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
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 meansSD, ns10 All-female diploids
All-female triploids ‘Rested’
Stressed ‘Rested’
Stressed Ž .
Hct 32.53.5
35.76.9 35.46.3
33.54.9
y1 a
a
Ž .
Hb g l 83.717.3
79.79.6 68.87.4
71.98.7
6 y1
a a
Ž .
RBCC 10 l 0.940.15
0.970.12 0.690.13
0.660.09
y1
Ž .
MCHC g l 259.957.8
227.529.7 201.849.4
214.315.2
a a
Ž . MCV fl
35.46.7 36.96.4
47.86.1 51.14.0
a a
Ž .
MCH pg 90.823.2
82.810.0 102.624.9
111.012.9
b b
Ž .
Glucose mM 3.30.7
4.71.2 4.01.1
5.31.5
b b
Ž .
Lactate mM 0.890.13
3.670.79 0.790.14
3.731.1 Ž
. Protein g
2.370.92 1.830.72
1.930.62 2.400.53
y1 b
b
Ž .
Cortisol ng ml 24.66.1
75.511.6 32.65.0
74.315.7
a
Ž .
Triploids significantly different from diploids P - 0.05 .
b
Ž .
Stressed significantly different from ‘rested’ P - 0.05 .
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
Ž .
P - 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 Ž
cell RNA values were similar for both diploid and triploid ‘rested’ fish mean SD s
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
P , Hill’s cooperativity coefficient n
, and
50 50
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 P
, b cooperativity coefficient n , and c Root effect. Data for
50 50
Ž . Ž .
a and b are from blood pooled from five fish in each group and each point is the average of duplicate Ž .
determinations. Data from c are mean valuesSD for ns 5.
Ž .
Ž .
the Root effect P are shown in Fig. 1, where the Bohr factors Dlog P rD pH
100 50
Ž .
Ž .
Ž .
Ž .
were y0.48 2N and y0.40 3N , and n was 2.3 2N and 2.4 3N . In addition,
50 max
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
. s 1.78 0.08 and 1.58 0.37 mmol 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. Blood Õiscosity
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.
4. Discussion
