Blood is widely used to explore the physiology and stable isotope ecology of wild animals. Ecological immunologists utilize blood samples to investigate how immune function changes seasonally, under different environmental conditions, and between individuals, particularly in the context of sexual selection (Hasselquist 2007). Disease ecologists and veterinarians also rely on blood samples to measure specific antibodies that indicate prior infection with particular pathogens (e.g. Hoye et al. 2011). Furthermore, the concentrations of various stable isotopes in blood samples can also be used to reconstruct the composition of an individuals’ diet and determine its trophic position (Inger & Bearhop 2008), as well as assess reproductive investment strategies (Klaassen et al. 2001), patterns of habitat occupancy (Inger et al. 2008) and migration routes and timing (Hobson & Norris 2008). Each of these immunological and stable isotope applications often requires blood cells to be separated from the fluid component, plasma or serum, by centrifugation prior to analysis.
Prolonged storage of uncentrifuged human, reptilian and avian blood samples has been shown to result in significant changes to a wide range of biochemical assays owing to glucose depletion, the movement of water into cells and leakage of intracellular constituents (Boyanton & Blick 2002; Harr, Raskin, & Heard 2005). Immediate separation of plasma or serum from cells has therefore been advocated to optimize analytical results (Boyanton & Blick 2002). Yet, field conditions often result in a delay between blood collection and sample separation and storage, particularly when animals are captured in remote locations or in large numbers. Blood fluid biochemistry has also been shown to differ between samples in which clotting is prevented (producing plasma) and those in which clotting is allowed (producing serum) (Ceron et al. 2004; Mohri, Narenji Sani, & Masoodi 2008). Furthermore, collection-to-centrifugation delay may differentially affect analytical results depending on whether the blood is allowed to clot or not (Boyanton & Blick 2002). One challenge in investigating immune function and stable isotope ecology through blood sampling is, therefore, to recognize the extent to which different methodological procedures in processing wildlife blood after collection affect the biological factors of interest.
Given that wildlife studies are inconsistent in their use of plasma or serum, and in the time between collecting blood and separating the cells from the fluid fraction, this study investigates the potential for variation in blood processing to influence seven widely used measures of immune function and stable isotope composition. Specifically, this study addresses whether: (i) the length of time between taking a blood sample and separating the cellular and fluid components affects the results of immunological and stable isotope assays; (ii) the type of blood fluid (plasma or serum) affects these results; and (iii) identified methodological effects are consistent between species. Two waterfowl species – Bewick’s Swans (Cygnus columbianus bewickii, Yarrell) and Mallards (Anas platyrhynchos, Linnaeus) – are used because blood-based measurements of immune function, disease exposure, diet choice and foraging habitat have been investigated in a range of waterfowl species and because relatively large birds are required to furnish the volumes of blood needed for a repeated measures design.