Oecologia

, Volume 150, Issue 2, pp 190–201

Correlation of metabolism with tissue carbon and nitrogen turnover rate in small mammals

  • Stephen E. MacAvoy
  • Lynne S. Arneson
  • Ethan Bassett
Ecophysiology

DOI: 10.1007/s00442-006-0522-0

Cite this article as:
MacAvoy, S.E., Arneson, L.S. & Bassett, E. Oecologia (2006) 150: 190. doi:10.1007/s00442-006-0522-0

Abstract

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.

Keywords

Stable isotope Turnover Metabolism Blood Mammal 

Introduction

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.

Methods

Experimental design and tissue sampling

Adult female Sprague-Dawley rats (R. norvegicus) (n=6) and adult female BALB/c mice (M. musculus) (n=6) were allowed to equilibrate to a control diet for approximately 120 days (2018, Harlan Teklad, Madison, WI, USA). Four of each species began an isotopically distinct experimental diet, while two remained on the control diet. The control diet contains both corn and wheat, and contains 18.9% protein. The experimental diet is 21% protein from casein, 59% carbohydrate from beet sugar and 7% soybean oil, with the remainder of the diet composed of fiber, vitamins and minerals (Arneson and MacAvoy 2005). Carbon and nitrogen isotope values for each diet are given in Table 1. Four blood samples (lateral tail vein bleeding, ~100 μl) were taken from each animal during the equilibration period, and every seven days (R. norvegicus) and every ten days (M. musculus) during the experimental period, which lasted for 80 days. Blood samples were placed in a drying oven at 60 °C for three days and then refluxed for 35 min in dichloromethane for lipid extraction (Knoff et al. 2002). After air drying, ∼1 mg of tissue was measured and placed in tin capsules in preparation for stable isotope analysis. Blood tissue samples were analyzed for δ13C and δ15N using a Europa Hydra 20/20 (University of California, Davis, CA, USA) stable isotope ratio mass spectrometer (IRMS) to obtain δ13C and δ15N. Standards were run in duplicate every twelve measurements (within a run of 100 samples, which included 15 standards, the standard deviation of the standard material was 0.2‰ for nitrogen and 0.1‰ for carbon).
Table 1

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

 

δ13C

±SD

Frac

δ15N

±SD

Frac

Control diet (N=10)

−21.4‰

0.7

 

2.8‰

0.6

 

M. musculus (N=15)

−19.2

0.1

+2.3‰

6.1

0.1

+3.3‰

R. norvegicus (N=6)

−19.0

0.1

+2.4

6.1

0.1

+3.3

Experimental diet (N=8)

−26.8

0.1

 

5.5

0.4

 

M. musculus (N=8)

−25.6

0.1

+1.2

8.5

0.1

+3.0

R. norvegicus (N=12)

−25.2

 0.1

+1.6

8.4

 0.1

+2.9

Stable isotope analysis

When determining isotopic signatures, the heavy to light isotopic ratio of a particular element in the sample is measured relative to that of a standard. The equation for an isotopic signature is as follows:
$$\delta ^{{\text{H}}} {\text{X}} = {\left[ {\frac{{{\left( {{^{{\text{H}}} {\text{X}}} \mathord{\left/ {\vphantom {{^{{\text{H}}} {\text{X}}} {^{{\text{L}}} {\text{X}}}}} \right. \kern-\nulldelimiterspace} {^{{\text{L}}} {\text{X}}}} \right)}_{{{\text{sample}}}} }} {{{\left( {{^{{\text{H}}} {\text{X}}} \mathord{\left/ {\vphantom {{^{{\text{H}}} {\text{X}}} {^{{\text{L}}} {\text{X}}}}} \right. \kern-\nulldelimiterspace} {^{{\text{L}}} {\text{X}}}} \right)}_{{{\text{standard}}}} }} - 1} \right]} \times 1000.$$
(1)
Here X is any element, and H and L are the heavy and light mass numbers, respectively; units are in per mil (‰).

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.

Growth rates

Masses of individual animals were taken prior to each blood collection and metabolic measurement. The growth rate constant, k, for each group was determined using
$$k = {\ln {\left( {{M_{{\text{S}}} } \mathord{\left/ {\vphantom {{M_{{\text{S}}} } {M_{0} }}} \right. \kern-\nulldelimiterspace} {M_{0} }} \right)}} \mathord{\left/ {\vphantom {{\ln {\left( {{M_{{\text{S}}} } \mathord{\left/ {\vphantom {{M_{{\text{S}}} } {M_{0} }}} \right. \kern-\nulldelimiterspace} {M_{0} }} \right)}} t}} \right. \kern-\nulldelimiterspace} t, $$
(2)
where M0 is the initial mass in grams and MS is the mass in grams on day t of the experiment. This was done so that the contribution of growth to tissue turnover could be ascertained (see below, Modeling turnover).

Modeling turnover

The rate of isotope turnover in blood can best be described by the following equation
$$\frac{{{\text{d}}C}}{{{\text{d}}t}} = - {\left( {k + m} \right)} \times {\left( {C - C_{{\text{E}}} } \right)},$$
(3)
which describes the change in isotope value over time. C is the signature at day t, CE is the signature in equilibrium with the new diet, k is the specific growth rate, m is the metabolic tissue replacement rate, and k can be measured directly as a function of time (see Eq. 2 above). Integrating equation 3 results in
$$C - C_{{\text{E}}} = \alpha {\text{e}}^{{ - {\left( {k + m} \right)}t}} , $$
(4)
where α is the difference between the initial and final isotope signatures. Therefore, Eq. 4 becomes
$$C(t) = C_{{\text{E}}} + {\left( {C_{{\text{0}}} - C_{{\text{E}}} } \right)}{\text{e}}^{{ - {\left( {k + m} \right)}t}} ,$$
(5)
where C0 is the initial signature. This is an equation describing isotope change over time as used by Hesslein et al. (1993); m can be calculated by rearranging Eq. 5 so that
$$m = - {\left[ {\frac{{\ln {\left( {\frac{{C - C_{{\text{E}}} }}{{C_{0} - C_{{\text{E}}} }}} \right)}}}{t} + k} \right]}.$$
(6)
The relative importance of k to m changes during different life stages. For young, fast-growing organisms, the m component is negligible relative to k (Frazer et al. 1997; Fry and Arnold 1982; Herzka and Holt 2000). The opposite is true for very slow-growing or adult organisms. In these cases it has been shown that metabolic tissue replacement is an important driver of isotopic change. MacAvoy et al. (2005) found that, in adult mice, the rate of change is almost entirely (>90%) due to m, as little or no growth occurs. It is also clear that m (metabolic tissue replacement rate) is apparently higher in animals with high metabolic rates (measured as oxygen consumption or carbon dioxide consumption per gram). Using the growth and metabolic tissue replacement constants, the half-lives of various elements within a tissue can be calculated, yielding a measure of how quickly an organism resembles the isotopic signature of its diet.

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.

Half-life

In this study, half-life refers to the time it takes for half of the existing tissue to resemble the isotopic signature of the new diet. Half-life is calculated by the following equation:
$$t_{{1/2}} = \frac{{\ln 2}}{{m + k}}.$$
(7)

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).

Results

Growth rates

The animals used in this study were mature at the beginning of the experiment. Both the rats and the mice were a minimum of eight months old on day zero of the experiment, and were sexually mature. The mass of each animal was determined every seven (rats) or ten (mice) days to determine rate of growth (Fig. 1). Although some change in mass was seen in both the control and experimental groups of rats and mice, it was minimal. R. norvegicus in the experimental group (n=4) had an average growth rate of 8.62×10−5 g/day (±3.13×10−4), while the control group (n=2) grew at an average rate of 4.50×10−4 g/day. M. musculus on the experimental diet (n=4) had an average growth rate of −3.95×10−4 g/day (±6.89×10−4), while the control group grew at an average rate of 3.64×10−4 g/day. Any change in mass was most likely due to change in fat deposition from excess caloric uptake. This is suggested by the relatively sharp decrease in mass after 15–24 h of fasting prior to metabolic testing (Fig. 1).
Fig. 1

Mass of individual Rattus norvegicus and Mus musculus versus time

Turnover rates

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.

Blood samples were collected every ten (mice, Fig. 2) or seven (rats, Fig. 3) days and carbon and nitrogen stable isotope ratios were analyzed. Blood carbon and nitrogen turned over at similar rates within the same species. The half-life of tissue carbon and nitrogen was 17.3 and 15.4 days, respectively, for mice (Table 2). Blood tissue carbon and nitrogen turned over more slowly in rats, with a half-life of 24.8 and 27.7 days, respectively (Table 2). Diet–tissue discrimination values at the end of the experiment were 1.2 and 1.6‰ for carbon and 3.0 and 2.9‰ for nitrogen in mice and rats, respectively (Table 1). For both mice and rats, equilibrium of blood tissue with the experimental diet occurred by day 65. Values at the end of the experiment were used as the equilibrium values in Eq. 5 to determine turnover constants. The data do not allow the extent to which the animals were actually at equilibrium to be determined, and we cannot rule out the existence of a very long turnover pool (see Ayliffe et al. 2004). However, the values at the end of the experiment are likely to be close (within ±0.1‰) to the true equilibrium values, and calculated turnover rates are relatively insensitive to error in this parameter.
Fig. 2a–b

a Carbon and b nitrogen isotope change in Mus musculus blood over time. Controls are squares and experimentals are circles. Bars represent ±1 standard deviation

Fig. 3a–b

a Carbon and b nitrogen isotope change in Rattus norvegicus blood over time. Controls are squares and experimentals are circles. Bars represent ±1 standard deviation

Table 2

Average half lives (t1/2) and metabolic constants (m) for each isotope in each species

 

 

m

t1/2 (days)

M. musculus

13C

0.04

17.3

R. norvegicus

0.028

24.8

M. musculus

15N

0.0449

15.4

R. norvegicus

0.025

27.7

Table 3

Mass, metabolic rate (MR in ml O2/min), and isotope (13C and 15N) turnover rates of various tissues in four mammals

Species

Size (g)

MR (ml O2/min)

Tissue

13C

t1/2 (days)

15N

t1/2 (days)

(1) Turnover reference

(2) MR and mass reference

Mammalian: Rodents 10g

 Gerbil

 Meriones unguiculates

64.8

1.61

Liver

Fat

Muscle

Brain

Hair

6.4

15.8

27.7

27.7

46.2

ND

(1) Tieszen et al. (1983)

(2) Lovegrove (2003), White and Seymour (2003)

 Mouse

Mus musculus

18.55

18.55

0.84

0.84

Blood

Liver

Muscle

16.9

ND

16.5

19.3

7.3

24.8

(1) Macavoy et al. (2005)

(2) Lovegrove (2003), Heusner (1991)

 Mouse

Mus musculus

27.7

3.16

Blood

18.6

19.6

(1) This Study

(2) Lovegrove (2003); Heusner (1991)

Mammalian: Rodents 102g

   Rat

Rattus norvegicus

288

5.90

Blood

24.7

21.3

(1) This Study

(2) Hart (1971)

Mammalian: 105g

 Horse

Equus caballus

Equus asinus

488,636

409,778

1125.22

487.45

Hair

136.0

ND

(1) Ayliffe et al. (2004)

(2) Bromham et al. (1996), Heusner (1991)

Table 4

Mass, metabolic rate (in mL O2/min), and isotope (13C and 15N) turnover data for seven species of birds

Species

Size (g)

MR (ml O2/min)

Tissue

13C

t1/2 (days)

15N

t1/2 (days)

(1) Turnover reference

(2) MR and mass reference

Avian: Passerine

 Canvasback

Aythya valisineria

1,250

27.50

Blood (clam/corn Diet)

Blood (tuber diet)

Blood (clam diet)

Average

26.2

19.8

16.0

20.7

26.5

25.4

17.6

23.2

(1) Haramis et al. (2001)

(2) Woodin and Stephenson (1998)

 Garden warbler

 Sylvia borin

 

24.8

1.23

Blood (meal worm diet)

Blood (blk elderberry diet)

Average

5.0

5.8

5.4

11.2

5.0

8.1

(1) Hobson and Bairlein (2003)

(2) Mckechnie and Wolf (2004)

 Yellow-rumped warbler

Dendrorica coronta

 

 

11.5

0.56

Blood (49% insect diet)

Blood (73% insect diet)

Blood (97% insect diet)

Average

3.9

5.0

6.1

5.0

7.5

 

 

 

(1) Pearson et al. (2003)

(2) Mckechnie and Wolf (2004)

 

 

American crow

Corvus brachyrhynchos

384.8

9.70

Plasma

Blood cells

2.9

30.1

 

(1) Hobson and Clark (1993)

(2) Mckechnie and Wolf (2004)

Avian: Nonpasserine

 Japanese quail

Coturni japonica

 

 

115

2.89

Blood

Liver

Muscle

Bone

11.2

2.5

12.4

173.3

 

(1) Hobson and Clark (1992)

(2) Roberts and Baudinette (1986)

 

 

 Great skua

Catharacta skua

970

 

14.03

 

Blood

 

15.1

 

12.0

 

(1) Bearhop et al. (2002)

(2) Mckechnie and Wolf (2004)

 Dunlin

Calidris alpina pacifica

44

 

1.57

 

Blood

 

11.2

 

10.0

 

(1) Evans-Ogden et al. (2004)

(2) Lindstrom (1997)

Metabolic rates

Metabolic rate averages were determined using data from the experimental period, including day zero. Average whole-body metabolic rate for mice was 3.2 ml O2/min compared to 8.9 ml O2/min for rats. However, as expected, metabolic rate per gram for mice was considerably higher than that for rats (6.84 ml O2/h/g for mice compared to 1.84 ml O2/h/g for rats) (Fig. 4).
Fig. 4a–b

a Log metabolic rate (O2/h) versus log mass (g) and b metabolic rate per gram (O2/h/g) versus log mass (g) for Rattus norvegicus and Mus musculus

Discussion

This study examined the blood turnover rate in mice and rats using stable carbon and nitrogen isotopes. We have found that blood carbon and nitrogen turnover was roughly equivalent in mice and in rats, and that mouse blood turned over slightly faster than rat blood. We have also determined the resting metabolic rate (MR) of the animals used in this study. The measured MR and the relationship between mass and metabolic rate are consistent with what other researchers have observed (Fig. 5). Our findings are also in accordance with the long-known relationship between size and metabolism (Kleiber 1932, 1947). The mice exhibited a faster MR per gram tissue but slower whole-body MR relative to rats (Fig. 4), and our data indicate that faster isotope turnover is associated with higher MR/g (Fig. 7).
Fig. 5

Metabolic rates (ml O2/min)/g for mammals (filled diamonds) and all birds (open squares) in Tables 3 and 4 were plotted versus average mass of the animal. Trend lines are shown for both categories of animals. For mammals, y = −0.0265ln(x) + 0.0462, r2=0.9246, whereas for all birds, y = −0.0313ln(x) + 0.0529, r2=0.8854

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.

In this study we could not determine the type of correlation between tissue turnover and metabolism because two species were utilized and two data points cannot yield a predictive equation. Only blood was sampled in this study, so we could not compare our isotopic turnover values with those reported for other mammals (horse, Ayliffe et al. 2004; gerbil, Tieszen et al. 1983), as these studies did not measure blood isotope turnover. Therefore, we examined the literature for other studies determining whole blood turnover rate in species for which the metabolic rate has been determined (Table 4). These data, exclusively from studies of birds, describe a logarithmic relationship between isotope turnover and metabolic rate (Fig. 6) or metabolic rate per gram (Fig. 7) when plotted. The various studies pooled to examine this relationship examined birds ranging in mass from 11.5 to 1,250 g and metabolic rates ranging from 0.56 to 27.5 O2/min (warbler and canvasback, respectively) (McKechnie and Wolf 2004). Yet even with the order of magnitude differences in these variables, the correlation of metabolic rate per unit mass with 13C and 15N turnover is very high (0.87 and 0.9, respectively, Fig. 7).
Fig. 6a–b

Carbon (a) and nitrogen (b) stable isotope half-lives in blood for the birds given in Table 3 are plotted versus metabolic rate (closed diamonds). The trend lines for these relationships are shown, and the equations are ay = 3.802ln(x) + 6.978, r2=0.90 and by = 3.2919ln(x) + 8.2191, r2=0.72. The metabolic rates for mouse and rat are overlaid for blood stable carbon (a) and nitrogen (b) isotope turnover. Note that values for gerbil and horse are not included, as blood isotopic turnovers were not determined for these species

Fig. 7a–b

Carbon (a) and nitrogen (b) stable isotope half-lives in blood for the birds given in Table 3 are plotted versus metabolic rate per gram (closed diamonds). The trend lines for these relationships are shown, and the equations are ay = −7.5508ln(x) − 16.536, r2=0.87 and by = −3.2376ln(x) − 1.6114, r2=0.90. The metabolic rates for mouse and rat are overlaid for blood stable carbon (a) and nitrogen (b) isotope turnover. Note that values for gerbil and horse are not included, as blood isotopic turnover was not determined for these species

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.

Acknowledgments

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.

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Stephen E. MacAvoy
    • 1
  • Lynne S. Arneson
    • 1
  • Ethan Bassett
    • 1
  1. 1.Department of BiologyAmerican UniversityWashingtonUSA

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