- Email: [email protected]
Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar
Aquaculture 184 Ž2000. 349–361 www.elsevier.nlrlocateraqua-online
Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar Joanne Sadler b
a,)
, Rufus M.G. Wells b, Patricia M. Pankhurst a , Ned W. Pankhurst a
a 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 Diploid and triploid all-female Atlantic salmon Ž Salmo salar . smolts were challenged with 2.5 h of confinement stress in aerated seawater. Subsequent stress responses were quantified by plasma cortisol, glucose, and lactate, and by the haematological parameters haematocrit ŽHct., red blood cell count ŽRBCC., mean cell volume ŽMCV., blood haemoglobin concentration ŽHb., mean cell haemoglobin ŽMCH., mean cell haemoglobin concentration ŽMCHC., total protein and erythrocyte adenosine triphosphate ŽATP.. 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
)
Corresponding author. Tel.: q61-363-2438-62; fax: q61-363-2438-04; e-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 2 1 - X
350
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 escapees ŽBenfey, 1999.. All-female triploid Atlantic salmon Ž Salmo salar . remain sexually immature, subsequently energy investment in somatic growth is not hindered by metabolic costs of sexual maturation ŽBenfey, 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 and thereby improve commercial returns ŽJungawalla, 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 carrying out basic metabolic processes in enlarged cells Žreviewed 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 possess fewer and larger erythrocytes ŽBenfey and Sutterlin, 1984; Benfey et al., 1984., and thus erythrocyte count and haematocrit ŽHct. measurements are a less useful measure of oxygen carrying capacity than is haemoglobin concentration. Atlantic salmon triploids have lower blood haemoglobin concentration ŽBenfey and Sutterlin, 1984., although this is not always the case with triploids of other species, including some salmonids ŽBenfey, 1999.. Additional factors affecting blood oxygen transport which might be influenced by erythrocyte dimensions are the rheological flow characteristics of blood Žincluding viscosity and erythrocyte deformability., and blood oxygen affinity Žnotably 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 ŽJohnson 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 Žsee 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.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
351
2. Materials and methods 2.1. Fish production and maintenance All-female diploid Ž2N. and triploid Ž3N. smolts were produced using standard commercial techniques ŽJohnstone 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 4 weeks prior to experimentation. Mean body mass Ž"SE, n s 20. 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 Žs‘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 known to elevate plasma cortisol values ŽPickering, 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 2 EDTA anticoagulant, and analysed as described below. Ploidy status 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 acetate as described by Pankhurst et al. Ž1992.. Extraction efficiency, determined as the recovery of 3 H-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 variability Ž%CV., using a pooled internal standard, was 5.4% Ž n s 3.. 2.4. Haematology and plasma chemistry Haematocrit ŽHct., haemoglobin ŽHb., and red blood cell counts ŽRBCC. were determined using standard haematological techniques ŽDacie and Lewis, 1984. with the added step of centrifugation to remove cell debris for Hb determination. Mean cell haemoglobin concentration ŽMCHC. was calculated from wHbxrŽHctr100., mean cell volume ŽMCV. from HctrRBCC, and mean cell haemoglobin ŽMCH. from wHbxrRBCC. Plasma glucose was estimated using a standard enzymatic test kit based on the hexokinase reaction Ž15-u.v., Sigma, St. Louis, USA., and plasma lactate using the enzymatic test kit no. 826-u.v. ŽSigma.. Plasma protein was determined using the biuret reaction ŽSigma test kit no. 541.1. with a certified albuminrglobulin standard.
352
J. Sadler et al.r Aquaculture 184 (2000) 349–361
Total cellular RNA was isolated from 50 ml blood samples using Trizol reagent ŽLife Technologies, USA.. The single-step RNA isolation method is based on a phenol– guanidine thiocyanate reagent ŽChomczynski, 1993.. Purified RNA was quantified from the relationship A 260 nm of 1.0 s 40 mg RNA mly1 . All samples showed A 260:280 ) 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 using a series of 50 mmol ly1 Hepes buffers at constant Ž125 mmol ly1 . chloride concentration ŽWeber, 1992., and decreasing pH Ž6.76–7.99., in a modified tonometric system ŽWells 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, P50 , and cooperativity coefficient, n 50 , were 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, s Dlog P50 rDpH. The Root effect was estimated from the change in saturation Ž P100 . with decreasing pH using the method of Pelster and Weber Ž1990.. 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 triphosphates, but ATP is essentially the only NTP in salmonid erythrocytes ŽWells and Weber, 1990.. Isoelectric focusing of haemoglobin components from 2N and 3N populations was carried out on lysates prepared for the PhastGele ŽPharmacia. 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. Blood Õiscosity Viscosity was measured in 500 ml aliquots of erythrocyte suspensions from 2N and 3N fish using a cone-plate viscometer with a cone angle of 88 Žmodel LVTD CPr11, Brookfield Engineering Laboratories, USA., capable of shear rates from 2.3–450 sy1 . 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 molecules such as fibrinogen and globulins ŽFletcher and Haedrich, 1987. and was avoided by resuspending erythrocytes in Cortland’s solution ŽWolf, 1963., a physio-
J. Sadler et al.r Aquaculture 184 (2000) 349–361
353
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 plasma by light centrifugation Ž1200 = 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 analysis Ž P s 0.05. 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 for data with unequal variances ŽJMP 3.1 Software.. Student’s t-test Ž P s 0.05. was used to compare blood viscosity and corresponding Hct values between diploids and triploids ŽExcel 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 increased approximately two-fold above ‘rested’ values Ž P - 0.05, Table 1.. Thus, both diploid and triploid smolts displayed a significant primary endocrine response to stress, but there was no difference in the response between 2N and 3N fish Ž 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
Hct Ž%. Hb Žg ly1 . RBCC Ž10 6 ly1 . MCHC Žg ly1 . MCV Žfl. MCH Žpg. Glucose ŽmM. Lactate ŽmM. Protein Žg%. Cortisol Žng mly1 . a b
All-female triploids
‘Rested’
Stressed
‘Rested’
Stressed
32.5"3.5 83.7"17.3 0.94"0.15 259.9"57.8 35.4"6.7 90.8"23.2 3.3"0.7 0.89"0.13 2.37"0.92 24.6"6.1
35.7"6.9 79.7"9.6 0.97"0.12 227.5"29.7 36.9"6.4 82.8"10.0 4.7"1.2 b 3.67"0.79 b 1.83"0.72 75.5"11.6 b
35.4"6.3 68.8"7.4 a 0.69"0.13 a 201.8"49.4 47.8"6.1a 102.6"24.9 a 4.0"1.1 0.79"0.14 1.93"0.62 32.6"5.0
33.5"4.9 71.9"8.7 a 0.66"0.09 a 214.3"15.2 51.1"4.0 a 111.0"12.9 a 5.3"1.5 b 3.73"1.1b 2.40"0.53 74.3"15.7 b
Triploids significantly different from diploids Ž P - 0.05.. Stressed significantly different from ‘rested’ Ž P - 0.05..
354
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 lower RBCC, greater MCV, lower wHbx and higher MCH than did diploid fish Ž P - 0.05.. Confinement stress did not result in any changes to the haematological profile within ploidy groups Ž P ) 0.05.. Plasma glucose and lactate increased as a result of confinement stress Ž P - 0.05., but no differences were detected between ploidy groups Ž 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 Žmean " SD s 36.8 " 5.9 and 36.1 " 2.0 pg RBCy1 , respectively.. 3.3. Haemoglobin oxygen transport Functional studies failed to reveal differences between 2N and 3N fish. The effect of pH on haemoglobin–oxygen affinity Ž P50 ., Hill’s cooperativity coefficient Ž n 50 ., and
Fig. 1. Blood haemoglobin–oxygen binding data at 158C for diploid and triploid S. salar smolts showing Ža. the pH-dependence of oxygen affinity Ž P50 ., Žb. cooperativity coefficient Ž n 50 ., and Žc. Root effect. Data for Ža. and Žb. are from blood pooled from five fish in each group and each point is the average of duplicate determinations. Data from Žc. are mean values"SD for ns 5.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
355
the Root effect Ž P100 . are shown in Fig. 1, where the Bohr factors Ž Dlog P50rDpH. were y0.48 Ž2N. and y0.40 Ž3N., and n 50 max was 2.3 Ž2N. and 2.4 Ž3N.. In addition, whole blood ATP, the principal allosteric regulator of haemoglobin–oxygen affinity in salmonids ŽWells and Weber, 1990., was similar for both 2N and 3N fish Žmean " SD s 1.78 " 0.08 and 1.58 " 0.37 mmol ly1 , respectively.. Isoelectric focusing revealed four equivalent bands for both 2N and 3N fish and thus the expression of isohaemoglobin components was identical. 3.4. Blood Õiscosity Viscosity was strongly dependent on Hct with 3N salmon erythrocytes showing lower viscosity at high Hcts ŽFig. 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.
356
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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. Our results are in close agreement with those of Biron and Benfey Ž1994. obtained following acute handling stress in diploid and triploid brook trout Ž SalÕelinus fontinalis., and confirm that the primary endocrine response to stress in the triploid fish follows the typical salmonid response ŽPickering, 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 ŽBenfey 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 wHbx in triploid salmon is consistent with earlier observations from S. salar ŽBenfey and Sutterlin, 1984; Graham et al., 1985. and other salmonids ŽSmall and Randall, 1989; Yamamoto and Iida, 1994b.. Given the similarity in cortisol responses, but important differences in erythrocytes between the ploidy groups, we were surprised to find no significant haematological changes following confinement stress. In the only other study of biochemical and physiological responses to confinement stress in triploid fish, Biron and Benfey Ž1994. found no change in Hct, although other haematological characteristics were not reported. By contrast, Virtanen et al. Ž1990. 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 causing the cells to swell ŽWells 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 fish swimming freely ŽLowe 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 ŽGamperl et al., 1994.. Further, there occur complex interactions between the two endocrine responses at the level of the erythrocyte which appear to regulate oxygen transport in response to either acute or chronic stress ŽPerry and Reid, 1993.. Accordingly, 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
J. Sadler et al.r Aquaculture 184 (2000) 349–361
357
rise in plasma glucose for triploid brook trout under confinement stress ŽBiron 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 response ŽBarton, 1997; Morgan and Iwama, 1997.. This response is an important metabolic indicator of available energy reserves, and healthy rainbow trout may show elevated plasma lactate for up to 24 h post-stress ŽPankhurst and Dedual, 1994.. Disturbances to plasma protein, however, occur only under nutritional stress or extreme physical disturbance ŽWood et al., 1983; Wells et al., 1986., and were not noted for either diploid or triploid salmon in the present study. Given the higher DNA content of triploid cells Žreviewed 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 ŽArndt et al., 1994. and recent measurements confirm increased production of RNA in muscle cells of triploid rainbow trout, Oncorhynchus mykiss ŽSuresh 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 ŽWeber 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, P50 , and cooperativity coefficient, n 50 ŽFig. 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 dependence of oxygen saturation Ž P100 . on pH ŽRoot effect. allowing for oxygen secretion to the eye ŽIngermann, 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 ŽGraham 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 ŽSoivio 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 depth ŽBenfey, 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 ŽSadler, J., Pankhurst, P. and King, H., unpublished data.. The occurrence of jaw deformities in older triploid fish ŽSutterlin et
358
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 Õiscosity 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 ŽGraham 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 ŽWells 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 and lower erythrocyte viscosity ŽWells and Weber, 1991; Wells et al., 1991.. Further, erythrocytic deformability in rainbow trout increases with adrenergic swelling during exercise and hypoxia ŽHughes and Kikuchi, 1984.. Confinement stress was shown to reduce blood viscosity in a marine teleost ŽPankhurst et al., 1992.. The larger erythrocytes in triploid Atlantic salmon appear to parallel these effects and the most marked effects of triploid cell viscosity occurred at low shear rates Žequivalent 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 nature Ždependence on particle number. of viscosity. Given that blood vessel diameters do not appear affected by ploidy status ŽBenfey, 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 perturbations, although there is little evidence for this ŽOliva-Teles and Kaushik, 1987, 1990a,b; Yamamoto and Iida, 1994a; McCarthy et al., 1996; Stillwell and Benfey, 1996a,b, 1997..
J. Sadler et al.r Aquaculture 184 (2000) 349–361
359
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 Enterprises of Tasmania ŽSALTAS., 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 Allendorf, F.W., Leary, R.F., 1984. Heterozygosity in gynogenetic diploids and triploids estimated by gene-centromere recombination rates. Aquaculture 43, 413–420. Arndt, S.K.A., Benfey, T.J., Cunjak, R.A., 1994. A comparison of RNA concentrations and ornithine decarboxylase activity in Atlantic salmon Ž Salmo salar . muscle tissue, with respect to specific growth rates and diel variations. Fish Physiol. Biochem. 13, 463–471. Baldwin, J., Wells, R.M.G., 1990. Oxygen transport potential in tropical elasmobranchs from the great barrier reef: relationship between haematology and blood viscosity. J. Exp. Mar. Biol. Ecol. 144, 145–155. Barton, B.A., 1997. Stress in finfish: past, present and future — a historical perspective. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 1–33. Benfey, T.J., 1999. The physiology and behaviour of triploid fishes. Rev. Fish. Sci., in press. Benfey, T.J., Sutterlin, A.M., 1984. The haematology of triploid landlocked Atlantic salmon Ž Salmo salar .. J. Fish Biol. 24, 333–338. Benfey, T.J., Sutterlin, A.M., Thompson, R.J., 1984. Use of erythrocyte measurements to identify triploid salmonids. Can. J. Fish. Aquat. Sci. 41, 980–984. Biron, M., Benfey, T.J., 1994. Cortisol, glucose and hematocrit changes during acute stress, cohort sampling, and the diel cycle in diploid and triploid brook trout Ž SalÕelinus fontinalis Mitchill.. Fish Physiol. Biochem. 13, 153–160. Bushnell, P.G., Steffensen, J.F., Johansen, K., 1984. Oxygen consumption and swimming performance in hypoxia-acclimated rainbow trout Salmo gairdneri. J. Exp. Biol. 113, 225–235. Chomczynski, P., 1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–536. Dacie, J.V., Lewis, S.N., 1984. Practical Haematology, 5th edn. Churchill Livingstone, Edinburgh, 453 pp. Fletcher, G.L., Haedrich, R.T., 1987. Rheological properties of rainbow trout blood. Can. J. Zool. 65, 879–883. Gamperl, A.K., Vijayan, M.M., Boutilier, R.G., 1994. Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout Ž Oncorhynchus mykiss . following black-box confinement and epinephrine injection. J. Fish Biol. 45, 313–324. Graham, M.S., Fletcher, G.L., 1985. On the low viscosity blood of two cold water marine sculpins. J. Comp. Physiol. B 155, 455–459. Graham, M.S., Fletcher, G.L., Benfey, T.J., 1985. Effect of triploidy on blood oxygen content of Atlantic salmon. Aquaculture 50, 133–139. Hughes, G.M., Kikuchi, Y., 1984. Effects of in vivo and in vitro changes in PO 2 on deformability of red blood cells of rainbow trout Ž Salmo gairdneri .. J. Exp. Biol. 111, 253–257. Ingermann, R.L., 1982. Physiological significance of Root effect hemoglobins in trout. Resp. Physiol. 49, 1–10. Johnson, O.W., Dickhoff, W.W., Utter, F.M., 1986. Comparative growth and development of diploid and triploid Coho salmon, Oncorhynchus kisutch. Aquaculture 57, 329–336.
360
J. Sadler et al.r Aquaculture 184 (2000) 349–361
Johnstone, R., McLay, H.A., Walsingham, M.V., 1991. Production and performance of triploid atlantic salmon in Scotland. In: Pepper, V.A. ŽEd.., Proceedings of Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids. Dept. Fisheries and Oceans, St. Johns, Newfoundland, p. 15. Jungawalla, P., 1991. Production of non-maturing Atlantic salmon in Tasmania. In: Pepper, V.A. ŽEd.., Proceedings of the Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids. Dept. Fisheries and Oceans, St. Johns, Newfoundland, pp. 47–71. King, H., Lee, P., 1993. Progress report: jaw deformity and respiratory physiology of triploids. In: Seeking and Solving: Papers from the SALTAS Research and Development Review Seminar, 1993. SALTAS, Wayatinah, Tasmania, pp. 37–44. Lowe, T.E., Wells, R.M.G., 1996. Primary and secondary stress responses to line capture in the blue mao mao. J. Fish Biol. 49, 287–300. Mazur, C.F., Iwama, G.K., 1993. Effect of handling and stocking density on hematocrit, plasma cortisol, and survival in wild and hatchery-reared chinook salmon Ž Oncorhynchus tshawytscha.. Aquaculture 112, 291–299. McCarthy, I.D., Carter, C.G., Houlihan, D.F., Johnstone, R., Mitchell, A.I., 1996. The performance of all-female diploid and triploid Atlantic salmon smolts on transfer together to sea water. J. Fish Biol. 48, 545–548. McGeachy, S.A., O’Flynn, F.M., Benfey, T.J., Friars, G.W., 1996. Seawater performance of triploid Atlantic salmon in New Brunswick. Bull. Aquacult. Assoc. Can. 2, 24–28. Milligan, C.L., Wood, C.M., 1987. Regulation of blood oxygen transport and red cell pHi after exhaustive activity in rainbow trout Ž Salmo gairdneri . and starry flounder Ž Platichthys stellatus .. J. Exp. Biol. 133, 263–282. Morgan, J.D., Iwama, G.K., 1997. Measurements of stressed states in the field. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 247–268. O’Flynn, F.M., McGeachy, S.A., Friars, G.W., Benfey, T.J., Bailey, J.K., 1997. Comparisons of cultured triploid and diploid Atlantic salmon Ž Salmo salar L... ICES J. Mar. Sci. 54, 1160–1165. Ojolick, E.J., Cusak, R., Benfey, T.J., Kerr, S.R., 1995. Survival and growth of all-female diploid and triploid rainbow trout Ž Oncorhynchus mykiss . reared in chronic high temperature. Aquaculture 131, 177–187. Oliva-Teles, A., Kaushik, S.J., 1987. Metabolic utilisation of diets by polyploid rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 88A, 45–47. Oliva-Teles, A., Kaushik, S.J., 1990a. Effect of temperature on utilisation of endogenous energy reserves during embryonic development of diploid and triploid rainbow trout Ž Salmo gairdneri .. Aquaculture 84, 373–382. Oliva-Teles, A., Kaushik, S.J., 1990b. Growth and nutrient utilisation by 0q and 1q triploid rainbow trout Oncorhynchus mykiss. J. Fish Biol. 37, 125–133. Pankhurst, N.W., Dedual, M., 1994. Effects of capture and recovery on plasma levels of cortisol, lactate and gonadal steroids in a natural population of rainbow trout. J. Fish Biol. 45, 1013–1025. Pankhurst, N.W., Van Der Kraak, G., 1997. Effects of stress on reproduction and growth of fish. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 73–93. Pankhurst, N.W., Wells, R.M.G., Carragher, J.F., 1992. Effects of stress on plasma cortisol levels and blood viscosity in blue mao mao, Scorpis Õiolaceus ŽHutton., a marine teleost. Comp. Biochem. Physiol. 101A, 335–339. Pelster, B., Weber, R.E., 1990. Influence of organic phosphates on the Root effect of multiple fish haemoglobins. J. Exp. Biol. 149, 425–437. Perry, S.F., Reid, S.D., 1993. b-Adrenergic signal transduction in fish: interactive effects of catecholamines and cortisol. Fish Physiol. Biochem. 11, 195–203. Pickering, A.D., 1992. Rainbow trout husbandry: management of the stress response. Aquaculture 100, 125–139. Quillet, E., Gaignon, J.L., 1990. Thermal induction of gynogenesis and triploidy in Atlantic salmon Ž Salmo salar . and their potential interest for aquaculture. Aquaculture 89, 351–364. Randall, D.J., Perry, S.F., 1992. Catecholamines. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, Vol. XII, Part B. Academic Press, London, pp. 255–300.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
361
Small, S.A., Randall, D.J., 1989. Effects of triploidy on the swimming performance of coho salmon Ž Oncorhynchus kisutch.. Can. J. Fish. Aquat. Sci. 46, 243–245. Soivio, A., Nikinmaa, M., Westman, K., 1980. The blood oxygen binding properties of hypoxic Salmo gairdneri. J. Comp. Physiol. 136, 83–87. Stillwell, E.J., Benfey, T.J., 1996a. The swimming performance of diploid and triploid brook trout. Bull. Aquacult. Assoc. Can. 2, 41–43. Stillwell, E.J., Benfey, T.J., 1996b. Hemoglobin level, metabolic rate, opercular abduction rate and swimming efficiency in female triploid brook trout Ž SalÕelinus fontinalis.. Fish Physiol. Biochem. 15, 377–383. Stillwell, E.J., Benfey, T.J., 1997. The critical swimming velocity of diploid and triploid brook trout. J. Fish Biol. 51, 650–653. Suresh, A.V., Sheehan, R.J., 1998. Biochemical and morphological correlates of growth in diploid and triploid rainbow trout. J. Fish Biol. 52, 588–599. Sutterlin, A.M., Holder, J., Benfey, T.J., 1987. Early survival rates and subsequent morphological abnormalities in landlocked, anadramous and hybrid Žlandlocked=anadramous. diploid and triploid Atlantic salmon. Aquaculture 64, 157–164. Virtanen, E., Forsman, L., Sundby, A., 1990. Triploidy decreases the aerobic swimming capacity of rainbow trout Ž Salmo gairdneri .. Comp. Biochem. Physiol. 96A, 117–121. Weber, R.E., 1992. Use of ionic and zwitterionic ŽTrisrBisTris and HEPES. buffers in studies on hemoglobin function. J. Appl. Physiol. 72, 1611–1615. Weber, R.E., Wells, R.M.G., 1989. Hemoglobin structure and function. In: Wood, S.C. ŽEd.., Comparative Pulmonary Physiology, Ch.10, Series: The Lung in Health and Disease ŽExec. editor C. Lenfant.. Marcel Dekker, New York, pp. 279–310. Wells, R.M.G., Baldwin, J., 1990. Oxygen transport potential in tropical reef fish with special reference to blood viscosity and haematocrit. J. Exp. Mar. Biol. Ecol. 141, 131–143. Wells, R.M.G., Forster, M.E., 1989. Dependence of blood viscosity on haematocrit and shear rate in a primitive vertebrate. J. Exp. Biol. 145, 483–487. Wells, R.M.G., Weber, R.E., 1989. The measurement of oxygen affinity in blood and haemoglobin solutions. In: Bridges, C.R., Butler, P.J. ŽEds.., Techniques in Comparative Physiology, Society for Experimental Biology Seminar Series 37. Cambridge Univ. Press, Cambridge, pp. 279–303. Wells, R.M.G., Weber, R.E., 1990. The spleen in hypoxic and exercised rainbow trout. J. Exp. Biol. 150, 461–466. Wells, R.M.G., Weber, R.E., 1991. Is there an optimal haematocrit for trout? An interpretation of recent data based on blood viscosity measurements. J. Fish Biol. 38, 53–65. Wells, R.M.G., McIntyre, R.H., Morgan, A.K., Davie, P.S., 1986. Physiological stress responses in big gamefish after capture: observations on plasma chemistry and blood factors. Comp. Biochem. Physiol. 84A, 565–571. Wells, R.M.G., Davie, P.S., Weber, R.E., 1991. The effect of b-adrenergic stimulation of trout erythrocytes on blood viscosity. Comp. Biochem. Physiol. 100C, 653–655. Wolf, K., 1963. Physiological salines for freshwater teleosts. Prog. Fish-Cult. 25, 135–140. Wood, C.M., Turner, J.D., Graham, M.S., 1983. Why do fish die after severe exercise? J. Fish Biol. 22, 189–201. Yamamoto, A., Iida, T., 1994a. Oxygen consumption and hypoxic tolerance of triploid rainbow trout. Fish Pathol. 29, 245–251. Yamamoto, A., Iida, T., 1994b. Haematological characteristics of triploid rainbow trout. Fish Pathol. 29, 239–243.
Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar Joanne Sadler b
a,)
, Rufus M.G. Wells b, Patricia M. Pankhurst a , Ned W. Pankhurst a
a 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 Diploid and triploid all-female Atlantic salmon Ž Salmo salar . smolts were challenged with 2.5 h of confinement stress in aerated seawater. Subsequent stress responses were quantified by plasma cortisol, glucose, and lactate, and by the haematological parameters haematocrit ŽHct., red blood cell count ŽRBCC., mean cell volume ŽMCV., blood haemoglobin concentration ŽHb., mean cell haemoglobin ŽMCH., mean cell haemoglobin concentration ŽMCHC., total protein and erythrocyte adenosine triphosphate ŽATP.. 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
)
Corresponding author. Tel.: q61-363-2438-62; fax: q61-363-2438-04; e-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 2 1 - X
350
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 escapees ŽBenfey, 1999.. All-female triploid Atlantic salmon Ž Salmo salar . remain sexually immature, subsequently energy investment in somatic growth is not hindered by metabolic costs of sexual maturation ŽBenfey, 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 and thereby improve commercial returns ŽJungawalla, 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 carrying out basic metabolic processes in enlarged cells Žreviewed 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 possess fewer and larger erythrocytes ŽBenfey and Sutterlin, 1984; Benfey et al., 1984., and thus erythrocyte count and haematocrit ŽHct. measurements are a less useful measure of oxygen carrying capacity than is haemoglobin concentration. Atlantic salmon triploids have lower blood haemoglobin concentration ŽBenfey and Sutterlin, 1984., although this is not always the case with triploids of other species, including some salmonids ŽBenfey, 1999.. Additional factors affecting blood oxygen transport which might be influenced by erythrocyte dimensions are the rheological flow characteristics of blood Žincluding viscosity and erythrocyte deformability., and blood oxygen affinity Žnotably 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 ŽJohnson 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 Žsee 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.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
351
2. Materials and methods 2.1. Fish production and maintenance All-female diploid Ž2N. and triploid Ž3N. smolts were produced using standard commercial techniques ŽJohnstone 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 4 weeks prior to experimentation. Mean body mass Ž"SE, n s 20. 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 Žs‘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 known to elevate plasma cortisol values ŽPickering, 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 2 EDTA anticoagulant, and analysed as described below. Ploidy status 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 acetate as described by Pankhurst et al. Ž1992.. Extraction efficiency, determined as the recovery of 3 H-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 variability Ž%CV., using a pooled internal standard, was 5.4% Ž n s 3.. 2.4. Haematology and plasma chemistry Haematocrit ŽHct., haemoglobin ŽHb., and red blood cell counts ŽRBCC. were determined using standard haematological techniques ŽDacie and Lewis, 1984. with the added step of centrifugation to remove cell debris for Hb determination. Mean cell haemoglobin concentration ŽMCHC. was calculated from wHbxrŽHctr100., mean cell volume ŽMCV. from HctrRBCC, and mean cell haemoglobin ŽMCH. from wHbxrRBCC. Plasma glucose was estimated using a standard enzymatic test kit based on the hexokinase reaction Ž15-u.v., Sigma, St. Louis, USA., and plasma lactate using the enzymatic test kit no. 826-u.v. ŽSigma.. Plasma protein was determined using the biuret reaction ŽSigma test kit no. 541.1. with a certified albuminrglobulin standard.
352
J. Sadler et al.r Aquaculture 184 (2000) 349–361
Total cellular RNA was isolated from 50 ml blood samples using Trizol reagent ŽLife Technologies, USA.. The single-step RNA isolation method is based on a phenol– guanidine thiocyanate reagent ŽChomczynski, 1993.. Purified RNA was quantified from the relationship A 260 nm of 1.0 s 40 mg RNA mly1 . All samples showed A 260:280 ) 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 using a series of 50 mmol ly1 Hepes buffers at constant Ž125 mmol ly1 . chloride concentration ŽWeber, 1992., and decreasing pH Ž6.76–7.99., in a modified tonometric system ŽWells 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, P50 , and cooperativity coefficient, n 50 , were 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, s Dlog P50 rDpH. The Root effect was estimated from the change in saturation Ž P100 . with decreasing pH using the method of Pelster and Weber Ž1990.. 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 triphosphates, but ATP is essentially the only NTP in salmonid erythrocytes ŽWells and Weber, 1990.. Isoelectric focusing of haemoglobin components from 2N and 3N populations was carried out on lysates prepared for the PhastGele ŽPharmacia. 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. Blood Õiscosity Viscosity was measured in 500 ml aliquots of erythrocyte suspensions from 2N and 3N fish using a cone-plate viscometer with a cone angle of 88 Žmodel LVTD CPr11, Brookfield Engineering Laboratories, USA., capable of shear rates from 2.3–450 sy1 . 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 molecules such as fibrinogen and globulins ŽFletcher and Haedrich, 1987. and was avoided by resuspending erythrocytes in Cortland’s solution ŽWolf, 1963., a physio-
J. Sadler et al.r Aquaculture 184 (2000) 349–361
353
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 plasma by light centrifugation Ž1200 = 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 analysis Ž P s 0.05. 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 for data with unequal variances ŽJMP 3.1 Software.. Student’s t-test Ž P s 0.05. was used to compare blood viscosity and corresponding Hct values between diploids and triploids ŽExcel 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 increased approximately two-fold above ‘rested’ values Ž P - 0.05, Table 1.. Thus, both diploid and triploid smolts displayed a significant primary endocrine response to stress, but there was no difference in the response between 2N and 3N fish Ž 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
Hct Ž%. Hb Žg ly1 . RBCC Ž10 6 ly1 . MCHC Žg ly1 . MCV Žfl. MCH Žpg. Glucose ŽmM. Lactate ŽmM. Protein Žg%. Cortisol Žng mly1 . a b
All-female triploids
‘Rested’
Stressed
‘Rested’
Stressed
32.5"3.5 83.7"17.3 0.94"0.15 259.9"57.8 35.4"6.7 90.8"23.2 3.3"0.7 0.89"0.13 2.37"0.92 24.6"6.1
35.7"6.9 79.7"9.6 0.97"0.12 227.5"29.7 36.9"6.4 82.8"10.0 4.7"1.2 b 3.67"0.79 b 1.83"0.72 75.5"11.6 b
35.4"6.3 68.8"7.4 a 0.69"0.13 a 201.8"49.4 47.8"6.1a 102.6"24.9 a 4.0"1.1 0.79"0.14 1.93"0.62 32.6"5.0
33.5"4.9 71.9"8.7 a 0.66"0.09 a 214.3"15.2 51.1"4.0 a 111.0"12.9 a 5.3"1.5 b 3.73"1.1b 2.40"0.53 74.3"15.7 b
Triploids significantly different from diploids Ž P - 0.05.. Stressed significantly different from ‘rested’ Ž P - 0.05..
354
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 lower RBCC, greater MCV, lower wHbx and higher MCH than did diploid fish Ž P - 0.05.. Confinement stress did not result in any changes to the haematological profile within ploidy groups Ž P ) 0.05.. Plasma glucose and lactate increased as a result of confinement stress Ž P - 0.05., but no differences were detected between ploidy groups Ž 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 Žmean " SD s 36.8 " 5.9 and 36.1 " 2.0 pg RBCy1 , respectively.. 3.3. Haemoglobin oxygen transport Functional studies failed to reveal differences between 2N and 3N fish. The effect of pH on haemoglobin–oxygen affinity Ž P50 ., Hill’s cooperativity coefficient Ž n 50 ., and
Fig. 1. Blood haemoglobin–oxygen binding data at 158C for diploid and triploid S. salar smolts showing Ža. the pH-dependence of oxygen affinity Ž P50 ., Žb. cooperativity coefficient Ž n 50 ., and Žc. Root effect. Data for Ža. and Žb. are from blood pooled from five fish in each group and each point is the average of duplicate determinations. Data from Žc. are mean values"SD for ns 5.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
355
the Root effect Ž P100 . are shown in Fig. 1, where the Bohr factors Ž Dlog P50rDpH. were y0.48 Ž2N. and y0.40 Ž3N., and n 50 max was 2.3 Ž2N. and 2.4 Ž3N.. In addition, whole blood ATP, the principal allosteric regulator of haemoglobin–oxygen affinity in salmonids ŽWells and Weber, 1990., was similar for both 2N and 3N fish Žmean " SD s 1.78 " 0.08 and 1.58 " 0.37 mmol ly1 , respectively.. Isoelectric focusing revealed four equivalent bands for both 2N and 3N fish and thus the expression of isohaemoglobin components was identical. 3.4. Blood Õiscosity Viscosity was strongly dependent on Hct with 3N salmon erythrocytes showing lower viscosity at high Hcts ŽFig. 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.
356
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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. Our results are in close agreement with those of Biron and Benfey Ž1994. obtained following acute handling stress in diploid and triploid brook trout Ž SalÕelinus fontinalis., and confirm that the primary endocrine response to stress in the triploid fish follows the typical salmonid response ŽPickering, 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 ŽBenfey 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 wHbx in triploid salmon is consistent with earlier observations from S. salar ŽBenfey and Sutterlin, 1984; Graham et al., 1985. and other salmonids ŽSmall and Randall, 1989; Yamamoto and Iida, 1994b.. Given the similarity in cortisol responses, but important differences in erythrocytes between the ploidy groups, we were surprised to find no significant haematological changes following confinement stress. In the only other study of biochemical and physiological responses to confinement stress in triploid fish, Biron and Benfey Ž1994. found no change in Hct, although other haematological characteristics were not reported. By contrast, Virtanen et al. Ž1990. 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 causing the cells to swell ŽWells 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 fish swimming freely ŽLowe 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 ŽGamperl et al., 1994.. Further, there occur complex interactions between the two endocrine responses at the level of the erythrocyte which appear to regulate oxygen transport in response to either acute or chronic stress ŽPerry and Reid, 1993.. Accordingly, 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
J. Sadler et al.r Aquaculture 184 (2000) 349–361
357
rise in plasma glucose for triploid brook trout under confinement stress ŽBiron 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 response ŽBarton, 1997; Morgan and Iwama, 1997.. This response is an important metabolic indicator of available energy reserves, and healthy rainbow trout may show elevated plasma lactate for up to 24 h post-stress ŽPankhurst and Dedual, 1994.. Disturbances to plasma protein, however, occur only under nutritional stress or extreme physical disturbance ŽWood et al., 1983; Wells et al., 1986., and were not noted for either diploid or triploid salmon in the present study. Given the higher DNA content of triploid cells Žreviewed 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 ŽArndt et al., 1994. and recent measurements confirm increased production of RNA in muscle cells of triploid rainbow trout, Oncorhynchus mykiss ŽSuresh 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 ŽWeber 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, P50 , and cooperativity coefficient, n 50 ŽFig. 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 dependence of oxygen saturation Ž P100 . on pH ŽRoot effect. allowing for oxygen secretion to the eye ŽIngermann, 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 ŽGraham 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 ŽSoivio 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 depth ŽBenfey, 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 ŽSadler, J., Pankhurst, P. and King, H., unpublished data.. The occurrence of jaw deformities in older triploid fish ŽSutterlin et
358
J. Sadler et al.r Aquaculture 184 (2000) 349–361
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 Õiscosity 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 ŽGraham 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 ŽWells 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 and lower erythrocyte viscosity ŽWells and Weber, 1991; Wells et al., 1991.. Further, erythrocytic deformability in rainbow trout increases with adrenergic swelling during exercise and hypoxia ŽHughes and Kikuchi, 1984.. Confinement stress was shown to reduce blood viscosity in a marine teleost ŽPankhurst et al., 1992.. The larger erythrocytes in triploid Atlantic salmon appear to parallel these effects and the most marked effects of triploid cell viscosity occurred at low shear rates Žequivalent 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 nature Ždependence on particle number. of viscosity. Given that blood vessel diameters do not appear affected by ploidy status ŽBenfey, 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 perturbations, although there is little evidence for this ŽOliva-Teles and Kaushik, 1987, 1990a,b; Yamamoto and Iida, 1994a; McCarthy et al., 1996; Stillwell and Benfey, 1996a,b, 1997..
J. Sadler et al.r Aquaculture 184 (2000) 349–361
359
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 Enterprises of Tasmania ŽSALTAS., 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 Allendorf, F.W., Leary, R.F., 1984. Heterozygosity in gynogenetic diploids and triploids estimated by gene-centromere recombination rates. Aquaculture 43, 413–420. Arndt, S.K.A., Benfey, T.J., Cunjak, R.A., 1994. A comparison of RNA concentrations and ornithine decarboxylase activity in Atlantic salmon Ž Salmo salar . muscle tissue, with respect to specific growth rates and diel variations. Fish Physiol. Biochem. 13, 463–471. Baldwin, J., Wells, R.M.G., 1990. Oxygen transport potential in tropical elasmobranchs from the great barrier reef: relationship between haematology and blood viscosity. J. Exp. Mar. Biol. Ecol. 144, 145–155. Barton, B.A., 1997. Stress in finfish: past, present and future — a historical perspective. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 1–33. Benfey, T.J., 1999. The physiology and behaviour of triploid fishes. Rev. Fish. Sci., in press. Benfey, T.J., Sutterlin, A.M., 1984. The haematology of triploid landlocked Atlantic salmon Ž Salmo salar .. J. Fish Biol. 24, 333–338. Benfey, T.J., Sutterlin, A.M., Thompson, R.J., 1984. Use of erythrocyte measurements to identify triploid salmonids. Can. J. Fish. Aquat. Sci. 41, 980–984. Biron, M., Benfey, T.J., 1994. Cortisol, glucose and hematocrit changes during acute stress, cohort sampling, and the diel cycle in diploid and triploid brook trout Ž SalÕelinus fontinalis Mitchill.. Fish Physiol. Biochem. 13, 153–160. Bushnell, P.G., Steffensen, J.F., Johansen, K., 1984. Oxygen consumption and swimming performance in hypoxia-acclimated rainbow trout Salmo gairdneri. J. Exp. Biol. 113, 225–235. Chomczynski, P., 1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–536. Dacie, J.V., Lewis, S.N., 1984. Practical Haematology, 5th edn. Churchill Livingstone, Edinburgh, 453 pp. Fletcher, G.L., Haedrich, R.T., 1987. Rheological properties of rainbow trout blood. Can. J. Zool. 65, 879–883. Gamperl, A.K., Vijayan, M.M., Boutilier, R.G., 1994. Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout Ž Oncorhynchus mykiss . following black-box confinement and epinephrine injection. J. Fish Biol. 45, 313–324. Graham, M.S., Fletcher, G.L., 1985. On the low viscosity blood of two cold water marine sculpins. J. Comp. Physiol. B 155, 455–459. Graham, M.S., Fletcher, G.L., Benfey, T.J., 1985. Effect of triploidy on blood oxygen content of Atlantic salmon. Aquaculture 50, 133–139. Hughes, G.M., Kikuchi, Y., 1984. Effects of in vivo and in vitro changes in PO 2 on deformability of red blood cells of rainbow trout Ž Salmo gairdneri .. J. Exp. Biol. 111, 253–257. Ingermann, R.L., 1982. Physiological significance of Root effect hemoglobins in trout. Resp. Physiol. 49, 1–10. Johnson, O.W., Dickhoff, W.W., Utter, F.M., 1986. Comparative growth and development of diploid and triploid Coho salmon, Oncorhynchus kisutch. Aquaculture 57, 329–336.
360
J. Sadler et al.r Aquaculture 184 (2000) 349–361
Johnstone, R., McLay, H.A., Walsingham, M.V., 1991. Production and performance of triploid atlantic salmon in Scotland. In: Pepper, V.A. ŽEd.., Proceedings of Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids. Dept. Fisheries and Oceans, St. Johns, Newfoundland, p. 15. Jungawalla, P., 1991. Production of non-maturing Atlantic salmon in Tasmania. In: Pepper, V.A. ŽEd.., Proceedings of the Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids. Dept. Fisheries and Oceans, St. Johns, Newfoundland, pp. 47–71. King, H., Lee, P., 1993. Progress report: jaw deformity and respiratory physiology of triploids. In: Seeking and Solving: Papers from the SALTAS Research and Development Review Seminar, 1993. SALTAS, Wayatinah, Tasmania, pp. 37–44. Lowe, T.E., Wells, R.M.G., 1996. Primary and secondary stress responses to line capture in the blue mao mao. J. Fish Biol. 49, 287–300. Mazur, C.F., Iwama, G.K., 1993. Effect of handling and stocking density on hematocrit, plasma cortisol, and survival in wild and hatchery-reared chinook salmon Ž Oncorhynchus tshawytscha.. Aquaculture 112, 291–299. McCarthy, I.D., Carter, C.G., Houlihan, D.F., Johnstone, R., Mitchell, A.I., 1996. The performance of all-female diploid and triploid Atlantic salmon smolts on transfer together to sea water. J. Fish Biol. 48, 545–548. McGeachy, S.A., O’Flynn, F.M., Benfey, T.J., Friars, G.W., 1996. Seawater performance of triploid Atlantic salmon in New Brunswick. Bull. Aquacult. Assoc. Can. 2, 24–28. Milligan, C.L., Wood, C.M., 1987. Regulation of blood oxygen transport and red cell pHi after exhaustive activity in rainbow trout Ž Salmo gairdneri . and starry flounder Ž Platichthys stellatus .. J. Exp. Biol. 133, 263–282. Morgan, J.D., Iwama, G.K., 1997. Measurements of stressed states in the field. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 247–268. O’Flynn, F.M., McGeachy, S.A., Friars, G.W., Benfey, T.J., Bailey, J.K., 1997. Comparisons of cultured triploid and diploid Atlantic salmon Ž Salmo salar L... ICES J. Mar. Sci. 54, 1160–1165. Ojolick, E.J., Cusak, R., Benfey, T.J., Kerr, S.R., 1995. Survival and growth of all-female diploid and triploid rainbow trout Ž Oncorhynchus mykiss . reared in chronic high temperature. Aquaculture 131, 177–187. Oliva-Teles, A., Kaushik, S.J., 1987. Metabolic utilisation of diets by polyploid rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 88A, 45–47. Oliva-Teles, A., Kaushik, S.J., 1990a. Effect of temperature on utilisation of endogenous energy reserves during embryonic development of diploid and triploid rainbow trout Ž Salmo gairdneri .. Aquaculture 84, 373–382. Oliva-Teles, A., Kaushik, S.J., 1990b. Growth and nutrient utilisation by 0q and 1q triploid rainbow trout Oncorhynchus mykiss. J. Fish Biol. 37, 125–133. Pankhurst, N.W., Dedual, M., 1994. Effects of capture and recovery on plasma levels of cortisol, lactate and gonadal steroids in a natural population of rainbow trout. J. Fish Biol. 45, 1013–1025. Pankhurst, N.W., Van Der Kraak, G., 1997. Effects of stress on reproduction and growth of fish. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. ŽEds.., Fish Stress and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambridge Univ. Press, Cambridge, England, pp. 73–93. Pankhurst, N.W., Wells, R.M.G., Carragher, J.F., 1992. Effects of stress on plasma cortisol levels and blood viscosity in blue mao mao, Scorpis Õiolaceus ŽHutton., a marine teleost. Comp. Biochem. Physiol. 101A, 335–339. Pelster, B., Weber, R.E., 1990. Influence of organic phosphates on the Root effect of multiple fish haemoglobins. J. Exp. Biol. 149, 425–437. Perry, S.F., Reid, S.D., 1993. b-Adrenergic signal transduction in fish: interactive effects of catecholamines and cortisol. Fish Physiol. Biochem. 11, 195–203. Pickering, A.D., 1992. Rainbow trout husbandry: management of the stress response. Aquaculture 100, 125–139. Quillet, E., Gaignon, J.L., 1990. Thermal induction of gynogenesis and triploidy in Atlantic salmon Ž Salmo salar . and their potential interest for aquaculture. Aquaculture 89, 351–364. Randall, D.J., Perry, S.F., 1992. Catecholamines. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, Vol. XII, Part B. Academic Press, London, pp. 255–300.
J. Sadler et al.r Aquaculture 184 (2000) 349–361
361
Small, S.A., Randall, D.J., 1989. Effects of triploidy on the swimming performance of coho salmon Ž Oncorhynchus kisutch.. Can. J. Fish. Aquat. Sci. 46, 243–245. Soivio, A., Nikinmaa, M., Westman, K., 1980. The blood oxygen binding properties of hypoxic Salmo gairdneri. J. Comp. Physiol. 136, 83–87. Stillwell, E.J., Benfey, T.J., 1996a. The swimming performance of diploid and triploid brook trout. Bull. Aquacult. Assoc. Can. 2, 41–43. Stillwell, E.J., Benfey, T.J., 1996b. Hemoglobin level, metabolic rate, opercular abduction rate and swimming efficiency in female triploid brook trout Ž SalÕelinus fontinalis.. Fish Physiol. Biochem. 15, 377–383. Stillwell, E.J., Benfey, T.J., 1997. The critical swimming velocity of diploid and triploid brook trout. J. Fish Biol. 51, 650–653. Suresh, A.V., Sheehan, R.J., 1998. Biochemical and morphological correlates of growth in diploid and triploid rainbow trout. J. Fish Biol. 52, 588–599. Sutterlin, A.M., Holder, J., Benfey, T.J., 1987. Early survival rates and subsequent morphological abnormalities in landlocked, anadramous and hybrid Žlandlocked=anadramous. diploid and triploid Atlantic salmon. Aquaculture 64, 157–164. Virtanen, E., Forsman, L., Sundby, A., 1990. Triploidy decreases the aerobic swimming capacity of rainbow trout Ž Salmo gairdneri .. Comp. Biochem. Physiol. 96A, 117–121. Weber, R.E., 1992. Use of ionic and zwitterionic ŽTrisrBisTris and HEPES. buffers in studies on hemoglobin function. J. Appl. Physiol. 72, 1611–1615. Weber, R.E., Wells, R.M.G., 1989. Hemoglobin structure and function. In: Wood, S.C. ŽEd.., Comparative Pulmonary Physiology, Ch.10, Series: The Lung in Health and Disease ŽExec. editor C. Lenfant.. Marcel Dekker, New York, pp. 279–310. Wells, R.M.G., Baldwin, J., 1990. Oxygen transport potential in tropical reef fish with special reference to blood viscosity and haematocrit. J. Exp. Mar. Biol. Ecol. 141, 131–143. Wells, R.M.G., Forster, M.E., 1989. Dependence of blood viscosity on haematocrit and shear rate in a primitive vertebrate. J. Exp. Biol. 145, 483–487. Wells, R.M.G., Weber, R.E., 1989. The measurement of oxygen affinity in blood and haemoglobin solutions. In: Bridges, C.R., Butler, P.J. ŽEds.., Techniques in Comparative Physiology, Society for Experimental Biology Seminar Series 37. Cambridge Univ. Press, Cambridge, pp. 279–303. Wells, R.M.G., Weber, R.E., 1990. The spleen in hypoxic and exercised rainbow trout. J. Exp. Biol. 150, 461–466. Wells, R.M.G., Weber, R.E., 1991. Is there an optimal haematocrit for trout? An interpretation of recent data based on blood viscosity measurements. J. Fish Biol. 38, 53–65. Wells, R.M.G., McIntyre, R.H., Morgan, A.K., Davie, P.S., 1986. Physiological stress responses in big gamefish after capture: observations on plasma chemistry and blood factors. Comp. Biochem. Physiol. 84A, 565–571. Wells, R.M.G., Davie, P.S., Weber, R.E., 1991. The effect of b-adrenergic stimulation of trout erythrocytes on blood viscosity. Comp. Biochem. Physiol. 100C, 653–655. Wolf, K., 1963. Physiological salines for freshwater teleosts. Prog. Fish-Cult. 25, 135–140. Wood, C.M., Turner, J.D., Graham, M.S., 1983. Why do fish die after severe exercise? J. Fish Biol. 22, 189–201. Yamamoto, A., Iida, T., 1994a. Oxygen consumption and hypoxic tolerance of triploid rainbow trout. Fish Pathol. 29, 245–251. Yamamoto, A., Iida, T., 1994b. Haematological characteristics of triploid rainbow trout. Fish Pathol. 29, 239–243.