Development and evaluation of a minimally invasive sampling technique to estimate the age of living birds

Using pest species in initial studies of pentosidine (Ps) aging research for birds may be the catalyst to discovering more effective population control strategies for pest, invasive, and hard to manage birds. Pentosidine is an irreversible, stable, fluorescent, collagen cross-link, created through the Maillard reaction, which has been found to accumulate throughout the lifetime of an organism in various body parts such as skin, lens crystalline, and dura matter. Pentosidine assays are more accurate at determining the age of adult birds in comparison to plumage coloration, eye and mouth color, feather wear, and molt sequences due to the discovery that Ps accumulates with age in the skin of birds. Past studies, however, have only taken place on deceased birds. To be considered a more generally useful tool for wildlife management studies, a procedure to obtain skin samples from living birds is needed. The objective of this project was to develop a minimally invasive sampling technique to age live birds through Ps analysis by (1) determining if differences exist in Ps concentration between the breast and patagium of black vultures ( Coragyps atratus), monk parakeets (Myiopsitta monachus), and double-crested cormorants (Phalacrocorax auritus), (2) determining if differences exist in Ps concentration of 6 mm2 and 20 mm2 skin samples, and (3) determining if healing rates differ between the breast and patagium and between wounds closed with tissue glue and wounds closed with sutures. Pentosidine concentrations were similar between the breast (x¯ = 8.9 pmol/mg collagen, SE = 0.55) and patagium (x¯ = 8.9 pmol/mg collagen, SE = 0.51) of black vultures (P = 0.97) as well as the breast (x¯ = 11.2 pmol/mg collagen, SE = 1.10) and patagium (x¯ = 10.6 pmol/mg collagen, SE = 1.10) of deceased double-crested cormorants (P = 0.10). Pentosidine, however, was significantly higher in the breast (x¯ = 15.9 pmol/mg collagen, SE = 1.30) than the patagium (x¯ = 11.5 pmol/mg collagen, SE = 1.10) of monk parakeets (P \u3c 0.0001). The Ps concentration was marginally higher in 6 mm2 skin samples (x¯ = 12.6 pmol/mg collagen, SE = 1.19) when compared to 20 mm2 skin samples (x¯ = 11.3 pmol/mg collagen, SE = 1.23) of cormorants (P = 0.02). Four new age curves were developed for cormorants (our linear breast skin age curve, our curvilinear breast skin age curve, our linear patagial skin age curve, and our curvilinear patagial skin age curve) and compared to the original Fallon age curve. Age estimates and actual ages for cormorant breast and patagial skins were found to be similar when using our linear and curvilinear breast and patagial skin age curves, but there were significant differences between actual and estimated ages for breast and patagial skin when using the Fallon age curve (P \u3c 0.0001 and P \u3c 0.0001 respectively). The mean estimated ages for all 5 age curves were found to be accurate to within approximately 1½ years (17.4 months). For 6 mm 2 skins, there was a marginal difference between real and estimated age when using our curvilinear patagial curve (P = 0.04), but real and estimated ages were similar for 6 mm2 skins using our linear patagial curve and 20 mm2 skins using our linear and curvilinear patagial curve. The mean estimated ages for the 2 age curves were found to be accurate to within approximately 2½ years (28.3 months). Seven living cormorants were caught at Bluff Lake, part of Noxubee National Wildlife Refuge in Mississippi, and were used to test the live sampling protocol. After a 6 mm2 biopsy was removed from the breast and patagium, the birds had their wounds closed with either tissue glue (n = 3) or sutures (n = 4). Wounds closed with tissue glue ( x¯ = 14.5 days, SE = 1.12) healed significantly faster than those closed with sutures (x¯ = 17.3 days, SE = 0.66) (P = 0.0003) but the healing rate was similar for the breast (x¯ = 15.9 days, SE = 1.36) and patagium (x¯ = 15.8 days, SE = 1.85) (P = 0.79). Our finding is that live sampling can be safely done for live birds. Our recommendations are to live sample birds from the patagium with a 6 mm2 biopsy punch and to close the wounds with tissue glue. Use of this technique could provide insight into senescence, reproductive success, and behavioral changes for different adult age classes as well as improve management strategies for pest and endangered/threatened species

Refinement of Biomarker Pentosidine Methodology for use on Aging Birds

There is no reliable method for determining age for most species of long-lived birds. Recent success using the skin chemical pentosidine as a biomarker has shown promise as an aging tool for birds. Pentosidine levels have been determined only from the breast tissue of carcasses, and we sought to refine the procedure with respect to biopsy size and location for safe and effective use on living birds. We compared pentosidine concentrations in 4 skin-size samples (4, 6, 8, and 20-mm diameter biopsies) from the breast of black vulture (Coragyps atratus) carcasses. We also compared pentosidine levels from breast and patagial tissue to document potential differences among collection sites of deceased vultures (with unknown ages) and monk parakeets (Myiopsitta monachus; with actual, minimal, and unknown ages). Pentosidine concentrations (pmol pentosidine/mg collagen) were similar among the 4 sizes of vulture breast skin (P = 0.82). Pentosidine concentrations for the breast (x̄ = 8.9, SE = 0.55, n = 28) and patagium (x̄ = 8.9, SE = 0.51, n = 28) of vultures were similar, but in parakeets, pentosidine was higher in the breast (x̄ = 15.9, SE = 1.30, n = 105) than the patagium (x̄ = 11.5, SE = 1.10, n = 105). We made pentosidine-based age estimates for vultures and parakeets using a general age curve for wild birds. We also made vulture age estimates using plumage characteristics and a cormorant (Phalacrocorax auritus) age curve. Vulture pentosidine-based age estimates appear to correspond to plumage-based age estimates. Pentosidine-based age estimates for 88% of the known-aged parakeets (n = 17) were within 6 months of actual ages. Even though known ages were not available for all birds, we found a positive trend in pentosidine versus age for both species. We suggest that 6-mm diameter skin samples from the patagium of living vultures and other similar-sized birds will provide sufficient tissue for reliable age estimation and will not impair flight ability