Measurement of Whole-Body CO2 Production in Birds Using Real-Time Laser-Derived Measurements of Hydrogen (δ2H) and Oxygen (δ18O) Isotope Concentrations in Water Vapor from Breath

The doubly labeled water (DLW) method is commonly used to measure energy expenditure in free-living wildlife and humans. However, DLW studies involving animals typically require three blood samples, which can affect behavior and wellbeing. Moreover, measurement of H (delta H-2) and O (delta O-18) isotope concentrations in H2O derived from blood using conventional isotope ratio mass spectrometry is technically demanding, time-consuming, and often expensive. A novel technique that would avoid these constraints is the real-time measurement of delta H-2 and delta O-18 in the H2O vapor of exhaled breath using cavity ring-down (CRD) spectrometry, provided that delta H-2 and delta O-18 from body H2O and breath were well correlated. Here, we conducted a validation study with CRD spectrometry involving five zebra finches (Taeniopygia gutiata), five brown-headed cowbirds (Molothrus ater), and five European starlings (Sturnus vulgaris), where we compared delta H-2, delta O-18, and rCO(2) (rate of CO2 production) estimates from breath with those from blood. Isotope concentrations from blood were validated by comparing dilution-space estimates with measurements of total body water (TBW) obtained from quantitative magnetic resonance. Isotope dilution-space estimates from delta H-2 and 6180 values in the blood were similar to and strongly correlated with TBW measurements (R-2 = 0.99). The H-2 and O-18 (ppm) in breath and blood were also highly correlated (R-2 = 0.99 and 0.98, respectively); however, isotope concentrations in breath were always less enriched than those in blood and slightly higher than expected, given assumed fractionation values between blood and breath. Overall, rCO(2) measurements from breath were strongly correlated with those from the blood (R-2 = 0.90). We suggest that this technique will find wide application in studies of animal and human energetics in the field and laboratory. We also provide suggestions for ways this technique could be further improved.