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.