1. Introduction
Artificially-induced triploidy can be used to produce sterile salmonid fishes for Ž
. aquaculture and fisheries management Pepper, 1991; Benfey, 1996 . An important
physiological consequence of induced triploidy is the resultant increase in cell size and decrease in cell number observed for a variety of cell types, including circulating
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leucocytes Benfey, 1999 . Leucocytes are a critical part of the fish’s immune system, but the effects of changes in leucocyte size and number on immunocompetence in
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triploids has not been investigated. Small and Benfey 1987 and Yamamoto and Iida Ž
. 1995
suggested that the larger size of triploid leucocytes may confer increased phagocytic activity per cell, but any such effect is apparently offset by reduced leucocyte
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numbers Yamamoto and Iida, 1994, 1995 . Although it is well known that stress affects leucocyte numbers and reduces immuno-
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competence in diploid fishes Ellis, 1981; Barton and Iwama, 1991; Pickering, 1992 , virtually no information is available on the effects of stress on any aspect of triploid
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physiology. A previous study with triploid brook trout SalÕelinus fontinalis showed their stress response to be essentially identical to that of diploids, with respect to
changes in cortisol, glucose and hematocrit, when subjected to an acute handling and Ž
. confinement stress
Biron and Benfey, 1994 . Cortisol is the major corticosteroid produced by teleosts during stress-induced activation of the hypothalamic–pituitary–in-
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terrenal HPI axis, and is considered a principal component of the primary stress Ž
. response Mazeaud et al., 1977; Donaldson, 1981 . Increases in plasma glucose levels
and hematocrit are secondary responses to stress, associated with corticosteroid and Ž
. catecholamine release during the primary stress response Barton and Iwama, 1991 .
The purpose of this study was to investigate the effects of an acute handling and confinement stress on leucocyte numbers in triploid brook trout and rainbow trout
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Oncorhynchus mykiss , relative to diploid controls. In addition, various other hemato- logical and physiological measurements were made to verify and expand upon our
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earlier results Biron and Benfey, 1994 , namely plasma cortisol, glucose, and chloride levels, hematocrit, total blood hemoglobin levels, and erythrocyte numbers.
2. Materials and methods
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Fingerling diploid and triploid rainbow trout females only were purchased from La Ž
. Pisciculture des Alleghanys Saint-Philemon, Quebec and transported to the University
´
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of New Brunswick UNB aquaculture facilities in Fredericton. All fish originated from Ž
the same broodstock, and were produced by using homogametic sperm from hormon- .
ally-masculinized genotypic females to fertilize normal eggs. Diploids received no further treatment; triploids were produced by hydrostatic pressure shock. Details of the
treatments used for sex reversal and triploidy induction could not be obtained as they are considered a trade secret by the supplier.
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Diploid and triploid brook trout males and females were progeny from broodstock kept at UNB. Both groups came from the same lot of eggs; triploids were produced by
exposing the eggs to 9500 psi for 5 min, beginning 20 min after fertilization and incubation at 10
8C.
Rainbow trout and brook trout were 24 and 18 months old, respectively, at the time of experimentation. Ploidy level was confirmed for all experimental fish at the time of
Ž sampling, either by examining gonadal appearance under low magnification females;
. Lincoln and Scott, 1983 , or by measuring erythrocyte dimensions from blood smears
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under high magnification males; Benfey et al., 1984 . Two months prior to experimentation, fish were transferred to 1-m square fiberglass
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3
. tanks 0.15 m of water for acclimation. Each tank was separated from the others by a
system of partitions that allowed access to one tank without disturbing fish in the other tanks. In order to maintain suitable stocking densities, four tanks were used for rainbow
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trout two for diploids and two for triploids, with 12 fish per tank and six tanks for Ž
. brook trout three for diploids and three for triploids, with 24–26 fish per tank . All fish
were in apparent good health, and were fed to satiation twice daily with a pelleted dry Ž
. trout diet Corey Fed Mills, Fredericton, New Brunswick . Aerated and dechlorinated
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City of Fredericton water was used at a flow rate of 1.8–2.2 lrs rainbow trout or Ž
. 2.1–2.6 lrs brook trout . Water temperature decreased from 11.1
8C at the start of the acclimation period to 9.2
8C during experimentation for rainbow trout, and from 10.78C to 8.9
8C during this interval for brook trout. All fish were held under an artificial 12 Ž
. h:12 h light:dark photoperiod.
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Six fish unstressed were removed for sampling from a given tank prior to subjecting the remaining fish in the tank to an acute stress. The stress consisted of netting all the
remaining fish from the tank, holding them out of water for 10 s, and then placing them in a 45-l bucket half full of water. Sufficient aeration was supplied to the bucket to
prevent additional stress from oxygen depletion. Six fish were removed from the bucket
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for sampling after 30 min of confinement rainbow trout or after 20 and 40 min of Ž
. confinement brook trout . These sampling times were selected on the basis of earlier
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results Biron, 1993; Biron and Benfey, 1994 in order to minimize the number of fish killed. All four tanks of rainbow trout were sampled on the same day, whereas two tanks
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of brook trout one of each ploidy were sampled per day over 3 days. The sampling procedure consisted of capturing six fish by carefully netting them with
minimal disturbance to the others. These fish were immediately killed by a sharp blow Ž
to the head. Blood was collected from the caudal vein by vacutainer 22g1 needle, no. .
7110, and 4 ml sodium-heparinized tube, no. 6499, Becton Dickinson . Blood sampling was always completed within 2 min of first disturbing the fish. A blood smear was made
from fresh blood, and the weight and length of each fish was measured. A sub-sample of blood was transferred to sterile plastic vials and kept on ice until hemoglobin concentra-
tion and total and differential cell counts were determined. Three hematocrit tubes were also filled from each vacutainer tube and refrigerated in an upright position until their
centrifugation for the measurement of hematocrit. Thereafter, the vacutainer tubes were centrifuged and the plasma from each transferred in approximately equal volumes into
three sterile plastic vials and stored at y80
8C for later measurement of cortisol, glucose and chloride ion concentrations.
Total blood hemoglobin levels were determined using the Drabkin Austin cyanmethe- Ž
. Ž
. moglobin technique Kit 525, Sigma . Total red blood cell RBC and white blood cell
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WBC counts were made according to Klontz 1979 , using modified Yokoyama
diluting fluid and a Spencer Bright-line hemocytometer. Blood smears were air-dried,
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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
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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
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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
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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
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. 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
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