Correlation of metabolism with tissue carbon and nitrogen turnover rate in small mammals
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- MacAvoy, S.E., Arneson, L.S. & Bassett, E. Oecologia (2006) 150: 190. doi:10.1007/s00442-006-0522-0
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Stable isotopes have proven to be a useful tool for deciphering food webs, examining migration patterns and determining nutrient resource allocation. In order to increase the descriptive power of isotopes, an increasing number of studies are using them to model tissue turnover. However, these studies have, mostly by necessity, been largely limited to laboratory experiments and the demand for an easier method of estimating tissue turnover in the field for a large variety of organisms remains. In this study, we have determined the turnover rate of blood in mice and rats using stable isotope analysis, and compared these rates to the metabolic rates of the animals. Rats (Rattus norvegicus) (n=4) and mice (Mus musculus) (n=4) were switched between isotopically distinct diets, and the rate of change of δ13C and δ15N in whole blood was determined. Basal metabolic rates (as CO2 output and O2 consumption per unit time, normalized for mass) were determined for the rats and mice. Rats, which were an order of magnitude larger and had a slower metabolic rate per unit mass than mice (0.02 vs. 0.14 O2/min/g), had a slower blood turnover than mice for 13C (t1/2 =24.8 and 17.3 days, respectively) and 15N (t1/2 =27.7 and 15.4 days, respectively). A positive correlation between metabolic rate and blood isotopic turnover rate was found. These are the only such data for mammals available, but the literature for birds shows that mass and whole-body metabolic rates in birds scale logarithmically with tissue turnover. Interestingly, the mammalian data graph separately from the bird data on a turnover versus metabolic rate plot. Both mice and rat tissue in this study exhibited a slower turnover rate compared to metabolic rate than for birds. These data suggest that metabolic rate may be used to estimate tissue turnover rate when working with organisms in the field, but that a different relationship between tissue turnover and metabolism may exist for different classes of organisms.
KeywordsStable isotope Turnover Metabolism Blood Mammal
Stable isotope analysis has become an important tool used by ecologists when determining food webs, resource use, migration patterns, and species interactions. The basic premise behind using stable isotopes in dietary studies is that a consumer’s tissues will isotopically resemble what is consumed (Fry and Sherr 1984; Minagawa and Wada 1984; Peterson and Howarth 1987). Tissues can only be built from available nutrients, such as carbohydrates, proteins, and lipids, although the extent to which a tissue resembles the different dietary components depends on the percent composition of the food as well as the type of tissue examined (Hobson et al. 1995; MacAvoy et al. 2005; Tieszen et al. 1983; Tieszen and Farge 1993).
Stable isotope analysis can be a valuable tool in differentiating the relative importance of protein and carbohydrate in diet as long as the isotope ratios of the various nutrient components are known (Arneson and MacAvoy 2005; Hobson and Bairlein 2003; Koch and Phillips 2002; Phillips and Gregg 2003; Phillips and Koch 2002). By utilizing stable isotopes, studies have shown that protein is the primary source material used for building new tissues whereas carbohydrates are used mainly as an energy source (Hobson et al. 2000; MacAvoy et al. 2005). Because organisms resemble the isotopic signatures of their diets, many ecological studies use stable isotopes to gain insights into the food web of a particular community or ecosystem (see Hobson 1999; Hobson and Wassenaar 1999 for reviews). These studies often assume that consumers are in isotopic equilibrium with their diet. However, this may not always be the case in the event of a shifting diet or migration of consumer or prey (Gannes et al. 1997; Hobson 1999).
The isotopic turnover rates of various tissues have been published for several animals, including birds, mammals, and marine organisms. For example, it has been shown that garden warblers (Sylvia borin) and Japanese quails (Coturnix japonica) have similar blood δ13C half-lives at 5.4 and 11.4 days, respectively (Hobson and Bairlein 2003; Hobson and Clark 1992), while larger birds such as the American crow (Corvus brachyrhynchos) and canvasback duck (Aythya valisineria) have longer blood half-lives: approximately 30 and 21 days, respectively (Haramis et al. 2001; Hobson and Clark 1993). MacAvoy et al. (2005) found that blood δ13C turnover in mice (Mus musculus) has a half-life of approximately 17 days, while Voigt et al. (2003) found blood δ13C half-lives in bats were much longer, 113 or 120 days in two different species. Turnover in poikilotherms is much slower than that of homeotherms, which is likely due to low metabolic rates (MacAvoy et al. 2001). In cases where turnover in poikilotherms is relatively fast, it has been shown that the rapid turnover is due mostly (and in some cases exclusively) to growth (Tieszen and Farge 1993; Frazer et al. 1997; Herzka and Holt 2000; Maruyama et al. 2001; Vander Zanden et al. 1997).
Studies continue to show that large and significant differences exist among organisms in how quickly they incorporate the isotopic signature of their diet. Although an increasing number of studies are appearing in the literature, measurements of isotope turnover are still limited to a few species. However, if a relationship between isotope turnover and a more common, well-documented parameter, such as metabolic rate, could be established, then estimates of isotope turnover rates could be more widely applied. Researchers using isotopes to characterize food webs could evaluate when the organisms could be expected to be in isotope equilibrium.
Metabolism refers to the sum of all catabolic (degrading) and anabolic (synthesizing) processes that occur in a living system. Metabolic processes produce energy by consuming O2 in order to break down molecules that store energy, producing CO2 as a waste product. Anabolic metabolic processes use this energy to produce macromolecules, carbohydrates, proteins or lipids, in order to either increase tissue mass (resulting in growth) or to replace macromolecules that have been degraded (resulting in tissue replacement). Thus, metabolic processes contribute to both components of tissue turnover as measured by stable isotope analysis, growth and tissue replacement (MacAvoy et al. 2005). Metabolic rate, which can be measured by oxygen consumption per unit time, correlates to the body mass of the organism according to the “3/4 rule” (Kleiber 1932, 1947), in which metabolic rate is proportional to M0.75, where M is body mass.
In this study we examine the relationship between tissue turnover rate and resting metabolic rate for two species of small mammal, rats (Rattus norvegicus) and mice (Mus musculus). We also compile existing information on metabolic rate and tissue turnover for a variety of avian species in order to broadly describe the overall relationship between the two variables for birds. We found that rats, which were an order of magnitude larger and had a slower metabolic rate per unit mass than mice, had a slower blood carbon and nitrogen turnover than mice (carbon t1/2 = 24.8 vs. 17.3 days; nitrogen t1/2 = 27.8 and 15.4 days). Metabolic rates and blood turnover rates of different bird species show that mass and whole-body metabolic rates scale logarithmically with tissue turnover. However, mice and rats both had a slower blood isotopic turnover rate versus MR/gram than birds of similar body mass, suggesting that the relationship between these two variables may differ between classes of organism.
Experimental design and tissue sampling
Average tissue signatures at t0 and tend and diet-to-tissue discrimination averages for each diet. ±SD is the standard deviation and Frac stands for “fractionation,” the name given to the diet-to-tissue discrimination value
Control diet (N=10)
M. musculus (N=15)
R. norvegicus (N=6)
Experimental diet (N=8)
M. musculus (N=8)
R. norvegicus (N=12)
Stable isotope analysis
Metabolic rates (MR)
CO2 output was measured using a Qubit Systems (Kingston, ON, USA) S153 CO2 Analyzer attached to an open air respiration chamber. Two readings were taken per animal during the equilibration phase, and two more were taken prior to blood sampling throughout the course of the experiment. In this study, MR is measured as O2/min.
When solving Eq. 5 for the contribution of growth to tissue turnover, m was set to zero. The growth constant, k, used in Eq. 5 was the value obtained from the day equilibrium was reached, tE. Any isotopic turnover in excess of what was attributable to growth was considered metabolic tissue replacement. The metabolic constant, m, was determined using observed data in Eq. 6 from day 0 to day tE. The best estimate of m resulted in the least absolute sum of the differences between calculated and observed isotope values for each measurement up until the time of equilibrium (which was day 65 in all cases). We interpreted an approximate 0.1‰ fluctuation between measurements made at successive time intervals as being indicative of the animals reaching isotopic equilibrium. The reproducibility of tissue isotope (δ13C and δ15N) measurements usually has a standard deviation of ±0.2‰, so we accepted a 0.1‰ fluctuation as a reasonable approximation of isotope equilibrium. Earlier feeding experiments with this particular strain of mouse and diets similar to those in this study observed the time to isotopic equilibrium to be approximately 70 days. Therefore, the experimental mice were fed for 85 days, and we expected equilibrium to be reached during this time, although it was some months before all isotope data could be gathered and analyzed. Although we did not have isotope equilibrium time estimates for rats, weekly sampling was halted at 72 days. However, the experimental group continued to be fed the new diet, and a final blood sample was taken at 176 days to ensure there was no difference between the last weekly measurement and the one 100 days later. No significant difference (δ13C −25.1 vs. −25.1‰ and δ15N 8.3 vs. 8.4‰, respectively) was observed between the 72- and 176-day samples.
Metabolic rate and m
Equations were developed relating the rate of blood turnover (m and t1/2) to average mass, metabolic rate and metabolic rate per unit mass. To complete these models, a literature review was also performed with any published turnover data available. In addition to using data obtained in this study with R. norvegicus and M. musculus, data found in published papers on other species was also used to support/strengthen correlations (birds: Bearhop et al. 2002; Chamberlain et al. 1997; Evans-Ogden et al. 2004; Haramis et al. 2001; Hobson and Clark 1992a, 1993; Pearson et al. 2003; mammals: Ayliffe et al. 2004; Tieszen et al. 1983).
The animals used in this study were weaned on the control diet, and were also fed this diet until they were switched to the experimental treatment. The animals were maintained on the control diet for an equilibration period of four months to ensure that the different lots of the commercially available diet did not affect the stable isotope ratio. Blood samples taken during the equilibrium phase prior to the beginning of the experiment from both species of animals indicated that the carbon and nitrogen stable isotope ratios did not change during this time (data not shown).
On day zero of the experiment, two mice and two rats were maintained on the control diet, while the diets of four mice and four rats were changed to the experimental diet. Blood samples were collected from all animals in order to determine the starting carbon and nitrogen stable isotope ratios. The average blood δ13C at the start of the experiment (t0) was −19.2‰ (±0.1) in M. musculus (n=15), and −19.0‰ (±0.1) in R. norvegicus (n=6; Table 1). The average blood δ15N at the beginning of the experiment was 6.1‰ (±0.1) in M. musculus (n=15), and 6.1‰ (±0.1) in R. norvegicus (n=6; Table 1). The diet–tissue discrimination values at the beginning of the experiment were 2.3 and 2.4‰ for carbon and 3.3 and 3.3‰ for nitrogen in M. musculus and R. norvegicus, respectively.
Average half lives (t1/2) and metabolic constants (m) for each isotope in each species
Mass, metabolic rate (MR in ml O2/min), and isotope (13C and 15N) turnover rates of various tissues in four mammals
MR (ml O2/min)
(1) Turnover reference
(2) MR and mass reference
Mammalian: Rodents 101 g
(1) Tieszen et al. (1983)
(1) Macavoy et al. (2005)
(1) This Study
Mammalian: Rodents 102g
(1) This Study
(2) Hart (1971)
(1) Ayliffe et al. (2004)
Mass, metabolic rate (in mL O2/min), and isotope (13C and 15N) turnover data for seven species of birds
MR (ml O2/min)
(1) Turnover reference
(2) MR and mass reference
Blood (clam/corn Diet)
Blood (tuber diet)
Blood (clam diet)
(1) Haramis et al. (2001)
(2) Woodin and Stephenson (1998)
Blood (meal worm diet)
Blood (blk elderberry diet)
(1) Hobson and Bairlein (2003)
(2) Mckechnie and Wolf (2004)
Blood (49% insect diet)
Blood (73% insect diet)
Blood (97% insect diet)
(1) Pearson et al. (2003)
(2) Mckechnie and Wolf (2004)
(1) Hobson and Clark (1993)
(2) Mckechnie and Wolf (2004)
(1) Hobson and Clark (1992)
(2) Roberts and Baudinette (1986)
(1) Bearhop et al. (2002)
(2) Mckechnie and Wolf (2004)
Calidris alpina pacifica
(1) Evans-Ogden et al. (2004)
(2) Lindstrom (1997)
The carbon isotope turnover rate of the mice was slightly slower in this study than that reported for the same strain by MacAvoy et al. (2005) (half-lives for carbon: 17.3 vs. 16.9). However, the mice used in the previous study were younger than the adults used here and the shorter half-life likely reflects an increase in mass superimposed on turnover from metabolic tissue replacement. MacAvoy et al. (2005) estimated that growth accounted for approximately 10% of the observed isotope turnover, whereas in the current study mass gain was negligible.
In this study, as in our previous studies (MacAvoy et al. 2005; Arneson et al. 2006), the carbon and nitrogen tissue turnover rates are roughly similar within a tissue. This is in contrast to results from Carleton and del Rio (2005) in which they find that δ13C incorporation is approximately 1.5 times faster than δ15N in birds exposed to low temperatures. Although we do frequently see differences between nitrogen and carbon incorporation rates, we cannot apply significance to these differences.
In the final section of this study, we compared the tissue turnover rate to MR in rats and mice, and found that there is a correlation between these two measurements. Specifically, we found that a faster MR per gram tissue correlates with a faster rate of tissue turnover (Fig. 7). This relationship is not unexpected, as the equation describing stable isotope turnover rate contains both a growth and a metabolic component (Hesslein et al. 1993; MacAvoy et al. 2005). For adult animals with relatively high metabolic rates and very little mass gain over time, isotope turnover should be particularly highly correlated with MR. Indeed, Sponheimer et al. (2006) suggest the existence of just such a relationship between alpacas and gerbils. However, our results are in opposition to the findings of Carleton and del Rio (2005), which conclude that metabolism and isotope incorporation into tissues are not directly related in birds.
Unfortunately, almost all of the tissue turnover studies published have only examined avian species. While these data show that within a class of organism there is a high degree of predictability regarding how quickly an organism will resemble the isotope signature of its diet that comes from knowledge of its metabolic rate, it is clear from Figs. 6 and 7 that mammalian MR versus isotope half-life are not well-described by the relationship in birds. Both mice and rats have longer blood tissue turnover rates per gram body mass than birds (Figs. 6, 7). While we realize that the relationship between passerines and nonpasserine birds and metabolic rate are not equivalent (McNabb 1988), when the birds are grouped as a whole, they tend to have faster metabolic rates per gram tissue than mammals, especially in smaller animals (Fig. 5). Because mammals have a slower metabolic rate per gram than birds, and metabolic rate per gram is positively correlated to tissue turnover rate, it is not surprising that mice and rats have a slower turnover rate than similarly sized birds. Although the metabolic rate and blood turnover rate have only been correlated for two mammals in this study, the relationship seen in various avian species suggests that the correlation in mammals would also be logarithmic. However, it is important to stress that additional data points are needed to verify this relationship and construct a model.
It should also be noted that the relationship between isotope turnover and MR found in the study likely only holds for adults (where growth rate is effectively zero) and homeotherms. We postulate that in growing animals or poikilotherms there would be a markedly different relationship and likely a weaker correlation between the two variables than what we show here.
Determining the relationship between metabolic rate and tissue turnover rate could allow researchers to predict the turnover rate of the tissue in question based on knowledge of the metabolic rate of the animal being studied. Currently, tissue turnover rates are determined in laboratory studies by changing the diet of the organism and measuring the stable isotope ratios of the tissues over time as they come into equilibrium with the new diet. The relationship proposed in this study will be valuable for researchers seeking a way to predict time to isotope equilibrium for species for which metabolic rate is known but the isotope turnover is not. If a relationship between metabolic rate and turnover rate can be modeled, the researcher could determine the metabolic rate for the organism and use this measurement to predict the isotope turnover rate.
The main caveat to this work is that metabolic rate can change, and therefore likely cause the tissue turnover rate to be altered. Most published metabolic rates are reported as basal metabolic rates (BMR), which require the measurement of metabolic rate under standard conditions (McNabb 1988). These standard conditions require the animal be (1) resting during its normal time of rest (as in circadian rhythm); (2) in thermoneutrality; (3) postabsorptive; and (4) an adult (McNabb 1988). In contrast, metabolic rates measured in the field may be affected by photoperiod, temperature (ambient and body), animal activity, age and diet.
It has been shown that the metabolic rate that can be measured in the field (FMR) scales differently with body size than BMR (Kojeta 1991). Kojeta (1991) and Ricklefs et al. (1996) showed that a correlation does exist between BMR and FMR with mammals (rodents), but a weak correlation exists with birds, and none with marsupials. The lack of evidence supporting a relationship between FMR and BMR is probably due to the fact that MR is highly variable in the field. BMR, therefore, is important as a standardized tool that justifies comparisons between individuals and species.
The potential difference between FMR and BMR indicates that using BMR to predict the tissue turnover rate could result in inaccurate estimates. However, most current studies determining tissue turnover rate using stable isotope analysis do not conduct their experiments in a field-type setting. Animals are usually housed in a laboratory, with limited living space and food supplied ad libitum, likely leading to decreased activity levels. Laboratory temperatures and photoperiod are generally stably and optimally maintained. Thus, the tissue turnover rates experimentally measured in laboratories are also likely obtained using animals that do not exhibit metabolic rates similar to those observed in the field.
However, recent data from Carleton and del Rio (2005) suggest that a cold-induced change in metabolic rate in an avian species has a negligible effect on carbon and nitrogen incorporation into red blood cells. The metabolic rates measured in this study are not basal MR, but are affected by environmental components that could be expected to affect MR in the field. These results suggest that FMR, although different then BMR, may have only an indirect, and perhaps negligible, effect on the isotopic tissue turnover rate, allowing laboratory studies relating BMR and tissue turnover to apply to studies of animals in the field.
An additional interesting component of this work is that low nitrogen dietary intake may negate the relationship between MR and tissue turnover, as seen in bats (Voigt et al. 2003). Nectar-feeding bats have higher mass-specific metabolic rates than similar sized terrestrial mammals, and yet blood carbon turnover rates are considerably slower than those seen in terrestrial mammals of the same mass, seemingly negating the relationship found earlier in this study. However, the bats were fed sugar-water, with little nitrogen content (Voigt et al. 2003), and nectar-feeding bats in the wild could also be expected to take in low levels of dietary nitrogen. As protein turnover and replacement is a major driver in metabolism, and the bats are deficient in dietary nitrogen and thus would need to recycle tissue components, it seems likely that both tissue nitrogen and carbon are being recycled, resulting in longer tissue carbon half-lives, despite the faster MR. As seen in other species (Hobson and Stirling 1997; Arneson et al. 2006), the carbohydrate in the nectar or sugar-water is likely primarily oxidized to provide energy and expelled as breath CO2 (Sponheimer et al. 2006), rather then being incorporated into tissues. A similar disconnect between MR and tissue isotope turnover rate would also be expected in other mammals that consume low dietary nitrogen, as well as in animals experiencing starvation.
A second caveat to this work is that studies have shown that different tissues turn over at different rates (Arneson and MacAvoy 2005; Evans-Ogden et al. 2004; MacAvoy et al. 2005; Tieszen et al. 1983; Voigt et al. 2003). Therefore, the relationship between whole-body metabolic rate and blood carbon or nitrogen tissue turnover could not be used to predict the turnover rates of other tissues. The relationship will likely need to be modeled for each type of tissue studied before any predictions can be made.
In a 2004 paper, Ogden, Hobson and Lank observed that studies are needed to determine the relationship between metabolic rate and isotope turnover. They point out that variable metabolic rate, even among individuals of the same species, could have a significant impact on isotope turnover rates. The study reported here presents the first examination we are aware of relating tissue isotope turnover to metabolic rate in an effort to determine a predictive relationship between the two. While we understand that directly applying these results to field situations where different types of animals experience variable physiological stresses would not be advisable (Carleton and del Rio 2005), this examination has been a useful step towards a better understanding of the effects of metabolic rate on isotope turnover. Given the strong and predictive relationship between metabolism and isotope turnover in adult homeotherms apparent from this study, we believe that knowledge of the metabolic rates of organisms within an ecosystem will allow researchers to make well-grounded assumptions about the isotope equilibrium status of each system studied.
The authors would like to thank the COSMOS Foundation and the Mellon Fund (American University) for partial funding of this study, and two anonymous reviewers for their constructive comments. The experiments described in this paper comply with the current laws of the United States.