Caldarone et al.: Biological indices of growth rate and nutritional state of Salmo salar 
291 
a microfuge vial containing 150 jiL 1% N-lauroylsar- 
cosine. The vial was placed in an ice slurry and the 
sample was sonicated for three 5-second pulses fol- 
lowed by 45 minutes of vortexing at room temperature. 
From that point onward, we followed the protocol of 
Caldarone et al.^ for nucleic acid analysis. Results from 
duplicate plugs were averaged. The ratio of the slope 
of the DNA standards to the slope of the RNA stan- 
dards was mean=2.5, SD=0.05 {n-7 microplates). This 
value can be used to convert the reported RNA/DNA 
data for direct comparison with other published stud- 
ies (Caldarone et al., 2006). The remaining extract was 
stored frozen and later analyzed for protein content by 
using a bicinchoninic-acid-based assay adapted for a 
microplate format (Caldarone, 2005). Nucleic acids (pg) 
are expressed as a RNA to DNA ratio (RNA/DNA; pg/ 
pg), RNA to protein ratio (RNA/pro; pg/mg) and DNA 
to protein ratio (DNA/pro; pg/mg). 
Calculations of growth rates Individual instantaneous 
weight-based growth rates (per d) were calculated with 
the following formula: 
growth rate (G) = (In WWt 2 - lnWWti )/(/2 - /i) 
(Ricker, 1979), (1) 
where WW = the wet weight of an individual at time 
t (day). 
For comparison with previously published data, growth 
rates were converted to specific growth rates (% per d) 
with the following formula: 
SGR=100(eG - 1). (2) 
Growth rates for fish in the fed and fasted treatments 
were calculated from day 0 until the day the fish were 
sacrificed. Growth rates were calculated from day 0 
until day 11 for the fasted portion of the refed treat- 
ment fish; for the fed portion of the refed treatment 
fish, growth rates were calculated from the first day 
of refeeding (day 11) until the day the fish were sacri- 
ficed. Because there is inherent variability in measur- 
ing the wet weight of fish, coupled with small changes 
in weight over short time intervals, growth rates from 
time intervals <4 days were not included in any of the 
statistical analyses. 
Data analysis To examine the effect of the feeding 
treatment and sampling day on growth rate, RNA/ 
DNA, RNA/pro, DNA/pro and IGFl, a 2-way multivari- 
ate analysis of covariance (MANCOVA) for unbalanced 
design was used with WWjnit as the covariate. When 
interactions were significant, feeding treatment was 
nested in day, and follow-up comparisons were exam- 
ined by using Tukey’s HSD multiple range test. Lin- 
ear growth rate models with all combinations of the 
^ Caldarone, E. M., M. Wagner. J. St. Onge-Burns, and L. J. 
Buckley. 2001. Protocol and guide for estimating nucleic 
acids in larval fish using a fluorescence microplate read- 
er. Northeast Fish. Sci. Cent. Ref. Doc. 01-11, 22 p. [Avail- 
able at website] 
4 biochemical variables plus WWi^jt were constructed. 
Akaike’s information criterion for small sample sizes 
(AICc; Wagenmakers and Farrell, 2004) was used to se- 
lect the best candidate model from the 31 models test- 
ed. Because of high collinearity of RNA/DNA with the 
other 3 biochemical indices, all combinations of models 
without the RNA/DNA term were also investigated. To 
examine the response of the 4 biochemical indices and 
growth rate in individual fish to food withdrawal or in- 
troduction, paired initial and final data from individual 
fish from both the starved and refed fish were analyzed 
by using a repeated measure ^-test. 
Within each feeding group, a Dunnett 2-tailed ^-test 
with WWinit as a covariate was used to detect changes in 
growth rate and the 4 biochemical variables compared 
with those in a control. Day 0 values were specified as 
the control for the biochemical variables for both fasted 
and fed treatments. The average growth rate of all fish 
from the time they were tagged until day 0 (50 days) 
was used as the control growth-rate value with the un- 
derstanding that growth rates during this time would 
have been less than optimal. For the refed treatment, 
values for day 11 (day they were refed) were used as 
the control for all variables. An ANCOVA was used to 
test whether the slope of the relation of growth rate 
to the measured biochemical indices was significantly 
different between the fed and refed groups. All statisti- 
cal analyses were carried out with SAS software, vers. 
9.3 (Statistical Analysis Software Inst., Inc., Cary, NC) 
with a significance level set at P<0.05. 
Results 
At the start of the experiment (day 0) -78% of the 
fish exhibited frayed and -12% exhibited eroded dor- 
sal and pectoral fins. The frequency and severity of 
these conditions did not change throughout the experi- 
ment and there were no mortalities during the study. 
All fish appeared to be immature and sex was not a 
significant factor in any of the statistical analyses. On 
day 0, WWjnit ranged from 43 to 132 g and FL from 
18 to 23 cm. Initial size distributions (WWjnit) between 
feeding treatments were not significantly different (fed 
mean=76 g, SD=12; fasted mean=75 g, SD=13; fasted 
then refed mean=80 g, SD=4). 
Weight-based growth rates of the fish responded 
quickly to changes in food availability. Fasted fish lost 
weight beginning on day 7 and by day 11 their growth 
rates were statistically significantly less than the con- 
tinually fed fish (Fig. lA). Negative growth rates of 
the fasted fish remained constant throughout the ex- 
periment (Dunnett, P=0.747), whereas fed fish growth 
rates increased in relation to day 0 rates (Dunnett, 
P<0.0001). On day 19, fed fish had faster growth rates 
than refed fish, whereas the relation was reversed on 
day 23. Refed fish grew significantly faster than fasted 
fish beginning 4 days after refeeding (day 15). During 
the experiment we noted that the feeding intensity 
of the salmon visibly decreased when the total num- 
