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 m
y3
. 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, n s 20 for 2N and 3N fish was 78.1 11.1 and 76.4 15.6 g, respectively.
2.2. Stress induction Ž
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 status
2
of each fish sampled was confirmed by determining mean erythrocyte nucleus length Ž
. Benfey 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 of
3
H-labelled steroid extracted with plasma, was 94 and assay values were corrected accordingly. The assay detection limit was 2.4 ng ml
y1
plasma and interassay Ž
. Ž
. variability CV , using a pooled internal standard, was 5.4 n s 3 .
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 Hb r Hctr100 , mean cell
Ž .
Ž .
w x
volume MCV from HctrRBCC, and mean cell haemoglobin MCH from Hb rRBCC. 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
Ž .
reaction Sigma test kit no. 541.1 with a certified albuminrglobulin standard.
Ž Total cellular RNA was isolated from 50 ml 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 A
of 1.0 s 40 mg RNA ml
y1
. All samples showed A 1.7
260 nm 260:280
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 , were
50 50
determined by interpolation and slope of Hill plots according to Weber and Wells Ž
. 1989 . The effect of pH on oxygen transport at various oxygen partial pressures was
evaluated from the Bohr factor, Ø, s Dlog P rD pH. The Root effect was estimated
50
Ž .
from the change in saturation P with decreasing pH using the method of Pelster and
100
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 Ž
. Sigma, 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. Blood Õiscosity Viscosity was measured in 500 ml aliquots of erythrocyte suspensions from 2N and
Ž 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
. avoided by resuspending erythrocytes in Cortland’s solution Wolf, 1963 , a 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 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
Ž .
Ž .
for data with unequal variances JMP 3.1 Software . Student’s t-test P s 0.05 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
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