Differences in the range of thermoneutral zone between mouse strains: potential effects on translational research.

Laboratory mice are commonly used for studies emulating human metabolism. To render human energetics, their ratio of daily (DEE) to basal (BMR) energy expenditure of 1.7-1.8 should be maintained. However, the DEE/BMR ratio strongly depends on whether a given study using a mouse model is carried out above, or below the lower critical temperature (LCT) of the thermoneutral zone, which is rarely considered in translational research. Here, we used mice artificially selected for high or low rates of BMR along with literature data to analyze the effect of ambient temperature on possible systematic bias in DEE/BMR. We demonstrated that the estimated LCTs of mice from the high and low BMR lines differ by more than 7°C. Furthermore, the range of variation of LCTs of mouse strains used in translational research spans from 23 to 33°C. Differences between LCTs in our selected mice and other mouse strains are mirrored by differences in their DEE-to-BMR ratio, on average increasing it at the rate of 0.172°C-1 at temperatures below LCT. Given the wide range of LCTs in different mouse strains, we conclude that the energetic cost of thermoregulation may differ greatly for different mouse strains with a potentially large impact on translational outcomes. Thus, the LCT of a given mouse strain is an important factor that must be considered in designing translational studies.

Decay rates of arboreal and terrestrial nests of Eastern chimpanzees (Pan troglodytes schweinfurthii) in the Bugoma Central Forest Reserve, Uganda: Implications for population size estimates.

Chimpanzees were once thought to sleep primarily in the trees, but recent studies indicate that some populations also construct terrestrial night nests. This behavior has relevance not only to understanding the behavioral diversity of Pan troglodytes, but also to the conservation of the species, given that nest encounter rates are often used to estimate great ape population densities. A proper estimate of decay rates for ground nests is necessary for converting the encounter rate of nests to the density of weaned chimpanzees. Here we present the results of the first systematic comparative study between the decay rates of arboreal and terrestrial chimpanzee nests, from the Bugoma Central Forest Reserve in western Uganda. We followed the decay of 56 ground and 51 tree nests in eight nest groups between April 2020 and October 2021. For 15 of the ground and 19 of the tree nests, we collected detailed information on the condition of the nests every two weeks; we checked the remaining 73 nests only twice. On average, ground nests lasted 238 days versus 276 days for tree nests (p = 0.05). Of the 107 total nests surveyed, 51% of tree and 64% of ground nests had disappeared after six months. Based on our results, we propose a modification of the formula used to convert nest density into chimpanzee density. Our results highlight the importance of taking into account potential differences in decay rates between ground versus tree nests, which will likely influence our understanding of the distribution of ground nesting behavior in chimpanzee across tropical Africa, as well as our estimations of the densities of ground nesting populations.

Factors affecting nest height and ground nesting behaviour in Eastern chimpanzees (Pan troglodytes schweinfurthii) of the northern Democratic Republic of the Congo.

In order to achieve a better understanding of the factors that might have led our hominin ancestors to transition to a more terrestrial niche, including sleeping on the ground, we have conducted a study on the ground nesting behavior of chimpanzees (Pan troglodytes). Chimpanzees, like all other species of great apes, build nests in which to sleep each night, but little is known about regional differences in their nesting habits. Previously, nesting on the ground was considered typical of gorillas, but rare in most populations of chimpanzees. Using data acquired during our extensive chimpanzee nesting survey conducted between 2004 and 2013 across a > 50 000 km2 region in northern Democratic Republic of the Congo, we report a distinctive ground nesting behaviour of eastern chimpanzees (P. t. schweinfurthii). We have mapped the geographical distribution of ground nesting and compared its frequency at 20 survey areas on both sides of a large river, the Uele. We found that ground nests made up more than 1% of total nests at 15 of the 20 survey regions. For a subset of 16 of these regions, we utilized statistical models to investigate whether forest type and structure, as well as the abundance of carnivores and large herbivores, and the activities of humans impacted the frequency of ground nesting and nest height. We predicted that higher encounter rates of human and dangerous animal signs would be associated with lower rates of ground nesting as well as increased nest height. Overall, 10.4% of the Bili-Uéré chimpanzee nests were terrestrial, but the frequency of ground nesting varied extensively between the survey areas (0-29% of nests). The occurrence of ground nests was positively associated with denser forests (p = 0.004), herb patches (p < 0.001), and light gaps (p < 0.001). Light gaps (p < 0.001), herb patches (p = 0.044), and vine tangles (p = 0.016) also had a strong negative effect on nest height. Hunting by humans had a negative effect on the probability of the occurrence of ground nests (p = 0.001) and a positive one on nest height (p = 0.013), with a similar but likely marginal effect of large herbivores on nest height (p = 0.023). In addition, the chimpanzees nested at significantly lower heights with increasing distance from roads and settlements (p < 0.001). Carnivore encounter rates, however, had no significant impact on ground nest frequency or nest height. Our results indicate that ground nesting can no longer be considered a rare and patchily-occurring phenomenon in Pan troglodytes, but is instead a major component of the chimpanzee behavioural repertoire across a considerable fraction of the range of the Eastern subspecies. Our study highlights that neither the large body size of gorillas nor the taming of fire are necessary conditions for hominids to sleep overnight on the ground, even in areas inhabited by multiple species of large carnivore. Human hunting, however, appears to reduce the probability of ground nesting, or eliminate the behavior altogether.

Mice selected for a high basal metabolic rate evolved larger guts but not more efficient mitochondria.

Intra-specific variation in both the basal metabolic rate (BMR) and mitochondrial efficiency (the amount of ATP produced per unit of oxygen consumed) has profound evolutionary and ecological consequences. However, the functional mechanisms responsible for this variation are not fully understood. Mitochondrial efficiency is negatively correlated with BMR at the interspecific level but it is positively correlated with performance capacity at the intra-specific level. This discrepancy is surprising, as theories explaining the evolution of endothermy assume a positive correlation between BMR and performance capacity. Here, we quantified mitochondrial oxidative phosphorylation activity and efficiency in two lines of laboratory mice divergently selected for either high (H-BMR) or low (L-BMR) levels of BMR. H-BMR mice had larger livers and kidneys (organs that are important predictors of BMR). H-BMR mice also showed higher oxidative phosphorylation activity in liver mitochondria but this difference can be hypothesized to be a direct effect of selection only if the heritability of this trait is low. However, mitochondrial efficiency in all studied organs did not differ between the two lines. We conclude that the rapid evolution of BMR can reflect changes in organ size rather than mitochondrial properties, and does not need to be accompanied obligatorily by changes in mitochondrial efficiency.

Brain size, gut size and cognitive abilities: the energy trade-offs tested in artificial selection experiment.

The enlarged brains of homeotherms bring behavioural advantages, but also incur high energy expenditures. The 'expensive brain' (EB) hypothesis posits that the energetic costs of the enlarged brain and the resulting increased cognitive abilities (CA) were met by either increased energy turnover or reduced allocation to other expensive organs, such as the gut. We tested the EB hypothesis by analysing correlated responses to selection in an experimental evolution model system, which comprises line types of laboratory mice selected for high or low basal metabolic rate (BMR), maximum (VO2max) metabolic rates and random-bred (unselected) lines. The traits are implicated in the evolution of homeothermy, having been pre-requisites for the encephalization and exceptional CA of mammals, including humans. High-BMR mice had bigger guts, but not brains, than mice of other line types. Yet, they were superior in the cognitive tasks carried out in both reward and avoidance learning contexts and had higher neuronal plasticity (indexed as the long-term potentiation) than their counterparts. Our data indicate that the evolutionary increase of CA in mammals was initially associated with increased BMR and brain plasticity. It was also fuelled by an enlarged gut, which was not traded off for brain size.

Larger guts and faster growth in mice selected for high basal metabolic rate.

Postnatal growth in birds and mammals is the time of highest vulnerability and relatively high energy demands and therefore shapes the organisms' future outcomes. Several different factors might impose limitations on growth in juveniles, one of them being the efficiency of the digestive process and size of the gastrointestinal tract. We tested the gut size-growth rate relationship using a unique experimental model-mice from a selection experiment designed to produce two lines with divergent levels of basal metabolic rate (BMR): the high BMR (H-BMR) and low BMR (L-BMR) line types. These lines differ with respect to not only BMR, but also correlated traits-internal organ size and food intake. Applying a cross-fostering design and a thermoregulatory burden imposed by shaving the mothers, we demonstrated that the mass of intestine strongly affected the growth rate, with the H-BMR pups having larger intestines and growing faster, and with reduced growth rate of pups of both lines nursed by shaved L-BMR mothers. Our study also provides a functional link between high growth rate of neonates and high BMR of adults, partly reflecting metabolic costs of maintenance of their guts.

Not that hot after all: no limits to heat dissipation in lactating mice selected for high or low BMR.

Heat dissipation has been suggested as a limit to sustained metabolic effort, e.g. during lactation, when overheating is a possible risk. We tested this hypothesis using mice artificially selected for either high or low BMR that also differed with respect to parental effort. We used fixed size cross-fostered families and recorded litter mass daily until the 14th day of lactation. Midway through the experiment (day 8) half of the mothers from each group had fur from the dorsal body surface removed to increase their thermal conductance and facilitate heat dissipation. Our results showed that neither high nor low BMR mouse lines benefited from increasing their thermal conductance at peak lactation. On the contrary, growth of the litters reared by the low BMR females was compromised. Thus, our results do not support the heat dissipation limitation hypothesis.

The nexus of hair corticosterone level, immunocompetence, metabolic rates and overwinter survival in the root vole, Microtus oeconomus.

Although corticosterone (CORT) regulates many physiological mechanisms, the associations between CORT levels, immunocompetence, energy expenditures and overwinter survival have not been examined. Here, we studied individual variation in CORT level extracted from hair, immunocompetence quantified as the neutrophil-to-lymphocyte (N/L) ratio, total white blood cells (WBC) and natural antibody levels (NAbs), along with the resting (RMR) and peak metabolic rates (PMR) and mortality during three consecutive winter seasons in a natural population of the root vole, Microtus oeconomus. In early winter, hair CORT level was strongly positively associated with body mass and inversely related to voles' ability to survive. We suggest that the observed association between hair CORT level and body mass may be the key component of the physiological nexus driving the survivorship of individual rodents. Additionally, hair CORT was a significant predictor of variation of the whole body RMR, which in turn enhanced overwinter survival in the studied population. On the other hand, hair CORT was not significantly associated with changes in the blood indices. Interestingly, the analysis carried out only during the first year of study (2008), which was characterized by a high population density and prevalence of infestation with a blood protozoan, Babesia spp., showed that the intensity of the infestation was negatively correlated with both the hair CORT level and the N/L ratio. Because CORT is often considered immunosuppressive, we expected a positive association between its level and the N/L ratio. However, hair CORT did not significantly correlate with the N/L ratio. We suggest that the lack of an association between hair CORT and the N/L ratio resulted from a small inter-individual variation in the N/L ratio in 2008, which was much higher and less variable than in the other years of our study.

Heat dissipation does not suppress an immune response in laboratory mice divergently selected for basal metabolic rate (BMR).

The capacity for heat dissipation is considered to be one of the most important constraints on rates of energy expenditure in mammals. To date, the significance of this constraint has been tested exclusively under peak metabolic demands, such as during lactation. Here, we used a different set of metabolic stressors, which do not induce maximum energy expenditures and yet are likely to expose the potential constraining effect of heat dissipation. We compared the physiological responses of mice divergently selected for high (H-BMR) and low basal metabolic rate (L-BMR) to simultaneous exposure to the keyhole limpet haemocyanin (KLH) antigen and high ambient temperature (Ta). At 34°C (and at 23°C, used as a control), KLH challenge resulted in a transient increase in core body temperature (Tb) in mice of both line types (by approximately 0.4°C). Warm exposure did not produce line-type-dependent differences in Tb (which was consistently higher by ca. 0.6°C in H-BMR mice across both Ta values), nor did it result in the suppression of antibody synthesis. These findings were also supported by the lack of between-line-type differences in the mass of the thymus, spleen or lymph nodes. Warm exposure induced the downsizing of heat-generating internal organs (small intestine, liver and kidneys) and an increase in intrascapular brown adipose tissue mass. However, these changes were similar in scope in both line types. Mounting a humoral immune response in selected mice was therefore not affected by ambient temperature. Thus, a combined metabolic challenge of high Ta and an immune response did not appreciably compromise the capacity to dissipate heat, even in the H-BMR mice.

Milk output and composition in mice divergently selected for basal metabolic rate.

From an evolutionary perspective, the high basal metabolic rate (BMR) of homeotherms is hypothesised to be a by-product of natural selection for effective parental care. We estimated daily milk output during two consecutive lactation bouts in mice divergently selected for high/low BMR and applied a cross-fostered design to control for potential differences in the between-line suckling abilities of nursed juveniles. Additionally, to remedy the potential limitation imposed by the ability of mother mice to dissipate excess heat, we exposed them to an ambient temperature of 17°C during the most energetically demanding second week of lactation. We found that the mice selected for high BMR produced significantly more milk in a 24 h period in both reproductive bouts. The milk samples obtained from the high BMR females had lower protein concentration and did not differ with respect to fat. However, the concentration of the primary milk carbohydrate ­ lactose ­ was higher. Although all the above between-line differences were statistically significant, their magnitude was too small to unambiguously ascribe them as stemming from a positive genetic correlation between the physiological traits underlying BMR and lactation performance. Nevertheless, our study lends such support at least at the level of phenotypic variation.

Effect of the abrasive properties of sedges on the intestinal absorptive surface and resting metabolic rate of root voles.

Recent studies on grasses and sedges suggest that the induction of a mechanism reducing digestibility of plant tissues in response to herbivore damage may drive rodent population cycles. This defence mechanism seems to rely on the abrasive properties of ingested plants. However, the underlying mechanism has not been demonstrated in small wild herbivores. Therefore, we carried out an experiment in which we determined the joint effect of abrasive sedge components on the histological structure of small intestine as well as resting metabolic rate (RMR) of the root vole (Microtus oeconomus). Histological examination revealed that voles fed with a sedge-dominated diet had shorter villi composed from narrower enterocytes in duodenum, jejunum and ileum. Reduction in the height of villi decreased along the small intestine. Activity of the mucus secretion increased along the small intestine and was significantly higher in the ileum. The intestinal abrasion exceeded the compensatory capabilities of voles, which responded to a sedge-dominated diet by a reduction of body mass and a concomitant decrease in whole body RMR. These results explain the inverse association between body mass and the probability of winter survival observed in voles inhabiting homogenous sedge wetlands.

High basal metabolic rate does not elevate oxidative stress during reproduction in laboratory mice.

Increased oxidative stress (OS) has been suggested as a physiological cost of reproduction. However, previous studies reported ambiguous results, with some even showing a reduction of oxidative damage during reproduction. We tested whether the link between reproduction and OS is mediated by basal metabolic rate (BMR), which has been hypothesized to affect both the rate of radical oxygen species production and antioxidative capacity. We studied the effect of reproduction on OS in females of laboratory mice divergently selected for high (H-BMR) and low (L-BMR) BMR, previously shown to differ with respect to parental investment. Non-reproducing L-BMR females showed higher oxidative damage to lipids (quantified as the level of malondialdehyde in internal organ tissues) and DNA (quantified as the level of 8-oxodG in blood serum) than H-BMR females. Reproduction did not affect oxidative damage to lipids in either line; however, it reduced damage to DNA in L-BMR females. Reproduction increased catalase activity in liver (significantly stronger in L-BMR females) and decreased it in kidneys. We conclude that the effect of reproduction on OS depends on the initial variation in BMR and varies between studied internal organs and markers of OS.

Shaving increases daily energy expenditure in free-living root voles.

Experimental manipulation of energy expenditure has long been recognized as an effective means for identifying causative effects and avoiding confounded interpretations arising from spurious correlations. This approach has been successfully applied mainly to studies on birds, particularly on reproducing adults, whereas manipulations in mammals have proved more problematic. Here, we tested the hypothesis that shaving off 50% of the dorsal pelage should effectively increase energy expenditure in wild root voles (Microtus oeconomus) in their natural environment. We measured daily energy expenditure (DEE), using doubly labelled water in shaved and unshaved voles at the beginning of winter. The difference in DEE (corrected for body mass and year effects) between experimental and control group fluctuated from 11.5% to 17.3%. Probability of recapture over the 3 day DEE assay was strongly dependent on body mass, but did not differ between shaved and unshaved animals; however, a prevalence of larger (heavier) shaved individuals was observed. Shaved animals lost more weight between the first and second trapping. Shaving therefore appears to be an effective method of increasing the cost of total DEE in wild endotherms in their natural environment.

Immunocompetence and high metabolic rates enhance overwinter survival in the root vole, Microtus oeconomus.

Despite its presumed significance, the association between immune defence, energy expenditures and overwinter survival is rarely studied. We analysed individual variation in immunocompetence quantified as neutrophil-to-lymphocyte ratio (N/L), total white blood cells (WBC) and natural antibody levels, along with resting (RMR) and peak metabolic rates (PMR) and mortality during three consecutive winter seasons in a natural population of the root vole, Microtus oeconomus. In early winter, WBC count was negatively correlated with RMR, whereas N/L ratio was negatively correlated with swim-elicited PMR. We suggest that while the first correlation reflected the trade-off between energy allocation in immunocompetence and other metabolically demanding processes, the latter correlation stemmed from stress-induced immunosuppression elicited by the necessity to cope with swimming in frequently flooded habitat. In addition, the analysis carried out during the first year of study characterized by a high population density and prevalence of infestation with a blood parasite--Babesia spp., showed that its intensity was inversely correlated with the N/L ratio. In summary, our results suggest that elevated N/L ratio increases the winter survival of free-ranging rodents by increasing their ability to cope with parasitic infections.

Basal metabolic rate is positively correlated with parental investment in laboratory mice.

The assimilation capacity (AC) hypothesis for the evolution of endothermy predicts that the maternal basal metabolic rate (BMR) should be positively correlated with the capacity for parental investment. In this study, we provide a unique test of the AC model based on mice from a long-term selection experiment designed to produce divergent levels of BMR. By constructing experimental families with cross-fostered litters, we were able to control for the effect of the mother as well as the type of pup based on the selected lines. We found that mothers with genetically determined high levels of BMR were characterized by higher parental investment capacity, measured as the offspring growth rate. We also found higher food consumption and heavier visceral organs in the females with high BMR. These findings suggested that the high-BMR females have higher energy acquisition abilities. When the effect of the line type of a foster mother was controlled, the pup line type significantly affected the growth rate only in the first week of life, with young from the high-BMR line type growing more rapidly. Our results support the predictions of the AC model.

The energy cost of voluntary running in the weasel Mustela nivalis.

The small size and elongate shape of weasels (Mustela nivalis) probably evolved to facilitate movement within the burrow systems of prey species, but result in high energy costs of thermoregulation. In this study we measured metabolic rates of weasels during voluntary locomotion to determine whether energy costs of transport are also high in these unusually shaped mammals. In addition, we measured the lower and upper limits of aerobic metabolism [resting metabolic rate (RMR) and maximal oxygen consumption in forced exercise (V(O(2),max))], and used the wide size range of adult weasels to investigate the intraspecific scaling of energy metabolism. Finally, we combined measurements of energy use during running with radiotracking and doubly labeled water data from free-living weasels to estimate the importance of locomotor costs in daily energy budgets. We found that weasels have higher than predicted costs of running, largely because of an elevated intercept of the speed versus metabolic rate relationship. Running costs were strongly affected by the approximately fourfold range of body size in adults. As reported in other studies, the RMR of weasels was considerably higher than predicted from body mass. Maximal oxygen consumption was also higher than predicted, but factorial aerobic scope (V(O(2),max)/RMR) was within the normal range for mammals. Intraspecific mass scaling of RMR and V(O(2),max) did not differ from typical interspecific mammalian allometries. In wild weasels, locomotor costs comprised roughly 5% of daily energy expenditures; this low value was primarily a result of short travel times and distances.

Phylogenetic analysis of mammalian maximal oxygen consumption during exercise.

We compiled published values of mammalian maximum oxygen consumption during exercise ( ) and supplemented these data with new measurements of for the largest rodent (capybara), 20 species of smaller-bodied rodents, two species of weasels and one small marsupial. Many of the new data were obtained with running-wheel respirometers instead of the treadmill systems used in most previous measurements of mammalian . We used both conventional and phylogenetically informed allometric regression models to analyze of 77 'species' (including subspecies or separate populations within species) in relation to body size, phylogeny, diet and measurement method. Both body mass and allometrically mass-corrected showed highly significant phylogenetic signals (i.e. related species tended to resemble each other). The Akaike information criterion corrected for sample size was used to compare 27 candidate models predicting (all of which included body mass). In addition to mass, the two best-fitting models (cumulative Akaike weight=0.93) included dummy variables coding for three species previously shown to have high (pronghorn, horse and a bat), and incorporated a transformation of the phylogenetic branch lengths under an Ornstein-Uhlenbeck model of residual variation (thus indicating phylogenetic signal in the residuals). We found no statistical difference between wheel- and treadmill-elicited values, and diet had no predictive ability for . Averaged across all models, the allometric scaling exponent was 0.839, with 95% confidence limits of 0.795 and 0.883, which does not provide support for a scaling exponent of 0.67, 0.75 or unity.

Environmental and genetic influences on body mass and resting metabolic rates (RMR) in a natural population of weasel Mustela nivalis.

Body mass (BM) and resting metabolic rates (RMR) are two inexorably linked traits strongly related to mammalian life histories. Yet, there have been no studies attempting to estimate heritable variation and covariation of BM and RMR in natural populations. We used a marker-based approach to construct a pedigree and then the 'animal model' to estimate narrow sense heritability (h(2) ) of these traits in a free-living population of weasels Mustela nivalis--a small carnivore characterised by a wide range of BM and extremely high RMR. The most important factors affecting BM of weasels were sex and habitat type, whereas RMR was significantly affected only by seasonal variation of this trait. All environmental factors had only small effect on estimates of additive genetic variance of both BM and RMR. The amount of additive genetic variance associated with BM and estimates of heritability were high and significant in males (h(2) = 0.61), but low and not significant in females (h(2 ) =( ) 0.32), probably due to small sample size for the latter sex. The results from the two-trait model revealed significant phenotypic (r(P) = 0.62) and genetic correlation (r(A) = 0.89) between BM and whole body RMR. The estimate of heritability of whole body RMR (0.54) and BM corrected RMR (0.45) were lower than estimates of heritability for BM. Both phenotypic and genetic correlations between BM corrected RMR and BM had negative signals (r(P) = -0.42 and r(A) = -0.58). Our results indicate that total energy expenditures of individuals can quickly evolve through concerted changes in BM and RMR.

Effect of dietary restriction on metabolic, anatomic and molecular traits in mice depends on the initial level of basal metabolic rate.

Dietary restriction (DR)-related delay of ageing is hypothesized to be mediated by the reduction of the metabolic rate (MR). However, studies on the effect of DR on MR have produced equivocal results. We demonstrated that this lack of congruency can be due to a variation in the initial level of MR within a given pool of experimental subjects. We subjected laboratory mice from two line types divergently selected for basal MR (BMR) to 30% DR lasting 6 months to test whether the effect of DR depends on the initial variation in BMR and peak MR. BMR and peak MR were independently affected by DR. The effect of DR was stronger in line types with higher initial levels of MR. Line-type-specific changes in the proportions of body components explained contrasting effects of DR on the mass-corrected BMR, which decreased in the high-BMR line type and did not change in the low-BMR line type. We conclude that the initial variation in MR can significantly affect response to DR. However, we found no association between the level of MR and mechanisms underlying the susceptibility to or protection against oxidative stress.

Significance of variation in basal metabolic rate in laboratory mice for translational experiments.

The basal metabolic rate (BMR) accounts for 60-70% of the daily energy expenditure (DEE) in sedentary humans and at least 50% of the DEE in laboratory mice in the thermoneutral zone. Surprisingly, however, the significance of the variation in the BMR is largely overlooked in translational research using such indices as physical activity level (PAL), i.e., the ratio of DEE/BMR. In particular, it is unclear whether emulation of human PAL in mouse models should be carried out within or below the thermoneutral zone. It is also unclear whether physical activity within the thermoneutral zone is limited by the capacity to dissipate heat generated by exercise and obligatory metabolic processes contributing to BMR. We measured PAL and spontaneous physical activity (SPA) in laboratory mice from two lines, divergently selected towards either high or low level of BMR, and acclimated to 30 °C (i.e., the thermoneutral zone), 23 or 4 °C. The mean PAL did not differ between both lines in the mice acclimated to 30 °C but became significantly higher in the low BMR mouse line at the lower ambient temperatures. Acclimation to 30 °C reduced the mean locomotor activity but did not affect the significant difference observed between the selected lines. We conclude that carrying out experiments within the thermoneutral zone can increase the consistency of translational studies aimed at the emulation of human energetics, without affecting the variation in physical activity correlated with BMR.