Validation of the doubly labeled water method in Japanese Quail Coturnix c. japonica chicks: is there an effect of growth rate?

The Doubly Labeled Water (DLW) method was validated against respiration gas analysis in growing Japanese Quail chicks (between 1 week and 3 weeks of age) as well as in birds after having achieved sexual maturity (7 weeks of age). A comparison was made between a strain selected for high growth rates (P-strain, n=18), and a non-selected strain (C-strain, n=18). Relative growth rates of individual chicks during the measurement ranged from −13.8% day⁻¹ to 23.1% day⁻¹. When employing a single-pool model (eq. 34, Lifson and McClintock 1966), it was found that the relative error of the DLW method was sensitive to assumptions concerning fractional evaporative water loss. The best fit was obtained after taking a fractional evaporative water loss value of 0.33. When applying this value for all chicks, it was found that neither strain, relative growth rate of the chick during measurement, nor age significantly contributed to the explained variance. When employing two-pool models, it was found that the DLW method significantly underestimated the true rates of CO₂ production at all assumed levels of fractional evaporative water loss. Based on an evaluation of DLW validation studies in growing shorebirds, terns, and quail we recommend Speakman's Eq. 7.17 (Speakman 1997) for general use in young birds. Accepted: 14 April 2000     

Validation of a non-invasive blood-sampling technique for doubly-labelled water experiments

Two techniques for bleeding small mammals have been used in doubly-labeled water (DLW) studies, including vena puncture and the use of starved nymphal stages of hematophagous reduviid bugs (Reduviidae, Hemiptera). In this study, we tested the validity of using reduviid bugs in doubly-labeled water experiments. We found that the isotope enrichment in initial blood samples collected with bugs was significantly lower compared to isotope enrichment in blood samples obtained using vena puncture. We therefore used the desiccation method for estimating total body water (TBW) in DLW experiments because TBW calculated using the isotope dilution method was overestimated when blood samples were collected using reduviid bugs. In our validation experiment with nectar-feeding bats (Glossophaga soricina), we compared estimates of daily energy expenditure (DEE) using DLW with those derived from the energy balance method. We considered Speakman's equation (controlling for 25% fractionated water loss) as the most appropriate for our study animal and calculated DEE accordingly. On average, DEE estimated with DLW was not significantly different from the mean value obtained with the energy balance method (mean deviation 1.2%). We conclude that although bug hemolymph or intestinal liquids most likely contaminate the samples, estimates of DEE are still valid because the DLW method does not depend on absolute isotope enrichments but on the rate of isotope decrease over time. However, dilution of blood with intestinal liquids or hemolymph from a bug may lead to larger variation in DEE estimates. We also tested how the relative error of DLW estimates changed with varying assumptions about fractionation. We used three additional equations for calculating DEE in DLW experiments. The basic equation for DLW experiments published by Lifson and McClintock (LM-6) assumes no fractionation, resulted in an overestimate of DEE by 10%. Nagy's equation (N-2) controls for changes in body mass but not for fractionation. Using Nagy's equation, DEE was overestimated by 8%. Under the assumption that 50% of total water flux fractionates, the alternative equation by Lifson and McClintock (LM-35) DEE was underestimated by 5%. The best fit between estimates of DEE based on DLW and energy balance measurements was derived by assuming that 32% of total water flux (TWF) is fractionated. We conclude that the outcome of DLW experiments is sensitive to assumptions regarding evaporative water loss, and thus recommend Speakman's equation 7.17 for use with bats.     

Validation of the DLW method in Japanese quail at different water fluxes using laser and IRMS.

In Japanese quail (Coturnix c. japonica; n = 9), the doubly labeled water (DLW) method ((2)H, (18)O) for estimation of CO(2) production (l/day) was validated. To evaluate its sensitivity to water efflux levels (r(H(2))O(e); g/day) and to assumptions of fractional evaporative water loss (x; dimensionless), animals were repeatedly fed a dry pellet diet (average r(H(2))O(e) of 34.8 g/day) or a wet mash diet (95.8 g/day). We simultaneously compared the novel infrared laser spectrometry (LS) with isotope ratio mass spectrometry. At low r(H(2))O(e), calculated CO(2) production rate exhibited little sensitivity to assumptions concerning x, with the best fit being found at 0.51, and only little error was made employing an x value of 0.25. In contrast, at high r(H(2))O(e), sensitivities were much higher with the best fit at x = 0.32. Conclusions derived from isotope ratio mass spectrometry and LS were similar, proving the usefulness of LS. Within a threefold range of r(H(2))O(e), little error in the DLW method is made when assuming one single x value of 0.25 (recommended by Speakman JR, Doubly Labelled Water. Theory and Practice. London: Chapman & Hall, 1997), indicating its robustness in comparative studies.     

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 (r_G) with assumed values that provide DLW estimates of rCO₂ with zero error, and (4) to measure the effect of assumed r_G 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 (r_G) of 0.45 rather than an assumed r_G 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 r_G of 0.45 to compute rCO₂ of poultry, which is a compromise between the two schools of r_G useage, i.e., r_G=0.25 or 0.5, however we hesitate to recommend 0.45 for all birds in all settings. Close agreement between measured r_G and an assumed r_G that produced zero rCO₂ error supports the validity of using the pooled fractionation correction factors (f_pool) 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 r_G 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 (rH₂O_inf) and efflux (rH₂O_eff) estimated for day 1 from tritiated water washout and an assumed r_G of 0.5 was ?0.5 (SD=6.4) and 0.1% (SD=11.1), respectively. An r_G of 0.5 produced significantly less arithmetic error than an r_G of 0.25 or an r_G of zero (i.e., no fractionation correction), and less absolute error in rH₂O_inf. Errors were slightly more negative (underestimates) with an r_G 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 r_G of zero). Tritiated water estimates of water influx and efflux during the first day had no error when using an r_G of 0.57 and 0.48, respectively. With assumed r_Gs 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 r_G of 0.45 to compute rH₂O_eff for poultry. The error of rCO₂ was about 3 to 4 times more sensitive to values of assumed r_G than the error of water flux.     

Extreme water efficiency of Cape gannet Morus capensis chicks as an adaptation to water scarcity and heat stress in the breeding colony

Cape gannet Morus capensis chicks depend entirely on fish prey and metabolic water for water requirements during development. Water loss through evaporative cooling due to heat stress is substantial. We measured water flux and field metabolic rates (FMR) of Cape gannet chicks and adults to determine if gannets developed water saving strategies. The water economy index (WEI, g kJ^?1) decreased with chick age according to the model WEI = 0.676 – 0.272 × log₁₀(t), indicating that water efficiency increased with age. At fledging, the WEI of chicks was at the level expected of adult desert birds. Desert birds maintain a low WEI by also having a low FMR, whereas Cape gannet chicks have FMR comparable to other seabird species’ nestling requirements. We propose that maintaining low WEI is adaptive for Cape gannets because (1) chicks need to balance water loss through evaporative cooling, (2) fledglings need to overcome a period of up to a week when they cannot ingest any water and (3) adults spend extended periods in the breeding colony during which water can become a limiting factor. Understanding the physiological mechanism of maintaining low WEI will become increasingly important with future rising temperatures.     

Measurement of fractionated water loss and CO2 production using triply labelled water

The doubly labelled water (DLW) method for measuring CO2 production has recently been the subject of much interest since no other technique gives integrated values for CO2 production over long periods by free-living subjects. The importance of evaporative water loss and fractionation factors to the calculation of CO2 production using this technique is described. Present methods of estimating evaporative water loss and the use of fractionation factors are summarized together with a discussion of their limitations. A novel technique is proposed whereby water labelled with three isotopes can be used to measure evaporative water loss and CO2 production in completely free-living subjects, and the feasibility of the method is tested in simulations using experimental data. This technique has three advantages over existing methods of estimating evaporative water loss: (1) it can be used in completely free-living subjects without any additional experimental procedures (e.g. water-balance studies or physical trapping of water vapour); (2) it gives a direct estimate of fractionated evaporative water loss in each subject, since non-fractionated water lost as vapour is automatically compensated for; and (3) the routes of water loss do not have to be known. The appropriate calculations are presented together with a discussion of the difficulties of measuring oxygen-17 by mass spectrometry. It is estimated that the maximum theoretical error on calculated CO2 production is +/- 0.3%. Practical ways of achieving this theoretical level of accuracy are suggested. We conclude that the proposed technique will allow correction for evaporative water loss to be made more exactly, thereby increasing the accuracy of the heavy water technique for measuring CO2 production in free-living subjects.     

Validation of the doubly labeled water method under low and high humidity to estimate metabolic rate and water flux in a tropical snake (Boiga irregularis).

This study uses indirect calorimetry to assess the effects of humidity on the accuracy of the doubly labeled water (DLW) technique to predict metabolic rate and water flux in brown treesnakes (Boiga irregularis). The DLW technique accurately predicted total water efflux in brown treesnakes under low-humidity conditions and found that the total number of water molecules exchanged with the environment under humid conditions was not significantly different than maximum net total evaporative water loss under low humidity conditions plus fecal water loss. Because of changes of total body water of >12%, the DLW technique overestimated metabolic rate by a factor of 2.2 under low-humidity conditions. Under high-humidity conditions, the DLW technique overestimated metabolic rate in brown treesnakes by a factor of 4.6. Researchers using the DLW technique in humid or moist environments should be cautious because this study indicates that DLW estimates of metabolic rate may be inflated when large amounts of water vapor are exchanged through the skin or respiratory passages.     

Validation of the doubly labeled water technique for measuring energy metabolism in starlings and sparrows

I have tested the idea that doubly labeled water (DLW) can accurately predict CO2 production in savannah sparrows, song sparrows, white-throated sparrows, starlings, and a single house sparrow by comparing DLW estimates with those obtained simultaneously by capturing expired CO2 in Ascarite. In addition I used the energy balance method to see if metabolic rates generated from DLW measurements accurately reflected the actual metabolic rates of these birds. I found close agreement in DLW and the gravimetric and energy balance methods, with DLW underestimating CO2 production on average by -3.5% in sparrows, and -7.1% in starlings. Similarly, the energy balance method indicated a -3.1% underestimate by DLW for sparrows and a -5.1% for starlings. The DLW method can yield reasonable estimates of CO2 production in a variety of passerine birds.     

Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.     

Comparison of different approaches for the calculation of energy expenditure using doubly labeled water in a small mammal

The doubly labeled water (DLW) method is an isotope-based technique for the estimation of the CO(2) production, and hence energy expenditure, of free-living animals and humans. Several methods are available for the calculation of CO(2) production from the isotope fluxes, depending on different assumptions about the behavior of isotopes during the elimination process. We used the DLW method to estimate the daily energy expenditures (DEE) of 55 field voles (Microtus agrestis) held in a captive facility at 8 degrees C. We calculated DEE using both plateau and intercept approaches for estimating the sizes of the isotope dilution spaces, three different assumptions about fractionation processes, and two ways of treating the different dilution spaces of the oxygen and hydrogen isotopes. We compared the resultant DEE estimates with metabolizable energy intake (MEI) measured during a 3-d feeding trial immediately before the DLW measurements, during which the animals were in energy balance. By making different assumptions about the apparent energy absorption efficiency, we generated a range of direct estimates of MEI. When we compared DEE and MEI, we found that the two-pool model formulations consistently underestimated energy demands by up to 29.8%, depending on the assumptions made in the reference calculation. However, while our data suggest that some correction for fractionation is necessary, with the present data we were unable to separate the two most common treatments of fractionation. These data strongly support the previous suggestion that for small mammals single-pool models provide more accurate estimates of energy demands than two-pool formulation of the DLW method.     

Effect of bolus fluid intake on energy expenditure values as determined by the doubly labeled water method.

The doubly labeled water (DLW, 2H(2)18O) method is a highly accurate method for measuring energy expenditure (EE). A possible source of error is bolus fluid intake before body water sampling. If there is bolus fluid intake immediately before body water sampling, the saliva may reflect the ingested water disproportionately, because the ingested water may not have had time to mix fully with the body water pool. To ascertain the magnitude of this problem, EE was measured over a 5-day period by the DLW method. Six subjects were dosed with 2H2(18)O. After the reference salivas for the two-point determination were obtained, subjects drank water (700-1,000 ml), and serial saliva samples were collected for the next 3 h. Expressing the postbolus saliva enrichments as a percentage of the prebolus value, we found 1) a minimum in the saliva isotopic enrichments were reached at approximately 30 min with the minimum for 2H (95.48 +/- 0.43%) being significantly lower than the minimum for 18O (97.55 +/- 0.44, P less than 0.05) and 2) EE values calculated using the postbolus isotopic enrichments are appreciably higher (19.9 +/- 7.5%) than the prebolus reference values. In conclusion, it is not advisable to collect saliva samples for DLW measurements within approximately 1 h of bolus fluid intake.     

Embryonic osmoregulation: consequences of high and low water loss during incubation of the chicken egg

The rates of water loss of domestic chicken eggs were varied during incubation to measure the osmoregulatory ability of the avian embryo. Egg water loss was increased by drilling holes in the eggshell over the airspace on day 13 (I = 21 days) and then placing these eggs in a low relative humidity (r.h.: 0-10%) incubator until hatch. Egg water loss was decreased by placing other eggs in a high-r.h. (85-90%) incubator on day 0. Eggs with low water loss (approximately 6% of initial fresh mass [IFM]) produced embryos and yolks that were not different in wet or dry mass when compared to control eggs that lost approximately 12% of IFM. However, 1-4 gm of excess albumen were left in low-water-loss eggs on day 21. Hatching success was 71% and 89% for low and control eggs, respectively. Low egg water loss did not appear to disturb embryonic growth. The allantoic fluid volume and millimolar allantoic Na+ and Cl- ions declined faster with high and slower with low rates of water loss. Thus, excess water was lost as a result of increased movement of water out of allantoic fluid, which was due to increased active transport of Na+ ions by the chorioallantoic membrane (CAM). Eggs with high water loss had elevated Cl- levels after day 17 in plasma and amniotic fluid, which indicated a period of osmotic stress after depletion of allantoic fluid between day 18 and hatch. The decrease in wet embryo mass measured in embryos from high-water-loss eggs was due principally to dehydration of skin. Embryonic skin may serve as an emergency water reservoir during osmotic stress. Dehydrated chicks produced from high-water-loss eggs were 6 gm less in wet mass at hatch compared to controls. However, these chicks regained the water deficit 7 days after hatch and grew at a rate not different from control chicks through 6 weeks of age. Total egg water loss of 12% of IFM results in highest hatching success. However, water losses between 6% and 20% of IFM do not appear to affect adversely the growth or water content of the chick. Water losses above 20% of IFM cause early depletion of allantoic fluid, prolong the period of osmotic stress, and result in subsequent dehydration of blood, amniotic fluid, and embryonic skin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of Implanted Radiotransmitters to Estimate Survival of Greater Sage-Grouse Chicks

ABSTRACT Reduced chick survival has been implicated in declines of greater sage-grouse (Centrocercus urophasianus) populations. Because monitoring survival of unmarked sage-grouse chicks is difficult, radiotelemetry may be an effective technique to estimate survival rates, identify causes of mortality, and collect ecological data. Previous studies have used subcutaneous implants to attach radiotransmitters to hatchlings of several species of birds with precocial young. Previous researchers who used subcutaneous implants in free-ranging populations removed chicks from the capture location and implanted transmitters at an alternate site. Because logistics precluded removing newly hatched greater sage-grouse chicks from the field, we evaluated a method for implanting transmitters at capture locations. We captured 288 chicks from 52 broods and monitored 286 radiomarked chicks daily for 28 days following capture during May and June 2001–2002. Two (>1%) chicks died during surgery and we did not radiomark them. At the end of the monitoring period, 26 chicks were alive and 212 were dead. Most (98%, 207/212) radiomarked chick mortality occurred < 21 days posthatch and predation (82%, 174/212) was the primary cause of death. Necropsies of 22 radiomarked chicks did not indicate inflammation or infection from implants, and they were not implicated in the death of any chicks. Fate of 48 chicks was unknown because of transmitter loss (n = 16), radio failure (n = 29), and brood mixing (n = 3). Overall, the 28-day chick survival rate was 0.220 (SE = 0.028). We found that mortalities related to the implant procedure and transmitter loss were similar to rates reported by previous researchers who removed chicks from capture sites and implanted transmitters at an alternate location. Subcutaneous implants may be a useful method for attaching transmitters to newly hatched sage-grouse chicks to estimate survival rates, identify causes of mortality, and collect ecological data.     

A review of seabird energetics using the doubly labeled water method

The doubly labeled water (DLW) method has been essential for understanding animal energetics of free-ranging individuals. The first published studies on free-ranging seabirds were conducted on penguins in the early 1980s. Since then, nearly 50 seabird species with representatives from each major taxonomic order have been studied using DLW. Although the basic methodology has not changed, there are at least nine different equations, varying with respect to assumptions on fractionation and the total body water pool, to estimate field metabolic rate (FMR) from isotopic water turnover. In this review, I show that FMR can vary by as much as 45% depending on the equation used to calculate CO₂ production in five albatross species. Energy budgets derived from DLW measurements are critical tools for understanding patterns of energy use and allocation in seabirds. However, they depend on accurate and representative measurements of FMR, so analyses that include greater partitioning of activity specific FMR yield more realistic cost estimates. I also show how the combined use of DLW and biologging methods can 1) provide greater clarity for explaining observed variation in FMR measurements within a species and 2) allow FMRs to be viewed in a wider physiological, behavioral, or ecological context. Finally, I update existing allometric equations with new FMR data. These updates reaffirm that albatrosses have the lowest at-sea FMRs per equivalent body mass and that individuals of other seabird orders have FMRs ranging between 1.39 and 2.24 times higher than albatrosses.     

Application of a weighing system for measuring total evaporative water loses in large ruminants

A weighing system originally designed for monitoring changes in the mass of humans due to fluid loss in hospital beds has been adapted to measure the total evaporative water loss of large domestic animals in studies of temperature regulation. Mass loss was monitored on a charge recording and regressed against time. The adaptation resulted in an overall reduction of 73% in the standard error of the regression coefficient of rate of total evaporative water loss. Accuracy was 48% better in the climate chamber than outdoors.     

Estimation of Precision in DLW Studies Using the Two-Point Methodology

Previous attempts to estimate precision of doubly labeled water (DLW) estimates of CO₂ production, using propagation of error analyses, have necessarily made simplifying assumptions which may compromise the resultant error estimate. Using an empirical iteration approach, error distributions for the DLW calculation were generated which overcome these problems. The error distribution for CO₂ estimates generated by DLW is symmetrical but not normal. The distribution is significantly truncated such that the 99% inclusion limits are 2.034 SD and not 2.58 SD. The precision error (99% CI for mean as percent of the mean) in DLW experiments, when using duplicate analyses, varies between approximately 3% and 47% depending on the ratio of the elimination constants of the two labels (k_o/k_d), experimental duration and initial isotope dose. The error could be improved by approximately 10 fold by increasing the number of replicates at all six isotope determinations from 2 to 5. Estimating precision in actual experiments can be made using the same empirical approach. The resultant estimates can be of extreme value in evaluating the role of precision as a factor influencing deviations during validation studies, and also for weighting mean estimates in applications of the technique. The deviations of DLW estimates from those made simultaneously by indirect calorimetry in a small mammal validation study were generally greater than the precision of the DLW estimates of CO₂ production. This may indicate there are more problems with the technique than precision alone.     

Correction Approaches for Doubly Labeled Water in Situations of Changing Background Water Abundance

Doubly labeled water (DLW) is an accurate, portable method for measuring free-living energy expenditure. However, under certain conditions shifts in baseline abundance of deuterium and oxygen-18 tracers used in the method may produce errors in derivation of both turnover (k) rates and calculated energy expenditure. Present objectives were to examine during what experimental situations baseline errors arise and to address means of correcting for such baseline shifts so that consequent errors in energy expenditure calculations are minimized. Under conditions where shifts in baseline abundance for deuterium and oxygen-18 parallel abundances corresponding to the natural meteoric water ratio, self-compensating changes in k values for both deuterium and oxygen will result in minimal error to the DLW energy expenditure calculations, provided that the dose ratio of isotopes also mimics the meteoric water line. However, in situations where relative shifts in abundance of each isotope across the measurement period are not in parallel relative to the natural meteoric water line, then the potential for larger DLW errors exists. Optimally, subjects should equilibrate with the new water source. Failing this, correction for shifting baseline can be accomplished by measuring isotopic abundance changes in a control group of subjects not given the DLW dose, but performing similar tasks and consuming the same diet as the group given DLW. Alternatively, theoretically based correction values can be calculated given knowledge of the abundances of the final drinking water and the interval time that subjects consumed the new fluid.     

Body size establishes the scaling of avian postnatal metabolic rate: an interspecific analysis using phylogenetically independent contrasts

The avian postnatal metabolic rate literature is reviewed using power equations, Y = aM^b, to describe the relation between postnatal resting metabolic rate (RMR) and chick body mass (M) for 25 species. In altricial species, the relation between RMR and M from hatching to fledging can be described by a single power equation, whereas in most nonaltricial species two such equations are needed, one for chicks weighing less than about 25% of mature mass ( M _a) and a second for larger chicks. For altricial chicks and larger nonaltricial chicks, the body-mass exponent, b, of 25 intraspecific power equations ranged from 0.25 to 1.67 and varied inversely with M _a. The scaling of postnatal RMR is thus unlike that of either adult or hatchling metabolism in that it is size dependent. We examined the relationship between intraspecific b and M _a using Felsenstein's independent contrasts method to control for statistical complications due to the hierarchical nature of phylogenetic relationships. This "phylogenetic regression" technique yielded the relation b = 1.6 M _a^-015, in which mature mass explained 38% of the variation in b. The mass exponent of this equation (-0.15) did not differ significantly from that determined by nonphylogenetic methods (-0.17).
In altricial chicks and larger nonaltricial chicks, the scaling coefficient, a, of the interspecific power equations varied with adult mass according to the phylogenetically determined relation a (kj/h) = 0.0052M_a^0.65and was higher in fed than in fasted chicks. Equations derived in this analysis permit one to estimate the RMR of a growing chick from its mass and adult body mass and provide a basis for evolutionary and ecological comparisons.     

Validation of the doubly labeled water method in rats during isolation and simulated weightlessness

Total energy expenditure (TEE) of rats during simulated microgravity is unknown. The doubly labeled water method (DLW) reliably measures TEE, but the results depend on the methods of calculation. These methods were validated and appraised by indirect calorimetry in eight rats during isolation (7 days) and simulated microgravity (10 days). There were no effects on CO(2) production in the method used to derive constant flux rates as in the regression models. r(CO(2)) estimates were dependent on the assumed fractionation processes, the derivation of constant flux rate methods, and the selected pool models. Use of respiratory or food quotients did not influence TEE estimations, which were similar during isolation and simulation. During either isolation with growth or simulation with a stabilized mass, the one-pool model of Speakman (Speakman JR. Doubly Labelled Water. Theory and Practice. London: Chapman and Hall, 1997) resulted in the more reliable validation (0.8 +/- 2.2 and 2.2 +/- 3.4% vs. calorimetry, respectively). However, during simulation, agreement was also observed with the single pool model of Lifson (Lifson N, Gordon GB, and McClintock R. J Appl Physiol 7: 704-710, 1955) (-2.5 +/- 2.5%), and two two-pool models [Schoeller (Schoeller DA. J Nutr 118: 1278-1289, 1988) (0.5 +/- 3.1%) and Speakman (Speakman, JR. Doubly Labelled Water. Theory and Practice. London: Chapman and Hall, 1997) (-1.9 +/- 2.7%)]. This latter finding seems linked to the stable body mass and to fractionation consideration close to the single-pool model of Speakman. During isolation or simulated microgravity, the other equations underestimated TEE by 10-20%.     

Disruption of endoplasmic reticulum is the primary ultrastructural lesion of the pancreas in the selenium-deficient chick

Severe uncomplicated selenium (Se) deficiency was produced in chicks by feeding, from 1 day of age, a purified diet that contained 0.010 ppm Se but was adequate with respect to all other known nutrients. The deficiency was characterized by depressions in rate of growth and efficiency of feed utilization and by reductions in the plasma activity of Se-dependent glutathione peroxidase (SeGSHpx) by 85-97% from levels in chicks fed the basal diet supplemented with 0.20 ppm Se as Na2SeO3. Histological observations of the target organ of Se deficiency in the chick, i.e., the pancreas, using transmission electron microscopy, showed severe losses of endoplasmic reticulum (ER) and absence of secretory granules in acinar cells of Se-deficient animals. These effects were not uniform within individuals, as Se-deficient pancreases also showed areas of unaffected acini. By 14 days of age, Se-deficient pancreases contained many apparently undifferentiated cells, which were absent from pancreases of Se-fed chicks. It is noteworthy that abnormal mitochondria were not observed in any pancreas sections. It is concluded that the metabolic consequences of severe uncomplicated Se deficiency in the chick result from the disruption of ER and loss of functional acinar cells, rather than to damage to mitochondria as previously suggested.