Validation of the Use of Doubly Labeled Water for Measuring Metabolic Rate in Timber Rattlesnakes (Crotalus horridus)

The doubly labeled water method is an isotopic technique for measuring field metabolic rate and water flux rates of free-living animals. We present a validation of the use doubly labeled water for measuring metabolic rate and water loss in Timber Rattlesnakes (Crotalus horridus). For this study seven animals of varying body size were used with masses ranging from 148 to 650 grams. Prior to dosing, blood samples were taken to establish background isotope levels for each animal. Snakes were injected with water enriched with isotopes of oxygen (18O) and hydrogen (2H, deuterium). The injected isotopes were then allowed to equilibrate with the animals’ body water pool for 3 hours and then a second blood sample was taken. Following the second blood sample, animals were placed into metabolic chambers and metabolic rates (VCO2 and VO2) were measured using open-flow respirometry for 20 days. During metabolic measurements water loss for each snake was estimated via Drierite uptake. On the 20th day, final blood samples were taken to determine isotope turnover rates. Measured CO2 production by gas exchange will be compared to CO2 production calculated from isotopic data. Water flux measured by Drierite uptake will be compared to water flux calculated from isotopic data. Despite years of application of the DLW technique, validation studies are few: our data are the first such validation in pitvipers