Extraction Protocols
The UFP was divided into 30 equal replicates, with 10 replicates allocated to the methanol protocol (MP), 10 to the keratinase protocol (KP), and 10 to the OKP.
The MP extraction procedure followed published methods (
Bortolotti et al., 2008). Briefly, samples were placed into glass vials containing 10 ml of HPLC grade methanol and sonicated in a water bath for 30 min (
Bortolotti et al., 2008). After sonication, the feather mince was incubated overnight in a shaking water bath at 50 °C (
Bortolotti et al., 2008). The following day, the feather mince in methanol was filtered with vacuum filtration (Whatman, #4) into new glass tubes, and any remaining mince remnants and original vials were rinsed with 2-5 ml of HPLC grade methanol (
Bortolotti et al., 2008). The filtered samples were concentrated by evaporation using vacuum centrifugation at 40 °C and 1200 rpm (Jouan RC 10-10).
KP was performed as published (
Alba et al., 2019). The digestion medium was prepared with a ratio of 1 gram of keratinase (Cibenza: IND900, Novus) per 30 mL of phosphate buffered saline at a pH of 9 (
Alba et al., 2019). A ratio of 2 mg of feather mince per mL of digestion medium was used with 5 mL volumes for digestion. Extraction tubes were capped and incubated at 45 °C, and digestion progress monitored over the next 5 days. On day 5, when samples had liquefied into a slurry with slight particulates, extracts were subjected to SPE (C-18 Bond Elute, Varian) (
Alba et al., 2019). Solid-phase extraction cartridges were conditioned with 1 mL of methanol and water. The total feather extracts, including solid and aqueous components, were then loaded into sterile syringes, and passed through attached 25 mm, 0.45 µm syringe filters into extraction cartridges. Samples were eluted with two 0.5 mL washes of 2 % acetic acid in acetone into glass tubes. The original KP method took samples to complete dryness via heat and vacuum-based centrifugal evaporation (40 °C and 1200 rpm), respectively. At dryness a waxy adherent material remained in the sample tubes.
The OKP was performed identically to KP except for the drying step. For the OKP, SPE was followed by heat and vacuum-based centrifugal evaporation (40 °C and 1200 rpm), respectively, that was stopped when a 5-10 µL convex droplet of extract remained. The droplet was then utilized for analysis by ELISA by combination with 1X assay buffer from the kit to a final volume of 500 µL.
Optimized Protocol Validation
To ensure results of the OKP were accurate representations of actual feather CORT concentrations a spike and recovery test was conducted using UFP as the sample. Prior to start of enzymatic digestion sufficient CORT standard (Corticosterone ELISA Standard, Cayman Chemicals) was added to UFP to create a sample set that contained 4 replicates of the 5 most central points of the standard curve for the chosen ELISA assay kit, namely 0.3125 pg CORT/mg feather, 0.625 pg CORT/mg feather, 1.25 pg CORT/mg feather, 2.5 pg CORT/mg feather and 5 pg CORT/mg feather. The four replicate preparations were assayed in duplicate for each concentration to create 4 averaged values at each concentration. Four replicates of a blank, un-spiked, UFP sample were included to establish basal UFP CORT concentration. All feather/keratinase solution tubes contained a 2 mg feather per 1 mL of keratinase digestion solution with 5 mL used for each digestion. Enzymatic digests used the OKP extraction protocol in which extracts were taken to incomplete dryness, and the resultant 5-10 µL extract droplet was resuspended to a total volume of 500 µL in 1 X assay buffer provided within the manufacturer’s kit and used for ELISA assay. After ELISA analysis, the average CORT concentration of the unspiked UFP sample was subtracted from each spiked sample and measured concentrations were calculated against theoretical yield to determine percent return.