 (i.e., 50% inhibition), which was ~1 Torr in state 3 (Fig. 5.14). However, muscle intracellular O2 tensions ( PiO 2 ) during intense exercise in normoxia can be well below 5 Torr in mammals (Gayeski and Honig, 1988). If the same is true in bird muscle then their  ! be O2-limited during flight, particularly during environmental hypoxia mitochondria may or vigorous exercise. This emphasizes the importance of the enhanced mitochondrial O2 supply in bar-headed geese for sustaining respiration during high altitude flight. The conservation of mitochondrial P50 across species also clarifies why barheaded geese could benefit from having more oxidative fibers. In absence of any change in O2 kinetics, increases in muscle aerobic capacity can enhance the total mitochondrial O2 flux of the entire muscle at a reduced PiO 2 (Hochachka, 1985). In this way, having more oxidative fibers may counterbalance the inhibition of respiration by intracellular  ! This would make bar-headed geese less reliant on hypoxia in each individual fiber. anaerobic metabolism for sustaining ATP turnover during flight in hypoxia, thus improving fatigue resistance. Their muscle may also be less inclined towards carbohydrate oxidation, as normally occurs at higher exercise intensities (McClelland et al., 1998), which is not as sustainable a fuel source for long distance migration as fats.  5.3.3. Evolution of O2 transport for flight at high altitude Our results are consistent with the idea that muscle phenotype evolved in barheaded geese as an important adaptation for flying at extremely high altitudes. Increasing the capacity for O2 diffusion from the blood and reducing the intracellular diffusion distance should both improve O2 transport during exercise in hypoxia. The unique phenotype of bar-headed geese is clearly not a product of phylogenetic history:  158 differences among distantly-related low altitude species were generally small, particularly when compared to the large differences between these species and bar-headed geese; furthermore, the associations between these muscle traits and high altitude flight were independent of phylogeny. The unique features of bar-headed geese do not result from phenotypic plasticity either, because all species were born and raised in similar conditions at sea level. Differences in body size had small effects on muscle fiber size, but because bar-headed geese were intermediate in mass between the two low altitude species, allometric variation cannot explain their unique muscle phenotype. It is therefore likely that the changes in muscle phenotype were uniquely derived in bar-headed geese and enhanced flight performance at high altitude. However, evolutionary forces other than high altitude adaptation could account for this change, such as genetic correlations with other phenotypes or selection for performance traits other than high altitude flight (Lauder et al., 1993). A stronger case for adaptation could be made if other high altitudeadapted bird species had similar specializations for improving muscle O2 transport. The differences in muscle phenotype and O2 diffusing capacity are part of a suite of evolutionary changes in the O2 transport pathway of bar-headed geese that improve O2 flux in hypoxia. One of these alterations, an inherently higher hemoglobin O2 affinity (Petschow et al., 1977), is known to be caused largely by a single amino acid change at the interface between α and β subunits (Jessen et al., 1991; Zhang et al., 1996). This improves O2 transport in hypoxia by enhancing pulmonary O2 uptake (Scott and Milsom, 2007; Chapter 3) and assuring better O2 delivery throughout the body (Faraci et al., 1984). Pulmonary O2 uptake is also increased in bar-headed geese due to an enhanced hypoxic ventilatory response, which appears to be partly caused by alterations in the  159 respiratory chemosensitivity of breathing, as well as a more effective breathing pattern (Scott and Milsom, 2007; Scott et al., 2008; Chapters 3 and 4). Our present findings therefore imply that increases in pulmonary loading and delivery of O2 occur in conjunction with enhanced O2 extraction from the blood by the flight muscle in barheaded geese. In addition to helping explain how this unique species can fly in extreme hypoxia at high altitude, our present findings provide insight into how respiratory systems evolve. Early theories for how the O2 transport pathway evolved suggested that the capacity of every step in the pathway must increase equally to enhance overall capacity (i.e., symmorphosis) (Weibel et al., 1991). More recent theories of respiratory pathway flux argue that overall control arises from the summed influence of each step in the pathway, and that different steps have unequal contributions to control (analogous to metabolic control theory) (Darveau et al., 2002; Hochachka and Burelle, 2004). This implies by extension that changes in the overall capacity for O2 transport during evolution can involve changes of varying magnitudes at different steps. Our work with bar-headed geese supports the idea that respiratory system evolution occurs through changes at multiple interacting steps in the O2 transport pathway. In light of previous work in this species, our present data suggest that the physiological traits having the greatest control over O2 transport in hypoxia (Scott and Milsom, 2006; Chapter 2) are also the most likely to evolve and improve exercise performance at high altitudes.  160 5.3.4. Conclusions Our present findings suggest that O2 diffusing capacity in the flight muscle is enhanced in bar-headed geese, due to both an increase in the number of capillaries surrounding each muscle fiber and a redistribution of mitochondria towards the cell membrane within the fibers, and thus closer to capillaries. Muscle aerobic capacity is also enhanced by an increased proportion of oxidative fibers, but properties of mitochondrial O2 demand are otherwise conserved between species. The flight muscle of bar-headed geese has therefore evolved for exercise in severe hypoxia by enhancing muscle O2 supply, which may be especially important for this species’ incredible ability to fly high.  5.4. Summary of Chapter •  The flight muscle phenotype and mitochondrial properties of bar-headed geese were compared to those of low altitude birds.  •  Bird flight muscle contained only fast oxidative (type IIa) and fast glycolytic (type IIb) muscle fibers.  •  Bar-headed goose muscle had a higher proportion of type IIa fibers. This probably increased muscle aerobic capacity, because the mitochondrial volume densities of each fiber type were similar between species.  •  Bar-headed geese had more capillaries per muscle fiber than expected from this increase in aerobic capacity, and also had higher capillary densities and more homogeneous capillary spacing.  •  Mitochondria were redistributed towards the subsarcolemma (cell membrane) and adjacent to capillaries in bar-headed goose flight muscle.  161 •  Isolated mitochondria had similar respiratory capacities, O2 kinetics, and phosphorylation efficiencies across species.  •  The unique differences in bar-headed geese were much greater than the minor variation between low altitude species and existed without prior exercise or hypoxia exposure, and the correlation of these traits to flight altitude was independent of phylogeny.  162 Table 5.1. Histological measurements from the flight muscle of geese Bar-Headed Geese Oxidative fiber numerical density (%) 1 surface 83.5 ± 1.5* intermediate 92.5 ± 0.7† deep 95.1 ± 0.3 Myosin-ATPase IIa numerical density (%) 1 surface 81.9 ± 1.3* intermediate 91.3 ± 0.7† deep 96.2 ± 0.4 2 2 IIa fiber transverse area (µm ) 852 ± 78 2 2 IIb fiber transverse area (µm ) 1797 ± 202 Capillary domain area (µm2) 3 536 ± 25* CV for capillary spacing (%) 3 15.1 ± 0.4§ IIa fiber Vv(mito,f) 4 0.25 ± 0.01 IIb fiber Vv(mito,f) 4 0.053 ± 0.003 2 3 4 Sv(cristae,mito) (µm /µm ) 59.8 ± 0.9 4 IIa fiber Vv(lipid,f) 0.020 ± 0.005† IIb fiber Vv(lipid,f) 4 0.007 ± 0.001 4 Vv(lipid,m) 0.024 ± 0.007  Barnacle Geese  Pink-Footed Geese  76.0 ± 1.5 89.7 ± 1.0 93.9 ± 0.5  76.4 ± 2.1 84.2 ± 2.5 92.2 ± 1.5  75.0 ± 2.7 91.1 ± 0.8 94.6 ± 0.4 683 ± 43 1504 ± 119 648 ± 21 20.3 ± 1.8 0.27 ± 0.01 0.056 ± 0.005 61.5 ± 0.8 0.017 ± 0.003† 0.008 ± 0.002 0.020 ± 0.003  76.1 ± 2.8 84.3 ± 1.8 91.9 ± 0.9 798 ± 45 1892 ± 157 872 ± 28 17.8 ± 1.7 0.25 ± 0.01 0.054 ± 0.006 62.4 ± 1.3 0.051 ± 0.01 0.006 ± 0.003 0.019 ± 0.004  CV, coefficient of variation; Vv(mito,f), mitochondrial volume density within IIa (oxidative) or IIb (glycolytic) fibers; Sv(cristae,mito), mitochondrial cristae surface density; Vv(lipid,f) and Vv(lipid,m), intracellular and intramuscular lipid volume densities. 1 N and df as in Fig. 5.3. 2 N as in Fig. 5.3, df=21. 3 N=4 for each species, df=11. 4 N and df as in Fig. 5.12. *,†,§ Significant difference from both low altitude species, just pink-footed geese, or just barnacle geese, respectively.  163  Fig. 5.1. (a) Hypothesized phylogenetic relationships among all species examined in the present study. (b) Phylogenetic tree for the three species used for phylogenetically independent contrast analysis, with branches drawn proportional to their actual lengths. Both of the above were generated from the comprehensive phylogeny presented by Lee et al. (2008). [This figure is in the supplementary material of the published version of this chapter].  164  Fig. 5.2. Histochemical staining of goose pectoralis muscle, showing that only fast oxidative (type IIa) fibers (arrow) and fast glycolytic (type IIb) fibers (asterisk) are present. (a) Succinate dehydrogenase (SDH), a mitochondrial enzyme that depicts oxidative capacity. (b) Myosin-ATPase (pH 4.6) to identify different muscle fiber types. (c) Alkaline phosphatase to identify capillaries. Inset is a representative digitization of capillaries and their domain areas (blue), an analyzed region of interest (green), and Krogh cylinder areas for capillaries (magenta). Scale bar represents 100 µm.  165  Fig. 5.3. Muscle fiber composition is altered in the pectoralis muscle of bar-headed geese (N=6) compared to barnacle geese (N=8) and pink-footed geese (N=8). High succinate dehydrogenase (SDH) activity identified oxidative fibers, and high acid-stable myosinATPase activity identified type IIa fibers. * and † represent significant differences from both low altitude species or just pink-footed geese, respectively (2-way ANOVA, df=65).  166  Fig. 5.4. Succinate dehydrogenase staining of flight muscle from geese. Representative sections stained for succinate dehydrogenase activity from surface (a-c), intermediate (df), and deep (g-i) regions of the pectoralis muscle of bar-headed geese (a,d,g), barnacle geese (b,e,h), and pink-footed geese (c,f,i). Scale bar represents 100 µm. [This figure is in the supplementary material of the published version of this chapter].  167  Fig. 5.5. Myosin-ATPase staining of flight muscle from geese. Representative sections stained for acid-stable (pH 4.6) myosin-ATPase activity from surface (a-c), intermediate (d-f), and deep (g-i) regions of the pectoralis muscle of bar-headed geese (a,d,g), barnacle geese (b,e,h), and pink-footed geese (c,f,i). Scale bar represents 100 µm. [This figure is in the supplementary material of the published version of this chapter].  168  Fig. 5.6. Capillarity is enhanced in the pectoralis muscle of bar-headed geese. (a) capillary density and capillary to muscle fiber ratio at different muscle depths (symbols and statistical features as in Fig. 5.3). (b) Within low altitude species, capillary to fiber ratios were strongly predicted by average mitochondrial volume density across the 3 muscle depths, for both ordinary least squares (OLS) (solid line; y=(8.03±1.02)x + (0.068±0.213), R2=0.939) and reduced major axis (black dashed line; y=(7.29±1.48)x + (0.198±0.308), R2=0.863) regressions (df=5). However, bar-headed geese were above the OLS 95% prediction intervals (grey dashed lines) at surface and intermediate depths.  169  Fig. 5.7. Capillary staining of flight muscle from geese. Representative sections stained for alkaline phosphatase activity from surface (a-c), intermediate (d-f), and deep (g-i) regions of the pectoralis muscle of bar-headed geese (a,d,g), barnacle geese (b,e,h), and pink-footed geese (c,f,i). Scale bar represents 100 µm. [This figure is in the supplementary material of the published version of this chapter].  170  Fig. 5.8. Relationship between muscle fiber area and body mass in geese. Both type IIa and type IIb fiber area scales positively to body mass, using both ordinary least squares (solid lines; IIa: y=(140±56)x + (450±131), R2=0.238, df=21; IIb: y=(393±159)x + (827±373), R2=0.234) and reduced major axis (dashed lines; IIa: y=(288±56)x + (113±132), R2=0.238, df=21; IIb: y=(813±159)x + (132±373), R2=0.234) regressions (df=21). [This figure is in the supplementary material of the published version of this chapter].  171  Fig. 5.9. Regressions between standardized independent contrasts of muscle traits and migration altitude strategy. After phylogeny was taken into account in this way, there were positive relationships between migration altitude strategy and (a) the proportion of all mitochondria that are subsarcolemmal (R2=0.999), (b) capillary to muscle fiber ratio at surface (R2=0.996), intermediate (R2=0.978), and deep (R2=0.999) muscle depths, (c) oxidative (R2=0.993) or IIa (fast myosin-ATPase) (R2=0.999) fiber areal densities at the muscle surface, or (d) capillary density at the muscle surface (R2=0.987). Different colours represent different traits or muscle depths, and ordinary least squares regression lines (forced through the origin, R2 is shown above) are only plotted for those traits having a significant positive correlation with migration altitude strategy (coded by a dummy variable; see chapter text for other details). [This figure is in the supplementary material of the published version of this chapter].  172  Fig. 5.10. (a-b) Representative transmission electron micrographs from bar-headed geese of oxidative (a) and glycolytic (b) muscle fibers (Scale bar represents 2 µm). Arrow, capillary; arrowhead, mitochondria; black asterisk, lipid droplet. (c) Bar-headed goose mitochondria showing the high cristae surface density (Scale bar represents 500 nm). Arrowhead, mitochondrial cristae; white asterisk, myofibril. [This figure is in the supplementary material of the published version of this chapter].  173  Fig. 5.11. Relationship between mitochondrial abundance and capillary density across muscle depths in geese. Average mitochondrial volume density was not as strong a predictor of capillary density as was capillary to fiber ratio (Fig. 5.6b). Ordinary least squares (OLS) (solid line; y=(15157±4621)x + (1074±965), R2=0.729) and reduced major axis (dashed line; y=(17452±5399)x + (1574±1127), R2=0.617) regressions were performed excluding data for bar-headed geese (df=5). The dashed grey line represents the 95% prediction interval of the OLS regression. [This figure is in the supplementary material of the published version of this chapter].  174  Fig. 5.12. Mitochondria are redistributed towards the cell membrane in the oxidative fibers of bar-headed goose muscle. (a) The proportion of mitochondria that were subsarcolemmal was higher in bar-headed geese (N=5) (*) than in both low altitude species (N=4 each) (1-way ANOVA, df=12). (b-c) Representative transmission electron micrographs of oxidative fibers from bar-headed geese (b) and barnacle geese (c). Scale bar represents 2 µm. Arrow, subsarcolemmal mitochondria; arrowhead, intermyofibrillar mitochondrion.  175  Fig. 5.13. Mitochondria isolated from the flight muscle of bar-headed geese (N=6), greylag geese (N=4), barnacle geese (N=3), and mallard ducks (N=9) had similar respiration rates and phosphorylation efficiencies. Two different combinations of malate (mal), pyruvate (pyr), and succinate (succ) were used. (a) Significant increases in respiration occurred in all species as mitochondria were transitioned from state 2 (no ADP or ATP), to state 4 (all ADP converted to ATP), state 3 (maximal ADP stimulation), and TMPD (maximally stimulates cytochrome oxidase) treatment, but FCCP (mitochondrial uncoupler) did not increase respiration any further (2-way ANOVA within each substrate combination, df=109). (b) P/O ratios (ADP/oxygen atom consumed) were lower when succinate was present (2-way ANOVA, df=43).  176  Fig. 5.14. Oxygen kinetics of mitochondria isolated from the flight muscle was similar in bar-headed geese to other bird species (N as in Fig. 5.13). Mitochondrial P50 is the O2 tension that causes 50% inhibition of normoxic respiration, and was determined for state 3 (maximal ADP stimulation), state 4 (no ADP), and TMPD (electron donor that maximally stimulates cytochrome oxidase) treatment. Two different combinations of malate (mal), pyruvate (pyr), and succinate (succ) were used. P50 was significantly different between respiration states (2-way ANOVA within each substrate combination, df=65). 1 Torr = 0.133 kPa. ¶, **, and †† represent significant differences from mallard ducks, greylag geese and mallard ducks, or all other species, respectively.  177  Fig. 5.15. (a) Relationship between mitochondrial respiration rate and O2 tension for a representative bar-headed goose in state 4 with malate, pyruvate, and succinate as substrates of intermediary metabolism (P50 = 0.4 Torr). The grey dashed line represents a hyperbolic curve fit through the data. (b) There was a positive relationship between mitochondrial P50 and respiration rate of mitochondria isolated from the flight muscle of different bird species. P50 is the O2 tension that causes 50% inhibition of normoxic respiration, and was determined for state 3 (maximal ADP stimulation), state 4 (no ADP), or TMPD (electron donor that maximally stimulates cytochrome oxidase) treatment. Two different combinations of malate (mal), pyruvate (pyr), and succinate (succ) were used. 1 Torr = 0.133 kPa. (ordinary least squares regression: solid line, y=(0.00140±0.00006)x + (0.1033±0.0440), R2 = 0.813; reduced major axis regression: dashed line, y=(0.00153±0.00006)x + (0.0095±0.0440), R2 = 0.813; df=130). [This figure is in the supplementary material of the published version of this chapter].  178 5.5. References Andrienko, T., Kuznetsov, A. V., Kaambre, T., Usson, Y., Orosco, A., Appaix, F., Tiivel, T., Sikk, P., Vendelin, M., Margreiter, R. and Saks, V. A. (2003). Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells. J. Exp. Biol. 206, 2059-72. Bassel-Duby, R. and Olson, E. N. (2006). Signaling pathways in skeletal muscle remodeling. Annu. Rev. Biochem. 75, 19-37. Beall, C. M. (2007). Two routes to functional adaptation: Tibetan and Andean highaltitude natives. Proc. Natl. Acad. Sci. USA 104, 8655-8660. Biressi, S., Molinaro, M. and Cossu, G. (2007). Cellular heterogeneity during vertebrate skeletal muscle development. Dev. Biol. 308, 281-293. Bishop, C. M., Butler, P. J., Egginton, S., Elhaj, A. J. and Gabrielsen, G. W. (1995). Development of metabolic enzyme activity in locomotor and cardiac muscles of the migratory barnacle goose. Am. J. Physiol. Reg. Integr. Comp. Physiol. 38, R64-R72. Bohonak, A. J. and van der Linde, K. (2004). RMA: Software for reduced major axis regression, Java version 1.21: http://www.kimvdlinde.com/professional/rma.html. Chappell, M. A. and Snyder, L. R. G. (1984). Biochemical and physiological correlates of deer mouse α-chain hemoglobin polymorphisms. Proc. Natl. Acad. Sci. USA 81, 5484-5488. Cramp, S. and Simmons, K. E. L. (1977). Handbook of the birds of Europe, the Middle East, and North Africa: the birds of the Western Palearctic. Oxford, UK: Oxford University Press.  179 Darveau, C. A., Suarez, R. K., Andrews, R. D. and Hochachka, P. W. (2002). Allometric cascade as a unifying principle of body mass effects on metabolism. Nature 417, 166-170. Degens, H., Deveci, D., Botto-Van Bemden, A., Hoofd, L. J. and Egginton, S. (2006). Maintenance of heterogeneity of capillary spacing is essential for adequate oxygenation in the soleus muscle of the growing rat. Microcirculation 13, 467476. Degens, H., Turek, Z., Hoofd, L. J., Van't Hof, M. A. and Binkhorst, R. A. (1992). The relationship between capillarisation and fibre types during compensatory hypertrophy of the plantaris muscle in the rat. J. Anat. 180, 455-463. Deveci, D., Marshall, J. M. and Egginton, S. (2001). Relationship between capillary angiogenesis, fiber type, and fiber size in chronic systemic hypoxia. Am. J. Physiol. Heart Circ. Physiol. 281, H241-H252. Egginton, S. (1990). Numerical and areal density estimates of fibre type composition in a skeletal muscle (rat extensor digitorum longus). J. Anat. 168. Faraci, F. M., Kilgore, D. L. and Fedde, M. R. (1984). Oxygen delivery to the heart and brain during hypoxia: pekin duck vs. bar-headed goose. Am. J. Physiol. 247, R69-R75. Garland, T., Harvey, P. H. and Ives, A. R. (1992). Procedures for the analysis of comparative data using phylogenetically independent contrasts. Syst. Biol. 41, 1832.  180 Gayeski, T. E. J. and Honig, C. R. (1988). Intracellular Po2 in long axis of individual fibers in working dog gracilis muscle. Am. J. Physiol. Heart Circ. Physiol. 254, H1179-H1186. Gnaiger, E. (2001). Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128, 277-297. Gnaiger, E., Méndez, G. and Hand, S. C. (2000). High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97, 11080-11085. Hochachka, P. W. (1985). Exercise limitations at high altitude: the metabolic problem and search for its solution. In Circulation, respiration, and metabolism (eds. R. Gilles), pp. 240-249. Berlin: Springer-Verlag. Hochachka, P. W. and Burelle, Y. (2004). Control of maximum metabolic rate in humans: dependence on performance phenotypes. Mol. Cell. Biochem. 256, 95103. Hoppeler, H., Howald, H., Conley, K., Lindstedt, S. L., Claassen, H., Vock, P. and Weibel, E. R. (1985). Endurance training in humans: aerobic capacity and structure of skeletal muscle. J. Appl. Physiol. 59, 320-327. Jessen, T.-H., Weber, R. E., Fermi, G., Tame, J. and Braunitzer, G. (1991). Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering. Proc. Natl. Acad. Sci. USA 88, 6519-6522.  181 Kinsey, S. T., Hardy, K. M. and Locke, B. R. (2007). The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle. J. Exp. Biol. 210, 3505-3512. Lauder, G. V., Leroi, A. M. and Rose, M. R. (1993). Adaptations and history. Trends Ecol. Evol. 8, 294-297. León-Velarde, F., Sanchez, J., Bigard, A. X., Brunet, A., Lesty, C. and Monge, C. (1993). High altitude tissue adaptation in Andean coots: capillarity, fiber area, fiber type and enzymatic activities of skeletal muscle. J. Comp. Physiol. B. 163, 52-58. Maddison, W. P. and Maddison, D. R. (2009). Mesquite: A modular system for evolutionary analysis. Version 2.6. http://mesquiteproject.org. Mainwood, G. W. and Rakusan, K. (1982). A model for intracellular energy transport. Can. J. Physiol. Pharmacol. 60, 98-102. Mathieu-Costello, O. (2001). Muscle adaptation to altitude: tissue capillarity and capacity for aerobic metabolism. High Alt. Med. Biol. 2, 413-425. Mathieu-Costello, O., Agey, P. J., Quintana, E. S., Rousey, K., Wu, L. and Bernstein, M. H. (1998a). Fiber capillarization and ultrastructure of pigeon pectoralis muscle after cold acclimation. J. Exp. Biol. 201, 3211-3220. Mathieu-Costello, O., Agey, P. J., Wu, L., Szewczak, J. M. and MacMillen, R. E. (1998b). Increased fiber capillarization in flight muscle of finch at altitude. Respir. Physiol. 111, 189-199.  182 McClelland, G. B., Hochachka, P. W. and Weber, J. M. (1998). Carbohydrate utilization during exercise after high-altitude acclimation: a new perspective. Proc. Natl. Acad. Sci. USA 95, 10288-10293. Midford, P. E., Garland, T. and Maddison, W. P. (2008). PDAP Package of Mesquite. Version 1.14. http://mesquiteproject.org/pdap_mesquite/. Petschow, D., Würdinger, I., Baumann, R., Duhm, J., Braunitzer, G. and Bauer, C. (1977). Causes of high blood O2 affinity of animals living at high altitude. J. Appl. Physiol. 42, 139-143. Rosser, B. W. C., Davis, M. B., Brocklebank, J. R. and George, J. C. (1987). On the histochemical characterization and distribution of fast and slow muscle fibers in certain avian skeletal muscles. Acta Histochem. 81, 85-93. Scott, G. R., Cadena, V., Tattersall, G. J. and Milsom, W. K. (2008). Body temperature depression and peripheral heat loss accompany the metabolic and ventilatory responses to hypoxia in low and high altitude birds. J. Exp. Biol. 211, 1326-1335. Scott, G. R. and Milsom, W. K. (2006). Flying high: a theoretical analysis of the factors limiting exercise performance in birds at altitude. Respir. Physiol. Neurobiol. 154, 284-301. Scott, G. R. and Milsom, W. K. (2007). Control of breathing and adaptation to high altitude in the bar-headed goose. Am. J. Physiol. Reg. Integr. Comp. Physiol. 293, R379-R391. Storz, J. F. and Moriyama, H. (2008). Mechanisms of hemoglobin adaptation to high altitude hypoxia. High Alt. Med. Biol. 9, 148-157.  183 Suarez, R. K., Lighton, J. R., Brown, G. S. and Mathieu-Costello, O. (1991). Mitochondrial respiration in hummingbird flight muscles. Proc. Natl. Acad. Sci. USA 88, 4870-4873. Swan, L. W. (1970). Goose of the Himalayas. Nat. Hist. 70, 68-75. Torrella, J. R., Fouces, V., Palomeque, J. and Viscor, G. (1998). Comparative skeletal muscle fibre morphometry among wild birds with different locomotor behaviour. J. Anat. 192, 211-222. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weibel, E. R. (1979). Stereological Methods. Toronto: Academic Press. Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA, USA: Harvard University Press. Weibel, E. R., Taylor, C. R. and Hoppeler, H. (1991). The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Natl. Acad. Sci. USA 88, 10357-10361. West, J. B. (2000). Human limits for hypoxia: the physiological challenge of climbing Mt. Everest. Ann. N. Y. Acad. Sci. 899, 15-27. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493.  184 6. CONTROL OF RESPIRATION IN FLIGHT MUSCLE FROM THE HIGH ALTITUDE BAR-HEADED GOOSE AND LOW ALTITUDE BIRDS5  6.1. Introduction The bar-headed goose (Anser indicus) flies over the Himalayas twice a year on its migration between southern and central Asia. In doing so, this species flies over the highest mountains in the world, at altitudes of up to 9000m, where barometric pressure and oxygen availability can be extremely low (Swan, 1970). While most mammals cannot even sustain resting levels of metabolism in the hypoxia at these elevations (West, 2000), bar-headed geese can sustain the 10- to 20-fold increases in O2 consumption that are necessary for fuelling flight (Ward et al., 2002). The physiological basis for this impressive feat is not completely understood. While morphological alterations that enhance lift may be important (Lee et al., 2008), high altitude flight in bar-headed geese undoubtedly involves an elevated capacity for transporting O2 during hypoxia. The O2 transport pathway is composed of a series of cascading physiological steps, namely breathing, pulmonary O2 diffusion, circulation via the blood, tissue O2 diffusion, and mitochondrial O2 utilization (Weibel, 1984). Multiple steps in this pathway are enhanced in bar-headed geese (Petschow et al., 1977; Jessen et al., 1991; Scott and Milsom, 2007; Scott et al. 2008, 2009; Chapters 3-5), which should increase O2 supply to mitochondria in the flight muscle during hypoxia. In contrast, less is known about whether factors related to muscle O2 demand are altered in bar-headed 5  A version of this chapter has been accepted for publication: Scott, G. R., Richards, J. G., and Milsom, W. K. Control of respiration in flight muscle from the high-altitude bar-headed goose and low-altitude birds. Am. J. Physiol. Reg. Integr. Comp. Physiol. In press. DOI 10.1152/ajpregu.00241.2009 (used with permission)  185 geese to help sustain the high rates of ATP turnover needed for prolonged flight at high altitude. Mitochondria isolated from the flight muscle of this species have similar properties to those from low altitude birds (Scott et al., 2009; Chapter 5), but given that isolation can disrupt normal mitochondrial function (Andrienko et al., 2003), apparent alterations in the mitochondria of bar-headed geese may only emerge when studied in the in situ environment within muscle cells. An important property of highly oxidative muscle is an efficient coupling between energy supply and demand that promotes metabolite stability (Hochachka, 1993). Many different mechanisms contribute to metabolite stability during changes in muscle work rates (Balaban, 2002; Andrienko et al., 2003; Balaban, 2006), one of which is the creatine kinase shuttle (Ventura-Clapier et al., 1998). With this shuttle, oxidative phosphorylation is strongly coupled to mitochondrial creatine kinase, and phosphate equivalents are rapidly transferred between mitochondria and sites of ATP hydrolysis (i.e., myofibrils and sarcoplasmic reticulum) by sequential near-equilibrium creatine kinase reactions. At a cellular level this can manifest as a low sensitivity to cytosolic ADP and an enhanced sensitivity to creatine for mitochondrial respiration in permeabilized muscle fibers (Kuznetsov et al., 1996; Andrienko et al., 2003). These characteristics are particularly evident in muscle from athletic humans (Zoll et al., 2002), and are enhanced in humans after exercise training in hypoxia (Ponsot et al., 2006) and in rats that have been artificially selected for exercise endurance capacity (Walsh et al., 2006). However, the ADP and creatine sensitivities of bird flight muscle are unknown and it is conceivable that improved creatine kinase shuttling could benefit exercise performance at high altitude.  186 The objective of the present study was to compare the metabolic properties of flight muscle from bar-headed geese to that of strong-flying low altitude birds. Barheaded geese were compared to multiple low altitude species (Fig. 6.1) to account for the effects of phylogenetic history and small differences in body mass. We studied birds that had never flown or experienced high altitude to eliminate the potential effects of physiological plasticity. Uniquely derived differences in bar-headed geese are therefore inherent and may represent important evolutionary specializations for flying at high altitude.  6.2. Materials and Methods 6.2.1. Animals Experiments were performed on bar-headed geese (1.7-3.1 kg), greylag geese (Anser anser) (3.2-5.6 kg), barnacle geese (Branta leucopsis) (1.6-1.7 kg), and mallard ducks (Anas platyrhynchos) (1.1-1.3 kg). These species have a close phylogenetic relationship (Fig. 6.1) (Lee et al., 2008), but barnacle geese, pink-footed geese, greylag geese, and mallard ducks all live at low altitudes, and generally follow low to moderate altitude migration routes (Cramp and Simmons, 1977). Therefore, high altitude migration is probably a derived characteristic of bar-headed geese. All animals were bred and raised at sea level, either at the Animal Care Facility of the University of British Columbia (UBC) or by local suppliers, and were housed outdoors at UBC. All animal care and experimentation was conducted according to UBC animal care protocol #A04-1013.  187 6.2.2. Permeabilized muscle fiber experiments Birds were anaesthetized and maintained at a surgical plane with isoflurane. The pectoralis major muscle was sampled at a site approximately half way along the length of the sternum, 3-5 cm lateral to the carina. Feathers were removed and local analgesia (Lidocaine) was applied to the skin at the site of incision. Small biopsies (~50 mg) were taken from the muscle surface and transferred to ice-cold relaxing and preservation solution (concentration in mM: 2.77 CaK2EGTA, 7.23 K2EGTA, 1.38 MgCl2, 20 taurine, 0.5 dithiothreitol (DTT), 50 potassium-methane sulfonate (K-MES), 5.8 Na2ATP, 15 creatine phosphate, and 5.18 MgCl2; pH 7.1). The skin was then sutured closed and birds were allowed to recover from anaesthesia. After sampling each bird was returned to the Animal Care Facility. Saponin-permeabilized muscle fibers were prepared on ice as previously described (Saks et al., 1998; Ponsot et al., 2006). Fibers in the biopsy bundle were mechanically separated in relaxing and preservation solution using dissecting probes under a dissecting microscope. Separated fibers were permeabilized in the same solution containing 50 µg/ml saponin for 30 min, followed by three 10 min rinses in respiration solution (in mM: 2.77 CaK2EGTA, 7.23 K2EGTA, 1.38 MgCl2, 20 taurine, 0.5 DTT, and 100 K-MES, 3 KH2PO4, and 2 mg/ml fatty acid-free bovine serum albumin; pH 7.1) to wash out endogenous adenine nucleotides and creatine. Fibers were weighed by transferring wet fibers to respiration solution on a tared analytical balance. In situ mitochondrial respiration (10-15 mg wet mass of permeabilized fibers) was measured in 2 ml of respiration solution (with or without 20 mM creatine) with an Oxygraph-2k high-resolution respirometer (Oroboros) at 25°C under continuous stirring.  188 After 5 min, malate (2 mM) followed by pyruvate (5 mM) were added to stimulate state 2 respiration. Stepwise additions of ADP (from 5 to 700 µM), each separated by 3 min or more, resulted in maximal ADP-stimulated respiration (VADP) (Fig. 6.2). Respiration rate increased gradually after each ADP addition, but a stable rate was usually reached within 1-2 min. However, we always waited until a stable rate was expressed for 1 min before proceeding. Respiration was then measured for 3 min after adding succinate (25 mM), and then for 3 min after adding N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD; 0.5 mM) and ascorbate (0.5 mM). TMPD and ascorbate are commonly used to maximally stimulate cytochrome oxidase (COX, complex IV) (Ponsot et al., 2006). The apparent Km values for ADP were calculated by linear interpolation of the ADP concentration at 1/2 VADP and respiration rates are expressed relative to the wet weight of fibers. Because there can be significant barriers to O2 diffusion between the bulk respiration solution and the interior of the fiber bundle lattice, O2 concentration was maintained above 160 µM by re-oxygenating as necessary (preliminary experiments found that maximal respiration was not O2 limited above this concentration).  6.2.3. Enzyme activity assays on homogenized muscle Only bar-headed geese and greylag geese were analyzed for enzyme activities in homogenized muscle. Small muscle biopsies were taken as described above, immediately frozen in liquid N2, and stored at -80°C. The frozen biopsies were weighed and homogenized on ice in 20 volumes of homogenization buffer (in mM: 50 Hepes, 5 EDTA, 0.2 DTT, and 0.1% Triton-X-100; pH 7.4) using a ground glass-on-glass homogenizer. Some homogenate was stored on ice until COX, carnitine  189 palmitoyltransferase (CPT), and pyruvate kinase (PK) activities were assayed. The remaining homogenate was stored at -80°C for later determination of creatine kinase (CK), citrate synthase (CS), 3-hydroxyacyl-coA dehydrogenase (HOAD), and lactate dehydrogenase (LDH) activities. Maximal activites (Vmax) in muscle homogenates were assayed at 41°C in 50 mM Tris (pH 8.0), by measuring absorbance at 340 nm (CK, HOAD, LDH, PK), 412 nm (CPT, CS), or 550 nm (COX) for 5 min. Assays were performed under the following conditions (concentrations in mM unless otherwise stated): COX, 0.05 reduced cytochrome, 0.5% tween-20; CK, 5 creatine, 5 phosphoenolpyruvate, 0.5 NADH, 5 ATP, 10 MgCl2, excess coupling enzymes (PK and LDH); CPT, 5 carnitine, 0.035 palmitoyl-coA, 0.15 5,5-dithiobis-2-nitrobenzoic acid (DTNB); CS, 0.5 oxaloacetate, 0.15 acetyl-coA, 0.15 DTNB; HOAD, 0.1 acetoacetylcoA, 0.5 NADH, 0.2 DTT; LDH, 0.5 NADH, 25 pyruvate; PK, 5 phosphoenolpyruvate, 0.5 NADH, 5 ADP, 0.01 fructose-1,6-bisphosphate, 100 KCl, 10 MgCl2, excess coupling enzyme (LDH). All enzyme assays were run in triplicate. Background reaction rates were measured in control reactions that contained no substrate. Enzyme activities were determined by subtracting the background rate from the rates measured in the presence of substrate. These activities are expressed relative to protein concentration, which was determined using the BCA method (Sigma-Aldrich). Preliminary experiments determined that all substrate concentrations were saturating. The above paragraph also applies to the creatine kinase assays below.  190 6.2.4. Creatine kinase activity assays on isolated mitochondria Mitochondria were isolated from all four bird species as part of a another study (Scott et al., 2009; Chapter 5). Birds were terminally anaesthetized with an intravenous overdose of sodium pentobarbital. The pectoralis major muscle was dissected, and large sample (approximately 6g) from various muscle depths was removed, immediately transferred to 15 ml of ice-cold isolation buffer (in mM: 100 sucrose, 50 tris base, 5 MgCl2, 5 EGTA, 100 KCl, 1 ATP; pH 7.4), and minced. Muscle was digested for 5 min by adding 10 ml of isolation buffer containing proteinase (nagarse; 1 mg/g muscle) and homogenized gently with 5 passes of a Potter-Elvehjem homogenizer (100 rpm). Homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatent was filtered through cheesecloth and then centrifuged at 8700g for 10 min at 4°C (same for all further centrifugation). The pellet was re-suspended in 20 ml isolation buffer and centrifuged, resuspended in 10 ml storage buffer (in mM: 0.5 EGTA, 3 MgCl2, 60 K-methanesulfonate, 20 taurine, 10 KH2PO4, 20 hepes, 110 sucrose, 1 MgCl2, 0.02 vitamin E succinate, 2 pyruvate, 2 malate; pH 7.1), centrifuged, and finally re-suspended in 3 ml of storage buffer. Isolated mitochondria were stored at -80°C until assayed. The contralateral pectoralis major muscle was removed and weighed and twice this recorded value is reported to represent each species’ total flight muscle mass. Due to the presence of pyruvate in the storage buffer, creatine kinase in isolated mitochondria was assayed by a different method to that used for homogenized muscle. Triton-X (0.1%) was added to mitochondrial isolates, which were sonicated on ice for 5 seconds and then stored at -80°C until assayed. Vmax was determined at 41°C in 50 mM Tris (pH 6.8) by measuring absorbance at 340 nm for 5 min. Assay conditions were as  191 follows (in mM): 20 creatine phosphate, 1.5 ADP, 12 AMP, 1.5 NAD, 20 glucose, 6.5 DTT, 25 MgCl2, and excess coupling enzymes (hexokinase and glucose-6-phosphate dehydrogenase).  6.2.5. Data and statistical analyses Data are reported as means ± standard error. Statistical significance was assessed on log-transformed data within and between species using one or two factor (species and treatment) analysis of variance (ANOVA), as appropriate, and Student-Newman-Keuls post-hoc tests. These statistical tests were performed using Sigmastat software (version 4, Systat Software Inc.). We also performed phylogenetically independent contrast analysis (Midford et al., 2005; Maddison and Maddison, 2009) on muscle respiration traits, using a previously employed method that can successfully identified clade-specific differences that are independent of phylogeny (Garland et al., 1992; Scott et al., 2009; Chapter 5). We used the comprehensive phylogeny with branch lengths that we generated in a previous study (Lee et al., 2008), pruned to include only the species in question (Fig. 6.1). Low and high altitude flight strategies were coded as 0 or 1 dummy variable (Garland et al., 1992), respectively. We tested for significant positive relationships between the standardized independent contrasts of muscle respiration traits and flight altitude by determining the one-tailed p-values for correlations computed through the origin (degrees of freedom, df=2 for all). Because variation in branch lengths can influence the outcome of phylogenetic analyses, we also performed the same tests with uniform branch lengths (set  192 to 1). The creatine effect used in this analysis is the difference between the average Km with and without creatine. A significance level of p<0.05 was used throughout.  6.3. Results 6.3.1. Respiration of permeabilized muscle fibers Respiration rates of pectoral muscle fibers changed substantially with respiration state in all species (Fig. 6.3). Transition from resting state 2 respiration (with pyruvate and malate as substrates) to maximal ADP-stimulated respiration increased respiration ~5-fold in all species. This represents the acceptor control ratio (ACR: state 3/state 2), which differs from the more common respiratory control ratio (RCR: state 3/state 4). The presence of endogenous ATPases in permeabilized muscle fibers prevents the establishment of state 4, so RCR cannot be determined, but the high ACR we observed was a good indication that mitochondrial respiration was well coupled in these fibers (Fig. 6.4). Subsequent addition of succinate increased respiration by ~1.8-fold and cytochrome oxidase excess capacity (estimated by respiration after TMPD and ascorbate treatment) was 3- to 3.5-fold above state 3 respiration rates (Figs. 6.3 and 6.4). Resting (state 2) and maximally-stimulated (state 3, succinate, TMPD) respiration rates were similar overall between experiments without creatine (Fig. 6.3A) and with 20 mM creatine (Fig. 6.3B). Small differences in maximal respiration rates of fibers appeared to exist between bar-headed geese and low altitude species. When all low altitude species were grouped together, respiration rates in the presence of creatine were 20-40% higher overall for barheaded goose muscle fibers, and this was particularly evident at higher respiration rates  193 (Fig. 6.3C). These differences were less pronounced on average in the absence of creatine (10-20%), and were not statistically significant. Differences were also less pronounced when individual species were compared statistically (Figs. 6.3A-B), possibly due to the low number of individuals available. However, when aerobic capacity of the whole pectoral muscle mass was estimated, bar-headed geese were higher than all low altitude birds when grouped together and higher than both greylag geese and mallard ducks in pairwise comparisons (Table 6.1). The kinetics of ADP stimulation of muscle respiration were very different in barheaded geese compared to low altitude birds (Fig. 6.5). The ADP concentrations leading to half-maximal stimulation of respiration (i.e., the Km for ADP; inversely proportional to ADP sensitivity) were between 4 µM and 10 µM, and Km was approximately 2-fold higher in bar-headed geese than it was in the low altitude species. In addition, creatine caused a 30% decline of Km in bar-headed geese, but had no effect on Km in the low altitude species. The results of phylogenetically independent contrast analysis on muscle respiration traits largely confirmed the above results. We tested for positive relationships between the standardized contrasts of muscle respiration traits and flight altitude, using a phylogeny with branch lengths either as shown in Fig. 6.1 or set at a uniform length (see Table 6.2 and Materials and Methods). There were significant positive relationships between flight altitude and state 2 respiration in the presence of creatine, TMPDstimulated respiration in the presence of creatine, the Km for ADP, and the effect of creatine on Km (Fig. 6.6). These relationships were driven by the large differences between bar-headed geese and greylag geese. Therefore, the relationship between the  194 unique characteristics of bar-headed geese and their high altitude flight strategy are independent of phylogeny.  6.3.2. Muscle enzyme activities Enzyme activities measured in muscle or in isolated mitochondria were similar between bar-headed geese and greylag geese (Table 6.3), and were comparable to previous measurements in other goose species (Bishop et al., 1995). In surface pectoral muscle biopsies, activities of the mitochondrial enzymes cytochrome oxidase and citrate sythase were nearly equivalent, but these were higher than the activities of the fat metabolism enzymes 3-hydroxyacyl-coA dehydrogenase and carnitine palmitoyl transferase (the slightly lower activity of the latter enzyme in bar-headed geese approached statistical significance, p=0.08). The glyocolytic enzymes, lactate dehydrogenase and pyruvate kinase, as well as creatine kinase (CK) had much greater activities than the mitochondrial enzymes. CK activities in bar-headed geese and greylag geese were 7- to 8.5-fold higher in isolated mitochondria than in muscle. Although mitochondrial CK activity was similar in bar-headed geese, greylag geese, and mallard ducks, CK was reduced in muscle mitochondria from barnacle geese compared to the other two low altitude species (Table 6.3).  6.4. Discussion Bar-headed geese fly at altitudes of up to 9000m during their biannual migration over the Himalayas (Swan, 1970). Despite the potentially drastic reductions in O2 availability at these altitudes, bar-headed geese can sustain the 10- to 20-fold increases in  195 metabolism and O2 consumption that are necessary for flight (Ward et al., 2002). Our present findings suggest that the respiratory properties of flight muscle are altered in barheaded geese, in addition to several unique specializations for enhancing mitochondrial O2 supply during hypoxia (Petschow et al., 1977; Jessen et al., 1991; Scott and Milsom, 2006; Scott and Milsom, 2007; Scott et al., 2008; Scott et al., 2009; Chapters 2-5). Respiratory capacities of bar-headed goose muscle were higher than those of low altitude species (Fig. 6.3, Table 6.1) and the creatine sensitivity of mitochondrial respiration was increased (Fig. 6.5). These unique characteristics of bar-headed geese may promote fatigue resistance and a more efficient coupling of ATP supply and demand during hypoxia, which should be extremely beneficial for high altitude flight.  6.4.1. Aerobic capacity of bar-headed goose flight muscle The present study supports our previous work (Scott et al., 2009; Chapter 5) suggesting that aerobic capacity of bar-headed goose flight muscle is enhanced. The proportion of oxidative fibers in the flight muscle of bar-headed geese was increased compared to low altitude birds, particularly in superficial muscle regions, and this increased the overall mitochondrial abundance (Scott et al., 2009; Chapter 5). In the current study, permeabilized muscle fibers from superficial muscle regions of bar-headed geese also appeared to have slightly higher respiratory capacities (Fig. 6.3) while intrinsic functional properties of respiratory chain complexes were unchanged (Fig. 6.4). The higher respiratory capacity in muscle fibers of bar-headed geese was caused primarily by differences in fiber composition, because (i) the magnitude of interspecific differences in these traits were similar, and because both (ii) mitochondrial abundance within individual  196 fiber types and (iii) respiration of isolated mitochondria are conserved between species (Scott et al., 2009; Chapter 5). Furthermore, bar-headed geese had a higher mass-specific aerobic capacity when these values were extrapolated to the entire flight muscle mass (Table 6.1). These calculated aerobic capacities likely underestimate the true values however, because oxidative fiber proportion increases with muscle depth in bird flight muscle (Mathieu-Costello et al., 1998a; Scott et al., 2009; Chapter 5). Curiously, the activities of aerobic enzymes were not enhanced in bar-headed geese (Table 6.3), consistent with previous measurements in the locomotor muscle of some high altitude mammals (Sheafor, 2003), suggesting that there may be a disconnect between the maximal activities in homogenized muscle and the capacities that are realized in semiintact muscle. High altitude exposure has been shown to influence aerobic capacity in many different species. For example, some birds living at high altitude have enhanced mitochondrial abundance (an important determinant of aerobic capacity) in their flight muscle (Hepple et al., 1998; Mathieu-Costello et al., 1998b). High altitude acclimatization can also increase muscle aerobic capacity in mammals, although the generality of this phenomenon is controversial (Mathieu-Costello, 2001). However, the higher aerobic capacity of bar-headed geese exists before they ever fly or experience high altitude, so it probably has an inherent genetic basis rather than being a consequence of physiological plasticity. Exercise or acclimatization to high altitude could amplify the differences in muscle physiology, and it is possible that phenotypic plasticity in response to these factors has evolved in bar-headed geese.  197 The benefit of a higher aerobic capacity at high altitude is unclear, but it is suggested to be a strategy for increasing the total mitochondrial O2 flux of an entire muscle when intracellular O2 tensions ( PiO 2 ) fall (Hochachka, 1985). Mitochondrial respiration can become O2 limited at low PiO 2 (Gnaiger, 2003), and bar-headed goose  ! mitochondria are just as sensitive to low O2 as those from low altitude species (Scott et ! al., 2009; Chapter 5). The maximum attainable respiration of an individual fiber in the flight muscle of bar-headed geese could therefore be impaired if PiO 2 declines during flight at high altitude. Having more oxidative fibers (and thus a higher aerobic capacity) should counterbalance this inhibition of respiration by ! intracellular hypoxia in each individual fiber. In other words, higher maximum respiration in normoxia may represent a surplus capacity that protects against O2 limitation in hypoxia. This could explain why an enhanced aerobic capacity was favored in bar-headed geese, but other factors may be important as well. For example, flight in hypobaric air might be more energetically costly than flight at sea level (Lee et al., 2008), which could also favor an increase in aerobic capacity.  6.4.2. Control of respiration in bar-headed goose flight muscle Mitochondrial sensitivities to ADP and creatine are known to vary between muscle fiber types, as well as between muscles of similar fiber composition but different aerobic capacities (due to either inherent differences or exercise training) (Kuznetsov et al., 1996; Zoll et al., 2002; Ponsot et al., 2006; Walsh et al., 2006). The more oxidative muscle fibers of mammals (cardiac and slow oxidative) have much lower ADP sensitivities (typically Km ≥ 300 µM) and a high sensitivity to creatine (Kuznetsov et al.,  198 1996). These are thought to be important characteristics of the creatine kinase (CK) shuttle, which is one of multiple systems contributing to efficient coupling between energy supply and demand (Ventura-Clapier et al., 1998; Andrienko et al., 2003). Birds express a mitochondrial isoenzyme of creatine kinase (mi-CK) in their heart and skeletal muscle (Hossle et al., 1988), and like in mammals it is kinetically coupled to oxidative phosphorylation in cardiac cells (Ventura-Clapier et al., 1998). However, the cardiac muscle of birds lacks the functional coupling between cytosolic CK and myosin ATPase that is thought to be an important part of this system (Ventura-Clapier et al., 1998). Control of mitochondrial respiration by creatine kinase in the oxidative muscle of birds may therefore be important but operate by a distinct mechanism from that in mammals. The flight muscle of strong-flying birds is composed primarily of fast oxidative (type IIa) fibers, with a secondary contribution by fast glycolytic (type IIb) fibers and no slow oxidative (type I) fibers (Rosser et al., 1987; Mathieu-Costello et al., 1998a; Torrella et al., 1998; Scott et al., 2009; Chapter 5). Not surprisingly, the ADP sensitivities we determined (Fig. 6.5) were similar to those of fast-twitch mammalian muscle fibers (Kuznetsov et al., 1996). Flight muscle of low altitude birds was also similar to mammalian type II fibers in having negligible sensitivities to creatine, indicating a lack of functional coupling between mi-CK and oxidative phosphorylation. The high activity of creatine kinase in mitochondria from low altitude birds may therefore operate to regenerate ADP locally but not to control respiration. In contrast to low altitude birds, bar-headed geese were sensitive to creatine and had a lower sensitivity to ADP in their flight muscle (Fig. 6.5). This effect of creatine suggests that mitochondrial creatine kinase may control mitochondrial metabolism in bar-  199 headed geese but not in low altitude species, which may be important for helping match ATP supply and demand and improving fatigue resistance in hypoxia. Its potential value for enhancing performance in hypoxia is supported by previous studies in humans, where exercise training in hypoxia increased both endurance (time to exhaustion) and creatine sensitivity compared to training in normoxia (Ponsot et al., 2006). However, the Km for ADP in bar-headed geese is still in the range of those exhibited by type II fibers of mammals (Kuznetsov et al., 1996), which do not typically possess such a system of CKbased mitochondrial regulation, so this possibility should be interpreted with some caution. If this unique characteristic of bar-headed geese is indeed involved in sustaining ATP turnover it should be very important during flight at high altitude, where both hypoxia and the costs of generating lift in hypobaric air could challenge performance. The presence of creatine sensitivity in bar-headed geese may result from differences in creatine kinase function and/or localization within mitochondria. It is clearly not caused by in vitro differences in mitochondrial creatine kinase activity (Table 6.3) and it is unlikely that the relatively small increase in the oxidative fiber proportion of bar-headed goose muscle (Scott et al., 2009; Chapter 5) can account for a characteristic that is otherwise absent in low altitude waterfowl. Nevertheless, if an inherent difference in bar-headed goose mi-CK does exist, and if it is important for intracellular energy shuttling, it may compensate for the altered mitochondrial distribution in this species. Mitochondria are redistributed towards the cell membrane in oxidative fibers from barheaded goose flight muscle (Scott et al., 2009; Chapter 5), which could hinder the intracellular movement of ATP equivalents if compensatory systems are not in place (Kinsey et al., 2007). Furthermore, it is possible that these differences are causally  200 related, arising from functional differences between subsarcolemmal and intermyofibrillar populations of mitochondria (Krieger et al., 1980; Cogswell et al., 1993).  6.4.3. Evolution of hypoxia tolerance Adaptation to environmental hypoxia has occurred across vertebrates and has led to the evolution of various strategies for matching O2 supply and demand (Boutilier, 2001; Hopkins and Powell, 2001; Nilsson and Renshaw, 2004; Bickler and Buck, 2007; Ramirez et al., 2007). Perhaps the most pervasive strategy is a coordinated metabolic depression that reduces O2 demands during hypoxia (Hochachka et al., 1996). This strategy is extremely important for species that have the luxury of being inactive, but for animals like the bar-headed goose, exercise must continue in hypoxia. Therefore, evolutionary changes in O2 demand processes will only be beneficial if they do not limit rates of ATP turnover. Our present findings illustrate two such potential mechanisms. On the one hand, bar-headed goose flight muscle probably has an increased aerobic capacity, which may represent a surplus to counteract the depressive effects of hypoxia on mitochondrial respiration. On the other hand, creatine kinase is functionally coupled to oxidative phosphorylation in the muscle of bar-headed geese, which may promote a more efficient coupling of ATP supply and demand, and thus contribute to metabolite stability and fatigue resistance. These uniquely derived characteristics of bar-headed geese are not a product of phylogenetic history (as the associations between these traits and high altitude flight were independent of phylogeny; Fig. 6.6) or prior exercise or hypoxia exposure, and may represent important adaptations for exercising in hypoxia. However,  201 adaptation to other challenges at high altitude (e.g., generating lift in low density air) or evolutionary forces other than adaptation (Lauder et al., 1993) could also be involved, and future work on the performance outcomes of these differences in muscle physiology would be very instructive. The differences in muscle metabolism occur in conjunction with several evolutionary changes in the O2 transport pathway of bar-headed geese for improving O2 flux in hypoxia. Pulmonary O2 uptake is increased in this species due to an enhanced hypoxic ventilatory response and a more effective breathing pattern (Scott and Milsom, 2007; Scott et al., 2008; Scott and Milsom, In press; Chapters 3-4). Blood O2 carrying capacity is also increased in hypoxia by an elevated hemoglobin O2 affinity (Petschow et al., 1977; Jessen et al., 1991; Zhang et al., 1996), which assures higher O2 delivery throughout the body (Faraci et al., 1984; Faraci et al., 1985). The O2 diffusing capacity in the flight muscle is also enhanced by an increased capillarity and a redistribution of mitochondria closer to capillaries (Scott et al., 2009; Chapter 5), which should increase O2 extraction from the blood during hypoxia. Multiple evolutionary changes thus defend O2 supply in hypoxia, which probably work in concert with changes in muscle O2 demand processes to sustain ATP turnover and muscle performance. Overall, our work and that of others suggest that high altitude flight in bar-headed geese is associated with evolutionary alterations to a number of physiological systems, which may be essential for this species’ exceptional ability to fly high.  202 6.5. Summary of Chapter •  The respiratory properties of permeabilized muscle fibers were compared between bar-headed geese and several low altitude waterfowl species (greylag geese, barnacle geese, mallard ducks).  •  Respiratory capacities were generally higher in bar-headed geese when creatine was present. This represented a higher mass-specific aerobic capacity when respiration rates were extrapolated to the entire pectoral muscle mass.  •  The ADP concentration leading to half-maximal stimulation (Km) was ~2-fold higher in bar-headed geese (10 vs. 4-6 µM) when creatine was not present.  •  Creatine reduced Km by 30% in bar-headed geese, but had no effect on Km in low altitude birds. This suggested that mitochondrial creatine kinase regulates oxidative phosphorylation in bar-headed geese but not in low altitude birds, but this was not based on differences in creatine kinase activity.  •  The unique differences in bar-headed geese existed without prior exercise or high altitude exposure and were not a result of phylogenetic history, and may promote efficient coupling of ATP supply and demand during flight.  203 Table 6.1. Aerobic capacity of the flight muscle is enhanced in bar-headed geese  Bar-Headed Goose Greylag Goose Barnacle Goose Mallard Duck  Body Mass (kg) 2.27 ± 0.20 4.18 ± 0.14 1.68 ± 0.04 1.18 ± 0.04  Pectoralis Major Mass (g) 454 ± 42 643 ± 14 368 ± 10 226 ± 14  Muscle Aerobic Capacity (mmol O2/kg/min) 120 ± 6*,† 63 ± 7 98 ± 11 76 ± 10  Low Altitude Birds  2.22 ± 0.38  387 ± 52  77 ± 6  Muscle aerobic capacity was estimated from the product of the succinate-stimulated respiration in permeabilized muscle fibers (the higher of treatments with or without creatine) and body mass specific pectoral muscle mass. * and † denote significantly higher respiration for bar-headed geese compared to greylag geese or mallard ducks, and the overall average of all three low altitude bird species, respectively (P < 0.05) (N as in Fig. 6.3).  204 Table 6.2. Statistical results from phylogenetically independent contrast analysis Trait State 2 State 3 Succinate TMPD State 2 State 3 Succinate TMPD Km Creatine effect  Actual Branch Lengths Uniform Branch Lengths r Slope r Slope Respiration without creatine 0.991 0.014 0.887 ns 0.982 0.060 0.792 ns 0.956 0.093 0.569 ns 0.914 0.089 0.366 ns Respiration with creatine (20 mM) 0.994 0.023 0.932 0.020 0.987 0.080 0.807 ns 0.987 0.147 0.805 ns 0.993 0.208 0.923 0.182 ADP kinetics 0.999 5.62 0.982 5.43 0.997 -3.24 0.949 -3.35  Correlations of standardized contrasts of each muscle respiration trait to migration altitude strategy (coded by a dummy variable) were calculated through the origin (df=2). Ordinary least squares regression slopes are only shown for significant correlations. r, Pearson product-moment correlation coefficient; ns, not significant. See Materials and Methods and Fig. 6.6.  Table 6.3. Activities of metabolic enzymes from superficial biopsies of flight muscle  Cytochrome oxidase Citrate synthase 3-Hydroxyacyl-coA dehydrogenase Carnitine palmitoyl transferase Pyruvate kinase Lactate dehydrogenase Creatine kinase Creatine kinase  Bar-Headed Goose Greylag Goose Homogenized muscle 0.35 ± 0.04 0.38 ± 0.05 0.30 ± 0.02 0.28 ± 0.04 0.025 ± 0.006 0.043 ± 0.007 0.0059 ± 0.0005 0.0062 ± 0.0010 3.83 ± 0.26 4.27 ± 0.31 7.57 ± 1.20 8.84 ± 0.90 0.98 ± 0.05 0.97 ± 0.05 Isolated mitochondria 6.91 ± 0.70 8.50 ± 1.61  Barnacle Goose  Mallard Duck  -  -  4.85 ± 0.32†  8.96 ± 0.66  All activities were measured at 41°C and are reported as µmol substrate/mg protein/min. There were no significant differences in enzyme activities of muscle homogenates between bar-headed geese (N=8) and greylag geese (N=7). † denotes a significantly lower creatine kinase activity in muscle mitochondria from barnacle geese compared to greylag geese and mallard ducks (P < 0.05) (N as in Fig. 6.3).  205  206  Fig. 6.1. Hypothesized phylogenetic tree for the species examined in the present study, pruned from the comprehensive phylogeny that we generated in a previous study (Lee et al., 2008). The tree was used for phylogenetically independent contrast analysis, with branches set either at the lengths shown or to uniform length (i.e., all branch lengths=1).  207  Fig. 6.2. Representative experiment showing the respiration of permeabilized fibers from the flight muscle of a bar-headed goose. (A) Arrows represent additions of malate (m), pyruvate (p), or ADP (cumulative concentration of ADP is shown in µM), and O2 consumption rate (VO2) was measured from the change in O2 concentration over time. Artifactual spikes in the VO2 trace are often observed after additions, followed by gradual increases in respiration until a new steady rate is reached. (B) The resulting plot of VO2 against [ADP] illustrates how ADP stimulates respiration rate.  208  Fig. 6.3. Respiration of permeabilized fibers from the flight muscle of birds, measured in the absence (A) or presence of 20 mM creatine (B). Significant increases in respiration occurred in all species as mitochondria were transitioned from state 2 (no ADP or ATP), to state 3 (maximal ADP stimulation) with only malate and pyruvate, state 3 with succinate, and TMPD treatment (maximally stimulates cytochrome oxidase). There were no significant differences between bar-headed geese (N=6), greylag geese (N=4), barnacle geese (N=3), or mallard ducks (N=6). (C) Rates of respiration of fibers in the presence of creatine (Cr) were higher overall for bar-headed geese than for all low altitude individuals when grouped together. * denotes a significantly higher respiration for bar-headed geese in a pair-wise comparison to all low altitude birds (P < 0.05).  209  Fig. 6.4. Effects of changing respiration state on the relative changes in O2 consumption rate, measured in the absence (A) or presence of 20 mM creatine (B). ACR, acceptor control ratio (quotient of state 3 and state 2 respiration); VADP, state 3 respiration; VSucc, respiration after succinate addition; VTMPD, respiration after TMPD and ascorbate addition (N as in Fig. 6.3).  210  Fig. 6.5. ADP kinetics of permeabilized fibers from the flight muscle of birds, measured in the absence (open bars) or presence of 20 mM creatine (hatched bars). * denotes a significantly higher Km (the ADP concentration causing half-maximal stimulation of respiration) in bar-headed geese, and  denotes a significant reduction in Km due to creatine (P < 0.05) (N as in Fig. 6.3).  211  Fig. 6.6. Regressions between standardized independent contrasts of muscle respiration traits and flight altitude strategy. After phylogeny was taken into account in this way, there were positive relationships between flight altitude and (A) state 2 respiration in the presence of creatine (R2=0.988), (B) TMPD-stimulated respiration in the presence of creatine (R2=0.987), (C) Km for ADP (R2=0.998), and (D) the effect of creatine on the Km for ADP (R2=0.993). 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Exercise training in normobaric hypoxia in endurance runners. II. Improvement of mitochondrial properties in skeletal muscle. J. Appl. Physiol. 100, 1249-1257. Ramirez, J. M., Folkow, L. P. and Blix, A. S. (2007). Hypoxia tolerance in mammals and birds: from the wilderness to the clinic. Annu. Rev. Physiol. 69, 113-143. Rosser, B. W. C., Davis, M. B., Brocklebank, J. R. and George, J. C. (1987). On the histochemical characterization and distribution of fast and slow muscle fibers in certain avian skeletal muscles. Acta Histochem. 81, 85-93. Saks, V. A., Veksler, V. I., Kuznetsov, A. V., Kay, L., Sikk, P., Tiivel, T., Tranqui, L., Olivares, J., Winkler, K., Wiedemann, F. and Kunz, W. S. (1998). Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol. Cell. Biochem. 184, 81-100. Scott, G. R., Cadena, V., Tattersall, G. J. and Milsom, W. K. (2008). Body temperature depression and peripheral heat loss accompany the metabolic and ventilatory responses to hypoxia in low and high altitude birds. J. Exp. Biol. 211, 1326-1335. Scott, G. R., Egginton, S., Richards, J. G. and Milsom, W. K. (2009). Evolution of muscle phenotype for extreme high altitude flight in the bar-headed goose. Proc. R. Soc. B In press. Scott, G. R. and Milsom, W. K. (2006). Flying high: a theoretical analysis of the factors limiting exercise performance in birds at altitude. Respir. Physiol. Neurobiol. 154, 284-301.  217 Scott, G. R. and Milsom, W. K. (2007). Control of breathing and adaptation to high altitude in the bar-headed goose. Am. J. Physiol. Reg. Integr. Comp. Physiol. 293, R379-R391. Scott, G. R. and Milsom, W. K. (In press). Control of breathing in birds: implications for high altitude flight. In Cardio-Respiratory Control in Vertebrates (eds. M. L. Glass and S. C. Wood). Berlin: Springer-Verlag. Sheafor, B. A. (2003). Metabolic enzyme activities across an altitudinal gradient: an examination of pikas (genus Ochotona). J. Exp. Biol. 206, 1241-1249. Swan, L. W. (1970). Goose of the Himalayas. Nat. Hist. 70, 68-75. Torrella, J. R., Fouces, V., Palomeque, J. and Viscor, G. (1998). Comparative skeletal muscle fibre morphometry among wild birds with different locomotor behaviour. J. Anat. 192, 211-222. Ventura-Clapier, R., Kuznetsov, A., Veksler, V., Boehm, E. and Anflous, K. (1998). Functional coupling of creatine kinases in muscles: species and tissue specificity. Mol. Cell. Biochem. 184, 231-247. Walsh, B., Hooks, R. B., Hornyak, J. E., Koch, L. G., Britton, S. L. and Hogan, M. C. (2006). Enhanced mitochondrial sensitivity to creatine in rats bred for high aerobic capacity. J. Appl. Physiol. 100, 1765-1769. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA, USA: Harvard University Press.  218 West, J. B. (2000). Human limits for hypoxia: the physiological challenge of climbing Mt. Everest. Ann. N. Y. Acad. Sci. 899, 15-27. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493. Zoll, J., Sanchez, H., N'Guessan, B., Ribera, F., Lampert, E., Bigard, X., Serrurier, B., Fortin, D., Geny, B., Veksler, V., Ventura-Clapier, R. and Mettauer, B. (2002). Physical activity changes the regulation of mitochondrial respiration in human skeletal muscle. J. Physiol. 543, 191-200.  219 7. GENERAL DISCUSSION AND CONCLUSIONS  The general objective of this thesis was to determine the mechanisms underlying high altitude flight in bar-headed geese. The preceding chapters have clearly shown that multiple evolutionary changes in the O2 transport pathway underlie the ability of this species to migrate at extremely high altitudes. By incorporating both theoretical modelling and empirical observations, I have shown that the steps in the O2 cascade with the greatest potential for increasing O2 supply during hypoxia were enhanced in barheaded geese. These unique specializations should be of primary importance for sustained exercise in hypoxia, and they occur along with additional alterations in biochemical processes that could optimize O2 demands. In considering the multiple unique features of the respiratory physiology of bar-headed geese, this thesis provides important insight into the integrative nature of physiological evolution.  7.1. Mechanisms Underlying Extreme High Altitude Flight in the Bar-Headed Goose The ability to sustain large increases in metabolism and O2 consumption rate during flight at high altitude (Ward et al., 2002) indicates that bar-headed geese have an exceptional capacity to transport O2 during hypoxia. The theoretical modelling in Chapter 2 suggested that three specific traits in the O2 cascade have the greatest potential for increasing O2 transport during hypoxia: (i) a heightened capacity to increase ventilation; (ii) a high haemoglobin O2 affinity; and (iii) an enhanced O2 diffusing capacity in the muscle. Based on these findings, I predicted that the same traits would have evolved in bar-headed geese in a manner that supports flight at high altitude. This was indeed the  220 case, and the mechanisms accounting for these changes were often novel and unexpected. These are discussed below, as are additional evolutionary changes in this species associated with the properties of muscle O2 demand.  7.1.1. Ventilatory and pulmonary systems The ventilatory response to poikilocapnic (uncontrolled CO2) hypoxia was enhanced in bar-headed geese compared to low altitude waterfowl, and this was primarily due to higher tidal volumes rather than higher breathing frequencies (Chapter 3). This was particularly apparent in severe hypoxia, during which breathing and arterial O2 tension were elevated by ~50% and ~20% (6 Torr) compared to other waterfowl. This was probably caused by two main factors. Firstly, bar-headed geese experienced less metabolic depression during hypoxia. Although this could not be concluded from measurements of O2 consumption, it was strongly suggested by the delayed changes in body temperature and heat loss seen during hypoxia in this species (see Discussion in Chapters 3 and 4). This should minimize the strong inhibitory effects metabolic depression can have on the drive to breathe during hypoxia (Barros et al., 2006). Secondly, bar-headed geese were less sensitive to the reductions in CO2 that occur during poikilocapnic hypoxia. This was concluded because their ventilatory response to hypoxia was similar to that of pekin ducks when isocapnia (constant CO2) was maintained, indicating that their O2 sensitivities were similar (Chapter 3). Interestingly, this change in CO2 chemosensitivity was restricted to hypocapnic conditions, because their ventilatory response to high CO2 was still intact. This differs from human populations from high altitude, who express altered O2 (rather than CO2) chemosensitivity of breathing  221 (Brutsaert et al., 2005; Wu and Kayser, 2006). Nevertheless, these mechanisms allowed bar-headed geese to maintain higher rates of total ventilation and, more importantly for O2 transport, higher effective ventilation of the pulmonary gas exchange surface. Although the enhanced ventilatory response to hypoxia probably contributes to higher arterial O2 tensions and increased O2 loading, it is possible that an enlarged pulmonary O2 diffusing capacity also plays a role. Preliminary measurements suggest that bar-headed geese have larger lungs than low altitude waterfowl (G.R. Scott and W.K. Milsom, unpublished), which should lead to proportional increases in pulmonary surface area due to the rigid structure of avian lungs (Macklem et al., 1979; Maina, 2002). This is consistent with findings in many hypoxia-tolerant mammals (Widmer et al., 1997; Hammond et al., 2001; Hsia et al., 2005), but is somewhat surprising given the small influence of this variable on O2 transport in hypoxia determined from the modelling study of low altitude birds (Chapter 2).  7.1.2. Circulatory system The enhanced hypoxic ventilatory response and pulmonary O2 diffusing capacity in bar-headed geese will likely reduce but not eliminate the declines in arterial O2 tension during hypoxia. The higher haemoglobin-O2 affinity of this species compared to low altitude birds will therefore be essential for defending O2 saturation and increasing blood O2 content in hypoxia (Petschow et al., 1977; Chapter 3). This unique trait is caused largely by a single amino acid change at the interface between α and β subunits that is unique among birds (Jessen et al., 1991; Zhang et al., 1996). By increasing blood O2  222 content, O2 delivery to various tissues is enhanced during hypoxia (Faraci et al., 1984b; Faraci et al., 1985).  7.1.3. Oxygen diffusing capacity in the muscle Bar-headed geese have increased capillarity in their flight muscle compared to low altitude waterfowl, which was particularly apparent from the number of capillaries surrounding each muscle fiber (Chapter 5). Given that the size of the capillary to muscle fiber interface is the primary morphological determinant of muscle O2 transport (Hepple, 2000; Mathieu-Costello, 2001), this enhancement of capillarity will increase the O2 diffusing capacity from the blood. This alteration in the flight muscle should be extremely important for sustaining O2 flux during flight in hypoxia, but an increased capillarity is also present in other muscles of bar-headed geese: capillarity in the gastrocnemius muscle is higher in this species than in Canada geese (Branta canadensis), even after the latter were born and raised in moderate hypoxia (Snyder et al., 1984). The mitochondria of bar-headed geese are also redistributed towards the cell membrane and closer to the more abundant capillaries (Chapter 5). With a greater proportion of mitochondria in a subsarcolemmal location, the distance needed for O2 diffusion will be reduced. This should increase O2 tensions at mitochondria relative to the low and homogeneous O2 tensions that are generally maintained throughout the sarcoplasm (Gayeski and Honig, 1988; Wittenberg and Wittenberg, 2003). This specialization may explain why the abundance of myoglobin is not enhanced in the pectoralis, cardiac, or gastrocnemius muscles of bar-headed geese (Snyder et al., 1984;  223 Saunders and Fedde, 1991), in contrast to other high altitude and hypoxia-tolerant animals (Reynafarje et al., 1975; Lechner, 1976; Widmer et al., 1997).  7.1.4. Oxygen utilization in the muscle Aerobic capacity of the flight muscle is increased in bar-headed geese compared to low altitude waterfowl (Chapters 5 and 6). This species has a 5-15% higher proportion of oxidative fibers in its flight muscle, which appeared to result in slightly higher respiration rates for their muscle fibers in vitro. This evolutionary change is not based on differences in the inherent respiratory capacities of mitochondria, and is not accompanied by increases in phosphorylation efficiency. Although this alteration should enhance exercise capacity in normoxia, it is probably more important as a surplus aerobic capacity that counteracts the depressive effects of hypoxia on mitochondrial respiration (Hochachka, 1985). This is particularly important when considering that mitochondria isolated from bar-headed goose muscle are no better at sustaining respiration during hypoxia than those from low altitude birds (Chapter 5). Control of mitochondrial respiration is also altered in bar-headed goose muscle (Chapter 6). Respiration of muscle fibers was less sensitive to ADP and more sensitive to creatine in this species, suggesting that creatine kinase regulates oxidative phosphorylation in bar-headed geese but not in low altitude waterfowl. This may improve the efficient matching of ATP supply and demand (Ventura-Clapier et al., 1998; Andrienko et al., 2003), which would have important benefits for fatigue resistance and metabolite stability during flight in hypoxia.  224 7.1.5. Costs and tradeoffs associated with high altitude flight ability Although many of the evolutionary changes in bar-headed geese are likely beneficial for improving O2 transport in hypoxia, it is intriguing to consider whether there are costs or tradeoffs associated with these changes. This is probably the case for some of the unique characteristics of this species: (i) the elevated hypoxic ventilatory response should necessitate higher energy expenditure by respiratory muscles, and may also increase the need to compensate pH disturbances that arise from respiratory hypocapnia (Chapter 3); (ii) the higher haemoglobin O2 affinity may be detrimental at sea level because it impairs O2 unloading at the tissues (Chapter 2); and (iii) the redistribution of mitochondria towards the cell membrane could increase ATP diffusion distances (Chapter 5), which may have favoured compensatory mechanisms for shuttling ATP equivalents (Chapter 6). It is less clear whether there are costs associated with the other evolutionary changes in bar-headed geese, such as the higher muscle capillarity, but there could well be an added energetic expense for building and maintaining these features. Nevertheless, the potential costs of enhancing O2 transport capacity may justify why barheaded geese alone possess these unique traits, particularly when considering that the low altitude species studied in this thesis are strong fliers with lengthy migration routes who could otherwise benefit from these traits.  7.2. Evolution of Respiratory Systems Two different theories for how respiratory systems evolve and how they can be reconciled were discussed in Chapter 1. It was argued that individual steps in the O2 transport pathway can evolve to increase overall pathway flux, and that continued  225 enhancement of aerobic performance may increase the capacity of all steps in the pathway over longer evolutionary time scales. The pattern of how these steps change should be influenced by potential constraints on evolutionary change, selection against excess structural capacity (as previously suggested: Weibel et al., 1981; Weibel et al., 1991), and the likelihood that individual steps will only improve pathway flux to a limited extent without subsequent increases in the capacity of other steps (Gonzalez et al., 2006). In such a scenario, it then becomes important to understand which steps in the O2 transport pathway are most likely to change during the course of evolution. The overriding hypothesis in this thesis was that changes in the steps of the O2 pathway with the most control over O2 transport in hypoxia would have the greatest advantage for performance, and would therefore have evolved in bar-headed geese to support high altitude flight. This hypothesis was largely supported by the findings of this thesis and the work of others (discussed in section 7.1 above), as the three traits that should have the greatest influence over O2 transport in hypoxia were enhanced in barheaded geese. Furthermore, some other traits that should have less control over pathway flux in hypoxia, such as regional blood flow distribution (Faraci et al., 1984b; Faraci et al., 1985), blood haemoglobin concentration (Black and Tenney, 1980), or the Bohr effect of haemoglobin (Liang et al., 2001; Chapter 3), were similar between bar-headed geese and low altitude birds. However, the preliminary finding that bar-headed geese have larger lungs than low altitude waterfowl is at odds with the predictions from the theoretical modelling. It may be that the modelling underestimated how much control the O2 diffusing capacity in the lungs has over O2 pathway flux in hypoxia. Alternatively, this trait could have only a small controlling influence over O2 transport in low altitude  226 waterfowl, but gained more control at some point after bar-headed geese diverged from their lowland ancestors, potentially due to prior evolutionary changes in other steps in the O2 pathway (e.g., ventilatory control). What is clear from this thesis is that the ability to fly at high altitude is associated with a suite of evolutionary changes in bar-headed geese, which occur at multiple steps in the O2 transport pathway. Oxygen supply to the flight muscle should be enhanced during exercise in hypoxia by the changes in ventilatory control, haemoglobin O2 affinity, and O2 diffusing capacity in the muscle in this species. Oxygen demand processes are also altered in bar-headed geese, such as muscle aerobic capacity and the control of mitochondrial respiration by creatine kinase, which may help sustain ATP turnover in hypoxia. Altogether, these unique characteristics of bar-headed geese are consistent with an enhanced capacity for matching O2 supply and demand during flight at extremely high altitudes.  7.3. Future Directions Some additional traits in the O2 pathway must be studied in bar-headed geese before a comprehensive understanding of respiratory system evolution is attained. The preliminary evidence suggesting that pulmonary O2 diffusing capacity is enhanced in barheaded geese should be verified with physiological and/or morphometric measurements, and could be complemented by studies of pulmonary blood flow regulation (Faraci et al., 1984a; West et al., 2006; West et al., 2007). Sustaining cardiac performance is undoubtedly important for exercising in hypoxia, so measurements of heart function and O2 diffusing capacity would be extremely informative (Hussain et al., 2001). Studies of  227 other biochemical aspects of muscle and heart function could also be revealing, such as hypoxic signalling or the production and tolerance of reactive oxygen species. A major limitation to studying exercise performance in birds is the technical challenges associated with making instructive physiological measurements during flight. Oxygen consumption and heart rates of bar-headed geese have been measured during flight in normoxia in a wind-tunnel (Ward et al., 2002), but only one previous study has made sufficient measurements of multiple variables in birds during flight to appreciate the multiple steps in the O2 transport pathway (in pigeons, Butler et al., 1977). Making these measurements in birds as large as bar-headed geese, with the additional (nontrivial) task of making these animals hypoxic, would be extremely challenging. Surmounting these challenges will definitely shed great insight into the ability of barheaded geese to fly in hypoxia, and will be essential for truly appreciating how much the respiratory capacity of this species is enhanced compared to other birds. This thesis studied animals that had never flown or experienced high altitude so the inherent differences in bar-headed geese could be determined. This was an important starting point for understanding the genetically based specializations of this species, but it ignored by necessity the important role of plasticity in developing the high aerobic and O2 flux capacities needed for flight (Bishop et al., 1995). In this regard, we have recently begun studying the migration patterns of wild bar-headed geese, and have observed these animals spending many days at stopover sites at moderately high altitudes in the foothills of the Himalayas (L.A. Hawkes, G.R. Scott, P.B. Frappell, W.K. Milsom, P.J. Butler, and C.M. Bishop, unpublished). This species may therefore acclimatize to hypoxia before surmounting the high peaks, and it is possible that the physiological response to this  228 acclimatization period has also evolved in bar-headed geese to rapidly improve O2 transport capacity in hypoxia. These are just a few potential directions for future research aimed at better understanding the evolutionary physiology of bar-headed geese. My thesis has laid important groundwork for these and other future studies. It is my sincere hope that future work continues to explore the incredible ability of bar-headed geese to fly high.  229 7.4. References Andrienko, T., Kuznetsov, A. V., Kaambre, T., Usson, Y., Orosco, A., Appaix, F., Tiivel, T., Sikk, P., Vendelin, M., Margreiter, R. and Saks, V. A. (2003). Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells. J. Exp. Biol. 206, 2059-72. Barros, R. C. H., Branco, L. G. S. and Cárnio, E. C. (2006). Respiratory and body temperature modulation by adenosine A1 receptors in the anteroventral preoptic region during normoxia and hypoxia. Respir. Physiol. Neurobiol. 153, 115-125. Bishop, C. M., Butler, P. J., Egginton, S., Elhaj, A. J. and Gabrielsen, G. W. (1995). Development of metabolic enzyme activity in locomotor and cardiac muscles of the migratory barnacle goose. Am. J. Physiol. Reg. Integr. Comp. Physiol. 38, R64-R72. Black, C. P. and Tenney, S. M. (1980). Oxygen transport during progressive hypoxia in high altitude and sea level waterfowl. Respir. Physiol. 39, 217-239. Brutsaert, T. D., Parra, E. J., Shriver, M. D., Gamboa, A. and León-Velarde, F. (2005). Ancestry explains the blunted ventilatory response to sustained hypoxia and lower exercise ventilation of Quechua altitude natives. Am. J. Physiol. Reg. Integr. Comp. Physiol. 289, R225-R234. Butler, P. J., West, N. H. and Jones, D. R. (1977). Respiratory and cardiovascular responses of the pigeon to sustained, level flight in a wind-tunnel. J. Exp. Biol. 71, 7-26. Faraci, F. M., Kilgore, D. L. and Fedde, M. R. (1984a). Attenuated pulmonary pressor response to hypoxia in bar-headed geese. Am. J. Physiol. 247, R402-R403.  230 Faraci, F. M., Kilgore, D. L. and Fedde, M. R. (1984b). Oxygen delivery to the heart and brain during hypoxia: pekin duck vs. bar-headed goose. Am. J. Physiol. 247, R69-R75. Faraci, F. M., Kilgore, D. L. and Fedde, M. R. (1985). Blood flow distribution during hypocapnic hypoxia in pekin ducks and bar-headed geese. Respir. Physiol. 61, 2130. Gayeski, T. E. J. and Honig, C. R. (1988). Intracellular Po2 in long axis of individual fibers in working dog gracilis muscle. Am. J. Physiol. Heart Circ. Physiol. 254, H1179-H1186. Gonzalez, N. C., Howlett, R. A., Henderson, K. K., Koch, L. G., Britton, S. L., Wagner, H. E., Favret, F. and Wagner, P. D. (2006). Systemic oxygen transport in rats artificially selected for running endurance. Respir. Physiol. Neurobiol. 151, 141-150. Hammond, K. A., Szewczak, J. and Krol, E. (2001). Effects of altitude and temperature on organ phenotypic plasticity along an altitudinal gradient. J. Exp. Biol. 204, 1991-2000. Hepple, R. T. (2000). Skeletal muscle: microcirculatory adaptation to metabolic demand. Med. Sci. Sports Exerc. 32, 117-123. Hochachka, P. W. (1985). Exercise limitations at high altitude: the metabolic problem and search for its solution. In Circulation, respiration, and metabolism (eds. R. Gilles), pp. 240-249. Berlin: Springer-Verlag.  231 Hsia, C. C., Carbayo, J. J., Yan, X. and Bellotto, D. J. (2005). Enhanced alveolar growth and remodeling in Guinea pigs raised at high altitude. Respir. Physiol. Neurobiol. 147, 105-115. Hussain, S. O., Barbato, J. C., Koch, L. G., Metting, P. J. and Britton, S. L. (2001). Cardiac function in rats selectively bred for low- and high-capacity running. Am. J. Physiol. Reg. Integr. Comp. Physiol. 281, R1787-R1791. Jessen, T.-H., Weber, R. E., Fermi, G., Tame, J. and Braunitzer, G. (1991). Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering. Proc. Natl. Acad. Sci. USA 88, 6519-6522. Lechner, A. J. (1976). Respiratory adaptations in burrowing pocket gophers from sea level and high altitude. J. Appl. Physiol. 41, 168-173. Liang, Y. H., Hua, Z. Q., Liang, X., Xu, Q. and Lu, G. Y. (2001). The crystal structure of bar-headed goose hemoglobin in deoxy form: The allosteric mechanism of a hemoglobin species with high oxygen affinity. J. Mol. Biol. 313, 123-137. Macklem, P. T., Bouverot, P. and Scheid, P. (1979). Measurement of the distensibility of the parabronchi in duck lungs. Respir. Physiol. 38, 23-35. Maina, J. N. (2002). Some recent advances on the study and understanding of the functional design of the avian lung: morphological and morphometric perspectives. Biol. Rev. 77, 97-152. Mathieu-Costello, O. (2001). Muscle adaptation to altitude: tissue capillarity and capacity for aerobic metabolism. High Alt. Med. Biol. 2, 413-425.  232 Petschow, D., Würdinger, I., Baumann, R., Duhm, J., Braunitzer, G. and Bauer, C. (1977). Causes of high blood O2 affinity of animals living at high altitude. J. Appl. Physiol. 42, 139-143. Reynafarje, C., Faura, J., Villavicencio, D., Curaca, A., Reynafarje, B., Oyola, L., Contreras, L., Vallenas, E. and Faura, A. (1975). Oxygen transport of hemoglobin in high-altitude animals (Camelidae). J. Appl. Physiol. 38, 806-810. Saunders, D. K. and Fedde, M. R. (1991). Physical conditioning: effect on the myoglobin concentration in skeletal and cardiac muscle of bar-headed geese. Comp. Biochem. Physiol. A. Physiol. 100A, 349-352. Snyder, G. K., Byers, R. L. and Kayar, S. R. (1984). Effects of hypoxia on tissue capillarity in geese. Respir. Physiol. 58, 151-160. Ventura-Clapier, R., Kuznetsov, A., Veksler, V., Boehm, E. and Anflous, K. (1998). Functional coupling of creatine kinases in muscles: species and tissue specificity. Mol. Cell. Biochem. 184, 231-247. Ward, S., Bishop, C. M., Woakes, A. J. and Butler, P. J. (2002). Heart rate and the rate of oxygen consumption of flying and walking barnacle geese (Branta leucopsis) and bar-headed geese (Anser indicus). J. Exp. Biol. 205, 3347-3356. Weibel, E. R., Taylor, C. R., Gehr, P., Hoppeler, H., Mathieu, O. and Maloiy, G. M. O. (1981). Design of the mammalian respiratory system. IX. Functional and structural limits for oxygen flow. Respir. Physiol. 44, 151-164. Weibel, E. R., Taylor, C. R. and Hoppeler, H. (1991). The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Natl. Acad. Sci. USA 88, 10357-10361.  233 West, J. B., Watson, R. R. and Fu, Z. (2007). Major differences in the pulmonary circulation between birds and mammals. Respir. Physiol. Neurobiol. 157, 382390. West, J. B., Watson, R. R. and Fu, Z. X. (2006). The honeycomb-like structure of the bird lung allows a uniquely thin blood-gas barrier. Respir. Physiol. Neurobiol. 152, 115-118. Widmer, H. R., Hoppeler, H., Nevo, E., Taylor, C. R. and Weibel, E. R. (1997). Working underground: Respiratory adaptations in the blind mole rat. Proc. Natl. Acad. Sci. USA 94, 2062-2067. Wittenberg, J. B. and Wittenberg, B. A. (2003). Myoglobin function reassessed. J. Exp. Biol. 206, 2011-2020. Wu, T. and Kayser, B. (2006). High altitude adaptation in Tibetans. High Alt. Med. Biol. 7, 193-208. Zhang, J., Hua, Z. Q., Tame, J. R. H., Lu, G. Y., Zhang, R. J. and Gu, X. C. (1996). The crystal structure of a high oxygen affinity species of haemoglobin (barheaded goose haemoglobin in the oxy form). J. Mol. Biol. 255, 484-493.  

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