Figure 2.

Example serum used for comparison between the standard and modified FITC-d procedures. Serum samples were obtained from an experiment with a three treatment diets with ad libitum access to feed and water. Negative (no stress) control diet was a standard corn-soybean meal diet meeting the Aviagen nutrient recommendations (21% crude protein; 201g/kg). Both the positive stress control and Spirulina platensis algae feed treatment groups had a 15% reduction in crude protein (still meeting all amino acid requirements), but the Spirulina treatment group had 50% of available crude protein from soybean meal replaced with a protein equivalent amount of Spirulina algae meal (Pond Tech, Inc., Markham, Ontario, Canada). All groups (Ross-708 line broilers) received the negative control diet from 0-14 days-of-age as a crumble, followed by respective pelleted treatment feed for the remainder of the trial (up to 37 days-of-age). The FITC-d assay was performed and serum samples were collected at termination of the trial. (A) Color difference observed in normal control serum (top row) compared to Spirulina-fed/beta-carotene-containing broiler serum (bottom row). (B) Standard curve with imposed linear regression line and equation. This equation will be used to calculate the ng/mL of FITC-d in the unknown sample by solving for “x”. To calculated this, the sample fluorescence value (with background autofluorescence removed) will be the “y” value, divided by the slope (slope “m” is 0.0497 in this example). Example using this standard curve: if raw sample fluorescence is 11 and treatment group background autofluorescence is 3, (11-3)/0.0497 = 160.97 ng/mL of FITC-d in the sample. (C) Example data of Spirulina-fed broiler serum samples run with the standard protocol (blanked to serum pooled from all treatment groups) compared to the modified protocol (blanked to per treatment background autofluorescence).