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Acute stress response in triploid rainbow trout
ž
Oncorhynchus mykiss and brook trout Sal
/
ž
Õ
elinus
/
fontinalis
Tillmann J. Benfey
), Michel Biron
1Department of Biology, UniÕersity of New Brunswick, Bag SerÕice 45111, Fredericton, New Brunswick,
Canada E3B 6E1
Accepted 10 September 1999
Abstract
The purpose of this study was to determine whether triploid salmonid fishes differ from diploids in their hematological and physiological responses to an acute handling and confinement stress, induced by netting fish from their tanks and placing them in a bucket. Blood samples were
Ž .
collected from fish prior to the handling stress and at either 30 min rainbow trout or 20 and 40
Ž .
min brook trout confinement in the bucket. Plasma cortisol, glucose and chloride levels, Ž
hematocrit, hemoglobin concentration, total blood cell concentrations erythrocytes and leuco-.
cytes , and differential leucocyte concentrations and their relative proportions were measured. As expected, resting blood cell concentrations were significantly lower in triploids than in diploids. In all other respects, triploids showed little difference from diploids in either values prior to the stress or in changes in these values induced by the acute stress. Both diploids and triploids showed a marked stress-induced increase in hematocrit and plasma cortisol and glucose, accompanied by a decrease in lymphocyte concentrations. Other values showed minimal or no change resulting from the stress. Triploid salmonid fishes therefore appear to exhibit a typical acute stress response, as has been well described for diploids.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Triploidy; Stress response; Hematology; Salmonid
)Corresponding author. Tel.:q1-506-453-4583; fax:q1-506-453-3583; e-mail: [email protected]
1
Present address: Gulf Fisheries Centre, Fisheries and Oceans Canada, P.O. Box 5030, Moncton, New Brunswick, Canada E1C 9B6.
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
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
Ž .
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
Ž .
triploids has not been investigated. Small and Benfey 1987 and Yamamoto and Iida
Ž1995. 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
Ž .
numbers Yamamoto and Iida, 1994, 1995 .
Although it is well known that stress affects leucocyte numbers and reduces
immuno-Ž .
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
Ž .
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-Ž .
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 ŽOncorhynchus mykiss , relative to diploid controls. In addition, various other hemato-. logical and physiological measurements were made to verify and expand upon our
Ž .
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
Ž .
Fingerling diploid and triploid rainbow trout females only were purchased from La
Ž .
Pisciculture des Alleghanys Saint-Philemon, Quebec and transported to the University
´
Ž .
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.
Ž .
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
Ž .
under high magnification males; Benfey et al., 1984 .
Two months prior to experimentation, fish were transferred to 1-m square fiberglass
Ž 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
Ž .
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
Ž .
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 11.18C at the start of the acclimation period to 9.28C during experimentation for rainbow trout, and from 10.78C to 8.98C during this interval for brook trout. All fish were held under an artificial 12
Ž .
h:12 h light:dark photoperiod.
Ž .
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
Ž .
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
Ž .
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
Ž .
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 aty808C 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
ŽWBC. counts were made according to Klontz 1979 , using modified YokoyamaŽ .
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
ŽSumpter 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
. 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 mean"standard error; different superscript letters across a row denote statistically significant
.
Plasma glucose mmolrl 8.37"0.66 5.38"0.24 9.22"1.05 8.37"0.75
Ž .
Plasma chloride mEqrl 119.5"1.6 125.7"1.0 118.4"1.8 120.9"2.8
a ab b b
Ž .
Total blood hemoglobin grdl 6.70"0.28 7.10"0.16 8.07"0.28 8.15"0.29
6 3 a b a b
Lymphocyte proportion % 61.4"2.3 64.5"1.5 35.2"1.5 43.9"2.4
a a b b
Ž .
Thrombocyte proportion % 31.0"2.3 27.6"1.4 47.1"1.3 41.9"2.2
a a b c
Ž .
Heterocyte proportion % 6.5"1.0 6.5"0.7 15.9"0.5 12.3"1.0
Ž .
()
Hematological responses to acute handling and confinement stress in diploid and triploid brook trout, S. fontinalis mean"standard 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
Total blood hemoglobin grdl 4.83"0.23 5.15"0.15 5.51"0.22 5.50"0.23 5.36"0.28 5.45"0.27
6 3 a b a b a b
Thrombocyte proportion % 16.2"2.2 17.6"2.1 17.8"1.2 24.4"2.5 25.4"2.5 26.3"2.0
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
The purpose of this research was to determine whether triploid salmonid fishes differ from diploids in their hematological and physiological responses to an acute handling and confinement stress. Before discussing the results with respect to the stress response, however, results from unstressed fish will be discussed.
Ž
Considering that many factors diet, strain, age, sex, season, method of capture, state
. Ž
of sexual maturity, etc. can alter the values measured in this study McCarthy et al., .
1973 , all values obtained for unstressed diploids fell within the ranges previously Ž
established for these two species Stevens, 1968; McKim et al., 1970; McCarthy et al., 1973, 1975; Christensen et al., 1978; Dick and Dixon, 1985; Angelidis et al., 1987;
. Foott and Hedrick, 1990 .
The hematocrit values of unstressed triploids were not significantly different from diploid values in either species, a characteristic previously demonstrated for both these
Ž .
and other species of fishes Benfey, 1999 . Mean plasma cortisol, glucose and chloride levels were similar for unstressed triploids and diploids in both species, supporting
Ž .
earlier observations Biron and Benfey, 1994 . Hemoglobin levels were the same for unstressed triploids and diploids of both species; previous studies have either found
Ž .
triploids to have the same or lower hemoglobin levels as diploids Benfey, 1999 . Numerous studies have previously demonstrated reduced cell numbers in triploids
ŽBenfey, 1999 , including for erythrocytes and leucocytes as observed here. Although.
cell size was not measured in the present study, the observation that hematocrit and hemoglobin levels were not different between triploids and diploids in spite of decreased blood cell numbers in triploids implies that cell numbers were reduced in proportion to their increase in size.
The acute handling and confinement stress used in these experiments evoked a clear and similar stress response in triploids and diploids of both species, confirming and
Ž .
triploids exhibit the ‘‘typical’’ stress response, as well documented for diploid salmonid
Ž .
fishes Mazeaud et al., 1977; Donaldson, 1981; Barton and Iwama, 1991 . This includes rapid elevation of plasma cortisol levels, indicative of activation of the HPI axis, and increased plasma glucose levels, due to stress-induced mobilization of energy reserves. The acute stress caused a significant elevation in hematocrit for all fish, but had no effect on erythrocyte concentrations and only a minor effect on blood hemoglobin levels. There are three possible causes of elevated hematocrit and hemoglobin concentration
during stress: decreased plasma volume, swelling of erythrocytes, andror release of
Ž .
additional erythrocytes into the blood Witters et al., 1990; Pearson and Stevens, 1991 . All three factors can affect hematocrit values, but only the first and third can affect
Ž .
hemoglobin concentration Pearson and Stevens, 1991 . In the absence of any change in erythrocyte concentrations in the present study, it therefore seems most likely that the observed changes in hematocrit and hemoglobin concentration were due to decreased plasma volume. Were this the case, however, there should also have been pronounced increases in plasma ion levels; such an increase was only observed in diploid brook trout. Thus, although there was a clear stress-induced increase in hematocrit of diploids
Ž . Ž .
and triploids of both species, it is not clear what mechanism s was were responsible. Acute stress was followed by marked leucopenia in diploids and triploids of both
Ž .
species, due specifically to a decrease in lymphocyte numbers lymphopenia . Although the relative proportions of thrombocytes and heterocytes were increased by the acute stress, their actual numbers did not change. Similar results following an acute stress
Ž
have been reported for diploid rainbow trout Dick and Dixon, 1985; Angelidis et al.,
. Ž . Ž
1987; Pickering et al., 1987 , brown trout Salmo trutta Pickering, 1984; Pickering et
. Ž . Ž .
al., 1982, 1987 and Atlantic salmon S. Salar Pickering et al., 1987 . In female
rainbow trout the decrease in lymphocyte numbers in both diploids and triploids was more than 50% following the 30 min handling and confinement stress, while in diploid and triploid brook trout the decrease was 15%–25% after 20 min and 30%–40% after 40 min. The severity of lymphopenia depends on the nature of the acute stress and the species investigated, and decreases in lymphocyte concentration by more than 50% have
Ž .
been reported for brown trout Pickering et al., 1982 .
Two hypotheses have been suggested to explain the mechanisms by which lymphope-Ž
nia occurs: either there is a redistribution of lymphocytes within the fish mainly to .
lymphoid tissues , or the lymphocytes are destroyed following a cytolytic response to
Ž .
elevated cortisol levels Pickering, 1984; Angelidis et al., 1987 . Although
corticos-Ž .
teroids are known to induce lysis of some mammalian lymphocytes Pickering, 1984 , evidence from other species of fishes suggests that acute cortisol-induced lymphopenia
Ž .
is probably due to cell redistribution Angelidis et al., 1987; Maule and Schreck, 1990 .
Ž .
An investigation by Maule and Schreck 1990 demonstrated that following an acute
Ž .
stress in coho salmon O. kisutch , the number of leucocytes increased significantly in the thymus and anterior kidney, and decreased significantly in the blood and spleen. Chronic stress, on the other hand, causes depletion of lymphocytes from all major
Ž .
lymphoidal tissues, as well as from the blood McLeay, 1973a,b; Pickering, 1984 . In conclusion, our results have shown that triploid rainbow trout and brook trout
Ž .
triploids and diploids in blood cell numbers and sizes, the effects of which on immunocompetence, disease resistance, and ability to withstand chronic stress need to be determined. In light of abundant anecdotal evidence that triploids do not cope well with poor water quality, a common source of chronic stress in aquaculture, such additional research is warranted. Poorer survival under conditions of chronic stress may be reflected in reduced energy stores andror increased rates of depletion of these stores during stressful conditions. Although substrate utilization during aerobic metabolism
Ž .
does not differ between triploids and diploids Benfey, 1999 , it may be that triploids
Ž .
differ in their ability to withstand sustained anaerobic metabolism Ojolick et al., 1995 .
Acknowledgements
We thank Robyn O’Keefe for technical assistance. This work was funded by research grants from the Natural Sciences and Engineering Research Council of Canada and from
the CanadarNew Brunswick Subsidiary Agreement on Industrial Innovation and
Tech-nology Development. M. Biron received additional support from a Vaughan Graduate Fellowship and a UNB Graduate Research Assistantship. The experimental protocol was approved by the UNB Animal Care Committee, meeting Canadian Council of Animal Care guidelines.
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