Refining methods to add stable isotope analysis of migratory bird fat to the ornithological toolbox

Prior to migration, songbirds increase food consumption to deposit subcutaneous fat that helps fuel migratory flights, and many periodically pause at stopover sites to rest and refuel for the next flight. We conducted a three-phase, proof-of-concept study to move toward a standardized approach to collecting and preparing fat samples from wild, migrating songbirds, and to explore the feasibility of using stable-isotope analysis on fat tissue to estimate previous stopover locations. First, using fat obtained from a store-bought chicken breast, we conducted an experiment to test whether lipid purification method affects percent lipid yield and stable hydrogen (δ²H) and stable carbon (δ¹³C) isotope values. We found that purified lipids had significantly higher δ²H values and lower δ¹³C values than whole fat samples indicating the need for consistency in sample preparation. Second, to test for repeatability in δ²H and δ¹³C values in purified lipids obtained from wild songbirds, we held Swainson’s Thrushes (Catharus ustulatus; n = 9) in temporary captivity provided with the same diet and local water, and found remarkable consistency in both δ²H (SD = 2.6‰) and δ13C (SD = 0.3‰) values between individuals. Third, we sampled fat from seven species (Magnolia Warbler [Setophaga magnolia], Swainson’s Thrush, Hermit Thrush [Catharus guttatus], Wood Thrush [Hylocichla mustelina], Gray Catbird [Dumetella carolinensis], Red-eyed Vireo [Vireo olivaceus], Northern Waterthrush [Parkesia noveboracensis]) of wild, migrating songbirds at Powdermill Nature Reserve, Pennsylvania, USA as a preliminary investigation into the usefulness of stable-isotope analysis in estimating geographic location of fat assimilation. Among five species assumed to have deposited fat at distant latitudes during spring migration, the three thrush species had δ²H values consistent with latitudes farther south relative to Magnolia Warblers and Gray Catbirds. However, in the two species (Red-eyed Vireo and Northern Waterthrush) assumed to have assimilated fat locally, there was significant variation in δ²H values between the species, and δ²H values did not fit into the pattern observed in distant-origin birds. Although geographic assignment of fat assimilation site in migrating songbirds is more complicated than we expected, stable-isotope analysis of fat is a promising method with potential applications in stopover ecology, diet, and habitat use.