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Control of heart rate during diving in ducks Furilla, Robert Alan 1986

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CONTROL OF HEART RATE DURING DIVING IN DUCKS by ROBERT ALAN FURILLA B.A. University of Massachusetts, Boston, 1977 M.Sc. University of Alaska, Fairbanks, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Zoology, July 1986 Robert Alan F u r i l l a , 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^oocof/  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date ABSTRACT Forced submergence of redhead ducks (Aythya americana) caused heart rate to f a l l from 100 ± 3 beats'min ^ (mean ± s.e.m., N = 12) to a stable rate of 35 ± 4 beats'min ^ (N = 12) within 5 seconds after submergence. Bradycardia was unaffected by breathing oxygen before the dive or by denervation of the baroreceptors, but was v i r t u a l l y eliminated by l o c a l anaesthesia of the na r i a l region. When freely diving on a man-made pond, heart rate of redhead ducks and lesser scaup (A. a f f i n i s ) two seconds after submergence was 2 po s i t i v e l y correlated with the pre-dive rate (r = 0.71). Breathing oxygen before the dive and denervation of baroreceptors had l i t t l e effect on this r elationship. Chasing to induce submergence caused a sl i g h t enhancement of bradycardia, heart rate during the dive being about 10% lower than after a voluntary dive. Local anaesthesia of the na r i a l region inhibited voluntary diving, but heart rates in chase-induced dives after nasal blockade were s i g n i f i c a n t l y higher (10-30%) than those obtained from untreated ducks in chase-induced dives. Dive heart rate, at 2-5 seconds submergence, was l i n e a r l y related to the logarithm of the pre-dive rate for a l l voluntary and forced dives as well as dabbles. Even the heart rate which occurred 2-5 seconds after being trapped under water as a function of the rate immediately before trapping f i t t e d t h i s relationship. The function was described by the equation Y = -451 + 246 LOG X, where Y = dive (or trapped) and 2 X = pre-dive (or pre-trap) heart rate (r = 0.98). The relationship was unaltered by 3-blockade with propranolol. Data from stimulation of the cut d i s t a l ends of vagal and cardiac sympathetic nerves suggest that a similar increase in vagal a c t i v i t y occurs on submergence in a l l of these dives. The f i r s t cardiac i n t e r v a l in voluntary dives represents a lower heart rate, indicating a higher l e v e l of vagal a c t i v i t y . When dabbling ducks (Anas platyrhynchos) dabble, heart rate at two seconds submergence i s l i t t l e changed from the pre-dabble rate. When these birds dive, however, heart rate at two seconds submergence i s about 250 beats*min , regardless of the pre-dive rate. B i l a t e r a l denervation of a r t e r i a l baroreceptors s i g n i f i c a n t l y altered the dive:pre-dive relationship . These results have shown that nasal receptors are responsible for bradycardia in diving ducks when f o r c i b l y submerged, but that nasal receptors contribute l i t t l e to the change in heart rate when ducks dive v o l u n t a r i l y . The results also suggest that there i s a psychogenic modulation of the heart rate in voluntary dives which influences the pre-dive rather than the dive heart rate. F i n a l l y , dabbling ducks d i f f e r from diving ducks in their response to forced and voluntary diving. Chemoreceptors are responsible for the majority of the response in forced dives, and baroreceptors provide primary control in voJrunxa-ry dives. i v TABLE OF CONTENTS ABSTRACT . . i i LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i GENERAL INTRODUCTION 1 Cardiovascular Adjustments to Forced Submergence 2 Cardiovascular Adjustments in Freely Diving Animals 3 Efferent Control of the Cardiovascular Adjustments 5 Afferent Control of the Cardiovascular Adjustments 7 SECTION 1: Control of Heart Rate during Restrained and Unrestrained Dives by Diving Ducks 10 INTRODUCTION. 10 METHODS AND MATERIALS 14 Heart rate response to restrained diving 14 Heart rate response to free diving 15 Control of heart rate by vagal and cardiac sympathetic nerves 20 RESULTS 22 Heart rate response to restrained diving 22 Heart rate response to free diving 29 Control of heart rate by vagal and cardiac sympathetic nerves 49 DISCUSSION 55 V SECTION 2: The Heart Rate Response to Diving and Dabbling in a Non-diving Duck 65 INTRODUCTION 65 METHODS AND MATERIALS 68 RESULTS 72 Heart rate response during dabbling 72 Heart rate response during diving 72 DISCUSSION 83 GENERAL DISCUSSION 87 LITERATURE CITED 97 v i LIST OF FIGURES 1. The heart rate response to forced submersion in diving ducks 23 2. EKG traces of forced dived ducks 25 3. The relationship between pre-dive and dive heart rate on forced submersion 27 4. EKG traces of voluntary submersions by a diving duck...30 5. The relationship between pre-dive heart rate and the f i r s t cardiac i n t e r v a l in voluntary dives 33 6. The relationship between pre-dive and dive heart rate during voluntary diving 35 7. EKG traces of voluntary and chase-induced dives 37 8. The relationship between inspired oxygen and dive time or dive heart rate 40 9. The relationship between pre-dive and dive heart rate at various levels of inspired oxygen and after baroreceptor denervation 43 10. EKG traces of forced and trapped dives. 45 11. The relationship between pre-trap and trapped heart rate 47 12. The heart rate response to stimulation of the cardiac sympathetic and vagus nerves 51 13. The relationship of heart rate to stimulation of the cardiac sympathetic and vagus nerves 53 14. The relationship of the logarithm of pre-dive and dive heart rate for a l l categories of dives 60 v i i 15. The relationship between pre-submergence and submersion heart rate i n dabbling by mallards. 73 16. EKG and heart rate traces for mallards dabbling and diving 7 5 17. The relationship between pre-dive and dive heart rate of intact and barodenervated mallards.... 78 18. The relationship between pre-dive and dive heart rate of one mallard after u n i l a t e r a l denervation of baroreceptors 80 19. Log Survivor plot of dives and pauses of lesser scaup diving in the wild 89 20. Traces of isometric tension of the t i b i a l i s anterior muscle after a r t e r i a l occlusion and after a forced dive 93 V l l l ACKNOWLEDGEMENTS I owe a special thanks to Dr. David R. Jones for his assistance in planning and executing this project. I also thank Drs. David J. Randall, William K. Milsom, John M. Gosline and H. Dean Fisher for their advice and for reviewing this manuscript. F i n a l l y , I thank Frank M. Smith for his assistance with baroreceptor denervations. The cardiac stimulation study was done in collaboration with David R. Jones. This research was supported by grants from NSERC of Canada and The Canadian National Sportmen's Fund to David R. Jones. 1 GENERAL INTRODUCTION Diving animals subject themselves to periods of asphyxia during which many homeostatic processes are disrupted. Sustained interruption of a process essential for internal homeostasis w i l l inevitably lead to physiological dysfunction, yet diving animals can survive prolonged periods of asphyxia that would k i l l many of their more s t r i c t l y t e r r e s t r i a l analogues. Homeothermic animals are especially susceptible to the danger of oxygen deprivation because of the high energy cost for maintenance of c e l l function. As early as the mid-nineteenth century, s c i e n t i s t s became interested in diving physiology. In 1870, Paul Bert discovered that ducks experienced a f a l l in heart rate when fo r c i b l y submerged in water. He also showed that ducks could survive submersion asphyxia for up to 20 minutes, whereas chickens died within 3 minutes after submersion. Richet (1899) calculated the oxygen stores of a 1.5 kg duck and deduced that a concomitant reduction in metabolism was necessary for the duck to survive more than 3 minutes under water. In 1909, Burne, working on the walrus, suggested a re d i s t r i b u t i o n of blood away from the "vegetative organs" during submersion. Scholander (1940) demonstrated that l a c t i c acid concentration was greater in skeletal muscle than in blood during a dive, but blood lactate increased substantially upon surfacing. Thus, i t was shown that muscle perfusion was 2 inhibited and that the muscles were metabolizing anaerobically. Cardiovascular Adjustments to Forced Submergence Since these early observations, bradycardia during enforced submersion has been observed in many air-breathing vertebrates including the frog (Jones and Shelton, 1964), snake (Johansen, 1959), muskrat (Drummond and Jones, 1979), vole (Clausen, 1964), fur seal (Irving et_ al_. , 1963), porpoise (Irving et a_l . , 1941), hippopotamus (Eisner, 1966), cormorant (Mangalam and Jones, 1984), and duck (Butler and Jones, 1971). In fact, submersion bradycardia appears to be a universal t r a i t among air-breathing vertebrates including man. Accompanying a f a l l in heart rate i s a reduction of cardiac output. Clearly, stroke volume could not increase s u f f i c i e n t l y to compensate for the observed decrease in heart rate. The sea l i o n (Eisner e_t a_l. , 1964) and duck (Jones and Holeton, 1972) showed no or l i t t l e change in stroke volume even in the face of extreme bradycardia. If adequate perfusion of the brain i s to take place, i t i s essential that a r t e r i a l blood pressure be maintained. Irving ert sQ. , ( 1942) measured the blood pressure of a seal during experimental dives and showed that, despite an intense slowing of the heart blood pressure remained unchanged. Jones and West (1978) stated that "mean a r t e r i a l blood pressure i s seldom more than 20% changed from the pre-dive l e v e l . " To maintain blood pressure in the face of reduced cardiac output, peripheral resistance must increase. Ducks (Jones e_t a_l . , 1979) and muskrats (Jones e_t al_. , 1982) that have been forced to dive experienced a large reduction in the percent of cardiac output flowing to the kidneys, l i v e r , muscles, spleen and small intestine (and lungs in the duck) while flow increased to the brain, heart and, to a lesser extent, the eyes. These cardiovascular adjustments are c o l l e c t i v e l y referred to as the diving response and have been touted as oxygen conserving compensations. Cardiovascular Adjustments in Freely Diving Animals Scholander (1940) showed that a seal submerged but free to raise i t s head and breathe f a i l e d to sh ow the expected bradycardia. Extensive research aimed at freely diving animals has had to wait the technological advances which brought about the miniaturization of electronic components. Unfortunately, devices small enough to monitor physiological variables of small animals, are not yet available and many studies require that animals be confined in small tanks while wired to external equipment. For this reason, data from naturally or freely diving animals i s not nearly so extensive as that from f o r c i b l y submerged animals. Bradycardia i s highly variable in animals trained to dive. Sea lions trained to dive on command had a higher heart rate in these dives than during restrained dives (Eisner et a l . , 1964), but dolphins showed a more intense bradycardia after training (Eisner e_t a_l. , 1966). The Weddell seal (Kooyman and Campbell, 1972) exhibited a mild bradycardia during short dives (<20 minutes), but during longer dives the bradycardia was more intense. More interesting i s the fact that within 30 seconds following submersion the heart rate was lower in the eventually longer dives than in the shorter ones. The muskrat always showed an extreme bradycardia, but the f i r s t cardiac i n t e r v a l immediately following submergence was more protracted in longer dives than in shorter ones (Drummond and Jones, 1979). Cormorants diving naturally showed no bradycardia (Kanwisher e_t a_l . , 1981), and the heart rate of freely diving tufted ducks increased just before the dive, but during the dive, did not f a l l below that while swimming on the surface and was considerably higher than at rest (Butler and Woakes, 1982a). Kooyman et_ a_l. ( 1980) reported that blood lactate of Weddell seals did not increase s i g n i f i c a n t l y following dives of less than 20 minutes, indicating that muscle and organ perfusion was maintained; however, blood lactate rose dramatically following dives of more than 20 minutes. Mi l l a r d et a l . (1973) observed a reduction in blood flow to the leg of a naturally-diving penguin, but as Butler and Jones (1982) pointed out, this may r e f l e c t a r e d i s t r i b u t i o n of blood away from non-active tissues because the penguin swims with i t s wings. Kooyman e_t a_l. (1971) reported seeing an emperor 5 penguin freely bleeding from a wound at i t s wing t i p for nearly 2 minutes during a dive. Efferent Control of the Cardiovascular Adjustments The rapid onset of cardiovascular responses to forced submergence or free diving suggests that they are controlled by neural mechanisms. In birds and mammals, the pacemaker i s innervated by cardiac (sympathetic) and vagal (parasympathetic) nerves (Burnstock, 1969). B i l a t e r a l vagotomy of the muskrat (Drummond and Jones, 1979) and inje c t i o n of atropine in the duck (Butler and Jones, 1971) completely abolished the bradycardia associated with submergence, indicating that heart slowing during enforced submersion is governed by the vagus nerve. Butler and Woakes (1982a) showed that the reduction in heart rate from the pre-dive tachycardia of fre e l y - d i v i n g ducks was n u l l i f i e d following i n j e c t i o n of atropine, and they proposed that the pre- and post-dive tachycardia are brought about by i n h i b i t i o n of vagal tone. Butler and Jones (1971) demonstrated that the post-dive tachycardia of f o r c i b l y submerged ducks was unaffected by sympathetic (3) blockade; however, Folkow et a l . (1967) found that the post-dive tachycardia was attenuated following 0-receptor blockade. The nature of the sympathetic influence on the pre- and post-dive tachycardia in freely diving ducks i s unknown. It has been suggested by Kanwisher et a l . (1981) that cardiac rhythm i s under v o l i t i o n a l control. Butler and Woakes (1982a) further suggest that the pre-dive tachycardia and hyperventilation serve to increase the oxygen stores by saturating the a r t e r i a l blood and decreasing the a-v O2 difference. It i s tempting to assume that the animal i n i t i a t e s these cardiac adjustments to mitigate the stress that would otherwise lessen i t s a b i l i t y to complete the dive successfully, but no such evidence exists. Peripheral vasoconstriction i s under the control of the sympathetic nervous system acting through a-adrenoceptors (Butler and Jones, 1971); whereas, vasodilation i s brought about by stimulation of ^-adrenoceptors (Hillman and Lundvall, 1981) or by metabolic d i l a t o r s (Folkow and N e i l , 1971). During periods of metabolic a c t i v i t y certain organs have increased blood flow (Bevegard and Shepherd, 1967). Ducks exercising on a treadmill underwent a 25% reduction of vascular resistance (Grubb, 1982), presumably to augment perfusion of the working muscles. It seems l i k e l y that animals swimming under water should also require muscle perfusion. Folkow e_t aj_. (1966) demonstrated that metabolic d i l a t o r substances could not abate neurogenic vasoconstriction of the hindlimb of the duck, but the cat experienced reactive hyperaemia during muscular work even with strong sympathetic stimulation. Anatomical studies by these authors showed more dense adrenergic innervation of the large a r t e r i e s in the hindlimbs of ducks than of cats. If the animal i s to perfuse i t s muscles during a dive, i t i s clear that neurogenic 7 c o n s t r i c t i o n must be inhibited, but i t i s unclear whether muscle perfusion i s necessary for a 20 second dive by ducks. Afferent Control of the Cardiovascular Adjustments Questions concerning the sensory mechanisms which bring about the dive response have been the subject of investigation for more than a century. These studies have centered on (1) f a c i a l , nasal, laryngeal and g l o t t a l receptors, (2) central and peripheral chemoreceptors and (3) a r t e r i a l baroreceptors. Andersen and Blix (1974) considered the barostatic reflex to be of major importance; however, Jones (1973) and L i l l o and Jones (1982) demonstrated that bradycardia and peripheral vasoconstriction develop in barodenervated ducks, and Drummond and Jones (1979) could show no involvement of the baroreceptors in the dive response of muskrats. It i s probable that in ducks the baroreceptors are responsible for maintaining blood pressure in the face of large changes in cardiac output and vascular resistance once these adjustments are established in the dive, but do not contribute much to the development of the dive response per se in forced dives. "The progressive hypoxia and hypercapnia that develop during diving would tend to stimulate the central and peripheral chemoreceptors" (Butler, 1982). The role of a r t e r i a l chemoreceptors in freely diving animals is uncertain, but during f o r c i b l e submersion of domestic ducks, denervation of the carotid bodies prevented much of the bradycardia (Jones and Purves, 1970; L i l l o and Jones, 1982); moreover, ducks breathing an hyperoxic gas mixture before submersion displayed l i t t l e or no bradycardia during enforced submersion (Mangalam and Jones, 1984). Carotid body denervation had no effect on the i n i t i a t i o n and depth of bradycardia from the pre-dive levels of freely diving tufted ducks (Butler and Woakes, 1982b), and Butler (1982) reported that the carotid bodies were not involved in the bradycardia that developed when a tufted duck was prevented from surfacing. A r t e r i a l chemoreceptors are not essential in i n i t i a t i n g the diving response in seals (Daly ert a_l . , 1977) and muskrats (Drummond and Jones, 1979); however, i f the chemoreceptors were stimulated in f o r c i b l y submerged seals, heart slowing was considerably augmented (Eisner e_t a_l_., 1977 ; Daly e_t al_ . , 1977) . In many birds and mammals, the gradual changes in blood gases following submersion could not account for the rapid onset of cardiovascular adjustments. Tufted ducks (Butler and Woakes, 1982a) showed an immediate increase (about two-fold) in the cardiac i n t e r v a l after a voluntary dive, and the cardiac i n t e r v a l of muskrats was expanded from approximately 0.2 seconds to more than 1 second immediately after an unrestrained dive (Drummond and Jones, 1979). When the nasal, f a c i a l and laryngeal receptors of muskrats were extirpated by nerve section, there was no submersion bradycardia (Drummond and Jones, 1979). Furthermore, l o c a l anaesthesia of the face 9 of a r t i f i c i a l l y ventilated harbor seals completely abolished the bradycardia associated with submersion (Dykes, 1974). Angell-James and Daly (1972) showed that bradycardia and peripheral vasoconstriction could be e l i c i t e d in dogs by stimulation of the nasal passages with water. It i s clear from these observations that the control of cardiovascular adjustments in freely diving animals i s uncertain. Moreover, the complex responses brought about by reflex interactions have not been investigated in freely diving animals. 10 SECTION 1 The Heart Rate Response and i t s Control during Restrained and Unrestrained Diving by Diving Ducks INTRODUCTION The cardiac response to diving shown by diving ducks submerging voluntarily i s very di f f e r e n t from that obtained by force diving the same ducks in the laboratory (Butler and Woakes, 1979; 1982a&b). Involuntary diving, heart rate increases before the f i r s t dive in a series, and on, or even just before, submersion there i s a transient bradycardia. Heart rate then increases in the f i r s t few seconds of the dive to a steady rate, which i s often quite similar to that obtained in ducks resting quietly on the surface (Butler and Woakes, 1979; 1982a). This contrasts with forced dives in which heart rate f a l l s progressively, usually to very low levels (Butler and Woakes, 1976; 1979; 1982b). An "immersion ref l e x " has been suggested to play an important role in divers, although i t s existence has never-been unequivocally demonstrated in birds (Butler and Woakes, 1982a; Mangalam and Jones, 1984). When a bird or mammal dives, i f i t i s to remain aerobic, i t must sustain i t s e l f on the oxygen stored at the moment of submersion. That aerobic metabolism i s preferable to anaerobic metabolism has been demonstrated by Kooyman ert al . (1980). They pointed out that clearing anaerobic metabolites 11 takes considerably longer than simply replenishing the oxygen stores, and the dive-pause r a t i o f a l l s off rapidly as the animal begins to rely on anaerobiosis. If terminating the dive before relying heavily on anaerobic metabolism i s adaptive, then i t seems l i k e l y that diving animals are capable of sensing PaC^ either continuously or at some threshold l e v e l . Butler and Woakes (1982b) reported that dive times of tufted ducks (Aythya f u l i g u l a ) whose carotid bodies had been denervated were s i g n i f i c a n t l y longer than those of intact animals. When Butler and Woakes (1982b) f o r c i b l y submerged tufted ducks, they wrote that the "cardiac response to f o r c i b l e submersion of the head of tufted ducks was, sur p r i s i n g l y , not greatly affected by denervation of the carotid bodies." The difference in cardiac responses to submergence in voluntary and forced diving has led to a controversy about the basic concept of a "diving response." It has been proposed that in forced dives the response i s a product of, or i s accentuated by, fear or stress (Kanwisher e_t al . , 1981 ; Kanwisher and Gabrielsen, 1985), while in voluntary dives, there i s not so much a diving bradycardia as a tachycardia before the dive (Butler and Woakes, 1982a&b). These ideas have been promulgated despite claims that "calm" or "relaxed" ducks respond better (deeper bradycardia) in forced dives (Irving et. aJL . , 1941; Folkow et. a_l . , 1967), and because some birds submerging voluntarily show l i t t l e heart rate change 12 before, during or even after the dive (Butler and Woakes, 1984; Gabrielsen, 1985). Nevertheless, the central nervous system probably plays an important role in modulating diving responses, but no one has attempted to study the role of psychogenic and reflexogenic mechanisms in i n i t i a t i n g diving responses of diving birds, and to determine how one influences the other. One attempt has been made to quantify the extent of the psychogenic modulation of the diving response in dabbling ducks ( B l i x , 1985), but this approach was l i t e r a r y rather than experimental. An experimental approach to this problem i s d i f f i c u l t . As a st a r t , comparing the diving response in voluntary and forced dives in the same group of animals under a range of conditions, might c l a r i f y any relationship between heart rate responses in both types of dive. It i s important, however, that any relationship be supported by our knowledge of both the afferent and efferent neural mechanisms involved in the response. To this end, results of a limited investigation of the efferent control of the heart in the duck are reported. This study was undertaken to investigate the nature of "immersion reflexes" in diving ducks, and an attempt was made to measure their contribution to the cardiac response in restrained and free dives. An attempt was also made to determine whether diving ducks are capable of adjusting dive 13 time and heart rate in oxygen, and to examine cardiac control during response to altered levels of inspired the role of the baroreceptors in forced and voluntary diving. 14 METHODS AND MATERIALS Heart rate response to restrained diving Six redhead ducks (Aythya americana) were used to test the role of nasal receptors in the i n i t i a t i o n of the diving response. Heart rate was monitored using needle electrodes placed subcutaneously on the l e f t side of the abdomen and the right shoulder. The ducks were f o r c i b l y submerged by gently lowering the head into a container of water. This manoeuver was done care f u l l y to prevent the animals struggling on submersion. Each duck was submerged three times, and a mean heart rate was calculated for that animal. Xylocaine (Lidocaine USP, Astra Pharmaceuticals Canada Ltd., Mississauga, Ontario) in aerosol form was administered into the nares for l o c a l anaesthesia of n a r i a l receptors. To minimize the p o s s i b i l i t y of anaesthetizing the g l o t t i s , loosely packed cotton wool was placed over the g l o t t i s to shield i t from the spray. A s l i p of the cotton protruded out of the corner of the mouth, and the beak was taped closed to prevent the duck swallowing the cotton. Fifteen minutes after Xylocaine application the cotton wool was removed and another 5 minutes allowed before the f i r s t of a series of three dives on each animal was performed. Two hours l a t e r , after recovery from the application of Xylocaine, the birds were again tested for a diving response. 15 Another six redhead ducks were tested to determine the effect of high a r t e r i a l oxygen tension on the i n i t i a t i o n of the diving response. A p l a s t i c bag was placed over the duck's head, and either a i r or 100% oxygen was passed through the bag for at least 3 minutes before the animal was submerged. As above, three dives per animal for each condition were recorded . To elevate the heart rate of ducks before a forced dive, one redhead duck was encouraged to run on a treadmill and 2 others were chased along a corridor. Immediately after exercise ceased, the duck's head was f o r c i b l y submerged into a beaker of water for 15 seconds. Heart rate was determined from the cardiac i n t e r v a l taken at 1, 2, 5, 10, and 15 seconds in the dive. Dive duration was varied to minimize the effect of conditioning (Gabbott and Jones, in preparation), but a l l dives lasted at least 15 seconds. The data were analyzed using analysis of variance, and significance was set at P < 0.05. A l l values from this series of experiments are given as means ± S.E.M. and N = the number of animals contributing to the mean. Heart rate response to free diving To test the response to voluntary diving, heart rate was obtained telem e t r i c a l l y using EKG transmitters (Narco Biosystems, Downsview, Ontario, Canada). The transmitter was s t e r i l i z e d with benzalkonium chloride (Zephiran, 1:750, 16 Winthrop Laboratories, Aurora, Ontario). A midline i n c i s i o n was made in the skin and body wall over the abdomen after anaesthetizing the area by in j e c t i o n of Xylocaine. Bipolar loop electrodes were placed on the pericardium and the transmitter was put in the peritoneal cavity. The peritoneal cavity was then closed with surgical s i l k . After surgery, 125 mg of Ampicillin (Penbritin, Ayerst Laboratory, Montreal, Quebec) was administered I.M. and the birds were allowed one day to recover before being used in any experiments. The EKG signal was received on a Narco FM-Biotelemetry receiver, stored on magnetic tape, and displayed on a pen recorder. On playback, heart rates were either determined from measurement of the cardiac i n t e r v a l s or by using a cardiotachometer. The ducks were placed on a pond deep enough to allow them to dive for food. This pond was man-made and had a surface area of 3 x 5.5 m. The bottom of the pond was tapered so that the depth of the water ranged from 0.3 - 1.7 m. A platform (2.0 x 1.5 m) and a Plexiglas enclosure (1.0 x 1.5 x 0.8m high) were placed at the shallow end of the pond. The enclosure had a v e r t i c a l l y s l i d i n g door to seal i t s u f f i c i e n t l y so that oxygen level s in the enclosure could be altered. The surface of the water outside the enclosure was covered with netting stretched 2 over wooden frames. Each frame was 1 m . The frames floated on the water and prevented the birds from surfacing anywhere but within the enclosure; however, the birds could l i f t the netting to take a breath i f i t became necessary. At the corner of the pond farthest from the enclosure was a feeding station with a chute through which food could be dropped into a receptacle at the bottom. Nitrogen or oxygen was infused (20 l»min ^) into one side of the enclosure and removed from the opposite side. A fan was mounted inside the box to mix the a i r . Gas from the box was led to a Beckman paramagnetic oxygen analyzer which was calibrated with nitrogen and a i r . The 50% le v e l of oxygen was estimated from flow rate and enclosure volume and should not be taken as precise. The value does, however, represent a high concentration of inspired oxygen. Most voluntary dives occurred in the period after food was delivered into the feeding chute. To reduce the sympathetic contribution to the increase in heart rate before a voluntary dive, 1.5 mg'kg ^ of propranolol was injected into the muscles of three redhead ducks, and the animals were immediately returned to the pond. Xylocaine blockage of the nasal area was performed as described for restrained animals, and a short, forced dive was done to confirm the efficacy of the blockade before the animals were put back onto the pond. Ducks were reluctant to dive after their nasal area had been blocked with Xylocaine. So few voluntary dives were obtained that the birds had to be "chased" to make them submerge (Butler and Woakes, 1979). This was usually done by banging on the l i d of the enclosure with a stick or waggling a net at them. 18 Pre-dive heart rate was reduced by non-pharmacological means by encouraging 3 redhead ducks to dabble for food. The ducks were placed singly on a 25 cm deep pond with food on the bottom which the ducks reached by dabbling. To simulate the "forced" dive response by unrestrained ducks, 2 redheads were presented with a l l beaker of water on the fl o a t i n g platform at the end of the pond. They voluntarily submerged their heads in the beaker to obtain food which covered the bottom. Four redhead ducks and four lesser scaup diving voluntarily were prohibited from surfacing by lowering a v e r t i c a l panel at the only point with access to the surface just before the bird was to surface. The panel was removed usually after an additional 10 seconds of diving; however, on one occasion each duck was forced to remain under water u n t i l i t had fatigued. Three redhead ducks (two male and one female) were used to test the effect of baroreceptor denervation on the submersion response. A l l surgery was performed under general anaesthesia (20mg/kg sodium pentobarbital, Somnotol, MTC Pharmaceuticals, Mississauga, Ontario). Entry to the thoracic cavity was gained through the i n t e r c l a v i c u l a r a i r sac. The l e f t baroreceptor nerve was sectioned approximately 1.5 cm d i s t a l l y from the nodose ganglion, and the right nerve was sectioned near the pulmonary vein at a point where the nerve turns ventrally and medially toward the heart. After denervation, the a i r sac was closed with surgical s i l k and the skin was sutured over the repaired a i r sac. The ducks were allowed one week to recover and then placed on the diving tank. The effectiveness of denervation was tested by i n j e c t i n g 25ug of phenylephrine into the brachial vein while monitoring blood pressure from the brachial artery and noting the presence or absence of a f a l l in heart rate. Two of the barodenervated redhead ducks were f o r c i b l y submerged using the procedure described above, except only three dives per duck were used. Heart rate was determined from measuring the number of beats over the second before submersion, from the cardiac i n t e r v a l actually at or even just before submergence (1st cardiac i n t e r v a l ) , and from cardiac i n t e r v a l s after the heart rate s t a b i l i z e d , but not later than 5 seconds after submergence. Data were analyzed by plotting the relationship between pre-dive heart rate and the heart rate which occurred on submersion, or that occurring in the f i r s t 2-5 sec diving. Regression analysis was performed on the data using a curve f i t t i n g program. When animals were trapped under water, the heart rate was measured one second before and 2-5 seconds after the p a r t i t i o n was closed. The significance of any difference (at P < 0.05) between data from chase-induced dives by untreated and Xylocaine blocked ducks was assessed from the s t a t i s t i c s for linear regressions on both sets of data. In this series of experiments n = the number of observations and N = the number of animals used. The effects of various afferent neural inputs on the diving responses in voluntary dives, especially those when animals were prohibited from surfacing, were investigated in eight birds. Baroreceptors were denervated in three redhead ducks, and diving responses were investigated 3 weeks l a t e r . Four ducks (2 redhead and 2 scaup) were allowed to breathe oxygen before unrestrained dives by passing pure oxygen through the enclosure, to reduce a r t e r i a l chemoreceptor stimulation in the dive. Nasal receptors were anaesthetized in 2 redheads using Xylocaine. Control of heart rate by vagal and cardiac sympathetic nerves To study the properties of the efferent neural pathway, stimulating electrodes were implanted b i l a t e r a l l y on the cut peripheral ends of the cardiac and vagal nerves of three young White Pekin ducks (Anas platyrhynchos; average body mass 1 kg). Ducks were anaesthetized by intramuscular i n j e c t i o n of pentobarbital and the sternum was divided in the mid-line to expose the central cardiovascular area. Cardiac and vagal nerves were i d e n t i f i e d as they coursed towards the heart and were sectioned 1-2 cm from the heart. Loop electrodes, similar to those described by Jones e_t a_l. (1982), were threaded onto the d i s t a l cut ends of the nerves. The electrodes were connected, one pair from each pair of nerves, to two stimulators via stimulus i s o l a t i o n units (Grass Model PSIU6D; Grass Instruments Corporation, Quincy, Mass., U.S.A.). 21 The vagus nerves were stimulated using a constant current of 0.5 mA, and the cardiac sympathetic nerves were stimulated at a constant current of 1.0 mA. Increasing vagal current to 1.0 mA caused no further change in heart rate at any stimulation frequency. The stimulus duration was 2.5 ms for both sets of nerves. Heart rates were recorded over a wide range of stimulation frequencies of each pair of nerves (although the frequency of stimulation was always the same for both vagal or both cardiac nerves). These heart rates were plotted against stimulation frequencies of vagal and sympathetic nerves using the method of presentation of Levy and Zieske (1960). At the end of these experiments animals were k i l l e d with an overdose of pentobarbital administered intravenously. In t h i s study, forced-dive refers to submersion of the head only while the animal i s under r e s t r a i n t , voluntary face immersion refers to submersion of the head only performed voluntarily by the duck to retrieve food from the bottom of a beaker f i l l e d with water, dabbling i s the act of upending in the water with head and thorax submerged, voluntary dives are those involving underwater swimming i n i t i a t e d by the animal, chase-induced dives are those in which the animal dived to escape an apparently threatening situation, and trapped-dive refers to the part of the dive following blockade of the only area with access to the surface. 22 RESULTS Heart rate response to restrained diving Heart rate of restrained redhead ducks in the laboratory -1 -1 was between 90 and 110 beats'min (100 ± 3 beats'min ; N=12). On submergence, heart rate f e l l rapidly and progressively to a stable rate of 35 ± 4 beats'min ^ (N=12) within 5 seconds ( f i g . la & l b ) . Bradycardia occurred rapidly ( f i g . 2) and was unaffected by breathing oxygen before the dive ( f i g . l a ) , but application of Xylocaine to the n a r i a l region v i r t u a l l y eliminated diving bradycardia ( f i g . lb & 2). After Xylocaine, heart rate f e l l to only 80% of the pre-dive rate 15 seconds after submersion ( f i g . l b ) . The f i r s t cardiac i n t e r v a l was usually the shortest in the dive ( f i g . 2). The duck running on a treadmill had a pre-dive heart rate of over 300 beats'min ^, and heart rate f e l l on forced submergence, but not as low as in restrained animals ( f i g . 3). Ducks which were chased and then caught and dived had extremely high pre-dive heart rates (over 400 beats'min ) . Heart rate f e l l immediately and remained stable for 2-5 seconds after which the rate began f a l l i n g gradually but quickly over the remainder of the 15 second dive. Only the f i r s t 5 second period was used in the present analysis ( f i g . 3). Unfortunately, i t was not possible to obtain r e l i a b l e estimates of the f i r s t cardiac i n t e r v a l in dives involving exercising animals and the regression l i n e shown in 23 Figure 1. Heart rate response to submersion in diving ducks (mean ± 1 S.E.M.). (a)Response of animals breathing oxygen (open c i r c l e s ) or air ( s o l i d c i r c l e s ) before submersion. (b) Response of animals after n a r i a l anaesthetization (open c i r c l e s ) compared with untreated animals ( s o l i d c i r c l e s ) . These animals were breathing room air before submersion, N = 6. Heart Rate (min - 1) ro 4^  cn CD o o O O O O o 1 1 1 1 1 1 25 ure 2. Traces showing heart rates of restrained ducks before, during, and after forced submergence. In the upper trace the duck had breathed a i r before submergence. The lower trace shows the cardiac response to submergence after the application of Xylocaine to the nares. 'Down' refers to the time at which the beak entered the water and 'up' refers to the time the animal surfaced. o 27 Figure 3. The relationship between pre-dive and dive heart rate taken at the time the heart rate s t a b i l i z e d but before 5 seconds submergence in a restrained dive. The s o l i d c i r c l e s represent data taken while the animals were immobilized and had not exercised. the open c i r c l e s represent data taken after the duck was exercised on a treadmill (the two points at 300 beats'mjn pre-dive) or after being chased (above 400 beats'min pre-dive). The short l i n e with a slope greater than one was drawn from the regression of the f i r s t cardiac i n t e r v a l for restrained dives excluding those following exercise. 29 figure 3 pertains only to forced dives in the laboratory when the animal was strapped to the table. Both of the barodenervated ducks showed a strong diving response when f o r c i b l y submerged. One of the ducks produced dive heart rates in the range of those of intact ducks (dive heart rates at 5 seconds submergence were 28, 32 and 39 beats'min ) , even when pre-dive heart rate was high (160 to 200 beats'min ^. The other duck, however, showed a much stronger bradycardia. Immediately upon submergence, an extremely long cardiac i n t e r v a l occurred (the f i r s t or second in t e r v a l was either 24, 26 or 28 seconds long, corresponding to s l i g h t l y more than 2 beats'min ^ ) . Heart rate response to free diving Heart rates varied greatly during voluntary diving and dabbling, but there was a consistent pattern to the responses. Dives were usually performed in a series, and heart rate increased to at least 300 beats'min ^ before the f i r s t dive and continued to do so in the pauses between dives so that after 5 or 10 dives, heart rate approached 500 beats'min ^. Before and during diving, heart rates of redhead ducks were always higher (e.g. f i g . 4a) than when dabbling ( f i g . 4b) or ret r i e v i n g food from the beaker of water ( f i g . 4c), but in a l l three situations, heart rate dropped immediately upon submergence. The f i r s t cardiac i n t e r v a l in voluntary dives was usually the longest, and heart rates f e l l to between 100 30 Figure A. EKG traces of a redhead duck a) b) dabbling and c) immersing i t s head water to retrieve food. The downward approximate point of submergence, and the point at which the duck surfaced. diving v o l u n t a r i l y , into a bucket of arrow i s the the upward arrow i s a) Diving \ t 32 and 140 beats'min ^ ( f i g . 5). Heart rate at the f i r s t cardiac i n t e r v a l was p o s i t i v e l y correlated with pre-dive heart rate ( f i g . 5). Heart rate after 2-5 seconds submergence was more strongly correlated with pre-dive heart rate ( f i g . 6a) and was s i g n i f i c a n t l y above that occurring at the f i r s t cardiac i n t e r v a l at a l l pre-dive rates over 250 beats»min ^ ( f i g . 5 & 6a). 3-blockade prevented pre-dive heart rate from exceeding 300 beats'min ^ even in a series of dives, but on submergence both the f i r s t cardiac i n t e r v a l and heart rate after 2 and before 5 seconds f e l l in the same range as those obtained from untreated ducks with low pre-dive heart rates ( i . e . before voluntary face immersion or dabbling). In chase-induced dives, the length of the f i r s t cardiac i n t e r v a l was highly variable, and was usually shorter than in voluntary dives (e.g. f i g . 7b). In one duck, chasing had no apparent effect on dive heart rates compared with those in voluntary dives. In another duck, however, chasing caused a more pronounced f a l l in heart rate than in voluntary dives. Dive heart rates after chasing were 10% below those in voluntary dives, and this difference was s i g n i f i c a n t . The regression l i n e s describing the relationship between dive and pre-dive heart rates in chase-induced dives in 3 ducks were compared with those obtained from the same animals after blockade of the nasal area with Xylocaine. In a l l three cases, the elevations but not the slopes of these regression l i n e s were s i g n i f i c a n t l y d i f f e r e n t , with the heart rates from 33 Figure 5. The relationship between the pre-dive heart rate and the f i r s t cardiac i n t e r v a l on submergence. The broken l i n e i s the regression on these data. Combined data from four ducks after B-blockade (open c i r c l e s ) and untreated (closed c i r c l e s ) I n this and a l l other similar figures the l i n e through the o r i g i n represents the l i n e of i d e n t i t y . CM ( , - U i w ) 3 1 V H 1 U V 3 H 3AIQ 35 Figure 6. Relationship between pre-dive and dive heart rate in dives by unrestrained ducks. The l i n e passing through the origin represents equal pre-dive and dive heart rates. (a) Combined data from 5 ducks diving voluntarily ( s o l i d t r i a n g l e s ) , 3-blocked with propranolol (open t r i a n g l e s ) , untreated. (b) Data from chase-induced dives by a single female redhead after l o c a l anaesthesia of the nares ( s o l i d c i r c l e s ) , untreated (open c i r c l e s ) . Data for propranolol blocked dives for this duck are not i d e n t i f i e d by use of a separate symbol but are included in the regression analysis. The broken l i n e i s the regression l i n e for voluntary dives by this animal with no nasal blockade. The regression equations include the 95% confidence l i m i t s of the slope and the standard error of estimate. 37 Figure 7. EKG trace obtained from unrestrained ducks submerging vo l u n t a r i l y . (a) A diving duck diving voluntarily and (b) a chase-induced dive. The downward pointing arrow indicates the approximate time at which the beak entered the water, and the arrow pointing up indicates the approximate time the animal surfaced. 38 < OD DUDOU&WD DALIlAV Xylocaine treated ducks being above those from untreated animals. In the duck shown in figure 6b, dive heart rate was elevated after Xylocaine blockade by 15 to 30% depending on the pre-dive heart rate. In contrast, in another duck, the elevation in dive heart rate was 10%, regardless of pre-dive heart rate. The third duck showed an elevation in heart rate of 20 to 25% over a much more r e s t r i c t e d range of pre-dive heart rates. The data were not combined, and figure 6b i s included to demonstrate that chasing and nasal blockade account for a small portion of the dive response even in that animal where these factors had the greatest e f f e c t . A few voluntary dives were obtained from two of the ducks after nasal blockade, and these indicated less elevation in dive heart rates compared with voluntary dives before nasal blockade to those obtained in chase-induced dives. Changing the l e v e l of oxygen in the air breathed before diving affected dive duration ( f i g . 8a), but had no effect on heart rate after 2-5 seconds submergence ( f i g . 8b). Dive duration increased as the concentration of oxygen in the inspired air increased. The increase was linear at oxygen level s between 10 and 15%, with a c o e f f i c i e n t of determination ( r 2 ) of 0.98 ( f i g . 8a). At oxygen levels below 10%, a l l diving ceased, even i f food were withheld for one day before the t r i a l . At oxygen leve l s above 15%, dive time increased more slowly with an increase in oxygen concentration ( f i g . 8a); however, no correlation occurred between the dive heart AO Figure 8. (a) Relationship between inspired oxygen and dive time in voluntary dives. The numbers in parentheses above or below the points are n and N. (b) The range of heart rates obtained between 2 and 5 seconds into voluntary dives when the animals breathed a i r , low or high oxygen concentrations before diving. the bar at 21% oxygen represents the range of heart rates of 42 dives from 5 ducks with the greatest density of points occurring at 190 beats per minute. 41 22i-20 o ( A E a> > 18 16 14 12 (23,4) (21. 1.5) I (12.2) g J (13i3) • j 5 (21.4) (20.4) 10' (53.6) I I (34.4) -fh 260r- B •- 220 JE a> ^ I80[ D Q> X > I40f Q • • I001- 1 1 J_ 10 12 14 16 18 20 Inspired O2 22 24 50 42 rate and the l e v e l of oxygen breathed before the dive, and the relationship between dive and pre-dive heart rate remained unchanged ( f i g . 9a). Dive heart rates after breathing 50% oxygen were in the same range as those obtained after breathing a i r or 13-16% oxygen ( f i g . 8b & 9a). Baroreceptor denervation caused a small but s i g n i f i c a n t depression of dive heart rate compared with that of intact animals ( f i g . 9b), heart rate being 5 to 20% lower than intact animals. Preventing access to the surface caused a pronounced f a l l in heart rate when the duck returned to surface in the enclosure at the end of a voluntary dive. Heart rate f e l l immediately after the entrance to the chamber was blocked and this new rate was either maintained for the rest of the enforced submergence or slowly increased ( f i g . 10b). Heart rate two seconds after blockade was correlated with the rate immediately before blockade, and although the heart rates of restrained ducks before and after submergence were often lower than those of unrestrained ducks before and after access to the surface was prohibited, there was considerable overlap of the dive:pre-dive relationship ( f i g . 3 & 11). This overlap in the relationship was unaffected by 3-blockade, baroreceptor denervation, or anaesthetization of nasal receptors with Xylocaine. In two scaup, breathing 100% O2 before the dive had no eff e c t , whereas in another two scaup and one redhead whose baroreceptors were denervated, dive heart rate was not 43 gure 9. (a) The relationship between pre-dive and dive heart rate after breathing high or low levels of oxygen. The s o l i d c i r c l e s represent dives after breathing 14-16% oxygen. The open c i r c l e s are from dives after breathing air containing less than 14% oxygen, and the s o l i d triangles after breathing air containing 50% oxygen. The lin e through the data i s the regression li n e from figure 6a. (b) The relationship between pre-dive and dive heart rate after b i l a t e r a l denervation of the baroreceptors. The s o l i d c i r c l e s are from non-3-blocked dives, and the open c i r c l e s are from dives following 3-blockade with propranolol. The broken l i n e i s from figure 6a, and the s o l i d l i n e i s the regression of these data. 44 45 gure 10. EKG traces of a redhead duck a) f o r c i b l y submerged while under r e s t r a i n t , b) prohibited from surfacing during a free dive and c) trapped as in b, but the duck had breathed oxygen before the dive. The point marked "turn" i s the point at which the duck turned to return to the enclosure . Dive Surface A ) K i n e J 6 ttftHbf-W+^-t-H I II i n n H I Unrestrained ( • | Dive ! U r u ^ ' ^ Surface . Prohibited i Forced Dive I C) Unrestrained a.^agra l>I! i M'' 011I ^  U '• > |- j 11' > IM > ) > > • • i '• > ! V'M» >^ After Oxygen I Turn I l 10 sec 0> 47 Figure 11. The relationship between pre- and "trapped" heart rates when surfacing was prohibited during a dive. The so l i d c i r c l e s represent points for intact animals having breathed air before the dive. The open c i r c l e s represent points taken from ducks that had breathed oxygen before the dive. The s o l i d triangles are from barodenervated ducks, and the open triangles are from ducks whose nares had been anaesthetized with Xylocaine. The so l i d l i n e i s the regression of the f i r s t cardiac i n t e r v a l from voluntary dives (figure 5), and the broken l i n e represents the s t a b i l i z e d rate at two seconds submergence (figure 6a) . ( u i u i ) 3ivd 1UV3H Q3ddVHl 49 stable ( f i g . 11). In these birds, heart rate rose suddenly soon after the birds turned to return to the enclosure ( f i g . 10c) and when surfacing was prevented heart rate f e l l but not as low as before. In fact, heart rates after blockade f e l l into the range of those in voluntary dives made from similar starting heart rates ( f i g . 11). With each duck, once after breathing a i r and once after breathing oxygen before the dive, the entrance to the enclosure was not re-opened. Animals swam around the pond although wing propulsion replaced leg propulsion after 40 sec or so under water. Eventually a l l a c t i v i t y ceased and the ducks floated up under the netting, covering the pond, and breathed. Heart rate remained low throughout these manoeuvers except when the breath was taken. The t o t a l period spent under water u n t i l a c t i v i t y ceased was around 60 seconds and this was extended by 10 seconds, on average, i f the ducks had breathed oxygen before the dive. Control of heart rate by vagal and cardiac sympathetic nerves Heart rate was 283 ± 28(S.D.; n=12) beats'min ^ after b i l a t e r a l section of the vagal and cardiac nerves. Interestingly, this was also the heart rate observed in 2 redhead ducks after pharmacological blockade of cardiac and vagal nerves (propranolol, 1.5 mg»kg ^ and atropine, 2.5 mg»kg ^ ) . B i l a t e r a l stimulation of the vagal nerves resulted in a rapid f a l l in heart rate, a stable rate usually being 50 achieved within 1 or 2 seconds ( f i g . 12). Restoration of pre-stimulation heart rate was equally rapid when stimulation was stopped. In contrast, heart rate only rose slowly in response to b i l a t e r a l stimulation of the cardiac nerves. Usually i t took 30 seconds or so for heart rate to s t a b i l i z e in response to the maximum stimulus frequency used (8 Hz). When stimulation stopped heart rate f e l l slowly and reached pre-stimulation levels within 20 to 30 seconds. Heart rates versus stimulation frequencies of vagal and sympathetic nerves are shown in figure 13, and the surface in figure 13 describes a l l possible heart rates that can be produced by any combination of sympathetic and vagal stimulation. The stimulation frequencies used may not r e f l e c t the a c t i v i t y on the nerves of an intact animal, but the shape of the curves w i l l not be altered, and the heart rate at AO Hz vagal and 8 Hz sympathetic stimulation represent the cardiac response to maximal a c t i v i t y of these nerves. 51 Figure 12. The heart rate response to b i l a t e r a l stimulation of the d i s t a l cut ends of the a) vagus and b) cardiac sympathetic nerves. The horizontal bar represents the duration of the stimulus. 52 53 Figure 13. The relationship of heart rate to b i l a t e r a l stimulation of the d i s t a l cut ends of the vagus and cardiac sympathetic nerves. The figure was drawn through the points by approximation, not by regression. See the text for an explanation of points A, B, C, D and E. 54 Heart Rate (min"') o o o o o *~\ V (,_U!Ui) 9 |Dy M D 9 H DISCUSSION The present results have revealed some interesting insights about the v a r i a b i l i t y in cardiac responses in forced and voluntary dives by free and restrained ducks. The main question i s whether any genuine relationships exist between heart rate in a l l these types of dives, and whether these relationships are supported by our knowledge of the efferent and afferent neural mechanisms affecting cardiac control. Heart rate f e l l immediately on f o r c i b l e submergence, and giving the duck 100% oxygen to breathe before submergence had no effect on the bradycardia in the f i r s t 15 seconds of the dive, nor did denervation of the baroreceptors. Butler and Woakes (1982b) had shown that carotid body denervation did not alter the onset of bradycardia in tufted ducks, when f o r c i b l y submerged. Anaesthetization of the narial region in diving ducks completely eliminated diving bradycardia in forced dives, performed with care; however, i f the animal struggled, heart rate was lower than that just before the struggle. Diving mammals, such as seals and muskrats, also show a rapid heart rate response to forced diving which i s eliminated by neurotomy or anaesthesia of the f a c i a l or n a r i a l region (Dykes, 1974; Drummond and Jones, 1979). Hence, i t appears that rapid bradycardia in forced dives i s l i k e l y to be associated with a "nasal re f l e x " (Jones, 1981). Diving ducks showed marked changes in heart rate during 56 every voluntary and chase-induced dive, even after breathing air with 50% oxygen in the pre-dive period. This confirms the observation of Butler and Woakes (1982b) that after carotid body denervation heart slowing in voluntary dives by tufted ducks was l i t t l e changed from that of intact animals in the early part of submergence. That i s , information from carotid bodies i s not ignored in longer dives. Tufted ducks with their carotid bodies denervated have a mean dive duration 3 seconds longer than intact ducks, and a maximum dive time 6 seconds above that of intact animals (Butler and Woakes, 1982b). Furthermore, heart rate at the end of a dive i s higher in ducks with denervated carotid bodies than in intact ducks (Butler and Woakes, 1982b). In this study, dive time increased by 2 seconds following oxygen loading. It i s l i k e l y that the dive i s terminated not by oxygen shortage, but probably for behavioral reasons. In free dives, unlike in restrained dives, nasal blockade with Xylocaine did not greatly affect heart rate after 2-5 seconds submergence. Stimulation of nasal receptors in free diving appeared to cause between 10 and 30% of the heart rate adjustment. Heart rate f e l l lower in chase-induced than voluntary dives in two of the three ducks, but the results suggest that the contribution from nasal receptors i s similar in both voluntary and chase-induced submersions. Denervation of the baroreceptors caused an apparent, although s l i g h t , enhancement of the dive response, but these three ducks were not tested before denervation; therefore, i t i s not certain where the dive heart rates would f a l l i f these ducks had intact baroreceptors. Furthermore, the data on heart rates obtained in voluntary dives after breathing various lev e l s of oxygen confirm that chemoreceptors have l i t t l e effect on heart rate adjustments up to 5 seconds after submergence. Consequently, nasal receptors, baroreceptors and chemoreceptors combined only account for a minor component of the i n i t i a l cardiac responses to voluntary submersion. There i s no doubt that in free dives, other inputs predominate in causing the cardiac responses and are not displayed in forced dives. This supports claims of "a n t i c i p a t i o n " of the dive response. Butler and Woakes' (1976) o r i g i n a l claim for "a n t i c i p a t i o n " was compromised somewhat by their e a r l i e r statement that "the i n i t i a l bradycardia occurs just as the animal dives and not before" (Woakes and Butler, 1975). More recent data, however, linki n g cine films of submersion behavior to telemetric recordings of heart rate, have shown that lengthening of the cardiac i n t e r v a l occurs before the nasal area contacts the water (Butler and Woakes, 1982a). Regions of figure 13 which appear to pertain to intact animals can be i d e n t i f i e d on the surface describing heart rates resulting from combinations of vagal and sympathetic stimulation. For instance, maximal sympathetic a c t i v i t y in the absence of vagal stimulation gave heart rates of 500 58 beats'min ^ (point A in figure 13), which was the highest rate observed before voluntary dives. (3-blockade with propranolol gave heart rates around 300 beats'min just before voluntary dives, and this i s indicated by the point B on figure 13. F i n a l l y , before restrained dives, the heart rate was around 100 beats'min ^, and since 3-bloekade did not lower heart rate further (tested in two ducks), cardiac efferent control in these animals i s l i k e l y to be described by point C or lower on figure 13. If sympathetic a c t i v i t y does not decrease in a dive ( i t cannot when pre-dive rates are represented by B & C), then the r e l a t i v e increase of vagal a c t i v i t y required to cause the diving heart rates observed must be similar in voluntary dives, represented by points A and B, and in forced dives represented by point C. S p e c i f i c a l l y , an increase in vagal a c t i v i t y of about 20 Hz w i l l give the dive heart rates observed in voluntary dives, with and without propranolol, and in forced dives. In l i g h t of the above suggestion, the cardiac responses in a l l dives, which, might represent 20 Hz vagal a c t i v i t y , were re-investigated. Pre-dive heart rate was plotted against dive heart rate in (1) voluntary dives and dabbles before and after propranolol, (2) forced dives by restrained animals at rest and after exercise and (3) trapped dives by unrestrained animals. Regression analyses were performed on these data and relationships were established for each group and a l l groups combined. Individually, and in combination, a single linear 59 relationship could be f i t t e d to a plot of dive heart rate against the logarithm of pre-dive heart rate ( f i g . 14). Of course, a diminution in sympathetic a c t i v i t y in dives would confound this argument, especially when voluntary dives are made from high heart rates. However, Butler and Woakes (1982a) showed that even when pre-dive heart rate was as high as 400 beats'min ^ in ducks diving after atropine, no change in heart rate occurred, implying that sympathetic cardiac nerve a c t i v i t y i s unaltered in dives. A 20 Hz increase i n vagal a c t i v i t y in forced dives by restrained ducks w i l l result in maximal vagal a c t i v i t y . In voluntary dives, the f i r s t cardiac i n t e r v a l i s usually the longest and could also represent the heart rate at maximal vagal a c t i v i t y , although there may not be s u f f i c i e n t time for f u l l expression of the bradycardia because as can be seen in figure 12, neither the f i r s t nor second cardiac i n t e r v a l following vagal stimulation i s the longest that was achieved for that stimulation frequency. Nevertheless, i t might be expected that these points would be related on a plot of dive:pre-dive heart rate, but i t i s d i f f i c u l t to predict what form the relationship would take. This relationship, however, could be similar to that describing the influence of sympathetic a c t i v i t y on heart rate at maximal vagal a c t i v i t y ( f i g . 13). This relationship i s i l l u s t r a t e d by the curve DE in figure 13. 60 gure 1A. The relationship of the logarithm of pre-dive (or pre-trapped) heart rate and dive (or trapped) heart rate for a l l categories. The s o l i d c i r c l e s represent restrained dives including those following exercise. The open c i r c l e s represent heart rates for a l l trapped dives. The s o l i d triangles are from a l l voluntary dives including 3-blocked dives, and the open triangles are from dabbles and voluntary face immersions. 61 62 It seems unlikely that a single afferent mechanism could provide the necessary vagal a c t i v i t y in a l l types of dive i f only because vagal a c t i v i t y may be maximal, although declining rapidly, at the start of a l l voluntary submergences whether dives or dabbles. Surprisingly, Butler and Woakes (1979) claimed that the tufted duck showed no heart rate changes when freely dabbling; yet, 9 out of 10 of the dabbling episodes seen in their figure 5a are associated with rapid changes in heart rate, and in three cases, these changes are greater than 100 beats'min ^. Some of the heart rates associated with dabbling and emerging in the tufted duck resemble those depicted in figure 4(b) of this study for a redhead duck dabbling v o l u n t a r i l y . The termination of hyperventilation at the onset of a dive or dabble (Butler and Woakes, 1976; 1979) could enhance vagal outflow and reinforce an increase in vagal a c t i v i t y concomitant with the onset of submergence, giving a prolonged i n i t i a l cardiac i n t e r v a l . There i s no pre-dive tachycardia or hyperventilation in forced dives, and in these dives, heart rate declines rapidly and progressively. The description of the f i r s t cardiac i n t e r v a l in free and forced dives by two separate linear regression equations seems en t i r e l y appropriate. It seems plausible for a common afferent mechanism to produce a similar l e v e l of vagal a c t i v i t y in a l l types of dive. Unfortunately, no support for such a mechanism can be derived from this investigation of the role of afferent 63 reflexogenic neural mechanisms in the diving response which showed that a l l of the cardiac response in restrained dives could be attributed to nasal receptors, but nasal receptors account for only between 10 and 30% of the response observed in voluntary or chased-induced dives. Hence, another mechanism must be involved in voluntary dives, but this cannot be peripheral chemoreceptors because breathing elevated or reduced levels of oxygen in the a i r before diving had no effect on the dive:pre-dive heart rate relationship up to 5 seconds after submergence. So, although chemoreceptors undoubtedly influence heart rate in longer dives (Butler and Woakes, 1982b), any influence i s not expressed in the f i r s t 5 seconds of a dive. The animals' response to being trapped i s of considerable i n t e r e s t . As soon as the duck became aware that i t was not going to be allowed to surface, heart rate f e l l to levels which would have been seen in either a forced or voluntary dive depending on the heart rate immediately before being trapped. A similar response was described by Butler and Woakes (1982a) in tufted ducks, although they described i t as "progressive", as opposed to the sudden f a l l seen in these diving ducks. This then may be an expression of the " c l a s s i c a l " dive response in unrestrained diving, but the mechanisms involved in generating t h i s response are unknown. If the response to being trapped were to conserve oxygen, thereby prolonging dive time, then these animals c e r t a i n l y 64 should be able to extend dive time beyond 60 seconds, especially in l i g h t of that Dewar's (1924) report that a duck of similar size (Aythya marila) had a maximum dive time of 49 seconds in the wild. Even after oxygen loading, redhead ducks could extend dive time by only 10 seconds. It seems l i k e l y that anaerobic byproducts accumulating in the muscles having reduced blood flow are l i m i t i n g dive time, not t o t a l oxygen stores. The absence of any major reflexogenic influences on heart rate in voluntary dives suggests that psychogenic influences are much more l i k e l y to be expressed in free than in restrained dives in diving ducks. For instance, psychogenic influences affect dive heart rate both i n i t i a l l y , and after 2 to 5 seconds submergence, through their influence on the pre-dive rate. Anticipation of the dive response, described in voluntary dives by Butler and Woakes (1976, 1982a), also implies a profound psychogenic influence. F i n a l l y , that heart rate can immediately drop when the normal diving pattern i s disrupted, in unrestrained animals, suggests that integration occurs well above the brainstem l e v e l in free dives. These conclusions c o n f l i c t with those obtained from studying restrained and free head submersions by dabbling ducks (Kanwisher e_t a_l. , 1981; B l i x , 1985), in that psychogenic influences affect the former and not the l a t t e r . Obviously th i s c o n f l i c t w i l l only be resolved by further research. SECTION 2 65 The Heart Rate Response to Diving and Dabbling in a Non-diving Duck INTRODUCTION Ducks are commonly divided into two groups, divers and dabblers, but a l l dabbling ducks dive when they are very young, and even adult dabbling ducks dive for food far more frequently than i s generally acknowledged. Foraging dives by mallards (Anas platyrhynchos) , black ducks (A_. rubripes) , African black ducks (A. sparsa) , p i n t a i l s (_A. acuta) , Bahama p i n t a i l s (A., bahamensi s) , shovelers (Spatula clypeata) , New Zealand brown ducks (A^ . auklandica ch l o r o t i s) , gadwalls (A. strepera) , cape teal (_A. capensis) , gray teal (_A. gibberif rons) , wood ducks (Aix sponsa) and mandarin ducks (A_. galericulata) have been observed (Bourget and Chapdelaine, 1982; Chapman e_t a_l. , 1959; Dean, 1950; Kear and Johnsgard, 1968; Kutz, 1940; Mylne, 1954). They have been reported diving to depths greater than 3 meters (Kutz, 1940), having mean dive times of approximately 5 seconds (Bourget and Chapdelaine, 1982; Dean, 1950), and a maximum dive time of 10 seconds (Bourget and Chapdelaine, 1982; Chapman et a_l. , 1959). Bourget and Chapdelaine (1982) suggested that dabbling ducks spending the winter in northerly regions may be forced to dive for food because the shallow water areas are more susceptible to freezing over during cold s p e l l s . The cardiac response to f o r c i b l e submergence of a restrained dabbling duck is characterized by a gradual slowing of the heart and an increase of peripheral resistance (Butler and Jones, 1982). This response i s v i r t u a l l y eliminated by sectioning the carotid body nerve (Jones and Purves, 1970), indicating that i t i s brought on by the progressive hypoxemia and hypercapnemia associated with the breath-hold dive. Diving ducks, however, develop an immediate bradycardia when f o r c i b l y submerged, and although the bradycardia i s not affected by carotid body denervation (Butler and Woakes, 1982b), i t i s eliminated by anaesthetizing the nasal receptors with a l o c a l anaesthetic (figure 1, section 1). Mangalam and Jones (1984) and Jones e_t aJL. (1982) showed that breathing 100% oxygen before a dive greatly reduced the cardiac response to enforced submergence of the Pekin duck. When pre-dive heart rate i s lower than 150-190 beats»min ^, v i r t u a l l y a l l of the bradycardia in dabblers during forced dives, results from stimulation of peripheral a r t e r i a l chemoreceptors (Jones & Purves, 1970; Jones e_t a_l . , 1982). An immersion reflex has been claimed to exist in dabbling ducks, which i s more obvious the higher the pre-dive heart rate (Andersen 1963 a,b,c; F e i g l & Folkow 1963; Folkow e_t al . , 1967; Rey 1971; Butler & Jones, 1982; Blix & Folkow, 1984; B l i x , 1985). However, Jones e_t aj^. (1982) suggested that the i n i t i a l rapid f a l l in heart rate in dabblers may not necessarily mirror a similar change in cardiac output. In 67 fact, cardiac output i s most l i k e l y to be unchanged in this period. Furthermore, they also showed that there was a strong relationship between changes in heart rate in the f i r s t few seconds of submergence (y) and the pre-dive heart rate (x). The relationship was expressed by the formula y = x - 188 for pre-dive heart rates above 188 beats'min ^, so i f pre-dive heart rate were below 188 beats'min ^, there was no change in heart rate early in the dive. This or a similar relationship also appears to apply to the Canada goose during voluntary dabbles (Kanwisher e_t a_l. , 1981). Heart rate f e l l rapidly, from rates in the range of 230-290 beats'min before submergence, to a rate in the range of 140 to 150 beats'min ^. Therefore, when heart rates are very high, the potential exists for rapid cardiac adjustments on submergence even though a s p e c i f i c "immersion ref l e x " may not exist. This study was undertaken to investigate the nature of the cardiac adjustments in dabbling ducks (_A. platyrhynchos) dabbling and diving v o l u n t a r i l y , and the underlying mechanisms which bring about these adjustments. METHODS AND MATERIALS Two male mallards and one female Pekin duck (Anas  platyrhynchos) were used to record the cardiac response to voluntary dabbling. Five mallards (2 male and 3 female) were used in the voluntary diving observations. An EKG transmitter (Narco Biosystems, Downsview, Ontario) was placed in the peritoneal cavity as described in the previous sections. A l l surgery was performed under l o c a l anaesthesia (2% Xylocaine, Astra Pharmaceuticals, Mississauga, Ontario). The birds were allowed one day to recover before being placed on the water. For dabbling, the ducks were placed in a 0.8 X 2.8m Fiberglas tank with water 0.3m deep. Food was scattered on the surface and quickly sank. The EKG signal was received on an FM-1100-7 biotelemetry receiver (Narco Biosystems), recorded on magnetic audio tape and displayed on a pen recorder. Only dabbles l a s t i n g longer than 3 seconds were used in data analysis. This was done to ensure that the heart rate was not changing at the 2 second mark as the result of any a c t i v i t y associated with surfacing. For diving, four of the mallards were placed one at a time in a 0.8 X 1.8m Fiberglas tank with water 1.0m deep. Food was scattered on the surface and quickly sank. The condition of the birds was monitored, and i f a bird would not dive, i t was removed before excessive weight loss. For the f i r s t week, the birds ate only that food which could be reached before the food sank. If the bird did not dive after one week, a small amount of food was placed on the platform d a i l y . The amount of food was kept small to maintain the health of the animal and the hunger drive. The duck was removed from the tank i f diving had not begun by the end of the second week. The duck's' behavior was recorded on a Canon VR-40 VHS video recorder using a JVC GX-N4 camera. Once diving had begun, an EKG transmitter was implanted, and the signal was placed on one of the audio tracks of the video recorder. The video tape was reviewed at high speed. When a dive was observed, the tape was played at normal speed, and the audio signal was displayed on a pen recorder. To test the effect of sympathetic influences on the pre-dive and dive heart rates, a 3-blocker was used, but because the diving behavior i s easily disturbed, and the effects of this disturbance can l a s t for several hours, a (3-blocker having a long h a l f - l i f e was required. Propranolol has a h a l f - l i f e of 2 hours in mammals, whereas nadolol (Corgard, Squibb Montreal, Quebec), a non-selective 3-blocker, has a h a l f - l i f e of 24 hours in mammals (Gilman e_t a_l., 1980). One of the mallards was given 4 mg of Nadolol or a l l y on the f i r s t day followed by 2 mg daily for 2 additional days. The duck was then observed for another week without 3-blockade after which the drug was again administered using the same protocol. Only dives l a s t i n g longer than 4 seconds were used to avoid changes in heart rate associated with surfacing. The 70 transmitters were removed after data c o l l e c t i o n . The three mallards and one additional mallard were used to study the effect of baroreceptor denervation on the cardiac response to voluntary diving. Barodenervation was done using the procedure described i n section 1. Diving usually resumed within a week after barodenervation. The EKG transmitter was then implanted using the same procedure as for intact ducks. Chronic barodenervation i s usually accompanied by high heart rates (Jones et a_l . , 1983) , and nadolol was used to increase the range of pre-dive heart rates in this group. One of the ducks was used as a sham. The nerves were exposed and a loop of thread was placed around each nerve. One end of each thread was anchored to the skin high in the neck after the wound was closed. After heart rate data were collected for th i s condition, the duck was again anaesthetized, and the thread was slowly pulled through the wound sectioning the nerves. The duck was returned to the tank on the following day and data c o l l e c t i o n resumed. The effectiveness of denervation was tested in each duck by in j e c t i n g 25yg of phenylephrine into the brachial vein and noting the presence or absence of a f a l l in heart rate. The animals were then k i l l e d with a l e t h a l dose of sodium pentobarbital and baroreceptor nerve section checked post mortem. One of the four mallards underwent a bradycardia following administration of the vasoconstrictive drug. The post mortem inspection revealed that only the l e f t baroreceptor nerve was sectioned. This animal i s reviewed separately. Pre-dive heart rate was determined one second before submergence, and dive heart rate was determined two seconds after submergence. The data are shown as least squares regressions with the 95% confidence l i m i t s of the slope and the standard error of estimate. The slopes were tested for significance (P < 0.05) against slopes of either 0 or 1 usi the t - s t a t i s t i c . 72 RESULTS Heart rate response during dabbling When dabbling v o l u n t a r i l y , heart rate of mallards did not change noticeably, although the slope of the regression l i n e i s s i g n i f i c a n t l y different from unity ( f i g . 15 & 16a). The large changes were rare, and were not always related to the moment of submergence or surfacing. The mean duration of submersion was 3 ± 1 (S.D.) seconds with a maximum of 9 seconds; however, the large majority of dabbles were less than 3 seconds, and were therefore not used in the analysis. Heart rate response during diving The time required to make a return t r i p to the bottom of the diving tank was approximately 2 seconds. Dive time increased during the f i r s t week of diving from 2 seconds to 6 seconds, and the combined mean of a l l ducks after the f i r s t week was 6.2 ± 2.1 seconds with a maximum dive time of 11.6 seconds. When voluntarily diving, the ducks showed l i t t l e or no anticipatory increase in heart rate before the dive even i f the pre-dive heart rate was low ( f i g . 16b). The heart rate was lowest before the f i r s t of a series of dives, and increased progressively between subsequent dives. This increase was positively correlated with dive duration, and negatively correlated with the length of the intervening 73 Figure 15. The relationship between pre-submergence and submersion heart rate during voluntary dabbling when submersion time exceeded three seconds. The broken l i n e represents the regression of these data. In this and a l l other similar figures, the l i n e passing through the or i g i n represents the l i n e of i d e n t i t y . 75 Figure 16. EKG and heart rate traces from mallards a) dabbling and b) diving. The broken l i n e at 250 beats'min represents the t y p i c a l heart rate at two seconds into the dive. There i s no t y p i c a l heart rate for dabbles. o o o O o O O o O o i n ro cvl — O r~r i i i o o o o o 0 o o o o _ K T J f r o O J — O 1 I I I I I I I I I I I o o o o o o o o o o o m ^- ro C J — ( UIUI ) 8JDy I w o o o o o o * O O O O O W Ifi <f lO w -(.ww) apy jjoaiH 77 pauses. The range of pre-dive heart rates can be seen in figure 17a. In one duck, the mean dive time was 7.7 ± 1.2 seconds, the dive:pause r a t i o was one, and pre-dive heart rate rapidly increased to 500 beats'min ^ during a series of dives. The duck represented by the data in figure 18 ( s o l i d c i r c l e s ) had a low dive:pause r a t i o (<<1). The heart rate of this duck immediately after a long dive (>9 seconds) was usually above 400 beats'min ^, but the duck rested at the surface for more than 10 seconds and heart rate decreased, consequently the rate before the next dive was never higher than 350 beats'min . The heart rate i n the early stages of the dive was nearly the same regardless of the pre-dive rate. The combined data from 3 mallards (50 dives each) show that dive heart rate was approximately 250 beats'min ^ ( f i g . 17a). When the data for each animal were analyzed separately, the regression l i n e s of 2 of the 3 ducks were not s i g n i f i c a n t l y different from zero, but the third as well as the non-3-blocked dives of the duck shown in figure 18 were s i g n i f i c a n t l y different from zero. The regression l i n e s of a l l ducks, however, cross the l i n e of i d e n t i t y . Obviously heart rate increased in dives when the pre-dive heart rate was low, and decreased in dives when the pre-dive heart rate was high ( f i g . 16b, 17a & 18). Following B-blockade with nadolol, not only was the pre-dive heart rate low, but the dive heart rate was s i g n i f i c a n t l y depressed 78 Figure 17. The relationship between pre-dive and dive heart rate during a) voluntary dives of three intact mallards ( s o l i d c i r c l e s ) and one sham operated mallard (open c i r c l e s ) , and b) voluntary dives of barodenervated ducks without S-blockade ( s o l i d c i r c l e s ) and following 3-blockade with nadolol. The s o l i d l i n e s are regressions of the data, and the broken l i n e i s the regression of the data after adding 30 beats'min to the dive heart rate of 3-blocked dives (see text for an explanation). 79 PRE-DIVE HEART RATE (min-1) 80 Figure 18. The relationship between pre-dive and dive heart rate from one mallard intact ( s o l i d c i r c l e s ) , after 3-blockade with nadolol (open c i r c l e s ) and after denervation of the l e f t baroreceptor nerve only ( s o l i d t r i a n g l e s ) . These data are from the same bird and are not included in the previous figures. 82 compared with dives in which sympathetic a c t i v i t y was not antagonized ( f i g 18). Baroreceptor denervation altered the relationship between pre-dive and dive heart rate ( f i g . 17b). The regression l i n e now approaches, although i s s i g n i f i c a n t l y different from, the l i n e of identity ( f i g . 17b). The duck that had only one baroreceptor nerve sectioned ( f i g . 18) gave a response between that of intact ducks and those ducks whose baroreceptor nerves were sectioned b i l a t e r a l l y ( f i g . 17b). 83 DISCUSSION Mallards appear to be regulating heart rate during the early stages of voluntary dives, with the regulated rate being approximately 250 beats'min ^. This cardiac response to voluntary diving i s nearly eliminated by b i l a t e r a l l y sectioning the baroreceptor nerves, but not by u n i l a t e r a l sectioning. Information about cardiac rhythm i s carried in the baroreceptor nerves (Arndt e_t al^. , 1977), but there i s no evidence that animals use this information to adjust heart rate. In any event, the change in heart rate must be vagally-mediated because the cardiac sympathetic nerves are incapable of bringing about rapid changes in heart rate (figure 12, section 1); therefore, not only i s the amount of change of vagal a c t i v i t y variable, but the direction of change also depends on the pre-dive rate. When propranolol was given to diving ducks, dive heart rate f e l l to values in the same range as that of non-3-blocked dives having similar pre-dive heart rates (figures 5 & 6a, section 1); however, when given nadolol, dive heart rate was approximately 30 beats'min ^ lower than when given propranolol (personal observations). Figure 18 also shows dive heart rates approximately 30 beats'min ^ lower after administration of nadolol than would be expected i f sympathetic antagonism were the only effect of this drug. This c a l l s into question the points representing B-blockade in figure 17b and the slope of the regression. A new regression can be generated by 84 adding 30 beats'min ^ to dive heart rates of 3-blocked dives (indicated by the broken l i n e of f i g . 17b). Both of the slopes are s i g n i f i c a n t l y different from one. Obviously, other factors are responsible for the remainder of the cardiac adjustments to submergence (e.g. those rel a t i n g to exercise or the cessation of breathing). As with mallards, penguins also showed no anticipatory tachycardia before the dive, but unlike mallards, penguins showed l i t t l e or no cardiac adjustments to voluntary submergence (Butler and Woakes, 1984). If the pre-dive tachycardia seen in a l l diving ducks thus far examined i s in anticipation of a high l e v e l of exercise, i t certainly cannot explain the absence of such a tachycardia in mallards because dabbling ducks are more buoyant than diving ducks (Dehner, 1946 - in King, 1966), and have to work harder to dive. Two of the fiv e ducks used in the present study seemed unable to submerge f u l l y with out the aid of the wings, but once under water, they switched to leg propulsion. The cardiac response to voluntary dabbling exhibited by the mallard i s puzzling. On the one hand, a r t e r i a l chemoreceptors are responsible for the cardiovascular adjustments to forced diving, and i t i s surprising that l i t t l e or no cardiac adjustment occurred during short periods of head submersion. On the other hand, however, the evidence presented by Jones e_t aJ. ( 1982) showed cl e a r l y that, when the duck's heart rate was high just before a forced dive, heart rate f e l l rapidly to approximately 188 beats'min . The rapid changes in heart rate that occur on forced submergence do not appear when the duck freely dabbles. Ventilation was not recorded, but i f minute v e n t i l a t i o n i s similar when pre-dive heart rate i s high in forced and voluntary head submergence, then the rapid bradycardia may not be reflexogenic. Since that study, more data have been analyzed, and the equation i s now y = 1.01 x - 146. Evidence from voluntarily dabbling geese (Kanwisher e_t aJ., 1981) and Pekin ducks (Gabrielsen, 1985) indicates changes in heart rate to approximately 150 beats'min ^, with heart rates of 200-300 beats'min ^ during the short breathing period. Observations from this study reveal some change in heart rate during the dabble, but these were seen when the period of submersion was short (less than 3 seconds), and when the dabbles occurred in rapid succession (surface time usually less than 2 seconds). In any event, heart rate did not f a l l to 150 beats'min ^ with any regularity during these dabbles, and as can be seen from the la s t trace of figure 16a, the presence of a bradycardia was unpredictable, and the rate of f a l l of cardiac frequency was variable. It i s clear from this study, and from other studies of voluntary diving in birds that few generalizations can be made regarding the cardiovascular adjustments to submergence. Moreover, our knowledge of the adjustments to submergence i s limited to heart rate. Hypotheses about other aspects of the 86 cardiovascular system are based on assumptions, such as the maintenance of a r t e r i a l pressure. If a r t e r i a l pressure i s being maintained during the dive, i t i s l i k e l y that the system i s responding to and correcting for changes in pressure brought about by alterations in t o t a l peripheral resistance, and since mallards are leg-propelled divers, the change in t o t a l resistance may be largely the result of modifications in hindlimb perfusion. There i s no evidence, however, for or against a r t e r i a l pressure being maintained. If not, heart rate i t s e l f i s the l i k e l y regulated variable. 87 GENERAL DISCUSSION Diving birds foraging in nature spend a considerable amount of time under water. Pedroli (1982) stated that tufted ducks may spend as much as 5.9 hours out of 24 under water, and that nearly a l l of this foraging a c t i v i t y takes place within a 14 hour period that spans night time. Siegfried (1974) reported that female scaups spent 5.5 hours under water during a 12 hour period between 0800 and 2000. This represents 42.1 and 45.8% for tufted ducks and scaups respectively of the t o t a l foraging period being spent under water. Diving birds, however, do not dive continually in the foraging period. Long pauses occur among bouts of diving. In a series of dives interrupted by no more than 30-second pauses (a diving bout), i t i s clear that the animals are spending far more time under water than at the surface. A lesser scaup (Aythya a f f i n i s ) performed a series of 42 dives over a period of 20 minutes during which 72% (14.4 minutes) of the time was spent under water (unpublished observations). Most diving ducks spend about 63% of a diving bout under water ( i . e . a dive-pause r a t i o of 1.7); however oldsquaw (Clangula  hyemalis) spend more than 80% of their diving bouts under water (Dewar, 1924). It i s not certain how long the bird can maintain this high dive-pause r a t i o . In an effort to understand the behavior of the lesser scaup, a Log-Survivor plot (Fagan and Young, 1978) was prepared from over 200 dives and pauses of 17 birds diving in the wild ( f i g . 19). The abscissa represents the duration of the event, and the ordinate displays the logarithm of the percent number of events greater than a given duration. This function allows predictions because the slope of any segment of the function i s proportional to the probability that the event w i l l terminate. The curve shows that the probability that a dive w i l l terminate in less than 10 seconds i s near zero, but the probability i s very high above 20 seconds. There i s nothing especially revealing in this curve; however, the figure for between-dive pauses shows some interesting c h a r a c t e r i s t i c s . The greatest probability that a pause w i l l end and a dive begin occurs between 6 and 10 seconds, after which the probability decreases and i s near zero above 30 seconds. The s p e c i f i c s of this figure do not apply to a l l diving birds, and w i l l not even apply to the same species i f that bird i s diving in very deep water because, as Dewar (1924) showed, dive times and dive-pause ratios are p o s i t i v e l y correlated with the depth of the water in which the bird i s diving. Dive-pause ra t i o s less than one were recorded for lesser scaup and redhead ducks on the man-made pond used in this study. It i s l i k e l y that the cardiovascular adjustments observed in this study r e f l e c t the adjustments occurring in naturally diving ducks in the wild; however, observations on dive-pause ratios underscore the need to record from animals diving in a more natural setting. 89 Figure 19. Log-Survivor plot of 202 dives and pauses of 17 lesser scaup diving in the wild. The ordinate shows the logarithm of the percent number of dives greater than the time shown on the abscissa. 90 O.I -L 5 10 -I 1 . i ' | 15 20 25 30 35 T i m e (sec) 91 Plasma l a c t i c acid concentration of tufted ducks swimming on the surface increased by more than two-fold when swimming velocity increased from 0.3 to 0.7 m'sec ^ (Woakes and Butler, 1986). The underwater velocity of redhead ducks in th i s study was 0.8 m'sec ^, and i f l a c t i c acid increases when swimming at these v e l o c i t i e s even while breathing, then i t i s l i k e l y that l a c t i c acid w i l l also increase during a dive. The short pauses may not be s u f f i c i e n t to clear this anaerobic byproduct, thereby causing a slow accumulation of l a c t i c acid during the diving bout. This may explain the termination of the diving bout; of course, there may well be other reasons for terminating diving, such as s a t i a t i o n , or to allow time for the bird to restore feather condition by preening. To extend this study w i l l require sampling a r t e r i a l blood, allowing us to estimate the influence of plasma lactate on the duration of a diving bout. Circumstantial evidence from section 2 suggests that muscle oxygen stores may not maintain contraction for more than 30 seconds i f ducks are prevented from gaining access to the surface at the end of a voluntary dive. In fact, leg propulsion was succeeded by an even shorter period of wing propulsion before a l l locomotor a c t i v i t y ceased and the animal floated passively to the surface. Heart rates telemetered from these animals suggest that cardiovascular adjustments similar to those observed in forced dives ( i . e . bradycardia and r e d i s t r i b u t i o n of blood flow) may occur when the animal 92 becomes aware of i t s plight. If so, then muscle oxygen stores are not ef f e c t i v e in prolonging muscle a c t i v i t y in submerged ducks. In a preliminary study, the t i b i a l i s anterior muscle of Pekin ducks was e l e c t r i c a l l y stimulated and isometric tension measured. The muscles of the hindlimb of the duck maintained their strength of contraction for less than 30 seconds and completely fatigued in about 90 seconds after muscle blood flow was occluded ( f i g . 20). In forced dives, contraction strength of the hindlimb decreased with a similar time course once extreme bradycardia occurred ( f i g . 20), but contraction strength was unaltered for 45 minutes while the animal was breathing. Diving ducks d i f f e r from dabbling ducks in their response to both forced and voluntary diving. When f o r c i b l y submerged, dabbling ducks respond to a f a l l in a r t e r i a l oxygen tension by slowing heart rate and increasing peripheral resistance, whereas diving ducks reduce heart rate before any change in a r t e r i a l oxygen tension occurs. This "immersion response" may be adaptive in the wild because oxygen conservation could extend dive time; however, nasal receptor stimulation caused only a small change in the dive response of freely diving redhead ducks. Furthermore, when trapped under water, diving ducks showed an intense bradycardia, yet dive time was not greatly increased. The pre-dive tachycardia and hyperventilation, seen when diving ducks voluntarily dive, w i l l increase oxygen stores thereby maximizing aerobic dive 93 Figure 20. Traces of the isometric tension, in arbitrary units, developed when the t i b i a l i s anterior i s stimulated at 3 pulses per second. A decrease in contraction strength when the i s c h i a t i c artery i s occluded can be seen within 10 seconds following occlusion. A r t e r i a l blood pressure i s included for the dive as an indication of cardiovascular performance in the dive. The spikes seen in the tension trace of the dive represents struggles. 95 time as suggested by Butler and Woakes (1982a). The bradycardia and probable r e d i s t r i b u t i o n of blood flow (Heieis and Jones, personal communication) occurring on submergence would then aid in conserving oxygen for the exercising muscles. Baroreceptor denervation does not a l t e r the cardiac response in diving ducks diving v o l u n t a r i l y , so i f a r t e r i a l pressure i s being maintained in intact ducks, then t o t a l peripheral resistance must be the controlled variable. During voluntary diving, diving ducks appear to control heart rate by producing a constant increase in vagal a c t i v i t y . In contrast, dabbling ducks diving voluntarily appear to regulate heart rate using their baroreceptors, but i t i s more l i k e l y that a r t e r i a l pressure i s being maintained. In this case, dabbling ducks may be c o n t r o l l i n g peripheral flow, and adjusting heart rate accordingly to regulate blood pressure. This explanation, however, hinges on assumptions that a r t e r i a l pressure i s being maintained and that stroke volume i s constant. Jones e_t aJ^. (1983) reported that stimulation of the proximal cut ends of the baroreceptors during submersion of a force-dived Pekin duck caused l i t t l e change in peripheral resistance. The reason i s unclear, but i t may be that the baroreflex i s incapable of overriding the intense vasoconstriction brought about by chemoreceptor act i v a t i o n , and may not r e f l e c t a central i n h i b i t i o n of the baroreflex. Chemoreceptors w i l l not be activated above resting levels at 96 the moment of submersion, and the baroreflex i s probably f u l l y operative and could regulate a r t e r i a l pressure through changes in peripheral resistance or cardiac output. Clearly, further research i s necessary to uncover the role of baroreceptors in voluntary diving of both groups of ducks. It i s t r a d i t i o n a l to report the heart rate response to submersion as means and standard errors in the manner presented in figure 1 (section 1). This presentation, however, suggests that there i s a ty p i c a l pre-dive and dive heart rate. This i s an acceptable conclusion when there i s l i t t l e v a r i a b i l i t y in pre-dive and dive heart rates among dives, but i t i s clear from this study that some information regarding the heart rate response to diving i s lost using t h i s form of analysis. It i s also common in the diving l i t e r a t u r e to report either percent change or difference in heart rate. A common mechanism, however, can lead to differences in percent change or absolute f a l l in heart rate depending on the i n i t i a l value. Clearly, an understanding of the contributions of cardiac sympathetic and vagal nerves i s necessary for a complete analysis of the heart rate response to forced and voluntary diving. 97 LITERATURE CITED Andersen, H.T. (1963a). Factors determining the circulatory adjustments to diving. I. Water immersion. Acta physio1. scand. 58, 177-185. Andersen, H.T. (1963b). Factors determining the circulatory adjustments to diving. I I . Asphyxia. 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