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.     

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.     

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.     

Doubly labeled water measurement of human energy expenditure during strenuous exercise

The energy expenditures (EE) of 23 adult male Marines were measured during a strenuous 11-day cold-weather field exercise at 2,200- to 2,550-m elevation by both doubly labeled water (2H2 18O, DLW) and intake balance methods. The DLW EE calculations were corrected for changes in baseline isotopic abundances in a control group that did not receive 2H2 18O. Intake balance EE was estimated from the change in body energy stores and food intake. Body energy-store changes were calculated from anthropometric [-1,574 +/- 144 (SE) kcal/day] and isotope dilution (-1,872 +/- 293 kcal/day) measurements made before and after the field exercise. The subjects kept daily logbook records of ration consumption (3,132 +/- 165 kcal/day). Mean DLW EE (4,919 +/- 190 kcal/day) did not differ significantly from intake balance EE estimated from food intake and either anthropometric (4,705 +/- 181 kcal/day) or isotope dilution (5,004 +/- 240 kcal/day) estimates of the change in body energy stores. The DLW method can be used with at least the same degree of confidence as the intake balance method to measure the EE of active free-living humans.     

Effect of long-term fertilization and cropping on the potassium supplying capacity of soils

Soil samples from a long term field experiment, to study the K depletion pattern, were analysed for K. The values of supply parameters pK?1/2p (Ca+Mg) and free energy δG _r ⁰ were calculated using the data on water soluble and exchangeable cations. Correlation coefficient values between pK?1/2p (Ca+Mg) vs. total K removed, K removal by pearl millet in 1980 and soil available potassium were positive and statistically significant. This indicated that these soils have reached the stage where they would start responding to potassium application. Also, negative but significant correlation of δG _r ⁰ with pK?1/2p (Ca+Mg) and available K, were found. δG _r ⁰ values were positive indicating the affinity of such soils for K and the values, for K fertilized plots were lower in comparison to plots where no K was applied. pK?1/2p (Ca+Mg) parameter was found to be a better measure of K availability over δG _r ⁰ due to either fixation of K or greater contribution of nonexchangeable K fraction towards plant utilized K.     

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     

水分对苜蓿叶片光合特性的影响

采用田间试验, 对每茬灌水3次(W₃)、2次(W₂)、1次(W₁)和不灌水(W₀)四种条件下的土壤水分, 苜蓿(Medicago sativa)叶片的叶绿素荧光参数、气孔导度(G_s)、净光合速率(P_n)和蒸腾速率(T_r)进行测定。结果表明, 灌水提高了苜蓿叶片的原初光能转换效率(F_v/F_m)、P_n和T_r, 并随着灌水量的增加而增加。苜蓿叶片的F_v/F_m、P_n和T_r的日均值与土壤含水量均呈极显著正相关关系。苜蓿叶片的P_n与F_v/F_m和光合有效辐射(PAR)的乘积呈正相关关系。灌水还改变了苜蓿叶片P_n的日变化格局。灌水较多的处理(W₃和W₂), 苜蓿叶片没有出现光合“午休”现象,P_n的日变化趋势呈现“单峰”型。而灌水较少和不灌水的处理(W₁和W₀), 苜蓿叶片出现了明显的光合“午休”现象, 其P_n的日变化进程呈现“双峰”型。在相同的水分条件下, 初花期苜蓿叶片的P_n高于再生期的, T_r则相反。     

Physical activity-related energy expenditure with the RT3 and TriTrac accelerometers in overweight adults

Objective: The objective was to evaluate two accelerometers, the RT3 and the TriTrac‐R3D for their ability to produce estimates of physical activity‐related energy expenditure (PAEE) in overweight/obese adults. Research Methods and Procedures: PAEE estimates from both accelerometers were obtained in two experiments. In Experiment 1, 13 overweight/obese subjects (BMI 34.2 ± 6.4 kg/m²) were monitored over 2 weeks in everyday life, PAEE being simultaneously measured by the doubly labeled water method (DLW). In Experiment 2, 8 overweight/obese subjects (BMI 34.3 ± 5.0 kg/m²) and 10 normal‐weight subjects (BMI 20.8 ± 2.1 kg/m²) were monitored during a treadmill walking protocol, PAEE being simultaneously measured by indirect calorimetry. Results: In Experiment 1, there was no significant difference between methods in mean PAEE (DLW: 704 ± 223 kcal/d, RT3: 656 ± 140 kcal/d, TriTrac‐R3D 624 ± 419 kcal/d). The relative difference between methods (accelerometer vs. DLW) was ?17.1% ± 16.7% for the RT3 and ?20.0 ± 44.6% for the TriTrac‐R3D. Correlation for PAEE between RT3 and DLW was higher than between TriTrac‐R3D and DLW (r = 0.67, p < 0.05 and r = 0.36, p = 0.25, respectively). The 95% confidence interval (CI) (kcal/d) of the mean difference between methods was large, amounting to ?385 to 145 for the RT3 and ?887 to 590 for the TriTrac‐R3D. In Experiment 2, both accelerometers were sensitive to the changes in treadmill speed, with no significant difference in mean PAEE between methods in overweight/obese subjects. Conclusions: Although both accelerometers did not provide accurate estimates of PAEE at individual levels, the data suggest that RT3 has the potential to assess PAEE at group levels in overweight/obese subjects.     

Contributions of evaporation, isotopic non-steady state transpiration and atmospheric mixing on the delta18O of water vapour in Pacific Northwest coniferous forests

Changes in the ²H and ¹⁸O of atmospheric water vapour provide information for integrating aspects of gas exchange within forest canopies. In this study, we show that diurnal fluctuations in the oxygen isotope ratio (δ¹⁸O) as high as 4‰ were observed for water vapour (δ¹⁸O_vp) above and within an old‐growth coniferous forest in the Pacific Northwest region of the United States. Values of δ¹⁸O_vp decreased in the morning, reached a minimum at midday, and recovered to early‐morning values in the late afternoon, creating a nearly symmetrical diurnal pattern for two consecutive summer days. A mass balance budget was derived and assessed for the ¹⁸O of canopy water vapour over a 2‐d period by considering the ¹⁸O‐isoflux of canopy transpiration, soil evaporation and the air entering the canopy column. The budget was used to address two questions: (1) do δ¹⁸O values of canopy water vapour reflect the biospheric influence, or are such signals swamped by atmospheric mixing? and (2) what mechanisms drive temporal variations of δ¹⁸O_vp? Model calculations show that the entry of air into the canopy column resulted in an isotopically depleted ¹⁸O‐isoflux in the morning of day 1, causing values of δ¹⁸O_vp to decrease. An isotopically enriched ¹⁸O‐isoflux resulting from transpiration then offset this decreased δ¹⁸O_vp later during the day. Contributions of ¹⁸O‐isoflux from soil evaporation were relatively small on day 1 but were more significant on day 2, despite the small H₂¹⁶O fluxes. From measurements of leaf water volume and sapflux, we determined the turnover time of leaf water in the needles of Douglas‐fir trees as ≈ 11 h at midday. Such an extended turnover time suggests that transpiration may not have occurred at the commonly assumed isotopic steady state. We tested a non‐steady state model for predicting δ¹⁸O of leaf water. Our model calculations show that assuming isotopic steady state increased isoflux of transpiration. The impact of this increase on the modelled δ ¹⁸O_vp was clearly detectable, suggesting the importance of considering isotopic non‐steady state of transpiration in studies of forest ¹⁸O water balance.     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.     

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.     

Milk intake and carbon dioxide production of piglets determined with the doubly labelled water technique

The present study was undertaken to study different methodological aspects of quantifying CO2 production and milk intake of suckling piglets using the doubly labelled water (DLW) technique. In total, 37 piglets were enriched intraperitoneally with DLW to study equilibration time of 18O (n = 3), to validate the estimation of milk intake and CO2 production (n = 10) of piglets fed milk replacer and to quantify milk intake and CO2 production of piglets nursed ordinarily by sows (n = 24). Enrichment of 18O in expired air was analysed without any sample preparation, whereas enrichment of 18O in serum was analysed after a minimum step of sample preparation, which included pipetting of the sample, blowing gaseous CO2 into the vial for 3 s and equilibrating for 24 h. The 18O enrichment of CO2 in expired air was constant within 30-40 min of intraperitoneal injection, suggesting that DLW was equilibrated within the body water by that time. For piglets fed milk replacer, the estimation of the daily CO2 production by the DLW method (64.0 ± 2.7 l CO2/day) was in agreement with that obtained by respiration trials (64.7 ± 1.8 l CO2/day). Furthermore, the intake of milk replacer (891 ± 63 g/day) determined by deuterium oxide (D2O) dilution was similar in magnitude to that found by weighing the milk disappearance (910 ± 58 g/day). The milk intake of piglets fed milk replacer was comparable with that of sucking piglets, but sucking piglets had a remarkably higher CO2 production than artificially reared piglets, which likely was caused by a higher intake of milk solids and a higher activity level. For sucking piglets, the daily CO2 production increased curvilinearly with increasing live weight (LW) in kg: piglet CO2 production (l/day) = 25.75 × LW - 1.01 × LW2. In conclusion, 18O equilibrates fast within the body water pool when administered intraperitoneally, and the accuracy of assessing milk intake and rate of CO2 production using the DLW technique is promising. Assessment of excess enrichment of 18O in serum proved to be robust. Finally, the CO2 production of piglets fed milk replacer differs considerably from that of sucking piglets.     

Tissue-specific turnover rates of the nitrogen stable isotope as functions of time and growth in a cyprinid fish

Ecological applications of stable isotope data require knowledge on the isotopic turnover rate of tissues, usually described as the isotopic half-life in days (T _0.5) or the change in mass (G _0.5). Ecological studies increasingly analyse tissues collected non-destructively, such as fish fin and scales, but there is limited knowledge on their turnover rates. Determining turnover rates in situ is challenging, with ex situ approaches preferred. Correspondingly, T _0.5 and G _0.5 of the nitrogen stable isotope (δ¹⁵N) were determined for juvenile barbel Barbus barbus (5.5 ± 0.6 g starting weight) using a diet-switch experiment. δ¹⁵N data from muscle, fin and scales were taken during a 125 day post diet-switch period. Whilst isotopic equilibrium was not reached in the 125 days, the δ¹⁵N values did approach those of the new diet. The fastest turnover rates were in more metabolically active tissues, from muscle (highest) to scales (lowest). Turnover rates were relatively slow; T _0.5 was 84 (muscle) to 145 (scale) days; G _0.5 was 1.39 × body mass (muscle) to 2.0 × body mass (scales), with this potentially relating to the slow growth of the experimental fish. These turnover estimates across the different tissues emphasise the importance of estimating half-lives for focal taxa at species and tissue levels for ecological studies.     

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition

Water dynamics in samples of ceramide tetrasaccharide (G_g4Cer) vesicles and G_M1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (²H-NMR). G_M1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10^?10 s, typical of water molecules near to the polar groups. Pure G_M1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from ?18 to 32°C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to G_M1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into G_M1 micelles substantially increases the mobility of water molecules compared with that found in pure G_M1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid–lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.     

Tritiated water measurements of water flux in nestling and adult zebra finches (Poephila guttata)

• 1.1. Total body water volume (TBWV) and daily water turnover rates (WTR) were measured in nestling and adult zebra finches using tritiated water (TOH).
• 2.2. TBWV and daily WTR increased with age up to 13 days post hatching.
• 3.3. TBWV and WTR approached adult levels after 13 days of age. WTR varied among ages and nest mates. All nestlings turned over at least 50% of their body water pool per day.
• 4.4. The WTR data for adult birds are consistent with natural history data suggesting zebra finches are dependent on water for breeding and survival.

     

Validation of the doubly labeled water method in growing precocial birds: the importance of assumptions concerning evaporative water loss

The doubly labeled water (DLW) method was validated against respiration gas analysis in growing precocial chicks of the black-tailed godwit (Limosa limosa) and the northern lapwing (Vanellus vanellus). To calculate the rate of CO2 production from DLW measurements, Lifson and McClintock's equations (6) and (35) were employed, as well as Speakman's equation (7.17) (all single-pool models). The average errors obtained with the first two equations (+7.2% and -11.6%, respectively) differed significantly from zero but not the error obtained with Speakman's equation (average: -2.9%). The latter error could be reduced by taking a fractional evaporative water loss of 0.13, instead of the value of 0. 25 recommended by Speakman. Application of different two-pool models resulted in relative errors of the DLW method of -15.9% or more. After employing the single-pool model with a fractional evaporative water loss value of 0.13, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. Recalculation of previously published results on Arctic tern (Sterna paradisaea) chicks revealed that the fit of the validation experiment could be considerably improved by employing a single-pool model and assuming a fractional evaporative water loss of 0.20 instead of the value of 0.50 taken originally. After employing the value of 0.20, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. This suggests that isotope incorporation into new body substances does not cause a detectable error. Thus, the DLW method seems to be applicable in young birds growing as fast as 20% d-1, after making adjustments for the fractional evaporative water loss. We recommend Speakman's equation (7.17) for general use in growing birds when evaporation is unknown.     

Adaptation to Water Stress in Wheat

Three experiments were designed to investigate to what extent adaptation to water stress take place. Wheat (Triticum aestivum L. cv. Kolibri) was grown in water culture at constant temperature, air humidity, and light intensity. When the plants were 16 days old, the potential of the root medium (ψ_r) was lowered by 1 bar every second day by means of polyethyleneglycol 1500 down to ?4 or ?7 bar and then remained at these levels. As a control one experiment was grown at ?0.7 bar. By regression it was found that when ψ_r was lowered by I bar, osmotic potential in leaf (ψ_π) decreased 1.46 bar, and leaf water potential (ψ_t) 0.68 bar, which mean an increase of turgor of 0.78 bar. At the same time the leaf water content did fall 0.30 g per g dry matter. Specific transpiration rate increased significantly after ψ_r was kept constant, but the increase in area of fresh leaves was strongly reduced due to wilting of old leaves. After an “adaptation” period during which ψ_r remained at ?0.7, ?4, and ?7 bar, respectively, for at least 1 week. ψ_r was altered so as to cover the range from 0 to ?14 bar and ψ_π, ψ_r, transpiration and diffusion resistance in stomata (r_s) were measured. The levels of ψ_π and ψ₁ were lower (more negative) and turgor potential higher in plants grown at low ψ_r. The transpiration in pre-stressed plants showed less sensitivity to the alteration of ψ_r than in the non-stressed plants. The values of ψ_r at which r_s increased greatly, were found to be about ?13, ?15, and ?18 bar for plants grown at ?0.7, ?4, and ?7 bar, respectively.     

Deuterium stable isotope ratios as tracers of water resource use: an experimental test with rock doves

Naturally-occurring deuterium stable isotope ratios can potentially be used to trace water resource use by animals, but estimating the contribution of isotopically distinct water sources requires the accurate prediction of isotopic discrimination factors between water inputs and an animals body water pool. We examined the feasibility of using estimates of water fluxes between a bird and its environment with a mass-balance model for the deuterium stable isotope ratio of avian body water (D_body) to predict isotopic discrimination factors. Apparent fractionation and thus discrimination factors were predicted to vary with the proportion of an animals total water losses than could be attributed to evaporative processes. To test our ability to predict isotopic discrimination, we manipulated water intake and evaporative water loss in rock doves (Columba livia) by providing them with fresh water or 0.15 M NaCl solution in thermoneutral or hot environments. After we switched the birds from drinking water with D=–95 VSMOW (Vienna Standard Mean Ocean Water) to enriched drinking water with D=+52 VSMOW, steady-state D_body was approached asymptotically. The equilibrium D_body was enriched by 10–50 relative to water inputs. After isotopic equilibrium was reached, the degree of enrichment was positively related (r²=0.34) to the fraction of total water loss that occurred by evaporation supporting the major prediction of the model. The variation we observed in discrimination factors suggests that the apparent fractionation of deuterium will be difficult to predict accurately under natural conditions. Our results show that accurate estimates of the contribution of different water sources to a birds body water pool require large deuterium isotopic differences between the sources.     

Daily energy expenditure and water turnover in two breeds of laying hens kept in floor housing

Laying hens are increasingly kept in barn or free-range systems, which not only allows birds to move freely but also potentially entails higher energy expenditures due to higher locomotor activity. Therefore, the aim of our study was to quantify the daily energy expenditure (DEE) and water turnover in freely moving laying hens. For that purpose, 10 Lohmann Selected Leghorn (LSL) and 10 Lohmann Brown (LB) hens were obtained from a conventional breeding company at 17 weeks of age. The trial started when birds reached an age of 34 weeks. All 20 birds were kept together in the same littered floor pen (12.1 m²). The pen was equipped with perches, a nest box, feeding and nipple drinkers. The DEE was determined individually for all experimental birds (n = 20) for a total of nine days using the doubly labelled water (DLW) method. Lohmann Brown hens were heavier than LSL hens, but laying rate did not differ between the two breeds, that is, one egg per hen and day during the study period. Average egg mass was 63.1 ± 0.20 g in LB and 61.7 ± 0.12 g in LSL hens, which converted to an egg energy content of 420 and 410 kJ/egg, respectively. Dilution spaces for oxygen and hydrogen differed between the breeds but not the respective turnover rates. Total body water as a percentage of body mass (LB: 54.4%, LSL: 53.8%; SEM = 0.7, F_1,18 = 0.41, P = 0.513) and total water intake (TWI) per day (LB: 275 ml/day, LSL: 276 ml/day; SEM = 20, F_1,17 = 0, P = 0.994) did not differ between LB and LSL hens. Individual DEE increased with body mass in LB but not in LSL hens. Average DEE did not differ between the two breeds (LB: 1 501 kJ/day; LSL: 1 520 kJ/day; SEM = 32.1, F_1,17 = 2.54, P = 0.131). However, when comparing the DEE on a metabolic mass basis, LSL hens expended with 984 kJ/kg^0.75 on average significantly more energy per day than LB hens (895 kJ/kg^0.75; SEM = 20.3, F_1,18 = 10.1, P = 0.005). Our results suggest that the DLW technique is a viable method to measure the energy expenditure and water turnover over several days in laying hens. Furthermore, we show that laying hens kept in floor pens fit into the general pattern of DEE among wild birds.     

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.