Materials and methods
Prey items were obtained from RodentPro in April 2023 and stored frozen until analysis. Smaller prey including smaller sized rats, pup Guinea pigs and all sizes of mice were sold in bulk in packs of 10-25, five individuals of each species group were processed. Quail were sold in a pack of five and all five were processed and larger prey (medium rabbits and extra large rats) were sold individually, and we ordered three individuals of each. Each individual was weighed, and then the hair was removed using a Remington Ultimate Precision Detail Trimmer Black NE3160, and feathers were plucked manually. The individual was reweighed to determine contribution of keratin outer covering to total organism weight. The trimmer was cleaned between individuals using water and 70% isopropyl alcohol. The individual was then homogenized using a OXO Good Grips Meat Tenderizer and VEVOR Hand Operated 304 Stainless Steel Multifunction Manual Meat Mincer (pup rats and large mice were too small for the meat grinder and were only homogenized with the meat tenderizer). Between individuals, the meat grinder was fully disassembled and each component plus the meat tenderizer were cleaned with soap and water, then sanitized in 95% EtOH. A subsample of hair or feathers was taken from each individual to analyze separately, and the remainder of this material was blended back into remaining homogenized mass with the meat grinder. All samples were dried at 60°C for at least 24 hours, then ground into powder using a mortar and pestle or finely diced with a scalpel blade.
We had three tissue types for analysis: 1) homogenized mass of whole body with keratin included (WB, three replicates per individual); 2) homogenized mass of whole body with keratin removed (KR, three replicates per individual), and 3) keratin tissue only (K, one replicate per individual). Isotopic values from the three replicates of WB and KR were averaged, and the mean value was used in statistical analysis for each tissue type.
Samples weighing between 0.400 and 0.600mg were placed into 5mm tin capsules and analyzed at the Stable Isotope Mass Spec Lab at the University of Florida on one of two systems. Samples were combusted on N.C. Technologies 8020 Elemental Analyzer or a Costech 4010 Elemental Analyzer at 1000°C interfaced to a ConFlo IV before passing to a Thermo Electron Delta V Advantage isotope ratio mass spectrometer. Sample gas was measured relative to laboratory reference N2 and CO2 gases. All are expressed in standard delta notation relative to Vienna Pee Dee Belemnite (carbon) or AIR (nitrogen). Analytical precision was calibrated by the standard deviation of the USGS40 replicates (n = 30, 0.08 ‰ [δ 13C] and 0.07 ‰ [δ 15N]).
Some mixing model scenarios have demonstrated that lipid-correction can substantially alter the estimated diet composition and interpretation of diet reconstruction, and lipid extraction may not be appropriate when using the isotopic composition of keratinous tissues to examine diet21,22. Therefore, samples in this study were not lipid extracted to best represent the isotopic values of the whole prey item.