Experimental Timeline
On September 21st we randomly assigned groups of 5
starlings to each of twenty cohorts (Figure 1) although this cohort
selection was stratified by extent of Pre-basic I molt (Ginn and
Melville 1983) – birds furthest along in molt were flight-trained
earliest. This stratified random sampling of individuals ensured that
all birds had completed their flight feather molt prior to flight
training. One cohort from each of the four diet groups were tested
sequentially over 10 days although the sampling order of the diet groups
was randomly assigned so a diet group was not consistently sampled first
or last. Thereafter another set of cohorts from each of the four diet
groups were tested and so forth until the 20 cohorts of starlings had
been tested by early December.
On September 23rd, and continuing every three days,
the 5 individuals from each selected cohort were removed from their
aviaries, and we randomly assigned 2 birds as untrained birds and 3
birds as flight-trained birds. Each selected cohort was initially placed
in individual cages (0.6m x 0.5m x 0.5m) for two days (days -9 and -8)
to measure food intake and another two days (days -7 and -5) to measure
basal and peak metabolic rates (citation redacted for initial review).
On day -5 we returned the two untrained birds to their original aviary
and moved the 3 flight-trained birds to a 0.8m x 1.5m x 2m flight
aviary.
Each cohort was blood sampled at consistent time points throughout the
25-26 day experimental period (Figure 1: Background, BG; Pre-Flight, PF;
After-Flight, AF; Recovery, RC). Birds were fasted for at least 1 hour
before all blood sampling time points and were bled within 30 minutes of
capture. Blood samples were taken from the brachial vein, and within 10
min of blood sampling the plasma was separated from the red blood cells
following centrifugation at 11,000 g for 10 minutes (Damon/IEC
Division, IEC MB centrifuge, micro hematocrit). Plasma was stored at
-80°C until OXY-adsorbent test and d-ROM analyses. A blood sample was
obtained 9 days prior to the start of flight-training at 8:00 hr in the
morning to obtain background oxidative measurements (BG) for each
individual. Measuring indices of circulating oxidative status is
important since an unbalanced oxidative state in the plasma would
indicate damage to crucial molecules transported by the plasma (e.g.
fatty acids, hemoglobin, cytokines) to muscles and organs.