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.