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www.elsevier.nlrlocateraqua-online

Blood oxygen transport, rheology and

haematological responses to confinement stress in

diploid and triploid Atlantic salmon, Salmo salar

Joanne Sadler

a,)

, Rufus M.G. Wells

, Patricia M. Pankhurst

,

Ned W. Pankhurst

School of Aquaculture, UniÕersity of Tasmania, Launceston, Tasmania 7250, Australia

School of Biological Sciences, The UniÕersity of Auckland, PriÕate Bag 92019, Auckland, New Zealand

Accepted 20 September 1999

Abstract

Ž .

Diploid and triploid all-female Atlantic salmon Salmo salar smolts were challenged with 2.5 h of confinement stress in aerated seawater. Subsequent stress responses were quantified by

Ž .

plasma cortisol, glucose, and lactate, and by the haematological parameters haematocrit Hct , red

Ž . Ž . Ž .

blood cell count RBCC , mean cell volume MCV , blood haemoglobin concentration Hb , mean

Ž . Ž .

cell haemoglobin MCH , mean cell haemoglobin concentration MCHC , total protein and

Ž .

erythrocyte adenosine triphosphate ATP . The magnitude of stress responses was similar between diploid and triploid smolts. Triploid smolts displayed a higher MCH, but lower Hb than diploid smolts. The blood oxygen affinity of diploid and triploid fish was similar at 158C over the pH range 6.76–7.99 and the isohaemoglobin components were identical. The enlarged triploid erythrocytes showed reduced shear-dependence on blood viscosity at constant Hct and are therefore unlikely to contribute to greater peripheral vascular resistance. The results show that despite having fewer, larger erythrocytes, triploids have very similar oxygen carrying capacity and haematological response to stress as diploids. This suggests that the higher farm mortality reported for triploids in response to stress is not generated by their failure to show respiratory homeostasis in the face of stress.q_{2000 Elsevier Science B.V. All rights reserved.}

Keywords: Atlantic salmon; Haematology; Stress; Triploidy; Oxygen transport; Viscosity

Ž .

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1. Introduction

The induction of triploidy in salmonid fish has become a valuable tool in the Tasmanian aquaculture industry. In Canada, the production of triploid salmonids has been proposed to address concerns regarding the environmental impact of domestic

Ž . Ž .

escapees Benfey, 1999 . All-female triploid Atlantic salmon Salmo salar remain sexually immature, subsequently energy investment in somatic growth is not hindered by

Ž .

metabolic costs of sexual maturation Benfey, 1999 . As a result, somatic growth continues in triploids and flesh quality is maintained over the period when diploid fish are sexually maturing and undergoing associated reduction in flesh quality. In Tasmania, all-female triploid Atlantic salmon are routinely used to extend the annual harvest period

Ž .

and thereby improve commercial returns Jungawalla, 1991 .

In order to accommodate the extra genetic material, triploid fish are composed of a smaller number of larger cells, though outwardly the fish cannot be distinguished from natural diploids. Several researchers have called attention to the theoretical problem of

Ž .

carrying out basic metabolic processes in enlarged cells reviewed by Benfey, 1999 . Diffusion processes and receptor binding chemistry, for example, may be affected by the reduced surface area to volume ratio of enlarged cells.

Fish erythrocytes are nucleated and contain the oxygen-binding protein haemoglobin, which is critical for the delivery of oxygen to metabolising tissues. Triploid salmonids

Ž .

possess fewer and larger erythrocytes Benfey and Sutterlin, 1984; Benfey et al., 1984 ,

Ž .

and thus erythrocyte count and haematocrit Hct measurements are a less useful measure of oxygen carrying capacity than is haemoglobin concentration. Atlantic salmon

Ž .

triploids have lower blood haemoglobin concentration Benfey and Sutterlin, 1984 , although this is not always the case with triploids of other species, including some

Ž .

salmonids Benfey, 1999 .

Additional factors affecting blood oxygen transport which might be influenced by

erythrocyte dimensions are the rheological flow characteristics of blood including

. Ž

viscosity and erythrocyte deformability , and blood oxygen affinity notably regulation

by pH and erythrocyte ATP . These aspects are poorly understood in triploid fish, yet they are likely to be important in regulating oxygen delivery during the stresses imposed by handling and exercise, confinement, and temperature and oxygen perturbations.

It appears that triploid salmonids may be more sensitive to the stresses imposed by handling and seawater transfer during routine farm management, and suffer higher

mortality rates under sub-optimal environmental conditions Johnson et al., 1986; Quillet

and Gaignon, 1990; Jungawalla, 1991; Yamamoto and Iida, 1994a; Ojolick et al., 1995 . Therefore, we examined the above-aspects of the blood oxygen transport system with a view to discerning whether differences in the respiratory component of the stress response might account for higher mortality in triploids. Accordingly, we have subjected

diploid and triploid Atlantic salmon to handling and confinement stress see Pickering,

1992 , and compared haematological variables, and the plasma metabolites glucose and lactate before and after the stress regime. Further, the response of blood oxygen affinity to pH, and the effect of shear rate on blood viscosity, were assessed to evaluate functional differences, if any, between the two groups.

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2. Materials and methods

2.1. Fish production and maintenance

Ž . Ž .

All-female diploid 2N and triploid 3N smolts were produced using standard

Ž .

commercial techniques Johnstone et al., 1991 carried out at the SALTAS Wayatinah Hatchery in Tasmania. At 14 months post-fertilisation, smolts were transferred to the School of Aquaculture, University of Tasmania, where 2N and 3N populations were each maintained in a 2000-l tank with a closed recirculating seawater system at 12.5"0.58C and a stocking density of 15 kg my3. Water quality and nutritional regimen followed commercial protocols. Fish were acclimated to seawater conditions for

Ž .

4 weeks prior to experimentation. Mean body mass "SE, ns20 for 2N and 3N fish was 78.1"11.1 and 76.4"15.6 g, respectively.

2.2. Stress induction

All fish were left undisturbed and fasting for 24 h prior to manipulations s‘rested’

fish . Ten ‘rested’ 2N and 10 ‘rested’ 3N fish were randomly sampled by scoop net from each tank. A further 10 2N and 10 3N fish were exposed to a stress protocol

Ž .

known to elevate plasma cortisol values Pickering, 1992 . These fish were separated according to ploidy status, subjected to handling and confined for 2.5 h, in one of two 20-l containers of aerated seawater, prior to blood sampling. Approximately 500 ml of blood was sampled immediately from the caudal vein and placed on ice in paediatric vials containing K EDTA anticoagulant, and analysed as described below. Ploidy status₂ of each fish sampled was confirmed by determining mean erythrocyte nucleus length

ŽBenfey et al., 1984 ..

2.3. Plasma steroid measurement

Plasma cortisol was measured by radioimmunoassay after extraction with ethyl

Ž .

acetate as described by Pankhurst et al. 1992 . Extraction efficiency, determined as the recovery of3H-labelled steroid extracted with plasma, was 94% and assay values were corrected accordingly. The assay detection limit was 2.4 ng mly1 _{plasma and interassay}

Ž . Ž .

variability %CV , using a pooled internal standard, was 5.4% ns3 .

2.4. Haematology and plasma chemistry

Ž . Ž . Ž .

Haematocrit Hct , haemoglobin Hb , and red blood cell counts RBCC were

Ž .

determined using standard haematological techniques Dacie and Lewis, 1984 with the added step of centrifugation to remove cell debris for Hb determination. Mean cell

Ž . w x Ž .

haemoglobin concentration MCHC was calculated from HbrHctr100 , mean cell

Ž . Ž . w x

volume MCV from HctrRBCC, and mean cell haemoglobin MCH from HbrRBCC. Plasma glucose was estimated using a standard enzymatic test kit based on the

Ž .

hexokinase reaction 15-u.v., Sigma, St. Louis, USA , and plasma lactate using the

Ž .

enzymatic test kit no. 826-u.v. Sigma . Plasma protein was determined using the biuret

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Total cellular RNA was isolated from 50ml blood samples using Trizol reagent Life

Technologies, USA . The single-step RNA isolation method is based on a phenol–

Ž .

guanidine thiocyanate reagent Chomczynski, 1993 . Purified RNA was quantified from the relationship A260 nm of 1.0s40 mg RNA mly1. All samples showed A260:280)1.7 and were therefore judged to be free from protein contamination. Total cellular RNA for each blood sample was divided by RBCC to obtain red cell RNA. The contribution of white cell RNA to total cellular RNA was thought to be negligible.

2.5. Haemoglobin oxygen transport

Functional evaluation of the blood oxygen transport system was carried out by determination of oxygen affinity using pooled blood from five individuals from diploid and triploid populations. Blood was taken from undisturbed fish by rapid caudal venepuncture into a heparinised syringe. Oxygen equilibrium curves were measured

y1 _Ž y1_.

using a series of 50 mmol l Hepes buffers at constant 125 mmol l chloride

Ž . Ž .

concentration Weber, 1992 , and decreasing pH 6.76–7.99 , in a modified tonometric

Ž .

system Wells and Weber, 1989 . Oxygen saturation was determined for each pH level at 158C with a 5-nm bandpass Novaspec II spectrophotometer for at least six points of equilibration, and the affinity coefficient, P , and cooperativity coefficient, n , were50 50

determined by interpolation and slope of Hill plots according to Weber and Wells

Ž1989 . The effect of pH on oxygen transport at various oxygen partial pressures was.

evaluated from the Bohr factor, Ø, sDlog P₅₀rDpH. The Root effect was estimated

Ž .

from the change in saturation P₁₀₀ with decreasing pH using the method of Pelster and

Ž .

Weber 1990 .

Total nucleoside triphosphates were extracted from red blood cells using cold 12% trichloroacetic acid and the extract was analysed using an enzymatic NTP test kit

ŽSigma, no. 366-u.v. . The method does not distinguish ATP from other nucleoside. Ž

triphosphates, but ATP is essentially the only NTP in salmonid erythrocytes Wells and

Weber, 1990 .

Isoelectric focusing of haemoglobin components from 2N and 3N populations was

Ž .

carried out on lysates prepared for the PhastGele _{Pharmacia system in the pH range}

3–9. Gels were unstained in order to eliminate non-haemoglobin proteins from analysis and immediately scanned for analysis.

2.6. BloodÕiscosity

Viscosity was measured in 500ml aliquots of erythrocyte suspensions from 2N and

3N fish using a cone-plate viscometer with a cone angle of 88 model LVTD CPr11,

. y1

Brookfield Engineering Laboratories, USA , capable of shear rates from 2.3–450 s . The temperature range of the sample cup was regulated to 15.0"0.28C using a circulating water bath. Calibration of the viscometer was checked with Brookfield standards, and found to be within specification. The technical problem of erythrocyte aggregation at low shear rates arises from the bridging effects of large plasma protein

Ž .

molecules such as fibrinogen and globulins Fletcher and Haedrich, 1987 and was

Ž .

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physio-logical saline. The viscosity of blood samples with adjusted Hct values was also measured. Erythrocytes pooled from five fish in each ploidy group were separated from

Ž .

plasma by light centrifugation 1200=g and resuspended in Cortland’s solution to provide a range of Hct values.

2.7. Statistical analysis

Data from blood haematology experiments were tested for normality and equal variances within treatments using Bartlet’s test prior to analysis. A two-way ANOVA

Ž .

analysis Ps0.05 was used to determine the effects of ploidy and confinement stress on respective parameters. We acknowledge the experimental design did not preclude possible tank effects, however, precluding fluctuation of parameters due to photoperiod, feeding status and disturbance was of greater concern. A Welch ANOVA was also used

Ž . Ž .

for data with unequal variances JMP 3.1 Software . Student’s t-test Ps0.05 was used to compare blood viscosity and corresponding Hct values between diploids and

Ž .

triploids Excel Software . A three-way ANOVA was used to compare the effects of ploidy, Hct and shear rate on viscosity.

3. Results

3.1. Plasma steroid measurement

Plasma cortisol levels of all fish exposed to 2.5 h of confinement stress were

Ž .

increased approximately two-fold above ‘rested’ values P-0.05, Table 1 . Thus, both diploid and triploid smolts displayed a significant primary endocrine response to stress,

Ž .

but there was no difference in the response between 2N and 3N fish P)0.05 . These results indicate it is unlikely that there was any tank effect within treatments.

Table 1

Haematology and plasma metabolites in diploid and triploid Atlantic salmon subjected to confinement stress. Data are expressed as means"SD, ns10

All-female diploids All-female triploids

‘Rested’ Stressed ‘Rested’ Stressed

Ž .

Hct % 32.5"3.5 35.7"6.9 35.4"6.3 33.5"4.9

y1 a a

Ž .

Hb g l 83.7"17.3 79.7"9.6 68.8"7.4 71.9"8.7

6 y1 a a

Ž .

RBCC 10 l 0.94"0.15 0.97"0.12 0.69"0.13 0.66"0.09 y1

Ž .

MCHC g l 259.9"57.8 227.5"29.7 201.8"49.4 214.3"15.2

a a

Ž .

MCV fl 35.4"6.7 36.9"6.4 47.8"6.1 51.1"4.0

a a

Ž .

MCH pg 90.8"23.2 82.8"10.0 102.6"24.9 111.0"12.9

b b

Ž .

Glucose mM 3.3"0.7 4.7"1.2 4.0"1.1 5.3"1.5

b b

Ž .

Lactate mM 0.89"0.13 3.67"0.79 0.79"0.14 3.73"1.1

Ž .

Protein g% 2.37"0.92 1.83"0.72 1.93"0.62 2.40"0.53

y1 b b

Ž .

Cortisol ng ml 24.6"6.1 75.5"11.6 32.6"5.0 74.3"15.7

Ž .

Triploids significantly different from diploids P-0.05 .

Ž .

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3.2. Haematology and plasma chemistry

Comparisons of haematological measurements, plasma glucose, lactate, and protein for ‘rested’ and stressed 2N and 3N fish are summarised in Table 1. Triploid fish had

w x

lower RBCC, greater MCV, lower Hb and higher MCH than did diploid fish

ŽP-0.05 . Confinement stress did not result in any changes to the haematological.

Ž .

profile within ploidy groups P)0.05 . Plasma glucose and lactate increased as a result

Ž .

of confinement stress P-0.05 , but no differences were detected between ploidy

Ž .

groups P)0.05 . Total plasma proteins remained constant in all groups. Red blood

cell RNA values were similar for both diploid and triploid ‘rested’ fish mean"SDs

y1 _.

36.8"5.9 and 36.1"2.0 pg RBC , respectively .

3.3. Haemoglobin oxygen transport

Functional studies failed to reveal differences between 2N and 3N fish. The effect of

Ž . Ž .

pH on haemoglobin–oxygen affinity P50 , Hill’s cooperativity coefficient n50 , and

Ž .

Fig. 1. Blood haemoglobin–oxygen binding data at 158C for diploid and triploid S. salar smolts showing a

Ž . Ž . Ž . Ž .

the pH-dependence of oxygen affinity P50, b cooperativity coefficient n50, and c Root effect. Data for

Ž .a and b are from blood pooled from five fish in each group and each point is the average of duplicateŽ . Ž .

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Ž . Ž .

the Root effect P100 are shown in Fig. 1, where the Bohr factors Dlog P50rDpH

Ž . Ž . Ž . Ž .

werey0.48 2N andy0.40 3N , and n50 max was 2.3 2N and 2.4 3N . In addition, whole blood ATP, the principal allosteric regulator of haemoglobin–oxygen affinity in

Ž . Ž

salmonids Wells and Weber, 1990 , was similar for both 2N and 3N fish mean"SD

y1 _.

s1.78"0.08 and 1.58"0.37mmol l , respectively .

Isoelectric focusing revealed four equivalent bands for both 2N and 3N fish and thus the expression of isohaemoglobin components was identical.

3.4. BloodÕiscosity

Viscosity was strongly dependent on Hct with 3N salmon erythrocytes showing lower

Ž .

viscosity at high Hcts Fig. 2 . The effect was most marked at low shear rates. Viscosity readings could not be obtained at high shear rates for the highest Hct samples.

Fig. 2. Dependence of viscosity on shear rate and Hct for erythrocyte suspensions at 158C from diploid and triploid S. salar smolts. Blood sample for each ploidy group pooled from five fish.

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4. Discussion 4.1. Haematology

Atlantic salmon subjected to confinement stress showed elevated levels of plasma cortisol, and the magnitude of the increase was similar in both diploid and triploid fish.

Ž .

Our results are in close agreement with those of Biron and Benfey 1994 obtained

Ž .

following acute handling stress in diploid and triploid brook trout SalÕelinus fontinalis ,

and confirm that the primary endocrine response to stress in the triploid fish follows the

typical salmonid response Pickering, 1992; Mazur and Iwama, 1993; Pankhurst and

Van Der Kraak, 1997 .

The haematological profile of fewer, and larger, erythrocytes in the triploid salmon is

also consistent with findings for other triploid species Benfey and Sutterlin, 1984; Graham et al., 1985; Small and Randall, 1989; Biron and Benfey, 1994; Yamamoto and

Iida, 1994a,b . The effect of triploidy on haemoglobin concentration, a direct measure of oxygen carrying capacity of the blood, appears equivocal and varies between species

Žsee Benfey, 1999 . Nonetheless, our finding of reduced Hb in triploid salmon is. w x Ž

consistent with earlier observations from S. salar Benfey and Sutterlin, 1984; Graham

. Ž

et al., 1985 and other salmonids Small and Randall, 1989; Yamamoto and Iida,

1994b . Given the similarity in cortisol responses, but important differences in erythro-cytes between the ploidy groups, we were surprised to find no significant haematological changes following confinement stress. In the only other study of biochemical and

Ž .

physiological responses to confinement stress in triploid fish, Biron and Benfey 1994 found no change in Hct, although other haematological characteristics were not reported.

Ž .

By contrast, Virtanen et al. 1990 reported a markedly greater increase in Hct, and reduction in MCHC, from triploid rainbow trout forced to swim in a flume. These responses to exercise are well-described in salmonids and are adrenergically mediated, resulting in splenic contraction, and activation of an erythrocyte surface proton exchange

Ž .

causing the cells to swell Wells and Weber, 1990, 1991; Randall and Perry, 1992 . The relative contributions of the initial and rapid adrenergic flush, and the slower, more persistent cortisol response are complex and depend on the nature and duration of stress imposed. The responses are qualitatively and quantitatively different in confined fish and

Ž .

fish swimming freely Lowe and Wells, 1996 . We would therefore anticipate that adrenergic responses are fully manifested in ‘rested’ fish acutely sampled, and that cortisol effects will persist beyond the restoration of baseline catecholamine levels

Ž_{Gamperl et al., 1994 . Further, there occur complex interactions between the two}.

endocrine responses at the level of the erythrocyte which appear to regulate oxygen

Ž .

transport in response to either acute or chronic stress Perry and Reid, 1993 . Accord-ingly, it is most likely the corticosteroid rather than the adrenergic stress response which has been evaluated in our study and is relevant to fish husbandry protocols.

4.2. Plasma metabolites

The increase in plasma glucose and lactate with handling and confinement stress of both diploid and triploid fish indicated a typical salmonid response, and confirms the

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rise in plasma glucose for triploid brook trout under confinement stress Biron and

Benfey, 1994 . The persistence of altered states of glucose, lactate, and protein, and recovery rates following stress are well-defined indicators of the robustness of the stress

Ž .

response Barton, 1997; Morgan and Iwama, 1997 . This response is an important metabolic indicator of available energy reserves, and healthy rainbow trout may show

Ž .

elevated plasma lactate for up to 24 h post-stress Pankhurst and Dedual, 1994 . Disturbances to plasma protein, however, occur only under nutritional stress or extreme

Ž .

physical disturbance Wood et al., 1983; Wells et al., 1986 , and were not noted for either diploid or triploid salmon in the present study.

Ž .

Given the higher DNA content of triploid cells reviewed by Benfey, 1999 , we were interested to see whether RNA content indicated a significant post-translational activity in the triploid erythrocytes of the Atlantic salmon. Total RNA content in the epaxial

muscle of S. salar appears to be a useful index of somatic growth rate Arndt et al.,

1994 and recent measurements confirm increased production of RNA in muscle cells of

Ž .

triploid rainbow trout, Oncorhynchus mykiss Suresh and Sheehan, 1998 . The similarity of RNA content in 2N and 3N erythrocytes was therefore unexpected. However, protein turnover in erythrocytes is unlikely to match that of muscle cells during phases of rapid growth.

4.3. Blood oxygen transport

Multiple haemoglobin components are functionally heterogeneous in salmonids and

their oxygen binding is regulated by the intracellular concentration of ATP Weber and

Wells, 1989 . Analysis of the haemoglobin components and erythrocyte ATP in Atlantic salmon revealed no differences between diploid and triploid populations. The oxygen transport characteristics of blood from diploid and triploid salmon were similar with respect to the pH-dependence of the oxygen affinity coefficient, P , and cooperativity50

Ž .

coefficient, n50 Fig. 1 . These data indicate that in response to oxygen demand, tissue oxygen unloading is effectively regulated in both groups by the Bohr effect in response to carbon dioxide and lactate loading. Moreover, both groups showed similar

depen-Ž . Ž .

dence of oxygen saturation P100 on pH Root effect allowing for oxygen secretion to

Ž .

the eye Ingermann, 1982 . An earlier observation reporting similarity in P50 for diploid and triploid S. salar was determined at a single pH value above the physiological range

Ž_{Graham et al., 1985 .}.

Accordingly, the responses of the oxygen transport system in triploids to stresses induced by exercise, anaesthesia, acute or chronic hypoxia, and hypercapnia can be compensated by allosteric phosphate regulation, and the Bohr effect, as is the case in

diploid salmonids Soivio et al., 1980; Bushnell et al., 1984; Milligan and Wood, 1987;

Weber and Wells, 1989 .

Triploid erythrocytes are greater in length and width than those of diploids but not in

Ž .

depth Benfey, 1999 , hence, it is likely that oxygen diffusion across the erythrocyte surface is compromised neither in the gills nor the tissues. Nevertheless, in a different experiment, a high incidence of branchial abnormalities was observed in the triploid

population sampled in the present study Sadler, J., Pankhurst, P. and King, H.,

. Ž

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al., 1987; King and Lee, 1993; McGeachy et al., 1996; O’Flynn et al., 1997 and branchial abnormalities might be expected to result in compensation in haematological and oxygen transport characteristics.

4.4. BloodÕiscosity

Blood viscosity contributes to vascular resistance and hence is a determinant of the cardiac output required to generate adequate peripheral blood circulation and oxygen delivery to tissues. Fish erythrocytes in suspension show complex, non-Newtonian

behaviour thus, viscosity decreases with increasing shear rate and reducing Hct Graham

and Fletcher, 1985; Fletcher and Haedrich, 1987 . Moreover, fish display a remarkable interspecies diversity in erythrocyte dimensions which is reflected in their rheological behaviour such that smaller erythrocytes tend to show less shear-dependence on

viscosity Wells and Forster, 1989; Wells and Baldwin, 1990; Baldwin and Wells,

1990 . However, erythrocyte size is not constant in salmonids, and adrenergically stress-mediated increases in MCV are thought to modulate haemoglobin–oxygen affinity

Ž .

and lower erythrocyte viscosity Wells and Weber, 1991; Wells et al., 1991 . Further, erythrocytic deformability in rainbow trout increases with adrenergic swelling during

Ž .

exercise and hypoxia Hughes and Kikuchi, 1984 . Confinement stress was shown to

Ž .

reduce blood viscosity in a marine teleost Pankhurst et al., 1992 . The larger erythro-cytes in triploid Atlantic salmon appear to parallel these effects and the most marked

effects of triploid cell viscosity occurred at low shear rates equivalent to blood flow in

vivo , and higher Hct. Aside from shear dependence, the lower viscosity of triploid compared to diploid cells at constant Hct is in part the consequence of the colligative

Ž .

nature dependence on particle number of viscosity. Given that blood vessel diameters

Ž .

do not appear affected by ploidy status Benfey, 1999 , the improved shear dependence of larger triploid erythrocytes should not result in increased vascular resistance in capillary beds or in the branchial lamellae.

4.5. Conclusion

Despite having fewer and enlarged erythrocytes, all-female triploid Atlantic salmon showed only slightly reduced oxygen carrying capacity and haematological responses to a confinement stress challenge were similar to diploids. The stress response, as indicated by plasma cortisol, glucose, and lactate concentrations, was equivalent for both groups. Further, haemoglobin–oxygen affinity, Bohr and Root effects, and the isohaemoglobin pattern were similar for both diploids and triploids. The larger triploid erythrocytes, however, showed lower shear dependence on blood viscosity, and thus oxygen transport is unlikely to be compromised. The similarity of the haematological response to stress in diploid and triploid fish suggests that the higher mortality reported for triploids reared under farm conditions is not generated by their failure to show respiratory homeostasis in the face of stress. The greater heterozygosity displayed by triploid salmonids

ŽAllendorf and Leary, 1984 may result in different responses to environmental perturba-. Ž

tions, although there is little evidence for this Oliva-Teles and Kaushik, 1987, 1990a,b; Yamamoto and Iida, 1994a; McCarthy et al., 1996; Stillwell and Benfey, 1996a,b,

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Acknowledgements

This research was funded by an APAIR grant awarded to P.M. Pankhurst, ARC infrastructure funding allocated to N.W. Pankhurst and was supported by Salmon

Ž .

Enterprises of Tasmania SALTAS , Wayatinah, Tasmania. We thank Polly Hilder for conducting RIAs and for technical assistance in fish husbandry, Mark Hilder for technical assistance in fish husbandry, and the staff of SALTAS for their assistance and provision of the smolts.

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4. Discussion

4.1. Haematology

Atlantic salmon subjected to confinement stress showed elevated levels of plasma

cortisol, and the magnitude of the increase was similar in both diploid and triploid fish.

Ž

.

Our results are in close agreement with those of Biron and Benfey 1994 obtained

Ž

.

following acute handling stress in diploid and triploid brook trout Sal

Õ

elinus fontinalis ,

and confirm that the primary endocrine response to stress in the triploid fish follows the

Ž

typical salmonid response Pickering, 1992; Mazur and Iwama, 1993; Pankhurst and

.

Van Der Kraak, 1997 .

The haematological profile of fewer, and larger, erythrocytes in the triploid salmon is

Ž

also consistent with findings for other triploid species Benfey and Sutterlin, 1984;

Graham et al., 1985; Small and Randall, 1989; Biron and Benfey, 1994; Yamamoto and

.

Iida, 1994a,b . The effect of triploidy on haemoglobin concentration, a direct measure of

oxygen carrying capacity of the blood, appears equivocal and varies between species

Ž

see Benfey, 1999 . Nonetheless, our finding of reduced Hb in triploid salmon is

.

w

x

Ž

consistent with earlier observations from S. salar Benfey and Sutterlin, 1984; Graham

.

Ž

et al., 1985 and other salmonids Small and Randall, 1989; Yamamoto and Iida,

.

1994b . Given the similarity in cortisol responses, but important differences in

erythro-cytes between the ploidy groups, we were surprised to find no significant haematological

changes following confinement stress. In the only other study of biochemical and

Ž

.

physiological responses to confinement stress in triploid fish, Biron and Benfey 1994

found no change in Hct, although other haematological characteristics were not reported.

Ž

.

By contrast, Virtanen et al. 1990 reported a markedly greater increase in Hct, and

reduction in MCHC, from triploid rainbow trout forced to swim in a flume. These

responses to exercise are well-described in salmonids and are adrenergically mediated,

resulting in splenic contraction, and activation of an erythrocyte surface proton exchange

Ž

.

causing the cells to swell Wells and Weber, 1990, 1991; Randall and Perry, 1992 . The

relative contributions of the initial and rapid adrenergic flush, and the slower, more

persistent cortisol response are complex and depend on the nature and duration of stress

imposed. The responses are qualitatively and quantitatively different in confined fish and

Ž

.

fish swimming freely Lowe and Wells, 1996 . We would therefore anticipate that

adrenergic responses are fully manifested in ‘rested’ fish acutely sampled, and that

cortisol effects will persist beyond the restoration of baseline catecholamine levels

Ž

_{Gamperl et al., 1994 . Further, there occur complex interactions between the two}

.

endocrine responses at the level of the erythrocyte which appear to regulate oxygen

Ž

.

transport in response to either acute or chronic stress Perry and Reid, 1993 .

Accord-ingly, it is most likely the corticosteroid rather than the adrenergic stress response which

has been evaluated in our study and is relevant to fish husbandry protocols.

4.2. Plasma metabolites

The increase in plasma glucose and lactate with handling and confinement stress of

both diploid and triploid fish indicated a typical salmonid response, and confirms the

(2)

Ž

rise in plasma glucose for triploid brook trout under confinement stress Biron and

.

Benfey, 1994 . The persistence of altered states of glucose, lactate, and protein, and

recovery rates following stress are well-defined indicators of the robustness of the stress

Ž

.

response Barton, 1997; Morgan and Iwama, 1997 . This response is an important

metabolic indicator of available energy reserves, and healthy rainbow trout may show

Ž

.

elevated plasma lactate for up to 24 h post-stress

Pankhurst and Dedual, 1994 .

Disturbances to plasma protein, however, occur only under nutritional stress or extreme

Ž

.

physical disturbance Wood et al., 1983; Wells et al., 1986 , and were not noted for

either diploid or triploid salmon in the present study.

Ž

.

Given the higher DNA content of triploid cells reviewed by Benfey, 1999 , we were

interested to see whether RNA content indicated a significant post-translational activity

in the triploid erythrocytes of the Atlantic salmon. Total RNA content in the epaxial

Ž

muscle of S. salar appears to be a useful index of somatic growth rate Arndt et al.,

.

1994 and recent measurements confirm increased production of RNA in muscle cells of

Ž

.

triploid rainbow trout, Oncorhynchus mykiss Suresh and Sheehan, 1998 . The similarity

of RNA content in 2N and 3N erythrocytes was therefore unexpected. However, protein

turnover in erythrocytes is unlikely to match that of muscle cells during phases of rapid

growth.

4.3. Blood oxygen transport

Multiple haemoglobin components are functionally heterogeneous in salmonids and

Ž

their oxygen binding is regulated by the intracellular concentration of ATP Weber and

.

Wells, 1989 . Analysis of the haemoglobin components and erythrocyte ATP in Atlantic

salmon revealed no differences between diploid and triploid populations. The oxygen

transport characteristics of blood from diploid and triploid salmon were similar with

respect to the pH-dependence of the oxygen affinity coefficient, P , and cooperativity

Ž

.

coefficient, n

Fig. 1 . These data indicate that in response to oxygen demand, tissue

oxygen unloading is effectively regulated in both groups by the Bohr effect in response

to carbon dioxide and lactate loading. Moreover, both groups showed similar

depen-Ž

.

Ž

.

dence of oxygen saturation P

100

on pH Root effect allowing for oxygen secretion to

Ž

.

the eye Ingermann, 1982 . An earlier observation reporting similarity in P

for diploid

and triploid S. salar was determined at a single pH value above the physiological range

Ž

_{Graham et al., 1985 .}

.

Accordingly, the responses of the oxygen transport system in triploids to stresses

induced by exercise, anaesthesia, acute or chronic hypoxia, and hypercapnia can be

compensated by allosteric phosphate regulation, and the Bohr effect, as is the case in

Ž

diploid salmonids Soivio et al., 1980; Bushnell et al., 1984; Milligan and Wood, 1987;

.

Weber and Wells, 1989 .

Triploid erythrocytes are greater in length and width than those of diploids but not in

Ž

.

depth Benfey, 1999 , hence, it is likely that oxygen diffusion across the erythrocyte

surface is compromised neither in the gills nor the tissues. Nevertheless, in a different

experiment, a high incidence of branchial abnormalities was observed in the triploid

Ž

population sampled in the present study

Sadler, J., Pankhurst, P. and King, H.,

.

Ž

(3)

.

al., 1987; King and Lee, 1993; McGeachy et al., 1996; O’Flynn et al., 1997 and

branchial abnormalities might be expected to result in compensation in haematological

and oxygen transport characteristics.

4.4. Blood

Õ

iscosity

Blood viscosity contributes to vascular resistance and hence is a determinant of the

cardiac output required to generate adequate peripheral blood circulation and oxygen

delivery to tissues. Fish erythrocytes in suspension show complex, non-Newtonian

Ž

behaviour thus, viscosity decreases with increasing shear rate and reducing Hct Graham

.

and Fletcher, 1985; Fletcher and Haedrich, 1987 . Moreover, fish display a remarkable

interspecies diversity in erythrocyte dimensions which is reflected in their rheological

behaviour such that smaller erythrocytes tend to show less shear-dependence on

Ž

viscosity Wells and Forster, 1989; Wells and Baldwin, 1990; Baldwin and Wells,

.

1990 . However, erythrocyte size is not constant in salmonids, and adrenergically

stress-mediated increases in MCV are thought to modulate haemoglobin–oxygen affinity

Ž

.

and lower erythrocyte viscosity Wells and Weber, 1991; Wells et al., 1991 . Further,

erythrocytic deformability in rainbow trout increases with adrenergic swelling during

Ž

.

exercise and hypoxia Hughes and Kikuchi, 1984 . Confinement stress was shown to

Ž

.

reduce blood viscosity in a marine teleost Pankhurst et al., 1992 . The larger

erythro-cytes in triploid Atlantic salmon appear to parallel these effects and the most marked

Ž

effects of triploid cell viscosity occurred at low shear rates equivalent to blood flow in

.

vivo , and higher Hct. Aside from shear dependence, the lower viscosity of triploid

compared to diploid cells at constant Hct is in part the consequence of the colligative

Ž

.

nature dependence on particle number of viscosity. Given that blood vessel diameters

Ž

.

do not appear affected by ploidy status Benfey, 1999 , the improved shear dependence

of larger triploid erythrocytes should not result in increased vascular resistance in

capillary beds or in the branchial lamellae.

4.5. Conclusion

Despite having fewer and enlarged erythrocytes, all-female triploid Atlantic salmon

showed only slightly reduced oxygen carrying capacity and haematological responses to

a confinement stress challenge were similar to diploids. The stress response, as indicated

by plasma cortisol, glucose, and lactate concentrations, was equivalent for both groups.

Further, haemoglobin–oxygen affinity, Bohr and Root effects, and the isohaemoglobin

pattern were similar for both diploids and triploids. The larger triploid erythrocytes,

however, showed lower shear dependence on blood viscosity, and thus oxygen transport

is unlikely to be compromised. The similarity of the haematological response to stress in

diploid and triploid fish suggests that the higher mortality reported for triploids reared

under farm conditions is not generated by their failure to show respiratory homeostasis

in the face of stress. The greater heterozygosity displayed by triploid salmonids

Ž

Allendorf and Leary, 1984 may result in different responses to environmental perturba-

.

Ž

tions, although there is little evidence for this Oliva-Teles and Kaushik, 1987, 1990a,b;

Yamamoto and Iida, 1994a; McCarthy et al., 1996; Stillwell and Benfey, 1996a,b,

.

(4)

Acknowledgements

This research was funded by an APAIR grant awarded to P.M. Pankhurst, ARC

infrastructure funding allocated to N.W. Pankhurst and was supported by Salmon

Ž

.

Enterprises of Tasmania SALTAS , Wayatinah, Tasmania. We thank Polly Hilder for

conducting RIAs and for technical assistance in fish husbandry, Mark Hilder for

technical assistance in fish husbandry, and the staff of SALTAS for their assistance and

provision of the smolts.

References