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.