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The effect of ingested NACL on body temperature in two avian species : the domestic fowl and the glaucous-winged… Kojwang, David Ogweno 1991

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THE EFFECT OF INGESTED NACL ON BODY TEMPERATURE IN TWO AVIAN SPECIES; THE DOMESTIC FOWL AND THE GLAUCOUS-WINGED GULL. By DAVID OGWENO KOJWANG B.Ed (Sc), KENYATTA UNIVERSITY COLLEGE, NAIROBI, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the requ i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA Zoology, 1991 (g) David Kojwang, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writteh permission. Department of Z O Q UQGTV The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The r e l a t i v e e f f e c t s of ingested sodium c h l o r i d e (NaCl) on body temperature were examined i n two avian s p e c i e s ; the domestic fowl (Gallus domesticus), a t e r r e s t r i a l b i r d which excretes NaCl r e n a l l y , and the Glaucous-winged g u l l (Larus qlaucescens), a marine b i r d which excretes NaCl both r e n a l l y and e x t r a r e n a l l y . Plasma sodium ([N a + ] p l ) , potassium ( [ K + ] p l ) , c h l o r i d e ( [ C l " ] p l , i o n i z e d ([Ca 2 +] p l) and t o t a l calcium ([Ca] p l) c o n c e n t r a t i o n s , plasma o s m o l a l i t y (Osmol p l), body temperature, evaporative water l o s s (EWL), t o t a l body water (TBW), r e s p i r a t o r y frequency ( f ) , minute v e n t i l a t i o n (VE) , and panting thresholds were determined i n r o o s t e r s and g u l l s exposed to i n c r e a s i n g d i e t a r y NaCl l e v e l s . High d i e t a r y NaCl s i g n i f i c a n t l y r a i s e d g u l l r e s t i n g body temperature (40 . 4°C +. 0 . 4°C) to 41. 0°C +. 0 . 5°C (P<0.05) but d i d not a l t e r r o o s t e r body temperature (41.0°C +. 0.2°C) s i g n i f i c a n t l y . [Na +] p l increased by 5.4% (P<0.01) i n g u l l s and by 3.7% (P<0.01) i n r o o s t e r s , and was c o r r e l a t e d w i t h body temperature i n g u l l s (r = 0.497, n = 25) . . [Ca 2 +] p l increased ( r o o s t e r s , 5.5%, P<0.01; g u l l s , 11.8%, P<0.01) when [Na +] p l was high. Plasma sodium to calci u m r a t i o s were not a l t e r e d and were u n r e l a t e d to body temperature i n both species. High d i e t a r y NaCl el e v a t e d the i n t e r n a l t h r e s h o l d f o r panting i n roosters from 41.95 +. 0.41°C to 42.35 +.-0.3 6°C (P<0.05), and the increase was a s s o c i a t e d w i t h an in c r e a s e i n Osmol p l (303.6 +. 4.0 mosmol/kg to 319.5 +. 4.3 mosmol/kg, P<0.01). In g u l l s Osmol p l d i d not in c r e a s e , n e i t h e r d i d panting t h r e s h o l d . Under thermal s t r e s s , body temperature rose more r a p i d l y i n both roosters and g u l l s on high s a l t d i e t s . VE, f, and w e i g h t - s p e c i f i c EWL at 30°C, were un a f f e c t e d i n both species by high s a l t i n t a k e . TBW was not r e l a t e d to r e s t i n g body temperature and d i d not change s i g n i f i c a n t l y w i t h h i g h NaCl i n t a k e i n e i t h e r species. I t i s concluded that r e s t i n g body temperature i s ele v a t e d by inc r e a s e d [Na +] p l i n concentration-dependent f a s h i o n r a t h e r than a r a i s e d Na : Ca r a t i o i n b i r d s w i t h and without s a l t glands. [ C a 2 + ] p l i s elevated when [Na +] p l i s high. The a d d i t i o n a l Ca 2 + probably o r i g i n a t e from a source other than the bound f r a c t i o n i n plasma; p o s s i b l y from the i n t r a c e l l u l a r Ca 2 + p o o l . The a b i l i t y of a b i r d to maintain a s t a b l e panting t h r e s h o l d i s r e l a t e d to i t s a b i l i t y to maintain a s t a b l e Osmol p l. EWL, VE, and f are una f f e c t e d by NaCl intake unless severe heat s t r e s s i s imposed. i v T A B L E OF CONTENTS A b s t r a c t i i L i s t of t a b l e s v i L i s t of f i g u r e s v i i L i s t of Appendices i x Acknowledgements x 1 INTRODUCTION 1 1.1 S a l t loading and plasma e l e c t r o l y t e s t a t u s 1 1.2 Cations and body temperature 2 1.3 The hypothalamic s e t - p o i n t theory 3 1.3.1 Species d i f f e r e n c e s 4 1.4 Plasma o s m o l a l i t y and evaporative water l o s s 5 1.5 Hypotheses 6 2 MATERIALS AND METHODS 8 2.1 Animal care 8 2.2 Transmitter c a l i b r a t i o n and i m p l a n t a t i o n 9 2.2.1 Anaesthesia 10 2.2.2 Surgery 10 2.3 Experimentation 11 2.3.1 NaCl a c c l i m a t i o n 12 2.3.2 Blood sampling 13 2.3.3 Plasma a n a l y s i s 14 2.3.4 T o t a l body water 15 2.3.5 Evaporative water l o s s 16 V 2.3.6 V e n t i l a t i o n and panting t h r e s h o l d 18 2.4 Data a n a l y s i s 22 3 RESULTS 24 3.1 Plasma i o n i c and osmotic concentrations '24 3.2 Body temperature 32 3.3 Body mass and t o t a l body water 33 3.4 Hematocrit 38 3.5 Evaporative water l o s s 38 3.6 R e s p i r a t o r y r a t e and panting t h r e s h o l d 38 3.7 R e l a t i o n s h i p s between v a r i a b l e s 40 4 DISCUSSION '. 47 4.1 Plasma sodium and osmotic concentrations 47 4.2 Plasma calcium 49 4.3 Plasma sodium to calcium r a t i o s and body temperature 51 4.4 T o t a l body water and body temperature 55 4.5 Evaporative water l o s s 56 4.6 V e n t i l a t i o n 59 4.6.1 S a l t loading and v e n t i l a t i o n 59 4.6.2 Panting t h r e s h o l d 61 4.6.3 Panting p a t t e r n 64 4.7 Conclusion 66 5 References 68 6 Appendices ••• 83 v i L I S T OF T A B L E S I Plasma sodium, c h l o r i d e , potassium, i o n i z e d and t o t a l c a l c i u m c o n c e n t r a t i o n s , sodium to c a l c i u m r a t i o s , o s m o l a l i t y and deep body temperature i n 6 r o o s t e r s f e d normal and h i g h NaCl d i e t s r e s p e c t i v e l y 25 II Plasma c o n c e n t r a t i o n s of sodium, i o n i z e d calcium, t o t a l c a lcium, c h l o r i d e , potassium; plasma o s m o l a l i t y , sodium to c a l c i u m r a t i o s , and body temperature of g u l l s d r i n k i n g 0 mM NaCl, and d u r i n g p r o g r e s s i v e a c c l i m a t i o n to 475 mM NaCl 28 I I I Body mass, t o t a l body water, hematocrit and e v a p o r a t i v e water l o s s i n : (i) 6 r o o s t e r s fed normal and h i g h NaCl d i e t s r e s p e c t i v e l y , and ( i i ) 5 g u l l s d r i n k i n g 0 mM NaCl and d u r i n g p r o g r e s s i v e a c c l i m a t i o n to 475 mM NaCl 35 IV R e s t i n g body temperature and p a n t i n g t h r e s h o l d i n r o o s -t e r s and g u l l s f ed normal and high NaCl d i e t s r e s p e c t i -v e l y 42 V R e s p i r a t o r y frequency and minute v e n t i l a t i o n at r e s t and at maximum pan t i n g i n 6 r o o s t e r s and 5 g u l l s f e d normal and h i g h NaCl d i e t s r e s p e c t i v e l y 43 v i i L I S T OF F I G U R E S 1 Diagrammatic re p r e s e n t a t i o n of the Open-flow system used f o r measurements of evaporative water l o s s at 3 0°C 2 0 2 Diagrammatic re p r e s e n t a t i o n of the plethysmograph and rec o r d i n g instruments f o r the determination of v e n t i l a t o r y p a t t e r n 21 3 Plasma concentrations of sodium, c h l o r i d e , potassium, i o n i z e d and t o t a l calcium i n roo s t e r s fed normal NaCl d i e t f ollowed by two weeks of high NaCl d i e t 26 4 Plasma o s m o l a l i t y and evaporative water l o s s (measured at 3 0°C) i n 6 roosters fed normal and high NaCl d i e t s r e s p e c t i v e l y , and i n 5 g u l l s on pro g r e s s i v e a c c l i m a t i o n from 0 mM NaCl to 475 mM NaCl 29 5 Trends i n plasma sodium, c h l o r i d e , potassium, i o n i z e d and t o t a l calcium concentrations i n g u l l s a c climated to i n c r e a s i n g concentrations of NaCl 30 6 Plasma sodium to calcium r a t i o s i n g u l l s p r o g r e s s i v e l y acclimated from 0 mM to 475 mM NaCl 31 7 Re s t i n g body temperature and panting t h r e s h o l d i n 6 ro o s t e r s fed normal then high NaCl d i e t s , and 5 g u l l s on p r o g r e s s i v e a c c l i m a t i o n from 0 mM to 475 mM NaCl... 34 8 A comparison between the trends of ambient and body temperature i n 5 g u l l s during p r o g r e s s i v e a c c l i m a t i o n 0 mM to 475 mM NaCl d r i n k i n g s o l u t i o n 36 v i i i 9 Mean t o t a l body water and hematocrit i n 6 r o o s t e r s and i n 5 g u l l s . The r o o s t e r s were fed normal NaCl f o r one week fol l o w e d by 2 weeks high NaCl d i e t s . G u l l s were p r o g r e s s i v e l y acclimated from 0 mM to 475 mM NaCl 37 10 T i d a l volume and r e s p i r a t o r y frequency i n r o o s t e r s fed normal NaCl d i e t s (Week 0), and a f t e r 2 weeks on high NaCl d i e t s 44 11 L i n e a r regressions of simultaneously measured plasma concentrations of sodium and i o n i z e d calcium i n roos-t e r s and g u l l s before and during exposure to hi g h NaCl d i e t s 45 12 L i n e a r r e g r e s s i o n l i n e s of simultaneously measured plasma concentrations of i o n i z e d and t o t a l c a l c i u m i n r o o s t e r s and g u l l s before and during exposure to hig h NaCl d i e t s 46 i x L I S T OF A P P E N D I C E S A Composition of l i q u i d v i t a m i n supplement given to Glaucous-winged g u l l s , Larus glaucescens 83 B Plasma i o n i z e d calcium concentration as a percentage of t o t a l calcium concentration, and c o r r e l a t i o n coef-f i c i e n t s of simultaneously measured plasma concentra-t i o n s of sodium, i o n i z e d and t o t a l calcium i n r o o s t e r s and g u l l s fed normal and high NaCl d i e t s 84 C O s c i l l o g r a p h recordings of panting thresholds and pan-t i n g p a t t e r n s of roosters and g u l l s 85 X Acknowledgements I wish to acknowledge the i n v a l u a b l e support, i n a l l forms, given to me by my supervisor, Dr. Maryanne R. Hughes. I would a l s o l i k e to thank my supervisory commitee; Drs. B i l l Milsom, L e s l i e Hart and Peter Hochachka fo r t h e i r advise throughout t h i s p r o j e c t . My a p p r e c i a t i o n to Claudia Kassera f o r her c o n s t r u c t i v e questions and to C h r i s t i n e Yungen f o r her help w i t h word pr o c e s s i n g . S p e c i a l thanks to Mark Roberts f o r h i s s e l f l e s s , and help w i t h computers. I very deeply thank my brother, Ochieng, and s i s t e r - i n - l a w , Anna who s t a r t e d me o f f i n B r i t i s h Columbia. L a s t l y , I acknowledge my dear f r i e n d s and colleagues, C r i s t i n a de Sobrino and Tania Zenteno-Savin f o r t h e i r closeness and support. I dedicate t h i s work to my parents; Rhoda and Walter Ojwang who have given me a l l the support parents could g i v e . This p r o j e c t was supported by N a t u r a l Science and Engineering Research Council of Canada (NSERC) grant A-3442 to Dr. Maryanne Hughes. 1 1 I N T R O D U C T I O N 1.1 S a l t l o a d i n g and plasma e l e c t r o l y t e s t a t u s Increased sodium c h l o r i d e (NaCl) in t a k e e l e v a t e s plasma o s m o l a l i t y (Osmol p l) and concentrations of sodium ([Na +] p l) and c h l o r i d e ( [ C l " ] p l ) i n the fowl (Thomas and Skadhauge, 1982; Skadhauge, 1981; Skadhauge et a l , 1983). A f t e r 8 days on high-or low- NaCl d i e t s , Osmol p l, [Na +] p l, and [ C l " ] p l seem to s t a b i l i z e (Thomas and Skadhauge, 1982). Likewise, i n ducks (Bradley and Holmes, 1972; Roberts and Hughes, 1984; Hughes and Roberts, 1988) and g u l l s (Hughes, 1970; Roberts and Hughes, 1984), s a l i n e a c c l i m a t i o n elevates [Na +] p l even though these species possess an e x t r a - r e n a l s a l t - s e c r e t i n g mechanism that most t e r r e s t r i a l b i r d s , such as domestic fowl, l a c k . The nasal s a l t glands of g u l l s s e c r e t e f l u i d much higher i n Na+ and CI- concentrations than in g e s t e d (Hughes, 1970; 1972), thus making fr e e water a v a i l a b l e to the b i r d f o r other functions such as r e n a l e x c r e t i o n of nitrogenous waste and thermoregulation. The e x c r e t i o n of Na* i n the fowl i s confined l a r g e l y to the r e n a l o u t l e t . The fowl has a low r e n a l concentrating a b i l i t y (Skadhauge, 1981) and loses much water i n e x c r e t i o n . Chronic exposure to high d i e t a r y NaCl in c r e a s e s fowl [Na +] p l and Osmol p l, and causes copious c l o a c a l f l u i d discharge (Skadhauge, 1981). G u l l s , on the other hand, have a r e l a t i v e l y high r e n a l concentrating a b i l i t y (Skadhauge, 1981) which coupled w i t h e x t r a r e n a l NaCl e x c r e t i o n allows them to maintain s t a b l e plasma osmotic and i o n i c l e v e l s when exposed to d r i n k i n g water s a l i n i t i e s as great as seawater (Hughes, 1970; 2 Gray and Erasmus, 1989) . Thus g u l l s have a l l round b e t t e r water r e t e n t i o n and excrete excess s a l t loads more e f f i c i e n t l y than the domestic f o w l . 1.2 Cations and body temperature A l t e r e d body f l u i d Na+ and Ca 2 + l e v e l s cause changes i n body temperature such that high [Na+] r a i s e s w h i l e high [Ca 2 +] lowers body temperature. High d i e t a r y NaCl increased fowl Osmol p l, [Na +] p l, [ C l - ] p l and body temperature at thermoneutral temperatures, and there was a s i g n i f i c a n t c o r r e l a t i o n between body temperature and plasma Na+ : Ca 2 + r a t i o (Arad and Skadhauge, 1986) . Increased body temperature, caused by dehydration and heat s t r e s s , a l s o c o r r e l a t e d w i t h increased plasma Na + : Ca 2 + r a t i o s (Arad et a l , 1983; 1985). As i n d i e t a r y a c c l i m a t i o n , intravenous loads of excess Na + or Ca 2 + ions r a i s e or lower core temperature r e s p e c t i v e l y . Hensel and Schafer (1974) noted that Ca 2 + s t i m u l a t e d p e r i p h e r a l warm re c e p t o r s , depressed p e r i p h e r a l c o l d receptors, and caused panting i n c a t s . As a consequence, body temperature f e l l . In another study, elevated e x t r a c e l l u l a r [Ca 2 +] a b o l i s h e d burst a c t i v i t y and i n h i b i t e d responses of ouabain-treated cat l i n g u a l c o l d receptors in vitro (Pierau et a l , 1983). Intravenous i n j e c t i o n of Ca 2 + ions e l i c i t e d panting and decreased core temperature i n b r o i l e r cockerels (Edens, 1976). In the same study, heat exposure increased body temperature and decreased [ C a 2 + ] p l ; the decrease i n [Ca 2 +] was greatest i n Na +-treated chickens. The thermoregulatory responses to acute c a t i o n loads seem to depend on ambient temperature. In b r o i l e r c o c k e r e l s , intravenous Ca 2 + induced a f a l l i n deep body temperature only at 3 or below 24°C whi l e Na+ evoked thermoregulatory responses only at h i g h (45°C) ambient temperatures (Edens, 1976). I n t r a v e n t r i c u l a r l y i n fused Na4 and Ca 2 + increased and decreased body temperature r e s p e c t i v e l y at 28°C; at 37°C Na + caused an incr e a s e i n the body temperature of l a y i n g hens but Ca 2 + had no e f f e c t (Maki et a l , 1988) . 1.3 The hypothalamic s e t - p o i n t theory The two c a t i o n s , Na+ and Ca 2 +, are thought to f a c i l i t a t e changes i n body temperature by a l t e r i n g the hypothalamic 'set-p o i n t ' f o r temperature r e g u l a t i o n (Myers and Veale, 1970). P e r f u s i o n of the p o s t e r i o r hypothalamic re g i o n of an unanaesthetized cat w i t h a Na+ s o l u t i o n equal to or exceeding e x t r a c e l l u l a r f l u i d (ECF) [Na+] caused a r i s e i n body temperature i f Ca 2 + was not i n the perfusate (Myers and Veale, 1970; 1971). At the same s i t e , a Ca 2 + s o l u t i o n equal to or exceeding ECF [Ca 2 +] caused a f a l l i n body temperature i f Na+ was absent from the p e r f u s a t e . Thus the r e l a t i v e excess of e i t h e r c a t i o n caused the animal to thermoregulate around a new ' s e t - p o i n t ' ; Na+ causing an e l e v a t i o n and Ca 2 + a depression. This concept of 'set-point' r e g u l a t i o n by Na+ and Ca 2 + i n the p o s t e r i o r hypothalamus has been demonstrated i n many homoetherms i n c l u d i n g avian species. I n t r a v e n t r i c u l a r i n f u s i o n s of Ca 2 + caused a f a l l i n c l o a c a l temperature i n p r o p o r t i o n t o perf u s a t e [Ca 2 +] i n pigeons (Saxena, 1976), and chickens (Denbow and Edens, 1980; 1981), and i n v a r i a b l y caused behavioural sedation, intense feeding, aggressive behaviour, and v a s o d i l a t i o n . S i m i l a r l y i n f u s e d sodium ions (Saxena, 197 6; Denbow and Edens, 1980, 1981; Maki et a l , 1988) i n v a r i a b l y increased body temperature. R e c t a l 4 temperature of b r o i l e r cockerels (Denbow and Edens, 1980), chicks (Denbow and Edens, 1981) and l a y i n g hens (Maki et a l , 1988) increas e d f o l l o w i n g s h i v e r i n g and p e r i p h e r a l v a s o c o n s t r i c t i o n . In a d d i t i o n , p i l o e r e c t i o n , digging, and r e s t l e s s n e s s , a l l c h a r a c t e r i s t i c of a c o l d animal, were recorded as responses to high Na+ i n f u s i o n . The i n j e c t i o n of e i t h e r d i s t i l l e d water or a r t i f i c i a l c e r e b r o - s p i n a l f l u i d (CSF) of p h y s i o l o g i c a l c a t i o n c oncentrations i n t o the l a t e r a l c e r e b r a l v e n t r i c l e s d i d not a f f e c t thermoregulatory responses (Saxena, 1976) thus r u l i n g out expanded volume as a stimulus f o r the thermoregulatory responses. Cati o n e f f e c t s are i o n - s p e c i f i c (Feldberg et a l , 1970; Denbow and Edens, 1980), and are unaffected by the anion i n the i n f u s a t e . Potassium (K+) and magnesium (Mg2+) ions have only very weak and equivocal e f f e c t s i n a l l species examined (monkey, Myers and Yaksh, 1971; r a b b i t , Veale and Cooper, 1973; cat, Myers et. a l , 1976; and pigeon, Saxena, 1976) . The replacement of CI" ions i n NaCl w i t h toulene-p-sulphonate (Denbow and Edens, 1981) d i d not a f f e c t the responses of young chicks to the i n j e c t i o n of Na +. 1.3.1 Species di f ferences Species d i f f e r e n c e s may e x i s t i n the extent to which animals respond to a l t e r e d c a t i o n l e v e l s i n t h e i r hypothalami. In pigeons, an increase i n e x t r a c e l l u l a r [Ca 2 +] i s h i g h l y e f f e c t i v e i n decreasing body temperature but a p r o p o r t i o n a l r i s e i n [Na +J i s l e s s e f f e c t i v e i n i n c r e a s i n g body temperature (Saxena, 1976). P e r f u s i o n of the p o s t e r i o r hypothalamus w i t h excess Na" caused in t e n s e hyperthermia i n the unanesthetized cat (Myers and Veale, 1971), but only a weak e f f e c t was obtained i n the r a b b i t 5 (Feldberg and Saxena, 1970) . The pigeon resembles the r a b b i t i n t h i s respect but not the cat hence the suggestion that the s e n s i t i v i t y to a l t e r e d Na+ : Ca 2 + r a t i o i n the c e n t r a l nervous system (CNS) may be species dependent. 1.4 Plasma o s m o l a l i t y and evaporative water l o s s E l e v a t e d body f l u i d o s m o l a l i t y suppresses evaporative water l o s s (EWL) i n many heat-stressed mammals (Senay, 1968; Baker and Do r i s , 1982), b i r d s (Arad, 1983; Arad et a l , 1984), and amphibians (Shoemaker et al., 1989) . I t i s thought that the osmotic s t a t e of the animal acts to a l t e r the responsiveness of the hypothalamus to i n c r e a s i n g temperature (Kozlowski et a l , 1980; Baker and Doris, 1982). In pigeons t r a i n e d to open a window t o a l l o w cool a i r i n t o the animal chamber when ambient temperature was high, i n t r a g a s t r i c i n f u s i o n of hypertonic s a l i n e i n c r e a s e d the frequency of window opening and lowered brea t h i n g frequency to l e s s than 15% of the preload maximum (Rautenberg et, a l , 1980) . Intravenous i n f u s i o n of 30% s a l i n e i n t o hydrated cats caused a s i g n i f i c a n t d e c l i n e i n evaporative heat l o s s and el e v a t e d body temperature at an ambient temperature of 38°C but not at 25°C (Baker and Doris, 1982). Intravenous and i n t r a c a r o t i d i n f u s i o n of hypertonic s a l i n e decreased r e s p i r a t o r y frequency to below p r e i n f u s i o n l e v e l s i n heat s t r e s s e d pigeons (Brummermann and Rautenberg, 1989), and increased Osmol p l i n dehydrated heat s t r e s s e d fowls tended to depress minute v e n t i l a t i o n (VE) thereby reducing EWL, a major pathway f o r evaporative heat l o s s (Arad, 1983; Arad et a l , 1984). The a b i l i t y of a b i r d to maintain s t a b l e [Na +] p l depends on i t s a b i l i t y to excrete ingested NaCl e f f i c i e n t l y . When d i e t a r y 6 NaCl i s high, b i r d s w i t h s a l t glands are b e t t e r able to r e g u l a t e [Na +] p l than b i r d s that lack s a l t glands. Increased [Na +] p l should a l t e r plasma Na+ : Ca 2 + r a t i o (Edens, 1976), exert i n f l u e n c e s on p e r i p h e r a l thermoregulatory responses (Hensel and Schafer, 1974; Edens, 1976) and elevate the hypothalamic s e t -p o i n t f o r temperature r e g u l a t i o n (Myers and Veale, 1971) i n p r o p o r t i o n to the changes i n plasma c a t i o n c o n c e n t r a t i o n s . In a d d i t i o n , s a l t gland a c t i o n may favour greater a v a i l a b i l i t y of f r e e water f o r thermoregulation i n the g u l l compared to the domestic fowl thus one might expect a higher EWL i n h e a t - s t r e s s e d g u l l s . S i m i l a r l y , the change i n plasma o s m o l a l i t y f o l l o w i n g s a l t i n t a k e may determine the amount of evaporative water l o s t pulmocutaneously so that the smaller the increase i n Osmol p l during h i g h d i e t a r y NaCl exposure, the higher EWL would be upon heat s t r e s s . Consequently, body temperature would be i n f l u e n c e d . This study sought to: i ) compare the e f f e c t s of high d i e t a r y NaCl on plasma Na+ : Ca2* r a t i o and body temperature i n the domestic fowl and the Glaucous-winged g u l l . i i ) assess the r e l a t i o n s h i p s between TBW, EWL, panting t h r e s h o l d , minute v e n t i l a t i o n (VE) and plasma [Na+] i n the two s p e c i e s . 1.5 Hypotheses During exposure to high d i e t a r y NaCl: 1. Plasma Na + : Ca 2 + r a t i o i s higher i n domestic fowl than i n g u l l s . 2. W e i g h t - s p e c i f i c EWL upon moderate heat s t r e s s i s higher i n g u l l s than i n domestic fowl. The i n t e r n a l t h r e s h o l d f o r thermal panting i s lower i n g u l l s than i n domestic fowl. R e s t i n g body temperature of domestic fowl increases more than that of the g u l l . 8 2 MATERIALS AND METHODS 2.1 Animal care Roosters S i x White Leghorn r o o s t e r s , obtained from a l a r g e r cohort at the P o u l t r y U n i t , UBC South Campus Animal Science Farm, were used. They were a l l seven months o l d and body mass ranged from 1.43 kg to 1.9 kg (1.7 _+ 0.17 kg). The b i r d s were kept indoors i n • i n d i v i d u a l , adjacent mesh wire cages (100cm X 70cm X 50cm). Room temperature was regulated at approximately 21.5°C and a 12 L, 12 D photoperiod was imposed. R e l a t i v e humidity was monitored on a d a i l y b a s i s . The b i r d s were maintained on P u l l e t developer crumbles (Na, 0.92 g/kg; K, 4.16g/kg; and CI, 1.53g/kg; O t t e r Co-op, Langley, B.C.), hereby designated as normal d i e t , and tap water ad l i b i t u m . Faeces and ur i n e were gathered under the mesh wire f l o o r and disposed of r e g u l a r l y . The b i r d s were acclimated to the room c o n d i t i o n s and handled r e g u l a r l y f o r 2 weeks before the i m p l a n t a t i o n of temperature t r a n s m i t t e r s . G u l l s F i v e 3-4 year o l d unsexed Glaucous-winged g u l l s , Larus  glaucescens, h e l d f o r at l e a s t 3 years i n c a p t i v i t y , were used. Body mass ranged from 815 g to 1060 g (961 + 95 g). The b i r d s were housed outdoors i n larg e metal cages (1.7 x 3.7 x 6.9 m3) at the UBC South Campus Animal Care f a c i l i t y , under the n a t u r a l photoperiod. R e l a t i v e humidity was not recorded but ambient temperature was measured whenever body temperature of the b i r d s was recorded. The g u l l s were fed, ad l i b i t u m , w i t h b a i t h e r r i n g (Clupea p a l a s i i ) , i o n i c concentration: Na, 82 mmol/L; K, 124 mmol/L; CI, 69 mmol/L (Hughes, 1972), supplemented twice weekly 9 w i t h l i q u i d v i t a m i n mixture (Paramette, Ayerst L a b o r a t o r i e s , Montreal) (Appendix A) . This d i e t was supplemented w i t h dog food (Tec h n i - c a l , Canada) .•Freshwater was provided, ad l i b i t u m , i n 20 g a l l o n c a p a c i t y wading pools. The b i r d s were maintained i n these c o n d i t i o n s u n t i l the implantation of temperature t r a n s m i t t e r s . 2.2 Transmitter c a l i b r a t i o n and implantation P r i o r to i m p l a n t a t i o n 6 temperature t r a n s m i t t e r s (model T-M-Dis c , M i n i m i t t e r Co., Oregon), each powered by an 850 mah L i t h i u m b a t t e r y w i t h an a c t i v e l i f e of 3-4 months, were coated w i t h s e v e r a l l a y e r s of p a r a f f i n / E l v a x f o r waterproofing and to cover sharp edges. Each coated t r a n s m i t t e r , weighing an average of 16.5 grams, was c a l i b r a t e d separately i n a water bath (Labline Instruments i n c . , I l l i n o i s ) using a p r e c i s i o n c a l i b r a t i o n (0.05°C accuracy), thermometer (Minimitter Co. , Oregon). Each t r a n s m i t t e r emits pulsed r a d i o s i g n a l s , the frequency of which i s l i n e a r l y c o r r e l a t e d to temperature over the range of p h y s i o l o g i c a l temperatures. The s i g n a l s were r e c e i v e d on a Model CH-6 (Minimmiter Co., Oregon) FM r e c e i v e r , counted manually, and timed w i t h an e l e c t r o n i c c l o c k (Casio Computer Co., L t d . , Japan), model ML-88. Time ( i n seconds) per 100 s i g n a l s was measured at s e l e c t e d temperatures. Three counts were taken at approximately 5 degree i n t e r v a l s from 25°C to 50°C. I n d i v i d u a l c a l i b r a t i o n curves were p l o t t e d f o r each t r a n s m i t t e r . Food and water were removed from the cages at l e a s t 24 hours before the s u r g i c a l procedure. P r i o r to surgery, body weight was measured using a weighing balance (Model 1N-12, C h a t i l l o n , N. Y.) to the nearest 25 g. 10 2.2.1 Anaesthesia Roosters Approximately 0.25 ml of a 1 : 1 Ketamine h y d r o c h l o r i d e (Rogar/STB i n c . , Canada)/ X y l a z i n e h y d r o c h l o r i d e (Lloyd L a b o r a t o r i e s , Iowa) combination was administered over 3-4 minutes by s y r i n g e i n t o the medial metatarsal v e i n . Anaesthesia was considered s u f f i c i e n t i f there were no motor responses when secondary feathers were p u l l e d from the abdominal r e g i o n . S u f f i c i e n t dose, determined e m p i r i c a l l y , was approximately p r o p o r t i o n a l to body weight (Mean = 0.166ml/kg). G u l l s G u l l s were anaesthetized w i t h approximately 0.32-0.52 ml/kg (mean = 0.434 ml/kg) sodium p e n t o b a r b i t a l (Somnotol, M.T.C. Pharmaceuticals, Mississauga, Ontario) i n j e c t e d i n t o the medial m e t a t a r s a l v e i n . In a d d i t i o n , approximately 0.1 ml L i d o c a i n e h y d r o c h l o r i d e (Astra Pharmaceuticals, Mississauga, O n t a r i o ) , a l o c a l a n a e s t h e t i c , was a p p l i e d subcutaneously, at the area of i n c i s i o n . Dose s u f f i c i e n c y was monitored as i n r o o s t e r s . 2.2.2 Surgery F o l l o w i n g anaesthesia, secondary feathers were removed from the lower abdominal region to expose the s k i n which was then s t e r i l i z e d w i t h 70% a l c o h o l . A m i d l i n e i n c i s i o n was made below the k e e l , i n r o o s t e r s , to approximately 5 mm a n t e r i o r to the c l o a c a . In g u l l s , the 6 cm v e n t r a l i n c i s i o n was not as caudal as 11 i n the r o o s t e r (The domestic fowl has a much deeper k e e l ) . The s k i n was f o l d e d to e i t h e r s ide of the i n c i s i o n to expose the peritoneum. The membrane was l i f t e d to avoid c u t t i n g the u n d e r l y i n g organs and another m i d l i n e i n c i s i o n was c a r e f u l l y made through the peritoneum. A temperature t r a n s m i t t e r , s t e r i l i z e d i n 70% ethanol, was placed l a t e r a l to the l i v e r , on the r i g h t s ide of the p e r i t o n e a l c a v i t y . A f t e r i m p l a n t a t i o n , a s o l u t i o n of t e t r a c y c l i n e was poured i n the v i c i n i t y of the i n c i s i o n to prevent i n f e c t i o n . The p e r i t o n e a l suture was closed before the o v e r l y i n g s k i n was sewn usi n g s u r g i c a l thread (Ethicon I n c . ) . The f r e s h wound was then dubbed w i t h cotton-wool soaked i n 70% ethanol. The b i r d was l a i d i n a hardware c l o t h cage under an i n f r a -red lamp. The f r e s h l y implanted t r a n s m i t t e r s were used to monitor recovery. A l l the b i r d s came out of anaesthesia w i t h i n two hours. S u f f i c i e n t time was allowed, under s u r v e i l l a n c e , f o r f u l l recovery before the b i r d s were returned to t h e i r o r i g i n a l cages i n which food and water were replaced. Body temperature was recorded once (at approximately 1.00 p.m.) every day during the recovery p e r i o d . A minimum of 7 days were allowed a f t e r surgery before experimentation. 2.3 Experimentation Evaporative water l o s s (EWL), r e s p i r a t o r y frequency ( f ) , t i d a l volume (VT) , panting t h r e s h o l d (PT) , t o t a l body water (TBW), plasma o s m o l a l i t y (Osmol p i ) , body temperature, and plasma 12 concentrations of t o t a l calcium ( [ C a ] p l ) , i o n i z e d c a l c i u m ( [ C a 2 + ] p l ) , sodium ([Na + ] p l ) , and c h l o r i d e ( [ C l " ] p l ) were measured w h i l e r o o s t e r s were maintained on normal p u l l e t developer crumbles, and repeated when the same b i r d s were acc l i m a t e d to the high s a l t d i e t . In g u l l s , body temperature was monitored d a i l y and plasma concentrations measured whi l e the b i r d s were on freshwater and repeated when the b i r d s were acclimated to 112.5 mM, 225mM, 337.5 mM, and 475 mM NaCl s o l u t i o n s r e s p e c t i v e l y . Measurements of EWL, TBW, PT and f were done when the b i r d s drank f r e s h water and repeated when they drank 225 mM and 475 mM NaCl ( s l i g h t l y more concentrated than seawater) r e s p e c t i v e l y . 2.3.1 NaCl a c c l i m a t i o n Roosters The b i r d s were held on the normal d i e t and were caught and handled r e g u l a r l y f o r 3 weeks to accustom them to the process. I n i t i a l measurements were made while the b i r d s were maintained on the normal NaCl d i e t . Then 990 g po r t i o n s of p u l l e t developer crumbles were sprayed w i t h 500 ml of 2% NaCl. The wet crumbles were thoroughly mixed and a i r - d r i e d . This mixture contained the same organic and i o n i c contents as the normal d i e t except Na and CI which were increased from 0.9 g/kg and 1.5 g/kg, to 5.0 g/kg and 8.7 g/kg r e s p e c t i v e l y . In a d d i t i o n , 0.5% NaCl was given as d r i n k i n g water. The b i r d s were maintained on t h i s regime designated the 'high NaCl d i e t ' ad libitum. On the f i f t h day, a 2 . 0 ml blood sample was taken from the median met a t a r s a l v e i n f o r 13 the determination of i o n i c and osmotic c o n c e n t r a t i o n s . Body temperature was measured every day at approximately 1.00 p.m. A f t e r the e i g h t h day, EWL, panting t h r e s h o l d , VT, and f were determined. TBW was redetermined a f t e r 12 days of exposure to the high NaCl d i e t . G u l l s The d r i n k i n g water concentration was increased to 475 mM NaCl s o l u t i o n i n 4 f a i r l y equal increments. The b i r d s were h e l d i n each s a l t w a t e r concentration f o r one week except 225 mM NaCl at which they were h e l d f o r two weeks to a l l o w s u f f i c i e n t time to complete determinations of v e n t i l a t i o n and evaporative water l o s s . The d r i n k i n g s o l u t i o n was prepared every morning and presented i n the wading pool. The d r i n k i n g water c o n c e n t r a t i o n was r e g u l a r l y monitored, and f o r the most p a r t , was as d e s i r e d (The highest concentration was s l i g h t l y higher (475 mM NaCl) than that of seawater (450 mM NaCl). 2.3.2 Blood sampling A 2.0 ml blood sample was c o l l e c t e d f o r the determination of plasma osmotic and i o n i c concentrations. The blood was t r a n s f e r r e d i n t o 1.5 ml c e n t r i f u g e tubes and microhematocrit tubes ( B l u - t i p , Monoject S c i e n t i f i c , St. Louis) were immediately f i l l e d , and both sets of tubes were c e n t r i f u g e d f o r 3 minutes at 15,000g i n an Eppendorf c e n t r i f u g e (Model 5412). Plasma was t r a n s f e r r e d i n t o capped v i a l s and immediately analyzed f o r i o n i z e d calcium, u n t i l analyzed .for 14 The remaining plasma was sealed and frozen plasma i o n i c and osmotic c o n c e n t r a t i o n s . 2.3.3 Plasma analysis (i) Ionized [Ca J +] p l. Measurements of [ C a 2 + ] p l were done between 1 to 3 hours a f t e r sampling to avoid l a r g e changes i n pH. U n d i l u t e d plasma samples were introduced i n t o a Ca 2 + i o n - s e l e c t i v e e l e c t r o d e (ICA 1, Radiometer, Copenhagen), and 50 u l were a u t o m a t i c a l l y a s p i r a t e d f o r a n a l y s i s . Values were read as c o n c e n t r a t i o n (mmol/1) at the a c t u a l pH but were i n t e r n a l l y c o r r e c t e d to give [Ca 2 +] at a standardized pH of 7.4. Corrected values were reported. ( i i ) [Na +] p l and [K +] p l. A l i q u a n t s of plasma (20 u l ) were a u t o m a t i c a l l y a s p i r a t e d i n t o 2 ml cesium d i l u e n t f o r simultaneous determination of [Na +] p l and [ K + ] p l using a flame photometer (Model IL 943, Instrumentation ab. Inc., Milan, I t a l y ) . ( i i i ) [ C l ~ ] p l . To determine [ C l " ] p l , 10 u l a l i q u a n t s of plasma were t i t r a t e d e l e c t r i m e t r i c a l l y u sing a d i g i t a l c hloridometer (Buchler Instrumentation, Fort Lee, N.J.). (iv) Total [Ca] p l. A l i q u a n t s of plasma (50 u l ) were p i p e t t e d , made up to 2.0 ml i n 0.1% lanthanum, and analysed f o r t o t a l [Ca] p l by atomic absorption spectrophotometry (Perkin-Elmer, Model 560; Perkin-Elmer Corporation, Norwalk, CT., USA). (v) Osmol p l. A l i q u a n t s of plasma (10 ul) were used i n the determination of Osmol p l using a vapour pressure osmometer (Model 5500, Wescor, Logan, Utah). 15 2.3.4 T o t a l body water (TBW) The b i r d s were f a s t e d overnight and deprived of water f o r a minimum of one hour p r i o r to the experiment. Each b i r d was weighed, r e s t r a i n e d using a v e l c r o band, and a 2.0 ml blood sample was taken from the median metatarsal v e i n u s i n g a he p a r i n i z e d t u b e r c u l i n syringe (Becton D i c k i n s o n Canada, Mississauga) f o r plasma ion determination. Approximately 24,000,000 d i s i n t e g r a t i o n s per minute (DPM, roosters) and 20,000,000 DPM (g u l l s ) t r i t i a t e d water (TOH, New England Nuclear, Lachene, Quebec), i n 1.0 ml of i s o t o n i c (0.9%) s t e r i l e s a l i n e were i n j e c t e d i n t o the c o n t r a l a t e r a l median met a t a r s a l v e i n , t a k i n g care to prevent backflow of l a b e l l e d blood. One hour was allowed f o r e q u i l i b r a t i o n of TOH wit h body f l u i d s a f t e r which a 1.0 ml blood sample was taken. This r o u t i n e was repeated f o r r o o s t e r s a f t e r 2 weeks of a c c l i m a t i o n to a high NaCl d i e t . For g u l l s , i t was c a r r i e d out during FW a c c l i m a t i o n and repeated when the b i r d s were acclimated to 225 mM and 475 mM NaCl s o l u t i o n s r e s p e c t i v e l y . Plasma a l i q u a n t s (100 ul) were t r a n s f e r r e d to 10 ml aquasol s c i n t i l l a t i o n f l u i d c o c k t a i l (New England Nuclear, Lachene, Quebec) and a c t i v i t y determined using an LS 9000 Beckman L i q u i d s c i n t i l l a t i o n counter w i t h programmed window s e l e c t i o n s . TOH space was c a l c u l a t e d as: TOH space = I n j e c t a t e dpm/ dpm.ml"1 i n plasma x 93.7% (1) where 93.7% i s the percentage of water i n plasma. TBW was 16 reported as % body mass (%BM) assuming 1 ml water = 1 g. 2.3.5 Evaporative water l o s s (EWL) Evaporative water l o s s was measured using a m o d i f i c a t i o n of the open flow technique ( C h r i s t i a n , 1978). The b i r d was placed i n an a i r t i g h t p l e x i g l a s s animal chamber (Fig 1) , dimensions 60 x 45 x 45 cm3, equipped w i t h a mesh wire f l o o r suspended 3 cm above a 1 cm l a y e r of mineral o i l to prevent evaporation of water from u r i n e and feaces. The t e s t chamber had been checked before experimentation f o r a i r t i g h t n e s s by v e r i f y i n g that i n f l u e n t and e f f l u e n t a i r flow rates were equal. The animal chamber was then placed i n an environmental chamber which maintained ambient temperature at 3 0-31°C. The i n t e r i o r of the environment chamber was kept i n darkness to minimize a c t i v i t y of the b i r d . The b i r d was allowed to get accustomed to the chamber and the chamber to e q u i l i b r a t e f o r two hours at the designated temperature (30-31°C) , and humidity (50-55%), before use i n experimentation. E q u i l i b r a t i o n time, t, can be p r e d i c t e d by the equation: t = 2.3V/D. l o g l / l - z (2) where t i s the time i t takes f o r the chamber to reach z per cent of e q u i l i b r a t i o n , V i s the volume ( i n l i t r e s ) of the chamber, and D i s the a i r flow r a t e (L/min) (Christensen, 1947) . This formula can be s i m p l i f i e d to obt a i n the time r e q u i r e d to reach 99% e q u i l i b r a t i o n as f o l l o w s : t = 4.6V/D (Heusner, 1955) (3) E q u i l i b r a t i o n time was determined using equation (3). A i r flow i n t o the chamber was monitored by a Gilmont flowmeter (Model, 622 PBX, Matheson, Whitby, Ont.) p r e v i o u s l y c a l i b r a t e d v o l u m e t r i c a l l y using a spirometer. The a i r was d r i e d by passing i t through a set of drying columns c o n t a i n i n g s i z e 8 mesh anhydrous CaS0 4 ( D i e r i t e , W. A. Hammond D i e r i t e Co., Xenia, Ohio) . I t had been e s t a b l i s h e d that no moisture was added to the system by i n f l u e n t a i r as long as the d r y i n g columns were r e f i l l e d w i t h oven-dried d r i e r i t e p r i o r to every experiment. Oven-drying was done f o r s e v e r a l hours at temperatures exceeding 100°C. A small fan was used to ensure mixing of a i r i n the chamber, and r e l a t i v e l y high flow rates (up to 4.0 l i t r e s per minute f o r a 1000 g bird) were used to minimize the e f f e c t s of chamber humidity on EWL. Temperature of the animal chamber was monitored by a thermistor probe introduced through one of the po r t s and read on a Tele-thermometer (Yellow Springs Instruments Co., I n c . ) . Chamber humidity was monitored using a r e l a t i v e humidity and temperature i n d i c a t o r (model M2 A4, Abbeon Inc., West Germany), usable i n temperatures up to 110°C. Downstream a i r was d i r e c t e d through pre-weighed columns of d r i e r i t e to c o l l e c t water vapour l o s t pulmocutaneously by the b i r d . At the end of two hours the apparatus was dismantled and the columns were reweighed to the nearest 0.01 g to ob t a i n the amount of water r e l e a s e d pulmocutaneously over that p e r i o d by the animal. The b i r d was reweighed and w e i g h t - s p e c i f i c EWL c a l c u l a t e d based 18 on the mean body mass of the animal. EWL (g H 2 0/Kg b o d y m a s s/hr) = 1/2 (MH20/BM) x 1000, (4) where M H 2 0 = mass of water released pulmocutaneously over 2 hours by the animal, and BM = mean body mass of the b i r d . 2.3.6 V e n t i l a t i o n and panting threshold The b i r d was placed i n an a i r t i g h t p l e x i g l a s s body box (Fig 2) w i t h the head pr o t r u d i n g through an opening i n the f r o n t w a l l . The primary feathers were l o o s e l y taped against the body to prevent f l a p p i n g w h i l e the b i r d was i n the plethysmograph. A rubber c o l l a r and petroleum j e l l y were used to make an a i r t i g h t s e a l around the neck and p l a s t i c cheeks were used to prevent the rubber membrane from o s c i l l a t i n g or the b i r d from p u l l i n g i t s neck up and down. The body box was used as a plethysmograph f o r v e n t i l a t o r y measurements. Before experimentation, the system was c a l i b r a t e d at room temperature, while c o n t a i n i n g a r i g i d body, by a sy r i n g e connected through a port to the plethysmograph and d r i v e n by hand, repeatedly i n t r o d u c i n g and withdrawing known volumes (10, 20, 30, 40, 50, and 60 ml r e s p e c t i v e l y ) of a i r at a frequency approximating the b i r d ' s r e s p i r a t o r y r a t e . Pressure changes due to a l t e r n a t e i n h a l a t i o n and e x h a l a t i o n were conducted by a d o r s a l l y placed pneumotach connected by cable to a pressure transducer (Validyne, Model DP 103-18 , Northridge, Ca), a m p l i f i e r (Validyne, Model CD 16, Northridge Ca.), i n t e g r a t o r (Gould Inc., Cleveland, Ohio), and f i n a l l y recorded on an o s c i l l o g r a p h (Havard Apparatus L t d . , Kent.). Separate c a l i b r a t i o n s were c a r r i e d out at the s t a r t and conclusion of each experimental s e s s i o n . 20 F i g 1. Diagrammatic representation of the Open-flow System. The arrows i n d i c a t e the d i r e c t i o n of a i r flow. D are d r i e r i t e columns; F, gas flowmeter; H, moisture sensor; P, t h e r m i s t o r probe; and T, telethermometer. The animal chamber measures 60 x 45 x 45 cm3, has 2 cm of mineral o i l below the mesh wire f l o o r to prevent evaporation of water from c l o a c a l discharges, and a small fan to ensure mixing of a i r . F i g 2. Diagrammatic representation of the body plethysmograph and r e c o r d i n g instruments f o r the determination of v e n t i l a t o r y p a t t e r n . The arrows i n d i c a t e the d i r e c t i o n of water flow, i n the outer j a c k e t of the body box, used to r e g u l a t e chamber temperature. S i s the c a l i b r a t i o n syringe; P, pneumotach; PT, pressure transducer; I, i n t e g r a t o r ; and 0, o s c i l l o g r a p h . The b i r d ' s neck protrudes through a hole i n dental dam sealed w i t h petroleum j e l l y and i s secured by p l a s t i c cheeks to prevent the b i r d from moving i t s head up and down. 22 Recordings of t i d a l volume (VT) and f were determined at room temperature. V T (ml) was taken as the mean amplitude of 10 successive e x h a l a t i o n s at every temperature i n t e r v a l . Likewise, f was c a l c u l a t e d based on the time taken over 10 successive r e s p i r a t i o n c y c l e s at every temperature i n t e r v a l . Chamber temperature, monitored continuosly by a t h e r m i s t o r probe introduced through one of the ports was g r a d u a l l y i n c r e a s e d by i n t r o d u c i n g warm water i n t o the outer compartment. The r a t e of increase of ambient temperature was not c o n t r o l l e d but was gradual, and was recorded. Panting th r e s h o l d , hereby d e f i n e d as the lowest body temperature at which thermal panting s t a r t s , was determined. Thermal panting i s i t s e l f d e f i n e d here as polypneic b r e a t h i n g o c c u r r i n g under a thermal load, at a d i s t i n c t l y depressed amplitude and a f a s t e r frequency than normal, normal b r e a t h i n g being assumed at 2 6°C ambient temperature. Ambient temperature, i n °C, was simultaneously recorded. 2.4 Data a n a l y s i s . Values are reported as means w i t h standard d e v i a t i o n . Comparisons between roosters and g u l l s , panting t h r e s h o l d , EWL, r e s p i r a t o r y values, and roos t e r TBW at normal and high NaCl d i e t s were made using p a i r e d t - t e s t . Rooster values a f t e r 2 weeks of hi g h d i e t a r y s a l t i n t a k e were compared w i t h g u l l values taken dur i n g exposure to 475 mM NaCl s o l u t i o n (unless otherwise s t a t e d ) . Tukey's post-hoc t e s t was used f o r m u l t i p l e comparisons of blood i o n and osmotic l e v e l s , body temperatures, and g u l l TBW i n the d i f f e r e n t stages of s a l t exposure. Simple r e g r e s s i o n a n a l y s i s was done using the m u l t i v a r i a t e general l i n e a r model i n SYSTAT. 24 3 RESULTS 3.1 Plasma i o n i c and osmotic concentrations Roosters Plasma sodium concentration ( [Na+]pl) increased s i g n i f i c a n t l y (P < 0.05) from 154.7 +_ 2.3 mmol/L, i n the normal d i e t , to 160.5 +.3.4 mmol/L by the end of the f i r s t week of high s a l t i n t a k e (Table I ) . In the second week [Na+] p l d e c l i n e d to a l e v e l that was n e i t h e r s i g n i f i c a n t l y higher than i n the normal s a l t d i e t nor lower than i n the f i r s t week of NaCl intake (Table I, F i g 3) . T o t a l c a l c i u m concentration ([Ca] p l) d i d not change. The [Na +] p l i n c r e a s e d but not s u f f i c i e n t l y to s i g n i f i c a n t l y change plasma Na : Ca r a t i o . I onized calcium concentration ([Ca 2 +] p l) i n c r e a s e d s i g n i f i c a n t l y (p < 0.05) from 1.54 +. 0.03 mmol/L at the normal NaCl d i e t to 1.63 +. 0.03 mmol/L a f t e r one week of high NaCl i n t a k e (Table I, F i g 3) and returned to normal (1.57 ± 0.08 mmol/L) by the end of the second week. The [C a 2 + ] p l was c o r r e l a t e d w i t h [Na +] p l (r = 0.664, n=18). Plasma Na+ : Ca 2 + d i d not change s i g n i f i c a n t l y (Table I ) . The percent increase of [Ca 2 + ] p l (5.5%) was, i n f a c t , greater than that of [Na +] p l (3.7%) a f t e r the f i r s t week of NaCl int a k e so that Na+ : Ca 2 + decreased s l i g h t l y . However, the r a t i o tended to normal, as had [Na +] p l and [Ca 2 + ] p l , i n the second week. 25 Table I Plasma sodium ( [Na+] ) , c h l o r i d e ( [ C I - ] ) , potassium ( [K +] ) , i o n i z e d and t o t a l calcium ( [Ca 2 +] and [Ca] ) , sodium to calcium r a t i o s , o s m o l a l i t y (Osmol), and deep body temperature (Tb) i n 6 r o o s t e r s fed normal NaCl d i e t , and f o l l o w i n g high NaCl i n t a k e f o r one and two weeks r e s p e c t i v e l y . Data are presented as means w i t h standard d e v i a t i o n . Diet [Na+] [Ca 2 +] [Ca] Na +:Ca 2 + Na + :Ca mmol/L mmol/L mg/100 ml Normal 154 .7 1.54 11.5 100.2 , 13 .5 ±2.3 ± 0.03 ± 0.7 ± 2.9 ± 0.9 High Na Week 1 160.5" 1.63" 11 .4 98.8 14.1 ± 3.4 ±0.03 ± 0.8 ±2.9 ± 1.0 Week 2 158 . 9 1.57 11.8 101.4 13 .9 ± 5.5 ± 0.08 ± 0.6 ± 2.6 ± 1.4 [CI'] [K+] Osmol [Ca2*] Tb mmol/L mmol/L mosmol/kg (% [Ca]) °C Normal 113 .7 3 .19 303 . 6 53 . 9 41.0 ± 3.4 ± 0.42 ± 3.9 ±2.8 ± 0.2 High Na Week 1 117 .7 3 . 92" 319.5" 57 .2 41.3 ± 2.4 ± 0.28 ± 4.3 ± 3.1 ± 0.3 Week 2 118.9* 3.39 315.9 53 . 0 41.2 ± 3.8 ± 0.21 ± 13.8 ±2.0 ± 0.3 S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from normal NaCl d i e t : *-P<0.05; **-P<0.01. [Ca] p l was converted to mmol/L f o r c a l c u l a t i o n of [ C a 2 + ] p l as a percentage of [Ca] p l. F i g 3. Plasma concentrations of: Top; sodium ( [Na+] , open bars) and c h l o r i d e ([CI"], s o l i d bars, second y - a x i s ) , and Bottom; potassium ( [K +] , s o l i d bars) and i o n i z e d calcium ( [Ca 2 +] , hatched bars, second y - a x i s ) , and t o t a l calcium ([Ca], open bars) of r o o s t e r s fed normal NaCl d i e t (Week 0) followed by two weeks (Weeks 1 and 2) of high NaCl d i e t . E r r o r bars represent standard d e v i a t i o n from the mean. ** denotes s i g n i f i c a n c e at P<0.01 i n comparison w i t h Week 0; n = 6. Plasma concentration (mmol/L) 7^ r-* f~ t- r ro rso W O* Oi CO o £5 55 ro ao o Plasma |Ca2+l (mmol/L) Plasma [Na+I (mmol/L) o o o o • • 27 The [ C a 2 + ] p : was 55% +. 3.1% of [Ca] p l but no c o r r e l a t i o n (r = 0.385, n = 18) was evident between [Ca] p l and [ C a 2 + ] p l . Plasma [Ca 2 +]/[Ca] r a t i o d i d not change w i t h high d i e t a r y NaCl i n t a k e . [ C l - ] p l increased s t e a d i l y from 113.7 + 3.4 meq/L at normal NaCl d i e t reaching s i g n i f i c a n c e (P < 0.05) at 118.9 +.3.8 meq/L a f t e r two weeks of high NaCl exposure (Fig 3) . [ K + ] p l rose s i g n i f i c a n t l y (P < 0.01) a f t e r one week from 3.19 +. 0.42 mmol/L to 3.92 +. 0.28 mmol/L, but f e l l s i g n i f i c a n t l y , a f t e r the second week, to a value not d i f f e r e n t from that at normal NaCl d i e t (Table I, F i g 3) . V a r i a b i l i t y i n [ K + ] p l decreased w i t h h i g h NaCl exposure. Osmol p l increased s i g n i f i c a n t l y (P < 0.05) by 5.2% from 304 +3.9 mosmol/kg at normal NaCl d i e t to 319.5 +_ 4.3 mosmol/kg a f t e r the f i r s t week of high NaCl i n t a k e . A f t e r 2 weeks of high s a l t i n t a k e , the value tended to f a l l though not s i g n i f i c a n t l y (Table I, F i g 4). V a r i a b i l i t y i n Osmol p l among i n d i v i d u a l s i n c r e a s e d w i t h i n c r e a s i n g NaCl exposure. Plasma o s m o l a l i t y was h i g h l y c o r r e l a t e d w i t h [Na +] p l (r = 0.956, n = 18). G u l l s In g u l l s [Na +] p l was 154.9 +_ 2.4 mmol/L i n freshwater (FW, Table I I ) . [Na +] p l increased s i g n i f i c a n t l y when the g u l l s drank s a l i n e e quivalent i n concentration to h a l f - s t r e n g t h seawater (225 mM NaCl) . There was a steady r i s e i n [Na +] p l u n t i l 475 mM NaCl was given at which p o i n t , the increase was very dramatic (Table I I , F i g 5). [Na +] p l peaked at 163.3 +. 1.5 mmol/L, a value s i g n i f i c a n t l y higher (P < 0.01) than at a l l the lower d r i n k i n g 28 T a b l e I I : Plasma concentrations of sodium ([Na] p l), i o n i z e d c a l c i u m ( [ C a 2 + ] p l ) , t o t a l calcium ( [ C a ] p l ) , c h l o r i d e ( [ C l - ] p l ) , potassium ( [ K + ] p l ) ; plasma o s m o l a l i t y (osmol p l) , sodium to calcium r a t i o s , and body temperature (T b), of g u l l s d r i n k i n g freshwater (0 mM NaCl), and during progressive a c c l i m a t i o n to f u l l - s t r e n g t h seawater (475 mM NaCl) . Data are presented as means w i t h standard d e v i a t i o n . [NaCl] [Na] [Ca: 2 + ] [Ca] Na+ :Ca2 Na + :Ca (mM) mmol/L mmol/L mg/100 ml 0 154 . 9 1. 36 9.6 114.8 16.2 ± 2 . 4 + 0 . 10 ± 0.8 ± 9.7 ± 1.5 112 .5 156. 4 + 1. 36 9.8 115.2 15 . 9 ± 0. 5 ± 0 . 04 ± 0.2 ± 3.0 ± 0.3 225 158 . 3* 1. 37 10.0 115 .8 15 . 9 ± 1. 6 + 0. 3 ± 0.5 ±3.4 ± 0.7 337 .5 158 . 9* 1. 38* 10.2 115 .4 15.6 ± 1. 3 + 0. 05 ± 0.3 ± 3.1 ± 0.3 475 163 . 3** 1. 52** 10 . 6* 107.4* 15 .3 ± 1. 5 + 0 . 05 ± 0.4 ± 2.4 ± 0.6 [NaCl] [ c i - •] [K+] Osmol [Ca 2 +] T b mmol/L mmol/L mosmol/kg (% [Ca]) °C 0 109 . 0 1. 31 314.1 56.5 40.4 ± 5. 4 + 0 . 10 ±3.5 ± 2.1 ± 0.4 112 .5 115. 9* 2 . 06 322 .2 55.2 40.5 ± 1. 9 ± 0-31 ± 3.7 ± 2.2 ± 0.4 225 116 . 0* 1. 63 313 .1 54 . 9 40.7* ± 2 . 2 + 0 . 81 ± 4.7 ± 3.8 ± 0.3 337 .5 116. 7 1. 55 306.9 54 .2 40 . 6 ± 1. 4 + 0 . 22 ± 2.5 ± 1.2 ± 0.5 475 112 . .9 1. 35 314.0 57 .3 41.0* ± 2 . 4 + 0 . 12 ± 7.5 ± 3.0 ± 0.5 S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from FW; * i s P<0.05; ** i s P<0.01. [Ca] p l was converted to mmol/L f o r c a l c u l a t i o n of [Ca2*"]pl as a percentage of [Ca] p l. F i g 4 . Plasma o s m o l a l i t y and absolute evaporative water l o s s (EWL measured at 3 0°C) i n 6 roosters fed normal (Week 0) and high (Week 2) NaCl d i e t (top), and i n 5 g u l l s on p r o g r e s s i v e a c c l i m a t i o n from freshwater (0 mM NaCl) to 475 mM NaCl s o l u t i o n (bottom) . Plasma o s m o l a l i t y (mmol/kg) , s c a l e d on the l e f t y - a x i s i s represented by s o l i d pentagons, while EWL (g H 20/kg/hr) s c a l e d on the second y - a x i s i s represented by the open bars. E r r o r bars represent standard d e v i a t i o n from the mean. S t a t i s t i c a l s i g n i f i c a n c e i s denoted by * at P<0.05, and ** at P<0.01 i n comparison w i t h Week 0. Drinking saline concentration (mM) 30 F i g 5. Plasma sodium ([Na +] , s o l i d c i r c l e s , l e f t y - axis) and c h l o r i d e (CI"] , open c i r c l e s , second y-axis) c o n c e n t r a t i o n i n g u l l s (top), and trends i n plasma potassium ([K +] , s o l i d squares), i o n i z e d calcium ( [Ca 2 +] , s o l i d t r i a n g l e s ) , and t o t a l c a lcium ([Ca], s o l i d c i r c l e s ) i n g u l l s acclimated to i n c r e a s i n g c o n centrations of seawater as f o l l o w s ; 0, 112.5, 225, 337.5, and 475 mM NaCl i n weekly i n t e r v a l s (The b i r d s were given 225 mM d r i n k i n g s a l i n e f o r two weeks). A l l concentrations are i n mmol/L. E r r o r bars represent standard d e v i a t i o n from the mean. S t a t i s t i c a l s i g n i f i c a n c e i s denoted by * at P<0.05, and ** at P<0.01 i n comparison w i t h 0 mM NaCl values. 3 0 a o.lCH • lNa+] Drinking saline concentration (mM) F i g 6. Top: Plasma sodium to i o n i z e d calcium (Na* : Ca 2 +) r a t i o ; Bottom: Plasma [Ca] ( s o l i d t r i a n g l e s ) , and [Na+] to [Ca] r a t i o ( s o l i d squares, second y-axis) i n g u l l s on p r o g r e s s i v e a c c l i m a t i o n from 0 mM to 475 mM d r i n k i n g NaCl s o l u t i o n . R a t i o was c a l c u l a t e d based on t h i s value and the [Na +] p l i n mmol/L. E r r o r bars represent standard d e v i a t i o n from the mean. S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from 0 mM NaCl; * P < 0.05 31a 16 n 8 J 1 1 •—i — i ' 0. 113. 225. 338. 475. Drinking (NaCll concentration (mM) NaCl c o n c e n t r a t i o n s . T o t a l calcium ([Ca] p l) i n c r e a s e d g r a d u a l l y from 9.6 +. 0.8 mg/100 ml (FW) , to 10.6 +. 0.4 mg/100 ml (P < 0.05) when the g u l l s drank 450 mM NaCl s o l u t i o n (Table I I , F i g 6) . This i n c r e a s e p a r a l l e l e d the increase i n [Na +] p l so that Na : Ca d i d not change (Fig 6) . Ionized [Ca 2 +] p l d i d not change u n t i l the b i r d s drank 475 mM NaCl. At t h i s p o i n t , [Ca 2 +] p l i n c r e a s e d (P < 0.01) by 11.8% from 1.36 +, 0.10 mmol/L (the FW value) to 1.52 + 0.05 mmol/L. [Ca 2 +] p l was c o r r e l a t e d w i t h [Na +] p l (r = 0.635, n = 25) . Plasma Na+ : Ca 2 + r a t i o was steady but tended to f a l l at 475 mM NaCl d r i n k i n g s o l u t i o n (Fig 6). Plasma [Ca 2 +] was p o s i t i v e l y (r = 0.639) c o r r e l a t e d w i t h and was 57% ±2.1% [Ca] p l. A c c l i m a t i o n to 475 mM NaCl d i d not a l t e r [Ca 2 +] p l/[Ca] p l r a t i o . [ K + ] p l tended to be hi g h at 112.5 mM d r i n k i n g [NaCl] but remained l a r g e l y unchanged ( F i g 5). [ C l - ] p l increased (P<0.05) from 109 meq/L at FW to 116 meq/L at 225 mM NaCl. This c o n c e n t r a t i o n was maintained u n t i l 475 mM NaCl s o l u t i o n when a small d e c l i n e occurred ( F i g 5). Osmol p l rose by 2.6% (n/s) a f t e r one week of a c c l i m a t i o n to 112.5 mM NaCl from 314 +.3.5 mosmol/kg to 322 +_ 3.7 mosmol/kg but d e c l i n e d to 314 +.7.5 mosmol/kg at 475 mM NaCl (Table I, F i g 4) . V a r i a b i l i t y i n Osmol p l rose between i n d i v i d u a l s at h i g h d r i n k i n g s a l i n e concentrations. 3.2 Body temperature In r o o s t e r s , i n t r a p e r i t o n e a l temperature tended to i n c r e a s e from 41.0 +_ 0.2°C to 41.3 +. 0.3°C a f t e r one week of NaCl a c c l i m a t i o n but t h i s was not s i g n i f i c a n t (Table I, F i g 7). The 33 time course of change was s i m i l a r f o r temperature, [Na +] p l, [ C a 2 + ] p l , and Osmol p l, but no strong p a i r w i s e c o r r e l a t i o n s were evident between body temperature and these v a r i a b l e s . In g u l l s , i n t r a p e r i t o n e a l temperature remained unchanged u n t i l the b i r d s drank 225 mM NaCl (Table I I , F i g 7). Mean normal core temperature was 40.4 +_ 0.4 °C and increased to 41.0 + 0 .5°C (P < 0.05) at 475 mM d r i n k i n g [NaCl] (Table I I ) . Ambient temperature f l u c t u a t e d (Fig 8) but there was no r e l a t i o n s h i p between ambient and body temperatures. The time course of temperature change was s i m i l a r to that of [Na +] p l, and [Ca 2*] p l, but p a i r w i s e r e g r e s s i o n a n a l y s i s d i d not r e v e a l any c o l l i n e a r i t y (There was high c o r r e l a t i o n (r = 0.978) between mean [Na +] p l and grouped temperature data). 3.3 Body mass (BM) and t o t a l body water (TBW) Before high NaCl exposure, ro o s t e r TBW (74 +.4.6% BM) was higher (P<0.001) than g u l l TBW (65 +. 1.1% BM). High NaCl d i e t s d i d not a f f e c t TBW i n e i t h e r r oosters or g u l l s (Table I I I ) . In both g u l l s and ro o s t e r s , body mass d i d not change s y s t e m a t i c a l l y upon exposure to high NaCl d i e t s . G u l l s tended to i n c r e a s e body mass when exposed to h a l f - s t r e n g t h seawater, but a reverse trend was evident when they drank more concentrated s a l i n e . N e i t h e r species was kept on high s a l t f o r a prolonged p e r i o d . F i g 7. Resting body temperature ( s o l i d squares) and panting t h r e s h o l d ( f i l l e d c i r c l e s ) i n 6 roosters (top) fed normal NaCl d i e t (up to Day 5), and high NaCl d i e t (Day 5 to Day 12), and i n 5 g u l l s (bottom) on progressive a c c l i m a t i o n from 0 mM to 475 mM NaCl d r i n k i n g s o l u t i o n . S t a t i s t i c a l s i g n i f i c a n c e i s denoted by * at P<0.05. 34 a 43 -i e 42 K 3 E I. 41 I 40 -Normal NaCl diet High NaCl diet 39 - — i — i — i — i — i — i — i — i — i — i — i — r L 2. a 4. 6. a. 7. a 9. 10. 1L 12. DAY 44 i 43 -42 -2 A 1 8. 4 1 £ 40 -39 -36 Panting threshold Body temperature ~i 1 1 1 1— 0. 113. 225. 338. 475. Drinking [NaCl] (mM) 35 Table I I I : Body mass (BM), t o t a l body water (TBW), hematocrit (Hct), and evaporative water l o s s (EWL measured at 30°C) i n : (i) 6 r o o s t e r s fed a normal NaCl d i e t , and f o l l o w i n g two weeks of high NaCl i n t a k e , and ( i i ) 5 g u l l s d r i n k i n g freshwater (0 mM NaCl), and during a c c l i m a t i o n to 225 mM and 475 mM NaCl. Data are presented as means with standard d e v i a t i o n . Diet BM TBW EWL Hct [NaCl] g % BM g/kg/hr ROOSTERS Normal 1809 74 .0** 1. 5 37 .1 ± 127 ± 4 .6 + 0. 1 ± 4.0 High 1890 73 .2 1. 9 41.4 ± 124 ± 3 .5 ± 0 . 3 ± 3.4 GULLS 0 mM 985 65 .2 2 . 3** 44.0 ± 101 ± 1 . 1 + 0 . 3 ± 3.0 225 mM 1012 63 .3 - — 43 .0 ± 97 ± 1 .7 ± 3.9 475 mM 995 64 .4 2 . 6** 45.5 ± 106 ± 2 .4 ± 0 . 3 ±2.0 S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s , r o o s t e r s compared to g u l l s , * P<0.05; ** P<0.01 (Paired t - t e s t ) . F i g 8. A comparison between the trends of ambient ( s o l i d t r i a n g l e s ) and body temperature ( s o l i d squares) i n 5 g u l l s during p r o g r e s s i v e a c c l i m a t i o n from 0 mM (Day 0 to 10) to 475 mM NaCl d r i n k i n g s o l u t i o n (Day 28 to 32) . S a l i n i t y (mM NaCl, open bars) i s s c a l e d on the second y - a x i s . 36 a 45 -i 0. 2. 4. 6. a 10. 12. U. 16. ia 20. 22. 24. 2a 2a 30. 32. Day 37 F i g 9. Mean t o t a l body water (TBW), and hematocrit (Hct, f i l l e d diamonds) i n 6 roo s t e r s (top) and i n 5 g u l l s (bottom) . Roosters were fed normal NaCl (week 1) followed by high NaCl d i e t (weeks 1 and 2). G u l l s were p r o g r e s s i v e l y acclimated from 0 mM to 475 mM NaCl d r i n k i n g s o l u t i o n . TBW i n roosters ( f i l l e d pentagons) and g u l l s ( f i l l e d c i r c l e s ) i s given as % body mass. E r r o r bars represent standard d e v i a t i o n from the mean. 37a • TBW •Hct r- r- 1 1 ' 0. 113. 225. 338. 475. Drinking saline concentration (mM) 38 3.4 Hematocrit I n i t i a l r o o s t e r Hct (37.1 +_ 4.0%) was s i g n i f i c a n t l y lower (P<0.05) than i n i t i a l g u l l Hct (44.0 + 3.01). High NaCl d i e t d i d not change hematocrit i n e i t h e r species, but the d i f f e r e n c e between species (roosters, 41.4 ± 3.4%; g u l l s , 45.5 +. 2.0%) became i n s i g n i f i c a n t (Table I I I ) . 3.5 Evaporative water l o s s (EWL) During normal d i e t intake, r o o s t e r EWL was 1.5 +. 0.1 g H 20.kg- 1 .hr- 1. A f t e r two weeks of exposure to high s a l t , EWL rose (P<0.05) to 1.9 + 0.3 g H20. kg- 1. h r - 1 (Table I I I ) . Chamber humidity at normal s a l t d i e t remained s t a b l e during experimentation. A f t e r two weeks NaCl a c c l i m a t i o n , the b i r d s passed wet faeces, c o n s t a n t l y wetting feathers around the c l o a c a l opening. Consequently, i t was not always p o s s i b l e to maintain chamber humidity. G u l l s , on freshwater, had a higher (P<0.01) w e i g h t - s p e c i f i c EWL (2.3 ± 0.3 g H^.kg-^hr- 1) than r o o s t e r s (1.5 ± 0.1 g H 20.kg-^ h r - 1 , Table I I I ) . NaCl a c c l i m a t i o n d i d not a f f e c t g u l l EWL. 3.6 R e s p i r a t o r y r a t e and panting t h r e s h o l d Roosters R e s t i n g r e s p i r a t o r y rate ( f ) , at normal NaCl d i e t , was 15 +. 3 breaths/min (Table V) . Under thermal challenge, the b i r d s panted at an i n t r a p e r i t o n e a l temperature of 41.95°C, approximately 1° above t h e i r normal r e s t i n g body temperature. 39 The ambient temperature at which panting s t a r t e d ranged between 35°C to 43°C. Minute v e n t i l a t i o n (%) increased up to 9- f o l d during the t r a n s i t i o n from normal breathing to panting. The t r a n s i t i o n was e i t h e r gradual or sudden w i t h i n the group (Appendix C) . V E decreased as panting continued. Breathing frequency 10 minutes a f t e r the onset of panting v a r i e d but reached a maximum of 170 cycles/min. F o l l o w i n g NaCl a c c l i m a t i o n , r e s t i n g f, t i d a l volume (VT) and V E tended to decrease. Panting t h r e s h o l d increased to 42.3 +_ 0.3 °C (Fig 7). The d i f f e r e n c e between r e s t i n g core temperature and panting t h r e s h o l d remained constant at approximately 1°C. The ambient temperature at which panting s t a r t e d d i d not change, and the value of f at maximum panting was unaffe c t e d i n r o o s t e r s (Table V). G u l l s R e s t i n g f (31 +. 2 breaths/min) at FW was higher (P<0.001) than that of ro o s t e r s eating the normal NaCl d i e t (Table V). FW g u l l s s t a r t e d panting at a mean i n t r a p e r i t o n e a l temperature of 41.9 ± 0.6°C, about 1.6°C +. 0.4 above t h e i r r e s t i n g body temperature ( F i g 7, Table IV). Following a c c l i m a t i o n to 475 mM NaCl, f increased s l i g h t l y , but was more v a r i a b l e s i n c e the b i r d s were very i r r i t a b l e and h i g h l y anxious during exposure to high d i e t a r y NaCl. Panting s t a r t e d at a mean i n t r a p e r i t o n e a l temperature of 42.4 +. 0.9°C, 1.4°C +. 0.5 above r e s t i n g body temperature (Fig 7, Table IV). The d i f f e r e n c e between r e s t i n g 40 and panting i n t r a p e r i t o n e a l temperature was s i m i l a r to that during FW a c c l i m a t i o n , and tended to be higher than that of ro o s t e r s under both normal and high NaCl d i e t s . Body temperature rose f a s t e r and panting was e l i c i t e d sooner than i n FW. There was no d i f f e r e n c e i n panting rates between g u l l s d r i n k i n g freshwater and seawater. 3.7 Relat ionships between var iables Plasma o s m o l a l i t y and [Na +] p l were c l o s e l y c o r r e l a t e d i n ro o s t e r s (r = 0.956, n = 18). Ionized calcium ([Ca 2 +] p l) rose (Fig 11) w i t h i n c r e a s i n g [Na +] p l i n both roosters (r = 0.664, n = 18) and g u l l s (r = 0.635, n = 25), and the slopes of the regr e s s i o n s were not s i g n i f i c a n t l y d i f f e r e n t (P<0.05). There was a c l o s e r p o s i t i v e c o r r e l a t i o n (Fig 12) between [Ca] p l and [ C a 2 + ] p l i n g u l l s (r = 0.639, n = 25) than i n roosters (r = 0.385, n = 18). In the l a t t e r , [Ca] p l d i d not f l u c t u a t e (Table I) . There was no c o r r e l a t i o n between i n t r a p e r i t o n e a l temperature and e i t h e r plasma Na : Ca ( g u l l s , r = 0.114, n = 25; r o o s t e r s , r = 0.097, n = 18) or Na + : Ca 2 + r a t i o ( g u l l s , r = 0.046, n = 25; r o o s t e r s , r = 0.458, n = 18) . There was a high p o s i t i v e c o r r e l a t i o n (r = 0.978) between mean [Na +] p l and grouped mean body temperature i n g u l l s . In r o o s t e r s , body temperature was p o s i t i v e l y c o r r e l a t e d w i t h panting t h r e s h o l d (r = 0.841, n = 18). In g u l l s , EWL and panting t h r e s h o l d showed weak c o l l i n e a r i t y (r = 0.648, n = 25). The r o o s t e r s passed wet feaces r e g u l a r l y during exposure to the hi g h NaCl d i e t . They a l s o vomitted f l u i d having a [Na+] of 41 141.3 mmol/L. Gulls exposed to 475 mM NaCl solution exhibited aggressive behaviour and were h y p e r i r r i t a b l e . Vigorous headshaking was observed. The birds spent longer periods i n t h e i r wading pools. As acclimation proceeded t h e i r feathers became r u f f l e d , the f a c i a l feathers appeared freckled, and t h e i r eyes, e n c i r c l e d by an increasingly v i s i b l e black ring, protruded outwards. 42 Table IV: Resting body temperature (Tb) , panting t h r e s h o l d (PT) , temperature d i f f e r e n c e (T d i f f ) between Tb and PT i n r o o s t e r s and g u l l s fed normal and high NaCl d i e t s r e s p e c t i v e l y . Tb, PT, and T diff are i n °C. High NaCl d i e t i n g u l l s c o n s t i t u t e d 475 mM NaCl d r i n k i n g s o l u t i o n . Diet Tb PT T d i f f ROOSTERS Normal 41 .0 42 .1 1.0 ± 0.2 ± 0.5 ± 0.4 High 41.2 42 .3* 1.0 ± 0.3 ± 0.3 ± 0.2 GULLS Normal 40.5 41.9 1.6 ± 0.5 ± 0.6 ± 0.4 High 41.2* 42 .4 1.4 ± 0.8 ± 0.9 ± 0.5 S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between normal and high NaCl d i e t s ; *-P<0.05. 43 Table V: Re s p i r a t o r y frequency if) and minute v e n t i l a t i o n (VE) at r e s t and at maximum panting i n 6 r o o s t e r s and 5 g u l l s fed normal and high NaCl d i e t s r e s p e c t i v e l y (VE data f o r g u l l s on high NaCl d i e t s are mis s i n g ) . High NaCl d i e t f o r g u l l s c o n s t i t u t e d 475 mM d r i n k i n g NaCl s o l u t i o n . Data are presented as means w i t h standard d e v i a t i o n . Diet Resting f Maximum f Resti n g V E Panting V E (ml) (ml) ROOSTERS Normal 15 .0 83 .7 • 455 586 ± 3 .2 ± 10 .2 ± 109 ± 181 High 13 .1 73 .7 349 615 ± 1.7 ± 2 2 . 5 ± 53 ± 221 GULLS Normal 30.7*** 128.1*** 878*** 1788*** ± 1.7 ± 18 .1 ± 225 ± 582 High 40.6*** 128.1*** ± 3 .4 ± 24 .2 G u l l values are s i g n i f i c a n t l y higher (P<0.001) than r o o s t e r v a l u e s . F i g 1 0 . T i d a l volume (VT, open bars) and r e s p i r a t o r y frequency (f , hatched bars) i n roosters fed normal NaCl d i e t (Week 0) and a f t e r 2 weeks i n high NaCl d i e t (Week 2 ). V T i s i n ml; f, i n cycles/min. V o l u m e ( m l ) o _i i i i i i_ ro o i i o o o j i i i i i i i i i t_ -i—i—i—|—i—i—i—i—p -i—i—i—r T T — i — i — I — i — i — i — r Respiration rate (cycles/mi n) ro o ro F i g 11. L i n e a r regressions of [Ca 2 +] p l and [Na +] p l r o o s t e r s (n = 18, s o l i d squares) and g u l l s (n = 25, open c i r c l e s ) before and during exposure to high NaCl d i e t s . C o r r e l a t i o n c o e f f i c i e n t s ( r ) , and r e g r e s s i o n equations are presented i n the diagram. 4 5 a Plasma [Na+] ( m m o l / L ) 46 F i g 12. L i n e a r r e g r e s s i o n l i n e s of [Ca 2 +] p l and [Ca] p l i n r o o s t e r s (n = 18, s o l i d squares) and g u l l s (n = 25, open c i r c l e s ) before and during exposure to high NaCl d i e t s . C o r r e l a t i o n c o e f f i c i e n t s ( r ) , and r e g r e s s i o n equations are presented i n the diagram. 46 a P l a s m a [Ca] ( m m o l / L ) 47 4 DISCUSSION 4.1 Plasma sodium and osmotic concentrations The [Na +] p l and Osmol p l of roosters rose maximally w i t h i n one week of high d i e t a r y NaCl exposure but d e c l i n e d i n the second week of NaCl i n t a k e (Fig 3, 4) suggesting an i n i t i a l i n c r ease i n i n t e s t i n a l NaCl absorption g r a d u a l l y countered by r e n a l e x c r e t i o n . High d i e t a r y NaCl t y p i c a l l y e levates [Na +] p l and Osmol p l i n domestic fowl (Arad and Skadhauge, 1986; Thomas and Skadhauge, 1989). Net feed consumption i n fowl given high NaCl d i e t i s higher than i n those given low NaCl d i e t , and absolute a b s o r p t i o n of Na+ i n the a n t e r i o r gut i s twice as high i n high Na compared to low Na b i r d s (Hurwitz et, a_l. , 1970) . U l t i m a t e l y water absorption i s higher i n the a n t e r i o r gut (Hurwitz et a l . , 1970) during high Na d i e t a c c l i m a t i o n . E x t r a c e l l u l a r f l u i d volume (ECFV) would be expanded due to a sustained increase i n foregut t r a n s f e r ; t u b u l a r Na+ and H20 reabsorption would decrease,, and r e n a l f l u i d discharge would increase. At the same time d i e t s c o n t a i n i n g high NaCl suppress NaCl and s o l u t e - l i n k e d water r e a b s o r p t i o n (Thomas and Skadhauge, 1979; Rice and Skadhauge, 1982) i n the coprodeum and colon. The u l t i m a t e r e s u l t would be the passage of watery c l o a c a l f l u i d as was demonstrated by the constant passage of wet faeces by the r o o s t e r s . The r o o s t e r s i n t h i s c o n d i t i o n were probably n a t r i u r e t i c . I t i s c l e a r however, that i n the f i r s t week of high d i e t a r y NaCl exposure, increased foregut absorption was not o f f s e t by decreased hindgut r e a b s o r p t i o n and p o s s i b l e increased r e n a l s o l u t e l o s s as Osmol p l 48 and plasma i o n concentrations remained high (Table I ) . The decreasing trends of both [Na +] p l and Osmol p l i n the second.week of high NaCl i n t a k e i n roosters suggest greater r e n a l s a l t - e x c r e t i n g c a p a c i t y presumably augmented by a much reduced p o s t e r i o r gut sodium r e t r i e v a l . In g u l l s d r i n k i n g s a l i n e which was s l i g h t l y more concentrated than seawater, [Na +] p l rose s i g n i f i c a n t l y ( F i g 5). Previous s t u d i e s have reported maximum plasma s o l u t e e l e v a t i o n at qu a r t e r - s t r e n g t h seawater (Hughes, 1970; Gray and Erasmus, 1989). The a b i l i t y of g u l l s to maintain f a i r l y constant plasma l e v e l s over a wide range of d r i n k i n g water s a l i n i t i e s i s a t t r i b u t a b l e to the presence of e f f i c i e n t l y f u n c t i o n i n g s a l t glands (Hughes, 1970; Roberts and Hughes, 1984). The maximum concentrations of d r i n k i n g s a l i n e presented i n those s t u d i e s were 450 mM NaCl. G u l l s a l t gland s e c r e t i o n (SGS) [Na+] can be as high as 800 mmol/L (Hughes, 1970). However, maximum volume of s e c r e t i o n per day may be small compared to f l u i d ingested (Hughes, 1972), t h e r e f o r e the absolute ingested s o l u t e load may exceed the absolute excreted amount and account f o r the observed i n c r e a s e i n [Na"] p l (Table I I ) . Mucosal water and Na + t r a n s f e r , measured i n everted sacs of another s a l t - s e c r e t i n g b i r d , the duck, Anas platyrynchos, increased i n a l l segments of the i n t e s t i n e throughout a 4-day seawater a c c l i m a t i o n p e r i o d (Crocker and Holmes, 1971). The r e l a t i o n s h i p between the amount of f r e e water made a v a i l a b l e by s a l t gland f u n c t i o n and the co n c e n t r a t i o n of ingested water may not be a simple a r i t h m e t i c one but one i n 49 which the f u n c t i o n i s a l t e r e d by f a c t o r s such as io n pump d e n s i t y which may l i m i t SGS. I f SGS rate f o l l o w s s a t u r a t i o n k i n e t i c s , s e c r e t i o n may not o f f s e t absorption, at very high d r i n k i n g s a l i n e c o n c e n t r a t i o n s , and [Na +] p l may not be maintained. The e x t r a -r e n a l e x c r e t o r y mechanism may therefore be inadequate to r i d plasma of a l l excess s a l t i f and when the d r i n k i n g s a l i n e i s very concentrated. The increase i n [Na +] p l recorded at 475 mM d r i n k i n g [NaCl] (Fig 5) suggests the i n s u f f i c i e n c y of the s a l t glands, at that c o n c e n t r a t i o n , to r i d plasma of any more ingested NaCl. 4.2 Plasma calcium The [Ca] p l was not a f f e c t e d by high NaCl i n t a k e i n r o o s t e r s . In g u l l s , there was a gradual increase as d r i n k i n g s a l i n e c o n c e n t r a t i o n increased. On the other hand, [ C a 2 + ] p l increased d r a m a t i c a l l y as [Na +] p l increased i n both species. Approximately 50% of t o t a l calcium i s i n the i o n i z e d form i n humans (Fogh-Andersen et -al.. , 1978), rainbow t r o u t (Andreasen, 1985), and european e e l (Hanssen et al.. , 1989). Roosters and g u l l s have approximately 55% plasma calcium i n the i o n i z e d form (Table I, II) thus the s i g n i f i c a n t increase i n g u l l [Ca] p l i s a t t r i b u t e d to the 11.8% increase i n i o n i z e d [Ca 2 +] p l. In r o o s t e r s , [ C a 2 + ] p l i n c r e a s e d by a modest 5.5% and the increase i n [Ca] p l was not s i g n i f i c a n t . Reuter and S e i t z (1968) observed that Ca 2 + i s extruded from sheep, guinea-pig, and c a l f c a r d i a c muscle c e l l s by a mechanism which was g r e a t l y dependent on e x t r a c e l l u l a r [Na+] . In c e l l s of guinea-pig t a e n i a c o l i , s u b s t i t u t i o n of Na+ i n the 50 bathing medium slowed down Ca 2 + e f f l u x and increased i n t r a c e l l u l a r Ca 2 + i n the absence of t i s s u e c o n t r a c t i o n , suggesting the exi s t e n c e of a N a - s e n s i t i v e c e l l u l a r Ca 2 + pool (Aaronson and Van Breemen, 1981). This Na +-dependent Ca 2 + e f f l u x may not be r e s t r i c t e d to muscle c e l l s and may be a more widespread phenomenon that could i n f l u e n c e e x t r a c e l l u l a r [Ca 2 +] when e x t r a c e l l u l a r [Na+] i s a l t e r e d . Plasma [Ca 2 +] was l i n e a r l y c o r r e l a t e d w i t h [Na +] p l i n g u l l s (r = 0.635, n = 25). This supports the suggestion that Ca 2 + may be l e a v i n g the i n t r a c e l l u l a r compartment as a consequence of increased [Na+] i n the e x t r a c e l l u l a r f l u i d . Furthermore, i n c u l t u r e d r a t glomerular mesangial c e l l s , a r g i n i n e vasopressin (AVP) induces r a p i d concentration-dependent increases i n Ca 2 + e f f l u x (Takeda et a l . , 1988). Since AVP and i t s avian equivalent, a r g i n i n e v a s o t o c i n (AVT), are secreted i n response to increased ECF o s m o l a l i t y which i s mainly a t t r i b u t a b l e to increased [Na +] p l (Mohring et a l . , 1980; S t a l l o n e and Braun, 1986; Raveendran, 1987), i t may be that i n c o n d i t i o n s of high Osmol p l, AVT may cause Ca2" e f f l u x from c e l l s that have AVT-receptors thus c o n t r i b u t i n g to the apparent in c r e a s e i n [Ca 2 + ] p l . The m o b i l i z a t i o n of Ca 2 + from the bound form i n plasma was not a l i k e l y cause of increased [ C a 2 + ] p l s i n c e the r a t i o of plasma i o n i z e d to t o t a l calcium, and the c o n c e n t r a t i o n of bound calcium stayed unchanged. An exchange of Na+ and K+ and/or outward leakage of K+ a l s o appears to occur. In both g u l l s and r o o s t e r s , [ K + ] p l increased, though not s i g n i f i c a n t l y i n the former, during the second week of 51 high NaCl i n t a k e . Since d i e t a r y [K+] was not manipulated, the incre a s e i n [ K + ] p l i n d i c a t e s that there was e x t r u s i o n of K + from i n t r a c e l l u l a r space i n t o the i n t e r s t i t i u m presumably i n exchange f o r Na +. Plasma [CI"] increased i n g u l l s during the f i r s t week of a c c l i m a t i o n and remained high when [Na +] p l appeared unchanged. In r o o s t e r s , [ C l " ] p l continued to r i s e as [Na +] p l d e c l i n e d i n the second week of a c c l i m a t i o n . This i n d i c a t e s that Na+ and CI" tra n s p o r t were not always coupled and strengthens the argument that Na+ t r a n s p o r t i n t o the c e l l s i s coupled w i t h the e x t r u s i o n of c a t i o n s such as Ca 2 + from c e l l s . Both i n t r a c e l l u l a r Ca 2 + and K + seem to be extruded i n exchange f o r Na+, and Na +/Ca 2 + exchange probably occurs only when [Na4] i n the e x t e r n a l medium i s very-h i g h ( F i g 5). 4.3 Plasma sodium to calcium r a t i o and body temperature The plasma sodium to calcium r a t i o d i d not incr e a s e w i t h increased s a l t i n t a k e i n e i t h e r species i r r e s p e c t i v e of whether [Na +] p l was compared to [Ca] p l or [Ca 2 +] p l. Plasma sodium to i o n i z e d calcium r a t i o , i n f a c t , tended to decrease s l i g h t l y when [Na +] p l was highest i n both species as high [Na +] p l was always a s s o c i a t e d w i t h a very high [Ca 2"] p l. A 5.4% r i s e i n [Na +] p l was asso c i a t e d w i t h a s i g n i f i c a n t e l e v a t i o n of body temperature i n g u l l s . This i n c r e a s e in,body temperature was unrelated to plasma sodium to calci u m r a t i o . Several s t u d i e s have reported changes i n body temperature when s o l u t i o n s of v a r y i n g Na+ and Ca 2 + concentrations were perfused i n 52 the v e n t r i c l e s or p o s t e r i o r hypothalami of d i f f e r e n t mammalian and avian species- (Myers and Veale, 1971; Myers, 1974; Hannegan and W i l l i a m s , 1975; Saxena, 1976; Denbow and Edens, 1980; 1981; Maki et. al.. , 1988). In every case, perfusate [Na+] and [Ca 2 +] were s e v e r a l times the p h y s i o l o g i c a l maxima. The t r a n s p o r t of ions from plasma to the c e r e b r o s p i n a l f l u i d (CSF) i s c o n t r o l l e d by the c h o r o i d plexus such that a 10% increase i n CSF [Na+] or [Ca 2 +] i s not achievable by e l e v a t i n g plasma i o n concentrations (Bradbury and Kleenan, 1969) . Furthermore, the exchange of ions between CSF and b r a i n t i s s u e i s f u r t h e r d e l i m i t e d by the b r a i n ' s ependymal and g l i a l surfaces (Cserr, 1971). Sodium does accumulate i n the b r a i n i n response to osmotic dehydration of the i n t e r s t i t i u m (DePascuale and Cserr, 1988; Cserr, 1988) but only at the l e v e l of 1 mEq of s o l u t e f o r a 3 osmol increase i n Osmol p l. I t i s even more u n l i k e l y that increased NaCl i n g e s t i o n would a l t e r the CSF Na + : Ca 2 + r a t i o when ECF [Ca 2 +] increases as a r e s u l t of increased ECF [Na+] . Edens (1976) i n j e c t e d 8 week o l d b r o i l e r c o c k e r e l s w i t h an intravenous NaCl load but was unable to demonstrate an e l e v a t i o n of body temperature under thermoneutrality. His i n j e c t a t e contained only 5% more Na+ than i n plasma and was administered i n one i n j e c t i o n at the l e v e l of about 2 ml per kg of body weight. At 8 weeks b r o i l e r cockerels have a high ECF (2 5% body mass, Newell and Shaffner, 1950) and plasma volume (70% blood volume, H a r r i s and Koike, 1977) and weigh approximately 1 kg. An a d d i t i o n a l 5% Na+ i n 2 ml perfusate, d i l u t e d by 250 ml ECF, would 53 r e s u l t i n a 4.4% increase i n [Na +] p l. This degree of [Na +] p l e l e v a t i o n was surpassed by that achieved i n my g u l l s through d i e t a r y NaCl a c c l i m a t i o n (5.4%) . In my r o o s t e r s , a 3.7% increase i n [Na +] p l was not a s s o c i a t e d w i t h a s i g n i f i c a n t r i s e i n body temperature. I t i s not s u r p r i s i n g then that Edens (1976) recorded no change i n body temperature under t h e r m o n e u t r a l i t y . An acute intravenous load of NaCl i s l e s s s t a b l e than a chronic one because i t s clearance s t a r t s immediately without replacement. A chronic load of s i m i l a r concentration i n plasma may exert a cumulative and sustained response thus be more e f f e c t i v e . At the end of two weeks of high d i e t a r y s a l t i n t a k e , r o o s t e r [Na +] p l and body temperature approached p r e - a c c l i m a t i o n l e v e l s . The e l e v a t i o n of body temperature seems to depend upon the magnitude of plasma [Na +] p l e l e v a t i o n suggesting that [Na +] p l e x e r t s a s t i m u l a t o r y e f f e c t on the body's thermogenic processes or an i n h i b i t o r y e f f e c t on some heat l o s s mechanism(s). I t must be argued that the increase i n core temperature i s not n e c e s s a r i l y due to an e l e v a t i o n of the hypothalamic 'set-p o i n t ' f o r temperature r e g u l a t i o n as suggested by Arad and Skadhauge (1986). F i r s t , the increase i n [Na +] p l was not enough to cause an e l e v a t i o n of CSF [Na+] that would s h i f t the Na+ : Ca 2 + r a t i o by a magnitude s u f f i c i e n t to elevate body temperature as reported i n previous s t u d i e s (Saxena, 197 6; Denbow and Edens, 1980; 1981; Maki et a l . , 1988)). Second, i t i s q u i t e c l e a r that [ C a 2 + ] p l increased by a l a r g e r percentage than [Na +] p l so that i f plasma Na + : Ca 2 + r e f l e c t s that of CSF, then the r a t i o was 54 u n l i k e l y to be higher i n the CSF. I t i s true that b r a i n calcium i s more c l o s e l y regulated than sodium (Bradbury and Kleenan, 1969; Cserr, 1971), but even i n the absence of any i n c r e a s e i n [C a 2 + ] p l , the i o n i c imbalance necessary i n b r a i n t i s s u e to el e v a t e body temperature could not have been achieved. Roosters and g u l l s do not d i f f e r q u a l i t a t i v e l y i n t h e i r body temperature s h i f t s i n response to increased d i e t a r y NaCl. In s p i t e of t h e i r e x t r a - r e n a l s a l t s e c r e t i n g a b i l i t y , g u l l s accumulated more Na* i n plasma above t h e i r normal l e v e l s than d i d r o o s t e r s . This may be because the s a l i n e g u l l s drank i n week 5 contained more NaCl than the rooster high NaCl d i e t . Owing to the d i f f e r e n c e s i n d i e t items of these two species i t was imposs i b l e to standardize t h e i r d i e t a r y c a t i o n i n t a k e . G u l l s ate b a i t h e r r i n g of v a r i a b l e i o n i c concentrations, and which s u p p l i e d much of t h e i r d a i l y water requirements e s p e c i a l l y when they drank s a l i n e more concentrated than seawater. Food and water in t a k e was not q u a n t i f i e d . Roosters, on the other hand, ate dry crumbles of constant NaCl concentration and drank water that was hypotonic to t h e i r blood. The r e s u l t s however exemplify the a b i l i t y of g u l l s to withstand very high increases i n [Na*] p l. I n t e r e s t i n g l y enough, g u l l Osmol p l remained r e l a t i v e l y constant even when [Na*] p l rose sharply. The d i f f e r e n c e s between the e f f e c t s of ingested NaCl on body temperature of the two species were q u a n t i t a t i v e and apparently r e l a t e d to the increase i n [Na +] p l. Body temperature rose more i n g u l l s than i n r o o s t e r s , but [Na*] p l too had r i s e n by a greater percentage i n g u l l s than i n 55 r o o s t e r s . 4.4 T o t a l body water and body temperature T o t a l body water (TBW) of roosters was 74.1 _+ 4.6%, a value c o n s i s t e n t w i t h that of Chapman and Mihai (1972). G u l l s on freshwater had TBW of 65.2 +. 1.1%. This value i s lower than those p r e v i o u s l y reported (Ruch and Hughes, 1975; Walter and Hughes, 1978), but i s w i t h i n the range of TBW of ad u l t b i r d s (Skadhauge, 1981; Hughes et a l . , 1987). . In g u l l s , TBW v a r i e d i n a d i r e c t i o n opposite to that of body mass (r = -0.642, n = 15) . TBW remained r e l a t i v e l y unchanged i n both species during exposure to high d i e t a r y NaCl. Increases i n TBW of up to 15% were reported f o r roo s t e r s under intravenous NaCl loads (Ruch and Hughes, 197 5) even though no d r i n k i n g water was s u p p l i e d during the course of the experiment. This suggests the s a l t load e l i c i t e d metabolic processes that produced s u f f i c i e n t metabolic water to increase TBW by 15%. In the same study intravenous NaCl l o a d i n g d i d not increase TBW of g u l l s . TBW does not appear r e l a t e d to body temperature, but the d i s t r i b u t i o n of water i n the two e x t r a c e l l u l a r body compartments, i . e . , i n t r a v a s c u l a r and i n t e r s t i t i a l spaces under a s a l t load may be c r u c i a l to the amount of water r e a d i l y a v a i l a b l e f o r evaporative c o o l i n g . A d i s p r o p o r t i o n a t e l y large water s h i f t from the i n t e r s t i t i a l compartment would reduce EWL and impair heat d i s s i p a t i o n l e a d i n g to an e l e v a t i o n of body temperature. In t h i s study, no r e l a t i o n s h i p was found between EWL and TBW. In ducks, Anas 56 platy r y n c h o s , plasma volume increased f o l l o w i n g s a l i n e a c c l i m a t i o n (Ruch and Hughes, 1975), p o s s i b l y at the expense of i n t e r s t i t i a l volume since TBW decreased by a percentage higher than the measured increase i n ECFV. Hematocrit values remained f a i r l y unchanged i n both g u l l s and ro o s t e r s suggesting that plasma volume remained constant assuming t o t a l red blood c e l l volume remained the same. I n t r a v a s c u l a r p r o t e i n s account f o r a s u b s t a n t i a l p o r t i o n of Osmol p l (Horowitz, 1984). In the event of an in c r e a s e i n Osmol p l body water d i s t r i b u t i o n would favour the i n t r a v a s c u l a r space e s p e c i a l l y i n the absence of high h y d r o s t a t i c pressure which would otherwise cause seepage of p r o t e i n s i n t o i n t e r s t i t i a l space as i n ex e r c i s e (Horowitz, 1984). Consequently, l e s s water w i l l be a v a i l a b l e i n the i n t e r s t i t i u m f o r evaporative c o o l i n g . I f , on the other hand, NaCl i n g e s t i o n merely r e s u l t s i n hypertonic normovolemia without any e f f e c t on water d i s t r i b u t i o n , the body's s e n s i t i v i t y to increased Osmol p l or [Na+] must r e s u l t i n reductions i n heat t r a n s f e r s u f f i c i e n t to cause an increase i n body temperature. 4.5 Evaporative water loss G u l l s had a higher w e i g h t - s p e c i f i c EWL at 30°C than r o o s t e r s . That EWL i s i n v e r s e l y p r o p o r t i o n a l to body weight i n adu l t b i r d s has been known since Bartholomew and Dawson (1953). G u l l s a l s o maintained a lower r e s t i n g body temperature (40.4 ± 0.4°C) than r o o s t e r s (41.0 +_ 0.2°C). In g u l l s , d i e t a r y NaCl a c c l i m a t i o n d i d not cause any changes i n EWL at 3 0°C. High 5V Osmol p l concurrent w i t h high [Na +] p l i s known to .minimize EWL i n many mammals (Senay, 1968; Baker and D o r i s , 1982; T u r l e j s k a and Baker, 1986). At 38°C, i n f u s i o n of 30% s a l i n e (1.5 ml.kg" 1) s i g n i f i c a n t l y i n c r e a s e d body temperature ' and decreased e v a p o r a t i v e heat l o s s i n cats (Baker and D o r i s , 1982); these e f f e c t s were not demonstrable at thermoneutral temperatures (25°C). I t seems, t h e r e f o r e , that osmotic changes i n plasma may not a f f e c t EWL mechanisms unless they are a c t i v a t e d beyond t h e i r r e s t i n g l e v e l s , f o r inst a n c e , d u r i n g thermal s t r e s s and/or d e h y d r a t i o n . I t was not p o s s i b l e to determine EWL of h i g h NaCl-a c c l i m a t e d r o o s t e r s , w i t h a good degree of accuracy, because f e a t h e r s around the vent were permanently dampened thus c o n s t a n t l y adding humidity to the open flow system. As a r e s u l t much of the moisture added to the D i e r i t e (CaCl 2) was of no thermoregulatory v a l u e . At thermoneutral temperatures, cutaneous e v a p o r a t i v e water l o s s (CEWL) reached 57% of t o t a l EWL i n ducks (Bouverot et a l . , 1974), exceeded 60% i n pigeons (Webster and King, 1987), and remained a s u b s t a n t i a l f r a c t i o n of t o t a l EWL at lower and h i g h e r temperatures. In gene r a l , CEWL amounts to between 40% and 75% of t o t a l EWL j at thermoneutral temperatures (Dawson, 1982). Temperature-dependent changes i n the r e l a t i v e c o n t r i b u t i o n s of r e s p i r a t o r y and cutaneous evaporation to t o t a l EWL are b e l i e v e d to be a s s o c i a t e d w i t h vasomotor adjustments or h y d r a t i o n of the stratum cornuem of the s k i n (Webster et al.. , 1985). No de t e r m i n a t i o n s were made, i n t h i s study, of d i f f e r e n t i a l 58 q u a n t i t i e s of water l o s t from cutaneous and r e s p i r a t o r y s u r f a c e s , but body f l u i d s h i f t s induced by o s m o l a l i t y changes may cause a l t e r a t i o n s i n the vasomotor tone and/or the h y d r a t i o n s t a t e s of d i f f e r e n t ECF compartments, and change the r e l a t i v e or a b s o l u t e c o n t r i b u t i o n s of CEWL and REWL to t o t a l EWL. High d i e t a r y NaCl d i d not change V E s i g n i f i c a n t l y i n r o o s t e r s . I f the moisture content of e x p i r e d a i r remained constant, the r e l a t i v e c o n t r i b u t i o n of CEWL under s a l t d i e t must have remained unchanged s i n c e t o t a l EWL remained c o n s t a n t . T o t a l EWL depends on deep body temperature (Dawson, 1982) . High [Na +] p l i n c r e a s e s deep body temperature perhaps by a ge n e r a l e l e v a t i o n of the body's metabolism. Body temperature rose by as much as 0.6°C i n g u l l s . T h i s i n i t s e l f may have e n t a i l e d an i n c r e a s e i n a b s o l u t e q u a n t i t y of water l o s t i n e v a p o r a t i v e c o o l i n g above that at normal core temperature under normal NaCl d i e t s . I t cannot be concluded on the b a s i s of these r e s u l t s that EWL necessary to keep body temperature at i t s normal l e v e l was constant w i t h h i g h NaCl i n t a k e s i n c e body temperature i t s e l f was maintained above normal. T h i s can only be e s t a b l i s h e d i f EWL i s measured i n s a l t loaded b i r d s i n the absence of an e l e v a t i o n i n deep body temperature. Ambient temperature i n f l u e n c e s EWL, but i t i s i n c r e a s e d body temperature that s t i m u l a t e s i n c r e a s e d EWL by re d u c i n g s k i n vapour pressure r e s i s t a n c e (Webster et_ a_l, 1985) . Webster et a l . , (1985) a l s o suggested that the s a t u r a t i o n vapour d e n s i t y on which CEWL depends i s an ex p o n e n t i a l f u n c t i o n of s k i n temperature, one that was arguably h i g h e r i n NaCl a c c l i m a t e d 59 b i r d s . An endogenous increase i n body temperature, as i n NaCl b i r d s , may be expected to cause an increase i n EWL. This , by no means, i n v a l i d a t e s the suggestion that high [Na +] p l and Osmol p l i n c r e a s e body temperature by reducing the c a p a c i t y f o r evaporative heat d i s s i p a t i o n . . R e l a t i v e l y speaking, more water may be l o s t i n evaporation when the body temperature of a freshwater-acclimated b i r d i s r a i s e d by 0.6°C compared to a sa l t w a t e r - a c c l i m a t e d one. Cutaneous EWL increased from 3.9 mg.m" 2 . s _ 1 at 30°C s k i n temperature (Tb = 40.6°C) to 9.0 mg.nf2.s'1 at 40°C s k i n temperature (Tb = 41.6°C) i n the pigeon; an EWL increase of over 100% c o i n c i d i n g w i t h a 1°C increase i n deep body temperature (Webster et a l . , 1985) . An increase i n deep body temperature of as much as 0.6°C may cause a tremendous increase i n EWL. I t can be argued that plasma h y p e r o s m o l a l i t y depressed EWL to the measured value which was not as larg e as i t might have been at the p r e v a i l i n g body temperature. 4.6 V e n t i l a t i o n 4.6.1 Salt loading and v e n t i l a t i o n The r e s p i r a t o r y frequencies and t i d a l volumes recorded f o r both normal NaCl roosters and FW g u l l s agreed w e l l w i t h those of b i r d s i n t h e i r s i z e ranges (Lasiewski and Calder, 1971). In keeping w i t h the inverse r e l a t i o n s h i p between f and body s i z e (Lasiewski and Calder, 1971), g u l l s had a higher f than r o o s t e r s . V E was a l s o higher i n g u l l s than i n roo s t e r s i n d i c a t i n g a much higher w e i g h t - s p e c i f i c oxygen consumption i n g u l l s . Again t h i s 60 agrees w i t h the p r e v i o u s l y reported body mass/metabolic r a t e r e l a t i o n s h i p (Lasiewski and Dawson, 1967). F o l l o w i n g high NaCl a c c l i m a t i o n , the brea t h i n g p a t t e r n was more v a r i a b l e between i n d i v i d u a l s , but r o o s t e r s tended to have a lower V E (P=0.08) than before a c c l i m a t i o n . Both f and V T tended to d e c l i n e although Osmol p l increased. In dogs a p o s i t i v e l i n e a r c o r r e l a t i o n was shown between a r t e r i a l hydrogen i o n c o n c e n t r a t i o n ([H + ] J and Osmol p l (Anderson and Jennings, 1988a; 1988b). The i n c r e a s e i n [H +] a was a t t r i b u t e d to a decrease i n a r t e r i a l strong i o n d i f f e r e n c e ([SID] a) caused by the s t a b i l i t y of a r t e r i a l potassium concentration ([K +] a) while [ C l " ] a i n c r e a s e d . In t h i s study, there was a l s o no change i n [ K + ] p l during the p e r i o d of v e n t i l a t o r y measurements before and a f t e r exposure to high d i e t a r y NaCl. No measurement was made of plasma l a c t a t e c o n c e n t r a t i o n , so i t was not p o s s i b l e to c a l c u l a t e [SID] p l. However, a c c l i m a t i o n of dogs (Anderson and Jennings, 1988b) to NaCl never a f f e c t e d the a r t e r i a l p a r t i a l pressure of carbon d i o x i d e (PaC0 2) though i n r e l a t i o n to Osmol p l, P aC0 2 would be considered to decrease. This would counterbalance the decrease i n [SID] a and maintain the r e l a t i o n s h i p between [H +] a and Osmol p l. The blood acid-base adjustments'would i n e f f e c t have no impact on v e n t i l a t i o n . In f a c t Anderson and Jennings (1988a, 1988b) saw no e f f e c t of 'decreased' P aC0 2 on v e n t i l a t i o n . In r o o s t e r s , there were no e f f e c t s of high d i e t a r y NaCl on the venous p a r t i a l pressure of carbon d i o x i d e (PvC02) and pH (Deyhim and Teeter, 1990) . Even i f , i n the present study, NaCl a c c l i m a t i o n could 61 have s l i g h t l y lowered ro o s t e r blood SID, i t would not e x p l a i n the s l i g h t decrease i n VE. The outcome suggests a decrease i n PC02, a c o n d i t i o n that i n d i c a t e s blood pH increased r a t h e r than decreased as a lowered SID would suggest. Furthermore, Hurwitz et a l . (1973) proposed that a d i e t a r y Na/Cl r a t i o ranging from 0.6 to 2.0 had no e f f e c t on blood pH at thermoneutral temperatures. The weight to weight r a t i o of d i e t a r y Na/Cl i n t h i s study was 0.6 f o r r o o s t e r s , and at l e a s t 0.65 f o r g u l l s . At these r a t i o s blood l e v e l s of C0 2 and H+ would not have been a l t e r e d beyond normal ranges i n the absence of thermal s t r e s s or e x e r c i s e . The s l i g h t decrease i n V E may not be d i r e c t l y a t t r i b u t a b l e to changes i n blood acid-base balance but to a general depressant e f f e c t of high Osmol p l or [Na"] p l on r e s p i r a t o r y f u n c t i o n perhaps at the c e n t r a l c o n t r o l . G u l l s were extremely i r r i t a b l e a f t e r s a l t a c c l i m a t i o n and when handled r e s p i r e d at frequencies that were s i g n i f i c a n t l y h igher than during FW a c c l i m a t i o n . Despite a body temperature i n c r e a s e of 0.6°C, e l e v a t i o n of t o t a l EWL was minimal. 4.6.2 Panting threshold The i n t e r n a l t h r e s h o l d f o r panting i n NaCl acclimated r o o s t e r s was s i g n i f i c a n t l y higher than the p r e - a c c l i m a t i o n average but i n g u l l s the change was not s i g n i f i c a n t . Nonetheless the panting response was e l i c i t e d at a higher ' deep body temperature. I n t r a p e r i t o n e a l temperature rose 1.4°C above the r e s t i n g value to e l i c i t panting i n g u l l s w h i l e i n r o o s t e r s only 62 a 1°C r i s e was s u f f i c i e n t before and a f t e r NaCl a c c l i m a t i o n . G u l l s thus seemed to withstand l a r g e r increases i n core temperature before panting occurred. Panting t h r e s h o l d s i n both species were v i r t u a l l y i d e n t i c a l d e s p i te the d i f f e r e n c e s i n r e s t i n g body temperature (The domestic fowl maintains i t s body temperature at a l e v e l higher than the g u l l ) . Upon high d i e t a r y NaCl i n t a k e the panting thresholds of each species increased r e s p e c t i v e l y (although i n g u l l s the increase was not s i g n i f i c a n t ) by roughly the same magnitude. FW g u l l s had a c l e a r thermoregulatory advantage over roosters s i n c e they t o l e r a t e d a gre a t e r accumulation of thermal energy before they s t a r t e d e l i m i n a t i n g heat by panting which i s e n e r g e t i c a l l y c o s t l y . I t i s w e l l known that a high Osmol p l e i t h e r delays panting or r a i s e s the i n t e r n a l body temperature at which i t i s e l i c i t e d (Crawford and Schmidt-Nielsen, 1967; Brummermann and Rautenberg, 1989). These r e s u l t s confirm t h i s phenomenon i n the domestic fowls. G u l l s had a much higher r e s t i n g V E than r o o s t e r s which may have been adequate to d i s s i p a t e excess body heat u n t i l core temperature rose by more than 1.4°C. The g u l l s were sm a l l e r i n s i z e than r o o s t e r s , and t h i s may have favoured greater s i g n i f i c a n c e of non-evaporative heat l o s s than i n r o o s t e r s . Furthermore g u l l s are l e s s s c a l y on the legs hence l e s s i n s u l a t e d . These heat l o s s avenues may take longer to exhaust before panting i s employed as a heat l o s s mechanism. A s h i f t i n the panting threshold may not n e c e s s a r i l y mean a s h i f t i n the hypothalamic 'set-point' f o r temperature r e g u l a t i o n . Panting i s thermally induced but may be i n f l u e n c e d by non-thermal f a c t o r s . C l e a r l y the increase i n fowl panting t h r e s h o l d f o l l o w i n g NaCl a c c l i m a t i o n i m p l i c a t e s body f l u i d h y p e r o s m o l a l i t y and/or incre a s e d [Na +] p l as f a c t o r s a l t e r i n g the responsiveness of the panting centre and/or resonating organs to thermal i n p u t s . Both r e s t i n g body temperature and panting t h r e s h o l d are r a i s e d by NaCl a c c l i m a t i o n , but nothing i s known about the core temperature at which s h i v e r i n g would be evoked. Thus to a s s e r t that the 's e t - p o i n t ' i s r a i s e d would be true f o r panting as a heat l o s s mechanism but not f o r thermogenic processes. There i s no b a s i s i n these s t u d i e s f o r a s s e r t i n g that the ' s e t - p o i n t ' f o r temperature r e g u l a t i o n was r a i s e d i n e i t h e r species although the t h r e s h o l d f o r a c t i v e EWL was elevated. The increase i n [Na +] p l r e a l i z e d i n t h i s study was not comparable to those obtained by hypothalamic and i n t r a v e n t r i c u l a r i n f u s i o n s (Saxena, 1976; Denbow and Edens, 1980; 1981; Maki et a l . , 1988). That a much sma l l e r c h r o n i c i n c r e a s e i n [Na +] p l elevated body temperature and panting t h r e s h o l d suggests that e l e c t r o l y t e s may have p e r i p h e r a l e f f e c t s t h a t augment the c e n t r a l imbalances imposed by a l t e r e d CSF [Na+] and [Ca 2 +] that the previous studies d i d not address. Perhaps i n the presence of an equal plasma s h i f t i n e l e c t r o l y t e r a t i o , s m a l l e r q u a n t i t i e s of Na+ and/or Ca 2 + i n hypothalamic per f u s a t e would be s u f f i c i e n t to a l t e r body temperature. Panting thresholds i n both species were a f f e c t e d s i m i l a r l y but to v a r y i n g degrees by NaCl a c c l i m a t i o n . In r o o s t e r s , v a r i a b i l i t y was minimal so s i g n i f i c a n c e was achieved. The 64 magnitude of increase of Osmol p l was r e l a t i v e l y g r eater i n ro o s t e r s than i n g u l l s . Roosters had a r e s t i n g Osmol p l of 303 mosmol/kg. A 15 mosmol/kg (5.2%, roosters) i n c r e a s e would be much more e f f e c t i v e than 8 mosmol/kg (2.6%, g u l l s ) i n a l t e r i n g the f u n c t i o n a l c h a r a c t e r i s t i c s of neurons i n the panting centre. G u l l s had a high i n i t i a l Osmol p l which was perhaps due to the NaCl content of t h e i r d i e t of b a i t h e r r i n g , Clupea p a l a s i i , but during high d i e t a r y NaCl intake, they were b e t t e r able to maintain Osmol p l due to the excretory a c t i o n of t h e i r s a l t glands. 4.6.3 Panting pat tern Roosters panted at frequencies that were as high as nine times the r e s t i n g f value while V T f e l l to as low as 1/4 the r e s t i n g value. The shallow breaths were broken by deeper i n h a l a t i o n s o c c u r r i n g every s e v e r a l seconds. This type of panting has been described as 'flush-out' panting (Johansen and Bech, 1984). The shallow breaths move a i r at or around dead space volume so l i t t l e or no a l v e o l a r v e n t i l a t i o n occurs between the deep breaths. This p a t t e r n i s designed to avo i d a l v e o l a r h y p e r v e n t i l a t i o n which could cause severe r e s p i r a t o r y a l k a l o s i s d uring prolonged panting (Calder and Schmidt-Nielsen, 1968; Kohne and Jones, 1975; Marder and Arad, 1989). The panting type was unchanged i n both species when they were exposed to excess NaCl. The values obtained f o r maximum panting r a t e s were lower than have been reported elsewhere (Richard, 1971b; Marder, 1973) . F i r s t , the b i r d s were heat-stressed f o r a maximum of only 10 65 minutes a f t e r the onset of panting. Secondly, the o s c i l l o g r a p h recordings obtained f o r f represented only the t h o r a c i c component of resonant breathing. The frequency of g u l a r f l u t t e r and the resonant v i b r a t i o n s of the upper airways were not r e f l e c t e d i n the recordings owing to the r e l a t i v e l y low p o s i t i o n i n g of the rubber c o l l a r ( Fig 2). Resting f i n g u l l s was double that of normal d i e t r o o s t e r s . Upon thermal s t r e s s g u l l s too e x h i b i t e d ' f l u s h - o u t ' panting. Breathing frequency rose as much as 5 - f o l d w h i l e V T f e l l to as low as 1/3 the r e s t i n g value i n FW b i r d s . High NaCl i n t a k e d i d not a f f e c t maximum f i n g u l l s . At SW, the r e s t i n g respiratory-amplitudes were r e l a t i v e l y l a r g e r than those of r o o s t e r s . The g u l l s were h y p e r i r r i t a b l e when s a l t i n t a k e was high and experienced s w i f t r i s e s i n body temperature when they were ther m a l l y s t r e s s e d . Panting occurred sooner than i n FW but at higher core temperatures, but body temperature rose more r a p i d l y upon thermal s t r e s s i n SW than i n FW g u l l s as p r e v i o u s l y observed i n chickens (Edens, 1975). G u l l s maintained a r a i s e d VT. The p e r s i s t e n c e of a high VT i n d i c a t e s that evaporative c o o l i n g may take p r i o r i t y over. acid-base balancing. The s p e c i f i c e f f e c t o:f v a r y i n g e x t r a c e l l u l a r f l u i d i o n i c or osmotic c o n c e n t r a t i o n on the f i r i n g of the panting centre neurons i s not c l e a r . There i s evidence however, that t h e i r s e n s i t i v i t y i s a l t e r e d by the e l e c t r o l y t e s t a t u s or o s m o l a l i t y of the bathing f l u i d s (Baker and D o r i s , 1982). The present study provides evidence that the e f f e c t of increased d i e t a r y s a l t on panting t h r e s h o l d i s not 66 d i f f e r e n t from that of infused s a l t loads. The magnitude of the e f f e c t was l e s s i n g u l l s since they were b e t t e r able to maintain ECF o s m o l a l i t y than ro o s t e r when exposed to high d i e t a r y NaCl. 4.7 Conclusion This study has shown that no fundamental d i f f e r e n c e s e x i s t between the domestic fowl and the Glaucous-winged g u l l i n the e f f e c t s of ingested NaCl on body temperature. NaCl a c c l i m a t i o n d i d not a f f e c t TBW s i g n i f i c a n t l y i n e i t h e r species, but body temperature rose w i t h [Na +] p l i n apparent concentration-dependent f a s h i o n . The increase i n rooster panting t h r e s h o l d - was a s s o c i a t e d w i t h an increase i n Osmol p l. Osmol p l d i d not increase i n g u l l s and t h e i r panting threshold was not a l t e r e d . Both plasma Na + : Ca and Na+ : Ca 2 + r a t i o s were not s y s t e m a t i c a l l y a l t e r e d by d i e t a r y s a l t a c c l i m a t i o n and were u n r e l a t e d to body temperature. The e l e v a t i o n of [Na +] p l caused an increase i n [ C a 2 + ] p l by a mechanism not i n v o l v i n g the m o b i l i z a t i o n of Ca 2 + from the bound plasma f r a c t i o n ; p o s s i b l y by f a c i l i t a t i n g the e x t r u s i o n of Ca 2 + ions from c e l l s i n t o the i n t e r s t i t i u m through a NaVCa 2 + exchange mechanism. The expected increase i n plasma Na : Ca r a t i o t h e r e f o r e d i d not occur. TBW was u n r e l a t e d to body temperature, was not a f f e c t e d by high d i e t a r y NaCl and was not higher i n b i r d s w i t h s a l t glands. EWL at moderate heat s t r e s s was not a l t e r e d by s a l t a c c l i m a t i o n i n the g u l l and d i d not account f o r the increase i n body temperature. 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S e i t z . 1968. The dependence of calcium e f f l u x of c a r d i a c muscle on temperature and e x t e r n a l i o n composition. J . P h y s i o l . 15: 451-470. Rice, G. E. , and E. Skadhauge. 1982. Col o n i c and coprodeal t r a n s e p i t h e l i a l transport parameters i n NaCl-loaded domestic fo w l . J . Comp. P h y s i o l . 147: 65-69. Richards, S. A. 1971. Bra i n stem c o n t r o l of polypnea i n the chicken and pigeon. Resp. P h y s i o l . 11: 315-326. Roberts, J . , and M. R. Hughes. 1984. Exchangable sodium pool s i z e and sodium turnover i n freshwater- and s a l t w a t e r - a c c l i m a t e d ducks and g u l l s . Can. J . Zool. 62: 2142-2145. Ruch, F. E., and M. R. Hughes. 1975. The e f f e c t s of i n j e c t i o n of sodium c h l o r i d e on body water d i s t r i b u t i o n i n ducks (Anas  p l a t y r n c h o s ) , g u l l s (Larus qlaucescens) and r o o s t e r s (Gallus  domesticus). Comp. Biochem. P h y s i o l . 52 A: 21-28. Saxena, P. N. 1976. 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Skadhauge. 1989. Function and r e g u l a t i o n of the avian caecal bulb: i n f l u e n c e of d i e t a r y NaCl and aldosterone on water and e l e c t r o l y t e f l u x e s i n the hen {Gallus domesticus) . J . Comp. P h y s i o l . B. 159: 51-60 T u r l e j s k a , E., and M. A.. Baker. 1986. Elevated c e r e b r o - s p i n a l f l u i d o s m o l a l i t y i n h i b i t s thermoregulatory heat l o s s responses. Am. J . P h y s i o l . 251 (4 Pt 2): R749-R754. Veale, W. L., and K. E. Cooper. 1973. Species d i f f e r e n c e s i n the pharmacology of temperature r e g u l a t i o n . In The Pharmacology of Thermoregulation. Ed. E. Schonbaum and P. Lomax. Basel : Karger: 289-301. Walter, A., and M. R. Hughes. 1978. To t a l body water volume and turnover r a t e i n f r e s h water and sea water adapted Glaucous-winged g u l l s , Larus qlaucescens. Comp. Biochem. P h y s i o l . 61 A: 233-237. 82 Webster, M. D., G. S. Campbell and J. R. King. 1985. Cutaneous resistance to water-vapour d i f f u s i o n i n pigeons and the role of the plumage. Physiol. Zool. 58 (1): 58-70. Webster, M. D., and J. R. King. 1987. Temperature and humidity dynamics of cutaneous and respiratory evaporation i n pigeons, Columba l i v i a . J. Comp. Physiol, b. 157: 253-260. 83 APPENDIX A COMPOSITION OF LIQUID VITAMIN SUPPLEMENT FOR GLAUCOUS-WINGED GULLS, LARUS GLAUCESCENS. COMPONENT QUANTITY/ML Vitamin A (Palmitate) 500 I.U. Vitamin D2 40 I.U. Vitamin B 1 2 (Cyanocobalamin) 0.4 meg Vitamin B : (Thiamine hydrochloride) 0.2 mg Vitamin B2 ( R i b o f l a v i n 5'phosphate sodium 0.2 mg Niacinamide 1.0 mg ; Vitamin B6 (Pyridoxine hydrochloride) 0.12 mg Vitamin C 4.0 mg Iron (Ferrous Potassium gluconate) 0 . 8 mg 0.005 mmol 84 APPENDIX B Plasma [Ca 2 +] as a percentage of [Ca] p l, and c o r r e l a t i o n c o e f f i c i e n t s (r) of simultaneously measured plasma [Ca 2 +] , [Ca] , and [Na+] i n ro o s t e r s and g u l l s . C o r r e l a t i o n c o e f f i c i e n t s (r) [Ca 2 +] [Ca 2 +] [Ca 2 +] [Ca] % [Ca] / [Ca] / [Na+] / [Na+] Roosters Normal NaCl d i e t G u l l s D r i n k i n g Week 0 (n=6) 53 . ± 0. 90 .03 0 .486 -0 , .567 -0 .347 Week 1 (n=6) 57 . ± 0. 20 .03 0 .772 -0. . 030 0 .064 Week 2 (n=6) 53 . ± 0, 00 .02 0 .714 0 . 877 0 .894 O v e r a l l (n=18) 54. ± 0. .70 .03 0 .385 0 , . 664 0 .264 [NaCl] 0 mM (n=5) 56. ± 0, .50 . 02 0 .897 -0 , .464 -0 .116 112 .5 (n=5) mM 55. ± 0, .20 .02 -0 .679 0 . .199 -0 .340 225.0 (n=5) mM 54 . ± 0. 90 .04 -0 .696 -0 , .426 -0 .784 337 .5 (n=5) mM 54 , ± 0. .20 . 01 0 .716 0 . 669 0 . 649 475.0 (n=5) mM 57 , ± 0, .30 . 03 -0 .367 0 .862 -0 .394 O v e r a l l 55, .60 0 .653 0 . 635 0 . 602 (n=25) 85 APPENDIX C A comparison of the o s c i l l o g r a p h r e c o r d i n g s of the p a n t i n g p a t t e r n of one r o o s t e r f e d normal NaCl d i e t (lower t r a c i n g ) , and f o l l o w i n g two weeks of a c c l i m a t i o n to hig h NaCl d i e t (upper t r a c i n g ) . Panting f r e q u e n c i e s i n each case are approaching maximum. Chart speed was constant, i n each case, at 1.0 mm/sec. Body temperature was h i g h e r i n the h i g h NaCl d i e t s t a t e . 86 APPENDIX C A comparison of the o s c i l l o g r a p h t r a c i n g s of the p a n t i n g p a t t e r n of one g u l l a c c l i m a t e d to freshwater (lower t r a c i n g ) , and l a t e r to f u l l - s t r e n g t h seawater (upper t r a c i n g ) . The p a t t e r n i s more r e g u l a r i n the freshwater s t a t e ; ' f l u s h - o u t ' phases are l e s s f r equent, and low amplitude phases are more s u s t a i n e d . These f r e q u e n c i e s both approach the maxima obtained. Chart speed was constant at 2.5 mm/sec and body temperature was h i g h e r i n the seawater regime. saltwater expiration Freshwater expiration 87 APPENDIX C The o n s e t o f t h e r m a l p a n t i n g . Top: A g r a d u a l t r a n s f o r m a t i o n f r o m h i g h a m p l i t u d e , low f r e q u e n c y b r e a t h i n g t o low a m p l i t u d e h i g h f r e q u e n c y b r e a t h i n g i n a r o o s t e r on n o r m a l N a C l d i e t . B o t t o m : An a b r u p t a d j u s t m e n t f r o m low f r e q u e n c y , h i g h a m p l i t u d e b r e a t h i n g , ; t o h i g h f r e q u e n c y , low a m p l i t u d e b r e a t h i n g i n a n o r m a l N a C l r o o s t e r . Panting ThreslTOW (Gradual) nsprntion Panting threshold (Sudden) 

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