Validation of the doubly-labeled water (DLW) method for estimating CO2 production and water flux in growing poultry chicks

This study is the first validation of the doubly-labeled water (DLW) method on birds (1) to evaluate the accuracy of 2 points versus multiple points for computing fractional isotopic washout rates (k) and CO2 production (rCO2), (2) to measure CO2 production and water flux each day over a 4-day period, (3) to compare measured fractional evaporative water loss (rG) with assumed values that provide DLW estimates of rCO2 with zero error, and (4) to measure the effect of assumed rG on the error of estimating water influx and efflux. Percent error of CO2 production of six growing poultry chicks estimated by the DLW method was not correlated with mean daily relative growth rates of up to 5% nor with daily rates of energy retained in growth of up to 320 kJ/day/kg, nor was it significantly reduced by using multiple points (5 points) rather than 2 points to compute fractional isotopic washout rates (k) and isotope pool sizes. Its seems clear from our study and the previous 5 validations on growing birds that average relative daily growth rates of up to about 20% do not increase the error of estimating rCO2 by the DLW method. Arithmetic error was significantly less when using one isotopic pool, rather than two pools, to compute rCO2 and was less when using an assumed fractional evaporative water loss (rG) of 0.45 rather than an assumed rG of 0.25 or 0.5 (the two values used predominantly in previous DLW studies). Our study supports Speakman's (1997) suggestion that the one-pool model is more appropriate than the two-pool model for birds weighing<1 kg. We recommend using an assumed rG of 0.45 to compute rCO2 of poultry, which is a compromise between the two schools of rG useage, i.e., rG=0.25 or 0.5, however we hesitate to recommend 0.45 for all birds in all settings. Close agreement between measured rG and an assumed rG that produced zero rCO2 error supports the validity of using the pooled fractionation correction factors (fpool) of 0.0339 for tritiated water and 0.0249 for deuterated water. Absolute error decreased with the percent washout of during measurement periods of 1 to 4 days. Accuracy of estimating rCO2 was not significantly different for durations of 2, 3, and 4 days using either tritiated or deuterated water. The arithmetic error of estimating rCO2 using a one isotopic pool model, 2 points, an rG of 0.5, and tritiated water was −1.9% (SD=13.5) for the first day of a 4-day period and −4.0% (SD=8.9) for the entire period. Percent arithmetic error of water influx (rH2Oinf) and efflux (rH2Oeff) estimated for day 1 from tritiated water washout and an assumed rG of 0.5 was −0.5 (SD=6.4) and 0.1% (SD=11.1), respectively. An rG of 0.5 produced significantly less arithmetic error than an rG of 0.25 or an rG of zero (i.e., no fractionation correction), and less absolute error in rH2Oinf. Errors were slightly more negative (underestimates) with an rG of 0.25, i.e., −2.2 and −2.0%, respectively and even more negative with no correction for isotopic fractionation (i.e., an assumed rG of zero). Tritiated water estimates of water influx and efflux during the first day had no error when using an rG of 0.57 and 0.48, respectively. With assumed rGs of 0.25 and 0.5, the errors of water influx were −7.8 and −5.9%, and the errors of water efflux were 3.4 and 5.6%, respectively, over 4 days. We recommend using an assumed rG of 0.45 to compute rH2Oeff for poultry. The error of rCO2 was about 3 to 4 times more sensitive to values of assumed rG than the error of water flux.