The alkaline tide and ammonia excretion after voluntary feeding in freshwater rainbow trout

Transcription

1 2533 The Journl of Experimentl Biology 211, Published by The Compny of Biologists 2008 doi: /jeb The lkline tide nd mmoni excretion fter voluntry feeding in freshwter rinbow trout Crol Bucking* nd Chris M. Wood McMster University, 1280 Min St. West, Hmilton, Ontrio, Cnd, L8S 4K1 *Author for correspondence (e-mil: Accepted 12 My 2008 SUMMARY We investigted the potentil cid bse nd nitrogenous wste excretion chllenges creted by voluntry feeding in freshwter rinbow trout, with prticulr focus on the possible occurrence of n lkline tide ( metbolic lklosis creted by gstric HCl secretion during digestion). Plsm metbolites (glucose, ure nd mmoni) were mesured t vrious time points before nd fter voluntry feeding to stition (pproximtely 5% body mss mel of dry commercil pellets), s ws the net flux of mmoni nd titrtble lklinity to the wter from unfed nd fed fish. Arteril blood, smpled by indwelling ctheter, ws exmined for post-prndil effects on ph, plsm bicrbonte nd plsm CO 2 tension. There ws no significnt chnge in plsm glucose or ure concentrtions following feeding, wheres plsm mmoni trnsiently incresed, peking t threefold bove resting vlues t 12 h fter the mel nd remining elevted for 24 h. The incresed plsm mmoni ws correlted with n increse in net mmoni excretion to the wter, with fed fish significntly elevting their net mmoni excretion two- to threefold between 12 nd 48 h post feeding. These prmeters did not chnge in unfed control fish. Fed fish likewise incresed the net titrtble bse flux to the wter by pproximtely threefold, which resulted in trnsition from smll net cid flux seen in unfed fish to lrge net bse flux in fed fish. Over 48 h, this resulted in net excretion of μmol kg 1 more bse to the externl wter thn in unfed fish. The rteril blood exhibited corresponding rise in ph (between 6 nd 12 h) nd plsm bicrbonte (between 3 nd 12 h) following feeding; however, no respirtory compenstion ws observed, s P CO 2 remined constnt. Overll, there ws evidence of numerous chllenges creted by feeding in freshwter teleost fish, including the occurrence of n lkline tide, nd its compenstion by excretion of bse to the externl wter. The possible influence of feeding ecology nd environmentl slinity on these chllenges, s well s discrepncies in the literture, re discussed. Key words: cid bse regultion, bse excretion, digestion, glucose, Oncorhynchus mykiss, plsm, ure. INTRODUCTION The gut is indispensble for multicellulr life, nd is responsible for meeting the nutritionl nd energy demnds of n orgnism. However, while digestion cn ffect numerous other physiologicl systems through ssocited processes, some of the most bsic consequences of feeding nd ssimiltion in fish hve only just begun to be discovered. For exmple, recently, the effects of digestion on overll osmotic nd ionic blnce in freshwter rinbow trout (Oncorhynchus mykiss) hve been ddressed (Bucking nd Wood, 2006; Bucking nd Wood, 2006b; Bucking nd Wood, 2007), reveling severl beneficil consequences to digestion in these teleosts in ddition to those offered by nutrition itself. However, digestion could lso potentilly crete chllenges for fish, especilly crnivorous fish such s rinbow trout, becuse of the formtion of excess mmoni during the ctbolism of dietry proteins (Hndy nd Poxton, 1993), s well s excess bse during the formtion of HCl by the stomch (reviewed by Hersey nd Schs, 1995; Niv nd Frser, 2002). In this regrd, mrked systemic lkline tide during digestion hs recently been described in crnivorous mrine elsmobrnch, the dogfish shrk (Wood et l., 2005; Wood et l., 2007; Wood et l., 2007b). As the mino cid surplus from protein-rich diets cnnot be directly stored in fishes, it is deminted nd converted into energetic compounds (Bllntyne, 2001; Stone et l., 2003), resulting in post-prndil increses in plsm totl mmoni levels (Kushik nd Teles, 1985) nd mmoni excretion rtes (vn Weerd et l., 1995; Dosdt et l., 1996; Gelineu et l., 1998; Leung et l., 1999). More thn 80% of this metbolic mmoni production is excreted cross the gills, portion of which my be in direct (NH 4 + ) or indirect (H + +NH 3 ) exchnge with N + uptke (reviewed by Evns et l., 2005). Indeed direct reltionship between protein intke nd mmoni excretion hs been found in fish (Li nd Lovell, 1992; Jyrm nd Bemish, 1992; Bllestrzzi et l., 1998; Medle et l., 1995; Ci et l., 1996; Chkrborty nd Chkrborty, 1998). The mino cid surplus is creted through the hydrolysis of dietry proteins, first initited in the stomch by pepsin nd completed by the combined ction of trypsin nd chymotrypsin in the intestine. Pepsin is the proteolyticlly ctive form of the enzyme pepsinogen, which is secreted by gstric cells nd utoctlyticlly ctivted in cidic environments. This is conserved mechnism cross species from fish (e.g. Bomgren et l., 1998; Hernndez et l., 2001; Lo nd Weng, 2006) to mmmls (reviewed by Kgeym, 2002), lthough the cells responsible for the production of pepsinogen vry, with mmmls possessing two distinct cid secreting cells (chief cells) nd pepsinogen secreting cells (prietl cells), wheres lower vertebrtes such s the rinbow trout posses only one secreting cell, the oxynticopeptic cell (Bomgren et l., 1998). Although HCl secretion is essentil for protein digestion through the forementioned pepsinogen ctivtion s well s by direct cid

2 2534 C. Bucking nd C. M. Wood hydrolysis, it cn lso dd to the chllenges creted by digestion by generting n lkline tide. Historiclly defined s the lkliniztion of the blood nd urine during the digestion of mel (Rune, 1965; Rune, 1966), the term lkline tide in essence refers to the increse in blood HCO 3 concentrtion tht occurs s consequence of incresed secretion of HCl t this time. It is believed tht gstric cells use bsolterl Cl /HCO 3 exchnger to import extrcellulr Cl needed for HCl formtion, nd simultneously export intrcellulr HCO 3 tht is formed vi the hydrtion of CO 2 by intrcellulr crbonic nhydrse [which forms proton nd HCO 3 ion (reviewed by Hersey nd Schs, 1995; Niv nd Frser, 2002)]. This ultimtely results in the equimolr secretion of H + into the lumen for HCl formtion, nd HCO 3 into the blood tht is responsible for the lkline tide (Rune 1965; Rune, 1966; Niv et l., 1993). To dte, the phenomenon hs been documented in mmmls, birds, reptiles nd elsmobrnchs (Wng et l., 2001b; Niv nd Frser, 2002; Wood et l., 2005; Wood et l., 2007; Wood et l., 2007b) but there is, s yet, no evidence tht it occurs in teleost fish. Indeed, neither Tylor nd Grosell (Tylor nd Grosell, 2006) using the mrine todfish, Opsnus bet, nor Tylor et l. (Tylor et l., 2007) using the euryhline Europen flounder, Pltichthys flesus, could detect post-prndil lkline tide in the blood of teleosts. Furthermore, in frogs there is tight correltion between the reduction in plsm Cl concentrtions nd the increse in plsm HCO 3 concentrtions following feeding (Busk et l., 2000), but Bucking nd Wood (Bucking nd Wood, 2006) found no such post-prndil reduction in plsm Cl levels in rinbow trout. Gill function my be one reson why it hs not been possible to see the symptoms of the lkline tide in teleost fish. In ddition to N + uptke nd NH 3 /NH 4 + nd H + excretion, the gills re the min site of bse (in the form of HCO 3 ) excretion nd concurrent Cl uptke (reviewed by Evns et l., 2005), believed to be fcilitted by n picl Cl /HCO 3 exchnger, either belonging to the SLC4 nion exchnger (AE) fmily (Cliborne et l., 1997; Wilson et l., 2002), or the SLC26 AE fmily (Piermrini et l., 2002). In recent review, Tresguerres et l. (Tresguerres et l., 2006) proposed model of Cl uptke by freshwter fish through n picl Cl /HCO 3 nion exchnger, cytoplsmic crbonic nhydrse nd bsolterl V-type H + -ATPse. If Cl /HCO 3 excretion cross the gills were fst enough to keep up with the HCl secretion nd ssocited Cl /HCO 3 exchnge t the stomch, then lklotic disturbnces of blood ph, HCO 3 nd Cl might be voided. However, net bse excretion into the wter should still be detected; this ws seen in the elsmobrnch Squlus cnthis (Tresguerres et l., 2007; Wood et l., 2007b), but not in the euryhline Europen flounder Pltichthys flesus (Tylor et l., 2007). Potentilly, the lrge mmoni excretion fter feeding could mke it difficult to detect net metbolic bse efflux, problem which would not occur in the ureotelic elsmobrnch. With this bckground in mind, we exmined the effect of feeding on cid bse exchnge with the environment using the originl single end-point titrtion methodology of McDonld nd Wood (McDonld nd Wood, 1981) to seprte mmoni nd metbolic bse fluxes, together with mesurements of systemic cid bse sttus nd plsm metbolites (glucose, ure nd mmoni) in freshwter rinbow trout. The overll hypothesis behind this study ws tht digestion of mel would crete numerous physiologicl chllenges to freshwter rinbow trout, including increses in plsm mmoni, increses in plsm ph nd HCO 3 concentrtion (n lkline tide), nd excretion of both the excess mmoni nd excess bse to the wter vi the gills. MATERIALS AND METHODS Rinbow trout (Oncorhynchus mykiss Wlbum) were obtined from commercil supplier (Humber Springs Trout Htchery; Orngeville, ON, Cnd) nd cclimted to lbortory conditions for 2-week period before experimenttion. The nimls (rnging in body mss from 300 to 400 g) were held in 500 l holding tnks t density of pproximtely 40 fish per tnk nd supplied with flow-through dechlorinted Hmilton (ON, Cnd) city tp wter [N + 0.6, Cl 0.7, K , C , Mg 2+, 0.1 mol l 1 ; titrtion lklinity (to ph 4.0) 1.9 mequiv l 1 ; totl hrdness 140 mg l 1 s CCO 3 ; ph 8.0]. Except during experimenttion, the nimls were fed 2% body rtion [crude protein 41%; crbohydrtes 30%; crude ft 11%; Mrtin Mills; Elmir, ON, Cnd] every 48 h. All experiments were crried out t 12 C. Post-prndil chnges in plsm mmoni, ure nd glucose Smpling occurred immeditely prior to (0 h), nd t severl time points following (2, 4, 8, 12, 24, 48 h) single feeding to stition of the trout (mounting to 5% of body mss) in the 500 l holding tnks. In prllel study, these stition feeding events resulted in the mjority of fish (95%) consuming between 80 nd 110% of the offered rtion (C.P.B. nd C.M.W., unpublished dt). The mel consisted of commercil trout pellets with mesured ionic composition of N + 215±15, Cl 188±16, K + 97±2, C ±3, Mg ±1 μmol g 1 originl food mss. The fish were netted nd smpled individully to reduce processing time (typiclly <60 s) nd ny resultnt stress. Ech trout ws rndomly netted from the holding tnk nd lightly nesthetized using MS-222 (tricine methne sulphonte; 0.03 g l 1 ; Sigm, St Louis, MO, USA) before blood smple ws obtined vi cudl puncture using no. 22 needle ttched to n ice-cold heprinized syringe. The whole blood ws immeditely centrifuged t g, nd the resultnt plsm ws removed, plced into liquid nitrogen, nd stored t 80 C for future nlyses. The fish were llowed to recover in fresh wter nd returned to seprte holding tnk to void repeted smpling. Plsm totl mmoni (T mm ) ws mesured enzymticlly (bsed on the glutmte dehydrogense/nad method) using commercil kit (Richem; Sn Diego, CA, USA) nd plsm totl ure ([ure] p ) ws mesured using colorimetric ure ssy modified from Rhmtullh nd Boyde (Rhmtullh nd Boyde, 1980). The plsm ws then deproteinized nd neutrlized before nlyzing for plsm totl glucose ([glucose] p ) by the hexokinse, glucose-6- phosphte dehydrogense method (Sigm, 301A). All smples were red on microplte reder (SpectrMx 340PC; Sunnyvle, CA, USA). Fluxes to the wter Individul trout were removed from the 500 l holding tnks immeditely before the scheduled feeding time to serve s unfed controls (N=6). The remining trout were then fed to stition (>5% body mss rtion) nd then more individul fish (fed fish, N=6) were removed from the holding tnk. The removed fish were plced in individul drkened flux boxes supplied with flow-through Hmilton city tp wter nd vigorous ertion. Flux mesurements were then performed over successive 6 h intervls for the next 48 h. For ech flux mesurement, the wter level ws set to 4 l (excluding the mss of the niml) nd the wter flow suspended. An initil wter smple ws then tken followed by nother wter smple 6 h lter, serving s strting nd finl flux smples, respectively. At the end of ech 6 h flux period, following the finl wter smple, the box ws thoroughly flushed with fresh wter by repetedly lowering

3 Feeding in freshwter rinbow trout 2535 nd rising the wter level, before the volume ws reset to 4 l. This procedure ws repeted every 6 h for 48 h. The fish were then returned to the holding tnks. The initil nd finl wter smples were tken in triplicte nd mesured for totl mmoni nd titrtble lklinity, the ltter by the single end-point technique of McDonld nd Wood (McDonld nd Wood, 1981) Titrtble lklinity ws determined by the titrtion of 10ml wter smples to ph3.8, using Rdiometer (Copenhgen, Denmrk) GK2401C glss combintion electrode coupled to Rdiometer PHM 82 stndrd ph meter. HCl ws dded to ech wter smple until the ph ws brought below ph5.0. The smple ws then erted for 15min to remove excess CO 2, nd then more HCl ws slowly dded to determine the totl quntity of cid needed to lower the ph of the wter smple to finl end-point ph of 3.8. Continul ertion ensured mixing nd CO 2 removl. A stndrdized cid (0.02moll 1 HCl; Sigm) ws used to lower the ph nd ws ccurtely delivered by Gilmont (Brrington, IL, USA) microburette. The mount of cid titrnt, fctored by the volume of the smple required to rech ph 3.8, represented the concentrtion of titrble lklinity in bsic equivlents. Wter totl mmoni concentrtion ([NH 3 /NH + 4 ] w ) ws mesured using the slicylte hypochlorite method (Verdouw nd Dekkers, 1978). Fluxes were clculted from chnges in concentrtion (i.e. from initil to finl smples), fctored by volume, time nd trout mss, nd expressed s μmolkg 1 h 1. The net cid bse flux ws clculted s the difference between the flux of titrtble lklinity (J TAlk ) nd the flux of totl mmoni (J Tmm ) to the externl wter (McDonld nd Wood, 1981). An overll net bse flux (i.e. HCO 3 equivlent flux; J net OH ) is shown by positive difference nd is plotted s negtive vlue (i.e. net bse loss from the niml), while net cid flux (i.e. H + equivlent flux; J net H + ) is shown by negtive difference nd is plotted s positive vlue (i.e. net bse uptke = net cid loss). The [NH 3 /NH + 4 ] w in the chmbers of the unfed fish did not exceed 150 μmol l 1 by the end of the 6 h flux period; however, some of the fed fish experienced [NH 3 /NH + 4 ] w close to 300 μmol l 1. To ensure tht this hd no influence on the outcome of the present experiment, vlidtion experiment ws conducted wherein the present flux study with fed fish ws repeted but J Tmm ws mesured over 3 h flux periods within intervening flushes, so tht the [NH 3 /NH + 4 ] w did not exceed 150 μmol l 1 in ny of the fish chmbers. The J Tmm over the 3 h periods were then combined nd compred with the 6 h fluxes in the present study. The results demonstrted tht the high mmoni levels hd no significnt effect on the overll net flux of mmoni or cid bse equivlents. At the sme time, n dditionl vlidtion experiment ws performed to ddress concerns tht the single end-point titrtion method used to mesure titrtble lklinity fluxes my hve incurred error if the buffer cpcity of the wter chnged over the flux period. Theoreticlly, this could occur s result of regurgittion of food or defection, lthough such events were never observed. In this prllel tril, fluxes were mesured t 0 6, 6 12 nd h post feeding (0 h). The wter smples were titrted s in the single end-point technique to below 3.8 with 0.02 mol l 1 HCl, nd the moles of cid dded to rech this single end-point ws clculted. However, the smples were then titrted bck up to ph 7 ( second end-point) with 0.02 mol l 1 NOH (which ws verified ginst the 0.0 mol l 1 HCl). The difference between the number of moles of cid nd bse dded ws used to clculte the totl titrtble lklinity of ech smple, which ws then used to determine the chnge from initil to finl wter smples to clculte the titrtble lklinity flux (i.e. double end-point titrtion). The titrtble lklinity fluxes tht were mesured, either with the single titrtion or the double titrtion method, were essentilly identicl (i.e. no significnt differences), lthough the ltter were more vrible (50% lrger stndrd error of the mens), s would expected for mesurements bsed on the difference between double end-points nd single end-points. We therefore conclude tht the single endpoint titrtion method used in this study is more ccurte nd pproprite for this type of investigtion. Systemic cid bse sttus Additionlly, following the cclimtion to lbortory conditions, 18 fish were trnsferred from the holding tnk to individul 25 l tnks supplied with flow-through dechlorinted Hmilton city tp wter nd individul ertion. These fish were fed dily t set time point to entrin feeding in the individul tnks nd ensure synchroniztion of ny feeding-ssocited ctivities. The fish were clened dily of ny wste ccumultion severl hours before feeding. Trining continued for severl weeks until ll the fish te redily when food ws supplied. Following trining, the fish were strved for 1 week to cler the gstrointestinl trct. After 1 week of strvtion, the trout were nesthetized with MS- 222 (0.07 g l 1 ) nd rtificilly ventilted on n operting tble. Dorsl ortic ctheters (Cly-Adms PE-50; Sprks, MD, USA) were then implnted ccording to the method of Soivio et l. (Soivio et l., 1972) nd filled with 0.3 ml of Cortlnd sline (NCl 120, KCl 5, CCl 2 2H 2 O 2, MgSO 4 7H 2 O 1, NH 2 PO 4 H 2 O 3, glucose 5 mmol l 1 ; djusted to ph 7.8 with NHCO 3 ) (Wolf, 1963) contining 50 i.u. ml 1 of lithium heprin (Sigm) nd seled. Ech trout ws then returned to its individul 25 l tnk nd llowed to recover for 1 dy. Following this recovery period, nine of the fish were then fed to stition (gin, typiclly 5% body mss mel). The other nine were used s unfed control nimls. Blood smples (250 μl) were tken from the dorsl ort ctheter, using n ice-cold pre-heprinised, gs-tight Hmilton syringe, before nd fter feeding t vrious time points ( 6, 3, 0, 3, 6, 9, 12, 24, 48 h). Approximtely 70 μl of the whole blood ws immeditely used to mesure rteril blood ph (ph) using Rdiometer GK2401C glss combintion electrode inserted into tightly seled chmber which ws thermosttted to 12 C. The remining whole blood ws centrifuged t g for 30 s to seprte plsm nd red blood cells. Plsm smples were then immeditely mesured for totl CO 2 (T CO 2; Corning 965 Totl CO 2 Anlyser; Lowell, MA, USA). Plsm CO 2 tension (P CO 2) nd bicrbonte concentrtion ([HCO 3 ] ) were clculted using rerrngement of the Henderson Hsselblch eqution with vlues of plsm pk nd CO 2 solubility coefficients for trout blood t 12 C (Boutilier et l., 1984). Sttistics All dt pssed normlity nd homogeneity tests prior to sttisticl investigtion, nd re reported s men ± s.e.m. (N=number of nimls) unless otherwise specified. Temporl chnges in T mm, [glucose] p, nd [ure] p were exmined with one-wy ANOVA followed by post-hoc HSD (Tukey s honest significnt difference) test. The temporl chnges in J Tmm, J TAlk, J net OH, nd J net H +, ph, [HCO 3 ] nd P CO 2 were exmined with repeted mesures twowy ANOVA followed by post-hoc HSD test. Vlues were considered significntly different t P<0.05. RESULTS Post-prndil chnges in plsm mmoni, ure nd glucose Feeding hd no significnt effect on [glucose] p, which ws mintined t n verge of 6.84±1.60 mmol l 1 (N=49) for the

4 2536 C. Bucking nd C. M. Wood T mm ( mol l 1 ) * * * * J TAlk ( mol kg 1 h 1 ) Unfed Fed Fig. 1. Chnges in totl plsm mmoni concentrtion (T mm ; μmol l 1 ) following feeding to stition. Feeding occurred immeditely following the time 0 h smpling, which ws used s the control. Vlues re mens ± s.e.m.; N=7. Ech vlue ws from seprte fish. *Significnt difference (P<0.05) from the control. Fig. 3. Flux of totl titrtble lklinity to the wter (J TAlk ; μmol kg 1 h 1 ) from fed nd unfed fish. Open symbols indicte unfed fish, filled symbols indicte fish tht were fed immeditely before the experiment begn. Dt were simultneously obtined from the fish used in Fig. 2. Vlues re mens ± s.e.m.; N=6 for ech tretment. Significnt difference (P<0.05) between fed nd unfed fish. durtion of digestion. Likewise, [ure] p concentrtions remined unchnged following feeding, verging 1.39±0.11mmoll 1 (N=49) over the 48 h of experimenttion. By contrst, feeding hd drmtic effect on T mm, which significntly incresed more thn threefold from pre-prndil vlues (109±44μmol l 1, N=7; 0 h), to pek 12 h following ingestion (335±82 μmol l 1, N=7), before subsequently returning to resting vlues by 48 h (116±38μmol l 1, N=7; Fig. 1). Fluxes to the wter from fed nd unfed fish Confined unfed fish (i.e. control fish) showed J Tmm tht remined unchnged over the course of experiment, verging 320±8 μmol kg 1 h 1 (N=48; Fig. 2). The control fish showed likewise unffected J TAlk, which verged 220±19μmol kg 1 h 1 J Tmm ( mol kg 1 h 1 ) Unfed Fed Fig. 2. Flux of totl mmoni to the wter (J Tmm ; μmol kg 1 h 1 ) from fed nd unfed fish. Open symbols indicte unfed fish, filled symbols indicte fish tht were fed immeditely before the experiment begn. Vlues re mens ± s.e.m.; N=6 for ech tretment. The sme six fish were mesured t ech intervl. Significnt difference (P<0.05) between fed nd unfed fish. (N=48; Fig. 3), slightly lower thn the J Tmm, resulting in stedy J net H + of 100±14 μmol kg 1 h 1 (N=48; Fig. 4). By contrst, while the confined pre-fed trout initilly showed J Tmm similr to tht of the unfed fish (between 0 nd 6 h; Fig. 2), the J Tmm incresed more thn twofold, eventully peking between 36 nd 42 h post-feeding, t rte of 817±133μmol kg 1 h 1 (N=6; Fig. 2). J Tmm then decresed to 760±156 μmol kg 1 h 1 (N=6) t 48 h post-feeding; however, it ws still significntly elevted when compred with the J Tmm of unfed fish (Fig. 2). Similrly, the J TAlk of fed fish incresed when compred to tht of unfed fish from initilly comprble vlues ( 456±112μmolkg 1 h 1 t 0 6h; Fig.3) Net cid/bse flux ( mol kg 1 h 1 ) Unfed Fed J net OH J net H Fig. 4. The overll net cid or bse flux to the wter from fed nd unfed fish. Positive vlues indicte net bse flux (J net OH ; μmol kg 1 h 1 ), wheres negtive vlues indicte net cid flux (J net H + ; μmol kg 1 h 1 ). Open symbols indicte unfed fish, filled symbols indicte fish tht were fed immeditely before the experiment begn. Dt were clculted from the difference between vlues shown in Fig. 2 nd Fig. 3 on n individul fish bsis (see Mterils nd methods for further explntion). Vlues re mens ± s.e.m.; N=6 for ech tretment. Significnt difference (P<0.05) between fed nd unfed fish.

5 Feeding in freshwter rinbow trout b A c ph ,b Fig. 5. Arteril blood ph (ph) in unfed nd fed fish. One set of fish (indicted by the filled symbols) were fed immeditely following the time 0 smple, while nother set of fish (open symbols) remined unfed. Vlues re mens ± s.e.m.; N=8 for ech tretment. Time points with the sme letters re not significntly different (P>0.05). to pek threefold higher t 30 h post-feeding t rte of 1161±189μmolkg 1 h 1, before decresing to 800±88μmolkg 1 h 1 t 48 h (Fig. 3). Hence, feeding ltered the net cid bse flux of the fed fish from n initil J net H + t 0 6 h ( 11±163 μmol kg 1 h 1 ) tht ws similr to tht of unfed fish (Fig. 4) to J net OH tht ws significntly different from control vlues until 42 h post-feeding. The J net OH peked between 24 nd 30 h fter the ingestion of the mel t 435±87μmol kg 1 h 1 (N=6; Fig. 4). Post-prndil rteril blood gses nd cid bse sttus Unfed cnnulted fish mintined their ph nd [HCO 3 ] unchnged over the course of the experiment, t n verge of 7.85±0.01 mmol l 1 (N=64; Fig. 5) nd 8.52±0.11 mmol l 1 (N=64; Fig. 6B), respectively. In the experimentl group, ph ws mintined t n verge of 7.86±0.01 (N=24; Fig. 5) before nd for 3 h fter the ingestion of the mel, which ws not significntly different from unfed fish. However, therefter, ph significntly incresed by over 0.15 ph units t 6 h (Fig. 5). This trnsient increse ws slowly dissipted over the next 12 h, flling bck to resting ph levels 18 h fter feeding (Fig. 5). This feeding-induced increse in ph ws mirrored by post-prndil increse in [HCO 3 ], which lso significntly incresed from pre-prndil vlues of 8.41±0.27 mmol l 1 (N=16; 3 to 0 h) s well s from unfed controls, to similrly pek 6 h following feeding t 12.19±0.43 mmol l 1 (N=8); 1.4-fold increse (Fig. 6A). Menwhile, there ws no pprent difference in the P CO 2 (2.48±0.08, N=64) between fed nd unfed controls, s well s no temporl chnge (Fig. 6B), indicting tht no respirtory compenstion ws mde during the metbolic lklosis. DISCUSSION The mjority of mino cids bsorbed fter the ingestion of protein, in excess of requirements for protein synthesis, re ctbolized in the liver (Cmpbell, 1991), resulting in n increse in plsm mmoni levels in fish (Fig. 1) (Kushik nd Teles, 1985; Wicks nd Rndll, 2002). In ssocition, there re chnges in mmoni excretion in fish during the postprndil period (Fig. 2) (vn Weerd et l., 1995; Dosdt et l., 1996; Alsop nd Wood, 1997; Gelineu et l., 1998; Tylor et l., 2007) nd plsm mmoni concentrtions,b [HCO 3 ] (mmol l 1 ) P CO2 (mmol Hg) B,b b,b,b typiclly pek 12 h following feeding in trout (Fig. 1) (Wicks nd Rndll, 2002). Therefore, it is not surprising tht n increse in dietry protein results in n increse in mmoni excretion in fish (Li nd Lowell, 1992; Jyrm nd Bemish, 1992; Bllestrzzi et l., 1998; Medle et l., 1995; Ci et l., 1996; Chkrborty nd Chkrborty, 1998). An erly study by Bemish nd Thoms (Bemish nd Thoms, 1984) on trout fitted with urinry ctheters nd trined to feed in smll flux boxes ttributed the mjority of the post-prndil increse in mmoni-n excretion to the gills (>96%), with the reminder excreted by the kidneys, emphsizing the role of the gills in mmoni regultion. In contrst to the present study on mmoniotelic rinbow trout, feeding nd digestion in the ureotelic dogfish resulted in only very smll rise in mmoni-n excretion, mounting to less thn 3% of the totl-n in the mel, which ws lso ccompnied by only modest increse in plsm mmoni-n concentrtion (Wood et l., 2005; Wood et l., 2007b). This present study, together with the simultneous investigtion of Cooper nd Wilson (Cooper nd Wilson, 2008), both on rinbow trout, present the first evidence for n lkline tide in ny teleost fish. Notbly, the fish in our study were feeding voluntrily, so there ws no confounding effect of disturbnce. Cooper nd Wilson (Cooper nd Wilson, 2008), working with smller rtion (1% vs the 5% used in the present study) compred voluntry nd forcedfeeding, nd found tht the ltter resulted in lrger, longer-lsting Fig. 6. (A) Arteril blood plsm bicrbonte concentrtion ([HCO 3 ] ; mmol l 1 ) nd (B) CO 2 tension (P CO 2; mmol Hg). One set of fish (indicted by the filled symbols) were fed immeditely following the time 0 smple, while nother set of fish (open symbols) remined unfed. Vlues re mens ± s.e.m.; N=8 for ech tretment. Time points with the sme letters re not significntly different (P>0.05).

6 2538 C. Bucking nd C. M. Wood cid bse disturbnce thn voluntry feeding. In our study, voluntry feeding clerly induced n lkline tide in the rteril blood of rinbow trout, evidenced by mrked increses in ph (Fig. 5) nd plsm [HCO 3 ] (Fig. 6A) from 3 to 12 h fter the mel, without chnge in P CO 2 (Fig. 6B) i.e. clssicl metbolic lklosis. This disturbnce ws lrger thn seen in the voluntrily feeding fish of Cooper nd Wilson (Cooper nd Wilson, 2008), probbly reflecting the difference in mel size between the two studies. As mentioned in the Introduction, the response lmost certinly reflects the ddition of metbolic bse to the blood by oxynticopeptic cells of the gstric mucos (reviewed by Hersey nd Schs, 1995) (Niv nd Frser, 2002). Upon stimultion, these cells secrete HCl into the stomch lumen to fcilitte digestion, s well s HCO 3 into the extrcellulr fluid comprtment in order to mintin intrcellulr ph. A K + -stimulted, H + -ATPse is responsible for the picl H + secretion, nd hs been identified in oxynticopeptic cells of elsmobrnchs (Smolk et l., 1994) s well s those of the rinbow trout (Sugiur et l., 2006). Although there is vigorous secretion of gstric cid t this time, the ph of the stomch fluid ctully increses substntilly due to the buffering ction of the ingested food (Sugiur et l., 2006; Bucking nd Wood, 2008). As Cl /HCO 3 exchnger is believed to be responsible for the bsolterl HCO 3 export nd Cl entry, the net trnsfer of HCl to the stomch cn led to reduction in plsm [Cl ] tht hs been correlted with n lkline tide in tods (Busk et l., 2000). Interestingly, post-prndil drop in plsm [Cl ] ws not seen by either Bucking nd Wood (Bucking nd Wood, 2006) or Cooper nd Wilson (Cooper nd Wilson, 2008) in rinbow trout tht hd fed voluntrily, but ws reported by the ltter uthors in rinbow trout tht hd been force-fed. Bsed on the results of the present study, probble explntion is tht during the removl of the excess bse to the wter by the gills, the brnchil Cl /HCO 3 exchnger is ble to compenste for the loss of Cl to the stomch lumen by uptke of Cl from the dilute externl environment. Interestingly, incresed ctivity of this brnchil exchnger my lso explin why our study reveled net excretion of bse to the wter to relieve the lkline tide, wheres Cooper nd Wilson (Cooper nd Wilson, 2008) found no such clernce of bse to the wter (in either of their feeding tretments) despite observing cler lkline tides in the bloodstrem. The lower Cl levels in the wter in the Cooper nd Wilson study my hve limited the exchnge of Cl for HCO 3 t the gills nd prevented the clernce of the metbolic lklosis to the wter, theory tht is further corroborted by the reduction in plsm [Cl ] t lest in the force-fed fish of the Cooper nd Wilson study (Cooper nd Wilson, 2008), becuse of the lck of environmentl Cl vilble for replcement. However, the extent to which the differences in wter chemistry cn contribute to the differences observed between the two studies is unknown. It hs been shown tht wter Cl concentrtions hd only modest effect on brnchil Cl /HCO 3 exchnge rtes in the flounder (Tylor et l., 2007). However, the higher ffinity nd cpcity of brnchil uptke kinetics of Cl in the rinbow trout [K m μmoll 1, J mx ~360μmolkg 1 h 1 (e.g. Goss nd Wood, 1990; Wilkie et l., 1999)] vs those in the flounder [K m ~650μmoll 1, J mx ~198 μmol kg 1 h 1 (Tylor et l., 2007)] suggest tht wter Cl concentrtions my ply lrger role in determining the rte of exchnge in the trout. The potentil limittions of low wter Cl concentrtions in relieving metbolic lklosis requires further investigtion. If this mssive bse excretion (Fig. 4) hd not occurred, t lest μmol kg 1 of bse (i.e. HCO 3 equivlents) would hve to hve been buffered in the body fluids over 48 h. It is not possible to precisely clculte the effect on blood ph without knowledge of how this 13.9 mmol kg 1 HCO 3 lod might distribute between intr- nd extr-cellulr comprtments. However, pplying the trditionl technique pioneered by Rune (Rune, 1965; Rune, 1966) nd now widely used in humns (Niv nd Frser, 2002), the ssumption is mde tht the excess bse of the lkline tide is distributed in blood buffer spce, equivlent to 0.3 body mss. Using blood non-hco 3 buffer cpcity of 10.8slykes for rinbow trout (Wood et l., 1982), the Henderson Hsselblch eqution, the pk 1 nd αco 2 constnts tbulted for trout blood plsm by Boutilier et l. (Boutilier et l., 1984) nd simple Dvenport (Dvenport, 1974) digrm nlysis, the blood ph would hve risen to bout 8.55, n increse of bout 0.7 units, in contrst to the 0.2 ph unit increse mesured here (Fig. 5). Thus the excretion of excess bse to the environment prevented bout 70% of the nticipted rise in blood plsm ph, n increse tht very likely would hve been ftl. Although the lkline tide ppered to be relieved by 18 h postfeeding (Fig. 5), this does not necessrily men tht gstric cid secretion hs subsided. Fsted fish exhibited smll net cid flux t ll time points, or n overll negtive bse excretion of 4344 μmol kg 1 over the 48 h of experimenttion (Fig. 4). By contrst, fed fish trnsitioned from net cid flux to net bse flux s the lkline tide progressed (Fig. 4), which remined significnt reltive to non-fed nimls during the 6 42 h post-feeding period (Fig. 4) nd resulted in the excretion of μmol kg 1 more bse thn by the unfed fish. In comprison, unfed dogfish exhibited pproximtely one hlf the net cid flux [ 2160μmol kg 1 (Wood et l., 2007b)], which is most likely due to the inherent differences in nitrogen metbolism between the two species (i.e. mmoniotelism vs ureotelism). However, following feeding, the dogfish showed net flux of bse to the wter tht ws similr, lthough quntittively smller [ μmol kg 1 (Wood et l., 2007b)], to tht seen in the current study, suggesting tht the rinbow trout hd lrger lkline tide thn the dogfish. These mesurements suggest substntil role for brnchil Cl /HCO 3 exchnge in lleviting the lkline tide through incresed bse excretion to the wter. The bility of freshwter teleosts to utilize brnchil ion trnsport mechnisms to correct cid bse disturbnces is well estblished (e.g. Perry et l., 2003; Evns et l., 2005; Tresguerres et l., 2006) nd the restortion of resting blood cid bse chemistry likely reflects the bility of brnchil bse excretion mechnisms to dequtely compenste for the metbolic lklosis creted during digestion, s in the elsmobrnch Squlus cnthis (Wood et l., 2005; Wood et l., 2007; Wood et l., 2007b; Tresguerres et l., 2007). The lkline tide, t lest for mmmls, is lso ccompnied by excretion of lkline urine (Rune, 1965; Rune 1966; Niv nd Frser, 2002) resulting from reduction in the metbolic cid lod normlly excreted in the urine (Brunton, 1933). In fct, Finke nd Litsenberger (Finke nd Litsenberger, 1992) determined tht postprndil ph of urine produced by cts ws linerly correlted with mel size. In humns, Johnson et l. (Johnson et l., 1990) observed correltion between chnges in postprndil urine cid output nd titrtble gstric cid output. While brnchil excretion in freshwter fish of cid bse equivlents generlly ccounts for the mjority of the totl exchnge, the urine cn ply n importnt supplementry role in the compenstion of metbolic cid bse disturbnces (Wood et l., 1999). The net fluxes of mmoni nd HCO 3 to the wter mesured in this experiment combined contributions from both brnchil nd urinry sources, nd the urinry contribution to both the excretion of bse to the wter nd the relief of the lkline tide cn only be speculted.

7 Feeding in freshwter rinbow trout 2539 The lkline tide observed in reptiles nd mphibins (e.g. Coulson et l., 1950; Wng et l., 2001; Andrde et l., 2004) results in only very modest increses in ph, s result of respirtory compenstion [i.e. n increse in P CO 2 (Wng et l., 1995; Wng et l., 2001; Overgrd et l., 1999; Busk et l., 2000; Busk et l., 2000b; Andersen nd Wng, 2003)] tht ppers to be cused by hypoventiltion (Hicks et l., 2000; Secor et l., 2000; Wng et l., 2001b). This phenomenon hs lso been observed in humns lthough to lesser degree (Higgins, 1914; Erdt, 1915; Vn Slyke et l., 1917; Ou nd Tenney, 1974). However, neither the freshwter rinbow trout of the present study, those studied by Cooper nd Wilson (Cooper nd Wilson, 2008) nor the mrine dogfish shrk (Wood et l., 2005) exhibited ny increse in P CO 2 during the postprndil period. Fish pper to hve no bility for respirtory compenstion of the metbolic lklosis creted by the lkline tide. In essence, the gills re believed to be hyperventilted with respect to CO 2 excretion becuse of the much lower solubility of O 2 reltive to CO 2 in wter. This results in miniml djustments of blood P CO 2 even if ventiltory chnges occur (Perry nd Wood, 1989). Although the lkline tide phenomenon is commonly reported in mphibins nd reptiles (Wng et l., 2001), it ppers to be more controversil in humns nd fish. Severl studies in humns hve filed to see lkline urine nd respirtory compenstion following feeding (e.g. Brunton, 1933; Johnson et l., 1995). These uthors hve even suggested tht ny respirtory or urinry compenstion for gstric cid secretion is too smll to be of physiologicl or clinicl significnce. When considering fish species, studies on the gulf todfish (Tylor nd Grosell, 2006) nd Europen flounder (Tylor et l., 2007) likewise reported no evidence for clssic lkline tide. By contrst, the present study nd tht of Cooper nd Wilson (Cooper nd Wilson, 2008) on the rinbow trout, s well s severl investigtions on the dogfish shrk (Wood et l., 2005; Wood et l., 2007; Wood et l., 2007b; Tresguerres et l., 2007) clerly demonstrte evidence for its existence. Differences in methodology my contribute to these discrepncies; for exmple, the study of Cooper nd Wilson (Cooper nd Wilson, 2008) demonstrtes tht the nture of feeding (voluntry vs forced) will lter the extent of the lkline tide. It is lso possible tht the differences re relted to differences in environmentl slinity (discussed subsequently) or feeding ecology mong species. For exmple, mny reptiles nd mphibins feed t irregulr intervls, but re ble to ingest mels tht re very lrge reltive to their own body mss (e.g. Greene, 1997; Shine et l., 1998). Digestion of these lrge mels is ssocited with considerble increments in oxygen uptke tht lsts for severl dys (Benedict, 1932; Secor nd Dimond, 1998; Wng et l., 2001b). By contrst, humns nd some fish ingest reltively smller mels more frequently, indicting possible role for mel size in the occurrence of n lkline tide. Fish tht hve exhibited n lkline tide lrge enough to elicit compenstion by excretion of bse to the environment pper to exhibit either spordic feeding ecology more similr to tht of reptile thn of mmml [dogfish (e.g. Jones nd Green, 1977; Hnchet, 1991; Tnsichuk et l., 1991)], or were strved for more thn week nd then consumed rtion of food >5% of body mss (rinbow trout, this study). Differences within species with similr feeding ecology lso probbly exist. The rinbow trout used in this study do not normlly fst for more thn one week, nd while their nturl feeding ptterns re probbly more similr to the todfish nd flounder thn the dogfish, our results suggest difference in cid bse disturbnces between the species s pointed out bove. The resons re uncler s of yet, but perhps it is relted to the diet itself. Indeed, s mentioned erlier the net flux of bse equivlents from fed trout in the current study were greter thn those seen from fed dogfish (Wood et l., 2007b), nd lthough both studies estimted feeding t >5% body mss, the food used in the current study ws commercil diet tht ws pproximtely 10 20% wter wheres the nturl diet fed to the dogfish ws ~80% wter. Commercil diets my in fct be digested in very different mnner thn nturl diets, s side from differing wter contents, commercil diets my possess higher buffering cpcity nd hence require greter cid secretion to rech the low ph required for protein digestion. In fct, the titrtion of commercil fish mel down to ph 3 (Cooper nd Wilson, 2008) required 10-fold more cid thn tht of nturl rgworm diet (Tylor et l., 2007). It is unlikely tht titrtion in vitro exctly duplictes the rel titrtion tht occurs s chyme is progressively digested nd diluted in vivo (Bucking nd Wood, 2006; Bucking nd Wood, 2008). Nevertheless, greter cid secretion with commercil diet my reflect the greter cid bse disturbnces observed in the present study when compred with studies using nturl diets (Wood et l., 2007b; Tylor et l., 2007). In fct, this my led to vriety of cid bse chllenges in the wild, where fish tht feed primrily on invertebrtes my secrete less gstric cid, thn fish tht et minly vertebrtes. The cuse(s) behind the incongruities between the bse excretion observed in this study nd the lck of bse excretion observed by Cooper nd Wilson (Cooper nd Wilson, 2008) nd Tylor et l. (Tylor et l., 2007) my be result of either different mel sizes or the vilbility of environmentl Cl to relieve the lkline tide through brnchil Cl /HCO 3 exchnge, s suggested erlier. Additionlly, the current study ws conducted on freshwter rinbow trout, however, mrine teleosts such s the flounder studied by Tylor et l. (Tylor et l., 2007) my very well rect differently to feeding becuse of ltered gstrointestinl nd brnchil trnsporter expression, s well s essentilly opposing physiologicl needs. Mrine teleosts secrete lrge quntities of HCO 3 into the intestine for purposes ssocited with osmoregultion [wter bsorption nd C 2+ precipittion, s reviewed by Grosell (Grosell, 2006)], so it is possible tht recycling of HCO 3 in this mnner will ttenute or prevent the systemic lkline tide nd/or bse excretion to the wter. Finlly, the diet itself, in its composition nd size, my ply strong role in determining the extent, durtion nd mechnism of compenstion for this metbolic disturbnce. Unlike plsm mmoni, plsm glucose ws not significntly ffected by feeding, which is symptomtic of poor utiliztion of crbohydrtes by rinbow trout. A rpid nd trnsient increse in plsm glucose concentrtions hs been reported in rinbow trout 1 h fter feeding (Wicks nd Rndll, 2002); however, this could hve been reflective of stress response to the experimentl procedure. Overll, crnivorous fish (such s rinbow trout) re recognized for their inefficiency in utilizing dietry crbohydrtes (Moon, 2001; Wilson, 1994). Crnivorous fish express lower bundnce of intestinl glucose trnsporters reltive to omnivorous nd herbivorous fish (Buddington et l., 1997). The current study lso reveled no post-prndil chnges in plsm ure concentrtions, s result of either lck of increse in ure production or mtching increse in ure excretion to mintin plsm levels. This is not the cse with glucose, s glucose is highly rebsorbed by the kidney, the primry site of glucose excretion (Bucking nd Wood, 2004). Previous studies (e.g. Brett nd Zl, 1975; Wiggs et l., 1989) reveled no significnt increse in ure excretion following feeding in severl fish species; however, these findings hve been contrdicted in other species (e.g. Alsop nd Wood, 1997; Wright, 1993). Resons for this discrepncy my reflect differences in metbolic pthwys

8 2540 C. Bucking nd C. M. Wood mong species nd/or diet composition. Notbly, Alsop nd Wood (Alsop nd Wood, 1997), working on juvenile rinbow trout, reported tht stedy feeding to stition incresed ure excretion rte bout fourfold reltive to tht of fsted fish, though ure-n excretion remined only bout 10% of the similrly elevted mmoni-n excretion. Future res of interest generted by this study include identifying the urinry contribution to the totl incresed mmoni nd bse excretion to the wter, s well s the detils of the brnchil bse excretion mechnism, s mentioned erlier. Additionlly, it my be possible to phrmcologiclly mnipulte gstric cid secretion using inhibitors, nd thereby evlute whether HCl production is the direct cuse of the lkline tide. In humns, Oder et l. (Oder et l., 2002) observed n increse in men gstric ph fter the dministrtion of proton pump inhibitors, nd urinry cid output significntly decresed when compred with control fed subjects. Holstein (Holstein, 1975) reported tht teleosten fish possess the histmine H 2 receptor in the stomch, which is believed to be responsible for stimulting gstric cid secretion, nd Trischitt et l. (Trischitt et l., 1998) demonstrted in vitro evidence for histmine stimultion of gstric cid secretion by the eel stomch s well s inhibition by crbchol ( histmine H 2 receptor ntgonist). Finlly, the effect of wter chemistry nd diet composition should be evluted. In summry, feeding nd digestion creted numerous physiologicl chllenges in the freshwter rinbow trout, including incresed plsm mmoni levels, incresed mmoni nd bse excretion to the wter, s well s n overll systemic metbolic lklosis. Although the metbolic lklosis cn be thought of s chllenge to fish creted by digestion, it my serve to mintin plsm ion concentrtions, especilly Cl through brnchil trnsport mechnisms. It hs long been known tht freshwter fish hve high cpcity for brnchil bse excretion, s usully demonstrted by NHCO 3 infusion (Perry et l., 2003; Evns et l., 2005; Tresguerres et l., 2006). The present demonstrtion of the lkline tide nd ssocited bse efflux provides nturl purpose (i.e. cid bse homeostsis following feeding) for the existence of this mechnism. We thnk Trvis Allen for excellent contributions, nd two nonymous referees for constructive comments. All procedures were in ccordnce with pproved McMster University niml cre protocols, nd conformed to CCAC guidelines. The work ws supported by n NSERC Discovery grnt to C.M.W., who is lso supported by the Cnd Reserch Chir Progrm. C.P.B. is supported by NSERC CG scholrship. REFERENCES Alsop, D. H. nd Wood, C. M. (1997). The interctive effects of feeding nd exercise on oxygen consumption, swimming performnce nd protein usge in juvenile rinbow trout (Oncorhynchus mykiss). J. Exp. Biol. 200, Andersen, J. B. nd Wng, T. (2003). 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