Experiment 1 –Timeline for Diet Manipulation Experiment
In the first experiment, zebra finches were given one of three diet
treatments that varied in protein and lipid content. We investigated how
diet influenced complement activity, baseline and stress-induced
corticosterone concentrations, and the gut microbiome. Birds were kept
on a 14 L: 10 D light cycle and individually housed in 24”x16”x16”
cages. Each cage had two perches, a water dish, and one food dish in
which birds were fed ad libitum . Diets consisted of hulled
millet, egg white, egg yolk, vegetable oil, and sorbic acid
(preservative) in agar blocks. Birds were placed on an acclimation diet
with equal ratios of lipid and protein for 5 days prior to experimental
treatment. Birds were then randomly assigned to one of three diet
treatments (Figure 1; day 0), which they remained on for eight
additional days. All diets were isocaloric and only varied in the ratio
of protein and lipid content. All diets contained similar concentrations
of carbohydrates (62-66 % of the total metabolizable energy of the diet
(TME)). Diet treatments consisted of a high fat diet (25% lipid and
13% protein of TME; high fat diet: n=11, 7 males, 4 females), a diet
with equally balanced ratios of lipid and protein (i.e., the acclimation
diet; 17% lipid and 17% protein of TME; equal ratio diet: n=12, 8
males, 4 females) and a high protein diet (13% fat and 25% protein of
TME; high protein diet: n=12, 8 males, 4 females). Three desiccation
controls for each diet type were also weighed daily and the average
desiccation values for each diet type (high fat, equal ratio, high
protein) were subtracted from feeding values to account for daily
changes in food mass due to desiccation.
To assess the effects of diet on physiological endpoints and gut
microbiota, blood samples and cloacal swabs were collected on the day
that acclimation diets were switched to manipulation diets and eight
days after the manipulation diets were implemented. Body mass and fat
score data were recorded for each bird at all sampling time points. All
blood samples were collected between 0830 – 0930 CDT to avoid
differences in physiological endpoints due to variation in daily
rhythms. Within 3 min of entering the room, a baseline blood sample of
120 uL was collected from the wing vein of each individual to be used to
determine baseline concentration of plasma corticosterone and hemolytic
complement activity. Next, we used sterile swabs to swab the cloaca of
each bird. Swabs were placed in 300 μl RNAlater (Invitrogen, Thermo
Fisher Scientific) and frozen at -80°C. Birds were then weighed and
placed into a paper bag. Thirty minutes after entering the room, a
second blood sample was collected to determine stress-induced
concentrations of plasma corticosterone. All blood samples were
collected into heparinized capillary tubes and immediately placed on
ice. Tubes were centrifuged for 3 minutes to separate blood plasma from
erythrocytes and plasma samples were stored at -20°C. All research
protocols were approved by the Oklahoma State University Institutional
Animal Care and Use Committee.