Electronic Supplementary Material
Egg injections:
Injection solutions were made by dissolving powdered testosterone (Steraloids,
Inc.) in ethanol, adding 200 µL of the ethanol solution to 5mLs of sesame oil, conducting
multiple rounds of vortexing, and incubation in a 37°C water bath until ethanol was
evaporated. On each egg, a site approximately 10mm below the intersection of the
longitudinal and latitudinal center lines was prepped with a 70% alcohol swab. A hole
was manually drilled through the shell using a small drill bit. A 100 µL Hamilton syringe
with a luer tip and a 25G, 1-inch disposable needle (BD #305125) was fully inserted at a
45 degree angle towards the airsac at the blunt end of the egg (a distance that places the
needle tip in the middle of the yolk), and 50 L was gently ejected. Holes were sealed
with a dot of superglue, eggs allowed to rest at room temperature for at least 5 hours, then
placed in an incubator. The effectiveness of this technique was validated by injecting red
food coloring into 20 practice eggs in the same manner, opening eggs and verifying that
the dye was found solely in the yolk.
172 unincubated, fertile eggs were injected as described above. Prior to injection
all eggs were weighed and allocated to alternating treatments by mass, such that there
was no difference in egg mass or distribution of egg mass between treatments (mean
±SEM): 58.44g + 3.92 for control and 58.54g + 3.94 for testosterone. Of these, 122
(71%) hatched. 28 of the unhatched eggs were control (56% of unhatched), 22 were
testosterone injected (44%). Upon hatching, chick sex (69 female (37 testosterone, 32
control) and 53 male (32 testosterone, 21 control) was determined morphologically,
based on wing length and comparison of the primary and secondary feather lengths.
Males were used in a separate experiment. The majority of the females were euthanized
at hatch for tissue collection; 10 females from each treatment were chosen randomly to be
included in this study. Hatch mass did not differ between females that were euthanized
and those that were retained for the experiment (mean ±SEM) euthanized versus retained:
44.32g ± 0.59 and 44.93g ±0.56 for control, 45.44g ± 0.63 and 45.06g ± 0.70 for
testosterone.
Reactive oxygen metabolites (ROMs) and Total plasma antioxidant capacity (TAC) :
We measured reactive oxygen metabolites (ROMs) using the d-ROMs test
(Diacron International, Grosseto, Italy), which measures the level of hydroperoxides,
compounds that signal lipid and protein oxidative damage. We diluted 10 l of plasma in
200 l of the provided acidic buffer solution, and then read the plate kinetically (one read
per minute) for the next 10 minutes. Absorbance was measured at 490nm (BioTek
ELx800, VT, USA) and we calculated change in ROMs concentrations (in mM of H2O2
equivalents) from these absorbencies by taking the difference between the reading at
minute 3 and minute 0, dividing by 3 minutes, and multiplying by the constant 9000 (all
per the manufacturers specifications).
The total plasma antioxidant capacity (TAC) was measured using the OXYAdsorbent test (Diacron International, Grosseto, Italy), which measures the effectiveness
of the blood antioxidant barrier by quantifying its ability to cope with oxidant action of
hypochlorous acid (HClO). We diluted 10 l plasma in 990 l of distilled water; we then
mixed 5 l of this diluted plasma with 195 l of the provided HClO solution and
continued by following the manufacture’s instructions. We measured absorbance at
490nm (BioTek ELx800) and we calculated TAC (in mM of HClO neutralized).
Single Cell Gel Electrophoresis (SCGE) assay:
Erythrocytes were diluted in 1X phosphate buffered saline, suspended in 0.75%
low melting point agarose, and plated onto Trevigen Flare slides (Gaithersburg, MD,
USA) for each of the three damage assessments. Slides measuring in vivo damage were
immediately exposed to lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1%
Triton X-100, 10% DMSO, pH 10.0). Challenge slides were exposed to 200µM H2O2 for
five minutes prior to lysis. Repair slides were exposed to 200µM H2O2 for five minutes
followed by incubation at 37°C and 5% CO2 in RPMI complete media for one hour prior
to lysis. After lysis, cells were digested by Endonuclease III and denatured prior to a 20
minute electrophoresis at 35 V and 300 mA in an 1X alkaline buffer (10 N NaOH, 200
mM EDTA, pH>13.0). During the electrophoresis, undamaged DNA remains
consolidated in the form of a distinct head while degraded strands migrate out of the head
leaving an elongated tail. Following electrophoresis all slides were neutralized (0.4 M
Tris base, pH 7.5), washed in 100% EtOH, air dried, and stored at room temperature until
visualization. To visualize, slides were stained with DAPI and imaged via confocal
microscopy. DNA damage to erythrocytes was quantified using Comet Score software.