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Influence of water and blood flow on gas exchange at the gills of rainbow trout, salmo gairdneri Davis, John Christopher 1971

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THE INFLUENCE OP MATER AND BLOOD PLOW ON GAS EXCHANGE AT THE GILLS OF RAINBOW TROUT, Salmo g a l r d n e r i . by JOHNCCHRISTOPHER DAVIS B . S c , Univers i ty of V i c t o r i a , 1966 M . S c , Un ivers i ty of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1971 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying o f th i s thes is f o r scho la r l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t i on o f th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT St u d i e s were c a r r i e d out to determine the i n f l u e n c e of water and blood f l o w p a t t e r n s on gas exchange a t rainbow t r o u t g i l l s . A v a r i e t y of g i l l water flows and p a t t e r n s of b l o o d d i s t r i b u t i o n w i t h i n the g i l l s e x i s t d u r i n g d i f f e r e n t p h y s i o l o g i c a l s t a t e s and hence a r e important to the gas exchange process. An e v a l u a t i o n of techniques f o r measuring mean e x p i r e d oxygen t e n s i o n was c a r r i e d out to determine a way f o r a c c u r a t e l y measuring v e n t i l a t i o n volume (VQ) i n t r o u t . Water from o p e r c u l a r and c l e i t h r a l cannulae had extremely v a r i a b l e P f ^ ' 8 * n ^ o t h ^ r e e -swimming and r e s t r a i n e d t r o u t . F i c k p r i n c i p l e c a l c u l a t i o n s of VQ based on data from such cannulae c o u l d be s u b j e c t to e r r o r . A method f o r d i r e c t l y measuring v e n t i l a t i o n volume i n r e s t r a i n e d t r o u t by means of a rubber membrane a t t a c h e d t o the mouth ( o r a l membrane) to separate i n s p i r e d and e x p i r e d water andppermlt c o l l e c t i o n of the l a t t e r i s d e s c r i b e d . F i s h ( 2 0 0 g, 8.6 C) f i t t e d w i t h o r a l membranes had r e s t i n g mean VQ'S of 3 7 - 1.8 ml/min and VQ c o u l d be i n c r e a s e d s e v e n - f o l d d u r i n g s t r u g g l i n g or excitement. I n c r e a s e s i n VQ were accomplished by a l a r g e r i s e i n v e n t i l a t o r y s t r o k e volume and a smal l i n c r e a s e l n v e n t i l a t i o n r a t e . U t i l i z a t i o n of oxygen from the water i n qu i e s c e n t f i s h was 4 6 - 1.5% and ranged from 26 - 6k%. The r a t i o between the volumes of water and blood p a s s i n g the g i l l / u n i t time ( v e n t i l a t i o n - p e r f u s i o n r a t i o ) was approximately 5. Area mean d i f f e r e n t i a l p r e s s u r e ( b u c c a l p r e s s u r e - opercular p r e s s u r e ) appeared t o be d i r e c t l y r e l a t e d to VQ over the V G range i i 4 0 - 1 6 0 ml/min as th i s f o u r - f o l d r i s e was accompanied by a pressure increase from 0 . 1 to 0 . 4 mm Hg. Oyer t h i s V G range the ca lculated res i s tance of the g i l l sieve d id not change Indicat ing that changes i n water s p i l l a g e past the g i l l s (anatomical deadspace) d id not occur. A r t i f i c i a l perfusion of the g i l l s with water by means of a mouth tube showed that trout could saturate t h e i r a r t e r i a l blood with oxygen and perfusion rates of 85 - 1 2 0 0 ml/min but could not do so at rates approximating normal r e s t i n g V Q (45 ml/min) . Such t perfusion l i k e l y provides a poor pattern of water flow over the g i l l s compared to normal i r r i g a t i o n . Infrared photographs of the g i l l s showed a large i n vivo increase l n the volume of blood i n the g i l l i r lamel lae fo l lowing adrenaline i n j e c t i o n but f a i l e d to detect small changes i n blood d i s t r i b u t i o n i n the g i l l s of uninfected f i s h . Rainbow trout responded to reductions i n g i l l surface area (bloodiasupply to some g i l l arches t i e d off) by e levat ing v e n t i l a t i o n volume and cardiac output. When the blood supply to the pseudobranch was i n t a c t the f i s h could maintain oxygen saturat ion of a r t e r i a l blood despite a 50% reduct ion i n g i l l area by adjust ing V Q and poss ib ly Q (cardiac output) . P i sh with the blood supply to the pseudobranch destroyed had low a r t e r i a l oxygen tensions and d id not increase t h e i r V Q to the same extent as those with i n t a c t pseudobranchs. The pseudobranch may contain chemoreceptors important l n the regulat ion of a r t e r i a l Pc>2* C i r c u l a t i o n times, determined by dye i n j e c t i o n , were about 1 minute i n quiescent trout ( 2 0 0 g, 1 0 C) . The responses of trout to reduced g i l l water i i i flow or hypoxia are too rap id to be i n i t i a t e d so l e ly by a venous receptor , at these c i r c u l a t i o n times?, as some authors have suggested, hence the pseudobranch i s a l o g i c a l s i t e for an oxygen receptor . A t h e o r e t i c a l ana lys i s showed that about 30% of the VQ was involved i n non-respiratory water shunts at the g i l l s during gentle to moderate breathing ( V G = 40 - 120 ml/min) and up to ?0% at the highest perfusion rates . Deadspace due to d i f f u s i o n a l problems i s small ( 2 - 5% of V G ) during gentle-moderate breathing but may r i s e to 30% of VQ at high perfusion rates . Anatomical deadspace i s probably large at high g i l l water flows and i s thus a major contr ibutor to the t o t a l water shunt at the g i l l s a M such flows. D i s t r i b u t i o n deadspace, r e s u l t i n g from unequal v e n t i l a t i o n and perfus ion of the g i l l s , may be an important part of the shunt at low flows but i s l i k e l y minimal at high flows when vasod i la t i on of the g i l l s occurs. Deadspace problems would not severely l i m i t oxygen extrac t ion over the 40 - 300 ml/min V G range measured for 200 g trout with o r a l membranes but may account for d e c l i n i n g % u t i l i z a t i o n of oxygen as VQ increases . The ca lcu la ted oxygen cost of breathing at t h i s V G range was only 1.4 - 6.7% of the t o t a l V Q 2 assuming the e f f i c i e n c y of the system i s only 1%. The system i s probably bet ter than 1% e f f i c i e n t so the oxygen cost of breathing over t h i s VQ range Is very low. i v TABLE OP CONTENTS PAGE INTRODUCTION 1 GENERAL METHODS EXPERIMENTAL ANIMALS . 4 OPERATING TABLE AND OPERATING PROCEDURE . 4 CANNULATION PROCEDURES 5 SYMBOLS AND ABBREVIATIONS USED 13 PART ONE - AN EXAMINATION OF OPERCULAR AND CLEITHRAL CATHETERIZATION AS TECHNIQUES FOR SAMPLING WATER EXPIRED BY TROUT. Introduction I 14 Methods I 15 Results I*. 18 Discussion I 19 Summary I 27 PART TWO - DIRECT MEASUREMENT OP VENTILATION VOLUME AND RELATED:PARAMETERS IN RAINBOW TROUT. Introduction II 29 Methods II 29 Results II,,.. 33 Discussion II 36 Summary II 41 PART THREE - THE RELATIONSHIP BETWEEN IRRIGATION OP THE GILLS AND PRESSURES IN THE MOUTH AND OPERCULAR CAVITIES OF TROUT. Introduction III 42 Methods III 44 Results III 45 Discussion III 48 V TABLE OF CONTENTS - Continued PAGE Summary III 55 PART FOUR - ARTIFICIAL PERFUSION OF THE GILLS AT DIFFERENT WATER FLOW RATES. Introduct ion IV , , 57 Methods IV 57 Results IV . . . . 59 Discussion IV 62 Summary IV 70 PART FIVE - BLOOD DISTRIBUTION AND FLOW WITHIN THE GILLS OF RAINBOW TROUT. Introduct ion V 72 Methods V 7^ Results V 79 Discuss ion V 83 Summary V 91 PART SIX - A THEORETICAL CONSIDERATION OF WATER AND BLOOD SHUNTING AT THE GILLS OF TROUT 93 Major Findings of the Thesis 109 L i t e r a t u r e Ci ted I l l v i LIST OF TABLES TABLE Fol lowing Page I A comparison of oxygen tensions In mixed expired water and water from c l e l t h r a l cannulae i n f i s h with o r a l membranes attached. 19 II A c o r r e l a t i o n ana lys i s on the data from Table I . 19 III V e n t i l a t o r y and c i r c u l a t o r y parameters from eighteen f i s h with o r a l membranes attached. 33 IV A c o r r e l a t i o n a n a l y s i s on the data from Table III .55 35 V A comparison of v e n t i l a t o r y parameters i n a group of trout before and a f t e r b r i e f , short-term hypoxia. 35 VI V e n t i l a t i o n volume data f o r a v a r i e t y of t e l eos t s as reported i n the l i t e r a t u r e , 36 VII Blood oxygen tensions from groups of trout whose g i l l s were l i g a t e d i n various ways to reduce t h e i r area , 77 VIII V e n t i l a t o r y response of groups of l i g a t e d f i s h . 81 IX A summary of c i r c u l a t i o n time estimates made by i n j e c t i n g dye in to the dorsa l aortae of quiescent t r o u t . 82 X Data used i n the c a l c u l a t i o n of deadspace phenomena at the g i l l s . 97 v i i LIST OF TABLES - Continued TABLE Fol lowing Page XI Calculated values f o r the magnitude of water shunt at the g i l l s . 97 XII Calculated values for the magnitude of the d i f f u s i o n deadspace at the g i l l s . 99 XIII Data used to ca lcu la te the metabolic cost of breathing . 105 XIV Calculated oxygen cost of breathing at d i f f eren t v e n t i l a t i o n volumes using various e f f i c i ency values of the branch ia l pump. 105 v i i i LIST OP FIGURES FIGURE Following Page 1) D e t a i l s of the buccal, c l e i t h r a l , dorsal a o r t i c , ventral a o r t i c and sublntestinal vein cannulae. 6 2) The three opercular cannula placements used to evaluate opercular catheterization as a method f o r sampling expired water. 7 3) Oxygen tensions recorded from the three opercular cannula placements from free-swimming f i s h . 18*: 4) D e t a i l s of the or a l membrane apparatus used to record v e n t i l a t i o n volume d i r e c t l y i n trout. 30 5) V e n t i l a t i o n volumes recorded from a single trout with an attached o r a l membrane. 3^ 6) The recording system used to monitor pressure from buccal and c l e i t h r a l cannulae. The method used to calculate area mean pressure i s i l l u s t r a t e d . 44 7) Ventilatory parameters measured from a group of trout over a range of v e n t i l a t i o n volumes. 45 8) Pressures recorded from buccal and c l e i t h r a l cannulae at a variety of v e n t i l a t i o n volumes. 46 9) Typical pressure recordings from the branchial chambers of a trout at three d i f f e r e n t v e n t i l a t i o n volumes. 46 10) The rel a t i o n s h i p between area mean d i f f e r e n t i a l pressure across the g i l l s and v e n t i l a t i o n volume. The calculated resistance of the g i l l sieve i s i l l u s t r a t e d . 46 i x LIST OF FIGURES - Continued FIGURE F o l l o w i n g Page 11) Area mean b u c c a l and c l e i t h r a l p r e s s u r e a t a v a r i e t y of v e n t i l a t i o n volumes. 46 12) The apparatus used to a r t i f i c i a l l y p e r f u s e the g i l l s of t r o u t w i t h water a t d i f f e r e n t f l o w r a t e s . 58 13) Oxygen t e n s i o n s r e c o r d e d from the t h r e e d i f f e r e n t o p e r c u l a r cannula placements a t a range of p e r f u s i o n r a t e s . 59 Ik) Oxygen t e n s i o n s i n i n s p i r e d and e x p i r e d water and the percent u t i l i z a t i o n of oxygen f o r the group of p e r f u s e d f i s h . 6 0 15) C a r d i o v a s c u l a r and r e s p i r a t o r y parameters recorded from a group of f i s h whose g i l l s were p e r f u s e d w i t h water a t v a r i o u s flow r a t e s . 60 16) Oxygen t r a n s f e r f a c t o r and a r e a mean d o r s a l a o r t i c p r e s s u r e f o r the group of p e r f u s e d f i s h . 62 17) The e f f e c t of r a p i d changes i n g i l l water flow on p r e s s u r e s recorded from the b u c c a l and v e n t r a l a o r t a of p e r f u s e d t r o u t . 62 18) D e t a i l s of the window implanted i n the operculum of t r o u t f o r i n f r a r e d photography of the g i l l s . The photographic apparatus Is i l l u s t r a t e d . 7k 19) The major b l o o d v e s s e l s on one s i d e of the head of a t y p i c a l salmon!d. 77 X LIST OF FIGURES - Continued FIGURE Following Page 20) A r t e r i a l oxygen tension, oxygen uptake rate and calculated cardiac output f o r f i s h whose g i l l area was reduced "by l i g a t i o n of the g i l l arches. 21) A t y p i c a l dye i n j e c t i o n curve f o r determination of c i r c u l a t i o n time i n quiescent trout. 82 22) Deadspace at the g i l l s calculated from a t h e o r e t i c a l analysis of gas exchange. 99 23) The calculated oxygen cost of breathing at d i f f e r e n t v e n t i l a t i o n volumes. 105 x i LIST OP PLATES PLATE Fol lowing Page 1) An i n f r a r e d co lor photograph of trout blood e q u l l i b r l a t e d to three d i f f eren t oxygen tensions. 79 2) An i n f r a r e d photograph of a por t ion of the g i l l before and a f t e r adrenal ine In jec t ion i n the sub ln te s t ina l v e i n . 79 3) An i n f r a r e d photograph showing a port ion of the g i l l of a trout with g i l l damage. 80 x i i ACKNOWLEDGEMENTS I should l i k e to thank my supervisor , Dr. David Randall f o r h i s advice and guidance during t h i s study and f o r h i s ass is tance i n preparing t h i s manuscript. My thanks a l s o , to Dr. Jim Cameron with whom I co l laborated i n the experiments invo lv ing d i r e c t measurement of water flow over the g i l l s . Dr. David Jones, Christopher Wood, Dr. J . R. B r e t t , Dr. R. L . Saunders, Miss S. Bourque and Miss M. Davie s prompted many f r u i t f u l discuss ions of t h i s work. The co-operation of Dr. Murray Newman and the s ta f f of the Vancouver Pub l i c Aquarium i s great ly appreciated. I am g r a t e f u l to the Nat ional Research Counci l of Canada and to the F i s h e r i e s Research Board of Canada f o r scholarship support. The B. C. Heart Foundation and the Nat ional Research Counci l of Canada provided f i n a n c i a l support for t h i s research through grants to Dr. Randal l . 1 INTRODUCTION F i s h are immersed i n a d © n s e , oxygen scarce environment i n comparison to a i r . A i r at 20 C contains approximately twenty-seven times the oxygen of an equivalent volume of water at that temperature (Fry , 1957) . In a d d i t i o n , the density of water i s about 600 times that of a i r at the same temperature. These phys ica l propert ies of the aquatic environment have no;-doubt d irec ted the evolut ion of f i s h re sp ira tory s tructures along cer ta in l i n e s . The g i l l i s the primary gas exchangerorgan i n most f i shes and i s so s tructured that a nearly continuous flow of water passes over i t i n a s ing le d i r e c t i o n . Thus most f i s h exhib i t a u n i d i r e c t i o n a l flow of water over the g i l l s which i s l i k e l y a r e f l e c t i o n of the slow oxygen d i f f u s i o n rate and low oxygen content of the environment. Mammals however, i n the oxygen-rich t e r r e s t r i a l environment, have adopted t i d a l v e n t i l a t i o n where the re sp ira tory gases are i n s p i r e d and then expired. I t i s l i k e l y that t i d a l v e n t i l a t i o n provides protect ion from dess lcat ion i n the t e r r e s t l a l environment as a large humidity gradient ex is ts w i th in the lung (Guyton, 1962) . Despite the l i m i t a t i o n s of t h e i r environment many f i s h exhib i t considerable a t h l e t i c a b i l i t y . Sockeye salmon are capable of Increasing t h e i r metabolic rate about ten times i n response to severe a c t i v i t y (Bre t t , 1964) and species §nch as tuna and mackerel can a t t a i n high swimming speeds. The a b i l i t y of f i s h to susta in high l eve l s of a c t i v i t y i n s as r e l a t i v e l y oxygen scarce environment i s due, i n part to the 2 s tructure of the g i l l s . The g i l l s have a large surface area and a counter-current arrangement of water and blood flow ( T r o i s , 1883 ; van Dam, 1938) to maintain an e f f ec t i ve gas tension gradient between water and blood. In most species water i s prope l led over the g i l l s by the ac t ions of the b r a n c h i a l musculature but i n some f i s h such as the tuna, mackerel and remora g i l l water flow i s provided by forward motion of the body through the water ( H a l l , 1930; Muir and Buckley, 1 9 6 7 ; Muir and Kendal l 1968g Brown and Muir , 1 9 6 9 ) . G i l l blood flow i s produced by the pumping a c t i o n of the heart . The s tructure of the g i l l s has been described i n d e t a i l (van Dam, 1938 ; B i j t e l , 1949 ; Rhodin, 1964 ; Hughes and Grimstone, 1965 ; Newstead, 1967 ; Hughes and Data Munshi, 1 9 6 8 ) . They are s i e v e - l i k e s tructures contained i n the branch ia l chambers d i r e c t l y i n the path of the water flow through these chambers. B a s i c a l l y they cons i s t of supportive g i l l arches conta ining the af ferent and efferent blood supply, g i l l f i laments and lamel lae . The wal ls of the lamel lae are only 2^m-thick (Hughes and Grimstone, 1965) and are the primary s i t e of gas exchange i n the g i l l . There i s evidence that c i r c u l a t o r y patterns w i th in the g i l l can a l t e r (Steen and Kryusse, 1964 ; Richards and Fromm, 1969) and a f f e c t the d i s t r i b u t i o n of blood to the lamel lae . In a d d i t i o n , the flow of water over the g i l l s and the p o s i t i o n i n g of the g i l l s ieve wi th in the b r a n c h i a l cav i ty can change according to the depth of breathing (Saunders, 1961) and the a c t i o n of adductor and abductor muscles attached to the f i laments (Pasztor and Kleerekoper, 1 9 6 2 ) . 3>, Gas exchange at the g i l l s w i l l be influenced&by the flow pattern and flow rate of f l u i d s wi th in and without the g i l l , by i t s surface area , the pressure gradients ex i s t ing across i t s wal ls and the b a r r i e r s to exchange across the walls (Randal l , 1970). The r e l a t i v e importance of each of these fac tors to the gas exchange process i s not w e l l known i n f i s h e s . This thes i s describes an attempt to l earn more about the importance of the various factors that inf luence gas exchange. In mammals the r a t i o of the flows of a i r and blood reachingethe gas exchanger (vent i l a t i on -per fus ion r a t i o ) i s approximately 1 and does not vary appreciably except under patho log ica l condit ions (Guyton, 1962). In f i s h v e n t i l a t i o n -perTuslon r a t i o s are extremely v a r i a b l e and have been reported to range between 10:1 and 80:1 (Randal l , 1970). Furthermore, un l ike mammals, v e n t i l a t l o n - p e r f u s i o n r a t i o can vary i n an i n d i v i d u a l f i s h and increases i n rainbow trout during hypoxia (Holeton and Randal l , 1967 b ) . This range of v e n t i l a t i o n - p e r f u s i o n r a t i o s i n an i n d i v i d u a l trout may have Important consequences f o r gas exchange at the g i l l s . Thus t h i s thes is w i l l examine the questions: What are the patterns and rates of water and blood flow across the g i l l s and how do these fac tors inf luence gas exchange? 4 GENERAL METHODS EXPERIMENTAL ANIMALS A l l the f i s h studied were rainbow t rout , Salmo g a l r d n e r i , weighing between 150 and 600 g that were purchased from the Sun Val ley Trout Farm i n Port Coquitlam, B . C . The f i s h were transported to the Vancouver Publ i c Aquarium at Vancouver, B . C . i n oxygenated 100 ga l tanks and were trans ferred to 250 ga l holding tanks suppl ied with running fresh water. The f i s h were fed 2 or 3 times weekly on a mixed d i e t of trout p e l l e t s ( J . R. Clark C o . , Sa l t Lake C i t y , Utah) and chopped horse heart . The f i s h were not fed for the l a s t 2 days p r i o r to use. Temperature i n the tes t apparatus and holding tanks var i ed seasonally over a range of 8 -16 C and was f a i r l y stable over any one experimental per iod . Any diseased f i s h were removed from the holding f a c i l i t y and destroyed. In the spring of 1969 & n / outbreak of disease suspected to be furunoulos ls (Davis, 1967) k i l l e d several trout and experiments were suspended for 2 weeks. During that time the f i s h were fed d a i l y a mixture of C l a r k ' s trout p e l l e t s suspended i n a ge lat in /water base to which chloramphenicol a n t i b i o t i c 1 g/kg food) was added. This treatment e l iminated a l l fur ther signs of the disease. OPERATING TABLE AND OPERATING PROCEDURE Experiments i n t h i s study often involved prolonged surgery on l i v e f i s h . To accomplish such surgery an operating table s i m i l a r to that described by Smith and B e l l (1964) was 5 used. The table was equipped with a pump and nozzles iro that 10 1 of aerated water containing 1;10,000 MS-222 anesthet ic ( t r i c a i n e methanesulfonate) could be d irec ted over the g i l l s . The nozzles were so designed that the g i l l s could be perfused e i ther from the mouth or thec&opercular openings. The usual operating procedure was i n i t i a t e d by p lac ing trout i n a bucket containing a so lu t ion of approximately 1;10,000 MS-222 anesthet ic i n water. A f t e r the f i s h had l o s t i t s equ i l ibr ium and i t s v e n t i l a t o r y movements had slowed (usual ly wi th in 2 minutes) i t was placed on the operating table and the nozzles were adjusted to provide a good water flow over the g i l l s . During the operation the f i s h were kept moist. A covering of wet paper towels was often used for t h i s purpose. At the end of each operation the g i l l s were usual ly perfused with water containing no anesthet ic to f a c i l i t a t e recovery. Most f i s h showed signs of a c t i v i t y and had s tarted regular breathing movements wi th in 1 to 5 minutes of the removal of anesthet ia . They were then placed In the appropriate experimental apparatus and allowed at l eas t 12 and often 24 hours recovery. Such recovery i s necessary as these operat ional procedures lead to considerable trauma (Houston, et a l , 1 9 6 9 ) . During the recovery and subsequent experimental period the apparatus was covered with black p l a s t i c to prevent the f i s h seeing the inves t iga tors . CANNULATI0N PROCEDURES Cannulae of various types were frequently employed i n these experiments. They allowed pressure measurements and 6 provided samples of blood and water afferent and efferent to the g i l l s . A l i s t of the cannulations routinely performed follows. Cannulation of the Buccal Cavity Lengths of polyethylene tubing, heat f l a r e d on one end, were implanted i n the buccal cavity as described by Saunders (1961). A hole was punched through the roof of the mouth using a number 13 gauge x 2-inch Lewisohn transfusion needle. 1 This hole was positioned on the midline about 4 mm posterior to the buccal valve. Care was taken to keep the hole on the midline and avoid the neural tissue serving the n o s t r i l region on each side of the f i s h . I f a dorsal a o r t i c cannula was implanted i n the same f i s h each cannula was positioned s l i g h t l y to one side of the midline but was s t i l l clear of the n o s t r i l innervation (Pig. 1). I f the buccal cannula was small (PE 60 tubing, I.D. = O.76 mm, O.D. = 1.28 mm) a 2.5 cm length of PE 200 tubing (I.D. = 1.4 mm, O.D. =1.9 mm) heat f l a r e d on one end was passed through the hole i n the snout from Inside to serve as an anchoring sleeve f o r the cannula. A t i g h t l i g a t u r e round t h i s sleeve at i t s point of emergence from the head constricted the sleeve on the cannula and held i t firm (Pig. 1). Alternately, the buccal cavity was cannulated with large heat f l a r e d polyethylene cannulae (Clay Adams PE 190, I.D. = 1.19 mm, O.D. =1.7 mm) without the use of an anchoring sleeve. Cannulae of t h i s size f i t t e d snugly through the hole i n the snout 1 Becton, Dickinson and Co., Rutherford, New Jersey. Figure 1 Deta i l s of the cannulae Implanted i n rainbow trout . The dorsa l a o r t i c and v e n t r a l a o r t i c cannulae provide samples of a r t e r i a l blood and venous blood respec t ive ly and allow recording of blood pressure. The buccal and c l e i t h r a l cannulae permit recording of pressures i n branch ia l chamber and sampling of i n s p i r e d and expired water. cleithral cannula cannula 7 and a l i g a t u r e wrapped around the cannula at i t s point of emergence from the head held I t secure. Buccal cannulae provided i n s p i r e d water samples and allowed measurement of buccal pressure. The small PE 60 cannulae were used on free-swimming trout where a minimum of drag was des irable while the large PE 190 cannulae were used f o r de l i ca t e pressure measurements as small diameter tubing reduces the frequency response of the recording system (Yanof, 1 9 6 5 ) . Opercular Cavity Cannulatlon Two types of cannulae were implanted i n the opercular c a v i t y . One type consisted of lengths of Clay Adams PE 60 tubing which passed through the operculum. These cannulae w i l l be r e f e r r e d to as "opercular cannulae". To Implant such a cannula the operculum was punctured with the t i p of a number 15 gauge hypodermic needle and a 3 mm length of PE 200 tubing, heat f l a r e d on one end was passed through the operculum from i n s i d e . The cannula was pushed into the sleeve from outside and held i n place by a t i gh t l i g a t u r e that cons tr i c ted the sleeve upon the cannula (P ig . 2 ) . Opercular cannulae were placed so that they penetrated the operculum about 5 from i t s pos t er ior margin. The other technique for cannulat lon of the opercular cavi ty consisted of passing a tube through the c l e i t h e r a l bone of a trout (Saunders, 1 9 6 1 ) . Such cannulae w i l l be termed " c l e i t h e r a l cannulae". A hole was bored through the c le i thrum with a number 13 gauge Lewisohn transfus ion needle and the cannula passed through the hole from Inside the opercular c a v i t y . Figure 2 Cannulae used f o r sampling expired water l n t r o u t . TJie three opercular cannula placements tested are I l l u s t r a t e d and the method used to at tach the cannulae to the operculum i s shown. 8 C l e i t h r a l cannulae consisted of 20 cm lengths of PE 190 tubing heat f l a r e d on one end. Care had to be taken during the cannulatlon procedure to avoid damaging the g i l l f i laments . The cannula was s t i t ched to the f i s h with four number 3 -0 s i l k sutures to anchor i t i n place ( F i g . 1 ) . Only one cannula was appl i ed to each f i s h and was pos i t ioned mid-way down the dorso-v e n t r a l ax i s of the c le i thrum and about 1 cm above the pec tora l f i n . C l e i t h r a l cannulae often become clogged with g i l l f i laments which slowed or hal ted the flow of water from the cannula or damped out the pressure trace . Cutt ing an "X" i n the heat f l a r e d port ion of the cannula l a r g e l y e l iminated t h i s problem (Holeton, 1 9 7 0 ) . Both opercular and c l e i t h r a l cannulae allowed sampling of expired water and measurement of pressure i n the opercular c a v i t y . Cannulatlon of the Dorsal Aorta The dorsa l aorta was cannulated i n a manner s i m i l a r to that described by Smith and B e l l ( 1 9 6 4 ) . The cannula consisted of a 70 cm length of PE 60 tubing t ipped with a 2 cm long number 21 Huber point needle (Becton, Dickinson and Co.) and f i l l e d with heparinized (10 I . U . / m l ) Coi t land sa l ine (Wolf, 1 9 6 3 ) . The cannula was secured by a PE 200 sleeve i d e n t i c a l to that used with the buccal cannula. A 1 cm length of c leaning wire from an 18 gauge disposable s p i n a l needle made a convenient plug for the free end of the cannula. Th i s cannulat lon allowed sampling of dorsa l a o r t i c blood f o r determination of a r t e r i a l P o 2 » p c © 2 a n d hematocrit . I t a l so allowed monitoring of dorsa l a o r t i c blood 9 p r e s s u r e . C a n n u l a t i o n of the V e n t r a l A o r t a The v e n t r a l a o r t i o cannula c o n s i s t e d of a 70 cm l e n g t h of P E 50 t u b i n g t i p p e d w i t h a number 22 s h o r t b e v e l p o i n t needle (Becton, D i c k i n s o n and Co.) 2 . 5 cm l o n g and bent a t an angle o f approximately 60 degrees 1-2 cm back from i t s p o i n t ( F i g . 1 ) . T h i s cannula was p o s i t i o n e d i n the v e n t r a l a o r t a by p u n c t u r i n g the isthmus a l o n g the v e n t r a l m i d l i n e as d e s c r i b e d by Holeton ( I 9 6 6 ) and Holet o n and R a n d a l l (196? a ) . The cannula was f i l l e d w i t h h e p a r i n i z e d C o r t l a n d s a l i n e and plugged w i t h a 1 cm l o n g p i e c e of d i s s e c t i n g p i n . I t was h e l d i n p l a c e w i t h number 3 -0 s i l k s u tures f i x e d to the s k i n . I m p l a n t a t i o n of the v e n t r a l a o r t i c cannula was f a r l e s s s u c c e s s f u l than the d o r s a l a o r t i c cannula. The v e n t r a l a o r t a was f r e q u e n t l y r u p t u r e d on the o p e r a t i n g t a b l e and the f i s h had to be abandoned. Oft e n c a n n u l a t i o n o f the v e n t r a l a o r t a appeared s u c c e s s f u l on the o p e r a t i n g t a b l e but a f t e r the f i s h was p l a c e d i n the apparatus and all o w e d to r e c o v e r the cannula was no. l o n g e r o p e r a t i v e . The technique was most s u c c e s s f u l w i t h l a r g e t r o u t and was very poor f o r f i s h l e s s than 200 g. The l a c k o f success w i t h s m a l l f i s h was probably due to the s m a l l s i z e o f the v e s s e l and the l a c k o f f i r m support f o r the needle o f f e r e d by the s m a l l isthmus. I f v e n t r a l a o r t i c cannulae a r e to be used w i t h Rainbow t r o u t i t i s recommended t h a t l a r g e f i s h (over 300 g) be chosen f o r c a n n u l a t i o n . The v e n t r a l a o r t i c cannula a l l o w e d sampling of bloo d a f f e r e n t t o the g i l l s f o r FQ2» and P C Q 2 10 measurement and permitted recording of ventral a o r t i c blood pressures. Subintestlnal Vein Cannulatlon The subintestlnal vein was cannulated as described by Stevens and Randall(1966) using a 40 cm length of PE 50 tubing f i l l e d with heparlnized saline which had been stretched at one end to produce a f i n e tapered point. To implant the cannula a 3 cm long i n c i s i o n was made along the ventral midline just posterior to the pel v i c g i r d l e . Once the skin was cut the opening was enlarged by blunt d i s e c t i o n to prevent excessive bleeding. Two number 3-0 s i l k sutures were placed under the vein and i t was t i e d o ff with a t h i r d l i g a t u r e posterior to the s i t e of cannulation. The anteriormost portion of the exposed vein was then clamped with a small serrafine clamp. A s l i t was cut i n the center of the vessel and the cannula was inserted and directed about 1 cm up the vessel towards the heart. The cannula was secured i n the vessel with two sutures and fastened to the posterior end of the i n c i s i o n and the skin of the f i s h with a d d i t i o n a l sutures. The i n c i s i o n was closed with closely spaced number 3-0 s i l k sutures. The subintestlnal cannula permitted intravenous i n j e c t i o n of substances such as adrenaline. DATA COLLECTION AND RECORDING Gas tensions l n water and blood afferent and efferent to the g i l l s were measured using a Radiometer electrode system. P 0 determinations were made with Radiometer type E5046 oxygen 11 electrodes while P C Q 2 measurements were attempted with a Radiometer type E5036 carbon dioxide e lectrode. Both types of electrodes were contained i n Radiometer type D6l6 thermostatted c e l l s (blood cuvettes) maintained at the same temperature as the experimental animal . The electrodes were connected to a Radiometer pH meter type 27b with a type PHA 927b gas monitor un i t or to a Radiometer EHM 71 Acid-Base Analyser . Both these instruments gave d i r e o t readout of P Q 2 ° R P C @ 2 from c a l i b r a t e d scales . C a l i b r a t i o n was c a r r i e d out by exposing the electrodes to moist gas samplers of known tension. Electrode c a l i b r a t i o n was frequently checked during any one experiment and the electrode membranes were replaced i f they appeared to produce d r i f t i n meter readings. Blood or water could be introduced to the cuvettes by i n j e c t i o n from a bubble-free syringe or by connecting the appropriate cannula d i r e c t l y to the thermostatted c e l l by means of a metal of p l a s t i c three-way valve . P lac ing the thermostatted c e l l s l i g h t l y below the water l e v e l i n the apparatus f a c i l i t a t e d the blood or water flow into the cuvette. P r i o r to in troduct ion of a blood sample, the cuvette was f i l l e d with heparinlzed (10 I . U . / c c ) Cort land sa l ine (Wolf, 1963). The valve was then opened and the blood d isp laced the sa l ine i n the cuvette. Once a sample was i n the cuvette and was observed to be bubble-free the valve was closed so that no hydrostat ic pressure was exerted on the e lectrode . The sample was l e f t l n the cuvette u n t i l the meter reading s t a b i l i z e d . To insure consistency of measurement the P Q 2 or P C Q 2 r e a d i n g was only taken a f t e r the 12 sample had remained i n the cuvette for a set time per iod . Th i s time period was k minutes at temperatures of 8-13 C and 3 minutes at temperatures above 13 C. Pressures from the appropriate cannulae were recorded using pressure transducers and Beckman R.S . Dynograph recorders . Statham P?3 BB transducers were used to measure a r t e r i a l and venous blood pressures and pressures i n the buccal and opercular c a v i t i e s . In one experiment a Sanborn type 267 BC d i f f e r e n t i a l transducer was used to record the water pressure d i f f e r e n t i a l between the buccal and^opercular c a v i t i e s of t r o u t . Hematocrits were determined by taking blood samples i n t r i p l i c a t e from the dorsa l aorta cannula a f t er some blood had previous ly been drawn out into a syringe to ensure that the sample was free of sa l ine contamination. The blood was placed i n Clay Adams microhematocrit tubes (75 mm x 0.55 mm I . D . or 75 mm x 1.1 - 1.2 mm I . D . ) . The tubes were spun for 3 minutes a t approximately 10,000 x g i n a Clay Adams microhematocrit centr i fuge . Hematocrits were determined on a Clay Adams microhematocrit tube reader and expressed i n packed c e l l as a percentage of t o t a l sample volume. At the end of each experiment the f i s h were k i l l e d and b l o t t e d dry , the cannulae were removed and the f i s h were immediately weighed. The t o t a l length was taken as the distance between the t i p of the snout to the t i p of the t a i l . Sex was determined by cut t ing open the f i s h and examining the gonads. 13 SYMBOLS AND ABBREVIATIONS USED IN THIS STUDY V G -volume of water passing over the g i l l s / u n i t time ( v e n t i l a t i o n volume) VR - v e n t i l a t i o n rate i n mouth or opercular c l o s u r e s / minute Vsv -volume of water pumped over the g i l l s / b r e a t h i n g cycle (vent i la tory stroke volume) % U -percentage of oxygen u t i l i z e d from Inspired water V o 2 -oxygen uptake/unit time P - p a r t i a l pressure of gas i n mm Hg - s o l u b i l i t y c o e f f i c i e n t f or gas i n water or blood Q - c a r d i a c output /uni t time S.D. -s tandard dev ia t ion S . E . -s tandard error Hct -packed c e l l volume of blood i n % (hematocrit) B - r e f e r r i n g to blood W - r e f e r r i n g to water Subscripts a - a r t e r i a l v -venous I - i n s p i r e d E -expired 14 PART ONE AN EXAMINATION OP OPERCULAR AND CLEITHRAL CATHETERIZATION AS TECHNIQUES FOR SAMPLING WATER EXPIRED BY TROUT INTRODUCTION I As one of the prime object ives bf th i s study was to evaluate the e f fect of water flow over the g i l l on gas exchange^ i t was v i t a l that the volume of water f lowing over the g i l l / u n i t time be accurate ly determined. Pre l iminary studies indicated that efferent water oxygen tensions recorded from s ingle cannulae placed mid-way down the pos ter ior margin of the operculum were qui te v a r i a b l e . A common prac t i ce has been to ca lcu la te values such as u t i l i z a t i o n and v e n t i l a t i o n volume using expired oxygen tensions measured from c l e i t h r a l or opercular cannulae (Saunders, 1 9 6 1 , 1962 ; Holeton and Randa l l , I 9 6 7 a ,b ; Stevens and Randa l l , 1967 a , b ) . I f mean expired oxygen tension was i n c o r r e c t l y estimated by these cannulatlon techniques such ca lcu la ted values would be i n e r r o r . Indeed, considerable data obtained by such techniques has been used l n t h e o r e t i c a l ana lys i s of the gas exchange process i n f i s h (Jones, et a l , 1970 ; T a y l o r , et a l , 1 9 6 9 ) . Thus i t seemed v i t a l to test whether or not such cannulae provide estimates of mean expired oxygen tension and to see i f cannula p o s i t i o n inf luenced the r e s u l t s . 15 METHODS I Hainbow trout were obtained and held during the spring and summer of 19^9 as previously described. The f i s h were placed on the operating table and 70 cm long PE 60 opercular cannulae were implanted. In addition, a PE 60 buccal cannula, also 70 cm long, was positioned as previously described. Three opercular cannula placements were tested and were termed "high", "mid" and "low" because of t h e i r p o sition r e l a t i v e to the dorso-ventral axis of the operculum (Fig. 2). The high opercular cannula was positioned immediately adjacent to the anterior end of the l a t e r a l l i n e , while the mid opercular cannula was located halfway down the dorso-ventral axis of the operculum. The low opercular cannula was inserted through a hole i n the 4th branchiostegal ray (counting from the most dorsal ray l n a ventral d i r e c t i o n ) . A l l the cannulae were placed so that they penetrated the operculum about 5 mm from i t s posterior margin. Occasionally a l l three cannulae were attached to the ri g h t operculum of a single f i s h although usually only 1 or 2 cannulae were employed. In some cases cannulae were placed on both sides of the animal's head. In order to study opercular PQ 2 l e v e l s i n free-swimming f i s h , cannulated animals were placed i n covered, darkened aquaria and were allowed at least 24 hours recovery from the operation. Experiments consisted of continuously monitoring inspir e d and expired oxygen tensions i n water from the various cannulae f o r approximately 1 hour while the f i s h remained undisturbed i n the darkened aquarium. The animal was then chased with a st i c k f o r a short period and P 0 o l e v e l s were recorded f o r 16 2 or 3 hours fo l lowing a c t i v i t y . Seven f i s h ranging i n weight from 382.5 to 426 g were studied i n t h i s manner. Temperature was constant during any one experiment ranged from 9.6 to 12C A over the ser ies of experiments. Oxygen tensions were measured by gently l i f t i n g the cannula t i p s over the side of the tank and a l lowing a flow of water to d r i p from t h e i r t i p s . The cannulae d i d not appear to be siphoning of f water as the flow was Intermittent and In time with the pressure pulses which occurred i n the branch ia l chamber of the f i s h as i t breathed. The cannula t i p s were p e r i o d i c a l l y connected to the Radiometer oxygen electrode system maintained at the same temperature as the tes t aquarium. Seven a d d i t i o n a l f i s h were used to evaluate the c l e i t h r a l cannulatlon technique as a method for measuring mean expired oxygen tensions. These f i s h were f i t t e d with a rubber membrane attached to the margin of the mouth which separated i n s p i r e d and expired water and allowed c o l l e c t i o n of the l a t t e r f o r d i r e c t determination of v e n t i l a t i o n volume. This technique i s described i n d e t a i l i n part II and i s i l l u s t r a t e d i n F i g . 4 ( p . 3 o ) . Expired water was c o l l e c t e d i n the rectangular body-holding box shown i n F i g . 4 so that a sample of mixed expired water was a v a i l a b l e f o r comparison with water c o l l e c t e d from c l e i t h r a l cannulae. The f i s h were f i t t e d with rubber membranes (ora l membranes) and PE 190 buccal and c l e i t h r a l cannulae as described i n the general methods. They were placed i n the apparatus as shown i n F i g . 4 f or overnight recovery fo l lowing 17 the operat ion. Then the f i s h were l e f t undisturbed over an 8-hour period while V Q ( v e n t i l a t i o n volume i n ml/min) , P i o 2 ( insp ired oxygen tension i n mm Hg), P g o 2 ( e x P i r e d - oxygen tension i n mm Hg), c l e i t h r a l P Q 2 and oxygen uptake r a t e , V Q 2 » were recorded per iod ica l ly . ; ; The method of c a l c u l a t i o n of V Q 2 was; ^0 2 = V P I 0 2 " W * W ° 2 where: V Q 2 = oxygen uptake i n ml /min / f i sh VQ = the v e n t i l a t i o n volume l n ml/min P J Q 2 = i n s p i r e d oxygen tension i n mm Hg PEC-2 = e x P i r e d oxygen tension i n mm Hg etw^Og = the oxygen s o l u b i l i t y c o e f f i c i e n t i n water £ at the t e s t temperature (ml O^/ml/mm Hg) C l e i t h r a l P Q 2 was measured by connecting the c l e i t h r a l cannula to the Radiometer oxygen electrode system. p i o 2 a n d m l x e d PgQ^ were obtained by c o l l e c t i n g samples of water l n a bubble-free syringe f i t t e d with a curved number 18 gauge x 3 - inch sp ina l needle as described i n f>art I I . Dye studies showed that the expired water was uniformly mixed by the v e n t i l a t o r y flow i n the rectangular body-holding box. In a d d i t i o n , oxygen tensions i n samples taken at a var i e ty of pos i t ions wi th in the chamber d i f f e r e d by no more than 2 mm Hg P Q 2 regardless of p o s i t i o n . Thus i t was f e l t that water co l l ec t ed from wi th in the chamber by means of a syringe and needle gave a representat ive sample of mixed expired water.;.. 18 RESULTS I Oxygen tensions i n opercular water samples from quiescent trout i n aquaria were extremely v a r i a b l e . Tensions recorded from any one cannula placement i n a s ingle f i s h v a r i e d with time and often changed abruptly ( F i g . 3 A ) . There were large d i f ferences i n tension between the three placements ( F i g . 3 B, C) and no one placement gave cons i s tent ly high or low P o 2 l s » Long term observations of the same i n d i v i d u a l ( c . f . F i g s . 3 A and 3 B) indicated that the degree of v a r i a b i l i t y at any one sampling s i t e d i f f e r e d from day to day. Often separate cannulae placed i n the same p o s i t i o n on e i ther side of the f i s h gave r e s u l t s that d i f f e r e d as much as 100 mm Egg* Chasing the f i s h usual ly resu l ted i n a r i s e i n opercular PQ 2 regardless of cannula p o s i t i o n . Some placements showed a greater PQ 2 r i s e fo l lowing a c t i v i t y than others . For example i n F i g . 3 B the oxygen tension i n water from the mid opercular placement rose to a l e v e l approaching that of i n s p i r e d water while tension from the higher opercular placement rose only s l i g h t l y . Thus although there was always an apparent decrease i n u t i l i z a t i o n of oxygen from the i n s p i r e d water fo l lowing a c t i v i t y , the magnitude of t h i s decrease and i t s durat ion were not uniform from placement to placement. A l l seven f i s h studied showed s i m i l a r v a r i a b i l i t y i n expired oxygen tensions with PEC>2 l e v e l s ( p a r t i a l pressure of oxygen i n expired water) varying from 5^ to 157 mm Hg. V e n t i l a t o r y data and oxygen tensions from the seven f i s h used to assess the r e l i a b i l i t y of the c l e i t h r a l cannula are Figure 3 Oxygen tensions recorded from free-swimming f i s h i n covered, darkened aquar ia . The three opercular cannulae are termed h igh , mid or low as i n F i g . 2 . Inspired oxygen tensions remained J between 155 and l 6 0 mm Hg. A and B represent two experiments c a r r i e d out on a s ingle 332.5 S t rout with a 48-hour i n t e r v a l between experiments; C i s f o r a 3^2 g f i s h . TIME ( h o u r s 19 given i n Table 1. Some f i s h were exc i table and had VQ'S that were p e r i o d i c a l l y high (numbers 3 , 5 and 7) while others were more quiescent. Inspired oxygen tension for the group remained at 150* 0.6 mm Hg PQ 2 and the mean VR was 81* 5 . ^ breaths/min. The mean expired oxygen tension l n water samples co l l ec t ed from ± the rectangular body-holding box was 79 .5 5.3 mm and ranged from 5 7 . 6 - 9 0 . 0 f or the group. C l e i t h r a l water P n averaged u 2 114.5 12.1 mm. This value was s i g n i f i c a n t l y d i f f e r e n t ("t-test", 0.05 l eve l ) from the mean mixed expired oxygen tension for the group. In two instances however, ( f i s h 7 and 8) mean c l e i t h r a l PQ 2 was s i m i l a r to the mean mixed expired water tens ion. Post-mortem examination of the cannulae on these two f i s h d id not reveal any p e c u l i a r i t y of cannula placement that would account for a d i f ference between these f i s h and the others with c l e i t h r a l cannulae. Table 3t shows a simple c o r r e l a t i o n ana lys i s c a r r i e d out on the means from Table 1. Thi s ana lys i s showed a s i g n i f i c a n t negative c o r r e l a t i o n between c l e i t h r a l PQ 2 and v e n t i l a t i o n ra te . Thus those f i s h with high VR's tended to have lower c l e i t h r a l PgQ^'s which were c loser to the mixed c l e i t h r a l PQ^ ( f i s h 7 and 9 , Table 1 ) . DISCUSSION I Results ind ica te that h ighly v a r i a b l e oxygen tensions were obtained from cannulae placed at d i f f e r e n t pos i t ions on the operculae of rtainbow trout swimming i n aquar ia . No cannula placement appeared subject to l e ss v a r i a t i o n than any other. The Table 1 A comparison of c l l e t h r a l oxygen tension (PgQg " o l * i ; ' f c ) and mixed expired oxygen tension (P EQ 2 ) f or seven f i s h with o r a l membranes a f f i x e d . Data are f o r 202 .5 * 1 0 . 8 g f i s h at 1 0 . 7 * Fish no. P l 0 2 P E 0 2 P E 0 2 " c l e i t V 0 ml.min VR #/min Vsv ml/breath 2 X 77 .9 1 5 0 . 9 88 .6 1 3 3 . 3 0 .19 MS* S.D. 4 5 . 5 1.9 4 .6 6 . 5 0 . 0 2 S.E. 1 6 . 1 0 . 7 1.7 0 . 0 2 0 . 0 1 n 8 7 7 7 7 3 x' 1 0 6 . 3 1 5 1 . 5 9 0 . 7 149 .7 0.24 7 3 . 5 1 . 1 S.D. 5 8 . 5 0 . 8 1 1 . 2 1 .9 0.04 3 . 7 0 . 1 S.E. 23 .9 0 . 4 4 . 6 0 . 8 0 . 0 2 1.5 0.04 n 6 6 6 6 5 6 6 4 X 42 .0 1 5 2 . 0 7 4 . 3 1 5 0 . 8 0 .16 6 9 . 0 0 .6 S.D. 6 . 1 1.2 6 . 4 1.7 0 .03 2 .6 0 . 1 S. E. 3 . 0 0 .6 3 . 2 0 .9 0 . 0 2 1.3 0 .05 n 4 4 4 4 4 4 4 5 X 144.3 148.3 9 0 . 0 1 1 3 . 5 0 .47 7 4 . 0 2 . 0 S.D. 90 .7 2 . 2 1 0 . 9 14 .5 0 .4 8 . 0 1.0 S.E. 3 2 . 1 1 . 1 5 . 5 7 .3 0 . 2 3 .3 0 . 4 n 8 4 4 4 4 6 6 6 X 8 6 . 0 147 .4 64 .8 1 0 2 . 8 0 .26 7 5 . 8 1.23 S.D. 5 1 . 0 3 . 3 5 . 2 2 9 . 3 0 .06 4 . 3 0 . 7 S.E. 1 9 . 3 1.5 2 .3 14 .7 0 .03 1.8 0 . 3 n 7 5 5 4 4 6 5 7 X 135.4 1 5 0 . 0 9 0 . 8 82 .3 0 . 3 5 9 0 . 0 1.7 S.D. 46 . 1 0 . 4 5 . 2 3 . 0 0 . 1 7 .0 0 . 4 S.E. 1 7 . 4 0 . 2 2 . 6 1 .5 0 . 0 5 3 . 2 0.14 n 7 5 4 4 4 5 7 8 X 5 5 . 1 1 5 0 . 0 5 7 . 6 6 9 . 4 0 .22 1 0 3 . 5 0 .53 S.D. 9 . 5 2 . 0 2 . 5 1 5 . 1 0.04 8 . 2 0 .06 S.E. 3 . 9 1.2 1 . 1 6 . 8 0 . 0 2 3 . 3 0 .03 n 6 3 5 5 5 6 6 X S.D. S.E. n 9 2 . 4 3 8 . 6 14 .6 7 1 5 0 . 0 1.7 0 . 6 7 7 9 . 5 H 4 . 5 14 .0 3 1 . 9 5 .3 1 2 . 1 7 7 0 .27 0 . 1 1 0.04 7 80.9 1 3 . 1 5 . 4 6 1.19 0 .57 0 .23 6 Table II Simple c o r r e l a t i o n ana lys i s on the data f o r the group of f i s h with o r a l membranes attached f o r comparison of mixed and c l e i t h r a l Pc^* Values given are c o r r e l a t i o n coe f f i c i en t s and the s ign i f i cance l e v e l s were obtained from a table of s i g n i f i c a n t corre la t ions i n Stee l and T o r r i e ( i 9 6 0 ) . P l 0 2 P E 0 2 P - c l VR Vsv Wt Leng Temp - * = s i g . at 0.05 l e v e l p i o . ns - ** = s i g . at O.Oi l e v e l Z p ns = not s i g . at 0.05 l e v e l E 0 2 .69* ns -P E 0 2 ' c l ns ns ns -\ . 89** ns ns ns -VR ns ns ns -G91** ns -Vsv ns ns -.81** ns ns ns -Wt ns . 71* ns ns ns ns ns Leng ns ns ns ns ns ns .74* .69* Temp ns -.80** ns -. 77* .68* ns ns ns ns 20 technique d i d ind ica te that expired P Q 2 rose when the f i s h was chased, but the magnitude of the P Q 2 increase was not constant from one cannula placement to another. Subsequent experiments which are described i n part IV of th i s thes is confirm these f ind ings . Furthermore, the experiments of part IV, i n which opercular # 0 2 ' s w e r e determined from the three placements i n f i s h whose g i l l s were a r t i f i c i a l l y perfused with water at d i f f e r e n t rates, show that t h i s v a r i a b i l i t y ex is ts over a wide range of g i l l water flows. In perfused f i s h PEO2 m e a s u r e { * from opercular cannulae was frequently i d e n t i c a l with B i o 2 ( t n e oxygen tension l n i n s p i r e d water). Of course u t i l i z a t i o n of oxygen was taking place but the opercular sampling technique f a i l e d to detect i t . The high v a r i a b i l i t y of opercular P Q 2 and lack of any consistency at any one sampling s i t e suggest that opercular cannulae may provide poor estimates of mean expired oxygen tension i n t rout . Although i t i s poss ib le to detect large changes i n u t i l i z a t i o n with t h i s technique the accuracy of measuring u t i l i z a t i o n i n th i s way i s quest ionable . There i s evidence that t h i s phenomenon i s not pecu l iar to Rainbow trout . Garey (1967) reported opercular P Q 2 v a r i a b i l i t y l n the carp, Cyprlnus oarplo , and i n i t i a l experiments i n our laboratory upon a 360 g s tarry f lounder , P la t i chys s t e l l a t u s , a l so ind ica te a considerable v a r i a b i l i t y i n th i s species expired oxygen tensions. C l e a r l y , any ca lcu la t ions based upon data c o l l e c t e d from opercular cannulae could be subject to considerable e r r o r . For example, Stevens and Randall ( I 9 6 7 ) reported that PEC-2 d i d n o t change from r e s t i n g l e v e l s during moderate swimming a c t i v i t y i n 21 rainbow trout. This r e s u l t was based upon data from a single mid opercular cannula and Is l i k e l y erroneous since PEO2 w o u - 1 - c i b e expected to increase when v e n t i l a t o r y flow goes up during a c t i v i t y (van Dam, 1 9 3 8 ) . In addition, the re s u l t s of parts I I , and IV of t h i s thesis show that u t i l i z a t i o n declines when ve n t i l a t o r y flow increases markedly and expired oxygen tension r i s e s as a re s u l t of t h i s decreased u t i l i z a t i o n . I t would appear that cannulae placed through the clelthrum may also y e i l d variable oxygen tensions. Table 1 i l l u s t r a t e s a marked difference i n the mean c l e i t h r a l P Q 2 and the mixed expired P Q 2 f o r the group. The two means are s i g n i f i c a n t l y d i f f e r e n t ("T-test", p a* 0 . 0 5 ) and the c l e i t h r a l P Q ^ was usually considerably higher than the mixed expired tension. Thus i f one used the mean c l e i t h r a l P 0 2 from Table 1 to calculate V G from the Pick p r i n c i p l e equation: p - p I 0 2 r E 0 2 the r e s u l t s obtained from that c a l c u l a t i o n would be misleadingly high. I t would appear that t h i s may have happened to some investigators. Saunders ( 1 9 6 2 ) , as pointed out by Holeton ( 1 9 6 6 ) , calculated a v e n t i l a t i o n volume of 1^-3 ml/second f o r a sucker, Catostomus commersonl, weighing 250 g. This c a l c u l a t i o n was based upon measures taken from a single c l e i t h r a l cannula. Even i f the v e n t i l a t i o n rate f o r t h i s f i s h was 150 mouth closures/minute i t would have to propel 5 7 . 2 ml/mouth closure over i t s g i l l s ; a considerable feat since the volume of the branchial cavity i n t h i s 22 species would be considerably l e ss than 57 ml . Indeed, K. Watters (personal communication), i n pre l iminary experiments has shown that c l e i t h r a l cannulae i n trout and salmon y i e l d P E O 2 ' s that are equal ly as v a r i a b l e as those from opercular cannulae. There are a number of poss ib le explanations f o r the observed v a r i a b i l i t y i n opercular ?E02 l e v e l s « F i r s t , the presence of cannulae on the operculae could prevent proper movement of these s tructures and thereby i n t e r f e r e with the normal pumping of water over the g i l l s . I f the operculae were held c losed by the cannulae then water would tend to remain stagnant i n the g i l l s and low expired oxygen tensions might r e s u l t . I f the cannulae held the operculae open, and prevented proper c losure of the opercular va lve , then residence time of the water i n the g i l l s would be reduced and water might tend to "leak" into the opercular cavi ty through the opercular opening. Under these l a t t e r condit ions the opercular PQ 2 l e v e l s would be h igh . I t would appear however, that the cause of the v a r i a b i l i t y i s not merely a simple case of mechanical interference of pumping caused by the cannulae. Expired oxygen tensions are ne i ther cons i s tent ly high or low i n f i s h with opercular cannulae nor are they cons i s tent ly high or low over the dorso-ventra l ax is of the operculum of a s ing le f i s h . In many instances the P E O 2 w a s kigfr and one placement on the operculum but low at another placement. Furthermore, Watters f i n d i n g s , and those of the present study, show v a r i a b i l i t y i n c l e i t h r a l PQ^ ex is t s i n salmonid f i s h when no opercular cannulae are present to hinder i t s movement. A more p l a u s i b l e explanation for the var i ed P E 0 ' s may 23 be r e l a t e d to the p a t t e r n of water and bloo d f l o w through the g i l l s i e v e . V a r i a t i o n s i n the p a t t e r n o§ v e l o c i t y of water f l o w a c r o s s the g i l l s would l i k e l y r e s u l t i n changes i n u t i l i z a t i o n . Indeed, water f l o w p a t t e r n might a l t e r r a p i d l y and produce changes i n u t i l i z a t i o n w i t h i n a s i n g l e b r e a t h i n g c y c l e . At some phases of the c y c l e t h e r e may be a p a t t e r n of laminar water flow a l o n g the i n n e r edge of the operculum and c l e i t h r u m . V a r i a t i o n s i n the depth of b r e a t h i n g o r changes l n p o s i t i o n i n g of the g i l l s i e v e may r e i n f o r c e o r break up t h i s l a m i n a r flow p a t t e r n . Thus the oxygen t e n s i o n s a l o n g the i n n e r edge of the operculum or c l e i t h r u m c o u l d be hig h o r low depending on the s t a b i l i t y of the boundary l a y e r and whether or not the water i n those r e g i o n s had co n t a c t e d the g i l l s . The f a c t t h a t c l e i t h r a l PQ 2 was s i m i l a r to mixed e x p i r e d oxygen t e n s i o n only when VR was h i g h supports t h i s argument. I f e l e v a t e d VR's and i n c r e a s e d depth of b r e a t h i n g d i s r u p t e d boundary l a y e r c o n d i t i o n s a t the i n n e r margin of the c l e i t h r u m then c l e i t h r a l P c ^ ' 3 t h a t were more r e p r e s e n t a t i v e of mean Pg02 w o u ^ d n a v e r e s u l t e d . Changeable water f l o w p a t t e r n s c o u l d r e s u l t from adjustments i n a c t i v i t y o f the v e n t i l a t o r y muscles o r a l t e r a t i o n s i n the p o s i t i o n of the g i l l s r e l a t i v e to the water flow. D i f f e r e n t muscles operate l n shallow v e n t i l a t i o n as compared to deep v e n t i l a t i o n ( B a l l i n t i j n and Hughes, 1965) and d e l i c a t e changes i n the a c t i v i t y of these v a r i o u s muscles c o u l d a l t e r the p a t t e r n of water over the g i l l s . In a d d i t i o n , P a s z t o r and Kl e e r e k o p e r (1962) have r e p o r t e d t h a t the adductor muscles between g i l l hemibranchs a c t to change the p o s i t i o n of the g i l l f i l a m e n t s d u r i n g 24 i d i f f e r e n t phases of the r e s p i r a t o r y c y c l e . They a l s o observed t h a t a t h i g h v e n t i l a t o r y flows these same muscles a c t to move ad j a c e n t g i l l f i l a m e n t t i p s a p a r t and a l l o w s p i l l a g e of water p a s t the g i l l s . They suggested t h a t t h i s s p i l l a g e i s advantageous as i t prevents damage to the g i l l l a m e l l a e a t h i g h water f l o w s . Such damage however, i s extremely u n l i k e l y as the d i f f e r e n t i a l p r e s s u r e a c r o s s the g i l l s i s only 1 to 2 cm H^O (Hughes and She l t o n , 1958) and would t h e r e f o r e not be s u f f i c i e n t t o damage the l a m e l l a e . I t i s more l i k e l y t h a t the g i l l adductor muscles r e g u l a t e the supply of water r e a c h i n g the g i l l l a m e l l a e and keep the g i l l apparatus p r o p e r l y o r i e n t e d t o the water flow as P a s z t o r and K l e e r e k o p e r have a l s o suggested. In a d d i t i o n , such adjustments c o u l d serve to a l t e r g i l l r e s i s t a n c e o r keep i t a t a constant l e v e l to reduce form drag when a f i s h swims. Such muscular adjustments c o u l d c e r t a i n l y a f f e c t u t i l i z a t i o n of oxygen from the i n s p i r e d water by changing the o r i e n t a t i o n of the g i l l s e i v e to the water f l o w i n g p a s t i t . V a r i a t i o n i n b l o o d d i s t r i b u t i o n t o any one r e g i o n of the g i l l s c o u l d r e s u l t i n changeable PEO2' S a * t n a t r e g i o n . Catecholamines are known to i n c r e a s e the bloo d flow through r e s p i r a t o r y p o r t i o n s of the e e l and t r o u t g i l l (Steen and Kryusse, 1964 ; R i c h a r d s and Fromm, 1 9 6 9 ) . C i r c u l a t i n g catecholamines (Nakano and Tomlinson, 196?)» or those r e l e a s e d l o c a l l y , c o u l d a c t on a d r e n e r g i c r e c e p t o r s which a r e thought to be l o c a t e d i n the g i l l s o f t r o u t ( R a n d a l l and Stevens, 196?) and cause i n c r e a s e s i n exchange a r e a . Adjustment of b l o o d f l o w to v a r i o u s p a r t s of the g i l l c o u l d p r o v i d e a means of v a r y i n g 25 oxygen ex trac t ion at any one s i t e and would r e s u l t i n v a r i a b l e PgQ^'s at that s i t e . I t i s i n t e r e s t i n g to note that Holeton and Skidmore (personal communication) observed dye accumulation i n only the f i r s t few g i l l lamellae located adjacent to the a f ferent branch ia l ar tery of an i s o l a t e d trout g i l l . They a lso showed that more lamellae became f i l l e d with dye i n the presence of adrena l in . Such experiments suggest that the ent i re exchange area of the g i l l may not always be supplied with blood and that c e r t a i n port ions of the g i l l t i s sue may be "Shut off" or "turned on" as required . The best way to get an accurate measure of t h e P m e a n expired oxygen tension i s to c o l l e c t a l l the water l eav ing the g i l l s and thereby obtain a mixed sample of ef ferent water. This can be accomplished using bags or tubes attached to the f i s h and works we l l with species such as p u f f e r - f i s h , Spheroides maculatus, ( H a l l , 1931) or the dragonet, Calllonymus l y r a (Hughes and Umezawa, 1968 b) due to the p a r t i c u l a r anatomy of these species . Such procedures are f a r more d i f f e r e n t l n t rout however, due to problems of interference with the proper funct ion of the v e n t i l a t o r y musculature. T h i n rubber or p l a s t i c membranes attached to the operculae of trout appear to load the re sp ira tory apparatus. In several experiments conducted i n our laboratory animals have died from apparant exhaustion and i n a b i l i t y to move s u f f i c i e n t water over the g i l l s when such bags were attached. Death occurred even though great care was taken to minimize interference with the v e n t i l a t o r y muscles and keep the res i s tance 26 of the c o l l e c t i n g bag as low as poss ib le . S i m i l a r l y , t h i n rubber membranes such as those used by van Dam (1938) may ser ious ly Interfere with v e n t i l a t i o n . These devices are stretched over the head to separate i n s p i r e d and expired water. Such structures may c o n s t r i c t ! t h e b r a c h i a l apparatus and prevent normal pumping. The so lu t ion to these problems consisted of the o r a l membrane technique described and discussed i n d e t a i l i n part I I . This device separates i n s p i r e d and expired water and allows c o l l e c t i o n of the l a t t e r . O r a l membranes appear preferable to devices that load the opercular apparatus and to ca lcu la ted V G ' s that are dependent on cannulation techniques. I t i s suggested that workers using such ca lcu la ted values should check t h e i r r e s u l t s by an independent technique. 27 SUMMARY OF PART I E x p e r i m e n t s w e r e c a r r i e d o u t o n r a i n b o w t r o u t t o s e e i f c a n n u l a e i m p l a n t e d a t d i f f e r e n t p o s i t i o n s o n t h e o p e r c u l u m y e i l d a c c u r a t e e s t i m a t e s of&mean e x p i r e d o x y g e n t e n s i o n . T h i s v a l u e i s f r e q u e n t l y u s e d t o c a l c u l a t e u t i l i z a t i o n o r v e n t i l a t i o n v o l u m e i n f i s h a n d s h o u l d t h e r e f o r e be a c c u r a t e l y d e t e r m i n e d . O p e r c u l a r o x y g e n t e n s i o n (Pgo 2) r e c o r d e d f r o m s e v e n q u i e s c e n t t r o u t (383 - 426 g, 10-12 C) i n d a r k e n e d a q u a r i a was h i g h l y v a r i a b l e w i t h r e s p e c t t o t i m e a n d c a n n u l a p l a c e m e n t . E x p i r e d o x y g e n t e n s i o n s f r o m c a n n u l a e i n t h e m i d d l e , l o w e r , o r u p p e r p a r t o f t h e p o s t e r i o r m a r g i n o f t h e o p e r c u l u m r a n g e d f r o m 54 - 157 mm Hg a n d w e r e s o m e t i m e s i d e n t i c a l w i t h i n s p i r e d o x y g e n t e n s i o n . When t h e f i s h w e r e c h a s e d e x p i r e d o x y g e n t e n s i o n r o s e b u t t h e m a g n i t u d e o f t h e r i s e a n d i t s d u r a t i o n w e r e n o t u n i f o r m f r o m c a n n u l a p l a c e m e n t t o p l a c e m e n t . I t i s s u g g e s t e d t h a t v a r i a b i l i t y l n o p e r c u l a r P Q 2 may b e r e l a t e d t o p a t t e r n s o f w a t e r a n d b l o o d f l o w t h r o u g h t h e g i l l s i e v e . The p a t t e r n o f w a t e r f l o w may be a f f e c t e d b y t h e a c t i o n o f t h e v e n t i l a t o r y m u s c l e s a n d t h e m u s c l e s t h a t s e r v e t o o r i e n t t h e g i l l f i l a m e n t s w i t h i n t h e b r a n c h i a l c a v i t y . A c o m p a r i s o n was made^between m i x e d e x p i r e d o x y g e n t e n s i o n s a n d t h o s e f r o m c l e i t h r a l c a n n u l a e i n f i s h f i t t e d w i t h o r a l membranes f o r d i r e c t m e a s u r e m e n t o f V G j 28 C l e i t h r a l P Q 2 was s i g n i f i c a n t l y higher than mixed expired P Q 2 and only approached mixed expired tensions l n two f i s h with high v e n t i l a t i o n ra tes . I t i s suggested that high VR*s may d i srupt a non-respiratory flow of water along the pos ter ior margin of the opercular chamber. I t i s suggested the c l e i t h r a l and opercular cannulae provide poor estimates of mean expired oxygen tensions under these experimental condi t ions . I t i s suggested that persons using such cannulae should check the r e s u l t s they give by using a d i f f e r e n t experimental method. 29 PART TWO DIRECT MEASUREMENT OF VENTILATION VOLUME AND RELATED PARAMETERS IN RAINBOW TROUT INTRODUCTION The P i c k p r i n c i p l e has often been used to estimate the volume flow of water per un i t time over the g i l l s of f i shes . A p p l i c a t i o n of the F i c k p r i n c i p l e requires accurate measurement of mean oxygen tensions i n water entering and leav ing the g i l l s and t o t a l oxygen uptake. Cannulae of various types have frequently been used to sample water entering and leav ing the g i l l s (van Dam, 1938; Holeton and Randal l , 1967 , a ,b; Randa l l , et a l , 1967; Saunders, I 9 6 I , 1962; Stevens and Randal l , 1967 a , b ) . The r e s u l t s of part I of t h i s d i s s e r t a t i o n and the work of Garey (1967) ind ica ted that these cannulation techniques y i e l d h ighly var iab l e r e s u l t s and may provide poor estimates of mean expired oxygen tensions. Procedures of t h i s type therefore could lead to inaccurate estimates of volume flow of water over the g i l l s . Hence i t was necessary to devise another method of measuring v e n t i l a t i o n volume for t h i s study. This sect ion describes such a method and r e s u l t s obtained with i t . METHODS Rainbow trout were obtained, held and maintained as previous ly described and placed on the operating table f o r 30 s u r g i c a l procedures. A ser ies of experiments was conducted to measure the volume of water flowing over the g i l l s per un i t t ime. The technique involved attachment of a port ion of a rubber surgeon*s glove to the mouth of a trout so that i t d id not i n t e r f e r e with breathing but al low d i r e c t measurement of v e n t i l a t i o n volume by seperating i n s p i r e d and expired water ( F i g . 4 ) . A PE 60 dorsa l a o r t i c cannula was implanted i n the f i s h as described previous ly and a 4 0 cm length of number 0 s i l k was t i e d through the a n t e r i o r por t ion of the upper jaw as a r e s t r a i n t f or the f i s h . Next, a 1 2 cm c i r c l e , which included the thumb, was cut from a number 8-1/2 l a tex surgeon's glove. The t i p of the thumb was cut to f i t the f i s h ' s head so that i t covered the dorsa l a o r t i c cannula at i t s point of emergence but l e f t the eyes uncovered. The membrane was then s t i t ched around the margin of the f i s h ' s mouth with number 0 0 0 s i l k sutures placed 2-3 mm apart . When properly cut and pos i t ioned the membrane exerted l i t t l e tension on the head or jaws and had s u f f i c i e n t s lack to permit easy movement of the branch ia l apparatus. On two of the subjects the membrane was too t i g h t and the f i s h were unable to open and close t h e i r mouths proper ly . Results from these two f i s h were d iscarded. F i s h with attached o r a l membranes were placed i n a l u c i t e box d iv ided into two chambers by a p a r t i t i o n with a hole through i t . The membrane was attached to the edge of the hole by a l u c i t e r e t a i n i n g r i n g and brass wing-nuts. Thus the membrane served as a b a r r i e r between the two chambers and separated i n s p i r e d and expired water. A rectangular l u c i t e box, open at both ends, ( F i g . 4 ) supported the f i s h while i t s head was anchored to the r e t a i n i n g Figure 4 The apparatus used f o r d i r e c t measurements of v e n t i l a t i o n volume i n rainbow trout . A port ion of a rubber surgeon's glove i s s t i t ched to the margin of the mouth to separate i n s p i r e d and expired water. V e n t i l a t i o n volume i s determined by c o l l e c t i n g the water that s p i l l s over the rear dra in as the f i s h breathes. A l u c l t e bar (not shown) across the r e t a i n i n g r i n g prevents the f i s h moving forward and a l i g a t u r e through the snout prevents i t moving too f a r backwards and tear ing loose from the membrane. ,3Water Level Overflow 1 31 r i n g by the s t r i n g through the upper jaw. Movement,was further r e s t r i c t e d by a l u c i t e bar across the re t a i n i n g r i n g so that the f i s h could not swim forward and tear loose from the membrane. When i n place a trout could move i t s head f r e e l y about 2 cm i n any one d i r e c t i o n but could not exert a d i r e c t p u l l on the membrane. The top and sides of the box were covered with black p l a s t i c to prevent the f i s h seeing the investigators. The drains at either end of the box could be set at various l e v e l s by s l i d i n g them up and down through rubber stoppers. Following the operation the drain at the head end of the box was raised to provide p o s i t i v e pressure i n the buccal cavity and f a c i l i t a t e v e n t i l a t i o n during recovery. At that time the membrane could be checked f o r leakage with dye. When the membrane was properly a f f i x e d very l i t t l e leakage was apparent with a pressure d i f f e r e n t i a l of 2-3 cm H2O across the membrane. The trout were allowed overnight reoovery following attachment of the membrane. Then the drains i n the box were l e v e l l e d and the f i s h was l e f t undisturbed f o r at least 1 hour. V e n t i l a t i o n rate (VR), v e n t i l a t i o n volume ( V G ) , dorsal a o r t i c oxygen tension (PaQ^), inspired oxygen tension ( P | - 0 ) t and expired oxygen tension (P E O 2 ) w e r e then measured at i n t e r v a l s . V e n t i l a t i o n volume was measured by c o l l e c t i n g the water s p i l l i n g over the rear drain as the f i s h breathed. Water was c o l l e c t e d f o r 1 minute and v e n t i l a t i o n rate was simultaneously determined by counting mouth or opercular movements through a small observation hole l n the black p l a s t i c cover. Often these measurements were carried out on the same i n d i v i d u a l f o r a period of 2 or 3 days. In a few cases 32 oxygen d e f i c i e n t water ( 5 6 - 6 5 mm Hg ^0^) w a s r u n * n ^ ° ^ e ^ox * ° study the f i s h ' s response. Oxygen tensions i n blood and water were measured with a Radiometer electrode system as previous ly descr ibed. Oxygen uptake was ca lcu la ted by the Pick p r i n c i p l e us ing the i n s p i r e d oxygen tens ion , the expired oxygen tension from the rectangular body-holding box (both samples co l l ec t ed i n a bubble-free 10 ml p l a s t i c syringe with a curved number 18 gauge sp ina l needle at tached) , the measured v e n t i l a t i o n volume and the oxygen s o l u b i l i t y c o e f f i c i e n t i n water at the tes t temperature (p .17). Cardiac output was ca lcu la ted by the F iek p r i n c i p l e from the oxygen uptake r a t e , the dorsa l a o r t i c P Q and an assumed venous oxygen tension of 3 2 mm Hg from: V 0 o Q = 2 Ca, - Cv, where: Q V o 2 <*02 <>o2 <»o2 C v n 2 where: «c B > and: = cardiac output i n m l / m i n / f l s h = oxygen uptake i n ml /min / f l sh = oxygen capaoity of a r t e r i a l blood (vols %\ = oxygen capacity of venous blood (vols %) = {% saturat ion of blood - from curve) X«cB = (% saturat ion of blood - from curve) X<*B = Hct ( . 3 1 1 ) + 0 . 7 1 0 2 0 2 1 (Holeton, 1 9 6 6 ) . 33 and: «<? B Q = the blood oxygen carry ing capacity at a given 2 hematocrit (ml 0 2 / m l blood) Hct = the hematocrit or packed volume of red blood c e l l s i n vo ls %. We be l i eve that;use of an assumed oxygen tension f o r venous blood of 32 mm Hg i s j u s t i f i e d as the r e s u l t s of Holeton and Randa l l , (I967 b) and part I V of t h i s study ind icate that P V Q 2 remains steady at 30-35 mm Hg when ?VC02 i s *>elow 3 mm Hg. Blood oxygen content ca l cu la t ions were based upon b l o o d c u r v e s for rainbow trout by (Cameron, 1971) and the r e l a t i o n s h i p between hematocrit and blood content determined by Holeton and Randall (I967 b) : ac B Q 2 = 0.311 Hct + 0 . 7 (where <=cB Q 2 and Hct are l n volumes %). Measurements of B 02 i n rainbow trout using Tucker's (1967) technique (Cameron, 1971) agreed with those predicted by Holeton and Randal l ' s formula. RESULTS II Mean v e n t i l a t i o n volumes and other parameters from 18 rainbow trout are summarized i n Table I I I . The number of observations used for each mean VQ , VR or Vsv i s given i n column 3 of the tab le . Mean v e n t i l a t i o n volume f o r the group, weight 210 - 3 . 0 g, at 8.6 - 0.2 C was 37 - 1.8 ml/min when the animals were qu ie t . In one i n d i v i d u a l V_ f e l l as low as 22 ml/min and i n another rose Table III V e n t i l a t o r y and c i r c u l a t o r y parameters recorded from eighteen trout with o r a l membranes attached. Each value i s a mean of a number of observations f o r each f i s h (shown as "n" i n the t h i r d column). + • • • • • • F i s h # x V G - S.E. n x VR x Vsv min. V max. V~ G G x % U Q V G/Q \ ml/min #/min ml ml/min ml/min mg/kg/hr 1 4 8 . 3 2 . 7 9 9 8 . 7 0 . 5 0 3 7 . 0 1 6 2 . 0 3 9 . 7 - - 6 4 . 8 2 3 8 . 4 3 . 3 6 9 4 . 7 0 . 3 9 3 0 . 0 1 4 4 . 0 4 4 . 8 - - 5 7 . 3 3 2 9 . 1 3 . 0 5 6 8 . 8 0 . 4 1 2 7 . 0 1 4 7 . 0 5 0 . 8 - - 4 9 . 3 4 3 1 . 4 2 . 0 7 67 . 0 0 . 4 7 2 8 . 0 4 6 . 0 4 7 . 9 7 v 5 4 . 2 5 2 . 4 5 2 6 . 0 0 . 9 9 6 8 . 3 0 . 3 4 2 4 . 0 2 7 . 0 4 9 . 5 - - 4 4 . 2 6 3 4 . 7 2 . 4 4 6 1 . 0 0 . 5 7 2 9 . 0 1 1 8 . 0 5 1 . 3 1 2 . 4 2 . 8 6 1 . 0 7 4 9 . 0 2 . 3 6 1 0 8 . 2 0 . 4 6 4 1 . 0 9 4 . 0 2 8 . 5 7 . 7 6 . 4 4 7 . 1 8 3 3 . 7 1 . 9 5 7 7 . 0 0 . 4 4 2 7 . 5 8 4 . 0 5 0 . 3 - - 5 9 . 6 9 4 1 . 1 1 . 6 6 8 6 . 5 0 . 4 8 3 6 . 0 1 2 9 . 0 4 8 . 0 1 3 . 0 3 . 2 6 8 . 3 1 0 4 4 . 6 4 . 0 1 1 6 2 . 4 0 . 7 1 4 0 . 0 1 3 8 . 0 3 9 . 0 6 . 4 7 . 0 5 0 . 2 1 1 3 5 . 3 0 . 6 5 7 3 . 8 0 . 4 8 3 4 . 0 3 8 . 0 4 1 . 0 5 . 7 6 . 2 5 0 . 5 1 2 3 1 . 1 2 . 4 6 6 7 . 8 0 . 4 7 2 4 . 0 4 3 . 0 4 8 . 9 5 . 4 5 . 8 4 7 . 8 1 3 2 7 . 1 1 . 8 1 0 5 9 . 1 0 . 4 6 2 2 . 0 4 0 . 0 4 8 . 7 - - 4 1 . 4 1 4 4 0 . 2 2 . 8 1 0 61. 7 0 . 6 5 3 2 . 0 5 7 . 0 3 7 . 9 5 . 5 7 . 6 5 3 . 8 1 5 4 9 . 0 2 . 3 4 6 8 . 0 0 . 4 8 2 7 . 0 5 9 . 0 4 6 . 5 7 . 3 6 . 7 5 3 . 0 1 6 2 9 . 1 1 . 5 7 5 9 . 0 0 . 5 0 2 6 . 0 1 1 4 . 0 4 9 . 6 5 v 7 5 . 1 5 4 . 4 1 7 3 7 . 6 2 . 2 5 7 1 . 2 0 . 5 3 3 3 . 0 7 6 . 0 5 0 . 8 6 . 5 5 . 8 6 3 . 7 1 8 3 9 . 6 1 . 5 6 7 5 . 0 0 . 5 3 3 5 . 0 7 1 . 0 5 5 . 4 1 1 . 6 3 . 4 7 5 . 9 X 3 7 . 0 7 3 . 8 0 . 4 9 3 0 . 7 8 8 . 2 4 6 . 0 , 7 . 9 5 . 4 5 5 . 3 n 1 8 1 8 1 8 1 8 1 8 1 8 1 2 1 2 1 8 S.D. 7 . 4 1 4 . 3 0 . 0 9 5 . 6 4 3 . 7 6 . 5 2 . 8 1 . 6 8 . 9 S.E. 1 . 8 3 . 4 0 . 0 2 1.-3 1 0 . 3 1 . 5 0 . 8 0 . 5 2 . 1 34 as h i g h as 162 ml/min. V G rose s h a r p l y when the f i s h s t r u g g l e d o r was d i s t u r b e d by t a p p i n g on the box ( F i g . 5 ) . V e n t i l a t o r y s t r o k e volume was normally about 0 .5 ml/ br e a t h and i n c r e a s e d markedly as v e n t i l a t i o n volume went up. During s t r u g g l i n g or when the animal was d i s t u r b e d i n some way Vsv was hi g h . V e n t i l a t i o n r a t e u s u a l l y v a r i e d l i t t l e f o r any one i n d i v i d u a l d e s p i t e the f a c t t h a t o b s e r v a t i o n s were o f t e n made on the same i n d i v i d u a l f o r two c o n s e c u t i v e days. There was c o n s i d e r a b l e v a r i a n c e i n VR between i n d i v i d u a l s however, and those f i s h w i t h h i g h mean V G * s tended t o have h i g h VR's ( c f . T a b l e I , Tab l e I I I ) . U t i l i z a t i o n of oxygen from the water p a s s i n g over the g i l l s , c a l c u l a t e d from: % U = P I 0 2 ~ P E 0 2 X 1 0 0 had a mean va l u e of 4 6 . 1 - 1.5 % and ranged from 2 8 . 5 - 6 4 $ . U t i l i z a t i o n decreased as VQ. i n c r e a s e d when the f i s h was d i s t u r b e d or s t r u g g l e d . The oxygen content of i n s p i r e d water was v i r t u a l l y c onstant f o r any one experiment and ranged between 157 and 163 mm PQ^ over the e n t i r e s e r i e s of experiments. A r t e r i a l PQ^ was q u i t e v a r i a b l e , r a n g i n g from 8 2 to 130 mm Hg. T h i s range of t e n s i o n s r e p r e s e n t s a s m a l l v a r i a t i o n i n b l o o d oxygen content. D i s s o c i a t i o n curves f o r rainbow t r o u t b l o o d (Cameron, 1971) show i t to be 95 to 100$ s a t u r a t e d with oxygen over t h a t P N range. Figure 5 A t y p i c a l v e n t i l a t i o n volume record taken from a s ing le 205 g trout at 9 .5 C. Only the points underl ined were used to determine mean r e s t i n g V r f or t h i s f i s h . 150 c E 100 > 50 t ap on b o x j 7 0 3 4 t i m e ( h o u r s ) 35 Calculated cardiac output values f o r the trout ranged between 5 .4 and 13.0 m l / m l n / f i s h and had a mean of 7.9 ~ 0.8 m l / m i n / f l s h . These cardiac outputs, together with the appropriate v e n t i l a t i o n volume values for each animal , were used to determine v e n t i l a t l o n - p e r f u s i o n r a t i o s , the r a t i o of water flow to blood flow over the g i l l s . V e n t i l a t i o n - p e r f u s i o n r a t i o (V„/Q) had a mean of 5 .4 and ranged from 2.8 to 7 . 6 . Oxygen uptake l eve l s f o r quiescent trout ranged between 41.4 and 75.9 mg/kg/hr and had a mean of 55 .3 " 2 .1 mg/kg/hr (Table I I I ) . Simple c o r r e l a t i o n c o e f f i c i e n t s between the various c i r c u l a t o r y and v e n t i l a t o r y parameters f o r quiescent f i s h are given i n Table IV. A s i g n i f i c a n t c o r r e l a t i o n ( S i g n i f i c a n t at the 0.01 l eve l ) i s shown between % U and VQ. There i s a l so a p o s i t i v e c o r r e l a t i o n between Vsv and weight ( 0 . 0 5 l e v e l ) . V e n t i l a t i o n volumes and other parameters for four f i s h subjected to hypoxia are summarized i n Table V. These data were recorded no l a t e r than f i v e minutes a f t e r the oxygen tension of the water was reduced rap id ly to approximately 60 mm P o 2 ' During hypoxia V G rose as high as 276 ml/mln l n one Ind iv idua l and v e n t i l a t o r y stroke volume went up to 3 .7 m l / s f koKe . Averaging the data for the four f i s h indicates that v e n t i l a t i o n volume Increased nearly seven-fold i n response to hypoxia. V e n t i l a t i o n rate rose only s l i g h t l y from the l e v e l accompanying normoxic condi t ions . Thus the increase i n v e n t i l a t i o n volume during hypoxia was l a r g e l y a r e s u l t of increased v e n t i l a t o r y stroke volume rather than a large increase i n v e n t i l a t i o n ra te . Table IV A simple c o r r e l a t i o n ana lys i s performed on the data from Table I I I . Only corre la t ions s i g n i f i c a n t at the 0 .05 l e v e l are inc luded . S igni f icance l e v e l s were obtained from a table of s i g n i f i c a n t corre la t ions i n Stee l and T o r r i e ( i 9 6 0 ) . * V G VR Vsv % U X Q V G/Q Hct Weight Temp V G -VR .68** * = s i g . at 0.05 l e v e l Vsv .46* ns - ** = s«ig/ at 0.01 l e v e l % U - .67** -.51* ns - ns = not s i g . at 0.05 l e v e l X .47* ns ns ns -Q ns ns ns ns . 73** -VG/Q ns ns ns - .69** - .62* - . 84** . -Hct ns ns ns ns ns -.76** .63* Weight ns ns .52* ns nns ns ns ns Temp ns .49* ns ns ns ns ns ns ns -Table V V e n t i l a t o r y parameters rec o r d e d from f o u r f i s h w i t h a t t a c h e d o r a l membranes b e f o r e and a f t e r the f i s h were s u b j e c t e d to b r i e f hypoxia (5 minutes a t P j Q o = 60-65 mm). W e i g h t g b e f o r e h y p o x i a d u r i n g h y p o x i a Temp o C x - v - i S.E. G m l / m i n x VR - S.E, #/min V s v m l m l / m i n VR #/min V s v m l i n c r e a s e VR V s v # o f t i m e s 230.0 7.8 35.8 3.4 58.8 4.9 0.61 . 190/0 72 2.64 1.22 4.33 232.0 7.8 27.5 0.7 72.0 0.8 0.38 276.0 74 3.73 1.03 9.82 182.3 7.5 36.4 1.4 70.4 1.7 0.52 194.0 96 2.02 1.36 . 3.88 2.38.6 7.1 24.4 0.5 72.0 0.6 0.34 185.0 76 2.43 1.06 7.15 x 220.8 7.6 31.0 68.3 0.46 211.3 79.5 2.71 1.17 6.30 36 DISCUSSION II B a s i c a l l y , two techniques have been used to determine v e n t i l a t i o n volume i n f i s h : 1) the d i r e c t technique where a l l the expired water was co l l ec ted and 2) i n d i r e c t methods where V G i s ca lcu la ted by the F i c k p r i n c i p l e or i s estimated by dye d i l u t i o n . A summary of l i t e r a t u r e values for te leos t V G and the methods used to obtain these values i s given i n Table V I . The re su l t s of experiments described i n parts I and IV of t h i s thes is and the work of Garey (1967) ind icated that expired oxygen tensions recorded from opercular cannulae i n both trout and carp were h ighly v a r i a b l e . I t was f e l t that t h i s v a r i a b i l i t y could lead to inaccurate estimation of mean expired oxygen tension. Mean expired tension had been frequently used to ca lcu la te v e n t i l a t i o n volume by the F i c k p r i n c i p l e . Owing to these l i m i t a t i o n s a d i r e c t method of V G measurement was adopted i n th i s study. Most estimates of V G l n Table VI are In ml /kg/hr although the studies were not done on 1 kg f i s h . There i s no evidence to • * show V G d i r e c t l y re la ted to weight so conversion of V G estimates to ml/kg/,1>F}, may introduce an a d d i t i o n a l source of e r r o r . Indeed, metabolic rate and g i l l surface area increase with body weight to the power of about 0 . 8 - 0 . 9 and the growth of other body parts i s a l l o m e t r i c (Prosser and Brown, 1962). Mouth s ize i n f i s h , and consequently v e n t i l a t i o n volume, l i k e l y fol low a s i m i l a r pat tern . Table VI reveals considerable di f ferences among v e n t i l a t i o n volume estimates made wi th in and between d i f f e r e n t species of Table VI A summary of v e n t i l a t o r y and c i r c u l a t o r y parameters for a v a r i e t y of t e leos ts recorded by a number of workers. The methods used to determine V n are: 1 - d i r . c o l l . = d i r e c t c o l l e c t i o n of a l l expired water, 2-Pick = Pick p r i n c i p l e c a l c u l a t i o n based on data from cannulae, 3-van Dam = separation of i n s p i r e d and expired water with a rubber membrane stretched over the head, 4 - ca l c = a ca lcu la ted value based on t h e o r e t i c a l cons iderat ions , 5 - o r a l mem. = the o r a l membrane technique used i n the present study. Species Temp Weight 0 2 Uptake Reference C kg mg/kg/hr % U Q V G/Q ml/kg/min Y Method of G V G ml/kg/min Determination Spheroides maculatus 1 12- 22 - 17- 103 45- 58 - - 17-117* 1- d i r . c o l l . Catostomus commersoni 2 20 . 215 90 2- 68 - - 391- 8063 2- F i c k I o t a l u r u s nebulosus 2 20 .181 71 8-78 - - 239- 2709 2- F i c k Cyprinus c a r p i o 3 10 2-6 28 73 18 12 214 2- F i c k Cyprinus c a r p i o 2 20 .174 100 5- 85 - - 331- 2891 2- F i c k Callionymus l y r a 4 11- 12 .07- . 14 65 40 - - 30 1- d i r . c o l l . M ugil cephalus 5 13- 21 .08- . 29 120 66 - - 20- 101* 3- van Dam I c t a l u r u s punctatus 6 23- 24 .007 206 47 - - 925 3- van Dam Tinea t i n e a 7 17- 20 .05- . 07 - - - - 39 = 164* 3- van Dam Tinea t i n e a 8 19- 20 .07- . 13 63- 140 67 - - 20- 55** 1, 3 A n g u i l l a v u l g a r i s 9 16- 18 .41- . 92 57- 77 65- 80 - - 90- 153 3- van Dam Katsuwonus pelamis 10 - 1.67 419 - - - 2014 4-c a l c . Mackerel 11 - - 51 53 120 15 1830 4-c a l c . Salmo g a i r d n e r i 12 9-19 . 21-1 .1 100- 135 55 65- 100 2.7- 55 274- 3560 2- F i c k Salmo g a i r d n e r i 13 5 .2-. 4 26- 131 10 6- 27 95- 106 571- 2855 2- F i c k Salmo g a i r d n e r i 14 9 . 21 55 46 7. 9 5. 4 37 * 5- o r a l mem. Salmo shas t a 9 10- 12 .90 67 80 - - 148 3- van Dam References: 1 - H a l l , (1931); 2-Saunders, (1962); 3-Garey, (1967); 4-Hughes and Umezawa, (1968); 5-Cech, (1970); 6-Gerald and Cech, (1970); 7-Hughes and Shelton, (1958); 8-Schumann and P i i p e r , (1966); 9-van Dam, (1938); 10-Brown and Muir, (1969); 11-Rahn, (1966); 12-Holeton and R a n d a l l , (1967 b ) ; 13-Stevens and R a n d a l l , (1967 b) ; 14-present study. * = ml/min. .**. = ml/100 g 37 t e l eos t s . These estimates were made over a wide range of experimental condit ions ( f i s h , s i z e , temperature, degree of a c t i v i t y , varying oxygen tensions , varying carbon dioxide tensions) and are hence not d i r e c t l y comparable without cons iderat ion of these condi t ions . V e n t i l a t i o n volume estimates f o r rainbow trout (Holeton and Randa l l , 1967) obtained by i n d i r e c t means (opercular cannulatlon) range from 274 to 3560, ml /kg/min. Holeton and Randal l ' s maximum VQ IS f o r a group of trout under severe hypoxia f o r a long period (35-40 mm a f t e r one to two hours progressive hypoxia). Stevens and Randall (1967) reported that V G ranged from 571 to 2855 ml/kg/min f o r 200-400 g rainbow trout at 5 C i n a tunnel-type respirometer with water f lowing past the f i s h . This range represents i n d i r e c t estimates of VQ f o r quiescent to moderately ac t ive trout i n the respirometer. In the present study the VQ of quiescent trout was 37 * 1.8 ml/min (171 ml /kg/min) , a value lower than the minimum values reported above. The di f ference may be re la ted to v a r i a t i o n s i n experimental condit ions or i n part to the techniques used to measure VQ. Van Dam ( 1 9 3 8 ) , using a d i r e c t technique of measurement on a s ingle 900 g t r o u t , obtained VQ values very s i m i l a r to the present study on a per kilogram bas i s . O r a l membranes could produce inaccurate measures of VQ i f they caused impairment of v e n t i l a t i o n . No such impairment was detected as the dorsa l a o r t i c blood remained 85 -100$ saturated and oxygen uptake values were consistent with those measured for s i m i l a r s ized rainbow trout at 4 - 8 C (Stevens and Randa l l , 1967 b ) . 38 Indeed, the membrane was designed so that the tension necessary to produce a seal was exerted l a t e r a l l y from the snout to the comer of the mouth along the m a x i l l a , and from the corner of the mouth along the mandible. There was v i r t u a l l y no tension exerted on the mouth l n a dorso-ventra l d i r e c t i o n . In a d d i t i o n , f i s h with o r a l membranes were capable of a seven-fold increase i n V G i n response to hypoxia (Table V ) . Since the f i s h were able to increase V^ to t h i s extent i t seems c l ear that the membrane d id not s i g n i f i c a n t l y r e s t r i c t pumping. Tests with dye showed that leakage was not a s i g n i f i c a n t source of e r r o r . In over 200 separate measurements of percent u t i l i z a t i o n from f i s h with o r a l membranes i t never rose as high as 80$ as reported by van Dam (1938). U t i l i z a t i o n ranged from 23 to 6>k% and was usual ly around 50$. I t i s poss ible that the t i g h t rubber membrane stretched over the head of van Dam's f i s h impaired pumping to some degree. Prel iminary experiments i n our laboratory ind ica ted that rubber or p l a s t i c membranes attached to the operculae of trout produced low V G ' s and high u t i l i z a t i o n s compared to f i s h with o r a l membranes. In some cases the r e s t r i c t i o n was severe enough to k i l l the f i s h . Van Dam's trout was large (900 g) and i t i s hard to say i f serious r e s t r i c t i o n of breathing existed i n a f i s h of that s i ze . Presumably, the r e s t r i c t i o n would be greater i n small f i s h owing to the s e n s i t i v i t y of the operculae to phys i ca l loading and the increased r e l a t i v e tension (Laplace's law). Furthermore, van Dam's V G estimates compare c lo se ly with ours on a per kilogram b a s i s . Indeed, the 80$ u t i l i z a t i o n reported by van Dam may be re la ted to the large s ize of h i s f i s h . 39 Holeton (personal communication) observed that larger trout tend to have high u t i l i z a t i o n s . Table I V , however, shows no co r r e l a t i o n between weight and % U, possibly because too small a size range of f i s h was examined. Oral membranes then, appear to be useful f o r measurement of VQ and other parameters i n quiescent trout providing r e s t r a i n t of the f i s h can be tolerated. They do not appear to produce any great r e s t r i c t i o n s of breathing i n 200 g f i s h . Oral membranes allow r e p e t i t i v e and accurate determinations of mean expired oxygen tension. Opercular catheterization, a technique used to determine mean ?E02* ° ^ ^ e n produces highly variable r e s u l t s and could lead to errors i n determining mean ^SQ^* Oral membranes are free of such l i m i t a t i o n s as they allow d i r e c t measurement of VQ and provide a mixed sample of a l l the expired water f o r determination of mean Pg 0 2* * s a d v i - s a D l e that the r e l i a b i l i t y of mean PEO2 m e a s u r e m e n t s from cannulae should always be checked using an independent method. The ventilation-perfuslon r a t i o f o r trout i n t h i s study was 5 . 4 * 0 . 5 . Holeton and Randall (1967 b) reported that VQ/Q ranged from 2.7-55 (normoxic to serve, prolonged hypoxia) and Stevens and Randall (1967 b) reported a VQ/Q range of 95-106 f o r trout subjected to a range of swimming a c t i v i t y l n a respirometer. The large v a r i a t i o n i n ventilation-perfusion r a t i o reported f o r trout i n these three studies could have resulted from the d i f f e r e n t conditions l n each experiment and/or the techniques«used to determine V G/Q. In each case Q was determined by the Pick p r i n c i p l e , a procedure which requires 40 accurate knowledge of a r t e r i a l and venous oxygen and carbon dioxide tensions and a representat ive oxygen d i s s o c i a t i o n curve for the blood. I f the blood curve was s l i g h t l y i n error or the magnitude of the Bohr s h i f t was not accurate ly estimated then inaccurate values for Q would r e s u l t . In the present study i t was not poss ible to measure blood P c c ^ * T n u s o u r ca lcu la ted Q's depend upon the assumption that venous and a r t e r i a l blood of quiescent trout contained 2 . 5 and 1 to 1.5 mm P C Q r e spec t ive ly , as reported by Holeton and Randall {1967 b ) . V G / Q estimates based on t h e o r e t i c a l considerations (Jones7A1970; Rahn, 1966) or determined for other f i s h species . (Baumgarten-Schumann and P l i p e r , 1 9 6 8 ; Hanson and Johansen, 1970; P i l p e r and Schumann, 1967; Robin, _et a l , 1966; and Garey, 1967) range from 8 to 20. Although there was only a small increase i n v e n t i l a t i o n rate during hypoxia, Table II shows VQ s trongly corre la ted with VR. This i s because Indiv iduals showed very l i t t l e v a r i a t i o n i n v e n t i l a t i o n rate but some f i s h had higher rates than others . Those i n d i v i d u a l s with high rates a l so had high V Q ' S , hence the p o s i t i v e c o r r e l a t i o n between these two parameters. 41 SUMMARY OP PART I I V e n t i l a t i o n volume was measured d i r e c t l y i n rainbow t r o u t u s i n g a rubber membrane a t t a c h e d to the mouth which separated i n s p i r e d and e x p i r e d water and allowed c o l l e c t i o n of the l a t t e r . Mean v e n t i l a t i o n volume a t 8,6 C f o r eigh t e e n t r o u t weighing approximately 200 g was 37 " 1 .8 m l / m i n / f i s h . Mean v e n t i l a t i o n r a t e and v e n t i l a t o r y s t r o k e volume averaged 74 breaths/min and 0 . 5 ml/breath r e s p e c t i v e l y . V e n t i l a t i o n volume c o u l d be i n c r e a s e d n e a r l y s e v e n - f o l d d u r i n g moderate, short-term hypoxia as a r e s u l t of a l a r g e i n c r e a s e i n v e n t i l a t o r y s t r o k e volume and a small i n c r e a s e i n v e n t i l a t i o n r a t e . The r a t i o between the flow rattes of water and b l o o d through the g i l l s was approximately, 5 . Percent u t i l i z a t i o n o f oxygen from i n s p i r e d water had a mean of 46 * 1 .5 % and ranged from 26-64$. I t i s suggested t h a t o r a l membranes pr o v i d e d an e x c e l l e n t means of measuring V G d i r e c t l y i n q u i e s c e n t t r o u t . T h i s technique appears preferable, to P i c k p r i n c i p l e c a l c u l a t i o n s of v e n t i l a t i o n volume based upon data from o p e r c u l a r o r c l e i t h r a l cannulae. 42 PART THREE THE RELATIONSHIP BETWEEN IRRIGATION OP THE GILLS AND PRESSURES IN THE MOUTH AND OPERCULAR CAVITIES OP TROUT INTRODUCTION III Water Is propel lecl over the g i l l s of most f i s h by the ac t ions of the muscular pumps i n the mouth and opercular c a v i t i e s . In f i s h such as the rainbow trout the pumping mechanism consis ts of a buccal pressure pump l inked to an opercular suct ion pump (Woskoboinikoff, 1932; Woskoboinokoff and B a l a b a i , 1937) with valves (Gudger, 1935» 19^6) i n the mouth and opercular opening to prevent backflow. Other species exh ib i t modif icat ions of th i s scheme of pump arrangement, (Hughes and Shelton, 1 9 6 2 ) . In bottom dwellers the opercular suction pump i s often more h ighly developed than the buccal pressure pump ( B a g l i o n i , 1910 ) . Others, such as the tunas and mackerel, have abandoned pumping i n favor of ram v e n t i l a t i o n — a process l n which the f i s h makes use of i t s forward movement through the water to f l u s h the g i l l s , much the way Jet engines r e l y on a "ram" intake of a i r (Brown and Muir , 19&9; Muir and Buckley, 1 9 ^ 7 ; Muir and K e n d a l l , 1 9 6 8 ) . Van Dam (1938) f i r s t demonstrated that there was a nearly continuous flow of water from the buccal to the opercular c a v i t i e s of t r o u t . He connected these c a v i t i e s with a glass tube pos i t ioned external to the f i s h and observed a nearly continuous flow of water and dye through th i s tube i n a s ing le 900 g Salmo 43 Shasta. Hughes and Shelton (1957. 1958) simultaneously recorded pressures i n the buccal and opercular c a v i t i e s and movements of the mouth and opercular apparatus and thus provided a d e t a i l e d ana lys i s of the pressure r e l a t i o n s h i p s across the g i l l s during the breathing cycle i n tench (Tinea t inea) roach (Leuolsus r u t l l u s ) and trout (Salmo t r u t t a ) . T h e i r r e s u l t s confirmed those of van Dam that the bucca l iopercu lar water flow was nearly continuous as the buccal cavi ty pressure exceeds the opercular cavi ty pressure during a l l but about one tenth of the breathing cyc le . Although the pressure r e l a t i o n s h i p s wi th in the branchia l pumps of trout are we l l documented there i s l i t t l e information on the r e l a t i o n s h i p between these pressures and the volume flow of water over the g i l l s (Hughes and Shelton, 1958; Hughes and Saunders, 1970). The o r a l membrane technique appeared usefu l f or studying pressure/f low re la t ionsh ips as i t permitted continuous recording of VQ and pressures i n the buccal and opercular cav i ty . This sect ion deals with experiments designed to simultaneously measure * V Q , buccal cav i ty pressure, opercular cavi ty pressure and d i f f e r e n t i a l pressure (buccal minus opercular) across the g i l l s of rainbow trout . I t was hoped that t h i s approach would provide information on changes i n the res i s tance of the g i l l s ieve at d i f f e r e n t VQ'S . The o r i e n t a t i o n of the g i l l f i laments to the water flow influences the amount of water passing between the t i p s of the f i laments and the res i s tance of the g i l l sieve to water flow. Large changes l n g i l l res i s tance may be i n d i c a t i v e of changes i n non-respiratory water s p i l l a g e . Such changes would reduce 0 2 u t i l i z a t i o n and would be r e f l e c t e d by an Increase i n 44 P £ 0 . Thus Pg Q , V G , buccal and opercular pressure were measured In order to gain ins igh t into changes i n the amount of non-. . . . . * - -resp ira tory water flow associated with v a r i a t i o n s i n V Q . METHODS III For the study of pressures and re la ted v e n t i l a t o r y parameters, a group of eleven rainbow trout ( 2 1 7 . 3 - 6.5 g) were f i t t e d with o r a l membranes and placed i n the v e n t i l a t i o n boxes as described i n part I I . These f i s h had 20 cm long PE 190 polyethylene cannulae implanted l n the buccal cav i ty and through the c le i thrum as out l ined i n the general methods sec t ion . These cannulae, when connected to a pressure transducer, permitted recording of bucca l , c l e i t h r a l (opercular cavi ty) and d i f f e r e n t i a l pressure. Pressures were recorded on a Sanborn 267 BC d i f f e r e n t i a l pressure transducer and a Beokman type B Dynograph recorder . The pressure recording apparatus i s i l l u s t r a t e d i n F i g . 6 . Turning the appropriate taps allowed recording to zero , bucca l , c l e i t h r a l and d i f f e r e n t i a l pressure. The tubing on the transducer was 0 . 4 7 cm bore tygon tubing. Equivalent 20 cm lengths of t h i s tubing were used dn opposite s ides of the transducer. Cannulae (PE 1 9 0 , 20 cm long) were connected to the large bore tygon tubing by a male/flale l u e r adaptor and the hub of a number 18 gauge sp ina l needle. Frequency response of the system with the cannula attached, measured using the trans ient ana lys i s of Yanof ( I 9 6 5 , p. 2 7 6 ) , was 1 0 . 2 cyc l e s / sec . The experimental procedure was as fo l lows . F i s h were oannulated, f i t t e d with o r a l membranes, and placed i n the V r Figure 6 A The pressure recording system used to record buccal, c l e i t h r a l and d i f f e r e n t i a l pressure from trout with o r a l membranes attached. Turning the appropriate taps allowed recording of zero, c a l i b r a t i o n pressure, buccal, c l e i t h r a l or d i f f e r e n t i a l pressure. B A hypothetical pressure trace showing how area mean pressure was determined. The area mean pressure, h, i s the l i n e that divides the stippled and oross-hatched areas such that the area under the l i n e equals the area above the curve. Areas were determined by planimetry or by outting out the pressure traces and weighing them. I t was found that the area mean pressure could be determined f a i r l y accurately by v i s u a l Inspection, hence on some of the lower a m p l i f i c a t i o n records v i s u a l Inspection was used. 45 boxes for overnight recovery. The fo l lowing morning the pressure recording system was zeroed with the water l e v e l i n the V G box and c a l i b r a t e d by apply ing a known pressure with a manometer. The cannulae were then connected to the recording system and the f i s h was l e f t undisturbed for an hour i n i t ' s black p l a s t i c covered box. Buccal pressure, opercular cavi ty pressure, d i f f e r e n t i a l pressure, P i 0 2 » P E02 a n d V G w e r e then recorded at i n t e r v a l s during the day. D i f f e r e n t i a l pressure and V G were always simultaneously recorded and the recorder zero was checked a f t e r each pressure measurement. As the object ive of t h i s study was to obtain pressure/f low data at a var ie ty of V G ' s , attempts were made to obtain a range of V G ' s for each f i s h . Some f i s h struggled p e r i o d i c a l l y and thus produced a range of v e n t i l a t o r y flows while others had to be stimulated by tapping on the side of the box. Water temperature i n the apparatus ranged from 12 to 15.5 C ,during the study (x temp. = 14.0 - 0 .5 C ) . RESULTS III The r e l a t i o n s h i p between v e n t i l a t o r y parameters and flow are i l l u s t r a t e d i n F i g s . 7, 8 and 9. At the lowest x V G , 44 m l / m i n / f i s h , v e n t i l a t i o n rate was 77.3 - 2 . 4 breaths/mln and Vsv was 0 .59 * °*°3 ml/breath ( F i g . 7 ) , while buccal pressure, c l e i t h r a l pressure and d i f f e r e n t i a l pressure ( s y s t o l i c / d i a s t o l i c ) were 0 . 7 1 / - 0 . 2 3 , 0 . 6 2 / - 0 . 3 , 0 . 5 9 / - 0 . 3 9 mm Hg respec t ive ly . Over the V\, range 40-120 ml/min these pressures increased s l i g h t l y i n amplitude. At v e n t i l a t o r y flows above 120 ml/min the amplitude of bucca l , c l e i t h r a l and d i f f e r e n t i a l pressure increased Figure 7 Ventilatory parameters reoorded f o r the group of trout with o r a l membranes attached. Data f o r eleven f i s h , 217.3 * 6.5 g, T = 14.0 - 0.5 CY Values are means *' 2 standard errors. 46 appreciably (Pig . 8). A l l the pressures tended to vary considerably from one i n d i v i d u a l to another and a range of pressures was often recorded at any one V G for an i n d i v i d u a l f i s h . Fol lowing s t rugg l ing , pressure r a p i d l y rose to high l e v e l s . One or two minutes a f t e r a t t a i n i n g t h i s high l e v e l , t h e pressure usua l ly dropped appreciably towards r e s t i n g l e v e l s . T y p i c a l pressure traces are shown i n F i g . 9. Usual ly the d i f f e r e n t i a l pressure trace contained one b r i e f phase when opercular cav i ty pressure exceeded buccal pressure. The magnitude of t h i s negative pressure phase tended to decrease when V G went up. In one i n d i v i d u a l buccal pressure was high at very high V G ' s , and there was no negative pressure phase. The area mean d i f f e r e n t i a l pressure for each f i s h at each V"G was ca lcu la ted from the area of the d i f f e r e n t i a l pressure trace as out l ined i n F i g . 6. A p lo t of mean d i f f e r e n t i a l pressures and V_ ( F i g . 10) i l l u s t r a t e d that d i f f e r e n t i a l pressure increased l i n e a r l y with V Q . This r e l a t i o n s h i p i s described by the regress ion equations Y = -.0108 + .00263 X where: Y = the d i f f e r e n t i a l pressure l n mm Hg X = V G l n ml/min The slope of the regress ion l i n e i s such that as V G rose from 4-0 to 160 ml/min, mean d i f f e r e n t i a l pressure increased f o u r - f o l d from 0.1 to 0.4 mm Hg. S imi lar p lo t s of area mean buccal and c l e i t h r a l pressure vs V r ( F i g . 11) showed that a poor r e l a t i o n s h i p exis ted Figure 8 Maximum and minimum pressures (systolic/dlastollo) at different VQ*s recorded from six f i s h (224.6 * 7.0 g, 13.8 * 0.8 C) with oral membranes attached. Values are means * 2 standard errors. D Figure 9 T y p l o a l pressure records at d i f f e r e n t V »s recorded from a G 195 S female trout at 12 C. Figure 10 A Area mean pressure vs V_ for six trout (224.6 - 7.0 g, 13.8 * 0.8 C) with oral membranes attached. The regression line i s described by the equation: f * -.0108 + .00263 X. B Resistance of the g i l l sieve at different V ' s calculated Cr from the pressure data in the upper figure. Resistance i s expressed i n CGS units, a basic physical unit of resistance calculated from: B -j 1333 X differential pressure (in mm Hg) V- ml/second o O f -o oo o < O 3 O o R dyne: sec/cm^ o o -j— t O o o co o o O 00 O o o o x differential press- mm Hg — i — , . , .— > > T t o o o Figure 11 Area mean buccal and c l e i t h r a l pressure calculated according to the method of F i g . 6. Data f o r s i x f i s h (224.6 * 7 .0 g. 1 3 . 8 * 0 . 8 C) with o r a l membranes attached. Values are i n d i v i d u a l observations. The regression l i n e s f o r buccal and c l e i t h r a l pressures are described by the equations: Y = . 2015 + .00078 X and: Y = .028 + .0018 X respectively. 7 I . . i AREA M E A N PRESSURE to o -00 o 2. o o j. o fO o o o T -\ cleithral « ho T T - T — B a § 0 : B B E3 S S ta • 053 a •••!•• • ! : I i • B B O —i E3 O 00 o (O o o o (O o o buccal 1 ~i— > > > > > > > > > o 47 between those mean pressures and V^. There i s a large scat ter of points about the regress ion l i n e s i n F i g . 11. The res is tance of the g i l l sieve was ca lcu la ted from the mean d i f f e r e n t i a l pressure i n F i g . 10 using points 20 ml/min apart on the regress ion l i n e and c a l c u l a t i n g r e s i s t a n c e 1 from: R = 1333 X P F where: R = res is tance i n CGS u n i t s , a basic phys ica l res is tance un i t (expressed i n dyne seconds/cm^) P = the mean d i f f e r e n t i a l pressure i n mm Hg from F i g . F = the water flow over the g i l l s i n ml /sec . The g i l l res i s tance ca lcu lated i n t h i s way i s i l l u s t r a t e d l n F i g . 10. Over the V G range 40-160 ml/min res is tance increased only very s l i g h t l y , r i s i n g from 197 to 206 CGS u n i t s . Calcu lat ions of res i s tance using the method of Hughes and Shelton (1958) of p l o t t i n g the r a t i o of mean pressure/minute volume y i e lded i d e n t i c a l r e su l t s expressed i n d i f f e r e n t u n i t s . Thus the four-f o l d increase i n V G from 40 to 160 ml/min was accomplished by a f o u r - f o l d r i s e i n mean d i f f e r e n t i a l pressure with l i t t l e a l terat ion i n res i s tance . V e n t i l a t i o n rate ( F i g . 7) remained around 75 breaths/min over the V G range 40-120 ml/min and increased when V G rose above 120 ml/min. V e n t i l a t o r y stroke volume however, rose i n a nearly 1 (Guyton, 1962, p. 3^ -4) 48 linear fashion between 40-120 ml/min VQ, reflecting the rising V G with no change in VR. At VQ'S above 120 ml/min the rate of increase of Vsv in relation to VQ declined due to the rising VR. The highest Vsv recorded was 1.93 * 0.03 ml/breath. Oxygen uptake rate increased from 62.9 - 10.22 to 138 -25.7 mg/kg/hr as VQ Increased from 40-120 ml/mln. Percent u t i l i z a t i o n at a VQ of 44 ml/mln was 44.9 * 3.1 % and showed l i t t l e change over the 44-120 ml/mln VQ range (Pig. 7 ) . Oxygen uptake and u t i l i z a t i o n were not recorded at high VQ'S as high VQ'S were of a transitory nature. They occurred after a f i s h had been struggling and tended to drop off rapidly following struggling. * Thus samples of expired water used to determine V Q would not be representative 6f a single VQ but a range of declining VQ'S. Inspired oxygen tension remained between 148 and 153 mm Hg in a l l cases. DISCUSSION III The results of this experiment i l l u s t r a t e a number of important relationships between water pressure and flow across the g i l i s of trout. When VQ increased from a resting level of about 40-ml/mln/fish to 120 ml/min/fish the amplitude of the buccal, cleithral and differential pressure traces increased only slightly and ventilation rate did not change. Ventilatory stroke volume however, increased linearly with VQ over this range. The driving force behind this three-fold VQ increase was a three-fold increase in mean differential pressure. The Slevated differential pressure caused a greater flow of water to pass over the g i l l s per 4 9 breathing cycle and thus increased. Vsv. At V G ' s above 120 ml/min VR increased and the amplitude of bucca l , c l e i t h r a l and d i f f e r e n t i a l pressure increased markedly. Mean d i f f e r e n t i a l pressure (as expressed by the regress ion l i n e i n F i g . 1 0 ) , continued to increase and res is tance of the g i l l sieve remained constant. The rate of increase i n Vsv, i n r e l a t i o n to VQ , dec l ined due to the r i s i n g VR. Thus the major features of the pressure/f low r e l a t i o n s h i p over the VQ range of 40-160 ml/min appeared to be constant g i l l res i s tance and a d i r e c t r e l a t i o n s h i p between mean and d i f f e r e n t i a l pressure across the g i l l s and v e n t i l a t i o n volume. As v e n t i l a t i o n volume rose from 40-120 ml/min, oxygen uptake approximately doubled and u t i l i z a t i o n of oxygen showed l i t t l e change. Hughes and Saunders (1971) and Marvin and Heath (1968) observed a s i m i l a r VQ^ r i s e i n f i s h with elevated breathing a c t i v i t y i n response to hypoxia. Hughes and Saunders concluded that the elevated oxygen uptake was due to the increased metabolic cost of breathing but, as pointed out by Cameron and Cech (1970), a var i e ty of fac tors may contribute to the elevated V Q 2 . I t i s l i k e l y that a c t i v i t y and rest lessness accounted, to a large extent, for the VQ increase i n th i s study as the higher * VQ'S were recorded a f t e r the f i s h struggled spontaneously or had been dis turbed by tapping on the box. The g i l l s themselves l i e pos i t ioned between the buccal and opercular c a v i t i e s and therefore act a s a res is tance between these c a v i t i e s . Flow between the c a v i t i e s w i l l be governed by the r e l a t i o n s h i p : 50 flow = d i f f e r e n t i a l pressure res is tance I t has been demonstrated that f i s h such as the trout possess the means for adjus t ing the res i s tance of the g i l l sieve to water flow. Pasztor and Kleerekoper (1962) have described the a c t i o n of small adductor and abductor muscles which contro l the p o s i t i o n i n g of the g i l l sieve wi th in the branchia l chamber during the breathing cyc le . These muscles a l so inf luence the spacing of adjacent hemibranchs. Saunders (1961) photographed the pos ter ior margin of the g i l l f i laments i n white suckers (Catostomus  commersoni) brown bullheads ( Icta lurus nebulosus) , and carp (Cyprinus carpio) through the opercular aperture during moderate and heavy breathing. He observed that " s l i t s " appeared between adjacent hemibranchs during the breathing cyc le . Saunders was unable to observe whether the s l i t s were present during shallow breathing as the opercular aperture did not open s u f f i c i e n t l y to permit observat ion. He concluded that the "s l i t s" funct ion to reduce the pressure d i f f e r e n t i a l across the g i l l s during heavy breathing and slow the flow of water through the i n t e r l a m e l l a r spaces. Pasztor and Kleerekoper observed t h i s non-respiratory s p i l l a g e of water i n a number of species with one of the operculae removed or a p l a s t i c window inserted i n the operculum using the technique of Hof d i jk -Ekklaar ( i n B i j t e l , 1951). They reported that the g i l l c u r t a i n moved rhythmically due to the a c t i o n of the adductor and abductor muscles and that there were periods of low amplitude movements when no s l i t s appeared at a l l . They d id not 51 r e l a t e the appearance of s l i t s to the depth of breathing but i t i s reasonable to assume that the low amplitude movements were associated with shallow breathing. Thus i n shallow breathing there may be l i t t l e or no non-respiratory s p i l l a g e of water through "s l i t s" between adjacent hemibranchs. The r e s u l t s of t h i s study f i t we l l with th i s l i n e of reasoning. I f the "s l i t s" between adjacent hemibranchs appear only during moderate and heavy breathing then i t i s only then that g i l l res is tance w i l l a d e c l i n e . The r e s u l t s of the present study suggest that non-respiratory s p i l l a g e of water d id not increase over the VQ range of 4 0 - 1 6 0 ml/min as res is tance d i d not dec l ine . These re su l t s are somewhat i n disagreement with those of Hughes and Shelton (1958) and Hughes and Saunders (1971) for tench and rainbow trout., r e spec t ive ly . They reported a decrease i n res is tance of the g i l l s with increas ing VQ. In the tench when VQ went up from 4 0 to 160 ml/min, mean d i f f e r e n t i a l pressure doubled and res is tance decreased by 50$. S i m i l a r l y , Hughes and Saunders observed a decrease i n res i s tance of the g i l l s of rainbow trout during severe hypoxia. The VQ of t h e i r f i s h rose from 0 .2 1 /kg/min (4:00-600 g f i s h , 13.5 C) to 1-5 1 /kg/min i n response to hypoxia. Thus Hughes and Saunders' study encompassed a f a r greater range of VQ'S than the present study. Indeed, they reported that the V Q l e v e l at which the res is tance f e l l markedly was 300 ml/min—a l e v e l nearly twice that of the maximum VQ i n the present study. Thus large decreases i n g i l l res is tance may occur at high VQ'S i n rainbow trout and there may be a range.of VQ values during shallow breathing during which res i s tance remains 52 constant. During shallow breathing u t i l i z a t i o n i s high and may not a l t e r appreciably u n t i l g i l l res i s tance s tar t s to drop. At high VQ'S the "s l i t s" may open between adjacent hemibranchs, s p i l l a g e of water past t h e i r t i p s would occur, g i l l res is tance would drop and u t i l i z a t i o n of oxygen would be reduced. G i l l res is tance may not remain constant when the f i s h are swimming r a p i d l y . Although we have observed that rainbow trout continue to v e n t i l a t e when swimming hard i t i s l i k e l y that they r e l y , i n p a r t , on ram v e n t i l a t i o n to f lush the g i l l s ; w i t h water. Under these condit ions the s ize of the mouth and opercular apertures during swimming as we l l as the p o s i t i o n i n g of the g i l l apparatus i n the branchia l chamber,wil l be important i n regu la t ing the pressure and flow through the system. (Muir and Buckley, 1971). This ana lys i s has r e l i e d heavi ly on the concept of mean d i f f e r e n t i a l pressure during the breathing cycle as an important fac tor i n governing water flow over the g i l l s . Indeed there i s considerable spread of points around the regress ion l i n e i n F i g . 10 and I t i s evident that a range of pressures ex i s t s for any one V G . I t should a l so be emphasized that g i l l res is tance probably f luctuates during a s ingle resp ira tory cycle as pointed out by Hughes and Shelton (1958). Thus the r e l a t i o n s h i p between pressure and flow during a s ingle breathing cycle may be more complicated than t h i s ana lys i s suggests. What i s needed i s information on the instantaneous r e l a t i o n s h i p of d i f f e r e n t i a l pressure, res i s tance and flow across the g i l l s during a s ing le c y c l e . Experiments are planned using an anamometer-type flow probe and d i f f e r e n t i a l transducers to study t h i s problem. 53 The o r a l membrane technique appears very usefu l f or recording v e n t i l a t o r y parameters i n res tra ined f i s h * i t does not however, al low study of v e n t i l a t i o n i n free-swimming f i s h . I t was hoped that the present study would provide clues as to how that might be done. In other words, i f there was a c l e a r l y defined r e a l t l o n s h i p between pressure i n the branchia l chambers 0 and V G then i t might be poss ib le to ca lcu la te the V Q of a swimming f i s h i f pressure records were obtained. F i g . 10 sugges that t h i s could be done using mean d i f f e r e n t i a l pressure but would be of l i m i t e d accuracy, p a r t i c u l a r l y at the lower VQ'S, due to the scat ter of points about the regress ion l i n e . However i f a number of pressure determinations were made at a sustained swimming speed then the average of these values might f a l l c lose to the regress ion l i n e . Of course, th i s assumes that the same pressure and flow r e l a t i o n s h i p s ex i s t i n swimming and non-swimming f i sh—an assumption that remains to be tes ted . The prime l i m i t a t i o n to using d i f f e r e n t i a l pressure measures as a means of c a l c u l a t i n g VQ i n swimming f i s h Is the high s e n s i t i v i t y of the recording system required . This system would be very sens i t ive to cannula movement which would occur during swimming and thus the pressure trace might we l l be un interpretab le . I f such t echn ica l d i f f i c u l t i e s could be overcome then there might be some merit i n th i s approach. Recordings of buccal and opercular cavi ty pressure can be made success fu l ly i n swimming salmon (Davis, 1968; Smith, B r e t t and Davis , 19&7) a n d rainbow trout (Stevens and Randa l l , I 9 6 7 ) . Unfortunately F i g . 11 ind ica te s that there i s a very poor r e l a t i o n s h i p between e i ther 5k area mean c l e i t h r a l or area mean buccal pressure and VQ hence there would be l i t t l e value i n th i s approach. The whole subject of VQ measurement i n swimming f i s h requires further examination. 5 5 SUMMARY OP PART III The relationship between pressure l n the branchial ohamber and g i l l water flow was examined in a group of eleven trout (217.3 * 6.5 g» 14.0 t 0.5 C) f i t t e d with oral membranes and buccal and cl e i t h r a l cannulae. Buccal, cl e i t h r a l and differential pressure traces increased in amplitude as V Q rose from 40 to 160 ml/min. This increase was most evident in the V G range 120-160 ml/mln. Mean ventilation rate remained steady at around 77 breaths/ min i n the V G range 40-120 ml/mln and ibhen rose to 90/breaths/ min at a V G of 175 ml/min. Ventilatory stroke volume rose steadily with V G u n t i l WR started to increase at which time the rate of Increase of Vsv f e l l off. Oxygen uptake approximately doubled as V G rose from feO-120 ml/ min. Util i z a t i o n of oxygen from the inspired water showed l i t t l e change over this V G range. It Is suggested that the elevated V Q 2 was largely the result of increased activity at high V G»s. Area mean differential pressure was direotly related to V G. As V Q Increased four-fold from 40 to 160 ml/mln, x differential pressure increased four times from 0.1 to 0.4 mm Hg. The greater pressure differential between buccal and opercular cavity led to a greater flow of water over the g i l l s per breathing oycle (elevated Vsv.). 56 Calculated res i s tance of the g i l l sieve to water flow remained constant over the VQ range examined. I t i s suggested that g i l l res i s tance would l i k e l y drop at V *s u higher then those recorded i n t h i s study. Area mean buccal and c l e i t h r a l pressure appeared poorly r e l a t e d to V G as a var i e ty of these pressures was recorded at each VQ. The p o s s i b i l i t y of us ing pressure measurements to ca lcu la te VQ i n swimming f i s h i s discussed. 57 PART FOUR ARTIFICIAL PERFUSION OF TROUT GILLS AT DIFFERENT WATER FLOW RATES. INTRODUCTION IV Part II of t h i s study showed that trout of approximately 200 g have a r e s t i n g v e n t i l a t i o n volume of approximately 40 ml/ min/fish and that t h i s V G can be increased about seven-fold i n response to hypoxia. Thus trout are capable of a considerable Increase l n water flow rate over the g i l l s and i t i s l i k e l y that the flow rate i s high when the f i s h i s active or i s stressed i n some way. Variations i n v e n t i l a t i o n volume Influence gas exchange across the g i l l s . Hughes (1966) and Saunders ( I 9 6 I ) discussed the e f f e c t s of high g i l l water flow, pointing out that anatomical deadspace increases under these conditions since portions of the v e n t i l a t o r y flow are shunted past the t i p s of adjacent g i l l filaments. I t was the objective of t h i s series of experiments to seefehow the v e l o c i t y of water flow over the g i l l s influenced a f i s h ' s a b i l i t y to exchange gases with the water. In addition, these tests would be ca r r i e d out with the "high", "mid" and "low" opercular cannulae implanted on some f i s h to see i f the v a r i a b i l i t y l n opercular PQ2 described i n part I was dependent on water flow rate over the g i l l s . METHODS IV To study the e f f e c t of varying water flow over the g i l l s i t 58 was necessary to devise a method of a r t i f i c i a l l y perfusing the g i l l s of intact trout with water at different flow rates. Cannulae were placed in the dorsal aorta, ventral aorta and buccal and opercular cavity ( a l l three opercular placements—high, mid and low) as previously described and a 4 cm length of s t i f f tygon tubing (9 mm I.D., 12 mm O.D.) was tied into the mouth. The tube was tied securely so that i t penetrated about 1 cm into the buccal cavity and directed a flow of water towards the g i l l s ln a plane parallel with the roof of Ihe mouth. The mouth tube eliminated any activity of the buccal pump but the opercular apparatus was free to move. Following cannulation the trout was placed in a lucite box similar to the constant flow system described by Fry (1957) (Fig. 12). Lucite pegs inserted in holes in a baseplate within the box held the fis h upright and kept i t s body straight. This form of restraint prevented the f i s h turning i t s head so that more water could flow over the g i l l s on one side df the body than the other. The top of the bo& was secured with a gasket and wlngnuts and cannulae passed out of the box through needles inserted in rubber stoppers. Black plastic covered the top and sides of the box. Oxygen tension in inflow water, outflow water, water from the opercular cannulae and dorsal and ventral aortic blood were measured with the Radiometer electrode system. A l l attempted measurements of P G O £ i n w a t e r a n d blood were unsuccessful as the P Q Q ^ S were below the 3 mm Hg limit of the C0 2 scale. Blood samples were returned to the animal by backflushing the cuvettes with heparinized Cortland saline (Wolf, 1963). Pressures in the Figure 12 Apparatus used for the a r t i f i c i a l perfusion of the g i l l s at different water flow rates. A tube was tied Into the mouth and cannulae pass out of the box top through rubber stoppers. The f i s h i s held upright by lucite pegs inserted In a perforated base-board in the box. 59 various cannulae were monitored with Statham model P 2 3 BB pressure transducers and a Beckman RS Dynograph recorder. The frequency response of the recording system, tested by the transient analysis technique of Yanof ( 1 9 6 5 , p. 2 ? 6 ) was 5.152 cycles/sec, (50 cm PE 60 cannula connected to transducer with 3-way p l a s t i c valve and 1 cm length of a number 2 1 gauge needle). The f i s h were allowed to reoover f o r 24 hours following cannulation since these procedures involve considerable trauma (Houston, et a l , 1 9 6 9 ) . Following recovery the g i l l s were perfused with water f o r a period of 1 hour at a given rate. At the end of that hour P 0 2 and blood pressure were recorded from the appropriate cannulae and the flow reset to a new l e v e l . Flows ranged from 4 ^ - 1 2 0 0 1 ml/min and were applied at random with the exception that the lowest flows were not used u n t i l the end of the experiment. In these experiments forty-one trout rangtag from 1 9 5 - 3 8 8 g were studied at temperatures of 1 0 - 13.5 C. RESULTS IV Expired oxygen tension measured from opercular cannulae was highly variable. As i n part I, no one placement was subject to l e s s v a r i a b i l i t y of P E Q than any other placement. Variable 2 PjBo's were evident l n a l l f i s h studied and the v a r i a b i l i t y extended over thw whole flow range examined (Fig. 1 3 ) . There was a tendency f o r P E O 2 t o b e n l S n e r a t high g i l l water flow, rates i n a l l three opercular placements studied. The v a r i a b i l i t y i n efferent POg f o r a l l three placements i s r e f l e c t e d by the large standard errors surrounding each point i n F i g . 1 3 . 1 Figure 13 Expired oxygen tensions recorded from the three opercular cannula placements (dark c i r c l e s , s o l i d l i n e s ) on trout whose g i l l s were a r t i f i c i a l l y perfused at a v a r i e t y of flow ra tes . Points are means~2 S . E . Oxygen tension i n the box outflow water (mixed expired water) i s shown as open c i r c l e s and dotted l i n e s . Standard errors for, the outflow tensions were too small to inc lude i n the diagram. 170 130 90 170 ; 7 fish 252-7+. 20-5 g 400 800 1200 . : : 130 P O 2 (mm Hg) : 90 _ _ - O - O MID 9 fish .297-3+ 11-6 g 170 130 90 L O W 10 fish 284-8 120-6 g 400 800 1200 per fus ion rate m l / m i n 60 Inspired oxygen tension measured l n the chamber inflow water was f a i r l y consistent over the entire flow range studied, ranging from 1 5 3 - 162 mm Hg. Oxygen tensions i n box outflow water reached a low of 8 5 mm P0 2 at the minimum flow rate applied ( 4 5 ml/min) and went up as perfusion rate increased, reaching a maximum value of 154 mm at the highest perfusion rate (Fig. 14). Percent u t i l i z a t i o n of oxygen from the water, calculated from the above data, dropped from 4 4 % at the lowest perfusion rate to a minimum value of 3.8 % as the perfusion rate Increased ( F i g . 14). Oxygen uptake rate, calculated by the Fick p r i n c i p l e (p. n ) rose from 5 6.2 to 104 . 9 mg/kg/hr as the flow rose to 7 5 0 ml/min (Fig. 1 5 ) . At higher g i l l water flows oxygen uptake remained around 100 mgAg/hr. Mean expired oxygen tension f o r the group, measured i n the water flowing out of the box) rose from 118 mm Hg at the lowest v e l o c i t y applied to a high of 15^ mm Hg at the highest flow rate. Thus mean expired P0 2 could be compared with the P 0 2 , s measured from the three opercular cannula placements. Figure 13 i l l u s t r a t e s t h i s comparison and shows that the mean expired P0 2 was often considerably higher than opercular P0 2 at the lower perfusion rates. Mean dorsal a o r t i c P0 2 ranged from 131-135 mm Hg at g i l l water flows above 5 0 0 ml/mln and diminished at lower perfusion rates, reaching 44 mm Pa0 2 at a perfusion rate of 4 5 ml/min (Fig. 15). Ventral a o r t i c P0 2 remained v i r t u a l l y unchanged over the entire flow range at 31 . 6 - 0 . 6 mm Hg. Blood pressure data was converted to area mean pressure F i g u r e 14 Oxygen t e n s i o n s i n box i n f l o w and outfl o w water f o r the group of f i s h whose g i l l s were a r t i f i c i a l l y p e r f u s e d a t d i f f e r e n t f l o w r a t e s . Values a r e means - 2 S.E. The percent u t i l i z a t i o n o f oxygen from the water, c a l c u l a t e d from fche t e n s i o n s i n box i n f l o w and o u t f l o w , i s i l l u s t r a t e d . Figure 15 Cardiovascular and v e n t i l a t o r y parameters recorded from the group of a r t i f i c i a l l y perfused f i s h . The values shown - 2 standard errors are means. V n and Q were ca lcu la ted using those u 2 means. 61 a c c o r d i n g to the formula: s y s t o l i c p r e s s u r e + 2 d i a s t o l i c p r e s s u r e a r e a mean pre s s u r e = — , Area mean p r e s s u r e g i v e s a good approximation of average p r e s s u r e s t h a t occur l n v e s s e l s d u r i n g p u l s a t i l e f low (Burton, 1 9 6 6 ) . Area mean b l o o d p r e s s u r e l n the v e n t r a l a o r t a remained steady a t around 35 mm Hg over the e n t i r e f l o w range. D o r s a l a o r t i c p r e s s u r e averaged 29 .3 - 1 . 9 mm Hg a t a p e r f u s i o n r a t e of 80 ml/min and dropped s l i g h t l y as p e r f u s i o n r a t e i n c r e a s e d , r e a c h i n g a low of 21 . 8 ± 2 . 0 mm Hg a t a f l o w of 1043 ml/min. A t - t e s t showed these two v a l u e s t o be s i g n i f i c a n t l y d i f f e r e n t a t the 0 . 0 5 l e v e l . C a r d i a c output, c a l c u l a t e d by the P i c k . p r i n c i p l e from the p r e c e d i n g data (p. 3 * . ) , rose i n accordance w i t h the i n c r e a s e i n p e r f u s i o n r a t e ( P i g . 1 5 ) . A t a f l o w of 85 ml/min c a r d i a c output was approximately 7 m l / m l n / f i s h w h i l e a t a f l o w of 750 ml/min Q was 14 .4 m l / m i n / f i s h . A t p e r f u s i o n r a t e s above 85 ml/min, h e a r t r a t e showed l i t t l e change, remaining around a mean v a l u e of 63 -3 . 4 ibeats/min. At the lowest flow t e s t e d (45 ml/min), c a r d i a c output, c a l c u l a t e d by the P i c k p r i n c i p l e u s i n g an assumed Pv@ 2 of 34 mm Hg, was 12 . 2 m l / m l n / f i s h . T h i s Q v a l u e i s c o n s i d e r a b l y h i g h e r than those a t i n t e r m e d i a t e p e r f u s i o n r a t e s . I n a d d i t i o n , h e a r t r a t e a t the minimum p e r f u s i o n r a t e was 49 beats/min, a r a t e somewhat lower than a t any o t h e r p e r f u s i o n r a t e . f i l e above data were used t o c a l c u l a t e the t r a n s f e r f a c t o r (TO2) f o r oxygen a c r o s s the g i l l s . T r a n s f e r f a c t o r i s a measure of the r e l a t i v e a b i l i t y of the r e s p i r a t o r y s u r f a c e t o exchange gases and may be c a l c u l a t e d by d i v i d i n g the oxygen uptake r a t e by 62 the pressure gradient for oxygen across the g i l l s (Randal l , et a l , 1967): T 0 2 • I / 2 (?I0 2 + P E 0 2 ) " 1 / 2 ( P A § 2 + P V 0 2 ) Transfer f a c t o r was 0.0078 ml 0 2/mln/mm Hg at ibhe the lowest flow rate and increased by about 50 % as the flow rose to i t s highest l e v e l (P ig . Opercular c losure rate was zero at perfusion rates i n excess of 580 ml/min but lnoreased r a p i d l y as flow dropped below t h i s l e v e l , reaching a maximum rate of 85 c losures/min at a perfus ion rate of 95 ml/min (P ig . 15). Rapidly dropping the perfus ion rate produced an almost immediate onset of opercular movements ( F i g . 17) which was frequently accompanied by bradycardia . P c o l eve l s i n both water and blood af ferent and efferent to the g i l l s were always below 3 mm Hg and could therefore not be measured. DISCUSSION IV These r e s u l t s confirm the high degree of v a r i a b i l i t y l n expired oxygen tension that can be measured from cannulae placed h igh , low or i n the middle of the pos ter ior margin of the operculum. Furthermore, they show that t h i s v a r i a b i l i t y ex i s t s over a wide range of a r t i f i c i a l v e n t i l a t o r y f lows, i n c l u d i n g the a r t i f i c i a l flow of 45 ml/min that corresponds to the ac tua l V G of quiescent t rout of about 40 / m i n / f l s h measured l n part I I . A comparison of F i g u r e J6 The oxygen t r a n s f e r f a c t o r a c r o s s the g i l l s and the a r e a meantdorsal a o r t i c b l o o d p r e s s u r e f o r the group of p e r f u s e d f i s h . The^pressures a r e shown as means i 2 standard e r r o r s w h i l e the t r a n s f e r f a c t o r was a c a l c u l a t e d v a l u e . Figure 17 Pressure records from 302.5 g trout at 10 C when perfus ion rate was r a p i d l y a l t e r e d . Flow rate could be changed very qu ick ly by applying or removing a clamp on the mouth perfusion tube. pressure M M Hg opercular dorsal aorta the mean expired oxygen tens ion, measured i n the box outflow water and the oxygen tension i n water from opercular cannulae (Pig . 13) indicated that the opercular tensions from a l l three placements were often considerably below the measured value. Thus opercular ca the ter i za t ion appears a poor technique f o r determining mean expired oxygen*tension l n t rout . The fac t that opercular tensions are lower than measured :V J mean expired oxygen tension over the range of perfusion rates approximately the normal range of VQ f o r trout suggests that laminar flow may indeed be present along the inner surface of the operculum. This laminar flow pattern would slow the water flow along the Inner opercular surface . Increasing i t s residence time l n the g i l l s and a l lowing a greater extract ion of oxygen. I t i s not poss ible to say however, If t h i s Is an a r t i f a c t of the way the g i l l s were a r t i f i c i a l l y perfused. The mouth tube might d i r e c t the water over the g i l l s i n a way that would provide a laminar flow s i t u a t i o n on e i ther side of the g i l l cavi ty that would not be present when the f i s h was v e n t i l a t i n g normally. The v e l o c i t y and pattern of water flow over the g i l l s must have a considerable ef fect on gas exchange. I f the mouth tube d irec ted water over the g i l l s i n a pattern of flow that was not optimum f o r gas exchange then impairment of exchange might r e s u l t . Perfused f i s h were unable to saturate t h e i r a r t e r i a l blood with oxygen at the lowest flow rate tested (Pig . 15) . This perfusion rate (4-5 ml/min) approximates the VQ of quiescent trout with o r a l membranes attached. Thus i t appears that a r t i f i c i a l perfusion of the g i l l s by means of the mouth tube provides less 64 e f f e c t i v e gas exchange conditions than those present during normal v e n t i l a t i o n . I t i s l i k e l y that the r e s t r i c t i o n of exchange resulted from a poor pattern of water flow over the g i l l s during perfusion. Probably only a portion of the g i l l sieve received adequate v e n t i l a t i o n at the lowest perfusion rate. A f l a r e d perfusion tube might have directed the water flow more uniformly over the g i l l s and resulted i n a more complete saturation of the blood at the lowest flows. At high perfusion rates the dorsal a o r t i c blood was saturated but u t i l i z a t i o n was reduced as a r e s u l t of the high flow rates. U t i l i z a t i o n at the lowest perfusion rate was 4 4 %% a value nearly i d e n t i c a l with the mean u t i l i z a t i o n ( 4 6 %) of trout with o r a l membranes at a mean V G of 3 7 ml/min/ Thus although the dorsal a o r t i c blood was not saturated with oxygen at t h i s low perfusion rate some compensation must have taken place within the f i s h to maintain a normal u t i l i z a t i o n when a poor pattern of flows existed at the g i l l s . Pig. 1 5 shows that cardiac output was high at the lowest perfusion rate l n comparison to Q*s at higher perfusion rates. This high Q value was calculated by the Pick p r i n c i p l e using an assumed PVQ of 3^ mm Hg and may therefore be subject to error. An increase i n Q i n response to diminished Pa@2 would f a c i l i t a t e u t i l i z a t i o n and maintain an adequate VQ2 despite the poor flow conditions at the g i l l s . Rainbow trout increasestheir cardiac output i n response to hypoxia (Holeton and Randall, 1 ° 6 7 b) or anemia (Cameron and Davis, 1 9 7 0 ) when environmental oxygen i s low or the oxygen carrying capacity of the blood i s reduced. 65 O p e r c u l a r movements a p p e a r e d a t p e r f u s i o n r a t e s b e l o w 700 m l / m i n a n d I n c r e a s e d i n f r e q u e n c y a s f l o w s d e c l i n e d ( F i g . 1 5 ) . T h e r e a r e two p o s s i b l e s o u r c e s f o r i n i t i a t i o n o f t h i s o p e r c u l a r a c t i v i t y . V e n t i l a t i o n c o u l d be c o n t r o l l e d b y i n f o r m a t i o n f r o m r e c e p t o r s i t e s o n t h e g i l l s s e n s i t i v e t o c h a n g e s i n w a t e r f l o w . S u t t e r l i n a n d S a u n d e r s (1969) d e s c r i b e d p r o p r i o c e p t o r s a s s o c i a t e d w i t h g i l l f i l a m e n t s a n d r a k e r s o f t h e s e a r a v e n a n d A t l a n t i c s a l m o n . T h e s e r e c e p t o r s r e s p o n d t o m e c h a n i c a l d i s p l a c e m e n t . I n d e e d , when t h e p e r f u s i o n r a t e d r o p p e d r a p i d l y ( F i g . 17) t h e r e was a n a l m o s t i m m e d i a t e i n i t i a t i o n o f o p e r c u l a r a c t i v i t y s u g g e s t i n g t h a t a n e x t e r n a l r e c e p t o r s y s t e m may be p r e s e n t o n t h e t r o u t g i l l . A l t e r n a t e l y , t h e o p e r c u l a r a c t i v i t y c o u l d be r e l a t e d t o a r t e r i a l o x y g e n t e n s i o n . O p e r c u l a r movement was i n i t i a t e d a t r o u g h l y t h e same f l o w r a t e a s t h a t when d o r s a l a o r t i c PQg b e g a n t o d e c l i n e ( F i g . 1 5 ) . I t i s p o s s i b l e t h a t t r o u t p o s s e s s a r e c e p t o r s y s t e m c a p a b l e o f a d j u s t i n g v e n t i l a t i o n i n r e s p o n s e t o c h a n g e s i n a r t e r i a l PQ^. The p s e u d o b r a n c h c o u l d be t h e s i t e o f s u c h a s y s t e m a s i t r e c e i v e s a n a r t e r i a l b l o o d s u p p l y a n d h a s b e e n shown t o e x h i b i t c h e m o r e c e p t o r a n d b a r o r e c e p t o r a c t i v i t y ( L a u r e n t , 1 9 6 7 ) . R e g a r d l e s s o f t h e p o s i t i o n o f t h e r e c e p t o r s y s t e m , F i g . 17 shows t h a t i t i s c a p a b l e o f a d j u s t i n g v e n t i l a t i o n v e r y r a p i d l y t o meet a c h a n g e i n w a t e r f l o w c o n d i t i o n s a t t h e g i l l s . C a r d i a c o u t p u t r o s e a p p r o x i m a t e l y 50$ a s p e r f u s i o n r a t e i n c r e a s e d f r o m 85 m l / m i n t o t h e h i g h e s t r a t e s t e s t e d . A s m e n t i o n e d e a r l i e r F i c k p r i n c i p l e c a l c u l a t i o n s o f Q i n t h i s s t u d y may b e somewhat I n a c c u r a t e due t o t h e l a c k o f PCO2 ^ a - t a . I n a d d i t i o n , t h e Q c a l c u l a t i o n s may b e a f f e c t e d ! i n p e r f u s e d f i s h b y 66 "washing out" of blood G0 2 due to the high s o l u b i l i t y of C0£ i n water and the pers i s tent gradient between blood and water at high perfus ion r a t e s . I t appears that Inaccuracies i n ca lcu la ted Q were present for the perfused f i s h as V Q ^ approximately doubled over the perfusion rate range while ca lcu la ted Q increased by only about $0%, Since arterio-venous oxygen d i f ference d i d not change apprec iably over the flow range (P ig . 15) s except at the lowest flow r a t e , the only means of increas ing V o 2 w a s by e levat ion of Q. Thus a doubling of V Q 2 should have been accompanied by a doubling of Q. These r e s u l t s show that the P i c k Q's are subject to the same l i m i t a t i o n s as P ick p r i n c i p l e ca l cu la t ions of V Q . Re l iab le P ick Q's require accurate knowledge of a r t e r i a l and venous P Q 2 and P c o 2 l e v © l s and a representat ive blood d i s s o c i a t i o n curve. E r r o r s i n Q i n the present study are l i k e l y a r e s u l t of lack of P c o 2 data. Oxygen uptake increased as perfus ion rate went up ( F i g . 15) despite the fac t that u t i l i z a t i o n f e l l ( F i g . 14) and anatomical deadspace i n the g i l l s had probably Increased (Hughes, 1 9 6 6 ) . Assuming our ca l cu la t ions of Q are s u f f i c i e n t l y accurate to show gross changes i n Q the r i s e i n V Q ^ can be a t t r i b u t e d to two fac tors : 1) an Increase l n cardiac output and 2) a v a s o d i l a t i o n of the g i l l s . Cardiac output rose i n accordance with perfusion rate but ibhere was no e levat ion of dorsa l a o r t i c pressure (P ig . 1 6 ) . In f a c t , blood pressure decreased s l i g h t l y as perfus ion rate went up. Since blood pressure d id not r i s e i n the presence of elevated Q there must have been a v a s o d i l a t i o n of blood vesse ls i n both the g i l l s * a n d per iphera l c i r c u l a t i o n . 67 V a s o d i l a t i o n of the g i l l s could open a d d i t i o n a l vascular exchange areas (Steen and Kryusse, 1964 ; Richards and Fromm, 1969) and lead to a reduct ion i n p h y s i o l o g i c a l deadspace (Hughes, 1 9 6 6 ) . A l s o , the 50 % increase i n t rans fer fac tor that occurred as flow went up suggests that adjustments favor ing the t rans fer of oxygen across the g i l l s had occurred. An increase i n t rans fer fac tor could he the r e s u l t of increased surface area of the g i l l s , a decrease i n d i f f u s i o n distance f o r oxygen, an Increase i n the d i f f u s i o n c o e f f i c i e n t f or oxygen or a combination of these fac tors (Randal l , et a l , 1 9 6 7 ) . The r i s e i n VQ 2» Q and v a s o d i l a t i o n could r e s u l t from the f i s h becoming dis turbed or exci ted at high flows. During excitement catecholamines l i b e r a t e d in to the blood (Nakano and Tomlinson, 1966) act on adrenergic receptors and may cause d i l a t i o n of the g i l l blood vessels (Randall and Stevens, 1 9 6 7 ) . These catecholamines could a l so elevate cardiac output by increas ing the rate and force of contract ion of the heart (Randal l , 1 9 6 8 ) . Another p o s s i b i l i t y i s that v e n t i l a t i o n and c i r c u l a t i o n are c o n t r o l l e d so that cardiac output and V G are matched. I t does not appear that V G increases i n response to elevated cardiac output alone i n trout since VQ does riot r i s e during anemia despite a ten-f o l d increase i n Q (Cameron and Davis , 1 9 7 0 ) . However, the capac i ty -ra te r a t i o was maintained i n anemic f i s h (the capac i ty -rate r a t i o i s the r a t i o between volume flow and s o l u b i l i t y of water and blood f lowing across the .g i l l s—Cameron and Davis , 1 9 7 0 ) . The present study showed that Q increased as perfus ion r a t e went up which would r e s u l t i n a tendency to maintain capac i ty -rate 68 r a t i o . This r i s e i n Q, accompanied by a v a s o d i l a t i o n of the g i l l s , would f a c i l i t a t e gas exchange at high VQ'S and would meet the increased exchange requirements associated with a c t i v i t y or excitement. U t i l i z a t i o n of oxygen from the i n s p i r e d water was exceedingly low at high perfusion rates ( F i g . l 4 ) . This i s a r e s u l t of the high flow rates and short residence time of the water i n the g i l l s and may a l so r e f l e c t poor exchange condit ions at the g i l l s at high flows. When flow rate i s high adjacent g i l l f i lament t i p s are drawn apart and a por t ion of the re sp ira tory flow s p i l l s past the t i p s without contact ing the lamellae (Saunders, 1 9 6 1 ; Pasztor and Kleerekoper, 1 9 6 2 ) . Hence a large port ion of the t o t a l flow would fol low a non-respiratory pathway and low u t i l i z a t i o n would r e s u l t . A l s o , i n t e r l a m e l l a r flow rate could be high enough to prevent water l n the centre of each i n t e r l a m e l l a r space from exchanging gases at a l l ( F i g . 2 i n Hughes, 1 9 6 6 ) . A c t i v e f i s h with high VQ'S have more c lo se ly spaced lamellae than s luggish f i s h (Hughes, 1 9 6 6 ) . Close ly spaced lamellae would reduce i n t e r l a m e l l a r anatomical deadspace at high flow ra tes . I t '-is a l so poss ib le that i n t e r l a m e l l a r flow decreases at high flows due to d i s t o r t i o n of the lamellae and abduction of the g i l l f i laments to al low non-respiratory s p i l l a g e of water. The fus ion of g i l l f i laments i n tuna (Muir and K e n d a l l , 1968 , 1969) may reduce lame l lar d i s t o r t i o n andMceep the lamellae c lose together to f a c i l i t a t e u t i l i z a t i o n of high flow ra tes . A r t i f i c i a l perfus ion of the g i l l s by means of a mouth tube appears to provide a l e s s than optimum flow pattern over the g i l l s . 6 9 The perfusion studies do provide useful Information on the control of c i r c u l a t i o n and v e n t i l a t i o n and emphasize the e f f i c i e n t d i s t r i b u t i o n properties of normal v e n t i l a t i o n . In addition they show that gas exchange i s not d r a s t i c a l l y i n h i b i t e d at high perfusion rates even though anatomical deadspace may be high at such rates. The limitatlon§pf mouth perfusion should be considered by those contemplating i t s use i n experiments or surgioalpprocedures. A f l a r e d perfusion tube might d i s t r i b u t e water more uniformly to the g i l l s . Care should always be taken to keep perfusion rates high to ensure saturation of the a r t e r i a l blood of- perfused f i s h . 70 SUMMARY OF PART IV A r t i f i c i a l perfusion of the g i l l s with water at d i f f e r e n t flow rates was achieved by ty ing a tube into the mouth of t r o u t . Perfused f i s h could not saturate t h e i r a r t e r i a l blood with oxygen at a perfusion rate of 45 ml/min but could do so at rates ranging from 85-12.^ 0 ml/min. Low a r t e r i a l oxygen tensions at a perfus ion rate approximating the mean V Q of f i s h with o r a l membranes are l i k e l y the r e s u l t of a poor pattern of water flow over the g i l l s during perfus ion . Opercular movements occurred only at perfusion rates below 700 ml/min and increased i n frequency as perfusion rate dropped. This v e n t i l a t o r y a c t i v i t y may have resu l ted from receptors sens i t ive e i ther to water flow over the g i l l s or to a r t e r i a l POg. Expired oxygen tensions measured from the three opercular cannula placements were h ighly v a r i a b l e and t h i s v a r i a b i l i t y extended over the en t i re range of perfusion rates . In a d d i t i o n opercular P 0 2 ' s measured from the cannulae were usual ly below the ca lcu la ted mean expired oxygen tens ion . These f indings support those of part I of t h i s study and suggest that opercular ca the ter i za t ion provides a poor estimate of mean expired oxygen tension i n t r o u t . As perfus ion rate went up cardiac output and oxygen uptake increased. These changes were accompanied by a drop i n dorsa l a o r t i c pressure which r e f l e c t e d v a s o d i l a t i o n of the 71 g i l l s and per iphera l c i r c u l a t i o n . This change i n the pattern of blood flow through the g i l l s contributed to a 50$ increase i n oxygen t rans fer fac tor at the g i l l s . At the highest perfusion rates there was no apparent i impairment of gas exchange even though anatomical deadspace was probably h igh . 72 PART FIVE BLOOD DISTRIBUTION AND FLOW WITHIN THE GILLS OF RAINBOW TROUT. INTRODUCTION V The volume, d i s t r i b u t i o n and flow rate of blood passing through the g i l l s w i l l a f f ec t the rate of gas t rans fer between blood and water. Vessels wi th in the g i l l s are arranged i n p a r a l l e l and can be grouped in to three anatomical categories; the c a p i l l a r i e s wi th in the secondary lamellae ( lamel lar c i r c u l a t i o n ) , fhe blood spaces wi th in the i n t e r l a m e l l a r region of the f i lament ( lacunar c i r c u l a t i o n ) , and the c a p i l l a r i e s running wi th in the margin of the f i lament ( f i lamentar c i r c u l a t i o n ) . In v i t r o observations have demonstrated that d i s t r i b u t i o n of blood flow to d i f f e r e n t parts of the g i l l c i r c u l a t i o n can be a l t e r e d (Steen and Kryusse, 19J4-: Richards and Fromm, 19^9). There i s a l so In v ivo evidence i n d i c a t i n g that the funct iona l area of the f i s h g i l l i s l e s s than i t s anatomical area and that the area may change under c e r t a i n condit ions (Stevens and Randa l l , 19^7 b; Stevens, 1968: Randa l l , 1970; part IV of t h i s study). Thus d i f f e r e n t port ions of the g i l l c i r c u l a t i o n may be perfused at d i f f e r e n t rates and may be adjusted to regulate the func t iona l area of the g i l l s i n proport ion to the rate of gas t r a n s f e r . In order to examine poss ible In v ivo shunting of blood at the g i l l s an i n f r a r e d photographic study was made. Infrared photography has been used i n medicine (Kodak p u b l i c a t i o n s , 1968; 73 1969) as a technique for studying s u p e r f i c i a l blood vesse l morphology. Surface vesse ls show up r e a d i l y on an Infrared p la te and veins and a r t e r i e s can be d i s t inguished according to t h e i r d i f f e r i n g ref lectance and absorption of Infrared l i g h t . Furthermore, Infrared co lor f i l m allows one to d i f f e r e n t i a t e between oxygenated and deoxygenated hemoglobin. Oxygenated blood appears reddi sh , deoxygenated blood yel low. I t was hoped that i n f r a r e d co lor p i c tures of the g i l l s would revea l patterns of blood d i s t r i b u t i o n and that the oxygen s a t u r a t i o n / c o l o r r e l a t i o n s h i p would provide information ON varying degrees of blood oxygenation wi th in the g i l l s . In accordance with the attempts to demonstrate blood shunting wi th in the g i l l s by i n f r a r e d photography a second ser ies of experiments was designed to study responses of t rout to a r t i f i c i a l manipulation of the surface area of t h e i r g i l l s . Various g i l l arches were robbed of t h e i r blood supply by l i g a t i o n thus reducing the g i l l area a v a i l a b l e for exchange. I t was hoped that t h i s approach would show how the animal adjusted c i r c u l a t i o n w i th in the g i l l and would give some idea of the minimal func t iona l area of the g i l l required by quiescent t r o u t . The v e l o c i t y of blood flow through the g i l l s w i l l a l so a f f ec t the rate of gas t r a n s f e r . In the absence of any d i r e c t measurements, Mott (1957) estimated a c i r c u l a t i o n time (the time the blood takes to make a c i r c u i t of the body) of nearly 2 minutes f o r the toadf lsh (Opsanus t a u ) , and observed that contrast medium took 5 .6 seconds to pass the g i l l s and 20 seconds to move from the heart to the v i s c e r a l veins of an anesthetized e e l . In these 74 experiments c i r c u l a t i o n time was measured i n the rainbow trout and the time blood takes to pass through the g i l l s was ca l cu la ted us ing estimates of cardiac output and g i l l blood volume (Stevens, 1968). METHODS V Infrared Photography of the G i l l s In preparat ion f o r photography of the g i l l s f i s h were anesthet ized with 1:10,000 MS 222 and placed on the operating tab le . In some f i s h 1 cm of the pos ter ior margin of the operculum was out away to revea l the g i l l s . In others an aparture 1 em square was cut i n the centre of the rear h a l f of the r i g h t operculum (P ig . 18 A) and a p l a s t i c window was inserted s i m i l a r to that used by Hofdi jk E n k l a r r ( in B i j t e l , 1951). In some i n d i v i d u a l s a s u b i n t e s t l n a l ve in cannula was implanted as described i n the general methods. The f i s h were then placed i n a flow-through p l ex ig la s s chamber (Pig . 18 B) and pos i t ioned so that the r i g h t operculum was wi th in 1 cm of the side of the chamber. P a r t i t i o n s (Ln the box prevented the f i s h turning around or moving forward so the area to be photographed always remained properly or iented towards the camera. Fol lowing the operat ion the f i s h were l e f t undisturbed i n the box overnight before photographs were taken. The box was covered with black p l a s t i c and a hole small enough to revea l the por t ion of the g i l l to be studied was cut l n the p l a s t i c . The photographic apparatus consisted of a Ze i s s Ikon or Asahi Pentax Spotmatic 35 mm r e f l e x camera attached to a Ze i s s Figure 18 A. D e t a i l s of the p l a s t i c window that was inserted i n a hole i n the operculum of trout f o r i n f r a r e d photography of the g i l l s . The window was held i n place by four number three "0" s i l k t i e s knotted on the ins ide of the operculum and pushed through holes i n the operculum bored with the t i p of a number 21 gauge needle. B . The photographic setup used to study the g i l l s . The f i s h was placed i n a p l ex ig la s s box with a p a r t i t i o n i n i t so i t could net turn around. The camera was mounted on a b inocular microscope and focussed on the side of the box. Two r e f l e c t i n g photofloods provided a good source of i n f r a r e d l i g h t . A Kodak Wratten number 12 f i l t e r excluded blue l i g h t from the i n f r a r e d co lor f i l m . '<H Y - p i a s t i c w i n d o w s i l k f a s t e n e r r e a r e d g e o f o p e r c u l u m 75 binocular Operating Microscope. A Kodak Wratten' number 12 (minus blue) f i l t e r was placed over the microscope object ive to exclude blue l i g h t from the f i l m . The cameras were loaded with Kodak ektachrome i n f r a r e d aero f i l m 8443 and the f i l m was processed by the standard E3 process. The l i g h t source was two 375 watt 3200 K r e f l e c t i n g photofloods mounted on the same plane as the f i s h and about 2 feet away from i t at an angle of 4 5 ° . The image reaching the camera passed through about 1 cm of water, 0 . 4 7 cm of p l ex ig las s and about 10 cm of a i r before reaching the microscope objec t ive . A number of photographic procedures were employed. For some f i s h sequence photographs were made every 10 minutes for a per iod of 200?sminutes. For others exposures were made before and a f t e r i n j e c t i o n of ep inephrine /sa l ine so lu t ion or known volumes of sa l ine so lu t ion i n the s u b i n t e s t l n a l v e i n . To study the co lor rendering of oxygenated or deoxygenated trout blood, samples were e q u i l i b r i a t e d f o r one hour i n a temperature contro l l ed shaking apparatus equipped with a gas supply as described by Cameron ( 1 9 7 1 ) . Deoxygenated blood (PQ 2 = 0 - 2 mm Hg) was obtained by e q u i l i b r a t i o n with ni trogen while oxygenated blood ( P o 2 = 1 1 1 3 1 1 w a s obtained by e q u i l i b r a t i o n with compressed a i r . Half saturated blood was made a v a i l a b l e by mixing equal port ions of oxygenated and deoxygenated blood anaerob ica l ly . A l l the samples were then trans ferred under o i l to 1 ml test tubes and photographed under water i n the apparatus at the same p o s i t i o n as the g i l l s were photographed. 76 L i g a t i o n of G i l l Arches In order to study trout that had a port ion of t h e i r g i l l area removed "by l i g a t i o n , two apparatuses were used. The f i r s t consisted of a darkened l u c l t e box s i m i l a r to that used i n part IV with a flow of water through i t (200 - 250 ml/mln) and a p a r t i t i o n to prevent the f i s h turning around. Twenty one f i s h ( 2 1 8 - 7 . 6 g, 12.0 - 0.2 C) l i g a t e d i n various wpys and equipped with dorsa l a o r t i c cannulae l n order to sample blood for a r t e r i a l P Q 2 determinations were placed i n d i v i d u a l l y i n the boxes. The appropriate g i l l arches were t i e d of f at t h e i r bases, c lose to the isthmus, by means of two number "0" s i l k l i ga tures t i g h t l y a p p l i e d . In one i n d i v i d u a l the g i l l blood supply was not disturbed but the r i g h t operculum was t i e d shut. The f i s h p a r t i a l l y recovered on the operating table and were then placed i n the darkened experimental boxes for overnight recovery. The fo l lowing day P a o 2t Pi02» P E 0 2 l n t n e b o 3 : o u t f , l o w a n d water flow rate through the boxes were recorded at i n t e r v a l s over a 6 - 8 hour per iod . Oxygen uptake rate was ca lcu la ted from t h i s information as previous ly described (p. n ) . The v e n t i l a t o r y responses of l i g a t e d f i s h were studied with the o r a l membrane technique. Nine trout ( 2 1 7 - 7 . 9 g 11 . 7 " 0 . 5 C) were l i g a t e d and equipped with o r a l membranes as described l n part I I . They were then placed i n the V G measurement boxes for overnight recovery. The fo l lowing day V G , VH, P j Q and PEO2 w e r e 2 recorded at i n t e r v a l s over an 8 -hour per iod . V Q 2 , % U and Vsv were ca l cu la ted as described i n part I I . 77 The blood supply to the g i l l arches was occluded by l i g a t u r e s at the base of a r c h e s * ! - 4 , as shown i n P i g . 1 9 . A number of patterns of l i g a t i o n were used and i n some cases involved a considerable reduct ion of g i l l surface area . Hughes ( l n P a l i n g , 1968) gave the fo l lowing f igures f o r the g i l l surface area of a s ingle 175 g brown t r o u t , Salmo t r u t t q : P a i r s of g i l l s Surface area , mm& 1 s t 1 6 , 3 6 4 2nd 17 ,422 3 r d 1 5 . 2 5 4 4 t h 1 0 , 1 6 4 59.204 These f igures could be used to ca lcu la te the percentage of t o t a l g i l l area removed by l i g a t i o n of various arches or groups of arches. I t was f e l t that such ca l cu la t ions would be reasonably accurate as Hughes* data was f o r s i m i l a r s ize and species of f i s h as that used i n the present study. To insure that a p a r t i c u l a r l i g a t i o n was successful the f i s h were k i l l e d a f t e r the experiment and the g i l l s were examined. Arches that were success fu l ly l i g a t e d appeared dark and contained hemorraglc spots i n the lamel lae . When the f i r s t arch was t i e d of f the pseudobranoh on that side a l so appeared dark and necro t i c on postmortem examination. The pattern of l i g a t i o n s used i s summarized i n Table V I I . C i r c u l a t i o n Time Determinations Dorsal a o r t i c cannulae were implanted l n trout anesthet ized with 1:10,000 MS 222 so lu t ion (Smith and B e l l , 1964) and the f i s h Figure 19 Diagram of the c i r c u l a t i o n on one side of the head of a Salmonid. The g i l l arches are numbered i n the same pattern described i n the text . This diagram was drawn from a sketch by Dr. L . S. Smith, of the c i r c u l a t o r y arrangement l n sockeye and coho salmon in jec ted with colored p l a s t i c by Dr. G. B e l l . Oxygenated blood passes through vessels shown as s o l i d l i n e s , deoxygenated blood through vesse ls shown as dotted l i n e s . Table. VII Data from groups of trout whose g i l l area was reduced by-tying-; of f various g i l l , arches and thus b locking t h e i r blood supply. A non- l igated contro l group i s Included f o r comparison. The g i l l arches are numbered as i l l u s t r a t e d i n P i g . 19. % G i l l R e m o v e d P a t t e r n o f L i g a t i o n s A r c h N o . 1 B l o o d S u p p l y N o . F i s h P a u 2 mm H g X m g / k g / h r Q m l / m i n H c t v o l s . % 0 n o n e - c o n t r o l s i n t a c t 4 X 1 0 0 . 2 4 4 . 9 6 . 2 1 7 . 2 S . D . 1 7 . 0 0 . 4 4 . 9 5 . 8 S . E . 4 . 1 0 . 1 1 . 2 2 . 6 n 17 19 1 7 5 14 A r c h N o . 1 o n i n t a c t o n 1 X 1 1 0 . 0 2 4 . 7 0 . 9 2 8 . 0 r i g h t s i d e l e f t s i d e S . D . 1 8 . 3 1 . 8 0 . 4 S . E . 1 0 . 6 0 . 8 0 . 3 i i 3 5 3 1 28 A r c h N o . . 1 d e s t r o y e d 4 X 5 6 . 8 4 9 . 2 2 0 . 8 b o t h s i d e s S . D . 2 6 . 0 3 . 0 6 . 0 S . E . 7 . 9 0 . 7 3 . 5 n 11 17 3 57 A r c h e s N o . 1 § d e s t r o y e d 5 X 4 0 . 3 6 0 . 5 2 3 . 5 2 b o t h s i d e s S . D . 2 1 . 9 2 . 4 5 . 8 S . E . 5 . 2 0 . 6 2 . 4 n 18 19 6 38 A r c h e s N o . 2 $ i n t a c t 2 X 9 0 . 9 6 7 . 3 2 1 . 3 1 9 . 5 4 l e f t , 2 r i g h t S . D . 9 . 6 1 . 2 3 . 3 S . E . 3 . 4 0 . 4 1 . 2 n 8 8 8 2 40 A r c h e s N o . 2 § i n t a c t 4 X 8 7 . 9 90 . 8 2 4 . 0 2 3 . 3 4 b o t h s i d e s S . D . 1 2 . 0 3 . 2 8 . 4 7 . 8 S . E . 3 . 5 0 . 8 2 . 4 4 . 5 n 12 16 12 3 0 n o n e - r i g h t i n t a c t 1 X 1 1 2 . 0 6 8 . 1 6 . 1 3 1 . 0 o p e r c u l u m s e w e d S . D . 1 . 7 1 . 5 1 . 9 s h u t S . E . 1 . 0 0 . 7 1 . 1 n 3 5 3 1 78 were placed In covered, darkened aquaria f o r 24-hours recovery, Nine f i s h from 190 - 235 g were s tudied at temperatures of 8 - 13 C i r c u l a t i o n time was measured by i n j e c t i n g 0 .3 ml of a 1 mg/ml stock so lu t ion of Cardiac-Green dye 1 (Indocaynine Green) in to the dorsa l a o r t a . The cannula was immediately f lushed fo l lowing dye i n j e c t i o n by a l t e r n a t e l y withdrawing and r e f i l l i n g i t s contents with heparinised Cortland sa l ine (Wolf, 1 9 6 3 ) . S e r i a l 0 .25 ml blood samples were taken at i n t e r v a l s for up to 5 minutes a f t e r dye i n j e c t i o n . Each time a sample was withdrawn an equivalent volume of sa l ine was returned to the f i s h . A p l a s t i c 3-way valve proved use fu l f o r blood sampling and sa l ine i n j e c t i o n . Usual ly 5 or 6 s e r i a l blood samples were taken f o r each run. The blood from each sample was t rans ferred to three 75 mm long Clay-Adams mierohematoorit tubes ( I .D . 1 . 1 - 1.2 mm). The tubes were sealed and spun f o r 3 minutes i n a microhematocrit centri fuge at 10 ,000 x g. The packed c o l l column was cut from each tube so that a 3 cm column of plasma remained. Then the contents of the three tubes f o r each sample were transferred to a s ingle cuvette. The plasma was d i l u t e d with 3 ml of d i s t i l l e d water and read at 800 mm i n a Turner model 330 spectrophotometer with an i n f r a r e d photo-diode and i n f r a r e d b locking f i l t e r . The meter was set to 100$ transmittanoe with a plasma blank obtained p r i o r to dye i n j e c t i o n . 1 Hynson, Westcott and Dunning, I n c . , Balt imore Maryland, 2 1 2 0 1 . 79 Heart r a t e and d o r s a l a o r t i c b l o o d p r e s s u r e were recorded immediately b e f o r e and a f t e r each c i r c u l a t i o n time run by connecting the cannula to a St/atham P 23 BB p r e s s u r e t r a n s d u c e r and a Beckman RS Dynograph r e c o r d e r . Average d o r s a l a o r t i c p r e s s u r e s were c a l c u l a t e d u s i n g the formula f o r a r e a mean pr e s s u r e (p. 6 1 ) . Only r e c o r d s from f i s h t h a t remained q u i e t d u r i n g the sampling p e r i o d a r e i n c l u d e d i n t h i s study. O c a s s l o n a l l y , f i s h became e x c i t e d and s t a r t e d to swim about d u r i n g a c i r c u l a t i o n time d e t e r m i n a t i o n . Data from such f i s h were d i s c a r d e d as changes i n c a r d i a c output a s s o c i a t e d w i t h a c t i v i t y would a l t e r the r a t e a t which dye r e t u r n e d to the i n j e c t i o n s i t e . RESULTS V I n f r a r e d Photography of the G i l l s L i k e mammalian b l o o d , t r o u t b l o o d showed an oxygen s a t u r a t i o n dependent c o l o r r e n d i t i o n on i n f r a r e d c o l o r f i l m . Oxygenated b l o o d was dark r e d , deoxygenated b l o o d , y e l l o w and 50% oxygenated b l o o d a s l i g h t l y l i g h t e r r e d than the oxygenated bl o o d ( P l a t e 1 ) . T h i s c o l o r d i s t i n c t i o n was not as evident however, when the blood was drawn i n t o a t h i n l a y e r , as shown by the m e n i s c i i n P l a t e 1. The t y p i c a l appearance of the g i l l s viewed through the o p e r c u l a r window of q u i e s c e n t t r o u t and r e c o r d e d on i n f r a r e d f i l m i s shown l n P l a t e 2 a. Note t h a t the g e n e r a l c o l o r of the secondary l a m e l l a e i s y e l l o w i s h and the l a m e l l a e a r e p a l e a t the f i l a m e n t t i p s . Time sequence photographs taken a t 10-minute i n t e r v a l s (Three f i s h , 11 C) over a 200-minute span f a i l e d to Plate 1 An i n f r a r e d photograph of trout blood samples e q u i l i b r a t e d to d i f f eren t oxygen tensions. The dark red blood on the l e f t was f u l l y saturated with oxygen while the yellow sample on the r i g h t was conplete ly unsaturated. The tube l n the centre contains a 50% saturated so lu t ion made by mixing equal volumes of oxygenated and deoxygenated blood. Samples were he ld under o i l and photographed i n a i r . The appearance of the samples was s i m i l a r when photographed under water. Plate 2 . v . ; A. T y p i c a l Infrared photograph of a por t ion of the g i l l of a quiescent trout taken through a window cut i n the operculum. Note the general co lor of the lamellae and the pale lamellae at the t i p s of each f i lament . B. The same f i s h a short time l a t e r photographed 5 minutes a f t e r adrenal ine was in jec ted i n the sub ln te s t ina l v e i n . The exposure and l i g h t i n g condit ions for A and B were I d e n t i c a l . Note that the co lor of the lamellae i s darker and that those lamellae at the t i p of each f i lament are no longer pale . The l i g h t e r "bars" i n each p i c ture are the g i l l f i laments and the lamellae protrude at r i g h t angles to them. 80 revea l any departure from t h i s bas ic appearance. In jec t ion of adrenal ine however, P late 2 b , produceda general reddening of the lamal lae , e spec ia l l y those at the t i p of the f i laments . The co lor of ...the g i l l sbecame i most red wi th in 2 to 4 minutes a f t er i n j e c t i o n and remained that way f o r about 10 minutes. A f t e r that the g i l l s gradual ly became more yellow and had resumed t h e i r pre-l n j e c t i o n appearance wi th in 45 minutes a f t e r adrenal ine admin i s tra t ion . Inject ions of equivalent volumes (0.3 - 0.5 ml) of s a l i n e , used as the veh ic l e f or epinephrine i n j e c t i o n , f a i l e d to produce any change i n appearance of the g i l l s . One i n d i v i d u a l (Plate 3) that had a port ion of the operculum removed damaged i t s g i l l s against the side of the box. In t h i s i n d i v i d u a l i n f r a r e d photographs showed that the g i l l was apparahtly_ gorged with blood at a l l times and adrenal ine i n j e c t i o n produced no a l t e r a t i o n i n J>he appearance of the g i l l s . I t was evident that the adrenal ine had entered the c i r c u l a t i o n of t h i s f i s h fo l lowing the i n j e c t i o n as i t "coughed" repeatedly and showed a 30$ r i s e i n VR. Unfortunately the adrenal ine a v a i l a b l e was o ld stock and had been exposed to l i g h t so determination of the dosage necessary to produce g i l l d i l a t i o n was not poss ib le . Had the adrenal ine been f u l l y ac t ive then the response shown i n Plate 2 would have been produced by 10/*g epinephrine d isso lved l n 0.5 ml s a l i n e . L i g a t i o n of G i l l Arches Cardiovascular and re sp ira tory parameters f o r 11gated f i s h i n the flow-through boxes are i l l u s t r a t e d i n Table yir. Ligated f i s h can be compared with non- l igated controls l n the tab le . Plate 3  Infrared photograph of the g i l l of a trout with a por t ion of the r i g h t operculum removed. This f i s h damaged i t s g i l l s on the sides of the box ( l i g h t a r e a ) , T h e appearance of the g i l l s d id not change fo l lowing adrenal ine i n j e c t i o n i n t h i s i n d i v i d u a l . 81 A r t e r i a l PQ 2 i n the contro l group was 100.2 - 4 .1 mm Hg and remained high as long as the blood supply to arch number 1 was i n t a c t , even when 40$ of the t o t a l g i l l surface area was removed by l i g a t i o n of arches 2 and 4 on both s ides . When arch number 1 was l l g a t e d both sides Pan,2 d r o P P e a to 56.8 - 7.9 mm Hg fo l lowing only a 28$ reduction i n g i l l area . When arches number 1 and 2 were t i e d o f f on both s ides , reducing the g i l l area by 57$, mean P a o 2 dropped to 40 .3 * 5 .2 mm Hg. As success ively greater areas were removed V o 2 rose ( F i g . X0) and the magnitude of the r i s e was most dramatic i n f i s h with eight hemibranchs removed and the blood supply to arch number 1 i n t a c t . Cardiac output, ca l cu la ted by the f l c k p r i n c i p l e using an assumed venous oxygen and carbon dioxide tension of 30 and 2 - 3 mm Hg respec t ive ly (part I I ) , rose i n accordance with reduct ion i n g i l l area ( F i g . 20). Q was not ca l cu la ted for f i s h with arch number 1 l i g a t e d on both sides as P a g 2 f e l l i n those i n d i v i d u a l s and i t was thought that P v o 2 m i S n t f a l l as PaQ^ dec l ined . Under these circumstances the assumption that P v o 2 w a s 30 1 1 1 1 1 1 ^ g might not ho ld . In f i s h with arch number 1 i n t a c t and eight hemibranchs removed, ca l cu la ted Q rose to 24 * 2.^ ml/min - roughly a four -f o l d increase above the cardiac output of the non- l igated contro l group. The r e s u l t s of the v e n t i l a t i o n study on l i g a t e d f i s h are given i n Table 7 m . Included are s i m i l a r values for non- l igated f i s h from part II f o r comparison. There i s a 3 degree temperature d i f ference between the non- l igated and l i g a t e d groups. The group with four arches l i g a t e d (eight hemibranchs removed) and arch F i g u r e 20 The r e l a t i o n s h i p between a r t e r i a l oxygen tension, oxygen uptake rate and ca lcu la ted cardiac output i n groups of f i s h with various port ions of the g i l l s l i g a t e d . Values are means and are shown - 2 standard e r r o r s . I f the standard errors were too small to include accurate ly on the graph, they were omitted. S o l i d squares are f o r groups with the blood flow to arch number 1 i n tac t on both s ides . Open squares are f o r groups with the blood supply to arch number 1 destroyed (only on one side f o r the group where two hemibranchs were l i g a t e d ) . .... -I ! ' • ! i L i - j - : : : : i J : | : | .^.r J :2 i . ; ; j : i t iTi ; 4rrj-»; 1:  6 .j .ij;ri:!|i^^;:::;:j-irt:;r;:. : 40r\ .;.-.:..! I., -j ....... J.. ;. .. . , . | . . . ! . . . 1.. . - R . . , , . . j ::' ...  i , , , ,.,.-| , ...... til' number' m-!&H': f -H of i ligated; TO hemibranchs:: • !. ! j i LM. f -1 T'i-.i:-!.!4i ].:.i:;: : j - | J ;4 : ; i i i . . N - j—. • . - I . 1 i,i .:• .i Table V I I I . Data from l i g a t e d trout i n the measurement box with o r a l membranes attached. Values f o r non- l lgated f i s h i n part II are included f o r comparison. % G i l l R e m o v e d P a t t e r n o f L i g a t i o n A r c h N o . 1 N o . B l o o d S u p p l y F i s h V R V s v m l / m i n # / m i n m l / b r e a t h U V ° 2 m g / k g / h r T e m p °C. 0 n o n e - c o n t r o l s i n t a c t 18 x : 3 7 . 0 7 3 . 8 0 . 5 4 6 . 0 5 5 . 3 8 . 6 S . D . 7 . 4 1 4 . 3 0 . 1 6 . 5 8 . 9 0 . 9 S . E . 1 . 8 3 . 4 0 . 02 1 . 5 2 . 1 0 . 2 n : 18 18 18 18 18 18 57 A r c h e s N o . 1 § d e s t r o y e d 5 X 8 3 . 3 9 3 . 8 0 . 9 3 4 . 1 7 1 . 9 1 1 . 6 2 b o t h s i d e s S . D . 4 3 . 5 9 . 0 0 . 4 9 . 7 2 . 2 0 . 1 S . E . 6 . 5 1 . 4 0 . 1 2 . 1 0 . 5 0 . 03 n 43 42 4 1 21 21 5 40 A r c h e s N o . 2 § i n t a c t 4 X 1 1 5 . 6 9 7 . 9 1 . 2 2 7 . 0 7 7 . 9 1 1 . 8 4 b o t h s i d e s S . D . 5 6 . 4 3 . 5 0 . 5 9 . 0 2 . 3 0 . 2 S . E . 9 . 7 0 . 6 0 . 1 1 . 8 0 . 5 0 . 08 n 34 33 33 25 25 4 82 number 1 in tac t had VQ'S that were about three times higher than the non- l igated group. In a d d i t i o n , percent util izationccwas 27$ and x VQ 2 W A S 42$ higher than the non- l igated group. The group with four arches l i g a t e d and the blood supply to arohssnumber 1 destroyed on both sides had a X V G that was 100$ above that of the contro l group and a x VQ 2 50$ above that of the contro l s . F i s h with arch number 1 i n t a c t and a s i m i l a r reduction i n g i l l area had VQ^ ' S that were 100$ above the contro l group and V G ' s that were 300$ that of the contro l s . Thus the group with arch number 1 i n tac t responded more f u l l y to the 50$ reduct ion i n g i l l area than the group with arch number 1 destroyed. C i r c u l a t i o n Time Determinations A t y p i c a l c i r c u l a t i o n time run i s i l l u s t r a t e d In F i g . 21. The dye returns to the i n j e c t i o n s i t e as a rather broad peak which, l n t h i s case, has a maximal o p t i c a l density 96 seconds a f t e r i n j e c t i o n . Results of determinations on nine f i s h are summarized i n Table ix. C i r c u l a t i o n times v a r i e d from 48 to 96 seconds with a mean value of 64.1 i 5 . 5 seconds. Heart rate had a mean value of 64,- 10.6 beats/minute p r i o r to dye i n j e c t i o n and d i d not r i s e s i g n i f i c a n t l y fo l lowing the sampling procedure ( t - t e s t , p = .05). Area mean dorsa l a o r t i c blood pressure was 26 - 0.9 mm Hg and d id not r i s e s i g n i f i c a n t l y ( t - t e s t , p = . 0 5 ) a f t er sampling. Figure 21 A t y p i c a l dye d i l u t i o n curve obtained ^^ f i n j e c t i o n of Cardio-Green dye i n the dorsa l aorta of a s ingle t r o u t . Data i s f o r f i s h number 4 i n table i x . Table IX Times for reappearance of Cardio-Green dye i n j e c t e d , i n the dorsa l aorta of nine rainbow t r o u t . Only f i s h that d i d not move about during the dye i n j e c t i o n and sampling procedure are inc luded. F i s h # W e i g h t g T e m p . ° C S e c o n d s t o D y e P e a k A p p e a r a n c e 1 2 3 5 12 90 2 2 3 0 " 12 60 2 2 3 0 12 65 3 2 2 8 12 54 4 2 2 9 12 96 5 2 3 0 1 3 48 6 1 9 0 8 49 7 1 9 1 8 55 8 200 8 . 5 60 9 1 7 4 8 60 X 2 1 1 . 9 1 0 . 4 6 4 . 1 n 9 9 10 S . D . 5 9 . 2 2 1 6 . 4 S . E . 1 9 . 7 0 . 6 5 . 5 83 t DISCUSSION V Infrared Photography of the © i l l s The experiments using in fraredphotography revea l that i t i s a use fu l technique f o r detect ing large changes supply to the g i l l s . Small changes i n blood d i s t r i b u t i o n wi th in a f i lament however, could not be resolved using t h i s technique. The lamellae were ye l lowish i n appearance but a f t e r adrenal ine i n j e c t i o n became red and swollen, Yellow co lors on i n f r a r e d f i l m are i n d i c a t i v e of e i ther deoxygenated hemoglobin or a very t h i n blood sample, Thea?e was no reason to suspect that blood i n the g i l l lamellae was deoxygenated as a r t e r i a l blood sampled from two f i s h was f u l l y saturated with oxygen p r i o r to adrenal ine i n j e c t i o n . The yel low co lor of the lamellae was therefore a r e f l e c t i o n of the small volume of blood i n the g i l l s . The lamellae appeared red and swollen a f t e r adrenal ine i n j e c t i o n probably because of an increase l n the volume of blood i n the g i l l s and not because of any change i n lamel lar blood oxygen sa turat ion . The r e s u l t s ind icate that adrenal ine increased the volume of blood i n the g i l l s , increas ing lamel lar blood f low, p a r t i c u l a r l y i n the d i s t a l regions of the f i lament . These i n v ivo observations are i n agreement with the i n v i t r o observations of Steen and Kryusse (196k) and Richards and Fromm (I969) who showed that adrenal ine increased lamel lar blood flow i n several t e l eos t species . 84 L i g a t i o n of G i l l Arches The oxygen uptake r a t e , v e n t i l a t i o n volume and ca lcu la ted cardiac output a l l Increased i n response to removal of success ively greater port ions of the surface area of the g i l l by l i g a t i o n of i t s blood supply. Thus the f i s h responded by increas ing the volume flow of both water and blood past the exchange surface. In so doing they d i d more work than normal non- l igated controls and thus had elevated oxygen uptake rates . As v e n t i l a t i o n volume increased percent u t i l i z a t i o n decreased, l i k e l y as a r e s u l t of the elevated water flow rate through the g i l l s ieve (see Part IV f o r a d i scuss ion of water flow a n d : u t i l i z a t i o n ) . G i l l area i n r e s t i n g trout can be reduced by nearly 50% and the f i s h can s t i l l maintain f u l l oxygen saturat ion of a r t e r i a l blood even i n the face of a doubling of Vo£» Thus adequate gas t rans fer can be maintained even i f the func t iona l area of the g i l l i s much l e s s than the anatomical area . The degree to which f i s h responded and the l e v e l at which they regulated t h e i r a r t e r i a l P 0 ^ appeared to be re la ted to the i n t e g r i t y of blood flow to the f i r s t g i l l a rch . I f there was jab-flow of blood to t h i s arch a r t e r i a l oxygen tension was l o w -p a r t i c u l a r l y i n those f i s h with eight hemibranchs l i g a t e d . V e n t i l a t i o n volume was approximately doubled and VR was up somewhat i n comparison to the controls In f i s h with arch pa i r s number 1 and 2 l i g a t e d but the Increased v e n t i l a t o r y flow d id not appear s u f f i c i e n t to saturate the blood. There are no ca lcu la ted 8 5 cardiac output f igures f o r t h i s group. F i s h with a s i m i l a r reduct ion i n g i l l s u r f a c e area but with blood flow to the f i r s t arch in tac t were able to maintain a r t e r i a l oxygen tension at saturat ion l e v e l . They responded to reduced g i l l area by e levat ing VQ about t h r e e - f o l d and Q about f o u r - f o l d . I t thus appeared that the l a t t e r group were able to saturate a r t e r i a l blood by producing the necessary adjustments of water and blood flow through t h e i r g i l l s . What i s unique about the d i s t r i b u t i o n of blood to arch number 1 ? These r e s u l t s could be explained i f t h i s arch was the s i t e for a large por t ion of the gaseous exchange l n these f i s h . Thus i f arch number 1 were removed, a drop i n a r t e r i a l tension would r e s u l t . Such a p o s s i b i l i t y seems u n l i k e l y and i n f r a r e d photographs of the whole g i l l on one side taken when apportion of operculum was removed show no spec ia l blood d i s t r i b u t i o n to arch number 1 . A more p laus ib le explanation may r e l a t e to the l o c a t i o n of the pseudobranch. Laurent ( 1 9 6 7 ) has found evidence f o r chemoreceptorand baroreceptor a c t i v i t y In the pseudobranch. Figure 1 9 shows that the pseudobranch receives i t s bipod supply from an extension of the f i r s t efferent a r t e r y . L i g a t i o n of arch number 1 on both sides would rob both pseudobranchs of t h e i r blood supply and lead to degeneration of t h e i r t i s s u e s . Thus those f i s h with arch number 1 l i g a t e d had no func t iona l pseudobranch. I t i s perhaps s i g n i f i c a n t that those animals without pseudobranchs were unable to maintain a r t e r i a l P Q 2 l e v e l s i n the face of reduced g i l l surface area . Thus the pseudobranch may w e l l serve as a s i t e important i n the regu la t ion of a r t e r i a l oxygen tens ion. 86 Hpw might the pseudobranch funct ion as a s i t e f o r regulat ing^ P 0 ? L i g a t e d f i s h w i t h i n t a c t pseudobranchs elevate C» V G and Q i n the face o f r e d u c e d g i l l surface a r e a . Thus i f a chemoreceptor 1s involved , as suggested by L a u r e n t ( 1 9 6 7 ) , information from t h i s receptor may stimulate c i r c u l a t i o n and v e n t i l a t i o n . Changes l n blood d i s t r i b u t i o n wi th in the remaining g i l l t i s sue may a lso have taken place as l i g a t e d f i s h elevated VQ 2 i n the face of a 50$ reduct ion i n g i l l area. R e d i s t r i b u t i o n of blood to a greater port ion of the remaining area i n the presence of elevated Q would f a c i l i t a t e an increased oxygen uptake. More experiments are planned to invest igate the r o l e of the pseudobranch as a receptor s i t e l n t rout . I t must be emphasized that although f i s h with no pseudobranch were unable to maintain high PaQ^'s, they d id respond to reduced g i l l surface area . V e n t i l a t i o n volume was double that f o r the contro l group. Therefore s i t e s as we l l as the pseudobranch may be involved i n the regulat ion of c i r c u l a t i o n and v e n t i l a t i o n i n t rout . The nature of these o t h e r r e g u l a t o r y s i t e s remains completely unknownat t h i s time. Tay lor et a l , (1968) have suggested, on the bas is of a computer model of the f i s h card iovascu lar -re sp i ra tory system, that a receptor s i t e may be located In the venous system. The f i s h with in tac t g i l l c i r c u l a t i o n but the r i g h t operculum sewed shut, was able to maintain P o g at saturat ion l e v e l but appeared to be breathing hard. This animal had an oxygen uptake rate about 50$ above that of the undisturbed c o n t r o l s , but i t s ca lcu la ted Q d id not appear to be d i f f eren t 87 from that of the contro l s (probably because t h i s f i s h had such a high hematpcrit----Table^ ^ ) . _ I t appeared that t h i s f i s h could maintain e f f ec t ive gas exchange i n the face of a poor pattern of water flow to one side of the g i l l s but that considerable energy expenditure was required f o r i t to do so. Perhaps t h i s f i s h u t i l i z e d a r e d i s t r i b u t i o n of bipod wi th in the port ion of the g i l l r ece iv ing water and thus achieved a be t ter v e n t i l a t i o n / p e r f u s i o n r e l a t i o n s h i p i n the por t ion of the g i l l that was f lushed with water. I t would appear then, that quiescent rainbow trout can t o l e r a t e a nearly 50$ reduction of t h e i r g i l l area and maintain e f f ec t ive gas exchange provided the blood flow to the f i r s t g i l l arch i s i n t a c t . These studies d id not ind ica te what e f fect such a reduced g i l l areawwould have on a swimming f i s h . I t i s l i k e l y that swimming f i s h require l a r g e r g i l l areas to support the increased metabolic demands of a c t i v i t y and that a 50$ reduct ion l n g i l l area might severely r e s t r i c t swimming a b i l i t y . Although trout may to lerate a considerable reduct ion i n g i l l area when unstressed, i t i s doubtful that they would survive extensive g i l l damage i n nature when exposed to g i l l damaging po l lutants or attacked by g i l l paras i t e s . A study of.swimming l i g a t e d f i s h would be most u s e f u l , p a r t i c u l a r l y . i f i t could be re la ted to g i l l area reductions that occur l n nature. 88 C i r c u l a t i o n Time Determinations Estimates of c i r c u l a t i o n time i n 200 g...trout with the dye i n j e c t i o n technique showed that these f i s h have a c i r c u l a t i o n time of about 1 minute during i n a c t i v i t y . Estimates of c i r c u l a t i o n tlfcme based on blood volume {k% body weight, Smith, 1966) and cardiac output ( 7 . 9 - 0 . 8 ml/min, part II) f or these f i s h were a l so of the order of a minute. C i r c u l a t i o n time reported i n t h i s study are mean values only. Blood, passing f . " •- ' through d i f f eren t port ions of the c i r c u l a t o r y system w i l l have d i f f e r e n t c i r c u l a t i o n times. Undoubtedly there are areas of the c i r c u l a t i o n such as white muscle that may have a more s luggish c i r c u l a t i o n . Some of the blood took l e s s than 1 minute to re turn to the heart and thus would pass through i t more often than the c i r c u l a t i o n time f igures would suggest. In mammals, 63$ of the t o t a l blood iteolume i s contained i n areas with vigorous c i r c u l a t i o n such as the heart , pulmonary vesse l s , large veins and a r t e r i e s (Guyton, 1 9 6 2 ) . In f i s h i t i s poss ib le that a s i m i l a r percentage of blood i s v igorous ly c i r c u l a t i n g as f i s h , although lack ing double c i r c u l a t i o n , have a much less extensive c a p i l l a r y network i n comparison to mammals (Stevens, 1 9 6 8 ) . The c i r c u l a t i o n times reported here provide information on the p o s i t i o n i n g of receptors f o r the contro l of c i r c u l a t i o n and v e n t i l a t i o n . Randall and Smith (1967) demonstrated that rainbow trout respond to low environmental oxygen tensions by r a p i d l y increas ing breathing rate and amplitude. When i n s p i r e d oxygen tension f e l l below 80-100 mm Hg, P o 2 » a bradycardia developed w i t h i n a few seconds. I f the water flow over the g i l l s was 89 stopped a bradycardia followed within 5 seconds. Thus trout respond very rapidly to such s t i m u l i . If the c i r c u l a t i o n time i s about 60 seconds then an oxygen receptor i n the venous system as suggested by Taylor et a l ( 1 9 6 8 ), cannot be responsible f o r the sole control of v e n t i l a t i d n a n d c i r c u l a t i o n , The responses to hypoxia or cessation of g i l l water flow occur too rapidly f o r blood to have reached a venous receptor at these c i r c u l a t i o n times. Thus a receptor s i t e must be located i n the a r t e r i a l system close to the g i l l s or on the surface of the g i l l s themselves as suggested by Hughes ( 1 9 6 4 ) . The pseudobranch i s the possible s i t e of such a receptor as discussed e a r l i e r . Since we know the cardiac output and Stevens (1968) using radioisotope i n j e c t i o n estimated the g i l l blood volume of rainbow trout at O.67 ml /100 g we can calculate the time i t would take f o r a l l the blood i n the g i l l to be replaced with new blood l e . the g i l l clearance time. For a quiescent 200 g trout, Q i s 7 .9 ml/min and the g i l l blood volume would be 2 x 0 . 6 7 a 1 .34 miy G i l l clearance time would be: i 34 J x 60 = 1 0 . 2 seconds 7 .9 ' ' _ I f we assume a l l the blood i n the g i l l s i s a c t i v e l y replaced then the maximum time a given red blood c e l l could remain i n the g i l l s i s 1 0 . 2 s e c o n d s . D u r i n g t h i s time^peripd the blood would have to pass through an afferent branchial vessel i n the g i l l arch, enter; the filament blood supply, pass through a secondary lamella, enter the efferent f'tlamentar vessel and exit the g i l l s v i a the efferent vessel i n the g i l l arch. Therefore the path followed 90 by an I n d i v i d u a l r e d b l o o d c e l l i s c i r c u i t o u s and t h a t c e l l p r o b a b l y s p e n d s only a sh o r t time i n the secondary l a m e l l a which i s the p r i m a r y s i t e of gaseous exchange. Hughes ( 1 9 6 6 ) c a l c u l a t e d t h a t i t t a k e s 1 . 0 - 1 . 9 s e c o n d s f o r the b l o o d to c i r c u l a t e through the g i l l c a p i l l a r i e s o f a mackerel w i t h a c a r d i a c output of 80 ml/kg/mln. I t must be s t r e s s e d t h a t the d y e i n j e c t i o n technique p r o v i d e s on l y a rough estimate of c i r c u l a t i o n time i n t r o u t . The dye returns to the i n j e c t i o n s i t e as a r a t h e r broad peak hence there a r e i n a c c u r a c i e s i n determining when the dye peak reaches i t s maximum i n t e n s i t y . Indeed, the poor time r e s o l u t i o n 0*f sampling may cause one t o miss the p e r i o d of maximum dye i n t e n s i t y . Some of the dye may r e t u r n v i a the coronary c i r c u l a t i o n and c o n t r i b u t e to the broadness of the a b s o r p t i o n peak. A l s o , the b l o o d sampling procedure may d i s t u r b , c i r c u l a t o r y dynamics d u r i n g sampling, a l t h o u g h b l o o d p r e s s u r e s and h e a r t r a t e d i d not change s i g n i f i c a n t l y d u r i n g sampling i n these experiments. However, d e s p i t e these l i m i t a t i o n s , the p r e s e n t t e c h n i q u e does p r o v i d e a rough estimate of c i r c u l a t i o n time i n t r o u t t h a t i s c o n s i s t e n t w i t h the known bl o o d volume and c a r d i a c output of the s p e c i e s . 91 SUMMARY V An Infrared photographic technique was used to study the patterns of blood d i s t r i b u t i o n within the g i l l s of intact trout. No change i n g i l l v a s c u l a r i z a t i o n could be detected from sequential photos taken over a 200-minute period i n quiescent f i s h . Adrenaline i n j e c t i o n i n the subintestlnal vein produced a marked change i n the photographic appearance of the g i l l . The g i l l looked engorged with blood and the lamellae appeared swollen. More blood appeared to have entered the lamellae at the t i p s of each filament. Reductions i n surface area of the g i l l were a r t i f i c i a l l y produced by l i g a t i n g various g i l l arches and occluding t h e i r blood supply. Trout responded to reduced g i l l surface area by increasing cardiac output and v e n t i l a t i o n volume and probably by r e d i s t r i b u t i n g the blood i n the remaining g i l l area. Fish with blood flow to the f i r s t g i l l arch could maintain a r t e r i a l Po 2 at around 90 - 100 mm Hg despite a reduction i n g i l l surface area of nearly 50$ . In trout with no blood flow to arch number 1 Pag- f e l l to around 40 mm Hg when eight hemibranchs were removed. I t i s suggested that the regulatory s i t e f o r maintaining a r t e r i a l PQ may be l n the psuedobranch which receives oxygenated blood from arch number 1. Thus f i s h with non-functional pseudobranchs were unable to regulate Pa02. The 92 f a c t t h a t these f i s h d i d respond i n d i c a t e s t h a t t h e r e i s another r e g u l a t o r y c e n t r e capable of s t i m u l a t i n g v e n t i l a t i o n and c i r c u l a t i o n . C i r c u l a t i o n time s t u d i e s based on t i m i n g the reappearance o f dye i n j e c t e d i n the d o r s a l a o r t a i n d i c a t e d t h a t q u i e s c e n t 200 g t r o u t had a c i r c u l a t i o n time of about 1 minute a t 10 C. T h i s was In agreement w i t h c i r c u l a t i o n time estimates based on c a r d i a c output and bloo d volume. C o n s i d e r i n g t h i s c i r c u l a t i o n time the responses of t r o u t t o hypoxia o r reduced g i l l water flow a r e too rap-id t o be i n i t i a t e d s o l e l y by a venous r e c e p t o r , as some authors have suggested. Receptor s i t e s must be l o c a t e d on the g i l l s o r a t some s i t e s i n the a r t e r i a l system c l o s e t o the g i l l s such as the pseudobranch. 93 PART SIX A THEORECTICAL CONSIDERATION OP WATER AND BLOOD SHUNTING AT THE GILLS OP RAINBOW TROUT. In t h i s sect ion we w i l l t reat the g i l l as a simple seive of prec ise dimensions placed i n the path of re sp i ra tory flow. This approach allows us to carry out a t h e o r e t i c a l ana lys i s to study the flow of water and blood past the g i l l . The nomenclature appl i ed to the g i l l s ieve w i l l be that of Hughes (1966) where: 1 = the maximum length of the secondary lamellae d = the i n t e r l a m e l l a r distance b = the average height of the lamellae Hughes (1966) has provided the most d e t a i l e d measurements of the dimensions and surface area of the trout g i l l s to date. His measurements were made on brown t r o u t , Salmo t r u t t a , weighing 175 g. The f i s h used i n t h i s study were rainbow trout and l i k e l y had very s i m i l a r g i l l dimensions to Hughes' brown t rout . As the average s ize of the f i s h used i n t h i s study was approximately 200 g and Hughes gives data for a 175 g f i s h i t i s reasonable to assume that h i s f i gures w i l l apply to our 200 g i n d i v i d u a l s . Thus the 94 f o l l o w i n g d i s c u s s i o n r e f e r s to 200 g rainbow t r o u t w i t h g i l l dimensions assumed to be i d e n t i c a l to Hughes 175 g brown t r o u t such t h a t : 1 = 0 . 7 mm d = 0.023 mm b = 0 . 4 mm Hughes (1966) gave the t o t a l l a m e l l a r a r e a of h i s f i s h as 339 mm2/g or a t o t a l l a m e l l a r a r e a of 67,800 mm2 f o r a 200 g f i s h . S ince t h e l l a m e l l a e a r e thought t o be the primary gas exchange u n i t s of the g i l l (Hughes, 1966; Muir and Hughes, I 9 6 9 ) we can assume t h a t the l a m e l l a r a r e a equals the t o t a l p o s s i b l e a r e a a v a i l a b l e f o r exchange. Furthermore, on the b a s i s of a study of the b l o o d pathways i n the g i l l , Hughes (1966) and Byczkowska-Smyk (19$7) concluded t h a t only about 65$ of the l a m e l l a r a r e a r e c e i v e s b l o o d and a c t s as an exchange s i t e . T h e r e f o r e the maximal f u n c t i o n a l a r e a f o r a 200 g f i s h would be 65$ of the t o t a l a r e a or 4 4 , 0 7 0 mm2. Many workers have observed t h a t oxygen u t i l i z a t i o n d e c l i n e s as V G i n c r e a s e s (van Dam, 1938: Saunders, 1962; Hughes, 1966; Holeton and R a n d a l l , 1967 b ) . Hughes ( 1 9 6 6 ) , Saunders (1962) and R a n d a l l (1970) have e x p l a i n e d t h i s d e c l i n i n g u t i l i z a t i o n w i t h V G i n c r e a s e as b e i n g dependant upon deadspace phenomena i n the g i l l s . B a s i c a l l y , t h e r e a r e t h r e e types of deadspace as d e s c r i b e d by R a n d a l l (197Q): 1) d i f f u s i o n deadspace where water remains w i t h i n the pores of the g i l l f o r too s h o r t a time f o r b l o o d and water gas t e n s i o n s to e q u i l i b r a t e because of the slow d i f f u s i o n of oxygen i n water, 2) d i s t r i b u t i o n deadspace 95 a s s o c i a t e d w i t h u n e q u a l v e n t i l a t i o n a n d p e r f u s i o n o f t h e g i l l s i e v e s u c h t h a t more o x y g e n i s d e l i v e r e d t o t h e s i e v e t h a n i s r e q u i r e d t o s a t u r a t e t h e a r t e r i a l b l o o d a n d 3) a n a t o m i c a l d e a d s p a c e w h e r e w a t e r t a k e s a n o n - r e s p i r a t o r y p a t h t h r o u g h t h e g i l l s b e t w e e n t h e t i p s o f a d j a c e n t f i l a m e n t s o r a r o u n d t h e e d g e s o f t h e a n t e r i o r a n d p o s t e r i o r - m o s t f i l a m e n t s . L e t u s c o n s i d e r t h e a n a t o m i c a l d e a d s p a c e f i r s t . The r e s u l t s o f p a r t I I I i n d i c a t e d t h a t t h e g i l l r e s i s t a n c e d i d n o t c h a n g e a s V Q r o s e f r o m 4 0 t o 160 m l / m i n . T h u s i t w o u l d a p p e a r t h a t a n a t o m i c a l d e a d s p a c e d i d n o t i n c r e a s e o v e r t h a t r a n g e f o r t h e r e s i s t a n c e o f t h e s i e v e w o u l d h a v e d r o p p e d a s t h e g i l l f i l a m e n t t i p s moved a p a r t t o a l l o w n o n - r e s p i r a t o r y s p i l l a g e o f w a t e r ( S a u n d e r s , 1 9 6 1 ; P a s z t o r a n d K l e e r e k p p e r , 1 9 6 2 ) . T h e s e i n c r e a s e s i n V Q w e r e n o t a s s o c i a t e d w i t h c h a n g e s i n o x y g e n u t i l i z a t i o n f r o m w a t e r p a s s i n g o v e r t h e g i l l s . L a r g e i n c r e a s e s i n V Q h o w e v e r , a r e o f t e n a s s o c i a t e d w i t h a r e d u c t i o n l n o x y g e n u t i l i z a t i o n ( S a u n d e r s , 19&2: H u g h e s , 1966) i n d i c a t i n g •> a n i n c r e a s e i n a n a t o m i c a l d e a d s p a c e a t h i g h v e n t i l a t i o n v o l u m e s . A n a t o m i c a l d e a d s p a c e t h e n , may r e m a i n c o n s t a n t o v e r l o w t o m o d e r a t e V Q l e v e l s a n d t h e n i n c r e a s e a t h i g h e r V Q l e v e l s . >l C R a n d a l l (1970) g a v e a n e q u a t i o n f o r c a l c u l a t i n g t h e m a g n i t u d e o f t h e n o n - r e s p i r a t o r y w a t e r s h u n t a t t h e g i l l s . T h i s w a t e r s h u n t r e p r e s e n t s t h e c o m b l n e d f v o l u m e s o f t h e a n a t o m i c a l , d i s t r i b u t i o n a n d d i f f u s l o n a l d e a d s p a c e a n d i s c a l c u l a t e d groms V D s h u n t = V G ( P J 0 2 " p v e q . 02) (x) P I 0 2 96 where: V D shunt = volume of the water shunt i n ml. VQ = v e n t i l a t i o n volume i n ml. PE02 = expired oxygen tension In mixed expired water i n mm Hg. P j 0 2 - inspired oxygen tension i n mm Hg. P V E Q = oxygen tension i n water having the 2 same P Q 2 as venous blood reaching the g i l l s . Thus i f PEC-2 = pveq ®2 T N E N V"Q shunt « 0 and there i s no non-respiratory s p i l l a g e of water from the g i l l s . However, P E O 2 i s always greater than P V e q °2 s o t n e r e * s always some water involved i n the shunt (and the d i f f u s i o n resistance of the g i l l s i s not zero). Randall (1970) modified (1) so that V D shunt could be calculated from: (PEOo " pveq <>2 ~ * p0 2> VD shunt = V G f _ (2 P I 0 2 where: P Q 2 = the oxygen gradient between blood and water across the lamellar wall. so f a r A P Q 2 has not been measured but Randall (1970) calculated i t to be between Q and 20 mm Hg using the following equation (Hughes, 1^66; Randall, 1970): * p0o = V 0 o d ' * 760 '0 2 D' X'S, where: V Q 2 = the oxygen uptake rate, ml/min/flsh \ Table X Data used f o r the c a l c u l a t i o n of deadspace at the g i l l s of trout. Values given are means f o r two groups of f i s h . One group has o r a l membranes attached f o r d i r e c t determination of V G while the other group had a tube t i e d to the mouth f o r a r t i f i c i a l perfusion of the g i l l s . The sect ion of the thes i s from which the data was taken i s ind ica ted . with o r a l membranes ml/min V n ml/min % U T C Source-part 44 86 120 .16 .26 .43 45 41 43 150 150 150 83 89 86 14 14 14 III I I I III 45 84 .14 .16 44 25 153 157 85 118 11 11 IV IV perfused f i s h 360 500 .18 .26 6 7 158 155 148 144 11 11 IV IV 752 986 .32 .29 6 4 160 160 151 154 11 11 IV IV 97 d 1 = the thickness of the g i l l epithelium Df = the oxygen permeation c o e f f i c i e n t i n g i l l epithelium. A = the area of the g i l l lamellae In ( 3 ) we can set the term; d« x 7 6 0 —• * K, a constant D« x A; since d', and 6 1 are f i x e d values and equation (1) expresses a l l the deadspace phenomena at the g i l l s so we can set A = to 65$ of the lamellar area (the maximum functional area of the g i l l s ) . Thus: and A P 0 2 i s therefore d i r e c t l y r elated to V Q 2 . Substituting ( 4 ) i n ( 2 ) we have: V D shunt = V G ( P E C - 2 " p veq ° 2 " v 0 2 K > ( 5 ) p I 0 2 and the magnitude of the shunt i s therefore dependent upon both V G and V o 2 . Data reported i n parts III and IV were used to calculate V D shunt from ( 4 ) and ( 5 ) . In order to determine A P 0 2 w a s assumed that d 1 = 2/i(Hughes, and Grimstone, 1 9 6 5 ) , was 1 . 0 1 x 1 0 " 5 at 1 4 C and 0 . 9 ^ 3 x 1 0 " - 5 at 1 1 C (Randall, 1 9 7 0 ) and A = 4 4 0 . 7 cm2. The r e s u l t s of these calculations are shown i n Table IX. In f i s h with o r a l membranes attached the shunt water was about 3 0 $ of the t o t a l V G and d i d not increase over the V G TABLE XI The ca lcu la ted water shunt involved i n deadspace phenomena at the g i l l s . with o r a l membranes Vg ml/min % U V D shunt, ml P n mm Hg u2 V n shunt, % V 44 86 120 45 41 43 13.8 25.8 31.4 5.6 8.9 14.7 30.3 30 26.1 pe r f u s e d f i s h 45 84 360 44 25 6 14.4 44 249 5.1 5.8 6.6 31.9 52.2 69.1 500 752 986 7 6 4 330 514 685 9.5 10.7 10.6 66.1 67.8 69.4 98 range 44 - 120 ml/min. In fact there was a slight decline in the magnitude of the shunt, possibly representing a declining 1 distribution deadspace as V G increased. In the perfused f i s h , when water flow went up u t i l i z a t i o n dropped markedly and the calculated V Q shunt was large. These large deadspaces may have resulted in part from a poor pattern of water flow over the g i l l s during perfusion. Some of the perfusion rates were considerably higher than any V Q ' S measured from f i s h with oral membranes. At high perfusion rates the g i l l sieve may have been severely distorted and the water shunfctwouldbe=large. We mentioned that the water shunt calculated in Table XI was composed of anatomical, diffusion and distribution deadspaces. As pointed out by Randall (1970) i t i s d i f f i c u l t to assess the relative contribution of each of these deadspace phenomena to the overall deadspace. Klystra, e_t a l , (1967) examined gas transfer i n water-breathing dogs and derived an equation for determining diffusion deadspace in f i s h based on the diffusion properties of the system: V D 4 1 f f = V°  X I + 3 Dt/a* <6> where: V D d i f f • the size of the diffusion deadspace, ml V Q = the ventilation volume, ml/min D = the rate of diffusion of oxygen in water, cm2 sec. t = the time 1 ml of water takes to pass the g i l l s , sec. a = the diffusion distance 9 9 L e t us take D = 1 .7 x I O " 5 a t 1 4 C and 1.5 x 1 0 ~ 5 a t 11 C (from R a n d a l l , 1970 , assuming a temperature c o e f f i c i e n t of 2$ per c e n t i g r a d e degree) and a = h a l f the d i s t a n c e between adj a c e n t secondary l a m e l l a e , . 0 0 2 3 / 2 cm. From ( 6 ) we get the r e s u l t s shown i n Table X I I . In f i s h w i t h o r a l membranes d i f f u s i o n deadspace amounts to o n l y 2 - 5 $ of the V and thus c o n t r i b u t e s o n l y s l i g h t l y to G the t o t a l water shunt. A t h i g h p e r f u s i o n r a t e s V D d i f f r i s e s to as much as 33$ of V G and thus i s a s i g n i f i c a n t c o n t r i b u t o r to the o v e r a l l shunt. We see then t h a t the c o n t r i b u t i o n of d i f f u s i o n deadspace i s minimal except a t the h i g h p e r f u s i o n r a t e s . A t the h i g h p e r f u s i o n r a t e s a l a r g e VQ shunt, composed of both anatomical and d i f f u s i o n a l deadspace, would account f o r the very low u t i l i z a t i o n s of oxygen observed a t those p e r f u s i o n r a t e s (Table X ) . I t would t h e r e f o r e be advantageous f o r t r o u t of t h i s s i z e t o keep VQ w i t h i n a range where V D d i f f was low. The o r a l membrane s t u d i e s showed t h a t VQ i n q u i e s c e n t t r o u t was 4 4 ml/mln. A t t h i s VQ the d i f f u s i o n deadspace was e q u i v a l e n t to only 1 .89$ of the t o t a l water flow. E x t r a p o l a t i o n of d i f f u s i o n deadspace c a l c u l a t i o n s t o a VQ of 300 ml/min (the maximum observed i n f i s h w i t h o r a l membranes) i n F i g . 22 shows t h a t d i f f u s i o n deadspace would not exceed 1 5 $ a t t h a t VQ l e v e l . I t would appear then, t h a t d i f f u s i o n does not p r o v i d e a s i g n i f i c a n t r e s t r i c t i o n t o oxygen uptake except a t the h i g h e s t g i l l water f l o w r a t e s . The o t h e r component that may c o n t r i b u t e to o v e r a l l deadspace Is t h a t r e l a t i n g t o unequal v e n t i l a t i o n and p e r f u s i o n of the g i l l s w i t h b l o o d and water i e . — the d i s t r i b u t i o n deadspace. Table XII The ca lcu la ted d i f f u s i o n deadspace at the g i l l s of two groups of trout at various V ^ ' s . w i t h o r a l membranes m l / m i n t s e c / m l V D d i f f , ml V D d i f f , % V G 44 86 1 2 0 1 . 3 5 . 7 . 5 . 8 3 3 . 0 8 5 . 9 5 1 . 8 9 3 . 6 4 . 9 5 p e r f u s e d f i s h 45 84 3 6 0 5 0 0 7 5 2 9 8 6 1 . 3 4 . 7 2 . 1 7 . 1 2 . 0 8 . 0 6 . 9 8 3 . 3 2 5 4 . 3 99 2 0 6 3 2 2 2 . 1 7 3 . 9 5 1 5 . 1 1 9 . 8 2 7 . 4 3 2 . 6 F i g u r e 22 C a l c u l a t e d deadspace v a l u e s a t the g l l l s . o o f p e r f u s e d t r o u t and those w i t h o r a l membranes a t v a r i o u s g i l l water flow r a t e s . 40 r O 0 C O O 80 r § 0) 0 *— » 1 L 0 400 800 1200 p e r f u s i o n r a t e m l / m i n 100 Vascular shunting at the g i l l s was discussed i n d e t a i l i n part V and i t was mentioned that the func t iona l area of the g i l l s may-vary according to the re sp ira tory requirements of the f i s h . I t was suggested that quiescent f i s h might have a smaller func t iona l area than a c t i v e or stressed f i s h . Steen and Kryusse (1964) have s tudied patterns of blood flow i n v i t r o i n lamellae and f i laments of the common e e l , A n g u l l l a v u l g a r i s , and Richards and Fromm (1969) performed s i m i l a r studies on rainbow trout g i l l s . These workers demonstrated that there were three bas ic paths f o r blood flow through the g i l l : 1) through the lamel lae , 2) through the f i l amenta l sinus beneath the lamellae and 3) by means of a d i r e c t connection of the af ferent and efferent f i l amenta l a r t e r i e s at the t i p of the f i lament . Adminis trat ion of ace ty lch lo ine reduced blood flow to the lamellae and acce lerated i t to the other pathways while adrenal ine increased blood flow through the lamel lar c i r c u l a t i o n . Steen and Kryusse suggested that the l ame l lar pathway was the prime r e s p i r a t o r y one and impl ied that blood going v i a the other chaurmels i n the g i l l followed a non-respiratory pathway. I f t h i s i s so only a small port ion of the blood must dp^l l3w..<Gircuits other than the lamellae as the a r t e r i a l blood i n quiescent trout i s always 90 - 100$ saturated with oxygen (Holeton and Randal l , I 9 6 7 b; Stevens and Randa l l , 1967 b; Cameron and Davis , 1970; t h i s study). I t seems l i k e l y that most of the blood must move through the lamel lar c i r c u l a t i o n i n order f o r a r t e r i a l PQ 2 to remain at such high l e v e l s . Whether some or a l l of the lamellae are f u n c t i o n a l i s the major fac tor determing the s ize of the d i s t r i b u t i o n deadspace. I t may be that i n t e r l a m e l l a r d i s t r i b u t i o n deadspace ex i s t s as we l l 101 as d i s t r i b u t i o n deadspace r e s u l t i n g from some of the lamellae not rece iv ing blood or from various lamellae being perfused at d i f f e r e n t ra te s . At low cardiac outputs blood may move mainly wi th in only apport ion of the lamel la and as Q increases the lamellae may become f i l l e d with blood. Indeed, the lamellae contain marginal blood channels as we l l as lacunar spaces (Hughes and Grimstone, 1965; Richards and Fromm, 19^9) so there may be p o t e n t i a l deadspace areas wi th in the lamel lae . Newstead ( I 9 6 7 ) and Hughes and Grimstone (1965) postulated that c o n t r a c t i l e elements w i th in the p i l l a r c e l l s ( c e l l s forming the supportive s tructure of the lamellae) could act to regulate blood flow wi th in thej lamel lae . Richards and Fromm found smooth muscle i n the a r t e r i o l e s which supply blood to the lamellae from the f i lamentar a r t e r y and suggested that t h i s muscle may regulate blood flow to the lamel lae . Unfortunately there appears to be no way of est imating d i s t r i b u t i o n deadspace wi th in the g i l l s of our f i s h . Stevens ( I 9 6 8 ) was unable to demonstrate an increase i n g i l l blood volume during moderate ; exercise i n rainbow trout by radio isotope i n j e c t i o n , poss ib ly because of lack of s e n s i t i v i t y of technique. From the foregoing d i scuss ion i t i s l o g i c a l that d i s t r i b u t i o n deadspace may be high i n quiescent f i s h and much reduced and poss ib ly absent i n s tressed, ac t ive f i s h . We cannot however, say what proport ion of the t o t a l deadspace i s involved i n d i s t r i b u t i o n deadspace. Muir and Brown (197,1) presented an equation r e l a t i n g the blood pressure drop across the g i l l s of f i s h to the phys ica l s tructure of the g i l l . The ir equation i s a modi f i cat ion of that 102 used f o r pressure drop i n any flow passage; K >V1 . 9 P drop = dynes/cm' 1 (7) where: P drop = the pressure drop i n the passage, mm Hg. K = a constant that ranges from 12 f o r a t h i n passage of i n f i n i t e width to 30 f o r a c y l i n d r i c a l passage. = v i s c o s i t y of f l u i d i n passage, poise v" = v e l o c i t y of flow l n the passage 1 - the passage length d = diameter of the passage Brown and Muir modified t h i s equation f o r the f i s h g i l l and a r r i v e d at : " . 8 K >u. Q l 2 760 P drop = — j (8) d 3 (10 6 ) where: Q 1 A d = g i l l blood f lowe ml /sec . = length of lamel lae , cm = surface area of the g i l l s , cm' = diameter of blood channel, cm Let us apply t h i s equation to the r e s t i n g f i s h with o r a l membranes, These animals have a cardiac output of 8.0 ml/mln = 0.134 ml / sec , K = 3 0 as the blood channels can be considered as tubular (Muir and Brown, 1971), 1 = 0.07 cm (Table X ) , A = the f u n c t i o n a l area of the g i l l s = 441 cm 2 and d = .00045 cm (Muir and Brown, 103 1971) f o r rainbow trout of t h i s s i ze . Since the mean hematocrit of. the f i s h i n Table XIII was approximately 20 vo ls % we can set the blood v i s c o s i t y of the f i s h equivalent to the v i s c o s i t y of human blood at that hematocrit , .025 poise (Guyton, 1962) by assuming that f i s h and mammalian blood have s i m i l a r v i s c o s i t i e s . Subs t i tu t ing these values i n ( 8 ) g ives: P drop = 2 3 . 9 mm Hg This pressure drop i s of?the r i g h t order of magnitude as that measured f o r rainbow•&trout. Holeton and Randall (I967 a) reported that mean a r t e r i a l pressure between the dorsa l and v e n t r a l aortae i n r e s t i n g 200 - 1050 g trout at 9 - 19 C d i f f e r e d by about 20 mm Hg. Stevens and Randall ( I 9 6 7 b) i n 200 - 600 g f i s h , at 10 - 12 C and the re su l t s of part IVftshowed that mean a r t e r i a l and venous pressures across the g i l l s d i f f e r e d by approximately 10 mm Hg. One would expect that the pressure drop across the lamellae would be somewhat less than the pressure drop between the dorsa l and v e n t r a l aortae as the pressure between the two cannula placements would be inf luenced by the res i s tance of other blood channels as we l l as the lamel lae . The pressure drop ca lcu la ted above suggests that the values used i n equation ( 8 ) may not accurate ly represent the dimensions of the trout g i l l . Accurate values of d and 1 are d i f f i c u l t to determine and i t may not be correct to fol low Muir and Brown's example and set K = 30 s ince the lamellae are much higher than they are broad (Hughes, I966) and treating'!?the i n t e r i o r of the lamel la as a tube 104 may not fee correc t . As mentioned e a r l i e r there i s both i n v ivo and i n v i t r o evidence that the func t iona l area of the g i l l s i s smaller than theamaximum func t iona l area when the f i s h i s not s tressed. Thus i f the equation and the g i l l dimensions used i n i t are correct then one would expect that se t t ing A = some percentage of the t o t a l area would give a ca lcu la ted P drop equivalent to that measured from r e s t i n g t r o u t . I f we set A = $0$ of the f u c t i o n a l area then P drop = 60 mm Hg and i f A i s 80$ of the func t iona l area then P drop = 2 9 . 9 mm Hg. These pressures are higher than any that have been)measured l n r e s t i n g t r o u t . Pressure drop ca lcu la t ions then, do not appear a r e l i a b l e way of c a l c u l a t i n g d i s t r i b u t i o n deadspace. I f a l l the values used l n the equation were absolute ly r e l i a b l e then the approach might be u s e f u l , provid ing there was some way of assess ing the contr ibut ion of blood channels other than the lamellae to the pressure d i f ference across the g i l l s . We mentioned that oxygen uptake rate increases as V G goes up. The r e s u l t s of parts III and IY show t h i s f o r both normally breathing and perfused f i s h . Hughes and Saunders (1971) and Schumann and P i i p e r (1966) observed s i m i l a r increases i n V Q 2 i n r e l a t i o n to VQ . They assumed that these VQ^ Increases were the r e s u l t of an increased cost of breathing at high VQ'S . This assumption has been questioned by £ a m e r o n and Cech (1970) who contended that i t was unreasonable to assume the ent ire increase i n V 0 £ ftt elevated VQ'S was a r e s u l t of increased cost of breathing. Since we measured pressure across the g i l l s at d i f f e r e n t VQ'S we can ca lcu la te the work done i n breathing by m u l t i p l y i n g pressure times flow. Alexander (19&7) out l ined such 10£ ca l cu la t ions f o r both r e s t i n g and a c t i v e f i s h . Fol lowing •Alexander's reasoning f o r the group of f i s h i n part I I I then at a VQ of 40 ml/min the mean d i f f e r e n t i a l pressure across the g i l l s was .094 mm Hg which equals . 122 cm H2O. Since a pressure of 1 cm H2O equals 1000 dynes/cm 2 and an erg i s a dyne-cm,then at a V G of 40 ml/min the work done was ( . 1 2 2 x 1000) 40 = 4880 ergs. This i s equivalent to .0001125 c a l o r i e s ( 1 c a l = 4 3 . 3 x 10^ ergs) . I t i s u n l i k e l y however, that the muscles w i l l be 100$ e f f i c i e n t . Alexander ( I 9 6 7 ) assumed that the maximum e f f i c i ency of the muscles could not exceed 20$ . Jones (1971) be l ieved that the e f f i c i e n c y of the re sp ira tory pump was lower (around 3 to 4$). No one has measured the e f f i c i e n c y of the branch ia l pump i n f i s h but i t i s l i k e l y low due to the morphology of the re sp ira tory musculature and the way these muscles apply force to the branch ia l chambers. (Jones, personal communication). I f we assume that the pump i s only 1$ e f f i c i e n t f o r the purpose of a sample c a l c u l a t i o n then .0001125 x 100 = .01125 c a l are needed to pump 40 ml H20/min through the g i l l chamber. Since 1 ml of oxygen y i e l d s about 5 c a l o r i e s (Alexander, I 9 6 7 ) then . 0 1 1 2 5 / 5 = .00225 ml of oxygen i s required to support a VQ of 40 ml/min. Expressed as a percentage of t o t a l oxygen uptake (VQ 2 = .16 m l / m i n / f l s h . at a VQ of 40 ml/min) then the cost of breathing Is . 0 0 2 2 5 / . 1 6 x 100 = 1 .4$ of the t o t a l oxygen uptake r a t e . Let us take t h i s l i n e of reasoning and apply i t to the r e s u l t s of part I I I . The data we w i l l need are given i n Table X I I I . We s h a l l do the ca l cu la t ions us ing e f f i c i e n c y f igures f o r the branch ia l pump of 1 , 3 , 5 , 104 and 20$. The re su l t s of these ca l cu la t ions are shown i n Table XIV and F i g . 23 . C l e a r l y the Table XIII Data from a group of six trout (224.6 ± 7.0 g, 13.8 ± 0.8 C) from part III used f o r c a l c u l a t i o n of the oxygen cost of breathing. area mean d i f f e r e n t i a l pressure 2 ml/min mm Hg cm H~0 m l / m i n / f i s h 40 .094 .122 .16 80 .2 .272 .26 100 .25 .340 .337 120 .305 .415 .342 Table X I V The ca lcu la ted oxygen cost of breathing f o r the f i s h i n the X I I I expressed as a percentage of the t o t a l V Q 2 . The d i f f eren t percentage e f f i c i e n c y values assigned f o r the branch ia l pump are inc luded . C o s t o f B r e a t h i n g as a P e r c e n t a g e o f V n a t V G 2 V a r i o u s E f f i c i e n c i e s o f t h e m l / r a i n B r a n c h i a l Pump 1% 3% 5% 10% 20% 40 1.4 .47 .28 .14 .07 80 3. 86 1.28 .80 .39 .19 100 4.66 1.55 .93 .47 .24 120 6. 72 2.23 1.33 .67 .34 Figure 23 A p lo t of the ca lcu la ted cost of breathing at various V„»s G using f i v e d i f f e r e n t e f f i c i ency values f o r the branch ia l pump. ! ! T'T 1 1 II ! i ' ! MM 1 I i ; i cost of b r e a t h i n g ! i i M i ! i. J.I . i - i : 106 oxygen cost of breathing obtained from these calculations i s very low, even i f the branchial pump i s only 1% e f f i c i e n t . Jones (1971) concluded that the e f f i c i e n c y of the system would be low at low V G's (3 - 4 %) and that i t would improve at intermediate V _'s (k0%) and decline markedly at high V G's, Thus from F i g . 23 we can see that the oxygen cost of breathing might not change appreciably during low to moderate breathing i f the e f f i c i e n c y of the system improved as VQ rose. Jones has suggested that a low e f f i c i e n c y , combined with considerable work output, could cause the metabolic cost of v e n t i l a t i o n to l i m i t active metabolism at high temperatures. The calculations i n Table XIV agree well with the low cost of v e n t i l a t i o n (0.5$ of Vo 2) estimated by Alexander (1967) f o r re s t i n g f i s h . Alexander used the same approach to calculate the cost of breathing f o r active f i s h and Cameron and Cech (1970) c r i t i c i z e d Alexander's calculations on the basis that he made the following assumptions that had not been proven to be correct; 1) that the pressure gradient across the g i l l s was constant, 2) that increases i n v e n t i l a t i o n are achieved by increases i n pressure and 3) that g i l l resistance was constant. For the purposes of our calculations we can s a t i s f y the above objections by examining the r e s u l t s of part I I I . Those studies showed that V G appeared d i r e c t l y related to area mean d i f f e r e n t i a l pressure and that Increasing pressure l e d to elevated V G. Furthermore, the calculated g i l l resistance did not decline oyer the V G range used f o r the calculations i n Table 14. The s i t u a t i o n may be somewhat more complicated than t h i s due to the f l u c t u a t i o n of pressures and a l t e r i n g resistance of the g i l l 107 sieve that l i k e l y occur during a s ing le breathing cyc le . Information on the instantaneous r e l a t i o n s h i p of pressure and flow across the g i l l s during a s ingle cyc le would prove u s e f u l . I t seems u n l i k e l y that the res i s tance of the mouth or opercular apartures are appreciable enough to increase the work of breathing at low to moderate VQ'S due to the s ize of these aparitires , and the r e l a t i v e l y slow flow of water through them. At very high VQ'S the res is tance of the apa^tiires' might be a f a c t o r important i n the res is tance of the o v e r a l l system. Since the metabolic cost of breathing i s low, at l eas t during low to moderate VQ'S , what leads to elevated V o 2 A S VQ increases? Excitement or increased muscle tone as VQ increased could be behavioral fac tors that might elevate V Q 2 . I f the func t iona l area of the g i l l s Increased as VQ went up then there would l i k e l y be an increased f lux of water o r ions across the g i l l (Randal l , 1970) and a resu l tant increase i n kidney funct ion would be required to maintain osmotic balance. Elevated kidney funct ion would increase V Q 2 s l i g h t l y . I f a r e d i s t r i b u t i o n of blood to other parts of the body as we l l as the g i l l s occurs as VQ r i s e s a general increase i n t i s sue metabolic rate could r e s u l t as the t i s sues that were f lushed with blood u t i l i z e d the oxygen * made a v a i l a b l e to them. The subject of elevated V Q 2 at elevated VQ'S requires more study and at present i s not understood. Regardless of the cause of elevated V Q 2 A S VQ goes up, i t seems doubtful on the bas is of these c a l c u l a t i o n s , that the cause of increased metabolic rate i s a large increase i n the oxygen cost of breathing. 108 In summary, these ca lcu la t ions showed that the water Involved l n non-respiratory s p i l l a g e from the g i l l s amounted to 30$ of the v e n t i l a t i o n volume during low and moderate breathing and rose as high as 70$ when the g i l l s were r a p i d l y perfused. The main contr ibutors to o v e r a l l deadspace are l i k e l y to be anatomical and d i s t r i b u t i o n deadspace. At low V Q ' S anatomical deadspace i s probably minimal and d i s t r i b u t i o n deadspace may be l arge . At high g i l l water flows d i s t r i b u t i o n deadspace probably f a l l s to zero and the majority of the t o t a l water shunt i s composed of anatomical deadspace. D i f fus ion deadspace was <$ ca lcu la ted to amount to 2 - 5 % of the t o t a l water shunt during gentle and moderate v e n t i l a t i o n and rose to 33$ at the highest perfusion r a t e . During normal v e n t i l a t i o n i t i s l i k e l y that d i f f u s i o n deadspace does not exceed 15$ of the v e n t i l a t i o n volume even at the highest measured g i l l water flows. These ca l cu la t ions Indicate that i t would be advantageous f o r 200 g t rout to keep V Q between 40 and 300 ml/min to minimize d i f f u s i o n and anatomical deadspace phenomena. The ca lcu lated oxygen cost of breathing over t h i s V Q range appears to be small even i f the branch ia l pump is . -highly i n e f f i c i e n t . Thus the metabolic cost of water breathing i n 200 g trout would not appear to ser ious ly r e s t r i c t the oxygen supply for other metabolic processes and contributes only s l i g h t l y to increased metabolic rate at elevated V G ' s . 109 MAJOR FINDINGS OF THE THESIS In the Introduct ion It was stated that the object ive of t h i s thes i s was to examine the pattern and rate of water and blood flow across the g i l l s and demonstrate t h e i r inf luence on gas exchange. The thes i s has evaluated methods of measuring vent i la t ion volume i n trout and has described a new method f o r directly-measuring VQ . I t would appear that the o r a l membrane technique i s a more accurate method for VQ determination i n trout compared to F lck p r i n c i p l e c a l c u l a t i o n of VQ us ing data from opercular or c l e i t h r a l cannulae. In a d d i t i o n , the technique has permitted a de ta i l ed ana lys i s of the pressure-flow r e l a t i o n s h i p s at the g i l l s during low to moderate V Q ' S . The data obtained by the o r a l membrane technique, combined with that from a r t i f i c i a l perfusion of the g i l l s at d i f f e r e n t water flow ra te s , has provided a de ta i l ed account of the i n t e r r e l a t i o n of VQ , oxygen u t i l i z a t i o n , gas tension gradients between blood and water across the g i l l s , res i s tance of the g i l l s ieve , pressures i n the a r t e r i a l , venous and branch ia l systems and oxygen uptake rate i n rainbow t r o u t . This data has been used for t h e o r e t i c a l ana lys i s of the exchange process at the g i l l s i n part s ix . The a b i l i t y of the rainbow trout to r e d i s t r i b u t e the blood within the g i l l s and a l t e r blood flow through the g i l l s was a l so demonstrated using an i n f r a r e d photographic technique. This data provides i n v ivo evidence of blood shunting i n the g i l l s i n the presence of adrena l in . Previous studies of such shunts had been made using a r t i f i c i a l l y perfused g i l l f i laments i n v i t r o (Steen and Kryusse, 1964: Richards and Fromm, 1969). L i g a t i o n studies showed that trout could survive and maintain a high a r t e r i a l P Q 2 i n the face of a considerable reduct ion i n g i l l surface area , 110 provided that the f lood flow to the f i r s t g i l l arch was i n t a c t . Rainbow t rout , therefore , can a l t e r the func t iona l area of the g i l l s by varying blood d i s t r i b u t i o n and s t i l l maintain adequate rates of gas exchange i n the face of reduced g i l l area. This study allowed a t h e o r e t i c a l ana lys i s of fac tors in f luenc ing gas exchange at the g i l l s . At low VQ'S the non-resp ira tory water shunt at the g i l l s i s small and i s composed of anatomical and d i s t r i b u t i o n deadspace and a small d i f f u s i o n deadspace. When V^ r i s e s to moderate l eve l s u t i l i z a t i o n does not change and anatomical deadspace does not increase . Increased oxygen uptake and a constant u t i l i z a t i o n i n the face of increased g i l l water flow at moderate VQ'S i s probably f a c i l i t a t e d by d e c l i n i n g d i s t r i b u t i o n deadspace. At high VQ'S u t i l i z a t i o n of oxygen drops due to a large anatomical and d i f f u s i o n deadspace but oxygen uptake rate i s h igh , probably because d i s t r i b u t i o n deadspace i s minimal or absent. Thus the patterns of both water and blood flow through the g i l l s are important to the gas exchange process i n rainbow trout . The f indings of these experiments allowed considerat ion of other fac tors important to gas exchange i n t rout . Pressure/f low relationsfein the g i l l cavi ty were used to ca lcu la te the oxygen cost of breathing, assuming various e f f i c i e n c i e s of the branchia l musculature. These ca l cu la t ions showed that the oxygen cost of breathing i s smal l , even i f the pump i s only 1% e f f i c i e n t . Some i n d i c a t i o n of how the r e s p i r a t o r y and c i r c u l a t o r y systems may be contro l l ed was a l so obtained. C i r c u l a t i o n times and l i g a t i o n studies Indicated that a receptor s i t e sens i t ive to a r t e r i a l PQ^ may be located i n the g i l l reg ion , poss ib ly wi th in the pseudobranch. I l l LITERATURE CITED Alexander, R. McN. (1970). Funct iona l Design i n F i shes . Hutchinson and Co. L t d . London. l 6 0 p. B a g l i o n i , S. (1910) Zur vergleichenden Physlo logie der Atembewegungen der W i r b e l t i e r e . 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