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.     

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.     

Comparison of methods for evaluating energy expenditure of incubating wandering albatrosses.

Measurements of incubation energetics can vary depending on the method used to measure metabolism of an incubating bird. Therefore, we evaluated the energy expenditure of six male and four female wandering albatrosses (Diomedea exulans Linnaeus) using doubly labeled water (DLW), the rate of mass loss, and estimates of metabolic water production derived from water influx rate (WIR). Incubation metabolic rates (IMR) determined with DLW (169+/-21 kJ x kg(-1) x d(-1) SD) were significantly lower than estimates derived from mass loss (277+/-46 kJ x kg(-1) x d(-1) SD) and WIR (males=289+/-60 kJ x kg(-1) x d(-1) vs. females=400+/-69 kJ x kg(-1) x d(-1) SD). Estimates of IMR from mass loss and WIR were similar to IMR (305+/-39 kJ x kg(-1) x d(-1) SD) determined by respirometry in a previous study, and IMR from DLW was similar to estimates based on heart rate (HR; 147+/-26 kJ x kg(-1) x d(-1) SD) determined in another study. Applying the different measurements of IMR to construct an energy budget, we estimate that a breeding pair of wandering albatrosses spends 124-234 MJ to incubate the egg for 78 d. Finally, IMRs determined with DLW and HR were similar to estimated basal metabolic rates derived from six different allometric equations, suggesting that heat production from adult maintenance metabolism is sufficient to incubate the egg.     

Energy and water flux during terrestrial estivation and overland movement in a freshwater turtle

The doubly labeled water (DLW) method for studying energy and water balance in field-active animals is not feasible for freshwater animals during aquatic activities, but several species of nominally aquatic reptiles leave wetlands for several critical and extended behaviors, where they face challenges to their energy and water balance. Using DLW, we studied energy and water relations during terrestrial estivation and movements in the eastern long-necked turtle (Chelodina longicollis), a species that inhabits temporary wetlands in southeastern Australia. Water efflux rates of 14.3-19.3 mL (kg d)(-1 ) during estivation were nearly offset by influx, indicating that turtles did not maintain water balance while terrestrial, though dehydration was slow. Estivation energy expenditure declined over time to 20.0-24.6 kJ (kg d)(-1) but did not indicate substantial physiological specializations. Energy reserves are predicted to limit survival in estivation to an estimated 49-261 d (depending on body fat), which is in close agreement with observed bouts of natural estivation in this population. The energy cost and water flux rates associated with overland movement behavior ranged from 46 to 99 kJ (kg d)(-1 ) and from 21.6 to 40.6 mL (kg d)(-1), respectively, for turtles moving 23-34 m d(-1). When a wetland dries, a turtle that forgoes movement to other wetlands can save sufficient energy to fuel up to 134 d in estivation. The increasing time in estivation with travel distance gained in this energy "trade-off" fits our previous observations that more turtles estivate when longer distances must be traveled to the nearest permanent lake, whereas emigration is nearly universal when only short distances must be traversed. The DLW method shows promise for addressing questions regarding the behavioral ecology and physiology of freshwater turtles in terrestrial situations, though validation studies are needed.     

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.     

Assessment of physical activity in older individuals: a doubly labeled water study.

We compared the accuracy of two physical activity recall questionnaires and a motion detector in 45- to 84-yr-old women (n = 35) and men (n = 32), using doubly labeled water (DLW) in conjunction with indirect calorimetry as the criterion measure. Subjects were administered the Yale Physical Activity Survey (YPAS) and Minnesota Leisure Time Physical Activity Questionnaire (LTA). Physical activity energy expenditure was determined over a 10-day period by using a Caltrac uniaxial accelerometer and DLW in conjunction with indirect calorimetry. In older women, Minnesota LTA (386 +/- 228 kcal/day) and Caltrac (379 +/- 162 kcal/day) underestimated physical activity by approximately 55% compared with DLW (873 +/- 244 kcal/day). No difference was observed between daily physical activity measured by the YPAS (863 +/- 447 kcal/day) and DLW in older women. In older men, Minnesota LTA (459 +/- 288 kcal/day) and Caltrac (554 +/- 242 kcal/day) underestimated daily physical activity by approximately 50-60% compared with DLW (1,211 +/- 429 kcal/day). No difference was found between physical activity measured by the YPAS (1,107 +/- 612 kcal/day) and DLW in older men. Despite no difference in mean physical activity levels between YPAS and DLW in women and men, Bland and Altman (Lancet 1: 307-310, 1986) analyses demonstrated poor concordance between DLW and YPAS (i.e., limits of agreement = -1,310-1,518 kcal/day). Our data suggest that the Minnesota LTA recall and Caltrac uniaxial accelerometer may significantly underestimate free-living daily physical activity energy expenditure in older women and men. Although the YPAS compares favorably with DLW on a group basis, its use as a proxy measure of individual daily physical activity energy expenditure may be limited in older women and men.     

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.     

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.     

Nectar intake and energy expenditure in a flower visiting bat

Summary In a coastal region of Venezuela the daily energy expenditure (DEE) and water turnover of the flower visiting bat Anoura caudifer was measured by using the doubly labeled water method. In flower visitors, this method allows independent measurement of energy intake and expenditure if the animals drink no additional water and if the nectar's energy content is known. An average DEE of 12.4 kcal/d and water exchange of 13.4 ml/d were found. Our data show a balanced energy budget when animals in the field imbibe nectar with a sugar concentration of 18–21%, which is roughly medial in the range of nectar concentrations of various bat flowers. The energy turnover of flower visiting bats is high compared with DEEs of other bat species, small mammals and birds; flower visiting bats seem to belong to those species having a fast spin of the life motor.     

Determinants of Free-Living Energy Expenditure in Normal Weight and Obese Women Measured by Doubly Labeled Water

Total free-living energy expenditure (TEE) was measured in 9 normal weight controls and 5 obese women using the doubly labeled water (DLW) method. Resting energy expenditure (REE) and the thermic effect of food (TEF) were measured by indirect calorimetry and the energy cost of physical activity (PA) calculated by deduction, in order to quantify the components and identify determinants of free-living TEE. Although REE was quantitatively the major component of TEE in both groups, PA best explained the variability, contributing 76% to the variance in free-living TEE. The obese women had elevated values for TEE (12397+/-2565 vs. 8339+/-1787 kJ/d, mean+/-SD; p<0.00S), compared with the control women. PA (5071+/-2385 vs. 2552+/-1452; p<0.0S) and REE (6393+/-678 vs. 5084+/-259; p<0.000S) were also raised in the obese, whereas TEF was not significantly different between the groups, accounting for 7.6% of energy expenditure for the obese and 8% for the control subjects. Body weight was the single best determinant of mean daily free-living TEE across both groups. We conclude that PA and body weight are the main determinants of free-living TEE .     

Energy expenditure adjusted for body composition differentiates constitutional thinness from both normal subjects and anorexia nervosa

Constitutional thinness (CT) is characterized by a low and stable body mass index (BMI) without any hormonal abnormality. To understand the weight steadiness, energetic metabolism was evaluated. Seven CT, seven controls, and six anorexia nervosa (AN) young women were compared. CT and AN had a BMI <16.5 kg/m(2). Four criteria were evaluated: 1) energy balance including diet record, resting metabolic rate (RMR) (indirect calorimetry), total energy expenditure (TEE) (doubly labeled water), physical activity; 2) body composition (dual-energy X-ray absorptiometry); 3) biological markers (leptin, IGF-I, free T3); 4) psychological profile of eating behavior. The normality of free T3 (3.7 +/- 0.5 pmol/l), IGF-I (225 +/- 93 ng/ml), and leptin (8.3 +/- 3.4 ng/ml) confirmed the absence of undernutrition in CT. Their psychological profiles revealed a weight gain desire. TEE (kJ/day) in CT (8,382 +/- 988) was not found significantly different from that of controls (8,793 +/- 845) and AN (8,001 +/- 2,152). CT food intake (7,565 +/- 908 kJ/day) was found similar to that of controls (7,961 +/- 1,452 kJ/day) and higher than in AN (4,894 +/- 703 kJ/day), thus explaining the energy metabolism balance. Fat-free mass (FFM) (kg) was similar in CT and AN (32.5 +/- 2.9 vs. 34.1 +/- 1.9) and higher in controls (37.8 +/- 1.6). While RMR absolute values (kJ/day) were lower in CT (4,839 +/- 473) than in controls (5,576 +/- 209), RMR values adjusted for FFM were the highest in CT. TEE-to-FFM ratio was also higher in CT than in controls. Energetic metabolism balance maintains a stable low weight in CT. An increased energy expenditure-to-FFM ratio differentiates CT from controls and could account for the resistance to weight gain observed in CT.     

Consumption of Atlantic salmon smolts and parr by goosanders: estimates from doubly-labelled water measurements of captive birds released on two Scottish rivers

The field metabolic rates (FMRs) of nine captive goosanders, Mergus merganser , released on two Scottish rivers were estimated using the doubly-labelled water (DLW) technique. Mean (± S.E.) FMR was 2.322±0.239ml CO₂ g⁻¹ h⁻¹ and daily energy expenditure (DEE) 1939 ± 184 kJ per day. This was significantly greater (x 1.5) than previous estimates which assumed DEE to be three times the basal metabolic rate (BMR) based on regression equations predicting BMR from body mass. FMR of captives and dietary data from previous studies were used to estimate daily consumption of salmon, Salmo salar , smolts and parr by natural populations of these ducks on the river North Esk, north-east Scotland. Goosanders are likely to consume 480-522 g fish per day of which two-thirds are juvenile salmon; equivalent to a daily intake of 10-11 smolts and 48-52 parr. Annual predation of smolts by goosanders was estimated to be between 8000 and 15 000 or 3 and 16% of annual production.     

Canadian recommendations underestimate energy needs of women over fifty years as determined by doubly-labelled water

Accurate estimations of energy requirements at the population level are crucial because of disease processes associated with energy imbalance. The present objective was to compare energy expenditure with existing Recommended Nutrient Intakes for Canadians (RNIC) and determine whether the RNIC provides a true index of energy requirement in middle-aged and elderly Canadian women. A second objective was to compare energy expenditure and the RNIC to Food and Agriculture Organization, World Health Organization, United Nations University (FAO/WHO/UNU) predictions. Seventy-six women were recruited for the study (67.3 +/- 11.5 y, 63 +/- 11.7 kg, BMI 24.8 +/- 4.4 kg x m(-2)). The two-point doubly-labelled water (DLW) method was used over 13 days to assess energy expenditure while subjects carried out their routine activities. Subjects were stratified to enable age specific requirements for middle-aged and elderly women. At weight maintenance, energy needs were underestimated using the RNIC (7.1 +/- 1.6 MJ x d(-1), 1698 +/- 391 kcal x d(-1)) compared to total energy expenditure (10.0 +/- 3.2 MJ x d(-1), 2395 +/- 746 kcal x d(-1)) as determined by DLW as a whole and for each age group. The RNIC recommendations were lower than the FAO/WHO/UNU estimations even for light activity. Results indicate that mean energy expenditure was 29% greater than the RNIC recommendations created using formulas based on age and weight, whereas the FAO/WHO/ UNU estimations closely approximated energy expenditure based on heavy activity in women 49-79 y and light activity in women over 80 y old. These data suggest a systematic underestimation of Canadian energy recommendations for women.     

Adequacy of food rations in soldiers during exercise in hot, day-time conditions assessed by doubly labelled water and energy balance methods

The energy requirements of people doing physical work in hot climates are not clearly understood. In particular, we know little about the combined effects of heat stress and muscular work on energy requirements. During military exercises in the African bush soldiers are supplied with standard rations, the adequacy of which is unknown. We have now assessed the adequacy of these food and water rations in 12 male Zimbabwean soldiers during 12 days of strenuous, heat-stress exercise in the field. We used two methods to measure energy expenditure: the double-labelled water method (DLW) and the energy balance method (i.e. comparing dietary energy with changes, if any, in body energy stores). Two groups were studied: one group (eight subjects) carried out field exercises; the control group consisted of four soldiers doing normal work. Mean daily energy expenditure as assessed by the DLW method was [mean (SE)] 23 (1.5) MJ · day⁻¹ for the field group and 14 (0.5) MJ · day⁻¹ for the control group (P<0.001). By the energy balance method, daily energy expenditure was calculated to be 26 (0.7) MJ · day⁻¹ and 15.5 (0.4) MJ · day⁻¹ for the field group and control group, respectively. Body mass loss was 3 (0.1) kg [4.6 (0.3)% of body mass] for the field group, but the control group gained 1.1 (0.1) kg. Mean daily fluid intake was 11 (0.5) 1 · day⁻¹, suggesting that the standard ration supplied was inadequate. Body mass loss was caused by both the energy deficit and total body water loss. These results suggest strenuous work in hot, dry field conditions imposes extra energy requirements. Accepted: 21 January 1997     

Sucrose hydrolysis does not limit food intake by Pallas's long-tongued bats

Nectarivorous bats include very dilute nectar in their natural diet, and recent work with Pallas's long-tongued bat Glossophaga soricina showed that sugar (energy) intake rate decreased at dilute sucrose solutions. However, chiropterophillous nectar is composed mainly of the hexoses glucose and fructose. Because bats fed hexose nectar would save the delay of hydrolyzing sucrose, we hypothesized that sugar intake rate should be higher on this diet than on sucrose nectar. We compared intake response in Pallas's long-tongued bats offered 1 : 1 glucose-fructose (hexose) and sucrose diets at 5%, 10%, 20%, 30%, and 40% (mass/volume) sugar solutions. We also tested the hypothesis that sucrose hydrolysis limits food intake in bats. Intake response was the same in bats fed both types of diet: sugar intake rate was lower in dilute solutions and then increased with sugar concentration. Similar intake responses in both diets indicate that sucrose hydrolysis alone does not limit food intake and support the idea that the burden of processing excess water in dilute solutions plays a major role.     

Influence of delayed isotopic equilibration in urine on the accuracy of the (2)H(2)(18)O method in the elderly.

Isotopic determination of total energy expenditure (TEE) by the doubly labeled water (DLW) method may be affected by urine retention in the elderly. The isotopic enrichments in urine and plasma sampled simultaneously 4 h post-DLW dose were compared in a subset of 281 subjects [139 women, 142 men, 75 +/- 3 (SD) yr] of the 3,075 participants in the Health, Aging, and Body Composition study. Based on analytic precisions, a +/- 2% urine-plasma difference was set as the cut-off value. Ten percent of the population presented a difference lower than -2%, suggesting a delay in urine isotopic equilibration. This -13 +/- 10% urine-plasma difference was not linked to analytic errors, illnesses, the sampling time, or the time and quantity of water intake, suggesting that urine retention may be the main factor. The consequences are an 18 +/- 13 and 21 +/- 16% overestimation of the total body water and the TEE, respectively. Unexpectedly, 21% of the population presented a urine-plasma difference higher than +/- 2% that resulted, however, in a nonsignificant TEE underestimation of -3 +/- 5%. In conclusion, the delayed isotopic equilibration observed in urine reduces the accuracy of the DLW method in the elderly. It is recommended, when blood sampling is impossible, to adopt the intercept method with urine sampling 24 h postdose.     

Termite digestibility and water and energy contents determine the water economy index of numbats (Myrmecobius fasciatus) and other myrmecophages

Digestibility by captive numbats for termites was determined by feeding trials to be 81%+/-1.2% for Coptotermes sp. and 64%+/-3.3% for Nasutitermes sp. Water, ash, and energy content of both the Coptotermes (0.96+/-0.099 mg(dry mass) individual(-1), 78.0%+/-0.36% water, 5.8%+/-0.31% ash, 23.1+/-0.19 kJ g-1dry total energy) and Nasutitermes (0.91+/-0.046 mg(dry mass) individual(-1), 76.7%+/-3.09% water, 7.5%+/-1.10% ash, 22.7+/-0.36 kJ g-1dry total energy) were similar to values measured previously for other termites and for ants and insects in general. Numbats have a slow passage time for termites (20-30 h), presumably to enhance the digestion of termites. The water economy index (WEI) was 0.2 for captive numbats feeding on Coptotermes and 0.25 for Nasutitermes, whereas the WEI measured for wild, free-living numbats was 0.29, which corresponds to a digestibility of 58%. The WEI of a myrmecophage diet is determined by the energy and water contents and digestibilities of termites and ants, in the absence of drinking. The WEI for numbats, and other termitivorous mammals as well as reptiles, is higher than would be expected for an animal-based diet because of their relatively low digestibility (58%-81%) for termites. A high WEI preadapts myrmecophages to survival in arid environments without having to drink.     

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.     

Effect of water restriction on energy and water balance and osmoregulation of the fruit bat Rousettus aegyptiacus

The energy budget, water balance and osmoregulation of the fruit bat, Rousettus aegyptiacus, were studied during normal hydration and during water restriction (oven-dried apple diet). The water input and output were balanced during both normal hydration and water restriction. The kidney of the fruit bat is well adapted to handle the water load from its fruit diet by excreting large volumes (14% of the body mass per day) of dilute urine (113±25 mosmol·kg H₂P^-1) as well as reducing urine volume (-95%) and increasing urine osmotic concentration (555±280 mosmol·kg H₂O^-1) during water restriction. The haematocrit, plasma haemoglobin and total protein concentrations did not increase during water restriction and heat exposure, suggesting the conservation of plasma volume. Gross energy intake was not alfected by water restriction. However, digested energy intake and digestibility were significantly reduced. The effective regulation of energy and water budgets during water restriction suggests that the fruit bat can cope with seasonal climatic changes and with variable fruit supply during various seasons.Abbreviations BM body mass - DEI digested energy intake - EWL evaporative water loss - GEL gross energy intake - NH normal hydration - T _a ambient temperature - WR water restriction     

Estimation of food intake and ingested energy in Daubenton’s bats (Myotis daubentonii) during pregnancy and spermatogenesis

We studied food intake of and estimated ingested energy in female and male Myotis daubentonii during the periods of pregnancy (period 1, 8 May–4 June) and of intense spermatogenetic activity (period 2, 24 July–22 August) over 8 years (1996–2003) in central Germany. We used radiotelemetry to determine the time spent foraging and marked animals with chemiluminescent light-sticks to determine prey attack rates. Body length, body mass, moisture content, and caloric content of chironomids, the main prey of Daubenton’s bats, were measured to estimate the nightly food intake and, in consequence, energy intake. Pregnant females spent significantly more time foraging than males during period 1 and females during the post-lactation period. In contrast, male foraged longer during the period of highest spermatogenetic activity than during late spring and also significantly longer than post-lactating females. Based on a mean number of 8.3 prey attacks per minute, the time spent foraging, and a capture success rate of either 50 or 92%, calculated intake values with a feeding rate of 7.6 insects per minute (=92% capture success) were more consistent with literature data for other insectivorous bats than that of values calculated on the basis of a capture success rate of 50%. In the high capture-success model, calculated insect intake of female bats was 8.0 g during pregnancy and 4.9 g per day during post-lactation, providing 5.0 and 3.0 kJ of ingested energy per gram body mass per day. Calculated intake of male bats was 3.6 g insects per day during late spring and 8.0 g during period of intensive spermatogenesis, providing 2.6 and 5.7 kJ of ingested energy per gram body mass.