The role of haematocrit in oxygen transport and swimming in

THE ROLE OF HAEMATOCRIT IN OXYGEN TRANSPORT AND SWIMMING IN SALMONID FISHES

by Patricia Elizabeth Gallaugher B.Sc. University of British Columbia

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of BIOLOGICAL SCIENCES

O Patricia Gallaugher 1994 SIMON FRASER UNIVERSITY August 1994

All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without pem~issionof the author.

APPROVAL

NAME:

PATRICIA ELIZABETH GALLAUGHER

DEGREE:

DOCTOR OF PHILOSOPHY

TITLE OF THESIS: THE ROLE OF HAEMATOCRIT IN OXYGEN TRANSPORT AND SWIMMING IN SALMONID FISHES

Examining Committee: Chair:

Dr. Leah Bendell-Young, Professor

Dr. A P F a p l l , Professor, sen& Supervisor, Sciences, SFU ~ e ~ a r b e nf Biological t

Dr. A.H.J. Burr, Aksociate Professor Department of Biological Sciences, SFU

Dr. B. McKeown, Professor Department of Biological Sciences, SFU

-

Dr. C. Kennedy, Assistant Profess Department of Biological SciencxSFU Public Examiner

-

Dr. Warren ~ u r ~ ~ rProfessor en, Department of Biology, Universitp bf Nevada Las Vegas External Examiner

Date Approved

PARTIAL COPYRIGHT LICENSE

I hereby g r a n t t o Simon F r a s e r U n i v e r s i t y t h e r i g h t t o l e n d my t h e s i s , p r o j e c t o r e x t e n d e d essay ( t h e t i t l e o f w h i c h i s shown below) t o u s e r s o f t h e Simon F r a s e r U n i v e r s i t y L i b r a r y , and t o make p a r t i a l o r s i n g l e c o p i e s o n l y f o r such u s e r s o r i n response t o a r e q u e s t from t h e l i b r a r y o f any o t h e r u n i v e r s i t y , o r o t h e r e d u c a t i o n a l i n s t i t u t i o n , on

I f u r t h e r agree t h a t permission

i t s own b e h a l f o r f o r one o f i t s u s e r s .

f o r m u l t i p l e c o p y i n g o f t h i s work f o r s c h o l a r l y purposes may be g r a n t e d by me o r t h e Dean o f Graduate S t u d i e s .

I t i s understood t h a t copying

o r p u b l i c a t i o n o f t h i s work f o r f i n a n c i a l w i t h o u t my w r i t t e n p e r m i s s i o n .

Author:

Patricia Gallaugher (name)

g a i n s h a l l n o t be a l l o w e d

ABSTRACT The optimal haematocrit (Hct) hypothesis suggests that vertebrate Hct is set to maximize the rate of oxygen transport in arterial blood (To2). Since T o 2 is equal to the product of the oxygen content of arterial blood (Cag2) and cardiac output (Q), this implies that Hct is adjusted to maximize blood oxygen carrying capacity without compromising blood flow and cardiac work through elevated blood viscosity (q). Thus, the Hct value which coincides with maximum T o 2 (ToZmax)represents the optimal Hct (Hctopt).

I tested this hypothesis in rainbow trout (Oncorhynchus mykiss) by altering Hct between extreme states of anaemia and polycythemia (Hct

=

8 - 55%). I predicted that

the effects of q on cardiac work would be greatest at peak aerobic exercise levels when Q would be maximum (emax). Moreover, I predicted that ToZmax and maximal oxygen uptake (Vo2,,,)

would peak at Hctopt. Therefore, my experimental approach was to

challenge fish to swim to their critical swimming velocity (Ucrit) in a swim-tunnel respirometer while oxygen uptake (Vo2) and other cardiovascular variables were measured.

Furthermore, since blood viscosity is higher at lower temperatures,

experiments were performed at both 5 O C and 13 O C . The mean normal Hct (normocythemia) for rainbow trout at rest was 27 - 30%. Cao2 was linearly related to Hct across the experimental range from 8 - 55%. Consistent with the Hctop, hypothesis, the decreased Cag2 in anaemic fish (Hct < 21%) caused a significant reduction in Uc,it and V02max. As expected, there was an exponential relationship between q, measured in vitro, and the experimental Hct, and Qmax was significantly lower in fish with the highest Hct values. Contrary to the Hctopl hypothesis, ToZmax was not compromised with polycythemia (Hct > 33%). Despite the effects of q , ToZmaxincreased with Hct, up to

Hct

=

55%, at both 5 "C and 13 "C, and moreover, polycythemia produced significant,

albeit small, increases in Ucrit Futhermore, peak V 02max occurred at an Hct (42%) well above normal Hct (normocythemic) values. It appears that both Ucrit and

VoZmax in

normocythemic fish are limited by the capacity for internal convection of 0 2 .

I suggest that while the lower limit for normocythemia is set by C q 2 , the regulation of the upper limit for normocythemia involves more than q effects on cardiac work. One factor which may set upper Hct is the Hct-dependent decrease in arterial oxygen tension ( P w 2 ) observed at Ucrit. This decreased Pao2 (indicative of diffusion limitations to O2 transfer at the gills) did not cause decreases in C w 2 at most Hct values, but the peak for

VoZmax may

coincide with an Hct at which this arterial hypoxaemia

begins. Other potential limiting factors for upper Hct were revealed in experiments with exercise-trained chinook salmon (0. tshawytscha).

Small (

80% increase in Hct, to an Hct value of 57% in the toad (Hillman et al. 1985) and the bullfrog (Withers et al., 1991), and these changes were inversely related to Hct-induced increases in q (see Chapter 1). Similar to the Antarctic icefish, amphibian hearts are known to have a poor homeometric capacity (Farrell, 1991a), and therefore may not be capable of compensating for the elevations in q and the associated increases in blood pressure which were associated with polycythemia in these studies. The Hctopt hypothesis has also been tested in a number of fish species, including rainbow trout, but the findings are equivocal, as in no case did the predicted HctOprmatch Hct, when measured in vivo (see Chapter 1). This lack of a tight matching may, in part, be attributable to the use of in vitro measurements of q to estimate oxygen transport capacity (OTC) (see Chapter I), rather than direct measurements of Q. It could also be that the viscosity effects on Q, in vivo, may not be significant, since unlike amphibians and icefish, rainbow trout hearts, and indeed those of many other temperate teleost species, have a broad range for homeometric regulation (Farrell, 1991a). In the experiments described in Chapter 2, I was unable to establish a relationship between Hct and exercise performance (as measured by Ucrit) within the normal range of Hct values for rainbow trout. These findings led to the suggestion that if indeed an Hctopt exists for this species, it must take the form of a plateau, rather than a discrete peak.

I also suggested that an Hctopr may become more evident using direct

measurements (i.e., V 02) of aerobic metabolic demands during exercise performance, and if the range of Hct values was broadened to include more extreme Hct values. Results from experiments with

conscious,

and

particularly

exercising,

polyc)ithemic mammals consistently demonstrate that mammalians can compensate for the detrimental effects of elevated q on cardiac function and To2, through vascular

adjustments (see Chapter 1). Moreover, there is indisputable evidence that V oZmax increases, and exercise performance improves, with "blood doping" in humans, although in most cases the increases in Hct are relatively small ( ~ 1 0 % (see ) Chapter 1). Indirect evidence of the magnitude of apparent increases in q which some mammalian species can tolerate, comes from observations of profound increases in Hct to values that are close to double the normal Hct, values, due to splenic transfusion during strenuous aerobic exercise (e.g., thoroughbred horses (see Chapter 1). Similarly, Hct has been observed to increase significantly during strenuous aerobic exercise in some fish species (see Chapter I), including rainbow trout (see Chapter 2, Appendix 1). Combined, the above observations indirectly argue against the Hctopt hypothesis. In view of the equivocal support for an Hctopt hypothesis, and to extend the findings of the work described in Chapter 2, I designed a more thorough set of experiments in which I adjusted Hct in rainbow trout over the range from 8% to 55%, and measured

Vo2 and

Q in addition to Ucrit. Moreover, I extended the putative viscosity

effects further, by performing the experiments at two temperatures, 5 OC and 13 OC. My working hypothesis was: If Hct is optimized for To2in rainbow trout, then experimental adjustment of Hct to extreme values (8 - 55%), and exposure to an exercise challenge which maximizes Q and oxygen demand, would reveal peak values for maximum swimming performance (Ucrit) and V 02rnaxwithin the range of normocythemia (i.e., 22 45%, as observed in Chapter 2).

Moreover, because q increases with decreasing

temperature, the Hctopt would shift to a lower Hct value at 5 OC, compared with 13 OC.

Materials and Methods Experimental animals Two groups of rainbow trout (Oncorhynchus mykiss) were used in these experiments. Both groups of fish (body mass = 500 g =

+ 9 S.E.M.; length = 33 - 38 cm; N

64) were obtained from West Creek Trout Fann, Aldergrove, B.C., and were held in

large outdoor tanks for at least two months prior to surgery. During this period, they were fed satiation levels of dry pellets (Moore Clarke, B.C.) once daily. The summer group of fish were acclimated to seawater (SW) (12

-

14 OC; salinity 30 ppt (parts per

thousand)) and the winter group of fish were maintained in freshwater (FW) at 4 - 6 OC. FW was chosen for the winter experimental medium because of the lower ambient water temperatures, compared with SW. Under winter conditions, the SW temperatures were never lower than 9 "C. All fish were starved for 24 hours prior to surgery.

Surgical Procedures Fish were netted and immediately anaesthetized in a 1:2000 solution of 2phenoxyethanol (Sigma Chemical Co., St. Louis, Missouri). Body dimensions were determined and the fish were transferred to an operating sling where their gills were constantly irrigated with a 1:4,000 solution of 2-phenoxyethanol during surgery. A cannula was inserted into the dorsal aorta as described by Soivio et al. (1975) using polyethylene tubing (PE50, Clay Adams, Parsippany, NJ) filled with heparinized (50 i.u. per mL) saline. In summer fish, relative cardiac output (Q) was measured using a pulsed Doppler flow probe (TMI, Iowa City, Iowa) which was implanted on the ventral aorta following the method of Thorarensen, Gallaugher and Farrell (see Appendix 3). The probe leads were secured on the body surface and anchored dorsally with the DA cannula.

Q was not measured in winter fish.

Experimental Protocol

Haematocrit adjustment Following surgery, summer fish were held for at least 24 h in 20 L circular tanks at ambient temperature.

They were then transferred to a Brett-type swim tunnel

respirometer (Kiceniuk and Jones, 1977), where they were allowed to recover for at least

4 h before resting values were recorded for heart rate CfH), relative Q, dorsal aortic blood pressure (PDA),Vo2,Pao2 and Caoz, Hct, [Hb], arterial blood pH (pH& and lactate concentration ([La]). Hct was adjusted (see below) and after an overnight recovery, another set of resting values were recorded for the above variables. Winter fish recovered from surgery for 24 h in black Plexiglass boxes which were continuously supplied with well-aerated dechlorinated freshwater (4 - 6

"c),after which

Hct, was determined. These fish were then transferred to a swim tunnel similar to that described by Gerkhe et al. (1990) and held with the water velocity at 10 cm-s-I for at least 1 h prior to Hct adjustment. After an overnight recovery, the new Hct, was recorded. For both summer and winter fish, Hct was adjusted in the following manner. Cannulated donor fish, held in a blackened Plexiglass box for at least 24 h after surgery, acted as either donor or recipient fish. Polycythemia was induced in recipient fish over the period of 48 h by infusion of small volumes of blood (1 - 2 mL at a time) from donor fish. The donor fish became the future anaemic fish. An equivalent volume of rainbow trout plasma was used to replace blood removed from the anaemia group of fish. The recipient fish, in turn, became polycythemic donor fish (Hct > 50%) to achieve a higher level of polycythemia for the polycythemic fish used in the experiments. The number of blood withdrawals or infusions, and the total volume of blood withdrawn or infused (1 - 2 mL per infusion per 2 h) in order to obtain a desired Hct, was calculated assuming a blood volume of 3.5 m L 100 g-I body mass (see Olson, 1992). Blood removal or infusion

was performed over a period of at least 5 min. Normocythemic fish that acted as controls. were prepared by first removing blood (2 mL), and then infusing an equivalent volume of blood from an equivalent-Hct donor fish. This process was repeated several times over 3 - 4 h. At no time was there evidence of cross-reactivity between the blood cells of

different individuals due to these manipulations.

Swimming trials and oxygen uptake measurements For both groups of fish, Ucrit was determined by increasing the water velocity, first in steps of 0.50 b1.s-I and then by 0.25 b1.s-I. Each velocity step was maintained for either 30 minutes or until the fish fatigued. Fatigue was evident when the fish could not swim off the rear grid (SW summer fish), or if they remained on the 8 V electrified rear grid for longer than several seconds (FW winter fish). Fatigue is a discrete event which Occurs when the fish can no longer swim against the imposed water velocity. Therefore, to get a representative blood sample, in some cases it was necessary to quickly reduce the swimming speed at fatigue by one step so that the fish continued to swim while the blood Was sampled. Uc,it values were calculated after appropriate adjustment for the blocking effect of the fish (Bell and Terhune, 1970). Water velocity was calibrated at least once per week with a flow meter. The O2 tension of the water in the swim tunnel (Pw02) was continuously monitored with an O2 electrode (Radiometer E5046, Copenhagen), thermostatically controlled at the experimental temperature and connected to a PHM 71 acid-base analyzer (Radiometer, Copenhagen). Calibration of the 0 2 electrode was achieved by setting the meter to zero when the electrode was disconnected and setting Pw02 using aerated water at the ambient temperature and barometric pressure.

Water from the tunnel was

c~ntinuouslydrawn past the electrode via a pressure head and the Pwo2 was recorded

every second by a computer. V o 2 was measured by closing off the tunnel for 6 min (summer fish) or at least 30 min (winter fish). Oxygen consumptior. was calculated as: V o 2 = V . ~ w o 2 . d r l. a . m - 1 where V is the tunnel volume (39 L summer fish; 130 L. winter fish). a is the solubility constant for O2 at the experimental temperature and salinity (pmol 0 2 . ~ - ' . k p a - ' ) (Boutilier er al., 1984), and m is body mass. At no time did the water temperature of the larger tunnel change by more than 1.0 "C during the V 0 2 measurement.

Blood sampling and analytical techniques Arterial blood samples (0.6 mL) were taken before and after Hct adjustment, a1 rest, once at a velocity of approximately 80% Ucrit, and at Uc,it. An equivalent volume of blood from an equivalent-Hct donor fish was used to replace blood removed for sampling. For some summer fish, blood samples (0.5 mL) were also withdrawn after Hct adjustment at rest and at Ucrit, and similarly replaced with blood from an equivalent-Hct donor fish, for measurements of q . Blood viscosity was not measured in winter fish. Measurement of Pag2 was made using a Radiometer E5046 Po2 electrode in a D616 cell and whole blood pHa was determined on samples injected into a Radiometer

pH microelectrode (type E5021). Both electrodes were regulated at the tunnel water temperature and linked to a Radiometer (Copenhagen) PHM 7 1 acid-base analyzer. Cag2 was measured in 30 pL samples using the method of Tucker (1967). Hct was measured in triplicate (20 pL samples drawn into microcapillary tubes) by spinning samples in a Haemofuge (Heraeus Sepatech, Netherlands) centrifuge at 12,000 rpm for 3 min.. Sigma diagnostic kits (Sigma Chemical Co., St. Louis, Missouri) were used to measure blood [Hb] (no. 525A) in 20 pL whole blood samples and [La] (no. 826-UV) in 100 pL plasma samples. Blood viscosity was determined at I5 "C using a cone-plate viscometer with

cone angle 0.8' (model LVT. Brookfield Engineering Laboratories, Massachusetts). Samples were used immediately, or within 12 h (in the latter case they were refridgerated at 4 'C until use). In summer fish, the dorsal aorta cannula was connected to a pressure transducer (Narco, LD 15). Signals from flow meters, pressure transducers, and oxygen meters were amplified and monitored by a Grass chart recorder (model 7CP B, Grass Instruments, Quincy, Massachusetts) and stored in a computer. The computer sampled Q and Pd, signals at a rate of 5Hz, and recordings were averaged over 6 min for each swimming velocity.

Labtech

Notebook

software

(Laboratory

Technology

Corporation,

Massachussetts) was used to convert the signals to digital form, to process the signals, and to calculate f H. Blood volume (BV) was measured in summer fish, immediately after fatigue, using the Evan's Blue dye dilution technique (Smith, 1966; Nikinmaa et al., 198 1). After an initial sample of blood was withdrawn to determine Hct and obtain plasma, a known amount of Evan's Blue solution (0.6 mL) was injected via the dorsal aorta cannula and the cannula was flushed with saline (0.4 mL). Timed (30, 60, 90, 120 min) serial blood samples (0.25 mL) were withdrawn. The total volume of blood withdrawn was equal to the volume of Evan's Blue injected

+ the volume of saline used to rinse the cannula.

The

blood was centrifuged, Hct was measured, and plasma was diluted with saline, and the optical density (O.D.) read against the original plasma sample (also diluted) at 600 nm wavelength using a Pye Unicam SP8 spectrophotometer. Plasma volume was calculated using the density of the dye in the plasma and the injected amount of dye as follows: Plasma volume (PV) = [EB] VEB/ 0 . D (where [EB] is the concentration of the injected Evan's Blue stock solution (0.6 g.L-I) and VEBis the injection volume, and O.D. was the value for Evan's Blue at time zero (obtained by regressing the relationship between O.D.

and time to zero). Since Hct did not change significantly during the sampling, BV was calculated as follows: BV = PV /((I - Hct/lOO).PV) (Nikinrnaa et al., 1981). BV was not measured in winter fish. Some variables were compared as discrete groups of fish. For summer fish, these groupings are: the most anaemic fish (mean Hct (mean Hct

=

=

12.4 %), the most polycythemic fish

47.9%), and normocythemic fish (mean Hct

=

26.3 %); for winter fish, the

most anaemic fish (mean Hct = 17.2%), the most polycythemic fish (mean Hct and normocythemic fish (mean Hct

=

32.5%) (see Table 1).

=

46.9%)

The significance of

differences in various parameters (PDA, Qrest, Qmax, pHa, [La]) between anaemic, normocythemic and polycythemic groups of fish was tested using ANOVA. The significance of changes in haematological variables at Ucrit, compared with rest, was tested using paired t-tests. For the data describing [Hb], Cag2, Ucrit, V 02rnax, To2, conductance, and Pag2 as functions of Hct, curves were fit with the use of a segmented polynomial model as modified by M. Zhan (Department of Mathematics and Statistics, Simon Fraser University) (Zhan et al., 1994).

Results Relationships between Hct (Hb) and CaO2 blood viscosity, Q, and To2 The initial mean Hct was 27.0% 30.0%

+ 0.8 S.E.M. (N = 35) for summer fish, and

+ 0.3 S.E.M. (N = 29) for winter fish.

The initial Hct range for all fish used in

these experiments was between 23% and 33%. For the rest of this chapter, this range of Hct values will define normocythemia. [Hb] increased proportionately with Hct, in both summer and winter fish (Figure 3). The relationship between [Hb] and Hct was not different in summer and winter fish (Figure 3) (P < 0.01, Student's t-test).

Figure 3. The relationship between [Hb] and Hct at Ucrit in SW summer (filled symbols) and FW winter rainbow trout (open symbols). The slopes of the lines were not significantly different for the two groups of fish (Equation for the regression line fitted to the combined sets of data: y = 0.3233.x, R2 = 0.912).

summer 0 winter

Figure 4. The relationship between Cag2 and [Hb] at Ucrit for SW summer (filled symbols) and FW winter (open symbols) rainbow trout. The solid lines show polynomials fitted to the data for the two groups of fish (y = 0.0092-x*+

1.306.+ ~ 0.013, R2 = 0.876, for summer fish; y R2 = 0.824,

= 0.023ax2 +

1.3701.~+ 0.013,

for winter fish). The dashed line represents the line of identity,

calculated assuming that 1 g of Hb binds 1.3 mol 02.

0

5

10

[Hb] U (go100 mL -')

15

20

C w 2 increased with increasing [Hb] (Hct), but the relationship between Cao2 and [Hb] was not directly proportional and was different, albeit not significantly so, in summer and winter fish (Figure 4) (P < 0.01, Student's t-test). At a [Hb] E 10 g.100 mL-1 (Hct r 30%), the relative increase in C w 2 , per unit increase in [Hb], was less than would be predicted by the theoretical line of identity (calculated by assuming that 1 g of Hb binds 1.3 mol of 02), indicating that the saturation of Hb with 0 2 declined as the [Hb] increased (Figure 4). This decrease was more profound in summer than in winter fish (Figure 4). Calculations for the amount of 0 2 bound to Hb (pmol-02.gHb-')(i.e., 0 2 bound to Hb = Cag2 - ((P~2.a02)(1-Hctl100))/[Hb], where Cag2 was converted fiom vol % to p mo1.L-I, and the fraction of 0 2 dissolved in the plasma was calculated using the tabulated solubility coefficient for 0 2 at the relevant temperature ( a ) (Boutilier et al., 1984) (see Milligan and Wood, 1987)), revealed a trend for desaturation in polycythemic summer, but not winter, fish at Ucrit, compared with rest (Table 1). However, the Hct-dependence for the amount of 0 2 bound to Hb was not significant. The amount of 0 2 bound to Hb was significantly higher in polycythemic winter, compared with polycythemic summer,

fish (Table 1). Blood viscosity changed predictably with changes in Hct in summer fish. Viscosity of blood sampled from fish under resting conditions was significantly higher (P < 0.05, N

=

5) in polycythemic, and lower (P < 0.05, N

=

6) in anaemic, compared with

normocythemic fish, at all shear rates (Figure 5, Table 2B). Blood viscosity was shearrate dependent (Figure 5). Viscosity values for blood sampled before and after Hct adjustment in normocythemic control fish were not different (Table 2A), indicating that the process of blood removal and reinfusion did not affect q. For the polycythemic fish, q was significantly lower at Ucrit, compared with rest, at the lowest shear rate (Table 2B).

Figure 5. Blood viscosity as a function of shear rate and Hct in SW summer rainbow trout. These data were obtained in collaboration with Dr. Mark Graham.

r

I

I

I

I

r

I

I

I

Hct

T

-

I

12.2*1.1% 26.5*3.1% v 42.2*1.9% 0

3

-

I

I

I

I

I

I

I

Shear r a t e ( s

I

I

-1

)

I

The Hct-dependent changes in q had a significant effect on relative Q. Relative Q at rest (i.e., Qrest, where Qrest

=

Q21Q1,and Q2 and Q1 are relative Q after Hct adjustment.

and initial Q, respectively) was significantly higher in anaemic (P poxia and anaemia on the swimming performance o f rainbow trout (Snlmo goirtinrri) J . exp. Blol. 55, 5-11-551. K I C E N I UJ. K , W. A N D JONES.D. R. (1977). The oxygen transport system in trout ( S d m o yairtineri) during sustained exercise. J. exp. Riol. 69. 2-17-2150, KITA.J. A N D ITAZAWA, Y . (1989). Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout. Jap. J. Ichthyol. 36, 38-52. M I L L I G AC. N . L. A N D WOOD.C. M. (1987). Regulation of blood oxygen transport and red cell pHi after exhaustive activity in rainbow trout (Salmo gairtinrri) and starry flounder (Platichthys srrlliirlts). J. rxp. Biol. 133. 263-282. NIEI.SEN. 0 . B. A Y D LYKKEBOE. G . (1992). Changes in plasma and erythrocyte K' during hypercapnia and different grades o f exercise in trout. J. appl. Physiol. 72, 1285-1290. NIKIVAA M., (1986). Control of red cell pH in teleost fishes. Ann. Zool. Fenn. 23, 223-235. N I ISSON.S. (1983). A ~ i t o r ~ o r n l.%'erw c Funcrion in rhe Vrrtebrcztrs. Berlin. Heidelberg, New York: Springer Verlag. 253pp. Nlr.sso~.S. A N D G R O V ED. , J . (1974). Xdrenergic and cholinergic innervation of the spleen of the cod: Gtitilis morhria. E w . J. Phnrrnr~c.28, 135-143. PE.AKSOY. bl. P. A N D STEVENS. E. D . ( 1 9 9 l a ) . Splenectomy impairs aerobic swim performance in trout. Can. J. Zool. 69. 2089 - 2 W . P I ~ A K S OM. N , P. A N D ST.EVENS. E . D . ( 19916). Size and hematological impact of the splenic erythroc)te reservoir in rainbow trout. Oncorhynch~ismykiss. Fish Physiol. Biochrm. 9, -39-50.

P I : K K S. \ ~ .F. A U D KINKF..\D. R. (1989). The role of catecholamines in regulating arterial oxygen content during acute hypercapnic acidosis in rainbotv trout (Snlnro gurrdrrrri). Rrsplr. Physiol. 7 7 , 365-378.

P ~ . R KS. I . F.

. A N D WOOD.C.

hl. (1989). Control and coordination of g3s transfer in fishes. Can. J.

Zool. 67, 2961 -2970.

P K I ~ ~ DI . -. R ~ A, Y D A L LD.. J . . kl.\z~.\cln,hl. \ Y D B O C T I I . I ~R. . R .G . (1986). The role of catecholaminc.~in erythroc)tc pH regulation and oxygen transport in rainbow trout (Salnlo gmrtineri) during exercise. J. exp. Biol. 122. 1-39- 1%. R I S T O Kbl. I , T . A N D L A U R ~ : NP.T (. I % ) . Plasma catecholamines and glucose during moderate c.tercisc in the trout: comparison with bursts of Liolent activity. Expl Biol. 4. 2-16-253. S O I V I OA.. . N I ~ ~ O I KA. . A N D W t s r h ~ ~ \ xK.. (1975). A technique for repeated sampling of the blood of individilal resting fish. J. r x p . Blol. 62. 107-217. S I I . \ I : Y SE. . D . A ~ KANDAI.I.. D D. J . (1967).C h a n ~ e sof gas concentrations in blood and water during moderate swimming activity in rainbou trout. J. r.rp. R i d . 46,3 3 - 3 7 . T H O M A S ,. , P O U P I UJ . . L ~ K K I : B G O .~A- . ~ JDo t i ~ ~ sK.t ~( IYS7). . Effects of graded exercise on blood gas tensions and acid-base charxteristics of rainbou trout. Rrspir. Physiol. 68. 85-97. T L C K ~V. K A. , (1967). A method for ox?gen content and dissociation curves on microliter blood samples. J. uppl. Physiol. 23, 407--110. WEI.I.S,R . M . G . A N D W E B E KR. , E. (IYYO).The spleen in hypouic and exercised rainbow trout. J. t,.rp. Biol. 150. -161-466. WF.LI.S.R . M . G . A N D WEBEK. R. E . (1991). 1s there an optimal haematocrit for rainbow trout, 0nc~orhynch~t.s mykiss (Walbaum)? An interpretation of recent data based on blood viscosity measurements'? J. Fish Biol. 38, 53-65. WOOD,C . M. A N D R A N D A I .D. ~ . .J . ( 1973). The influence of swimming activity on water balance in the rainbow trout (Sulnlo grrirtinrrl). J. cornp. Physiol. 82, 257-276. W o o r m ~ J.~ (1982). ~ . Plasma catecholamines in resting trout. Sulmo gairtinrri Richardson by high pressure liquid chromatography. J. Fish Biol. 21, 429-432.

YAMAMOTO. K. I. (1989). Contraction o f spleen in three perciformes and two tetraodontiformes during severe exercise. Comp. Biochem. Physiol. 9JA. 633-634. YAMAMOTO, K . (1991). Increase of arterial O2 content in exercised yellowtail (Seriola quinq~~eradiata). Comp. Biochem. Physiol. 98A, 43-46, Y-\\IA\IOTO. K . I . , ITAZAWA. Y. A N D KOBAYASHI, H. (1980). Supply of erythrocytes into the circulating blood from the spleen of exercised fish. Comp. Biochem. Physiol. 6 j A . 5-11.

Appendix 2. A summary of haematological parameters in rainbow trout and chinook salmon at rest and changes which occur when swimming to U,,it.

I)A ca~i~iula T r a ~ ~ s o ~flow i i c probe

[)A caliliula Slia~ii-opcratctl. ope11body wall

9 winter (1993)SW

10 winler (199l)SW

12 s1111111ier (1992)s~

winter (1993)FW

5

18 swiiliier ( I99O)FW

Seas011 (ycnr)l:W/SW

oc ("/.I

IIcl

'I'nblc I . I\lootl osygcn carrying capacity o f arterial (A) and

(~01%)

Ca02

MCI IC (g. 1,- I )

blood i n rainbow trout (a) and chinook salnion

[Clb] (g.dL- I)

VCI~OUS (V)

(I)) at

Ucril 55.6 (1.9) N=15 61.6 (2.0) N=IO 54.4 (2.4) N=5 49.7 (3.5) N-8)

51.0 (3.2) N=7 18.2 (3.5) N=7

53.5 (2.1) N=15 54.5 (2.5) N=12 52.9 (2.7) N=9 49.0 (3.6) N=9

55.0 (2.6) N=IO 34.0 (2.7) N=l l

15.3 (0.5) N-IS 15.2 (0.6) N=l l 11.2 (0.8) N=8 11.1 (1.1) N=7

-80% Rest

Rcc

Table 4. A summary of the mavimal swimming performance and oxygen uptake o f rainbow trout ( A ) and chinook salmon (B). A. RAMBOW TROUT weight (9)

length (cm)

Hct (&rid ("/.)

V~~rnax (umolkg Im in)

FW S ham-operated

530 73 8

36 - 41

34.4

FW 5 OC Adjusted Hct

466 (19) N=10

32.9 (1.4) N=9

33.1

SW (Winter 1991)

554 (2)

34.3 (0.5)

28.5

N=9

N=9

SW (Summer 1992) Adjusted Hct

514 (2 7 ) N=8

35.1 (0.6) N=8

30.1

176.3 (12.0) N=7

SW (Winter 1993) DA cannula only

550

34.7

30.7

138

25.6

121

DA and Transonic flow probe

126

DA and Doppler flow probe B.CHIKOOK SALMON Fall 1991 368 LS I (30) N=25

I87 (8.7) N=11

30.3 (0.3) N=25

24.5 (0.9) N= 15

250 (22) N= I0 290 (15.3) N= 10

30.1 (0.3) N=2 l

28.3 ( 1 .O)

N=15

318 (25) N-l l 330' (12.8) N= I0

Winter 1992

LSZ

D A only

e

442 (48, N=7

336 (18) N=8

Numbers in parentheses are +I- S.E.M.

*

represents unoperated fish.

Methods used for catecholamine analysis after Aota and Randall (1 994)

*

LS = exercise-trained at 0.5 bl-sec-I HS = exercise-trained at 1.5 blsec-1 Figures in parentheses represent S.E.M.

HS 1 training group

LS 1 training group

[Noradrenaline] (nrn01.L-1) [Adrenaline] (nmol-L-1)

Table 5. Concentrations of catecholamines (noradrenaline and adrenaline) at UCritin chinook salmon from TR1.

Appendix 3. Cardiac output in swimming rainbow trout.

Cardiac output in swimming rainbow trout, Oncorhynchus mykiss.

Helgi ~horarensen*,Patricia Gallaugher and Anthony P. Farrell

Cardiac output in swimming rainbow trout

Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6

Summary This is the first study to report absolute measurements of cardiac output (Q) with Transonic flow probes during prolonged swimming in a salmonid. Simultaneous measurements of hematocrit and arterial blood oxygen content in seawater-acclimated rainbow trout (Oncorhynchus mykiss) (body mass 40 1- 1025g) at 1

1 "C, indicated that

the fish were in a good physiological condition, while measurements of resting heart rate, arterial blood pressure, and systemic resistance suggested relatively low levels of stress. Maximum Q (Q,,,)

(48.7 mL.min-'.kg-') was reached at 97.3% of the maximum

prolonged swimming speed, and is similar to the Q,

estimated by the Fick principle

during swimming in freshwater rainbow trout (Kiceniuk and Jones, 1977).

em,,was

84% greater than the resting cardiac output (QresJ (26.6 mL.min-'.kg-') and was brought about by a 38% increase in heart rate V^,) and a 25% increase in stroke volume (SV,). Correlation analysis showed that individual variability in Q,

was mainly due to

variability in maximum SVH;maximum fH was relatively invariant among individuals. At rest, SV, andf, were negatively correlated. Individual fish with a high Q, characterized by a low resting fH and a high resting SV,.

were

Introduction

As salmonids increase swimming speed up to the maximum prolonged (= critical) swimming velocity (U,,,,; Hoar and Randall, 1978), the transfer of gasses between the environment and the locomotory muscles increases as a result of a host of concurrent adjustments to the cardio-respiratory system. An important adjustment during swimming in rainbow trout is an increase in Q (Stevens & Randall, 1967a, b; Kiceniuk and Jones, 1977). However, Q was estimated with the Fick principle in these earlier studies and this technique has been criticized in its application to fishes because of inherent errors related to gill blood flow shunts, gill oxygen consumption and skin oxygen uptake (Johansen and Petterson, 198 1; Metcalfe and Butler, 1982; Daxboeck et al. 1982; Randall, 1985). Compounding this technical problem, is the fact that Stevens and Randall, (1967a) only estimated Cw, from measurements of blood Po2. In support of the use of the Fick principle, Randall (1 985) argued that the errors tend to cancel each other out under resting conditions. Furthermore, our view is that the errors may become insignificant when rainbow trout are swimming and skeletal muscle oxygen consumption dominates the overall oxygen consumption of the animal. Given the importance of rainbow trout as a model for studying fish physiology, it seemed important to resolve the issue of the accuracy of previous Q measurements in swimming rainbow trout. In this study we used a technique that directly measures Q in rainbow trout engaged in prolonged swimming. A number of direct and indirect techniques have been used to measure Qrestin rainbow

trout, including flow probes (Wood and Shelton, 1980a,b; Xu and Olson, 1993; Garnperl, Pinder and Boutilier, 1994), dye dilution techniques (Neuman, Holeton and Heisler, 1983; Barron, Tarr and Hayton, 1987) and the Fick principle (Holeton and Randall, 1967; Stevens and Randall, 1967b; Cameron and Davis, 1970; Davis and Cameron 1971; Kiceniuk and Jones, 1977; Neuman, Holeton and Heisler, 1983). The past difficulties in

measuring QreStin rainbow trout are reflected in the very large range reported for erest, from 16 to 65 m ~ . m i n - .kg-l 1 (see Farrell and Jones, 1992). However, comparisons between studies are further complicated because of differences in the extent of the surgery, the state of the fish, and the experimental temperatures (5-1 8 "C) (Banon, ~m and Hayton, 1987). We were particularly interested in a direct comparison with the data of Kiceniuk and Jones (1 977), the most comprehensive data set for swimming rainbow trout, albeit on a small sample size.

Recently, ultrasonic flow probes (i.e., Doppler and Transonic) have been used to continuously measure Q in swimming fish (Axelsson and Nilsson, 1986; Axelsson et a/. 1 989; Axelsson and Fritsche, 199 1 ; Thorarensen et al. 1993; Kolok, Spooner and Farrell, 1993). We chose Transonic flow probes because they give readings of absolute blood flow as well as a zero flow signal, a feature not shared by either the Doppler or the electromagnetic flow probes. Our laboratory has developed techniques to use the Transonic flow probes to measure Q in swimming fish without affecting the rank order of swimming performance within a group of fish (Kolok, Spooner and Farrell, 1993; Kolok and Fmell, 1994). Qresthas been measured in rainbow trout with Transonic flow probes (Xu and Olson, 1993), but in this case, placement of the flow probe on the ventral aorta involved opening the pericardium. Rupturing of the pericardium is known to impair cardiac performance, since vis afionte filling of the heart can only occur if the pericardium is intact (Farrell, Johansen and Graham, 1988). We used a novel method was used in the present experiment to place a Transonic probe on the ventral aorta without opening the pericardium. We report the first absolute measurement of Q during sustained swimming in a salmonid, which are compared with earlier estimates of Q using the Fick principle..

Materials and methods Experimental animals. Rainbow trout, Oncorhynchus mykiss, of both sexes (mean weight 6 10 g, range 40 1- 1025 g) were transported from West Creek Trout Farm, Aldergrove, British Columbia, to the West Vancouver Laboratory (Department of Fisheries and Oceans) where the experiments were performed. The fish were acclimated to seawater (salinity 30 parts per thousand; 9-1 1 OC) for at least two months prior to the experiments which were conducted in November and December, 1992. During the seawater-acclimation period, the fish were kept outdoors in circular tanks, supplied with an ample flow of seawater, and were fed satiation levels of dry pellets (Moore Clarke, British Columbia) once daily. Prior to surgery, the fish were starved for at least 24 h. Surgery. The fish were anaesthetized in a 1 :2,000 solution of 2-phenoxyethanol (Sigma Chemical Co., St. Louis, MO) in seawater, and the anaesthesia was maintained by continuously irrigating the gills with a 1:4,000 solution of 2-phenoxyethanol in seawater. A cannula (PE 50) was inserted into the dorsal aorta (DA) as described by Thorarensen et al. (1993) and allowed measurements of blood pressure (P,,) and sampling of arterial blood. P,, was measured with a LDI5 pressure transducer (Narco, Houston, TX). To measure Q, a Transonic flow probe (Transonic Inc., Ithaca, NY) was placed around the ventral aorta (VA) of the trout, just distal to the bulbus arteriosus. The VA was accessed from the opercular cavity. When the operculum and the gills are folded forward, the VA is readily visible as it runs dorsad immediately below the gills. This site has been used previously for the cannulation of the VA, for ligation of the coronary artery (Farrell

and Steffensen, 1987), and for Doppler blood flow measurements in the VA and the coronary artery (Axelsson and Farrell, 1993). The VA was exposed by gently teasing apart overlying skin and connective tissue, and the flow probe was placed around the VA without rupturing the pericardi~unor obstructing the coronary artery. The flow probe was secured in place with crosswise stitches (3-0 silk suture) that were tied to the probe, and this ensured that the flow probe would not move while the fish swam. The probe leads were anchored with a 1-0 silk suture near the pectoral fin and again anterior to the dorsal fin. TWOsizes of Transonic flow probes were used, depending on the size of the fish; the larger probe was 9 rnm long had a 14.8 mm2 rectangular sensing window and the smaller probe was 8 rnrn long with a 4 mrn2 sensing window. The surgery was completed in less than 30 minutes and the fish were allowed to recover for 18-24 hours before swimming trials commenced.

Experimental procedures Fish were swum in the same swim tunnel (volume 39 L) used by Kiceniuk and Jones (1977). All experiments were performed at 1W1 O C . Prior to a swimming trial, the fish was allowed to habituate to the tunnel for at least 12 h. Q,,,, and P,, at rest were recorded while the water velocity was 5 - 10 body-length s-l (blasec-1) with the fish resting on the bottom of the chamber. An arterial blood sample was also taken for the measurement of resting values for haematocrit (Hct), haemoglobin concentration ([Hb]), Pag, and Cag2. Swimming velocity was then increased to approximately 1b1.s-1 and the same variables were recorded again. Subsequently, swimming speed was increased in steps of 0.25 b1.s-1, each step being maintained for 30 min, or until the fish fatigued. At each step, 10 min after the velocity had been increased, and when Q and P,, had reached a stable level, cardiovascular recordings were sampled for 6 minutes. At approximately

50% U,,,,, blood samples were taken at each velocity increment until the fish reached Ucri,. This minimized the amount of blood removed from the fish. After swimming to U,,, the fish were allowed to recover in the swim-tunnel at A water velocity of 0.25 b1.s-1. After 1 hour of recovery, the cardiovascular variables were recorded and a final arterial blood sample was taken.

Analysis of blood samples. Each blood sample was 1 mL. Some of the blood sample was used for analysis, and the remainder was reinjected into the fish along with enough saline to replace the fluid used for the haematological measurements. A maximum of six blood samples was taken from the fish while they swam, resulting in a total volume of 1-1.5 mL of blood being replaced with saline prior to the final sample. Replacing this amount of blood with saline has a minimal effect on Hct in rainbow trout (Gallaugher, Axelsson and Farrell, 1992). Pao2 was measured with a thennostatted electrode (E5046, Radiometer, Copenhagen), maintained at the experimental temperature and connected to a PM71 unit. CaOz was measured with the method of Tucker (1967). Triplicate samples for Hct were spun in micropipettes (10 pL) for 3 min at 12,000 rpm. Blood [Hb] and plasma [la~:dte]were analyzed with Sigma kits 52544 and 826-UV, respectively. Plasma osmolality was measured in triplicate on 10 pL samples using a calibrated Wescor (5 100) Vapour Pressure Osmometer (Wescor, Logan, Utah). Mean cell [Hb] was calculated as [HbIlHct. The saturation of haemoglobin in arterial blood was calculated as:

where a is the solubility of O2 in plasma (Boutilier, Hemming and Iwama, 1984) and it is assumed that l g of haemoglobin can bind 1.34 mL of oxygen.

Data acquisition and statistical analyses. The signals for P,, and Q were recorded directly on a Grass chart recorder (model 7PCP

B, Grass Instruments, Quincy, MA) and relayed to a computer for storage and on-line processing with Labtech Notebook (Laboratory Technology Corp., MA). At each velocity step, the computer sampled the signals for PDAand Q for 6 minutes at a rate of 5

Hz and calculated heart rate from the flow pulses. The mean values for the 6 min periods were used for analysis. Systemic vascular resistance (Rsys)was calculated from mean PDA and Q at each step ( R,,,

= P,,

I Q), with the assumption that changes in venous pressure

were negligible relative to those in arterial pressure (Farrell, 199 1). Statistical comparisons were performed using the General Linear Models Procedure and the Correlation Procedure in SAS (Version 6, SAS Institute). Data for variables recorded at different swimming velocities were compared using an analysis of variance (ANOVA) with repeated measures. Individual means were compared with Tukey's tests. To examine the variability in the cardiovascular variables, the coefficients of variation (CV) were calculated (CV = 100.standard deviation I mean). Residual correlations (Bennett, 1987) were performed on cardiovascular data. Values of P