North American Journal of Aquaculture 62:249–259, 2000

q Copyright by the American Fisheries Society 2000

Optimization of Thermal Shock Protocols for

Induction of Triploidy in Brook Trout
PETER F. GALBREATH* AND BARBARA L. SAMPLES
Mountain Aquaculture Research Center, Western Carolina University,
Cullowhee, North Carolina 28723, USA

Abstract.—Techniques for production of triploids are well described for commercially reared
salmonids (e.g., rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar). Among
less commonly used species, including brook trout Salvelinus fontinalis, these protocols have not
been optimized. As a prelude to investigating the regional potential for commercial culture of
brook trout with all-female and all-female triploid stocks, we conducted experiments to test the
effects of the primary variables of thermal shock used to induce retention of second polar bodies.
The objective was to identify a protocol that maximizes yield of both triploid and gynogenetic
diploid progeny. The first experiment measured survival of gynogenetic embryos produced by heat
shocks involving combinations of different temperatures (26, 27, 28, or 298C) and durations (7,
10, 13, 16, 19, or 22 min). In three subsequent experiments, production of triploids was assessed
for thermal shocks involving variable times of initiation (7, 10, 13, 16, 19, or 22 min postactivation)
in addition to the above temperatures and durations. Significant trends for decrease in survival
and increase in percent triploidy were observed with increase in temperature and duration of
thermal shock. Effects for time of initiation in two of three experiments were nonsignificant; in
the third, survival tended to increase and percent triploidy to decrease with increase in time of
initiation. With a preshock incubation temperature of 11–128C, the most generally effective protocol
involved a thermal shock of 288C for 10 min duration initiated at 10–16 min postactivation, for
which relative survival was 68–71%, percent triploidy was 79–99%, and triploid yield was 54–
70%. In a fifth experiment, the fertilized eggs of 14 females were treated separately with a 288C
shock of 10 min applied at 10 min postactivation. Although relative survival to initiation of feeding
ranged between 42% and 100%, percent triploidy was consistently 98–100%.

Introduction uration reduces the economic profitability of com-

Triploidy has been induced in many salmonid
species by application of thermal or pressure
shocks to newly fertilized eggs to prevent extru-
sion of the second polar body. Sterility in triploid
salmonids of both sexes and inhibition of sexual
maturation in triploid females have been exploited
in various situations in both commercial salmonid
culture and fisheries management (see reviews by
Thorgaard 1983; Ihssen et al. 1990; Pepper 1991;
Benfey 1996).
Brook trout Salvelinus fontinalis is the only sal-
monid species native to upland areas of the eastern
United States (MacCrimmon and Campbell 1969).
Although brook trout are highly appreciated for
sportfishing, the introduced rainbow trout Oncor-
hynchus mykiss is used almost exclusively in com-
mercial trout farming in this region. This is due
in part to the relatively young age and small size
at which brook trout become sexually mature
(Dubé et al. 1991; Flick 1991). Acquisition of sec-
ondary sexual characteristics by the fish at mat-
* Corresponding author: galbreat@wcu.edu
Received November 23, 1998; accepted April 21, 2000
mercial salmonid culture (Johnstone et al. 1979;
Bye and Lincoln 1986; Pepper 1991; Benfey
1996).
Commercial culture of brook trout as a food fish
will require use of all-female (diploid) and all-female
triploid stocks to circumvent maturation of the fish
before harvest, as is the case for commercially reared
rainbow trout. However, all-female and all-female
triploid brook trout are not readily available, nor are
commercial production procedures well established.
We therefore researched methods to reliably produce
groups of triploid and gynogenetic diploid fish. The
latter are produced through activation of eggs with
sperm having DNA rendered nonfunctional by ul-
traviolet (UV) irradiation, followed by thermal or
hydrostatic pressure shock. Gynogenetic trout are all-
female and are useful in experimentation on sex-
reversal techniques (Chourrout 1982; Lou and Pur-
dom 1984). Because thermal shock can be highly
effective and is easily applied (Levanduski et al.
1990; Palti et al. 1997), we chose this technique for
producing triploid and gynogenetic fish. However, a
clearly optimized thermal shock protocol for brook
trout has not been described. Scheerer and Thorgaard
(1983) and Gray et al. (1993) described successful

249
250 GALBREATH AND SAMPLES

TABLE 1.—Design for experiments 1–4, which were conducted to identify thermal shock protocols that optimize yield
of gynogenetic or triploid brook trout.

Experiment
1 2 3 4
Desired progeny Gynogens Triploids Triploids Triploids
Number of broodfish (females/males)a 13/0 4/4 16/5 14/8
Average number of eggs/replicate (range) 317 (217–430) 158 (90–208) 170 (90–243) 243 (136–323)
Preshock water temperature (8C) 11 12 11 11
Thermal shock treatment variables b
Time of initiation (min) 10 7, 10, 13, 16 7, 10, 13, 16 10, 13, 16, 19, 22
Temperature (8C) 26, 27, 28, 29 26, 27, 28, 29 26, 27, 28, 29 26, 27, 28
Duration (min) 7, 10, 13, 16, 19, 22 7, 10, 13, 16 7, 10, 13, 16 10, 13, 16, 19, 22
Number of treatments 16 64 64 75
Number of replicates per treatment 3 1 1 1
Number of control replicates 3 3 6
Average postshock water temperature (8C) 11 10.5 10.1 11
a Broodstock were from Ford State Fish Hatchery, Spokane, Washington (experiment 1), Armstrong State Fish Hatchery, Marion, North
Carolina (experiments 2 and 3), and Pisgah Forest State Fish Hatchery, Brevard, North Carolina (experiment 4).
b In experiment 1, the following combinations of temperature and duration treatment variables were tested: 268C for 13, 16, 19, or 22
min; 278C for 10, 13, 16, or 19 min; and 288C and 298C for 7, 10, 13, or 16 min. In experiments 2, 3, and 4, all possible combinations
of the indicated variables were tested.

induction of triploidy in brook trout using a single
protocol found to be effective in rainbow trout. Dubé
et al. (1991) provided a comparison between alter-
native thermal shock protocols for triploidy induc-
tion in brook trout; however, replication of treatments
that provided reasonably high yields of triploids was
minimal.
Optimizing thermal shock protocols is compli-
cated by the large number of variables involved.
The variables of principal interest are time of ini-
tiation following activation, temperature, and du-
ration of the shock. In addition, preshock incu-
bation water temperature, species or strain differ-
ences, and differences in egg quality among fe-
males within strains can alter the effectiveness of
a given protocol (Lou and Purdom 1984; Solar et
al. 1984; Johnstone 1985; Seeb et al. 1988; Lev-
anduski et al. 1990; Diaz et al. 1993; Palti et al.
1997). Inability to control all of these factors ac-
counts for the wide variability in thermal shock
protocols reputed as effective for salmonids (Lev-
anduski et al. 1990; Galbreath et al. 1994).
The objective of this study was to better un-
derstand the effects of the principal thermal shock
variables and to identify a protocol that produces
the maximum yield of gynogenetic and triploid
brook trout. Four separate experiments were con-
ducted using eggs obtained from three different
hatchery strains. Each experiment involved si-
multaneous testing of a range of values for mul-
tiple variables of thermal shock: time of initiation
following activation, temperature, and duration. In
a fifth experiment, a protocol deemed optimal was
applied to the eggs of several females separately
to determine if a maternal effect would effect var-
iation for percent triploidy.
Methods
Comparison of heat shock protocols.—On four
occasions, gametes were obtained for experimen-
tation from one of three state fish hatcheries. With-
in each experiment, eggs from several females
were pooled, sublotted, and sperm from one of the
available males was added to each of the sublots
along with an amount of activator solution (0.9%
NaCl, 0.01 M tris base, and 0.02 M glycine; Billard
et al. 1974) sufficient to cover the eggs. Fresh wa-
ter at 11–128C was then added to initiate sperm
penetration of the egg membrane and reactivation
of development in the eggs. At 2–3 min after ini-
tiation of water hardening, the eggs were repooled,
mixed, and sublotted into an appropriate number
of screen-bottomed incubation boxes. At 10 min
postactivation for experiment 1 and at 7–22 min
postactivation for experiments 2, 3, and 4 the eggs
were subjected to thermal shock by immersion in
heated water baths: temperatures of 26–298C and
durations of 7–22 min. The eggs were then trans-
ferred to vertical-tray incubators and reared at 10–
118C to the initiation of feeding stage, when num-
ber of surviving embryos per treatment replicate
was recorded. Details on numbers of broodstock,
numbers of eggs per treatment replicate, incuba-
tion water temperatures, and heat shock protocols
for each experiment are presented in Table 1.
Experiment 1 involved production of gynoge-
netic fish for which survival was a direct indicator
of successful retention of the polar body. Egg de-
 BROOK TROUT TRIPLOID INDUCTION
velopment was activated as follows: sperm from
a single rainbow trout was diluted 1:9 in an ex-
tender solution (135.5 mM NaCl, 40.0 mM KCl,
4.2 mM NaH2PO4, 1.25 mM MgSO4.7H2O, and
1.25 mM CaCl2; Galbreath et al. 1994), spread to
a thickness of approximately 0.5 mm in glass bak-
ing dishes (10 3 15 cm), and exposed for 10 min
to UV light at an intensity of approximately 240
mW/cm2 (modified from Chourrout 1982). Sperm
from a rainbow trout was used so that any sperm
cells that were not effectively irradiated would
produce nonviable hybrid embryos (Chourrout
1986; Gray et al. 1993). Survival data were arc-
sine-transformed (arcsine[proportion1/2]) and com-
pared by analysis of variance (ANOVA) for dif-
ferences among temperature–duration treatments
(Data Desk 5.0, Data Description Inc., Ithaca, New
York). Tests of significance in this and subsequent
ANOVAs were based on a 5 0.05.
In experiments 2, 3, and 4, triploid progeny were
produced by fertilizing the eggs with nonirradiated
sperm from brook trout males, followed by thermal
shock to induce retention of the second polar body.
At initiation of feeding the total number of em-
bryos and number and percentage that possessed
gross vertebral malformations were calculated for
each group. Deformities included scoliosis, lor-
dosis, ‘‘cork-screw,’’ and ‘‘Siamese twin’’ or ‘‘Y-
shaped’’ double-headed embryos. Also at this
time, a sample of 50 embryos per treatment (or all
remaining embryos when survival was less than
50) were placed in individual microcentrifuge
tubes and frozen at 2708C. Percent triploidy was
determined for each treatment following flow cy-
tometric analysis, in which the embryos were first
thawed and lightly ground in approximately 0.5
mL of 4,6-diamidino-2-phenylindole (DAPI)
DNA-staining solution (146 mM NaCl, 10 mM tris
base, 22 mM MgCl2, 2 mM CaCl2, 50 mg/L BSA,
0.1% Triton-X, 10 mg/L DAPI, and 10% DMSO;
S. K. Allen, Virginia Institute of Marine Science,
personal communication). Then, another 1 mL of
DAPI solution was added to each sample, the sam-
ple was vortexed, poured through a 50-mm filter,
and the filtrate was analyzed for ploidy level on a
Partec CCA flow cytometer using as standards
DAPI-stained blood samples from brook trout pre-
viously determined to be diploid or triploid.
Relative survival was calculated by dividing per-
cent survival for each treatment by the average per-
cent survival for the respective experimental control
replicates. Triploid yield was calculated for each
treatment as the product of relative survival 3 per-
cent triploidy. Relative survival, percent triploidy,
251
and triploid yield data were arcsine-transformed and
analyzed within each experiment by ANOVA for
main effects and interactions of time of initiation,
temperature, and duration. Single replication within
each treatment in the balanced factorial design for
these experiments required use of the three-way in-
teraction mean squares as the error term. Subsequent
ANOVAs to examine effects of time of initiation and
temperature within levels of duration, and of time of
initiation and duration within levels of temperature
were conducted for each experiment using the re-
spective two-way mean squares as the error term
(Sokal and Rohlf 1987).
Percent deformed data (excluding treatments for
which survival was of fewer than 10 individuals)
were arcsine-transformed and compared by AN-
OVA for differences between control and ther-
mally shocked fish.
Maternal effect.—Eggs obtained November 10,
1999, from 14 brook trout females and a single
brook trout male from Pisgah Forest State Fish
Hatchery, Brevard, North Carolina, were used to
test variability in triploid production and survival
among females produced by a standard heat shock
protocol (experiment 5). Approximately 500 eggs
per female were placed in separate bowls, fertil-
ized with milt from the single brook trout male,
and then divided in half. To induce triploidy, one
group of eggs per female was exposed to a heat
shock of 288C at 10 min postactivation and for a
duration of 10 min. The other half was placed di-
rectly into the incubation water to serve as diploid
controls. Incubation temperature averaged 11.6 8C.
Mortalities were removed and recorded every 3–
4 d. After 64 d the fry began feeding. Surviviors
were counted and percent survival was calculated
for each group. Twenty embryos from each control
group and 50 embryos from the heat-shocked
groups were placed in individual microcentrifuge
tubes and frozen at 2708C. Ploidy was analyzed
in these embryos as previously described, and per-
cent triploidy and triploid yield were calculated
for each female’s progeny. Percent survival of con-
trol eggs was correlated to relative survival of the
respective of heat-shocked eggs.
Results
Comparison of Heat Shock Protocols
Survival of gynogenetic progeny in experiment
1 ranged from 0% to 24% among replicates, and
from 0% to 12% among treatment averages.
Among the 16 temperature–duration treatments
ANOVA indicated a significant effect for treat-
252 GALBREATH AND SAMPLES

average survival for each temperature–duration

FIGURE 1.—Response surface curves for percent sur-
vival of gynogenetic brook trout embryos produced in
experiment 1 by thermal shock of eggs at 10 min post-
activation. Curves were calculated for every 2% survival
from the array of average survival values for the tem-
perature–duration thermal shock treatments.
ment, but a posthoc pairwise comparison (least-
squares differences) showed significant differenc-
es only between those treatments near the extremes
of the range, and no significant differences were
observed among the eight highest ranking treat-
ments. Because of imbalance in the factorial de-
sign, analysis for the main effects of temperature
and duration on survival was performed by using
subsets of the data for which a series of ANOVAs
examined the effect of temperature within each
level of duration and the effect of duration within
each level of temperature. Effects were not sig-
nificant except for temperature at 298C (survival
decreased with increase in duration) and for du-
rations of 13 and 16 min (survival decreased with
increase in temperature). To illustrate the results,
treatment was graphed and the array of values used
to calculate response surface curves (Figure 1;
Dubé et al. 1991). In this case the contour lines
were calculated for every 2% survival by direct
interpolation between adjacent points in the array.
In Experiments 2, 3, and 4, the tested range of
thermal shock variables effected a wide range of
values within each performance measure: relative
survival, percent triploidy, and triploid yield. The
effects of time of initiation, temperature, duration,
and their respective interactions on these measures
are illustrated in Table 2 by the probability values
calculated in the ANOVAs for each experiment.
In experiments 2 and 3, the main effects of tem-
perature and duration on relative survival, percent
triploidy, and triploid yield were highly signifi-
cant, whereas the main effect for time of initiation
was significant, or nearly so, only for percent trip-
loidy. Lack of replication within the factorial de-
sign for these experiments, however, necessitates
using the highest order interaction mean squares
as the error term, which is only valid presuming
the interaction is not significant. This was probably
not the case, in view of significant temperature 3
duration interaction terms in each analysis. So,
subsets of the data were used to perform additional
ANOVAs to examine effects of time of initiation
and temperature within levels of duration and for
time of initiation and duration within levels of tem-
perature. In these analyses, the effects of temper-
ature and of duration were generally highly sig-
nificant, whereas the effects of time of initiation
were consistently nonsignificant. Relative survival
decreased and percent triploidy increased with
both increasing temperature and duration of heat
shock. These counteracting trends lead to variable
results for triploid yield. Because the effects of
time of initiation on relative survival, percent trip-
loidy, and triploid yield were not significant, the

TABLE 2.—Probability values from analyses of variance for the effects of time of initiation (initiation), temperature
(temp), and duration of heat shock on relative survival (rel surv), percent triploidy (%3n), and triploid yield (3n yield)
for brook trout in experiments 2, 3, and 4. The three-way interaction mean squares was used as the error term within
each analysis. Statistical significance was based on a 5 0.05.

Experiment 2 Experiment 3 Experiment 4

Source Rel surv % 3n 3n yield Rel surv % 3n 3n yield Rel surv % 3n 3n yield
Initiation 0.450 0.031 0.851 0.718 0.050 0.387 ,,0.001 ,0.001 0.032
Temperature ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001
Initiation 3 temp 0.893 0.760 0.624 0.047 0.782 0.108 ,0.001 0.007 0.287
Duration ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,,0.001 ,0.001
Initiation 3 duration 0.562 0.305 0.293 0.676 0.153 0.022 0.863 0.739 0.982
Temp 3 duration ,,0.001 0.014 0.004 ,,0.001 ,,0.001 ,,0.001 0.003 0.213 ,,0.001
 BROOK TROUT TRIPLOID INDUCTION 253

FIGURE 2.—Response surface curves for percent relative survival (percent survival of treated embryos/percent
survival of control embryos) to initiation of feeding by brook trout fry that were subjected, as embryos, to thermal
shock to induce triploidization (experiments 2, 3, and 4). Curves were calculated for every 10% survival from the
array of average relative survival values for the temperature–duration thermal shock treatments (averaged for
respective time of initiation treatments).

four treatments involving different times of initi-
ation within each temperature–duration combina-
tion were considered as replicates and their values
were averaged. These average values per temper-
ature–duration treatment were then plotted and
used to create the response surface curves for rel-
ative survival, percent triploidy and triploid yield
presented in Figures 2, 3, and 4, respectively. The
curves were calculated by direct interpolation be-
tween adjacent points in the arrays for every 10%
difference (plus 95% for percent triploidy).
As in experiments 2 and 3, the main effects in
experiment 4 for temperature and duration on rel-
ative survival, percent triploidy, and triploid yield
were highly significant (Table 3). However, sig-
nificant effects for time of initiation were also ob-
served, as were significant effects for the time of
initiation 3 temperature and temperature 3 du-
254 GALBREATH AND SAMPLES

FIGURE 3.—Response surface curves for percent triploidy measured at initiation of feeding by brook trout that
were subjected, as embryos, to thermal shock to induce triploidization (experiments 2, 3, and 4). Curves were
calculated for every 10% (and 95%) from the array of average percent triploidy values for the temperature–duration
thermal shock treatments (averaged for respective time of initiation treatments).

ration interactions. Subsequent ANOVAs per-
formed within levels of duration indicated signif-
icant effects for temperature on relative survival,
percent triploidy, and triploid yield. The effect for
time of initiation was sometimes significant for
relative survival but not significant for percent
triploidy or triploid yield. In the three analyses
performed within levels of temperature, highly
significant effects for duration on relative survival
and on percent triploidy were consistently ob-
served; in two of the analyses a significant effect
for time of initiation was observed; and at one
temperature level a significant effect on triploid
yield was observed. These results are illustrated
by the probability values for the analyses presented
in Table 3. In summary, there was a consistently
significant trend for decreased relative survival
and increased percent triploidy with both increas-
ing temperature and duration of heat shock. Trends
were sometimes apparent, sometimes not, for in-
creasing relative survival and decreasing percent
triploidy with increase in the time of initiation of
 BROOK TROUT TRIPLOID INDUCTION 255

FIGURE 4.—Response surface curves for triploid yield (percent relative survival 3 percent triploidy) measured
at initiation of feeding by brook trout fry that were subjected, as embryos, to thermal shock to induce triploidization
(experiments 2, 3, and 4). Curves were calculated for every 10% from the array of average triploid yield values
for the temperature–duration thermal shock treatments (averaged for respective time of initiation treatments).

the thermal shock. To compare results with those
obtained in experiments 2 and 3, and because sig-
nificant effects for time of initiation were not con-
sistently observed in experiment 4, the data for the
time of initiation replicates within each tempera-
ture–duration treatment were averaged. The re-
sultant values were plotted and used to create the
response surface curves for relative survival, per-
cent triploidy, and triploid yield in this experiment
(Figures 2, 3, 4, respectively).
Average percentages of deformed fry among heat-
shocked fish relative to controls in experiments 2
and 3 were not significantly different (Table 4). In
experiment 4, however, there was a significantly in-
creased incidence of deformities among heat-
shocked embryos (P , 0.001; Table 4).
Maternal Effect
In experiment 5 (Table 5) relative survival of
the triploids ranged from 42% to 100%; however,
256 GALBREATH AND SAMPLES

TABLE 3.—Probability values from analyses of variance for the effects of time of initiation (initiation) and temperature
within levels of duration and for the effects of time of initiation and duration within levels of temperature on relative
survival (rel surv), percent triploidy (%3n), and triploid yield (3n yield) for brook trout in experiment 4. The two-way
interaction mean squares was used as the error term within each analysis. Statistical significance was based on a 5
0.05.

Temperature
268C 278C 288C
Source Rel surv % 3n 3n yield Rel surv % 3n 3n yield Rel surv % 3n 3n yield
Initiation 0.310 0.460 0.332 ,,0.001 0.014 0.995 ,,0.001 ,0.001 0.006
Duration ,0.001 ,,0.001 ,,0.001 0.002 0.002 0.004 ,,0.001 0.012 ,,0.001

TABLE 3.—Extended.

Duration
10 min 13 min 16 min
Source Rel surv % 3n 3n yield Rel surv % 3n 3n yield Rel surv % 3n 3n yield
Initiation 0.445 0.998 0.007 0.047 0.797 0.734 0.030 0.265 0.631
Temperature 0.164 ,,0.001 ,,0.001 ,,0.001 ,0.001 ,0.001 ,,0.001 ,0.001 0.006

TABLE 3.—Extended.

Duration
19 min 22 min
Source Rel surv % 3n 3n yield Rel surv % 3n 3n yield
Initiation 0.068 0.105 0.416 0.010 0.147 0.900
Temperature 0.001 ,,0.001 0.0349 ,0.001 ,0.001 0.325

percent triploidy was consistently 98–100%. Discussion

Among the 300 control (diploid) individuals an-
alyzed for ploidy, 3 were triploid, representing a
spontaneous triploidization rate of 1%. When dip-
loid survival was graphed against relative survival
of triploids within females, the correlation coef-
ficient was low (r250.143) and slope of the re-
gression line was not significantly different from
zero (P 5 0.225).
TABLE 4.—Average percent of deformed embryos 6 SD
for control versus thermal-shocked brook trout embryos
(averaged among all heat shock treatments) from experi-
ments 2, 3, and 4. Asterisk indicates statistical significance
(P , 0.05) relative to control as determined by analysis
of variance.
Experiment
2 3
Control 1.7 6 0.4 0.9 6 0.9
Thermal-shocked 1.0 6 1.5 2.3 6 4.6
Production of gynogenetic fry provides a direct
measure of successful retention of the second polar
body and avoids the need for numerous, time-con-
suming ploidy analyses. This technique has been
used previously to evaluate the efficacy of heat
shock protocols (Chourrout and Quillet 1982; Ref-
stie 1983; Levanduski et al. 1990; Quillet and
Gaignon 1990; Quillet et al. 1991; Palti et al.
1997). However, low survival and high treatment
variation for the production of gynogenetic brook
trout in experiment 1 limited confidence in our
ability to distinguish differences in efficacy be-
tween treatments. In subsequent trials we switched
to testing protocols for the production of triploids,
followed by flow cytometric analysis of the em-
bryos.
In experiments 2, 3, and 4, relative survival con-
4
sistently decreased and percent triploidy consis-
0.3 6 0.5 tently increased with increases in both temperature
1.9 6 1.8*
and duration of heat shock. These effects have
 BROOK TROUT TRIPLOID INDUCTION 257

TABLE 5.—Survival, relative survival (percent survival/percent survival of respective control group), percent triploidy,
and triploid yield for 14 separate females’ eggs exposed to a standard thermal shock of 288C for 10 min initiated at 10
min postactivation to induce second polar body retention.

Control Thermal-shocked
Number Percent Number Percent Relative Percent Triploid
Female of eggs survival of eggs survival survival triploid yield
1 290 0.91 173 0.91 1.00 0.98 0.98
2 280 0.89 200 0.68 0.76 0.98 0.74
3 307 0.86 140 0.52 0.60 1.00 0.60
4 293 0.85 183 0.70 0.82 1.00 0.82
5 263 0.91 169 a
6 249 0.92 181 0.83 0.90 1.00 0.90
7 382 0.82 191 0.68 0.83 1.00 0.83
8 178 0.98 153 0.69 0.70 1.00 0.70
9 155 0.57 184 0.31 0.54 0.98 0.53
10 218 0.37 182 0.30 0.81 1.00 0.81
11 231 0.45 208 0.19 0.42 1.00 0.42
12 312 0.85 226 0.39 0.46 1.00 0.46
13 195 0.95 169 0.71 0.75 0.98 0.74
14 243 0.54 156 0.56 1.00 1.00 1.00
a Discounted due to high mortality for reasons unrelated to experimental design.

been previously observed in the production of trip-
loid salmonids by thermal shock (Chourrout and
Quillet 1982; Solar et al. 1984; Johnstone 1985;
Guoxiong et al. 1989; Levanduski et al. 1990;
Quillet and Gaignon 1990; Dubé et al. 1991).
Effect of time of initiation of the shock on these
measures was inconsistent. In experiments 2 and
3 (time of initiation from 7 to 16 min), differences
within temperature–duration treatments were non-
significant. This is in keeping with findings of oth-
er studies that tested similar ranges for time of
initiation (Refstie 1983; Arai and Wilkins 1987;
Levanduski et al. 1990; Quillet and Gaignon 1990;
Dubé et al. 1991). In experiment 4, however, there
were significant trends towards increasing survival
and decreasing percent triploidy with increasing
time between egg activation and initiation of the
heat shock (from 10 to 22 min). Although time of
initiation of thermal shock may have some effect
on efficacy of a heat shock treatment, this effect
is apparently of much smaller magnitude than that
of shock temperature and duration, within the
range of values we examined.
Our experiments involved a narrow range of
preshock incubation temperatures (11–12 8C). Diaz
et al. (1993) provide evidence that the effect of
heat shock temperature on survival and percent
triploidy in rainbow trout is not solely one of ab-
solute temperature, but also of differential with the
preshock incubation temperature. However, their
observations are confounded by a possible effect
of egg quality associated with prespawning rearing
of the broodfish at the variable preshock incuba-
tion temperatures. Palti et al. (1997) found no sig-
nificant effect of preshock incubation temperature
(8.8–14.68C) in the production of gynogenetic
rainbow trout.
Genetic differences within and between strains
of a species may also have effected variation in
previous results for triploidization rate and triploid
yield experienced under similar heat shock pro-
tocols. Results from experiment 5 indicated con-
sistently high (98–100%) triploidization rates fol-
lowing an (near) optimal thermal shock in females
of the same strain. Differences in triploid yield
(0.42–1.00) solely reflected variation in survival
rate. However, dramatic individual differences in
triploidization efficacy for a given (sub-optimal)
heat shock have been observed in females within
rainbow trout strains (Diaz et al. 1993; Tipton
2000). Differences in triploidization rate and sur-
vival of thermally shocked eggs that experienced
similar protocols are probably related to variation
in egg quality caused by differences in prespawn-
ing rearing conditions between female broodfish
and to the time lag between ovulation, spawning,
and egg activation (Lou and Purdom 1984; Guo et
al. 1990; Levanduski et al. 1990; Diaz et al. 1993;
Palti et al. 1997).
Results for relative incidence of vertebral de-
formities at the initiation of feeding stage were
inconsistent among our experiments. Similarly, in
the only three studies that included measurement
of this trait, Solar et al. (1984) and Guoxiong et
al. (1989) observed increases in deformities
among triploid groups of fry, whereas Chourrout
and Quillet (1982) observed no differences.
In summary, increasing temperature and dura-
258 GALBREATH AND SAMPLES
tion of heat shock over the range of values we
tested, leads to an increase in rate of polar body
retention and a decrease in survival of triploid em-
bryos. Generally, efficiency of a given protocol
does not vary significantly relative to time of ini-
tiation of the shock, within the range of 7–22 min
postactivation. Depending on factors probably re-
lated to egg-quality characteristics, an optimal heat
shock protocol for production of gynogenetic or
triploid brook trout might vary between spawnings
by small differences in temperature (618C) and
duration (63 min). In our trials, the most generally
efficient protocol was a heat shock of newly fer-
tilized brook trout eggs in water at 288C for 10
min initiated at 10–16 min postactivation; this pro-
tocol resulted in relative survival of 68–71%, per-
cent triploidy of 79–99%, and triploid yield of 54–
70%.
Acknowledgments
The authors wish to express their appreciation
to Bob Johns of the Washington Department of
Fisheries and Wildlife and to Derek Boggs, Mal-
lory Martin, and Tom Ort of the North Carolina
Wildlife Resources Commission for contribution
of trout eggs and to Karen Adams, Shanna Farmer,
Brenna Farmer, John Steven Fisher, Chad Hally-
burton, and Kevin Hining for their participation in
the above described experiments. This study re-
ceived financial support from USDA/CSREES/
NRICGP grant: ‘‘Optimization of protocols for the
production of all-female triploid brook trout’’
#9504235.
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