Experimental Diets
Birds had ad libitum access to one of two semi-synthetic diets that had the same macronutrient content as a lipid-rich fruit diet (41% carbohydrate, 13% protein, 30% fat) and differed only in fatty acid composition. We manipulated the proportions of canola, sunflower, and palm oil so that the diets were either high (32%) or low (13%) in 18:2n-6 PUFA (linoleic acid) which was primarily traded off with 16:0 (palmitic acid). Thus, our experimental design requires us to attribute any observed dietary fat effects to both 18:2n-6 and 16:0 content. However, our interpretations focus on the potential effects of 18:2n-6 due to its demonstrated importance in metabolic signaling (Forman et al. 1997, Kennedy et al. 2007, Hamilton et al. 2018, Dick and Guglielmo 2019, Price et al. 2022). The complete list of diet ingredients and amounts have been previously published (citation redacted for initial review). Starlings in two aviaries received a 13% 18:2n-6 diet and two others received a 32% 18:2n-6 diet. The two diets have been shown to produce reliable differences in tissue fatty acid composition of starlings (citation redacted for initial review). On September 1, after we were confident birds were well acclimated to the fat quality in the semisynthetic diets, we began adding a supplementary water-soluble antioxidant, anthocyanin (elderberry powder; Artemis International, Fort Wayne, IN) to the diets of birds in one 13% 18:2n-6 aviary and one 32% 18:2n-6 aviary, producing a 2 X 2 factorial diet manipulation with four diet groups (Figure 1): 13% 18:2n-6, anthocyanin unsupplemented (N = 23), 13% 18:2n-6, anthocyanin supplemented (N = 23), 32% 18:2n-6, anthocyanin unsupplemented (N = 21), and 32% 18:2n-6, anthocyanin supplemented (N = 20). We chose the anthocyanin concentration used by researchers studying the effects of anthocyanin supplementation on food choice and immunocompetence in European blackcaps, Sylvia atricapilla, (Catoni et al. 2008, Schaefer et al. 2008). The anthocyanin supplement was equal to eating 2.8 mg per day which is equal to consuming 17 berries per day based on an average daily synthetic diet consumption of ca. 35 wet g day-1 (as observed in food intake trials in this study).