Ž .
stained with a prepared Wright-Giemsa stain C5434, Fisher Scientific
for 15 s, immersed in a bath of deionized water for 30 s, and rinsed with tap water. Once the
smears were dry, they were mounted with Permount and left on a slide warmer overnight. Differential leucocyte counts were made for lymphocytes, thrombocytes, and
Ž .
heterocytes, according to Klontz 1979 . All unidentified or other types of cells were classified as ‘‘other cells’’ during each count. Identification of leucocyte types was
Ž .
Ž .
Ž .
based on Conroy 1972 , Ellis 1977 and Christensen et al. 1978 . Plasma cortisol Ž
concentrations were determined by radioimmunoassay CA-529 and CA-549, Baxter .
Clinical . The assay has been validated for salmonid fishes of the genus Oncorhynchus Ž
. Sumpter and Donaldson, 1986 . Glucose concentrations were obtained by the Raabo
Ž .
and Terkildsen colorimetric glucose oxidase procedure Kit 510, Sigma . Chloride levels Ž
were determined by coulometric–amperometric titration with silver ions Buchler-
. Cotlove Chloridometer, Buchler Instruments .
Ž All statistical analyses were executed with GraphPAD InStat Version 1.14 Graph-
. Ž
PAD Software, San Diego . Differences among groups diploids and triploids at various .
time points for a given variable were tested using analysis of variance. In cases where a p value of less than 0.05 was found, Bonferroni t-tests were subsequently used to test
for significant differences between the means of pairs of groups. Values within groups Ž
. Ž
. for replicates both species and separate sexes brook trout only were combined after
first demonstrating that, in all cases, they were not significantly different.
3. Results
Ž .
At the time of sampling, there was no significant difference p 0.05 between
Ž diploid and triploid rainbow trout in either weight or length 593 36 g and 35.8 0.6
. cm vs. 543 23 g and 35.7 0.3 cm, respectively . Triploids had extremely small
ovaries showing no oocyte development, whereas diploids could be divided into two groups: those that were immature, with small ovaries full of oocytes at early stages of
Ž .
development six of 12 unstressed fish and six of 12 stressed fish , and those that were Ž
. at or near ovulation six of 12 unstressed fish and five of 12 stressed fish . No
significant differences were found between immature and ovulatory diploids for any of the variables examined, so the results from these two groups were combined. A single
Ž hermaphrodite
as evidenced macroscopically by the presence of both developing .
oocytes and regions of white, testicular tissue was found among the stressed diploids, and was eliminated from all analyses. It is assumed that this fish, likely the result of
hormonal treatment at the hatchery from which the fish were purchased, was inadver- tently transferred to the tank from which the experimental fish originated prior to their
delivery to UNB. Hemoglobin data and total and differential blood cell counts for two of the stressed triploids were removed from the analysis because of coagulation of their
blood samples, and a diploid sample from this group was destroyed during hemoglobin analysis.
Ž .
There was no significant difference p 0.05 in weight between diploid and triploid Ž
. brook trout at the time of sampling 263 7 g vs. 275 8 g, respectively but diploids
Ž were significantly shorter than triploids 26.4 0.2 cm vs. 27.6 0.3 cm, respectively,
. Ž
p - 0.001 . A single triploid fish was found among the diploids 40 min confinement .
group , and was removed from all analyses because triploids may be chronically stressed Ž
. by cohabitation with diploids Benfey, 1999 . Whether this triploid individual arose
Ž .
‘‘spontaneously’’ Benfey, 1989 or was the result of a fish being placed into the wrong tank is not known. The remaining diploids were comprised of 25 females and 28 males,
while the triploids were comprised of 36 females and 18 males. Blood samples from Ž
. four fish in the 40 min confinement group one diploid and three triploids showed signs
of coagulation, and were excluded from all analyses except cortisol, glucose and chloride.
Results are summarized in Tables 1 and 2 for rainbow trout and brook trout, respectively. Hematocrit, plasma cortisol and plasma glucose were all elevated by the
acute handling and confinement stress in both species, with no significant differences between 20 and 40 min confinement in brook trout. Hematocrit was not significantly
different between diploids and triploids of either species at any specific time interval. Plasma cortisol levels were not significantly different between diploid and triploid
rainbow trout at either time interval, but were consistently higher in diploid than in triploid brook trout at both post-stress time intervals. Plasma glucose levels were
significantly higher in unstressed diploid rainbow trout than in unstressed triploids, but were not different between stressed diploid and triploid rainbow trout or between diploid
and triploid brook trout at any specific time interval. Plasma chloride levels were lower in unstressed diploids than in unstressed triploids of both species, although this
difference was only statistically significant for brook trout. Stress had no significant
Table 1 Hematological responses to acute handling and confinement stress in diploid and triploid rainbow trout, O.
Ž mykiss
meanstandard error; different superscript letters across a row denote statistically significant .
differences at p- 0.05 Pre-stress
30 min post-stress Diploid
Triploid Diploid
Triploid Ž
. Ž
. Ž
. Ž
. ns12
ns12 ns10–11
ns9–12
a a
b b
Ž . Hematocrit
38.41.2 36.11.4
45.02.0 46.31.5
a a
b b
Ž .
Plasma cortisol ngrml 28.611.4
12.52.1 25328
23229
a b
a a
Ž .
Plasma glucose mmolrl 8.370.66
5.380.24 9.221.05
8.370.75 Ž
. Plasma chloride mEqrl
119.51.6 125.71.0
118.41.8 120.92.8
a ab
b b
Ž .
Total blood hemoglobin grdl 6.700.28
7.100.16 8.070.28
8.150.29
6 3
a b
a b
Ž .
RBC count =10 cellsrmm 1.240.04
0.730.03 1.190.04
0.890.07
4 3
a b
b c
Ž .
WBC count =10 cellsrmm 5.400.30
3.920.27 3.890.25
2.710.18
y2 ab
a b
b
Ž .
WBCrRBC =10 4.40.2
5.40.3 3.30.3
3.30.4
4 3
a a
b b
Ž .
Lymphocyte count =10 rmm 3.300.24
2.510.19 1.400.20
1.260.19
4 3
a b
a b
Ž .
Thrombocyte count =10 rmm 1.670.13
1.130.16 1.830.11
1.130.07
4 3
Ž .
Heterocyte count =10 rmm 0.380.12
0.260.05 0.610.10
0.310.04
4 3
Ž .
‘‘Other’’ WBC count =10 rmm 0.060.01
0.050.01 0.060.01
0.040.01 Ž .
a a
b c
Lymphocyte proportion 61.42.3
64.51.5 35.21.5
43.92.4
a a
b b
Ž . Thrombocyte proportion
31.02.3 27.61.4
47.11.3 41.92.2
a a
b c
Ž . Heterocyte proportion
6.51.0 6.50.7
15.90.5 12.31.0
Ž . ‘‘Other’’ WBC proportion
1.00.3 1.20.3
1.30.3 1.60.2
T.J. Benfey,
M. Biron
r Aquaculture
184 2000
167 –
176 172
Table 2 Ž
Hematological responses to acute handling and confinement stress in diploid and triploid brook trout, S. fontinalis meanstandard error; different superscript letters .
across a row denote statistically significant differences at p- 0.05 Pre-stress
20 min post-stress 40 min post-stress
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Diploid ns17–18 Triploid ns18
Diploid ns18 Triploid ns18
Diploid ns16–17 Triploid ns15–18
ab b
c ac
c abc
Ž . Hematocrit
31.31.3 31.00.7
36.80.8 35.40.9
36.40.8 35.01.0
a a
bd c
b cd
Ž .
Plasma cortisol ngrml 7.01.6
1.50.7 88.15.9
53.46.0 112.36.7
76.47.9
a a
b b
b b
Ž .
Plasma glucose mmolrl 5.730.27
5.310.20 6.780.19
6.870.17 7.360.22
7.240.31
a b
b b
b ab
Ž .
Plasma chloride mEqrl 119.21.2
125.10.7 125.31.0
126.40.8 128.41.2
123.81.4 Ž
. Total blood hemoglobin grdl
4.830.23 5.150.15
5.510.22 5.500.23
5.360.28 5.450.27
6 3
a b
a b
a b
Ž .
RBC count =10 cellsrmm 0.950.01
0.680.01 0.970.01
0.680.01 0.930.01
0.700.01
4 3
a abc
ab bc
abc c
Ž .
WBC count =10 cellsrmm 3.900.23
3.080.20 3.330.25
2.470.19 2.960.25
2.300.20
y2 ab
a ab
ab b
ab
Ž .
WBCrRBC =10 4.10.3
4.50.2 3.40.2
3.70.3 3.20.3
3.40.3
4 3
a ab
ab bc
bc c
Ž .
Lymphocyte count =10 rmm 3.040.21
2.430.20 2.580.20
1.820.17 2.060.21
1.530.14 Ž
4 3
. Thrombocyte count =10 rmm
0.630.10 0.540.08
0.590.06 0.580.05
0.730.07 0.590.07
4 3
Ž .
Heterocyte count =10 rmm 0.160.05
0.090.03 0.130.03
0.070.02 0.140.03
0.170.04
4 3
a ab
ab b
ab b
Ž .
‘‘Other’’ WBC count =10 rmm 0.060.01
0.020.01 0.030.01
0.010.01 0.030.01
0.010.01
a a
a ab
ab b
Ž . Lymphocyte proportion
78.52.4 78.62.4
77.31.6 72.02.7
68.81.6 65.32.6
a ab
ab ab
ab b
Ž . Thrombocyte proportion
16.22.2 17.62.1
17.81.2 24.42.5
25.42.5 26.32.0
ab a
ab a
ab b
Ž . Heterocyte proportion
3.91.0 3.01.0
3.80.5 2.90.7
4.90.7 7.91.8
a ab
ab ab
ab b
Ž . ‘‘Other’’ WBC proportion
1.40.3 0.60.2
1.10.3 0.70.2
0.80.2 0.30.1
effect on plasma chloride levels in rainbow trout of either ploidy or in triploid brook trout, but significantly elevated plasma chloride levels in diploid brook trout.
There was no significant difference in total blood hemoglobin levels between diploids and triploids of either species at any specific time interval. The stress caused a
significant elevation in total blood hemoglobin levels in diploid rainbow trout but in none of the other groups. Total erythrocyte and leucocyte counts were lower in triploids
than in diploids in all cases, although this difference was not statistically significant for leucocytes in the brook trout. The stress had no effect on erythrocyte counts but reduced
leucocyte counts, resulting in reduced leucocytererythrocyte ratios in both species.
Lymphocyte, thrombocyte, heterocyte and ‘‘other’’ leucocyte counts were lower in triploids than in diploids in virtually all cases, although not always significantly so.
Lymphocytes, which were the most abundant leucocytes observed, were significantly reduced in numbers by the stress in both species. No such effect was observed for the
other types of leucocytes.
4. Discussion
