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THE WILSON JOURNAL OF ORNITHOLOGY • Vol. 123, No. 3, September 2011 
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Days relative to first egg 
FIG. 1. Plasma testosterone concentrations in adult 
Tree Swallows relative to the date when the female of the 
pair initiated egg laying. (A) Data from males. (B) Data 
from females. Incubation begins -4 days after the first egg 
is laid and eggs hatch -14 days later. Testosterone 
concentrations are displayed on a logarithmic scale. 
concentration in plasma directly using I ,as labeled 
testosterone. The kit was modified by running all 
volumes at 25% of the suggested kit volume. 
Samples were run in two separate assays. The 
intra-assay and inter-assay coefficients of varia¬ 
tion were 0.11 and 0 . 07 , respectively. 
We calculated the time in days relative to the 
initiation of laying in that bird's nest for each 
sample. We assumed egg-laying began on the 
mean date of initiation of laying for the colony 
(18 May) for individuals sampled prior to egg- 
laying that did not remain to nest in our colony 
(n = 11). Varying this estimated egg lay date by 
±7 days did not affect our results significantly. 
Incubation in Tree Swallows typically begins with 
the penultimate egg (-4-5 days after laying 
initiation) and hatching occurs ~ 14-15 days after 
the onset of incubation (Zach 1982). 
Statistical Analyses ,—Plasma testosterone con¬ 
centrations were not normally distributed and we 
log-transformed testosterone concentrations for all 
TABLE 1. Mean testosterone (T) concentrations in 
adult male and female Tree Swallows during three stages ol 
the breeding season in nesting colonies near Ames. 
Iowa, USA. 
Breeding stage 
Mean ± SD T (ng/ml) 
n 
Males 
Nest establishment 
0.63 ± 0.86* 
7 
Incubation 
0.28 ± 0.26" 
7 
Nestling care 
0.03 ± 0.05” 
11 
Females 
Nest establishment 
0.06 ± 0.09 a 
10 
Incubation 
0.01 ± 0.01* 
11 
Nestling care 
0.02 ± 0.02* 
9 
Breeding slages with different superscripts have significantly different 
mean testosterone (T) concentrations in Tree Swallows (Tukev-Kramer HSD 
P < 0.05). 
statistical analyses. We performed an analysis of 
variance with plasma testosterone as the depen 
dent variable using a model that incorporated time 
in days relative to the first egg, males or females, 
and the interaction between the two variables. 
There was a significant interaction between males 
or females and time relative to egg lay; thus, wc 
used separate Tukey-Kramer honestly significant 
difference (HSD) tests to compare mean testos¬ 
terone concentrations among the different breed¬ 
ing stages for males and females. We used JMP 
statistical software for all statistical analyses (SAS 
Institute Inc. 2009). 
RESULTS 
Circulating testosterone concentrations in Tree 
Swallows ranged from 0.01 to 2.4 ng/ml in males 
and 0.01 to 0.23 ng/ml in females during the 
breeding season (Fig. 1). Gender (F, iS3 = 127 
P = 0.0008). days relative to the first egg tF/..v = 
5-7. P ~ 0.006), and the interaction between the 
two variables (F, 51 = 4.9, P = 0.01) significant!) 
affected testosterone concentration. Testosterone 
concentrations over the breeding season were 
about an order of magnitude higher in males than 
in females (male mean = 0.27 ng/ml: female 
mean = 0.03 ng/ml). 
Circulating testosterone concentrations of 
males during the nest establishment stage did 
not differ from those during the incubationstage, 
but male testosterone concentration decreased 
significantly (Tukey-Kramer HSD. q = 
P < 0.05) during the nestling stage (Table 0- 
Mean testosterone concentrations of females did 
