Flight Training
In order to assess the impact of diet and endurance flight on the endogenous antioxidant system, three flight-trained birds were flown in a wind tunnel for four days of pre-training followed by fifteen days of flight training. Such a flight training regime has demonstrated success at eliciting long-duration flights in starlings (Engel et al. 2006). The wind tunnel was set to 12 m/s windspeed, 15°C, and 70% humidity, and birds were fasted for 1 hr prior to all flights. Pre-training (PT; days -4 to -1) consisted of allowing training birds to fly between their flight cage and the wind tunnel followed by 20 minutes of habituation time per day in the wind tunnel with a perch. These initial four ‘pre-training’ days were not included in the reported overall training time, since birds could rest when needed. Starlings in the flight-training group then participated in a fifteen-day training regimen (FT) that consisted of increasing periods of flight (20 min – 180 min) in the wind tunnel as follows: days 1-4, 20 min each day; day 5-6, 30 min each day; day 7, 60 min; day 8, 90 min; day 9, 30 min; day 10, 120 min; day 11, 180 min; day 12, rest day; day 13, 60 min; and day 14, 30 min. This flight training culminated in a flight on day 15 that lasted as long as birds would voluntarily fly, up to 6 hrs. To determine fuel use during flight, body condition (fat and lean masses) was measured using a quantitative magnetic resonance machine (QMR; Echo Medical Systems, Houston, TX) immediately before and after the final flight. Energy expenditure during the flight was estimated by multiplying the mass of fat and lean tissue lost during flight by their respective energy densities, adding them, and dividing by fight duration (full methods and results reported previously, citation redacted for initial review). The final flight was on average 193 +/-71 (SD) min and the maximum was 360 min. In order to test the acute effects of flight, we blood sampled flight-trained birds on the morning of day 14 at 8:00 hr before their 30 min flight (Pre-flight, PF), this is at the same time that they would have been blood sampled before their longest flight on day 15. We sampled birds the morning prior to their long-flight to avoid excess stress associated with handling immediately prior to their long-flight. Since all experimental conditions were the same among the two days, we assume that this blood sample reflects the state of the flight-trained birds before a flight. An after-flight (AF) blood sample was taken immediately after the experimental flight (day 15) ranging from 11:00-14:00 hr. Flight-trained birds were returned to their flight cages for two days to recover from their last flight. At 1400hr – 1500hr on days 16 and 17 the untrained and trained birds, respectively, in each cohort were blood sampled for the final Recovery sample (RC). Birds were euthanized by cervical dislocation while under isofluorane anesthesia and the liver and pectoralis muscle samples were collected and immediately weighed. All tissues were flash frozen in liquid nitrogen and stored at -80°C until analysis. This sampling design allowed us to compare oxidative status in the liver and pectoralis of untrained (control) birds and flight-trained birds that had recovered (for 48 hrs) from their longest flight on day 15. The liver is a crucial food processing organ, especially for exercising birds that rely on fat to fuel flight (Guglielmo, 2018; Scott R. McWilliams, Guglielmo, Pierce, & Klaassen, 2004), and the pectoralis is the major skeletal muscle used to power flight (Biewener, 2011). Thus, these two metabolic tissues relied on by flying birds likely have high antioxidant capacities to protect against oxidative damage, yet it remains unknown how the oxidative status of both these tissues along with that of plasma respond to flight training, dietary fats, and dietary antioxidants or how these different classes of antioxidants work together to protect individuals against oxidative damage.