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Effect of arousal state and increased respiratory drive on cardio-respiratory variables in the harbour… Skinner, Lisa Ann 2002

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EFFECT OF A R O U S A L STATE A N D INCREASED RESPIRATORY DRIVE O N CARDIO-RESPIRATORY V A R I A B L E S IN THE H A R B O U R S E A L (Phoca vitulina richardsi) by LISA A N N SKINNER B . S c , The University College of the Cariboo, 1998 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Zoology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A August, 2002 ©Lisa Ann Skinner, 2002  In presenting degree  this thesis  in partial fulfilment  of the  requirements  for an advanced  at the University of British Columbia, I agree that the Library shall make it  freely available for reference and study. I further copying of this thesis for scholarly purposes department  or  by  his  or  her  representatives.  agree that permission for extensive  may be granted It  is  by the  understood  that  head of my copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  oecrAe^wypQr  l\ aoo^  Abstract In this study, I examined the cardio-respiratory patterns of Pacific harbour seal pups (Phoca vitulina richardsi) under normoxic/normocarbic (air), hypoxic/normocarbic (15.22% 0 ± 0.47 S E M , 11.99% 0 ± 0.29 S E M , and 8.63% 0 ± 0.22 S E M in air) and 2  2  2  normoxic/hypercarbic (2.04% C 0 ± 0.06 S E M , 4.16 % C 0 ± 0.06 S E M , and 5.66% C 0 2  2  ± 0.07 S E M in air) conditions while awake and sleeping on land. chronically instrumented  to record electroencephalogram  (EEG),  2  Animals were electromyogram  (EMG), and electrocardiogram (EKG) signals, and recordings of all variables, plus respiration (whole body plethysmography) and metabolic rate ( 0 consumption and C 0 2  2  production), were made on each gas for two to four hours on separate days. M y results show that, on air, metabolic rate was lower in sleep (7.71 ml 0 min"  1  2  kg" ± 0.93 SEM) than wakefulness (WAKE) (8.79 ml 0 min" kg" ± 0.70 SEM), with 1  1  1  2  the fall in metabolism accompanied by a decrease in breathing frequency (18.76 breaths/minute ± 0.23 S E M in W A K E to 10.70 breaths/minute ± 0.49 S E M in sleep) and an increase in the incidence of periods of apnea. The maximum apnea length recorded (~ 3 minutes) was similar in length to the average dive time of harbour seals (-3.2 minutes). Breathing was rarely seen in R E M sleep, appearing in only one or two animals, during one or two R E M sleep episodes. Tachypnea was present at all levels of increased respiratory drive, both hypoxia and hypercarbia, however hypoxia induced a dramatic bradycardia (up to 50% decrease in heart rate [/H]) on 8.63% 0 ± 0.22 S E M regardless of arousal state, while hypercarbia 2  produced a small tachycardia in slow-wave sleep (SWS) only. Metabolic rate ( V 0 2 ) was relatively unchanged in both hypoxia and hypercarbia, while total ventilation ( V E )  ii  increased significantly in both W A K E and SWS.  The hypoxic and hypercarbic  chemosensitivities of the harbour seal pups, therefore, are similar to those of terrestrial mammals. Unlike the situation seen in terrestrial mammals however, where hypoxic and hypercarbic sensitivities are often reduced in SWS, the sensitivity of harbour seal pups to both gases appears unchanged during the change in arousal state from W A K E to sleep.  iii  Table of Contents Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  List of Abbreviations  viii  Acknowledgements  ix  Chapter 1. General Introduction: Sleep and Respiration in Phocid Seals 1.1 Sleep 1.1.1 Slow-wave Sleep 1.1.2 Rapid eye-movement Sleep 1.1.3 Sleep in Marine Mammals 1.2 Respiration 1.2.1 Breathing Pattern 1.2.1.1 W A K E 1.2.1.2 Sleep 1.2.2 Oxygen Consumption 1.2.3 Oxygen Storage 1.3 Respiratory Drive 1.4 Heart Rate 1.5 Purpose of Study 1.5.1 Specific Hypotheses  --  2 3 3 7 9 9 9 11 13 16 18 22 26 28  Chapter 2. Effects of arousal state and respiratory drive on cardio-respiratory variables in harbour seal pups (Phoca vitulina richardsi). 2.1 Introduction 2.2 Methods - - 2.2.1 Animals and Housing 2.2.2 Surgical Procedures 2.2.3 Experimental Protocol 2.2.4 Data Analysis 2.2.5 Statistical Analysis 2.3 Results 2.3.1 Hypoxia 2.3.1.1 Respiration 2.3.1.2 Metabolic Rate 2.3.1.3 Heart Rate 2.3.2 Hypercarbia 2.3.2.1 Respiration 2.3.2.2 Metabolic Rate  iv  ---  '.  31 33 33 34 37 39 45 45 45 45 46 50 50 50 54  2.3.2.3 Heart Rate 2.3.3 R E M sleep 2.3.4 Breathing Pattern 2.4 Discussion 2.4.1 State of Arousal 2.4.2 Hypoxic Ventilatory Response 2.4.3 Hypercarbic Ventilatory Response 2.4.4 Heart Rate 2.5 Conclusions  55 55 59 59 59 67 69 71 74  Chapter 3. General Discussion and Conclusions 3.1 Critique of Methodology 3.2 State of Arousal 3.3 Increased Respiratory Drive 3.3.1 Hypoxic Ventilatory Response 3.3.2 Hypercarbic Ventilatory Response 3.4 Heart Rate -3.5 Conclusions  77 78 82 82 87 88 90  Literature Cited  --  93  v  List of Tables Table 2.1  Table 2.2  Mean V02, Vco2, and RQ values of harbour seal pups on air during both W A K E and SWS states  43  Values for various respiratory variables during W A K E and SWS in an hypoxic and an hypercarbic environment  48  Table 2.3  Frequency of occurrence of R E M sleep  58  Table 2.4  Effect of hypoxia and hypercarbia on respiratory variables during R E M sleep  60  vi  List of Figures Figure 1.1  Representative recordings of E E G and E M G during the three primary states of arousal: W A K E , SWS, R E M sleep  4  Oxygen stores at the beginning of a dive in various species of marine mammal  20  Figure 1.3  Electrocardiogram signal of a 11 -month old harbour seal  24  Figure 2.1  E E G , E K G , and E M G Electrode placement  36  Figure 2.2  WAKE-Sleep hypnogram  38  Figure 2.3  Experimental set-up  40  Figure 2.4  The effect of hypoxia on / R , average apnea length, V T , and V E ...  47  Figure 2.5  The effect of hypoxia on V 0 2 , %Eo , and A C R  51  Figure 2.6  The effect of hypoxia on / H , % change in / H , eupneic / H , and apneic / H  52  The effect of hypercarbia on / R , average apnea length, V T , andVe  53  Figure 2.8  The effect of hypercarbia on V 0 2 , %Eo , and A C R  56  Figure 2.9  The effect of hypercarbia on / H , % change in / H , eupneic / H ,  Figure 1.2  Figure 2.7  2  2  and apneic/H  57  Figure 2.10  Poincare plot of interbreath intervals  61  Figure 2.11  Representative breathing and heart rate traces during W A K E , SWS and R E M sleep 62 Comparison of heart rate and breathing traces in a harbour seal pup breathing air and 8%02 during SWS 73  Figure 2.12  vii  List of Abbreviations EEG  electroencephalogram  EMG  electromyogram  EKG  electrocardiogram  WAKE  wakefulness  SWS  slow wave sleep  REM  rapid-eye movement sleep  Fo  fractional composition of O2  2  Fco  fractional composition of CO2  2  FEo  fractional expired O2 content  2  FEco Flo  2  fractional expired CO2 content fractional inspired O2 content  2  Flco  2  fractional inspired CO2 content  /R  breathing frequency  /Rinst  instantaneous breathing frequency  VT  tidal volume  VE  total ventilation  V02  rate of oxygen consumption  VCO2  rate of carbon dioxide production  %Eo  % oxygen extraction  2  ACR  air convection requirement  Po  partial pressure of O2  2  Pco  2  partial pressure of CO2  viii  Acknowledgments First and foremost, I would like to take this opportunity to thank my supervisor Dr. Bill Milsom. Y o u accepted me into your lab knowing I had never worked on a "real" research project before, hopefully a decision you don't regret too much. And although I wanted to quit after the first week, and many weeks thereafter, your optimism and enthusiasm kept me interested enough to stick around, a decision I look upon fondly, and will never regret. Three years and eleven months later, not a day longer, I have finally completed my degree. Sorry it took so long Bill, but I hope you are happy with the final product, I know I am. Thank-you for all your support, advice, and encouragement. Dr. David Jones, although your primary role was committee member for my thesis, you were my supervisor when Bill was off traveling to distant parts of the world. Thank-you for your guidance and wisdom. Dr. Andrew Trites, your role on my committee was minor, however, your presence was a constant reminder to keep the ecological aspects of my project in perspective with the physiology. Thank-you. This thesis would not have been possible without the cooperation of seven very curious seals. Pepper, Curry, Poppy, and Chili, thank-you for letting me play and allowing for such a sharp learning curve with regards to training and seal husbandry. I am especially grateful to my baby boys: Hermes, Zeus, and Apollo, who, although didn't always want to work, were willing research subjects. And while they sometimes put up quite a fight, their contributions to this project, and my time in Vancouver, are very much appreciated. Dr. Stephane Lair, thank-you so very much for your help with surgeries, all your advice on husbandry and animal experimentation, as well as your friendship. You never seemed to mind answering my questions, no matter how trivial or silly they sounded. Without your help, and humour, things probably would not have gone as smoothly. Thank-you also for teaching me how to swear in French. Although I don't know when, I'm sure it will be useful someday! Merci beaucoup. Beth Zimmer and Glenn Tattersal, as the two senior members of the Milsom lab, you were both supportive and helpful in the analysis of my data, as well as the writing of my manuscript and thesis. Thank-you for your help and your friendship (and thank-you Chef Don for all your delicious meals!). And of course, the rest of the lab, Joyce Boon, Colin Sanders, and the newbies: Jon Chatburn, Charissa Fung, and Angie O'Neill. Thanks for your patience when I showed up stressed and frazzled. Your humour and compassion were very much appreciated. I owe an enormous amount of gratitude to all the people who made my experiments and data analysis run smoothly. Bruce Gillespie and Granville Williams made sure the equipment was safe from seal destruction by building my "research hut" and were always quick to fix the dome that Zeus insisted on breaking, while Don Brandys was exceptionally helpful in the soldering of the leads during the surgeries. Thank-you to the Zoology Mechanical Workshop for all your help.  ix  This project could not have been done without the help of some very dedicated volunteers. Danielle Brochu, you were my side-kick in the raising of the pups, a challenge you willingly took...without too much begging. James Sidney and Daniel Holloway, you helped me outsmart the seals (not as easy as it sounds) and patiently sat outside to make visual observations, even in the rain and snow. Thank-you all for your help and dedication. I hope you had fun. On a more personal level, I would like to take this opportunity to thank my family and friends who were instrumental in helping me keep my sanity and encouraging me to stick with this project. Dr. Tom Dickinson, Nancy Flood, Dr. Val Collins, and Dr. Ken Taylor, without your teaching skills, friendship, encouragement, and advice, I never would have attempted a Master's degree. Thank-you for knowing how much I would benefit from this experience. Nicole Elliott, you were there to help me through one of the most difficult times in my life; grad school. Thank-you for your help, support, encouragement, and most of all, your friendship. You have helped me in more ways than you probably know. Thankyou. Mom, Dad, Twila, Kuldeep, Kevin, Ieashia, and Brianne, you are my family and have been so unbelievably supportive through all the pains and pleasures of this project. You have taken the love of families to a whole new level. I know at times I was a basket-case, full of stress, anger, and hopelessness, but you always seemed to help me find the light at the end of the tunnel and get me laughing. Thank-you for all your love and support. And last, but definitely not least, although she is the smallest, I would like to take this opportunity to thank Storm, my little rattie. Although not quite the same as a dog, Storm has given me unconditional love and has provided many laughs, something that is very important when undertaking a thesis!  This research was funded through an operating grant awarded to W.K. Milsom by the National Science and Engineering Research Council (NSERC) Canada.  x  xi  Chapter 1 General Introduction Sleep and Respiration in Phocid Seals  1.1 Sleep Sleep is a state or period of reduced physical and neuronal activity accompanied by a suspension of voluntary movements and a complete or partial unconsciousness (Home 1988; McGinty and Szymusiak 1994).  Early scientists defined sleep and its  different states through either physiological variations in brain wave activity, detected via electroencephalogram (EEG) patterns, or changes in behaviour of the animal. E E G patterns are useful in identifying the different arousal states as they measure the amount of brainwave activity in both the right and left cerebral hemispheres, thereby acting as a measure of alertness of the brain. Aside from wakefulness, the two main states of arousal identified are slow-wave sleep (SWS) and rapid eye-movement (REM) sleep, or paradoxical sleep (PS). Although E E G patterns are useful and convenient in identifying the different arousal states, there are some instances, such as during certain drug treatments or after  neocortical lesions, where an animal is considered  asleep  behaviourally, but shows minimal sleep-like E E G patterns (McGinty and Szymusiak 1994).  Because of this, both behavioural and electrical brain activity are used in  conjunction to accurately identify and define W A K E and sleep states which, in most terrestrial mammals, cycles from W A K E to SWS to R E M sleep (Lydic 1987). Electromyograms (EMG), or physiological measures of muscle tone, are used in conjunction with E E G patterns to identify and distinguish the different states of arousal. This is especially useful in differentiating R E M sleep from W A K E , where R E M sleep is associated with muscle atonia (see below), while wakefulness is associated with relatively constant muscle tone (Lydic 1987; Home 1988; McGinty and Szymusiak 1994).  2  1.1.1 Slow-wave Sleep SWS (Fig. 1.1) is identified by low frequency, high amplitude, synchronized E E G patterns which typically comprise 75-80% of the average sleep cycle (Lydic, 1987; Home 1988; McGinty and Szymusiak 1994; Hunter and Milsom 1998).  In humans, four  different stages of SWS have been described (stage 1 to 4), each varying slightly in E E G pattern.  As the level of alertness decreases in an individual, E E G patterns become  progressively slower in frequency and higher in amplitude, with the most slow-wave activity associated with the deepest stages of SWS. In non-human mammals, SWS is not typically separated into the four different stages but is considered one stage. In addition to its distinctive E E G pattern, SWS is associated with some very distinctive physiological characteristics.  During SWS, it is not uncommon to see an  increase in the release of growth hormones and gonadotropic hormones from the anterior pituitary, or a decrease in blood pressure, heart rate, respiratory rate, and metabolic rate over W A K E state levels (Lydic 1987). Other physiological characteristics commonly observed during SWS include a drop in hypothalamic temperature sensitivity and body temperature as well as reduced cerebral blood flow, which can be correlated to the reduced cerebral metabolism and the reduced activity in a majority of brain neuronal sites (McGinty and Szymusiak 1994).  1.1.2 Rapid eye-movement sleep The second state of sleep commonly identified is rapid eye-movement (REM) sleep, or paradoxical sleep (PS). The name "rapid eye-movement sleep" originates from the fact that when this state was first identified in humans, it was characterized by rapid  3  eye-movements. Not all animals, however, show the rapid eye-movements characteristic of human R E M sleep.  Because of the desynchronized or activated E E G patterns  associated with R E M sleep (Fig. 1.1), identifiable through the high frequency, low amplitude EEG signals, similar to those observed during wakefulness (Fig. 1.1), a more universal classification may be paradoxical sleep, paradoxical in the sense that the E E G signal looks like W A K E , but the body is asleep (Home 1988). R E M sleep, as with SWS, is associated with many unique physiological characteristics. Body temperature is not constant due to reductions in thermoregulatory control, while blood pressure, heart rate, and respiratory rate are irregular, with metabolic rate and cerebral blood flow increased compared to SWS levels (McGinty and Szymusiak 1994).  The increased cerebral blood flow leads to an increase both in cerebral  metabolism and hypothalamic temperature.  R E M sleep is also associated with an  increased discharge in brain neuronal sites, leading to the characteristic activated W A K E like EEG patterns (Home 1988; McGinty and Szymusiak 1994). As with SWS, there are different stages associated with R E M sleep, specifically tonic R E M sleep and phasic R E M sleep, also referred to as tonic and phasic events, which are present in most mammals. The term "event" suggests these are not successive changes in arousal, as in SWS, rather they appear consistently throughout the R E M sleep episode.  Tonic R E M sleep lasts the duration of the R E M sleep episode and is  characterized by the desynchronization of the E E G signal (similar to W A K E ) and muscle atonia (Orem 1980; Home 1988). The degree of muscle atonia present and the amount of R E M sleep observed is species dependent. Some species, such as humans and other  5  animals with high degrees of encephalization (i.e. highly advanced cerebrum), exhibit a relatively high amount of R E M sleep, while animals with relatively low degrees of encephalization, such as ungulates, exhibit minimal amounts of R E M sleep (Home 1988). Regardless of the amount of R E M sleep present, or the degree of encephalization, muscle atonia, especially of head and neck muscles, is a constant feature of tonic R E M sleep. To allow for muscle atonia, without injury to the animal, R E M sleep is often observed in individuals lying down, and in the case of animals that sleep standing up (e.g. ungulates), individuals will lie down for a portion of the sleep episode, allowing for R E M sleep to occur (Home 1988).  There are some exceptions to the overall muscle atonia.  The  respiratory muscles, and in some instances, the eye muscles leading to the rapid eyemovements of some animals, maintain both muscle tone and the ability to contract (Orem 1980; Home 1988). Phasic R E M sleep is episodic in appearance and is characterized by the occurrence of pontogeniculooccipital (PGO) waves in the E E G signal, as well as myoclonic twitches, and in some animals, eye movements (Orem 1980; Home 1988; Hunt et al. 1998).  PGO waves are 200-300uV peaks found in the E E G signal,  approximately 100ms in length, occurring at a frequency of 1 to 2 per second (Orem 1980; Hunt et al. 1998).  PGO waves, aside from triggering bursts of firing from  geniculate and cortical neurons of the brain, as well as other signals originating in the brainstem that damp out sensory input and motor output, are associated with the activation of muscles surrounding the eye, and are positively correlated with respiratory neuronal activation (Orem 1980; Orem 1988; Hunt et al. 1998).  6  1.1.3 Sleep in Marine Mammals - Family Phocidae Although the exact reasons are unknown, sleep is important to survival (Home 1988) and to ensure that the physiological needs of the individual are met, and different strategies of sleep and sleep patterns have evolved in animals living in different environments. Sleep in marine mammals, especially those that cannot or do not spend any appreciable amount of time on land, present an interesting challenge. How can an animal living completely submerged in water sleep? It is a well-known fact that sleep consists of various levels of consciousness and that motor ability is dampened considerably during sleep (McGinty and Szymusiak 1994). During certain portions of sleep (tonic event of R E M sleep), postural muscles experience atonia, while at the same time, in marine mammals, some level of consciousness or motor ability has to be present to prevent sinking or drifting in the water and to allow the sleeping individual to breathe. The nineteen different species of seal from family Phocidae occupy numerous different niches, from estuarine and coastal to deep sea, spending an appreciable amount of time both on land and in water. Because of this, one can closely examine the evolution of some of the different sleep strategies developed in marine mammals. One of the more interesting strategies of sleep in marine mammals occurs in bottlenose dolphins Tursiops truncates), harbour porpoises (Phocoena phocoena), beluga whales (Delphinapterus leucas) and otariid seals, both on land and in water (Cape fur seal [Arctocephaluspusilluspusillus], northern fur seal [Callorhinus ursinus], Stellar sea lion [Eumetopias jubatus], and southern sea lion [Otaria byronia], where sleep occurs in one cerebral hemisphere at a time (Home 1988; Lyamin and Chetyrbok 1992; Lyamin et al. 2002a; Lyamin et al. 2002b).  Studies of sleep involving phocid seals reveal a  7  different, albeit equally interesting strategy. These animals follow the pattern of SWS and R E M sleep commonly associated with sleep in terrestrial mammals; both cerebral hemispheres sleep at the same time, regardless of arousal state, and individuals alter breathing pattern as an adaptation to sleep in water (see below). O f the nineteen different species of phocid seal, patterns of sleep have been extensively examined in gray seals (Halichoerus grypus) (Ridgway et al. 1975), Caspian seals (Pusa caspica) (Mukhametov et al. 1984), harp seals (Pagophilus groenlandicus) (Lyamin 1993; Lyamin et al. 1993), and northern elephant seals (Mirounga angustirostris) (Blackwell and Le Boeuf 1993; Castellini et al. 1994a; Castellini et al. 1994b; Castellini 1996; Milsom et al. 1996), while sleep in other phocid seals, such as southern elephant seals (Mirounga leonind) (Falabella et al. 1999), hooded seals (Cystophora cristata) (Castellini 1996), and Weddell seals (Leptonoychotes weddelli) (Castellini et al. 1992) has been observed behaviourally, although not examined at the neurophysiological level. While the pattern of cerebral brainwave activity is similar during sleep in all phocid seal species studied to date, the actual order of arousal state that have been reported varies.  The Caspian seal, harp seal, and northern elephant seal undergo an  arousal state pattern similar to terrestrial mammals: W A K E - SWS - R E M sleep (Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1994a). Studies of sleep in the gray seal however, reveal a different pattern of arousal state: W A K E - R E M sleep - SWS (Ridgway et al. 1975). This arousal state pattern has not been observed in any other mammal, marine or terrestrial (Ridgway et al. 1975). Because arousal state patterns have only been intensively studied in four species of phocid seal (gray seal, Caspian seal, harp seal, and northern elephant seal) and two  8  different patterns have been observed, it is difficult to predict the arousal state pattern of other phocid seals and to determine the underlying reasons for the variation in arousal state pattern. It is possible that the evolutionary relationships and ecological similarities of the phocid species in question affect the sleep state pattern.  1.2 Respiration 1.2.1 Breathing Pattern 1.2.1.1 WAKE Phocid seals are diving mammals. They live primarily in aquatic environments and remain submerged for extended periods of time.  While at the water's surface,  individuals are most vulnerable to predation and must maximize the total amount of time spent underwater.  The length of the dives and consequential apneas (cessation of  breathing), both average and maximum, vary from species to species, with some animals, such as the harp seal, exhibiting maximum apneas of 13 minutes (Lydersen and Kovacs 1993) while other animals, such as the southern elephant seal, exhibit maximum apneas as long as 120 minutes (Hindell et al. 1992). During dive bouts, breathing cannot be continuous due to repeated prolonged submergences, and therefore, is considered episodic with breaths clustered into distinct episodes separated by pauses of variable lengths (Irving et al. 1935; Butler and Jones 1982; Castellini 1996). This ability to maintain prolonged apneas, and exhibit episodic breathing patterns, suggests that phocid seals have specific adaptations allowing for either increased oxygen storage, decreased oxygen consumption, or both, and it leads to intriguing questions regarding the mechanisms of breathing in these animals.  9  Unlike other marine mammals (cetaceans and otariids), phocid seals take many breaths before re-submergence and are therefore considered "slow" breathers while at the surface (Kooyman 1973). The tidal volume ( V T ) , or the volume of an average breath, of phocid seals is similar in size to its total lung capacity (TLC), such that with each breath, most of the air in the lungs is renewed.  This aids in quickening the removal of  respiratory waste products such as carbon dioxide, as well as replenishing the used oxygen stores (Kooyman 1973). Because the tidal volume of phocid seals is so large and is near its maximum value (40-90% of TLC), the primary mechanism through which seals can meet increases in metabolic oxygen requirements is through increasing respiratory frequency, and hence, the total amount of time spent eupneic (Gallivan 1981; Butler and Jones 1997). Although primarily considered aquatic mammals, phocid seals also spend an appreciable amount of time on land, with the amount of time spent in water versus time spent on land varying greatly from species to species. It has been shown in many studies, on a variety of species of phocid seal (including the northern elephant seal [Castellini et al. 1994a; Castellini et al. 1994b; Milsom et al. 1996], the Weddell seal [Castellini et al. 1992], the harp seal [Lyamin 1993], the gray seal [Ridgway et al. 1975], the hooded seal [Castellini 1996], the leopard seal (Hydrurga leptonyx) [Williams and Bryden 1993], and the Caspian seal [Mukhametov et al. 1984]) that breathing patterns while on land and in an awake state are not episodic, but continuous and irregular (i.e. arrhythmic).  10  1.2.1.2 Sleep Research has shown that while on land, breathing in phocid seals is not always continuous. During certain states of sleep, breathing becomes episodic in pattern, similar to the diving animal (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1992; Castellini et al. 1994a; Milsom et al. 1996). Changes in breathing pattern associated with changes in arousal state are not unique to marine mammals. In terrestrial mammals, SWS is primarily accompanied by slower, yet regular breathing patterns, and changes in tidal volume (both increasing and decreasing), as well as decreases in total ventilation ( V E ) and breathing frequency (/R) over W A K E state levels (Lydic 1987). R E M sleep is associated with increases in breathing frequency, as well as the presence of apneas and hypopneas (decreased lung ventilation) (Lydic 1987). Current information regarding breathing patterns during sleep in marine mammals in general, and phocid seals in particular, is limited and ambiguous.  From the four  species of phocid seal that have been studied with regard to breathing patterns and different states of arousal (gray seal, Caspian seal, harp seal, and northern elephant seal) it appears that breathing pattern is not only specific to arousal state, but is possibly also specific to species (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1994a; Milsom et al. 1996). As mentioned previously, breathing during periods of wakefulness is either episodic (dive situations) or continuous and irregular (on land). During SWS, breathing becomes episodic in all phocid seal species studied to date, with the animal either on land or in the water (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Lyamin et al. 1993; Castellini et al. 1994a; Castellini et al.  11  1994b; Milsom et al. 1996). The variability arises in the breathing patterns seen in R E M sleep. In phocid seals, a conflict arises between sleep and swimming. It is known that during R E M sleep, in all mammals studied to date, muscle atonia occurs (Lydic 1987; Home 1988). If seals were to also experience muscle atonia during R E M sleep, while submerged, their swimming ability would be impeded. Therefore, i f a seal cannot swim to the surface to breathe during R E M sleep, it can do one of two things: 1) fail to enter R E M sleep or 2) alter its breathing pattern such that it can remain submerged for much, i f not all, of the R E M sleep episode. Because R E M sleep is so important to the survival of animals (Orem 1980; Home 1988), it would be difficult to remove it completely from the sleep cycle; therefore all species of phocid seal currently studied alter their breathing pattern, body posture, or both, during R E M sleep. In the gray seal, breathing during R E M sleep is continuous and regular, therefore the animal must be on land or positioned in the water such that it can maintain a continuous breathing pattern (i.e. floating with nostrils just above the surface of the water) (Ridgway et al. 1975). During R E M sleep in the ice-dwelling Caspian and harp seals, on land and fully submerged, breathing is sporadic and rare, with most R E M sleep episodes occurring within one respiratory pause (Mukhametov et al. 1984; Lyamin 1993; Lyamin et al. 1993). The northern elephant seal, an offshore species, does not breathe during R E M sleep. It maintains prolonged apneas for the duration of the R E M sleep episode, regardless of location, on land or in water (Castellini et al. 1994a; Castellini et al. 1994b; Milsom et al. 1996).  12  Thus, of the four phocid seal species studied to date, there are three very different state dependent patterns of breathing. It is difficult to determine, without further studies, whether different breathing patterns arise from differences in ecology, phylogeny, or adaptations.  1.2.2. Oxygen Consumption Oxygen consumption rate is a variable commonly used to determine the energy (metabolic) requirements of animals (i.e. how much energy must be consumed for an animal to survive). During dive and breath-hold situations, there are limited amounts of O2 available to the cells, tissues, and organs of phocid seals. According to Hochachka (1988) there are two ways in which to deal with finite oxygen stores during these apneic situations: 1) turn to alternate sources of energy such as anaerobic metabolism (Pasteur effect), or 2) decrease oxygen consumption rates in hypoxia tolerant tissues, such as skeletal muscle (reversed Pasteur effect or hypometabolism). Many studies have shown that during natural dives, phocid seals do not turn to anaerobic metabolism unless the dive is exceptionally long, as is evident by the lack of lactate build-up (see Hochachka and Guppy 1987, and Hochachka 1988 for reviews). Thus, hypometabolism of hypoperfused tissues allows the hypoxia-sensitive tissues (heart, brain, and lungs) to maintain normal, or near-normal oxygen consumption rates, as well as allows the seal to remain apneic (diving or voluntary) for prolonged periods without the build-up of waste byproducts. In this context, the changes in breathing pattern associated with changes in arousal state seen in phocid seals leads to some intriguing questions regarding oxygen  13  consumption rates.  Why is breathing on land in an awake animal continuous and  irregular, while breathing in the same animal during SWS is episodic?  Why is the  breathing pattern during R E M sleep species dependant? It has been suggested that when metabolic rate and oxygen requirements are low, the need to breathe continuously is eliminated and breathing becomes slow and/or episodic (White et al. 1985; Milsom et al. 1997). This implies that during wakefulness on land, where breathing is slow but continuous and irregular, phocid seals have a much higher metabolic rate than during SWS on land and in the water, where breathing is episodic.  Breathing during R E M sleep shows no apparent pattern or relationship to  metabolic rate. In the Caspian and harp seals, although sporadic and rare during R E M sleep, breathing remains episodic (Mukhametov et al. 1984; Lyamin 1993). If breathing patterns are a reflection of metabolic rate, the change in state of arousal from SWS to R E M sleep in these species does not appear to significantly affect the metabolic rate. In the gray seal, where R E M sleep occurs after W A K E and before SWS, breathing is continuous (Ridgway et al. 1975), again suggesting no change in metabolic rate with the onset of R E M sleep. It also suggests a decrease in metabolic rate as the animal enters SWS, illustrated by the change in breathing pattern from continuous to episodic. The complete absence of breathing during R E M sleep in the northern elephant seal (Castellini et al. 1994a; Castellini et al. 1994b; Milsom et al. 1996) suggests a dramatic decrease in metabolic rate as arousal state decreases.  These data lead to the question: is the  metabolic rate of phocid seals linked to state of arousal and breathing patterns? In terrestrial mammals, it is hypothesized that breathing during wakefulness is governed by both metabolic rate and behaviour (Phillipson 1978).  14  As arousal state  decreases, the factors affecting breathing change.  During SWS, it is suggested that  breathing pattern is controlled primarily through metabolic demands, while during R E M sleep, it is thought that behaviour, as well as the absence of many physiological control mechanisms, play the primary role and appear to act independent of the metabolic system (Phillipson 1978; Lydic 1987). According to Heller (1988), whole body metabolic rate is lowered during the change of arousal state from W A K E to SWS by about 10%, on average, supporting the hypothesis of Phillipson (1978). Because of its easy accessibility in the wild, the Weddell seal is one species in which numerous field metabolic studies have been performed (Kooyman et al. 1973; Castellini et al. 1992; Ponganis et al. 1993; Davis and Kanatous 1999). Castellini et al. (1992) closely examined the changes in the metabolic rate of Weddell seals in response to changes in activity, and behaviourally defined arousal levels. They found that as the level of activity decreased from relatively short dives to resting at the surface (wakefulness), metabolic rates remained relatively constant despite the decrease in activity. Interestingly, metabolic rates of short dives appeared to be 1.5 times higher than the metabolic rate observed in behaviourally defined sleep, while the metabolic rate of resting seals was no different from sleeping seals. Studies by Lavigne et al. (1986a and 1986b) and Worthy (1987) indicate that the resting metabolic rate of phocid seals, in an awake state, is the same, regardless if the seal is at the surface of the water or on land, allowing for comparisons of the patterns of change in metabolic rate between different arousal states of marine and terrestrial mammals, regardless of location. As mentioned previously, in terrestrial mammals, a decrease in both arousal and alertness levels is typically accompanied by a decrease in  15  metabolic rate (Phillipson 1978; Heller 1988). Data on the changes in metabolic rate of marine mammals, due to changes in arousal state, contradict this. Castellini et al. (1992) conclude that the metabolic rate of a behaviourally sleeping Weddell seal is no different from an awake, resting individual. Worthy (1987) examined changes in metabolic rate of harp and gray seals and found a 40.8 ± 10.6% decrease in metabolic rate during behavioural sleep when compared to resting, W A K E state levels. The differences in magnitude of change in metabolic rate between the different states of arousal, raises many questions regarding the control mechanisms.  One possibility is that there are  physiological differences associated with differences in behaviour, allowing for the different magnitudes of change in metabolic rate, associated with arousal state. Before any concrete conclusions or predictions can be made however, the effects of neurophysiologically defined sleep on metabolic rates in phocid seals need to be examined.  1.2.3 Oxygen Storage One of the most important adaptations of phocid seals, allowing them to remain apneic for prolonged periods of time, is their ability to store excess oxygen for use during long breath-hold periods. There are three potential sources for oxygen storage: lungs, muscle, and blood. In phocid seals, the lungs are not an optimal store for oxygen during dives.  First, an increase in lung oxygen stores would increase the buoyancy of the  animal, thereby increasing the energy expenditure required to dive below the surface (Kooyman 1973). Secondly, i f an animal diving deep beneath the surface of the water were to store large quantities of air in its lungs, the nitrogen gas that comprises most of that air (79%) would be forced into the blood as the lungs collapsed from the pressure of  16  the dive (1 atmosphere absolute for each 10m below sea level), resulting in nitrogen narcosis upon re-surfacing, a potentially deadly state (Gooden 1990). In diving phocid seals therefore, the lungs contain only a small portion of total oxygen stores, and in fact, most dives occur on exhalation with the lung volume 20 to 60% of the total lung capacity (for reviews, see Kooyman 1973 and Burggren 1988). While awake and resting quietly on the surface of the water, or on land, periodic, short-duration apneas are common. In situations such as these, the lungs would be beneficial as oxygen stores and, in fact, to utilize the oxygen stores of the lung, apneas in awake, resting phocid seals usually occur on inspiration (Kooyman 1973). Muscle, although not a major store for oxygen in most terrestrial mammals, is known for its increased oxygen storage capacity in seals (Lenfant et al. 1970), with approximately 3-10 times more oxygen stored than in terrestrial mammals (Polasek and Davis 2001). During apneic situations, vasoconstriction brought about by stimulation of carotid body chemoreceptors, a result of the dive response, decreases the amount of blood oxygen stores available to the hypoperfused tissues (Eisner et al. 1977; Daly et al. 1977). The increased myoglobin oxygen stores (MbC^) and mitochondrial densities of these hypoperfused muscle tissues however, prevent complete ischemia and provide sufficient oxygen to the tissues, allowing them to function with minimal reliance on anaerobic metabolism during prolonged periods of apnea (Kanatous et al. 1999). The primary oxygen store in phocid seals is the blood. There are many different physiological aspects of the blood that can be changed to increase its overall oxygen carrying capacity. Increases in blood volume, hematocrit, and hemoglobin concentration, compared to levels seen in terrestrial mammals, all aid in increasing the total oxygen  17  stores available to the diving animal during apneic situations (Lenfant et al. 1970; Kooyman 1973; Burggren 1988).  Studies have shown that seals that experience the  longest breath-holds have the highest blood oxygen stores, with the highest hemoglobin concentrations and hematocrit levels (Lenfant et al. 1970), thus indicating that increasing the oxygen storage capacity of the blood can be very beneficial to the animal without negative effects on its diving capability (Kooyman 1973; Burggren 1988). If we compare the average and maximum recorded dive times of various species of phocid seal with the total oxygen stores (Fig. 1.2), it is interesting to note that the longer the maximum attainable dive time, the less the lungs are used as an oxygen store and the more the blood and muscles are utilized.  1.3 Respiratory Drive Oxygen is required by all air-breathing vertebrates to maintain aerobic metabolism.  If oxygen supplies are depleted, either internally or externally, aerobic  metabolism cannot be utilized and the animal must turn to anaerobic metabolism, an inefficient source of ATP.  When hypoxic conditions, or conditions of low 0  carotid body peripheral chemoreceptors,  2  arise,  stimulated by low blood oxygen partial  pressures (Po ), stimulate respiratory control centres to increase breathing frequency as a 2  mechanism to increase O2 uptake and blood Po levels. The rate of oxygen consumption 2  however, is decreased during hypoxic conditions to conform to oxygen availability, while the rate of oxygen delivery to the blood, or total ventilation ( V E ), is increased (Frappell etal. 1992; Mortola 1999).  18  In hypercarbic conditions, where ambient levels of carbon dioxide are increased, the partial pressure of CO2 in the blood (Pco ) increases, with immediate results of 2  decreasing both blood and intracellular pH levels (Cameron 1989; Henderson-Smart 1994). This is primarily due to the bicarbonate reaction:  CO2 + H2O ^ C 0 2  3  *->HC0 + H  +  3  where any increase in CO2 shifts the equation to the right, increasing the overall [H ], and +  decreasing blood pH. Although there is some input from the peripheral chemoreceptors, the majority of the hypercarbic response is due to central chemoreceptor input (Cameron 1989; Henderson-Smart 1994).  Breathing frequency, total ventilation, and tidal volume all  increase significantly during hypercarbia, while oxygen consumption rates remain relatively unchanged (Mortola and Lanthier 1996). Studies on the effects of hypoxia and hypercarbia in marine mammals are primarily limited to phocid seals in the W A K E state (Irving 1938; Robin et al. 1963; Bainton et al. 1973; Pasche 1976a; Pasche 1976b; Craig and Pasche 1980; Parkos and Wahrenbrock 1987; Mortola and Lanthier 1989; Milsom et al. 1996; Kohin et al. 1999). From the initial studies on restrained animals, and the more recent studies on unrestrained animals, it is apparent that there is no difference in sensitivity to hypoxia or hypercarbia when most phocid seals are compared to terrestrial mammals, however there are increased thresholds for the hypoxic and hypercarbic ventilatory responses (i.e. animals react with the same intensity but do not show a response until higher levels of respiratory drive are reached) (Bainton et al. 1973; Pasche 1976a; Pasche 1976b; Gallivan 1980;  19  Figure 1.2. Oxygen stores at the beginning of a dive in the sea otter and various species of pinniped. Top row gives the total body store; the lower rows give the distribution between lung, blood (arterial-top half, venous-bottom half), and muscles respectively (figure adapted from Lenfant et al. 1970). Maximum dive times are noted at the bottom of the figure. Data for the harbour seal, South American fur seal (Arctocephalus australis), and walrus (Odobenus rosmarus) are from Riedman (1990), data for the California sea lion (Zalophus californianus) are from Reeves et al. (2002), data for the ribbon seal (Histriphoca fasciatd) are from Burt and Grossenheider (1980), and data for the sea otter (Enhydra lutris) are from Estes and Bodkin (2002).  20  Parkos and Wahrenbrock 1987; Milsom et al. 1996). The responses of respiratory sensitivity to hypoxia in juvenile Weddell seals are different from all other phocid seals studied to date. These data indicate that juvenile Weddell seals have a decreased hypoxic sensitivity compared to terrestrial mammals and other species of phocid seal (Parkos and Wahrenbrock 1987). Parkos and Wahrenbrock (1987) hypothesized that the decreased hypoxic sensitivity of the Weddell seal could be due to the exposure of the pups to hypoxia during diving early in life and therefore could be an adaptation for prolonged dives. No other phocid seal studied to date however, even the more precocial harbour seal, shows the decreased hypoxic ventilatory sensitivity that the Weddell seal does (Bainton et al. 1973; Pasche 1976a; Pasche 1976b; Gallivan 1980; Milsom etal. 1996). During periods of sleep, the threshold for medullary neuronal activation is increased over W A K E state levels, and although there is some discrepancy over the level of change in sensitivity associated with arousal state, lower levels of blood Po (higher 2  degree of hypoxia) and higher levels of blood Pco (higher degree of hypercarbia) are 2  required to stimulate increases in respiratory variables (Netick et al. 1984; Khoo et al. 1995; Daly 1997). The northern elephant seal is the only phocid seal in which the effects of hypoxia and hypercarbia have been examined during different states of arousal. Using / R as an indicator of sensitivity, Milsom et al. (1996) concluded that hypoxic sensitivity was increased and hypercarbic sensitivity was unchanged during SWS, when compared to W A K E state levels in this species. The increase in hypoxic sensitivity is similar to that seen in rats and ground squirrels, and the unchanged hypercarbic sensitivity is similar to that seen in goats (Milsom et al. 1996). Although there are similarities in the hypoxic and  21  hypercarbic sensitivities of phocid seals during the W A K E state, the species-specific differences in breathing pattern, related to changes in arousal state in phocid seals, make it difficult to predict the chemosensitivities of the different species of phocid seal, during SWS.  1.4 Heart Rate Heart rate is not static. As the internal and/or external environment in which the animal resides changes, physiological variables such as breathing and metabolism also change. These changes typically lead to simultaneous changes in heart rate; specifically tachycardia (increased heart rate) and bradycardia (decreased  heart rate).  An  electrocardiogram (EKG) is a tool designed to measure the variations in the electrical potential of the heart from various locations on the surface of the body. Using the E K G signal (Fig. 1.3a), there are two common techniques for calculating heart rate: 1) counting the number of beats per unit time, or 2) using the R-R peak interval (seconds) to determine instantaneous heart rate (1/R-R interval) (Fig. 1.3b). Although heart rate varies from species to species and individual to individual, average heart rate values can be used as an estimate of a normal heart rate for that species. In most terrestrial mammals, as well as marine mammals, heart rate accelerates during inhalation and decelerates during exhalation, leading to the presence of a respiratory sinus arrhythmia (Daly 1997). This is especially prevalent in phocid seals during both dive and sleep-related apneas. It is known that when seals dive, there is a subsequent bradycardia, similar to that present during resting and sleep apneas (Kooyman 1985; Castellini et al. 1994a).  Both the diving and sleep-apneic bradycardias are  22  therefore thought to be associated with the changes in respiratory pattern, and in fact, it has been suggested that the bradycardia of short dives and apneas (on land and in water) are extensions of the normal respiratory sinus arrhythmia and therefore are not "true" bradycardias (Castellini et al. 1994a). Kooyman (1985) suggests that in Weddell seals, a "true" diving bradycardia does not occur until dives longer than ten minutes, at which point the heart rate is lower than that observed in a resting apneic state. Although there is no correlation between swim speed and heart rate in phocid seals, there is a strong relationship between the strength of the bradycardia and the length of the apneic period, such that the lower the heart rate, the longer the dive and/or subsequent apnea (Kooyman 1985; Hill et al. 1987; Thompson and Fedak 1993). In reference to this, Thompson and Fedak (1993) suggested that phocid seals have a finite number of heart beats in apneic situations, such that once that number is reached, the seal must recommence respiratory activity: i.e. the lower the heart rate during the apneic period, the longer the animal can breath-hold. This appears true only for short dives and apneas (Hill et al. 1987; Butler and Jones 1997). During forced and prolonged dives, as well as extended sleep-related apneas, the bradycardia in some species of phocid seal can reach values as low as 2-3 beats min" , however most diving heart rates typically stay 1  above 10 beats min" (Andrews 1999). 1  In addition to lowering the heart rate during apneic periods, phocid seals also vasoconstrict blood vessels to selective, hypoxia resilient tissues (see Butler and Jones 1982 for review).  This serves to increase the peripheral resistance to blood flow,  allowing for the maintenance of constant blood pressure and blood flow to hypoxic  23  Figure 1.3. Electrocardiogram (EKG) signal of an 11-month old harbour seal. Gold-disc E K G electrodes were sutured just beneath the panniculus, one caudal and one cranial to the heart, on opposite sides of the dorsum of the seal. A ) illustrates the important deflections of the E K G signal, while B) illustrates the R-R interval commonly used for determining instantaneous heart rate.  24  sensitive tissues such as the brain, lungs, and heart (Hill et al. 1987; Thompson and Fedak 1993; Castellini and Zenteno-Savin 1997). As phocid seals surface, they show an anticipatory tachycardia and vasodilation of the hypoperfused tissues. It is thought that the increase in heart rate, and the increased blood flow act to remove waste products such as lactate and CO2 from the hypoperfused tissues, as well as allowing for the uptake of residual O2 from the blood (Fedak et al. 1988). The benefit of collecting respiratory waste byproducts and reducing blood Po  2  levels prior to any respiratory event is to minimize surface time due to increased efficiency in removing the waste products and oxygen loading (Fedak et al. 1988; Thompson and Fedak 1993; Butler and Jones 1997). Because heart rate is so intimately linked to respiration and metabolism, factors that affect these variables also affect heart rate. In terrestrial mammals, as arousal state decreases from W A K E to SWS, there is a subsequent decrease in heart rate due to decreases in both breathing frequency and vasomotor tone (Lydic 1987). In phocid seals, as arousal state decreases from W A K E to SWS, heart rate is also observed to drop, however in northern elephant seal pups, harp seal pups, and yearling gray seals, the decrease in overall heart rate is due to significant decreases in apneic heart rate only (Ridgway et al. 1975; Lyamin 1993; Castellini et al. 1994a). Hypoxia and hypercarbia also significantly increase breathing frequency in terrestrial mammals, thereby causing tachycardia and hypertension, increasing cardiac output, and reducing systemic vascular resistance (Daly 1997).  To maintain a  ventilation-perfusion match, allowing optimal delivery of oxygen to body tissues, heart rate increases with breathing frequency in both W A K E and sleep states, as respiratory  25  drive increases (Daly 1997). In phocid seals, the cardiovascular response to increased respiratory drive (hypoxia and hypercarbia) is seldom studied. In the northern elephant seal, Kohin et al. (1999) found an increase in heart rate during wakefulness in both hypoxia and hypercarbia, while Milsom et al. (1996) found no change in heart rate associated with hypoxia and an increase in heart rate during hypercarbia, while awake. Opposite to terrestrial mammals, there was no increase in heart rate during SWS in both hypoxia and hypercarbia (Milsom et al. 1996) in the northern elephant seal. The matching of the heart rate to breathing frequency in phocid seals is rarely studied, especially under conditions of increased respiratory drive and decreased states of arousal. To date there has only been one species examined, thus providing information regarding the cardiovascular response of that phocid seal, the northern elephant seal, to hypoxia and hypercarbia. From the studies on northern elephant seals (Milsom et al. 1996; Kohin et al. 1999), there are two different patterns of response, possibly due to low N-values. It is therefore difficult to understand and predict the mechanisms behind the cardiovascular response to variations in respiratory drive.  1.5 Purpose of Study Due to their aquatic and terrestrial lifestyles, as well as their ability to remain apneic for prolonged periods of time, phocid seals have many unique physiological adaptations. Because they spend so much of their time at sea, little is known about the respiratory physiology of phocid seals during the different states of arousal! Studies of gray seals, Caspian seals, harp seals, and northern elephant seals on land and in water reveal that the changes in respiratory variables, in response to arousal state, are species  26  dependent (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin et al. 1993; Castellini et al. 1994a). The data currently available however, do not provide enough information allowing for a potential model, possibly relating the cardio-respiratory physiology of the animal to its environment and/or ecology. Sensitivity to increasing levels of respiratory drive (i.e. hypoxia and hypercarbia) has been studied in phocid seals and data indicate that, although they are capable of prolonged breath-holds, seals are just as sensitive to changes in blood gas composition as terrestrial mammals.  Due to animal and/or equipment constraints, none of the  aforementioned arousal state studies, or hypoxic/hypercarbic respiratory drive studies, completed to date, give a complete picture of the changes in cardio-respiratory variables and breathing pattern. They focus rather, on one or two of these variables, typically V E and/or V 0 2 . This thesis, therefore, sets out to determine the cardio-respiratory and breathing patterns of the harbour seal, a small, short-duration diver, both during different states of arousal and different levels of respiratory drive. Specifically, male harbour seal pups were examined in each arousal state, under conditions of hypoxia and hypercarbia. A stimulus response curve, depicting the effects of increased levels of respiratory drive, on the cardio-respiratory and breathing patterns, during the different arousal states was produced. With the completion of these experiments, three main questions regarding state of arousal, increased respiratory drive, and the cardio-respiratory/breathing patterns of harbour seal pups were answered:  27  •  What is the sleep state pattern of the harbour seal? Does it follow the typical mammalian pattern of W A K E - S W S - R E M sleep?  •  What is the breathing pattern of the harbour seal during different states of arousal?  Is it state specific? How does it compare with other species of  phocid seal?  •  How do harbour seals respond to changes in respiratory drive during the different states of arousal? Do sleep and breathing patterns change during different levels of respiratory drive (i.e. in normoxia versus hypoxia versus hypercarbia)?  1.5.1 Specific Hypotheses Hypothesis #1. The sleep state pattern will follow the pattern typically seen in terrestrial mammals (awake-SWS-REM sleep). Hypothesis #2. The breathing pattern of the harbour seal, on land, will be state specific. During periods of wakefulness, breathing will be continuous and irregular, during periods of SWS breathing will be episodic, and during periods of R E M sleep, breathing will be sporadic and rare, similar to that seen in the Caspian and harp seals (Mukhametov et al. 1984; Lyamin 1993). Hypothesis #3. Metabolic rate will decrease as arousal state decreases, such that W A K E V 0 2 values will be greater than SWS V 0 2 values.  Hypothesis #4. Awake harbour seals will respond to increases in respiratory drive (i.e. hypoxia and hypercarbia) in a manner similar to terrestrial mammals. In hypoxia, V 0 2 will decrease, / R and V E will increase and V T will remain unchanged. In hypercarbia, V 0 2 will remain unchanged, while / R , V E , and V T increase significantly.  28  Hypothesis #5. During SWS and R E M sleep, both hypoxic and hypercarbic sensitivity will be reduced compared to W A K E state levels, however the change in sensitivity will remain similar to terrestrial mammals.  29  Chapter 2 Effects of arousal state and respiratory drive on cardio-respiratory variables in harbour seal pups (Phoca vitulina richardsi).  30  2.1 Introduction Since Irving (1938) suggested that the extreme dive capabilities of marine mammals were in part due to a reduction in their sensitivity to hypoxia and hypercarbia, numerous studies on phocid seals have suggested that this is not the case, at least while animals are awake and not diving. Under these conditions, phocid seals appear just as sensitive to increases in CO2 (hypercarbia) as terrestrial mammals, although the threshold level of CO2 required to elicit a response may be higher (Pasche 1976b; Craig and Pasche 1980; Parkos and Wahrenbrock 1987; Milsom et al. 1996; Kohin et al. 1999). Northern elephant seal pups (Mirounga angustirostris) (Milsom et al. 1996; Kohin et al. 1999), juvenile harbour seals (Phoca vitulina) (Craig and Pasche 1980), and hooded seal pups (Cystophora cristata) (Pasche 1976a) also appear just as sensitive to hypoxia as terrestrial mammals (with an increased threshold level for eliciting a response), while juvenile Weddell seals (Leptonyhotes weddelli) exhibit a decreased hypoxic sensitivity (Parkos and Wahrenbrock 1987). The effects of arousal state on breathing and responses to changes in respiratory drive in marine mammals have not been studied widely. Sleep is a state or period of reduced physical and neuronal activity accompanied by a suspension of voluntary movements and a complete or partial unconsciousness (Home 1988; McGinty and Szymusiak 1994). As such, it is also accompanied by a reduction in metabolic rate and breathing. In mammals, there are three main states of arousal: wakefulness (WAKE), slow-wave sleep (SWS), and rapid eye-movement (REM) sleep.  One of the key  characteristics of SWS is a decrease in muscle tone, while in R E M sleep there is a complete loss of muscle tone (Lydic 1987). Many marine mammals sleep for prolonged  31  periods while completely submerged in water suggesting they possess adaptations that either allow them to overcome the reduction or loss of muscle tone and float to/at the surface and breathe during sleep, or prolong breath-holding. The latter could entail a reduction in hypoxic/hypercarbic sensitivity.  While there is a growing body of  information regarding breathing patterns during sleep in phocid seals, information regarding sensitivity to changes in respiratory drive during different arousal states is limited. During routine dive bouts in awake phocid seals, breathing is episodic with periods of apnea during diving followed by short periods of breathing at the water's surface (Butler and Jones 1982). Studies on northern elephant seal pups (Bartholomew 1954; Castellini et al. 1994; Milsom et al. 1996), harp seal pups (Pagophilus groenlandicus) (Lyamin 1993; Lyamin et al. 1993), adult Caspian seals (Phoca caspicd) (Mukhametov et al. 1984), and juvenile gray seals (Halichoerus grypus) (Ridgway et al. 1975; Mortola and Lanthier 1989) have shown that while awake on land, breathing is continuous and irregular with only short periods of apnea present. In all phocid seal species studied to date, SWS has been shown to occur both on land and in the water, and breathing during SWS is always episodic with submerged individuals floating to the surface to breathe (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1994a). Gray seals (juvenile and newborn) (Ridgway et al. 1975; Mortola and Lanthier 1989), northern elephant seal pups (Castellini et al. 1994), harp seal pups (Lyamin 1993), and adult Caspian seals (Mukhametov et al. 1984) all exhibit R E M sleep both in and out of the water. Breathing is continuous during R E M sleep in gray seals, therefore animals must be positioned such that they can breathe, while in both harp seal  32  pups and adult Caspian seals, breathing is rare during R E M sleep, and when present, it is sporadic and irregular (Ridgeway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Lyamin et al. 1993). In northern elephant seal pups, breathing appears never to occur during R E M sleep (Castellini et al. 1994; Milsom et al. 1996). To the best of my knowledge, the information concerning respiratory sensitivity in phocid seals during sleep is restricted to one study. Milsom et al. (1996) examined the effects of both hypoxia and hypercarbia on breathing in northern elephant seal pups. In their study, animals were subjected to levels of hypoxia and hypercarbia that roughly doubled their breathing frequency (reported average: 13.3% O2 ± 0.15 S E M and 5.6% CO2 ± 0.31 SEM). No study to date has performed a detailed examination of the slopes and thresholds of the ventilatory responses to hypoxia and hypercarbia during sleep in phocid seals.  The purpose of my study, therefore, was to produce a more complete  stimulus response curve depicting the effects of hypoxia and hypercarbia on the cardiorespiratory and breathing pattern responses of the harbour seal during different states of arousal. Specifically I wished to determine whether the responses seen in harbour seal pups differed from those seen in terrestrial mammals, particularly during the transition from W A K E to sleep.  2.2 Methods 2.2.1 Animals and Housing Three male harbour seal pups, wild caught off the west coast of BC, under a license issued by the Department of Fisheries and Oceans, Canada, were housed in two connecting outdoor tanks located at the University of British Columbia (UBC), under  33  conditions of natural photoperiod and ambient temperature. They were raised in captivity and at the time of the experiments were 6 to 12 months old, weighing 36-42 kg. A large, wooden deck on which the seals could haul out surrounded the two tanks. The tanks were 3 meters in diameter and 1.5 meters deep, and fresh water continuously flowed through each. A l l seals were fed a diet of herring (Clupea sp.), which was supplemented daily with a vitamin tablet (Mazuri Vita-Zu Mammal Tablet, PMI Nutrition International) placed under the operculum of one fish. Experiments were conducted between January and June of 2000.  2.2.2 Surgical Procedures The following experiments were performed under the guidelines established by the Canadian Council for Animal Care, and were approved by the U B C Animal Care Committee (Permit # A98-0290). Each seal was manually restrained and anesthetized with 5% Isoflurane (Janssen) in oxygen (5 L/minute) administered via face mask. When a sufficient level of anesthesia was obtained, such that jaw muscle tone was minimal, the animal was intubated and anesthesia was maintained with 1 to 3% Isoflurane.  Since  apnea is common in seals under anesthesia, all animals were manually ventilated throughout the entire surgery. A l l surgical procedures were performed aseptically. Surgical sites were shaved and disinfected with 70% alcohol and an iodine-based solution (polyvinylpyrrolidineiodine complex 10%, Iodovet, Rougier Pharma).  To measure cerebral activity  (electroencephalogram [EEG]), four cutaneous incisions were made on the dorsal aspect of the head, two above the frontal and two above the parietal bones of the skull. At each  34  incision point, a small, stainless steel screw with accompanying lead wire was fixed into the skull, and secured with dental acrylic (Flexacryl, Lang Dental). To measure postural muscle tone (electromyogram [EMG]), two Teflon-coated, stainless steel wires were sutured into the right and left nuccal muscles respectively. On opposite sides of the dorsum of the seal, gold disc electrocardiogram (EKG) electrodes (Grass Instruments) (one caudal and one cranial to the heart) were sutured just beneath the panniculus (blubber layer). The leads from the E E G , E M G and E K G electrodes were then tunneled between the skin and panniculus to an exit point located on the back of the animal between the scapulae, where they were subsequently soldered to an eight-pin underwater computer connector (USI square miniconn, Underwater Systems) which was then secured to the fur on the back of the seal with epoxy glue (Evercoat Ten Set Epoxy Adhesive and Filler, Fibre Glass-Evercoat Co., Inc.). A diagrammatic representation of the electrode placement is shown in Figure 2.1. A l l incision sites were sutured and infiltrated with 1 ml of Bubivacaine hydrochloride (Marcaine, Sanofi Winthrop Pharmaceuticals) to provide post-operative analgesia.  After surgery, seals were given one intra-muscular injection of an anti-  inflammatory (1 mg/kg of Ketoprofen, Anafen, Merial) and allowed to recover in a dry pool for 24 hours. For two weeks post-surgery, seals were placed on oral antibiotics (2.5 mg/kg of Enroflaxacin; Baytril 50 mg/ml, Bayer Inc., Toronto, Ontario, Canada) to prevent infection of the cutaneous wounds. Of the three study animals, only one developed a minor infection at one of the incision sites. This was successfully treated with oral antibiotics (2.5 mg/kg of Enroflaxacin; Baytril 50 mg/ml, Bayer Inc.) with no long-term effects on the health of the animal.  35  Computer Connector  Figure 2.1. Electrode placement. Four E E G electrodes were placed in the skull o f each seal, two above the frontal and two above the parietal bones. E M G electrodes were sutured into the right and left nuccal muscles respectively, and on opposite sides o f the dorsum o f the seal, gold disc E K G electrodes (one caudal and one cranial to the heart) were sutured just beneath the panniculus. The leads from the E E G , E M G and E K G were then tunneled between the skin and panniculus to an exit point located on the back o f the animal between the scapulae, where they were subsequently soldered to an eight-pin underwater computer connector which was then secured to the back o f the seal with epoxy glue.  36  2.2.3 Experimental Protocol Prior to each series of experiments, seals were habituated to a 120 litre, custom built, clear, plexi-glass metabolic dome until they exhibited periods of sleep and quiet wakefulness with no signs of stress. Two to four hours before an experiment, seals were fed approximately 500g of herring. Just prior to the experiment, the relaxed, satiated seal was coaxed onto deck and entered a kennel, in which they were subsequently weighed (± 0.2 kg). After returning to the deck, the computer connector on the back of the seal was attached to the lead to a computer, and the seal was secured in the metabolic dome. Seals were allowed a ten-minute adjustment period before data recording began, and the length of the recording session varied from two to four hours, depending on co-operation levels of the seal. Preliminary observations indicated that the most optimal time in which to conduct experiments was at night, however due to limitations in visual acuity of the observer, experiments were performed during the second most optimal time, between 10am and 2pm. A hypnogram of this time period for one seal in one session (Fig. 2.2) illustrates that there was still a large proportion of time spent in sleep. Ambient air was pulled through the metabolic dome via a vacuum (6.5 Gallon, 3.0 peak horsepower Wet/Dry Vacuum, Shop Vac) at a rate of 28 to 30 litres/minute. Small holes placed at the back of the dome allowed for controlled inflow of ambient air, as well as the manipulation of that air by the addition of various gases. Sub-samples of the outflow air were dried with anhydrous calcium sulfate (Drierite, Drierite Company) and analyzed for fractional composition of oxygen (Fo ) and carbon dioxide (Fco ) using medical gas 2  2  analyzers (Beckman OM-11 oxygen analyzer [calibrated with ambient air] and Beckman LB-2 C 0 analyzer [calibrated with a 5% C Q , 95% N mixture]) (Fig. 2.3). Temperature 2  2  37  2  Wake H  Drowsy -  SWS  REM H —i  20  1  1  1  1  1  1  1  40  60  80  100  120  140  160  1  180  Time (minutes)  Figure 2.2. Wake-Sleep Hypnogram. The amount of time an animal spends in each state of arousal, during a specified period of time, is visible in this four-hour hypnogram of one harbour seal pup. Note that the appearance of R E M sleep is rare, and in all cases, is preceded by SWS.  38  within the metabolic dome was continuously monitored, and maintained near ambient temperature (10°C to 25°C), well within the thermal neutral zone for juvenile harbour seals (-2.5°C to 25.1°C, Hansen et al. 1995). EEG, E K G , and E M G signals, as well as a breathing trace (measured as the differential pressure between the metabolic dome and the outside) were amplified and filtered using Gould A C amplifiers, and, along with Fo , and Fco were recorded 2  2  continuously on computer using a data acquisition system (DataQ Instruments 1998; DI400, DataQ Instruments) sampling at a rate of 222 Hz/channel. Behaviour, monitored through visual observation, was also recorded for the duration of the experiment. To increase respiratory drive, the environment within the metabolic dome was made either hypoxic or hypercarbic. To decrease the oxygen content of the inflow air (Flo ) to the dome, 100% medical grade nitrogen was added to the metabolic dome at a 2  known flow rate, and mixed with ambient air to give three different levels of hypoxia: 15.22% 0  2  ± 0.47 S E M , 11.99% 0  2  ± 0.29 S E M , and 8.63% 0  2  ± 0.22 S E M .  To  increase the carbon dioxide content of the inflow air (Flco ) to the dome, 100% medical 2  grade carbon dioxide was added to the metabolic dome and mixed with ambient air to give three different levels of hypercarbia: 2.04% C 0 ± 0.06 S E M , 4.16% C 0 ± 0.06 2  S E M , and 5.66% C 0 ± 0.07 S E M . 2  2  Each gas level was presented to the animal on  separate days, in semi-random order.  2.2.4 Data analysis Arousal states were scored using standard sleep scoring techniques, as outlined by Rechtschaffen et al. (1968). Data traces were analyzed in 30-second epochs and divided  39  2  cd  o3  •B  s . °  £  00  42 £ & ° 42 W5 1 "> O  <D  *  |  03  £ o  .a s | w on CD  >  £ U . 3 *3 C 3 U 3  S-E S  CD  6 B  1?  OH  6? 03 CD  2 §  £  3  O * £ 2o 09o O  o  3  03  CD  TO  .2 4=  ©  CD  4= O  3  00 3  03  Ct) ^H CQ • £ O ,5 "3 d © CO so £ 3  so c3  SO  CD  o  CD  —  u u. T3 3  o3  03  c  CD  03  0 9  o  *  03  00 4S  T3  2o  3  3  03 QJ  _ CH  -H  £  ^  8 3  B Q  o 03  3  a o. •is CD  & 00  S ^ w  <•* O 03 U O "3  .2? o *  CD  d  -§ 5 i22  tH £  00 ^ 3 tCD 3  T t "  «g J co s © 5Hc od  O  3:  CO T3  03  °  o3  °  eu 4= ^3  ON  g  as  o'  .  G  CD  |  a  O  4=  -5  -H  *  >  "3 CL)  O.  So 5  £  £  Q  o & ®  Li-  2 3  3  >  CN  d  I 03  1  »i I  a  s a I g  CD  -  N J  " "2 03  4  ^ if ~  OH  O C* CN «  03  03  CD  _u  a  *J  00 oo  00 • JL t-T T3 X o 03  & n  4=  1  :£  is  «  42 CO •g NO  > o  5 32S >,3 O^ -32 s c  u  03  43 3 * g" "5 I © 00 » ^ OH 2 3 P o J"  T3  qj3  * £d  3 o  aa 4=  <  CCS  3  „  I s  •c S  ex X  HS  3 &  g  ^  •9  09  c  £I 1)  m 'o. 1  ©  .3 00 03  & 3 O DJ O > O C 5« ? ^ NO ^ (§ . O  00  3  « 3  M  i  °  " -O  03  03  £  03  £  .3  >  03  03 0  3 3 00.2 M  40  U  c3 C co  tH 0>  s-  L O  |  "00  >  «  c  4»d  § 3  2 o  w +i CO „ CN O CN Q O HH CM  O  $  O CN  into W A K E , drowsy, SWS, and R E M sleep based on E E G and E M G traces (Fig. 1.1), as well as behaviour. Only well-established W A K E , SWS, and R E M sleep periods were analyzed further. The breathing trace was analyzed for total frequency (/R) and tidal volume ( V T ) , which were subsequently used to calculate total ventilation ( V E ) in ml air/kg/minute. VE=/RXVT  eq. 2.1  To determine total breathing frequency (breaths/minute) for each state of arousal, the number of breaths per total time was calculated for each section of trace analyzed. Tidal volume (ml/kg) was calculated using the area under the differential pressure transducer trace, which is proportional to the product of (flow) X (time), for each breath. For each animal, ten breaths were analyzed in each state, at each level of respiratory drive, and V T was calculated using the following equation, adapted from McArthur and Milsom (1991). f (area ofbreath)x (volume of calibration b r e a t h ) x T b ( P S H 2 Q - P C H 2 0 ) | 1 (area of calibration breath)x [ T b ( P S H 0 - P C H 2 0 ) - T c ( P S H 0 - P C H 2 O ) ] j 2  eq. 2.2 V T = —  weight of seal  2  -  where T b is body temperature (°K), P S H 0 is water vapor pressure at sea level 2  (760mmHg), P C H 0 is water vapor pressure of the metabolic dome (corrected for dome 2  temperature), and T c is temperature inside the metabolic dome (°K). Because of the presence of prolonged end-inspiratory pauses or apneas during normal breathing, animals could increase breathing frequency by either reducing or eliminating the periods of apnea, or by reducing the length of individual breaths, and hence the inter-breath interval. To distinguish between these options, apnea length and inter-breath intervals were also determined from the breathing trace. A n apnea is defined as any pause in breathing exceeding the length of two missed breaths, in this case, any  41  pause greater than 5 seconds. The inter-breath interval is defined as the time, in seconds, between any two breaths not separated by an apnea, and was used to determine the instantaneous breathing frequency  (/Rinst  = 60/inter-breath interval). The total time spent  breathing (eupneic) versus not breathing (apneic) at each level of respiratory drive was determined as a percentage of the total time analyzed. Oxygen consumption  (V02)  and carbon dioxide production  (Vco2)  values were  calculated for W A K E and SWS states, on air, using equation 2.3 and 2.4 (adapted from Frappell et al. 1992, equations A5 and A8 respectively). A l l V02 and recorded in ml  02/kg  VCO2  values are  body weight, STPD. The average respiratory quotient (RQ) for  each seal, on air, was determined from the calculated values of  V02  and  Vco2  (Table  2.1). (F102 - FEo )air flow 2  V02 =  eg. 2.3  1  _  F  l  0  j  weight of seal (FECOI -  Fico2)air flow  Vco =  eg. 2.4  2  weight of seal Vco  eg. 2.5  RQ = -V02 ; ° C  To determine V02 during hypoxic conditions, the average RQ value for that animal on air, and the calculated  Vco2  (equation 2.6) for the gas level in question were used  (equation 2.7): eg. 2.6  (FECO: -  Vco = 2  42  Fico2)air flow  weight of seal  Table 2.1. Mean V02, Vco2, and RQ values on air, during both W A K E and SWS states. The calculated RQ values in air were used to calculate V02 during hypoxic conditions.  V02  WAKE  SWS  8.79 ±0.70  t7.71±0.93  6.37 ± 0.35  f 4.97 ± 0.28  ml 0 /kg/min 2  VC02 ml C02/kg/min  RQ 0.73 ± 0.03 0.66 ± 0.03 Mean values ± SEM. indicates a significant difference between W A K E and SWS values. (N=3)  43  eq. 2.7  Vo2 =  RQ To determine V02 during  where Flco is assumed to be 0.003% in ambient air. 2  hypercarbic conditions, Flo values (0.2094 in ambient air) and recorded values of FEO 2  2  were used in the following equation: (F102 - FEoiWr flow eq. 2.8  V02 = ^  weight of seal Equations 2.6 and 2.8 are adapted from Frappell et al. (1992; equations A 6 and A3 respectively). It is assumed, when calculating V02 and Vco2, that air is dried and CO2 free (Withers 1977; Frappell et al. 1992). Although air was dried prior to analysis in the current study, CO2 was not removed. According to Withers (1977), the errors associated with failure to remove CO2 are negligible under normal conditions. Failure to dry the outflow air prior to the measurement of flow rate (as was done in the present experiment) results in a maximum error of 6.2% in the calculated values of V02 (Withers 1977, Frappell et al. 1992). It should be noted however, that the air flow rates were recorded using a mass flow meter in litres/minute STPD, and therefore any errors associated with failure to dry the air prior to recording flow rate are minimal. Oxygen consumption values were also used to calculate percent oxygen extraction (%Eo ) and air convection requirement (ACR) (ml air/ml 0 ) at each level of respiratory 2  2  drive for each state of arousal using the relationships: eq.2.9  %Eo2= . ° V  2  VEXFIO  and  44  2  eq.2.10  ACR = - ^ Vo  2  To determine average heart rate (/H), the number of beats per total time (beats/minute) was calculated for each section of the trace.  Both eupneic and apneic  heart rates were also calculated independently (i.e. total # beats in eupnea/total time eupneic and total #beats in apnea/total time apneic). To assess the relative change in heart rate due to increases in respiratory drive, the % change in heart rate was calculated using air values as a baseline.  2.2.5 Statistical Analysis Statistical analyses were performed using Sigma Stat software (Jandel Scientific, version 1.0, 1993). Data for the different levels of respiratory drive were analyzed and compared to air values using a one-way Repeated measures A N O V A , followed by the Bonferroni t-test method of all pairwise multiple comparison. compared to W A K E values using similar statistical analyses.  Data for SWS were Data were considered  significantly different if P O . 0 5 . A l l results are presented as means ± S E M .  2.3 Results 2.3.1 Hypoxia 2.3.1.1 Respiration Decreasing Flo produced increases in / R during both wakefulness and SWS (Fig. 2  2.4), however the increase was only significant at levels of 8.63% 0 ± 0.22 S E M . 2  /Rinst  remained relatively constant in both states as Flo decreased (Table 2A) indicating that 2  the increases in / R were due solely to decreases in the length (Fig. 2.4) and frequency  45  (Table 2A) of the periods of apnea. As a consequence, the percent time apneic decreased dramatically in both W A K E and SWS (Table 2A). There was no change in V T in W A K E or SWS during hypoxia and, as a consequence, the changes in total ventilation ( V E ) during hypoxia reflected the changes in / R (Fig. 2.4D). / R was significantly lower in SWS than W A K E during air and 15.22% 0 ± 0.47 2  S E M due primarily to increases in the length and frequency of occurrence of apneas, and thus increases in the percent time apneic (Fig. 2.4, Table 2.2A). V T , on the other hand, increased in animals as state of arousal decreased from W A K E to SWS (this increase was however, not significant) (Fig. 2.4C) and thus, in general, levels of total ventilation remained constant regardless of arousal state at all levels of hypoxia Fig. 2.4D). Thus while there was no decrease in hypoxic sensitivity associated with the transition from W A K E to sleep (there was a strong hypoxic ventilatory response present in both states), breathing was always slower but deeper during SWS.  2.3.1.2 Metabolic rate Oxygen consumption ( V 0 2 ) was unaffected by hypoxia within each state (Fig. 2.5A).  Although the total amount of oxygen available in each breath decreased  substantially in hypoxia, there was no significant change in the relative amount of oxygen extracted per breath (%Eo ) in W A K E or SWS (Fig. 2.5B). Thus, since the amount of 0 2  2  in the ambient air decreased, while the V 0 2 and %Eo levels remained constant, the air 2  convection requirement (ACR) or total amount of ventilation required to meet oxygen demands, increased, although not significantly, during both arousal states (Fig. 2.5C).  46  Figure 2.4. The effect of hypoxia on breathing frequency (/R) (A), average apnea length ( B ) , tidal volume ( V T ) ( C ) , and total ventilation ( V E ) ( D ) as a function of state of arousal. * indicates a significant difference between a specific level of respiratory drive and air. f indicates a significant difference between W A K E and SWS values at that level of respiratory drive. (N=3 in all states of arousal, at all level of hypoxia, except 15.22% 0 ± 0.47 S E M where N=2). 2  47  -I—  ©8 co ^  . -H  -H  — r  oo  o  "8  NO  „ "H  *  CN  O "-O 0 0 t-;  -H  co co -H  i n co r- ^  o  O OO CN C-HN  N o C in  *  o o  •"5  •^r NO t-^ N©  ON  ^  CN  00  ©  in ON CN -H m  co  CN © C N in -t— -H ©  *  co  oo  CN O co  co  in  Q CN  o o r-- ^ - i  NO  NO  co  —" NO  o o-H  © o ©  NO  oo  ©  -H  ON CN ON ON  ©  -H  *  CN NO  +1  o >n  OO  < ino C-HN  ON  o  CN +1  -H  J—„ o o r-; CN o co CN +1 in +l  ~  ON  -H  ON — i  -H  o o NO NO od ON in C N  O  •t-- -H  ^r  CO CO  CO  co  o  ©  *  -H  -H  CO 00  p m  o  i—1  *  o  o  NO  ON CN  CO  -H  r-  CN CN  o '—1  NO  -H  >—1  u d»  in  © CN  C  OH 03  C 3fc  00  •  m  —,  »—H 00 ON  in ©°  +— -H  8 s r  -H  00  NO ON  co  o  ON  o-H  O  NO  -H  ©  CN 00  •«t CN  CN  -H  CN  o  in -H  00 "3-  +1  CN #  -H  in  o  o  NO  -H  o o o ON NO 00  co -H  o  o  *  co -H  NO ON  ON ^  o  00  CO CN CO  ^r CN  -H 00  CN  CO NO  *  00  •^j- in O ^  CN -H  NO  o o  o *o  o r-  CN -H  T—1  - ?! -H  00  oo ^r'  o ON -H  in m  -H  o >o m ^-1  CO CN co -H  O CN co O CN CN co -H o in 00 •*r CO o CO -H  o <u  Pi  03  <u C  03  a  A  o3  O  o  CN O CN  *  *—<  CN © in  ON  I 03  OH 03  e  o  <u  OH  a  6 2 [_  03  o3  C  03  1 -  22 &  3 <u  O O  O  -H  41  O  -H  ©  ©  CN *  CN  +1  ON  p^ *  CN  00 vo 4|  -H  oo co  CO J_  *  P~ —.  co 4)  -H  *  ON  p^ co -H  O  in co  *t  ON  ON P~ O  CN CN  CN  ©  00  co co  1  4  p* -H  VI  o  CN t-~ < = >  P^ CN  -  O  -H  CN 00  NO  ©  CN CN H—  -H  ON C N NO r n •^T H  *  -H  r- r-  o  o o in  p© © -H  00  #  ©  co  CN  , "H  -H  © © in -H  P~ 1 1 "H  NO  ©  NO  CN  ©  Os  p^  H—  -H  in  #  ©  M  a— ^1  I-  -H  ©  co  CO  CN  p- _j  -H  II  CN  NO  NO  ©  ©  CN  P-  41  CO  p-  o  4H  CN 00  CN OO  © © * +1  IT) ©  CN  NO *  CN *  ON  -H  in  P~;  4?  NO  r- ^  ON  -H  © PON C N  >/-)  m 4i  CN CN  CO  o  IT)  NO  NO O  4|  co ©  CN *  41  ON  4H co ON  CN NO © P-; CN  ^  r~  41  r  4(  2 ©  © ON  iri  oo ° °  m  CN  2°  -H  4H  o u  2 o  ©  O q  O  CN CN  ON P-  NO  © CN  41  CN  © m TJ-  ~  CO  ©  00  41 ©  CO © CN  CN  41  4i  CO  ©  CN  A  C O a C D c c^  .—i  ©  i—i  41  41  ,—i  cn  O  o > - Ho CN  2  CO CD e  in m  in NO CO vd  41  CN  CO  p-  ©  p~  in in 4i *—i  ^r 41  03 CD  C  a 3  ^r  C/l 03  CD  a,  m  ro © CO 41  2  o CD C  © 00  o 13 >  CO  oo  cu  pq  © p~ * -H  oo  NO ON CN  CN  CD  s  c  CD &, CO of; CD C  6 2  I—i ^  03 CD  Pi CD CO ^ CD g S3 w +J  co  5  13 1/3  13  CD  u<  e  CO  II  ON  Vo2 was significantly reduced during SWS in animals breathing air and 11.99% O2 ±0.29 S E M compared to levels during W A K E (Fig. 2.5A). There was, however, no difference in %Eo  2  or A C R as arousal state decreased from W A K E to SWS (Fig. 2.5B, C).  2.3.1.3 Heart Rate With increasing levels of hypoxia, / H decreased during W A K E and SWS (Fig. 2.6A). In general these decreases were only significant at the lowest levels of Flo . While the decrease was 2  not significant during R E M sleep, I have no data at the lowest level of Flo (Fig. 2.6A). 2  Interestingly, the relative decrease in / H was the same regardless of arousal state (Fig. 2.6B). / H was always higher during periods of breathing than during periods of apnea, and hypoxia led to decreases in heart rate under both conditions (breathing and apnea) (Fig. 2.6C, D). / H was significantly lower in SWS and R E M sleep compared to W A K E in air and at all levels of hypoxia tested. Since there were no significant differences between the / H observed during breathing in W A K E and SWS, this reflected the lower / H seen during periods of apnea in SWS and R E M sleep.  2.3.2 Hypercarbia 2.3.2.1 Respiration Hypercarbia also produced significant increases in / R in both W A K E and SWS (Fig. 2.7A). The response was, however, proportionately much greater in SWS compared to W A K E (a 220% versus an 8 8 % increase).  /Rinst  (Table 2.2B) did not change in either state as CO2 levels  50  "5 «  0  T3 -  0)  C  «  •>  /-v  <D  Cd  1/1 CD  =3  CD CD  -  o o  o o  CD  T3  CD  Q  o o  o ^  \m i JIB IUI) H 3 V  a «»  o o  (Zq  00  O  o o  OH  CD  ^-N  cd  —'  -T3  > C O  ~  O  ©  -H  cD CD CD  X)  o  CD  cd §  (o/ ) uoipBJjxa  X CD  2"  CD > CD  0  x c  cd  O  cd o  o O 0>  0  +j OH CD O  <+_ o 3 O O  "K §b  >»  <N •  'I JH—1  o "3 CO  pq 00  cD  fS m  00  O  c3 ra  CO Q  o  1—1  fl O  C/l  o •  *H  co CD  cd ' « J "cd ^  2 ^  5  co  O  '*  w  O  Cd ^ CD a >  -a 11 <D  g  o cd  CD  O  "cd  „  CN 0>  3 ox a S 8  >  o  « "E, > CD  *—1  CO <D * - i  Figure 2.6. The effect of hypoxia on total heart rate (/H) (A), % change in / H (B), eupneic / H (C), and apneic / H (D) as a function of state of arousal. Data for the % change in / H are calculated using air values as baseline levels. * indicates a significant difference between a specific level of respiratory drive and air. f indicates a significant difference between W A K E , SWS, and R E M sleep values at that level of respiratory drive. (N=3 in all states of arousal, at all level of hypoxia, except 15.22% 0 ± 0.47 S E M where N=2). 2  52  Figure 2.7. The effect of hypercarbia on breathing frequency (/R) (A), average apnea length ( B ) , tidal volume (VT) ( C ) , and minute ventilation ( V E ) (D) as a function of state of arousal. * indicates a significant difference between a specific level of respiratory drive and air. f indicates a significant difference between W A K E and SWS values at that level of respiratory drive. (N=3).  53  rose.  Thus, during wakefulness the increase in / R was due to a significant decrease in the  frequency of apneas while during SWS it was due to decreases in both the length and frequency of periods of apnea (Fig.2.7, Table 2.2B). As a consequence, there was a significant decrease in the percent time apneic in both W A K E and SWS (Table 2.2B).  V T also increased during  hypercarbia during both arousal states, as did V E , however due to low N-values, these increases were not significant (Fig. 2.7). As state of arousal decreased from W A K E to SWS, there were significant decreases in / R in air and 2.04% C 0 ± 0.06 S E M (Fig. 2.7A). The average length of apnea in SWS, although 2  always  greater than  WAKE  values, was  never  significantly greater (Figure 2.7B).  Correspondingly, there were no differences in the frequency of occurrence of apneas between the different states of arousal, although the frequency of long apneas (>20 seconds) was significantly higher in SWS (Table 2.2B).  Levels of V T were not different between the different arousal  states. The net result of these changes was that V E was not significantly different between the two arousal states (Fig. 2.7D) regardless of the level of C 0 present. Thus there was also no 2  decrease in hypercarbic sensitivity associated with the transition from W A K E to sleep (there was a strong hypercarbic ventilatory response present in both states), although breathing was slower but deeper during SWS at low levels of inspired C 0 . 2  2.3.2.2 Metabolic Rate V 0 2 did not change during hypercarbia in either state (Fig. 2.8A). As the level of C 0 in 2  air increased, and / R increased significantly, there were significant decreases in the amount of  54  oxygen extracted from each breath (%Eo ) SWS only (Fig. 2.8B). 2  This resulted in large,  although not significant, increases in the A C R (Fig. 2.8C). V 0 2 was significantly reduced during SWS in animals breathing air, while %Eo was not 2  significantly different between states and neither was the A C R (Fig. 2.8B, C).  2.3.2.3 Heart Rate There were no significant changes in / H during hypercarbia in awake animals or animals in SWS (Fig. 2.9A, B) but there were during R E M sleep. This increase in / H only occurred at 2% CO2. fa was always higher during periods of breathing than during periods of apnea while heart rate in hypercarbia remained constant during both eupniec and apneic periods (Fig. 2.9C, D). On average, / H was significantly reduced during SWS only in animals breathing air but was always reduced in R E M sleep. This primarily reflected the higher heart rate seen during periods of breathing in awake animals and the fact that animals rarely breathed in R E M sleep (Fig. 2.9D).  2.3.3 REM sleep R E M sleep was observed at all gas levels, but not in all animals (Table 2.3). At low levels of hypoxia there was a progressive increase in the percent of sleep episodes containing R E M sleep but no change in the average length of R E M sleep (Table 2.3). At the most severe level of hypoxia, however, there were reductions in both the incidence of R E M sleep and the length of the R E M sleep bouts. During hypercarbia, there was a progressive decrease in the percent of sleep episodes containing R E M sleep but the length of R E M episodes did not vary.  55  a t£ °  o  a 2  « 8 <* 0 S  o  (-1  8. S ^ 03  0  >  u go  _.  g| % o o  o o  o o  o o  o o  o o  o o  o o  o o  o o  ill u  >->.  O H 03  Sis g O  a o  o  u  fj u ID  o 03 <D  S8  "£ g § ° S O  C3  O  -J3  02  IT3  PI  (o/ ) U O i p B J J X a ZQ 0  o  >« Pi  H-t  O  23 .S  o ™  • I-H  c3  O U  <*H  r  -  I  "T"  o  90  i  ve  (3jnuiui/8>i/Zo W) o\ z  a  p PS  Pi  c2 . a o a) a> • J * J* B 00 9> i. S  OX)  .2  .  2> <D  <~ 2  °J >  &  150 100  2 -1  r  4 —i  6  8  1  1  125 ^  75 100  50 7S  a  75  ex 25  c  50 25 -25  8  -50  B  150  150  125  125  3  c  c  a ioo H  I  75 03  SU  W  75 H  50  e  100  C3  50  C  25  ® • A  Wake SWS REM  0  2  25  4  6  D  F l c o (%)  2  4  6  FICQ (%) 2  2  Figure 2.9. The effect of hypercarbia on total heart rate (/H) (A), % change in / H (B), eupneic / H (C), and apneic / H (D) as a function of state of arousal. * indicates a significant difference between a specific level of respiratory drive and air. | indicates a significant difference between W A K E , SWS, and R E M sleep values at that level of respiratory drive. (N=3).  57  o  o  m d CN  o3  CN  NO  O o d O -H O  q iri  N  03  N ©  +1  O  CN CN  < o  CN  NO  o  Q O 4 U  +1  NO  q  o  CN CN  00  CN  -H  o  co  CN  00 IT) ON  CN  d +1 ON ON  +1  O  oo  00  co co  NO  d +l  CN  f-;  +1  CN co CN  CN CN  NO  -H co ON  CN  CO CD  c CO  J D  w C / J  00  •fen  C «  CO  epi  00 .is <u  po  ^ 3  a, CD  1> CO  *—' PH  03  'I  8  co  >  a l  OH CD J D  -H  M  2  Breathing was rare during R E M sleep, occurring in 2 0 % of trials in one animal breathing air, and in 2 5 % and 3 3 % of trials in two animals breathing 1 1 . 9 9 % O2 ± 0 . 2 9 S E M and 2 . 0 4 % C 0 ± 0.06 S E M (Table 2.4). Both / R and V T were highly variable on 2  those rare occasions when breathing did occur during R E M sleep (Table 2.4).  2.3.4 Breathing Pattern The inter-breath intervals on air, and at the most extreme levels of hypoxia and hypercarbia are plotted on Poincare plots (N versus N - l ) for both awake animals and animals in SWS in Figure 2.10. While breathing air, the inter-breath interval was quite variable both in W A K E and SWS. During wakefulness, most inter-breath intervals were less than 5 0 seconds (Fig. 2.1 OA) while in SWS they were more variable and could be up to 110 seconds long. The increased variability of inter-breath intervals in SWS reflected the greater incidence of apneas and the presence of a more episodic breathing pattern compared to W A K E (Fig. 2.11).  As the level of respiratory drive increased, in both  hypoxia and hypercarbia, the length of the inter-breath interval was reduced and the amount of variability decreased. They were reduced to less than 2 0 seconds in length during W A K E and SWS in hypercarbia but could still last up to 7 0 seconds during SWS in hypoxia. Breathing during R E M sleep was too rare to include in these comparisons.  2.4 Discussion 2.4.1 State of Arousal The changes in respiration that accompany changes in arousal state in phocid seals have only been quantified in four species; yearling gray seals (Ridgway et al. 1975),  59  o o o o d  o  NO  en  >n  ON  NO  -H  O  o  -H ON  IT)  CN  -H  CN  NO  +1  O  o  NO  o oo d  -d-  o  ON ON  -H  oo  d  d  +! ON ON  -u  <n in  -H  o ON d  +i  o  ON  CN  en en  NO  10.03  136.10  CN  28.00  ON  0.80  CN  +i  o q  en  o NO  o  o o d ON oo  <  00  '—  ON  -H  o q in in CN  <n ON  1  -H  O  NO  d  -H  >n  ON  00 c3 <D  cd  if  >  C CO  OH  w -H C/J CL>  s  > CD  CO  03  OJD  C  5 sec A  62  Figure 2.11. Breathing patterns of harbour seal pups in air during (A) W A K E (continuous and irregular and (B) SWS (episodic).  63  adult Caspian seals (Mukhametov et al. 1984), harp seal pups (Lyamin et al. 1993), and northern elephant seal pups (Castellini et al. 1994a). The study on northern elephant seal pups is the most in-depth study, focusing exclusively on the changes that occur in respiratory and cardiac frequency, as well as breathing patterns during the different states of arousal. In the present study, I have extended these studies to harbour seal pups and have also quantified the accompanying changes that occur in V T , V E , and V02. In terrestrial mammals, decreases in state of arousal from W A K E to SWS are accompanied by decreases in both breathing frequency and metabolic rate, and the transition to R E M sleep is accompanied by irregular breathing and metabolic rates that are increased above SWS levels, but still decreased compared to W A K E (Lydic 1987; McGinty and Szymusiak 1994). Castellini et al. (1994a) determined that these statedependent changes in breathing pattern were also present, although to a greater degree in northern elephant seal pups, with continuous and irregular breathing during wakefulness, giving way to episodic breathing during SWS, and a complete cessation of breathing during R E M sleep. I found similar, but less profound changes in harbour seal pups. During W A K E , breathing was continuous and irregular with periodic apneas of shortduration. During SWS breathing was more episodic with longer apneas, and in R E M sleep breathing was rare. It has been suggested that the decrease in / R seen during sleep, a common attribute of all vertebrates studied to date, most likely reflects the 10-20% decrease commonly seen in V02 (Orem et al. 1978; Phillipson 1978; White et al. 1995, Milsom et al. 1997). M y results are consistent with this hypothesis; / R , V02 and Vco2 were all significantly lower in SWS than W A K E in the harbour seal pups (44.3%, 12.4%, and  64  22.0% respectively) and were accompanied by an increase in both the frequency and length of periods of apnea. Given life history strategies, it is difficult to obtain reliable measurements of basal metabolic rate (BMR) (post-absorptive, mature, non-pregnant, and quiescent) in marine mammals (notably phocid seals) since at the times of the year during which most are accessible, they usually violate at least one of the criteria required for establishing basal metabolism (Huntley 1987; Rosen and Renouf 1995). Most studies on marine mammals tend to report resting metabolic rates (RMR) where animals are resting quietly while one or more of the variables defining B M R are not met. In the present study, the seals were both immature and had been fed prior to the experiments, violating two of the criteria required for recording B M R . Despite this, the values for W A K E V02 that I collected were similar to the R M R reported by Miller and Irving (1975), and Rosen and Renouf (1995) for young harbour seals. Although there was a strong correlation between the fall in / R and the fall in V02 during SWS in harbour seal pups, the fall in / R was not accompanied by an equal fall in V E as it is in terrestrial mammals (Lydic 1987). In terrestrial mammals, V T can either increase or decrease during SWS compared to W A K E , however the decrease in / R is dramatic enough to produce a significant decrease in V E . In harbour seal pups, V T increased during SWS, offsetting the fall in / R such that the fall in V E between W A K E and SWS was not significant. Consequently, the change in V02 was not accompanied by an equal change in V E , leading to a small, but insignificant rise in the air convection requirement ( V E / V O ) . 2  In the harbour seal pups of this study, the air convection  requirement in W A K E (136.7 ml air/ml 0 ) was more than double that reported for small 2  65  terrestrial mammals (Frappell et al., 1992) (typically between 40 and 60 ml air/ml O2). This would appear to be partly due to the relatively low V02 of the harbour seals compared to other terrestrial mammals and the relatively high V E . There are few studies that examine the A C R of terrestrial mammals, and even fewer that compare the changes in A C R during different arousal states. Given the decreases that occur in both V02 and V E , during sleep in terrestrial mammals (Phillipson et al. 1976; White et al. 1985), it would seem reasonable that the A C R remain relatively constant, as both Phillipson et al. (1976) and White et al. (1985) demonstrated in their studies on dogs and humans, respectively. This is similar to my results. The net effect of the respiratory changes in the harbour seal pups was that SWS was primarily accompanied by a slowing and deepening of breathing with little effect on V E . The changes in respiration that accompany R E M sleep differ between species of terrestrial mammal. In most cases, R E M sleep leads to an irregular breathing pattern with increases in / R and V02 over levels seen in SWS (White 1985; Lydic 1987). Breathing in phocid seals during R E M sleep, however, is rare. Castellini et al. (1994a) suggested that the absence of breathing during R E M sleep in northern elephant seal pups might be due to an extension of the muscle atonia that occurs in R E M sleep to the respiratory muscles. While this may be the case for northern elephant seals, my data for harbour seal pups, along with data for yearling gray seals, adult Caspian seals, and harp seal pups (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin, 1993) indicate that these species of phocid seal can breathe during R E M sleep, even though most typically don't, with the majority of R E M sleep episodes occurring within one respiratory pause.  Further  evidence that phocid seals can breathe during R E M sleep comes from the report that  66  yearling gray seals breathe continuously during R E M sleep (Ridgway et al. 1975). The reason for the diversity that is seen in breathing patterns during R E M sleep between species is not clear.  2.4.2 Hypoxic Ventilatory Response During wakefulness in harbour seal pups, hypoxia led to increases in / R and V E , due primarily to significant decreases in the length and frequency of periods of apnea. In most (but not all) terrestrial mammals, under hypoxic conditions, increases in V E are due primarily to increases in / R with either small increases, no change, or decreases in V T (Gothe et al. 1982; Garland et al. 1994; Webb and Milsom 1994; Bisgard and Neubauer 1995). In other marine mammals, however, increases in V E during hypoxia have also been reported to stem from increases in breathing frequency alone (hooded seal, Pasche 1976a; Amazonian manatee (Trichechus inunguis), Gallivan 1980; newborn gray seals, Mortola and Lanthier 1989). In the case of the hooded seals, the increase in / R during hypoxia was due primarily to decreases in the length and frequency of periods of apnea (Pasche 1976a), similar to the harbour seal pups of the present study. Because of the variability in the relative contributions of changes in / R and V T to changes in total ventilation in response to hypoxia seen in different species, the sensitivity of animals to changes in respiratory drive is generally expressed as the slope of the ventilatory response curve ( • V E / Fl02). M y data indicate that, like other marine #  mammals, harbour seal pups exhibit a strong hypoxic ventilatory response and are just as sensitive to hypoxia as terrestrial mammals.  67  In general, the ventilatory response to hypoxia is blunted during SWS in terrestrial mammals compared to the response seen during W A K E (Henderson-Smart 1995;  Corfield et al. 1999). The threshold for medullary respiratory neuronal activation  is increased during SWS, and it requires lower levels of blood Po and higher levels of 2  blood Pco to stimulate increases in breathing (Daly 1997). Given their tolerance for 2  prolonged apnea during diving, the presence of episodic breathing during SWS, and the large fluctuations in blood Po and Pco this should produce, one might expect an even 2  2  greater decrease in hypoxic sensitivity during SWS in phocid seals. In harbour seal pups, however, this was not the case.  Hypoxic chemosensitivity was not reduced in SWS.  Milsom et al. ( 1 9 9 6 ) , using changes in / R alone as an indication of respiratory chemosensitivity, found that there was an increase in hypoxic sensitivity in northern elephant seal pups. In the present study, I also found a greater increase in / R during hypoxia in SWS compared to that seen while the animals were awake, however, when the changes in V T and V E during hypoxia in the two states were taken into account, there was no difference in chemosensitivity.  Given this, indications of changes in  chemosensitivity based on measure of respiratory frequency alone should be viewed cautiously. As the amount of oxygen available in ambient air decreases under hypoxic conditions, animals must compensate by either increasing the supply of oxygen to the blood or by decreasing the demand for oxygen. The former is done by increasing V E , increasing the efficiency with which oxygen is extracted from each breath, or both (Chappell and Roverud 1990), while the latter is done by reducing metabolic rate (Frappell et al. 1992). In the harbour seal pups, I saw increases in / R and V E , as well as  68  reductions in V02 at more extreme levels of hypoxia. I saw no change in the relative amount of oxygen extracted from each breath during hypoxia, consistent with reports for terrestrial mammals (Chappell and Roverud 1990). Due to the large increase in V E and small reduction in V02, the air convection requirement ( VE/V02) increased significantly during hypoxia in both W A K E and SWS, as has also been reported for other mammals (Frappell etal. 1992).  2.4.3 Hypercarbic Ventilatory Response Many studies on phocid seals have now shown that these, and other marine mammals are as sensitive to hypercarbia as terrestrial mammals, although their stimulus threshold is elevated (Bainton et al. 1973; Pasche 1976b; Gallivan 1980; Parkos and Wahrenbrock 1987). The increased threshold for hypercarbia appears to be due to an increased buffering capacity of the blood for CO2 (Gallivan 1980), which is felt to be an adaptation that allows these animals to tolerate the high levels of blood Pco that occur 2  due to the accumulation of the by-products of cellular metabolism during periods of apnea (either diving or voluntary). Retention of hypercarbic sensitivity is believed to be an adaptation that produces shorter surface intervals during diving (Gallivan 1980; Daly 1984).  From the present study, it is apparent that harbour seal pups also express  sensitivity to hypercarbia similar to that of other phocid seals and terrestrial mammals, but they did not exhibit the increased response threshold to hypercarbia that is seen in other phocid seals. Significant increases in / R , V T , and V E were evident at the lowest level of hypercarbia tested.  69  In harbour seal pups, hypercarbia led to increases in V E , due to increases in both /R and VT, at all levels tested. At the higher levels of CO2 tested, however, V T and V E did not increase as much as they did on lower levels of CO2, although / R increased more. The reasons for this are not clear. Pasche (1976b) found similar trends in female hooded seals breathing 6% CO2. These data may suggest that these animals had quickly reached the limit to which they could increase V T , and that for / R to increase any further as the levels of CO2 continued to climb, the time available for inspiration had to be reduced leading to the reduction in V T . Because of the large increase in total ventilation, the percentage of time spent breathing, rather than apneic, increased significantly. Despite this, there was no increase in V02 in the harbour seal pups in W A K E .  This is in contrast to reports for northern  elephant seal pups where there was a significant increase in V02 during wakefulness at inspired C 0 levels of 7% (Kohin et al. 1999). 2  Because of the large increase in  ventilation and constant metabolic rate, the air convection requirement increased dramatically while the amount of oxygen extracted from each breath fell. Similar results have been reported for terrestrial mammals (Barros et al. 2001). Along with the decrease in V E that accompanies SWS in terrestrial mammals, there is typically a decrease in sensitivity to hypercarbia, accompanied by slightly elevated blood Pco levels (Orem et al. 1977; White et al. 1985; Daly 1997). Just as with 2  the hypoxic sensitivity, while one might expect the hypercarbic sensitivity of the harbour seals during SWS to be reduced as much as, or more than, is typically seen in terrestrial mammals, there was no difference in sensitivity between the two states. Although / R was significantly lower in SWS compared to W A K E at air% and 2% C 0 , there were no 2  70  differences in V T , and hence, no changes in V E as arousal state decreased. The stable V02 that I recorded in both W A K E and SWS during hypercarbia in harbour seal pups is similar to what has been reported for terrestrial mammals (Mortola and Gautier 1995).  2.4.4 Heart Rate Respiratory-induced cardiac sinus arrhythmias are common in mammals, leading to an increase in heart rate during inspiration and a decrease in heart rate during expiration or during periods of apnea. This tends to match ventilation and perfusion and minimizes the work of the heart.  In the present study, harbour seal pups showed a  decrease in heart rate during apnea by as much as 31% in wakefulness and 68% in SWS under normoxic and normocarbic conditions. When seals breath-hold, they typically due so on expiration (Eisner et al. 1977). The act of lung deflation decreases pulmonary stretch receptor activity, increasing cardiac vagal tone, and thus allows a bradycardia to develop (Daly et al. 1977). Once an apnea has begun, blood Po levels begin to decrease, stimulating the peripheral carotid body 2  chemoreceptors. This should cause hyperventilation, but it is overridden by trigeminal and superior laryngeal nerve stimulation, by water in submerged animals, and by central mechanisms during apnea on land.  In seals (see below), it also contributes to the  bradycardia, and vasoconstriction (Eisner et al. 1977; Daly et al. 1977; Daly 1984). When breathing resumes, the low blood Po and high blood Pco continue to stimulate the 2  2  carotid bodies transiently, which would act to maintain the bradycardia. However, the decrease in vagal tone brought about by increases in excitatory input from the pulmonary stretch receptors causes / H to increase, overriding the carotid body induced bradycardia  71  and vasoconstriction (Eisner et al. 1977; Daly 1984).  For a representative trace  illustrating the degree of the hypoxic bradycardia in harbour seal pups, refer to Figure 2.12. In terrestrial mammals, hypoxia causes hyperventilation and tachycardia. Hypoxia in harbour seal pups however, caused hyperventilation and a significant bradycardia (up to a 5 0 % reduction in heart rate) during all states of arousal, while at the same time causing a significant increase in / R and V E (by up to 5 0 % ) .  Eisner et al.  (1977) have previously shown that stimulation of carotid body chemoreceptors in anesthetized harbour seals by hypoxia leads to decreases in / H and increases in / R . This was also the case in the harbour seal pups in the present study. It is interesting to note that while lung inflation appears to be able to suppress the cardio-inhibitory response arising from the carotid body stimulation during the resumption of breathing following diving and breath holding in seals (Eisner et al. 1977), it does not do so under conditions of chronic hypoxia. Previous studies performed on northern elephant seal pups (Milsom et al. 1996; Kohin et al. 1999) did not find a bradycardia present under hypoxic conditions (13% 0 , 2  and 14 % and 11% 0 respectively in the two studies). In the study by Milsom et al. 2  (1996) hypoxia had no effect on / H or / R during any state of arousal, while in the study by Kohin et al. (1999) there was a trend for / H to increase as hypoxia ensued, although this appeared to be primarily due to an increase in the amount of time spent breathing. Perhaps the levels of hypoxia used in the studies on northern elephant seal pups (Milsom et al. 1996; Kohin et al. 1999) were at the threshold of hypoxic sensitivity in these animals, and were not great enough to induce the hypoxic bradycardia seen in this study.  72  /H  |"|[  m m  |i  J-  © 1/5  a in 5 sec  >  /H  a  ©  IT,  5 sec B  Figure 2.12. Hypoxic bradycardia. In harbour seal pups, during W A K E , SWS (shown here) and R E M sleep, there was a dramatic hypoxic bradycardia with heart rate (/H) decreasing up to 50% on 8% O2 (B) when compared to air (A), while breathing frequency (/R) increased.  73  Currently there are no other studies in which the effects of hypoxia on fn are examined in phocid seals. Hypercarbia in most terrestrial mammals stimulates central chemoreceptors, causing increases in both / R and / H .  Milsom et al. (1996) found that the effects of  hypercarbia on fn were state dependent in northern elephant seals with increases in / H only being observed during wakefulness. In the harbour seal pups in the present study, there were no increases in / H during wakefulness, however in SWS / H was slightly elevated over levels obtained in air. Thus while these data support the idea that changes in / H during hypercarbia are state dependent in phocid seals, they also suggest that the pattern of change is species dependent, with some species increasing / H during wakefulness only, while others increase / H during SWS. More likely, however, the results from both studies reflect low sample size.  2.5 Conclusions M y data indicate that the breathing pattern of harbour seal pups is state dependent with animals exhibiting irregular breathing during wakefulness, episodic breathing during SWS, and sporadic, but rare incidents of breathing during R E M sleep. These data are in agreement with the data presented for other phocid seals (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1994; and Milsom et al. 1996). Harbour seals responded vigorously to large increases in respiratory drive, both in W A K E and SWS, and were just as sensitive to these changes in drive as terrestrial mammals. The hypercarbic ventilatory response in harbour seals was similar to that seen in terrestrial mammals, as was the hypoxic ventilatory response. Unlike the reductions  74  seen in hypoxic and hypercarbic sensitivity in many terrestrial mammals during sleep, the sensitivity to hypoxia and hypercarbia was unchanged during the transition from W A K E to sleep in the harbour seal pups. The changes in heart rate seen in harbour seal pups during both hypercarbia and hypoxia also did not follow the pattern seen in terrestrial mammals. In hypercarbia, heart rate remained relatively constant, while in hypoxia heart rate decreased significantly. Both changes occurred concurrent with slight increases in total ventilation. The retention of normal ventilatory sensitivity during sleep, despite the increased occurrence of periods of prolonged apnea, is reminiscent of the relatively normal ventilatory sensitivity seen in these animals (relative to their terrestrial counterparts) during W A K E despite their ability to dive for prolonged periods. Both may reflect an adaptation for rapidly and efficiently renewing lung gas stores following prolonged periods of breath holding, during which time this sensitivity must be suppressed.  75  Chapter 3 General Discussion and Conclusions  76  3.1 Critique of Methodology Although the data appear to be overwhelmingly convincing that arousal state and respiratory drive significantly affect the cardio-respiratory patterns of harbour seal pups in a manner similar to terrestrial mammals, the low N-values of this study should be acknowledged. Due to logistical constraints, only three male harbour seal pups were used as study subjects.  The variability of the data collected from the three animals  however, was minimal, suggesting that my data are true reflections of breathing patterns and cardio-respiratory responses to increased respiratory drive in harbour seals. Each animal underwent four experimental sessions per gas level, with each session lasting 2 to 4 hours in length, with 6 to 10 WAKE-sleep episodes being analyzed per seal, per gas level. Both the sex and age of the seals used in this study were kept constant (i.e. male harbour seal pups), thus eliminating the possibility of sex and age influencing results. Male harbour seals were used as opposed to females due to their relatively docile and friendly demeanor (personal observation). The pattern of response to changes in arousal state and increased respiratory drive in female mammals is similar to that seen in males, although the level of response tends to be somewhat lower (White et al. 1985; Mortola and Gautier 1995). Our data on male harbour seal pups, therefore, is likely representative of the responses of both male and female animals. It is well known that age influences both the hypoxic and hypercarbic ventilatory responses of mammals (Mortola and Lanthier 1996; Mortola 1999). Castellini et al. (1994b) and Blackwell and Le Boeuf (1993) suggested that in phocid seals there are also age related responses of the breathing pattern to changes in arousal state, due in part to  77  the development of the dive response. It was essential then, that all seals were at similar developmental stages and of similar age. In my study, harbour seal pups (6 to 12 months) were used as study subjects. The data from these animals can be compared with older phocid seals, regardless of age, as the dive response in precocial harbour seal pups should be fully developed by this stage.  3.2 State of Arousal The first objective of my thesis was to describe the breathing and cardiorespiratory patterns of a phocid seal, the harbour seal, during different states of arousal while on land. As mentioned in Chapter 1, as arousal state decreases from W A K E to SWS to R E M sleep in terrestrial mammals, many changes occur in both breathing and cardio-respiratory patterns.  These include decreases in breathing frequency, tidal  volume, metabolic rate, total ventilation, and heart rate (for a review see McGinty and Szymusiak 1994). In phocid seals, the effect of arousal state on breathing pattern has been studied in four species (gray seals (Ridgway et al. 1975), Caspian seals (Mukhametov et al. 1984), harp seals (Lyamin 1993), and northern elephant seals (Castellini et al. 1994a)), while the effect of arousal state on heart rate and cardiorespiratory patterns (total ventilation, metabolic rate, etc.) has never been examined. Comparing data from my study on harbour seal pups with that from other studies, it is apparent that arousal state does affect the cardio-respiratory system in the same manner in phocid seals as it does in terrestrial mammals. As arousal state decreases from W A K E to SWS, breathing pattern changes from continuous and irregular, to episodic, possibly as a result of decreases in V 0 2 . Although the sleep state pattern of the gray seal  78  is different from that of other phocid seals (i.e. W A K E - R E M sleep - SWS), the breathing patterns during both W A K E and SWS are similar to those reported for other seals (Ridgway et al. 1975). During SWS, there are decreases in overall / R (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; Castellini et al. 1994a; current study) and V 0 2 , as well as increases in V T , with no apparent change in V E (current study), when compared to W A K E state values. During R E M sleep, where muscle atonia is present, it is plausible that life history strategies and/or the environment of the phocid seal in question are key in determining the pattern of breathing observed during this arousal state. Northern elephant seals (subgroup Monachinae) spend eight to nine months of the year at sea, with 80-95% of that time spent submerged (Andrews et al. 1997).  Because of its importance  physiologically, R E M sleep must occur during this time at sea. The muscle atonia that is prominent during R E M sleep prevents submerged individuals from surfacing without waking.  The absence of breathing during R E M sleep in northern elephant seals  (Castellini et al. 1994a; Castellini et al. 1994b; Milsom et al. 1996) therefore, maybe an adaptation of the animal to its environment, allowing complete sleep cycles to occur while the animal is out at sea. The gray seal, harp seal, Caspian seal, and harbour seal are phylogenetically closely related members of the sub-group Phocinae, and all have similar life history strategies in that they haul out on a regular, almost daily basis, either on the mainland, islands, or ice flows, and therefore have almost constant access to land (Riedman 1990). Because of their similar life history strategies, one might expect similarities in their adaptations to sleep in the water. It is possible, therefore, that the breathing pattern of all  79  species is similar, during all states of arousal. This however, is not the case. Although all species exhibit an episodic breathing pattern during SWS, the pattern of breathing in R E M sleep is variable. During R E M sleep, the harp, Caspian, and harbour seals maintain a breathing pattern that is sporadic and rare, while the gray seal breathes continuously (Ridgway et al. 1975; Mukhametov et al. 1984; Lyamin 1993; current study).  The  reasons for the difference in breathing pattern during R E M sleep of seals with similar life history strategies are unknown and require further study and speculation. In terrestrial mammals, as state of arousal decreases, there are decreases in both V 0 2 and V E , with variable changes in V T . Although my study is the first to examine the effect of physiologically determined arousal state on V T , V 0 2 , and V E in phocid seals, the effects of behaviourally defined arousal states on V 0 2 have been previously measured in freely diving Weddell seals (Castellini et al. 1992), captive harp and gray seals (Worthy 1987), as well as captive harbour seals (Matsuura and Whittow 1973; Miller et al. 1976) and yield similar conclusions. These data, along with my data on harbour seal pups indicate that, similar to terrestrial mammals, V 0 2 does decrease with arousal state. Changes in V T during different arousal states in terrestrial mammals are highly variable, with some species, such as humans and dogs, showing an increase in V T (Phillipson et al. 1976; White et al. 1985), while other species exhibit no change in V T , such as golden-mantled ground squirrels (Spermophilus lateralis) (Hunter and Milsom 1998). As mentioned in Chapter 1, and confirmed by my study, while on land, in an awake state, phocid seals have a large V T (40-90% of TLC), especially when compared to terrestrial mammals (Kooyman 1973). In harbour seal pups, V T increases as the seal  80  enters SWS by as much as 61% (current study) suggesting that although the V T may be larger than in terrestrial mammals, it is probably closer to 40% than 90% of the TLC. Values of V E in phocid seals have not been reported during behaviourally or physiologically defined periods of sleep. In the harbour seal pups of my study, it appears that V E remains unchanged as arousal state decreases from W A K E to SWS, a pattern different from terrestrial mammals where V E typically decreases during SWS. The similarity in V E values for W A K E and SWS in the harbour seal are due, in part, to the significant increase in V T during SWS, offsetting the decrease in / R that is present as arousal state decreases. Because of the state-dependent differences in breathing pattern among other species of phocid seal, it is difficult to suggest what the changes in their V E during decreased arousal states might be. However, due to the similarities in the changes in / R in response to SWS, in all species of phocid seals studied to date, it is probable that as arousal state decreases, most seals maintain a constant V E . Due to the decrease in V 0 2 and the constant V E as arousal state decreases in harbour seal pups, one might expect the %Eo , or the relative efficiency of oxygen 2  extracted per breath, to decrease as well. In most terrestrial mammals, however, there is a relatively constant %Eo as V 0 2 values change (Chappell and Roverud 1990). In the 2  current study, there was also no change in %Eo as arousal state decreased from W A K E 2  to SWS, suggesting that the relative efficiency of oxygen extraction was maintained throughout the different states of arousal, regardless of the levels of V 0 2 or V E . Most of the data collected and information discussed in my thesis refer to changes in arousal state from W A K E to SWS. In phocid seals, R E M sleep episodes can be long, however most are relatively short (>20seconds) and therefore, due to equipment  81  constraints, it is difficult to obtain accurate, trustworthy measurements of most variables during this short window of time.  3.3 Increased Respiratory Drive The second objective of my thesis was to determine the effects of increased respiratory drive in phocid seals during both W A K E and SWS. Because of their prolonged breath-holding abilities, during which blood Po levels decrease and Pco levels 2  2  increase, it was initially thought that W A K E phocid seals were less sensitive to hypoxia and hypercarbia than terrestrial mammals (Irving et al. 1935; Robin et al. 1963). Later studies revealed, however, that most species of seal are in fact, just as sensitive to increases in respiratory drive as terrestrial mammals, however the threshold level at which they respond to changes in blood gases is increased (Bainton et al. 1973; Pasche 1976a; Pasche 1976b; Milsom et al. 1996; Kohin et al. 1999). Almost no data exists, however, describing the interplay of arousal state and level of respiratory drive on the various cardio-respiratory and breathing patterns (i.e. full stimulus response curve showing changes in V E , V 0 2 , /R, V T , and / H ) .  3.3.1 Hypoxic Ventilatory Response As mentioned in Chapter 1, freely diving juvenile Weddell seals have a decreased hypoxia sensitivity compared to terrestrial mammals, illustrated by little or no change in V E during exposure to hypoxia. Parkos and Wahrenbrock (1987) hypothesized that the decreased hypoxic sensitivity of juvenile Weddell seals could be due to the exposure of  82  the pups to hypoxia during diving early in life, and therefore could be an adaptation for prolonged dives. Data from more precocial species of seal that begin diving earlier in life, disagree with this hypothesis. M y data on hypoxia sensitivity in harbour seal pups agree with the data of Pasche (1976a) on diving hooded seals, of Craig and Pasche (1980) in diving juvenile harbour seals, and of Mortola and Lanthier (1989) in newborn gray seals, all precocial species. In these studies, hypoxia elicited increases in either / R , V T , or both, resulting in increases in V E . In harbour seal pups (current study), newborn gray seals (Mortola and Lanthier 1989), and yearling hooded seals (Pasche 1976a), hypoxia produced increases in / R and V E , with no change in V T . The increases in / R of all three studies appear to be primarily caused by increases in time spent breathing and decreases in time spent apneic. Craig and Pasche (1980) also noted that the primary cause of increases in V E in hypoxia was an increase in time spent breathing and a decrease in time spent apneic. Opposite to my study on harbour seal pups however, Craig and Pasche (1980) noted that in freely diving, captive, juvenile harbour seals, / R did not increase during hypoxia, but that V T and V E did. Just prior to diving, Craig and Pasche (1980) observed an increase in V T , possibly a final effort to remove CO2 and to "top-up" oxygen stores. It should be noted that there were also small increases in V T before and after prolonged voluntary apneas in my study, although this did not appear to affect overall changes in V T . In studies of hypoxia sensitivity of northern elephant seals by Milsom et al. (1996), values of V E were not reported, and changes in / R alone were used as a measure of respiratory sensitivity. In terrestrial mammals, as mentioned in Chapter 2, and phocid seals, as already noted, the relative contributions of / R and V T to changes in V E are  83  species and arousal state specific. To accurately compare the hypoxic sensitivity of different species, values of V E should be used instead of values of its individual components. Because my data on harbour seal pups indicate a significant rise in / R as well as a significant rise in V E as hypoxia levels increase, it is possible that the results from the northern elephant seals, where / R also increased with hypoxia levels, are an accurate reflection of changes in V E in phocid seals, however further measurements should be made. Under conditions of hypoxia, mammals can increase the supply of oxygen to the blood (increase V E / hyperventilation), decrease the demand for oxygen (decrease V02 / hypometabolism), or both (Mortola et al. 1989; Frappell et al. 1992; Kohin et al. 1999). The metabolic response to increasing levels of hypoxia (hypometabolism) is affected by numerous factors including size, age, and relative amount of exposure to hypoxia (Frappell et al. 1992; Kohin et al 1999; Mortola 1999). In normoxia, metabolic rate in both terrestrial and marine mammals is related to body size through the exponent 0.75 (Vo2=aM  0 7 5  where M is body mass) (Weibel 1984; Lavigne et al. 1986b; Huntley 1987).  This suggests that as body mass and size decrease, there is an increase in V02 and oxygen consumption rates such that smaller mammals have a higher mass specific V02 than larger mammals. Under conditions of hypoxia, due to their higher mass specific V02 and oxygen consumption rates, smaller mammals typically show a greater degree of hypometabolism than larger mammals (Frappell et al. 1992). During hypoxic exposure in very large mammals (above 50kg), metabolic down-regulation (hypometabolism) is absent, and is suggested to be an event limited to small mammals (Frappell et al. 1992; Kohin etal. 1999).  84  Newborn mammals also typically show a greater metabolic response to hypoxia than older animals, usually with a greater decline in V 0 2 and a decreased ventilatory response, possibly allowing for a greater tolerance to asphyxia during the transition from placental respiration to lung respiration (Kohin et al. 1999; Mortola 1999). Whether or not the hypometabolic response to hypoxia in newborn animals is related to size or age is still unknown, however it is most likely a combination of the two factors (Frappell et al. 1992; Mortola 1999). Harbour seal pups show small decreases in V 0 2 (9.9%) during hypoxia when compared to normoxia, however these are never significant (current study), while newborn gray seals show significant decreases in V 0 2 (23%) at Flo levels of 0.10 2  (Mortola et al. 1989). The difference in magnitude of the metabolic response to hypoxia between the gray and harbour seals may be due to the age at which the animals were tested, or the mass of the animals, as the harbour seal pups of the current study were twice the mass of the newborn gray seals (Mortola and Lanthier 1989). In phocid seals, this difference in magnitude of hypoxic response could also represent the inexperiences in diving between a newborn seal and a more experienced pup (i.e. newborn animals are not exposed to hypoxic conditions on a frequent basis while 6-12 month old pups frequently dive for prolonged periods) (Mortola and Lanthier 1989; Mortola et al. 1989). When I compare my results to those obtained in W A K E northern elephant seal pups exposed to 14% and 11% O 2 , the V 0 2 response is quite different. As Flo levels 2  decreased from normoxic levels to 0.11, V 0 2 increased by 38% in northern elephant seal pups (Kohin et al. 1999).  The differences in response to hypoxia in phocid seals  (increased V 0 2 in northern elephant seals, and decreased V 0 2 in harbour seals and gray  85  seals) may be a result of age, size, or both, as the northern elephant seal is, on average, four times the weight of the harbour and gray seals. Regardless, these data confirm that phocid seals do respond to hypoxia in a manner similar to terrestrial mammals, with different magnitudes of response dependent upon the size and age of the animals tested. During sleep in most terrestrial mammals, there is a decrease in both the sensitivity and threshold for the ventilatory response to hypoxia (Corfield 1999). In some terrestrial mammals however, such as ground squirrels and rats, there is an increase in hypoxic sensitivity during SWS (Milsom et al. 1996).  The sensitivity to increased  hypoxia during different arousal states in marine mammals has previously only been examined in the northern elephant seal (Milsom et al. 1996). Again, using changes in / R as a determinant of respiratory sensitivity, Milsom et al. (1996) concluded that as arousal state decreased from W A K E to SWS, there was a corresponding increase in sensitivity to hypoxia (13.3% O2 ± 0.15 SEM), illustrated by the greater magnitude of increase in / R during SWS as hypoxia ensued. In my study on harbour seal pups, similar to the northern elephant seal pups, as arousal state decreased, there was a greater magnitude of increase in / R during SWS than W A K E . However when V E values were compared, there was no difference between the two states of arousal, suggesting no change in hypoxic sensitivity as arousal state decreased. Because there are only two species of phocid seal studied with respect to hypoxic sensitivity in the different arousal states, it is impossible to make generalizations about the hypoxic sensitivity of phocid seals, and therefore more studies are needed.  86  3.3.2 Hypercarbic Ventilatory Response The primary stimulus to breathe in most mammals is the level of CO2 present in the blood (Pco ). As mentioned in Chapter 1, it is not the CO2 per se that causes the 2  increases in / R and V T , rather it is the increase in [ H ] , due to the bicarbonate reaction, +  that the central chemoreceptors are responding to (Phillipson 1978; Milsom 1992). B y increasing / R and V T in response to increased C 0 , the body removes the excess CO2 at a 2  faster rate, and the bicarbonate equation shifts to the left, removing [H+] and preventing acidosis of the blood. In phocid seals, the response to hypercarbia is similar to that seen in terrestrial mammals, with CO2 once again considered as the primary stimulus for breathing (Bainton et al. 1973; Pasche 1976b; Gallivan 1980; Milsom et al. 1996; Kohin et al. 1999).  While my study on harbour seal pups demonstrated a similar sensitivity to  hypercarbia, the threshold level of change in CO2 required to produce a change in V E was different from that observed in other marine mammals. Pasche (1976b), Gallivan (1980), and Parkos and Wahrenbrock (1987) found that changes in V E of unrestrained, freely diving phocid seals and manatees occurred at a higher threshold level than was observed in terrestrial mammals. In the harbour seal pups of my study, however, the threshold level of V E was similar to that of terrestrial mammals, with strong hypercarbic ventilatory responses occurring at the lowest level of CO2 tested. Similar results were found during SWS. No other study to date has quantified the changes in V E of phocid seals during different arousal states in response to changes in respiratory drive.  Milsom et al. (1996) showed that there was no change in the  magnitude of the change in / R in response to hypercarbia in northern elephant seal pups  87  during SWS, when compared to W A K E state values.  In my study, there were no  differences in the magnitude of the changes in V T and / R , or V E during SWS. This suggests that, similar to the northern elephant seal pups, there was no decrease in sensitivity to hypercarbia as arousal state decreased in harbour seal pups.  3.4 Heart Rate At times when lung function is not needed and there is no new intake of O2, such as seen in dives and respiratory apneas, the work done by the heart (i.e. pumping blood) is minimized through a decrease in heart rate, thereby keeping ventilation and perfusion matched. In this study, harbour seal pups, under normoxic and normocarbic conditions (i.e. air), showed a decrease in the heart rate during apnea by as much as 31% of the eupneic values in wakefulness and 68% of the eupneic values in SWS. When phocid seals breath-hold, either on land or in water, their nostrils close, stimulating the trigeminal and superior laryngeal nerves, as well as receptors in the upper respiratory tract (nasal mucosa and larynx). This causes a decrease in heart rate and re-enforces the inhibition of breathing (Daly 1984). When seals breath-hold, they do so either on expiration (during a dive apnea) or on inspiration (during a resting apnea) (Kooyman 1973; Eisner et al. 1977). The act of lung deflation (expiration) decreases pulmonary stretch receptor activity, increasing cardiac vagal tone, and allowing a bradycardia to develop (Daly et al. 1977). Once an apnea has begun, blood Po levels begin to decrease, stimulating the carotid body 2  peripheral chemoreceptors, causing hyperventilation (overridden by trigeminal and superior laryngeal nerve stimulation to sustain apnea), bradycardia, and vasoconstriction  88  (Eisner et al. 1977; Daly et al. 1977; Daly 1984). When breathing resumes, the low blood Po and high blood Pco are initially still present. 2  2  These stimuli should continue to  stimulate the carotid bodies and maintain the bradycardia, however the decrease in vagal tone brought about by increases in excitatory input from the pulmonary stretch receptors causes / H to increase,  overriding the  carotid body induced bradycardia and  vasoconstriction (Daly 1984). In terrestrial mammals, hypoxia causes hyperventilation and tachycardia.  In  northern elephant seal pups, hypoxia either has no effect on / H or / R (Milsom et al. 1996) or causes increases in / H , although this appears to be primarily due to an increase in eupneic time (Kohin et al. 1999). Hypoxia in harbour seal pups however, caused a significant bradycardia (up to 50%) during all states of arousal, while at the same time, / R and V E increased (up to 50%). Eisner et al. (1977) revealed that stimulation of carotid body chemoreceptors in anesthetized harbour seals by stimuli such as hypoxia led to decreases in / H , and increases in / R . In most terrestrial mammals and post-dive marine mammals, when / R and V E increase, lung inflation suppresses the carotid body's cardioinhibitory response through removal of vagal input, leading to an increase in / H regardless of the carotid body input (Eisner et al. 1977).  In harbour seal pups, the  removal of vagal input, caused by the substantial increase in / R and V E in hypoxia, does not appear to be enough to override the carotid body induced bradycardia, leading to the pronounced hypoxic bradycardia. Although the efficiency of oxygen removal remained unchanged during hypoxia, (~5%> at each level) the total amount of oxygen being removed from the air at 8.63% O2 ± 0.22 S E M was less than half the total amount of oxygen being removed when  89  atmospheric air was breathed. Perhaps the vasoconstriction brought about by the hypoxic environment, allowing vital organs to maintain normal V02 levels, is so profound in these seals that the fn must be kept at a low level to maintain constant cardiac output levels. Or, perhaps, as a mechanism to allow constant blood flow to the brain and other vital organs, there is some other centrally controlled neural mechanism preventing removal of the vagal input as the pulmonary stretch receptors are stimulated. Hypercarbia in most terrestrial mammals stimulates central chemoreceptors, causing increases in both / R and fn. Milsom et al. (1996) suggest that the effects of hypercarbia on fn are state dependent in phocid seals, with increases in fn observed only during wakefulness. In hypercarbic conditions, harbour seal pups show no increase in fn during wakefulness or in SWS. R E M sleep shows a significant increase in heart rate at 2.04% C 0 ± 0.06 S E M , which disappears at higher levels of C 0 . These data support 2  2  the idea of state dependent changes in fn during hypercarbia in phocid seals. These data also suggest, however, that the pattern of change is species dependent, with some species increasing fn during wakefulness only, while others increase fn during SWS and/or during R E M sleep.  Low n-values in both studies, however, could also explain the  differences.  3.5 Conclusions Little is known about the respiratory physiology of phocid seals, and even less is known about the relationship between arousal state and respiratory drive. M y data add support to the data of previous studies, and raise new questions regarding the cardiorespiratory sensitivity of harbour seals and other phocid seals to changes in both arousal  90  state and respiratory drive. It is apparent that the state-dependent changes in breathing pattern are universal to all species of phocid seal, however the exact pattern that is present is, as of yet, unpredictable. The differences in breathing pattern in R E M sleep may be associated with life history strategies and/or behaviour, however more studies are needed to predict possible respiratory patterns. The maximum observed sleep associated apneas are similar in length to average dive times (~3 minutes in harbour seal pups) possibly suggesting oxygen stores and central regulation play some role in determining breathing pattern and apnea length. Respiratory sensitivity in phocid seals is similar to that reported in terrestrial mammals, with increases in both / R and V T leading to increases in V E . Hypoxia is associated with a decrease in V 0 2 , while hypercarbia is associated with minimal changes in V 0 2 .  Although phocid seals are primarily aquatic, there are strong similarities  between their respiratory physiologies and those of terrestrial mammals. 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