Acta Zoologica (Stockh.), Vol. 68, No. 1, pp. 57-64, 1987 0001-7272/87$3.00+ .OO Printed in Great Britain Pergamon Journals Ltd.

0 1987 The Royal Swedish Academy of Sciences

Allocation of Matter in the Body of Growing Toads, Bufo bufo: Components of ‘Condition’ C . Barker J0rgensen and H . Wind-Larsen

Zoophysiological Laboratory A, August Krogh Institute, Copenhagen, Denmark (Received 14 July 1986)

Abstract Jbrgensen, C. B., Wind-Larsen, H . 1987. Allocation of matter in the body of growing toads, Bufo bufo: components of ‘condition’. (Zoophysiological Laboratory A, August Krogh Institute, Copenhagen, Denmark.)-Acta 2001. (Stockh.) 68, 57-64.

The condition, CI = [body mass (g)/length (~m)~]11000, tends to increase in toads growing in the laboratory on a diet of meal-worms. The allocation was therefore studied of ingested food to growth corresponding to maintenance of standard condition (AW,) and to increases in condition, expressing bulky growth. A condition index of 100, typical of toad populations, is chosen to characterize standard growth, higher values expressing bulky growth. The relationship between mass specific standard growth (AWJW,,) and actual growth in mass (AW/W,) was the same in controls and growth hormone-treated toads and was independent of sex and season. It could be described by the allometric equation AWJW,, = 0.28 (AW/W,)’ 36, r = 0.93. The composition of mass representing increases in bulk in excess of a CI = 100 was assessed from allometric relationships established between the indices of the chemical constituents and the overall condition index. The increases in condition were partitioned among the organs and parts of the body from allometric relationships established between indices of organs and body parts and the overall condition index. Treatment with growth hormone enhanced growth in mass and significantly increased the relative proportion of mass allocated to standard growth. However, the effect was not specific to growth hormone, but was an indirect effect of the increased growth rate. Water was the predominant constituent of the growth in bulk, amounting to about 70% of increases in CI. Fat and protein contributed about equally initially, 5-10%, with fat strongly increasing and protein slightly decreasing with increasing condition, fat to approach 2&30% when CI approached 200. Increases in water content largely reflected increases in volume of blood and lymph.

C. Barker Jmrgensen, Zoophysiological Laboratory A , August Krogh Institute, Univ- ersitetsparken 13, DK-2100 Copenhagen 0, Denmark.

Introduction

Toads growing in the laboratory tend to increase relatively more in bulk than in length and they develop larger fat bodies than are usually observed in toads in nature (Jorgensen 1983, 1986). The increase in bulkiness is thus cor- related with fattening, but it remained to be assessed to what extent fat represented the excess increase in body mass. In a previous paper (Wind-Larsen and Jorgensen 1986) growth in the toad Bufo bufo was analysed in terms of body constituents, including protein, fat and glycogen. It was thus possible to analyse the composition of the body as correlated with growth in bulk and length. In the present paper these data have been further used to assess the components of the excess bulk, as expressed by the body pro-

portions of the growing toads, and to analyse some of the factors that determine the pattern of allocation of matter in the body of growing toads.

Condition and its Components

The condition factor. The analysis is based on the concepts of allometric growth and condition. Growth may be described by the allometric equ- ation body mass = a (body length)b. In popu- lation studies the factor u has been referred to as the condition factor, because it may (as the name implies) reflect the nutritional condition of the individuals constituting the population. However, the value of a depends upon several other factors as well, including bodily stature,

57

58 C. B . Jmrgensen and H. Wind-Larsen

degree of filling of the digestive tract, state in reproductive cycle and degree of hydration, and it may be difficult to distinguish between the several factors that determine the value of a (Hile 1936, Kesteven 1947, le Cren 1951, Wea- therley 1972, Donaldson et al. 1979). The safest use of a as an indicator of nutritional condition of an animal is in the laboratory, where changes in other factors than the nutritional state can be eliminated or controlled.

Standard growth. The chemical constituents of the body presumably exhibit a similar allometric relation to body length as that applying to total body mass. It is therefore possible to derive allo- metric relationships for these constituents expressed by the general equation a, = con- stituent mass lLb. We may thus dissolve a = Wl Lb into its constituent factors, such as awater, aorganic matter, aprotein and afat. Theoretically, the sum of these factors equals a(= W/Lb).

Components of condition. When the condition in the growing toads deviates from an allometrically determined standard value these constituent fac- tors, or indices, presumably also deviate from their allometrically determined standard values. The nature of the relations between deviations of the constituent factors and the overall condition factor is, however, not self-evident. As shown below, these relationships could also be described by the allometric equation acOnstituent - asp, where f3 is an exponent that characterizes the relationship between the overall condition factor and the constituent factor.

The deposition of constituents that cause their deviations is not uniform among the parts and organs of the toad body. Fat is thus known to be deposited primarily in the fat bodies. How deviations of the parts from their allometrically determined relative sizes were related to the overall condition was therefore also studied. Again the relationships could be satisfactorily described by allometric equations of the general type, apart = a@.

In the equations that describe the relations observed between the constituent or component indices and the condition indices of the body, values of the exponent >1 indicate that the rela- tive share of the constituent or component increases with increasing condition. At a value of 1 the share remains constant and at values <1 the relative share decreases with increasing condition.

Toads grow isometrically (J@rgensen 1983), and the factor a is W(g)lL(~m)~. In Danish popu- lations of Bufo bufo a is typically 0.1. This value has therefore been used to express standard growth, AWs. The values of the ratios W/L3

-

obtained in the experiments were multiplied by lo3 in order to avoid decimals. The values are termed condition indices (CI).

Material and Methods

Two experiments were made on juvenile toads, one performed in the autumn, when toads discontinue growth before hibernating, and one in the spring, after hibernation when growth is resumed. The toads were kept at 21°C on a 12L112D regimen of illumination. They were fed meal-worms ad libitum for 24 h periods one, two or three times a week. In half the toads growth was stimulated by injection five days a week of 20 pg bovine growth hormone (USDA-bGH-B-I) dissolved in 0.1 ml frog Ringer. (Some toads were treated with thyroxin, alone or combined with growth hormone. Thyroxin had no obvious effects on growth or condition and the thyroxin-treated toads were there- fore pooled with the controls or growth hormone- treated.)

Each experiment lasted 10-11 weeks. At autopsy the fat bodies, liver, gonads and digestive tract were excised and these organs and the rest of the body, the carcass, were analysed separately. The difference between body mass at autopsy and the sum of the masses of the body parts represents fluid drained from the body during autopsy and is termed ‘blood’.

In the spring experiment at autopsy most of the females contained vitellogenic ovaries which con- tributed significantly to the condition index. Non- vitellogenic indices were obtained by correcting body mass for vitellogenic mass, assuming non-vitellogenic ovarian mass to correspond to 2% of the body mass (J0:gensen et a / . 1979). In the males gonadal mass is insignificant.

Further details about the experiments may be found in a previous paper (Wind-Larsen and J~rgensen 1987).

Results

Organic Matter: Constituents and Allocation

Organic matter amounted to about 20% of the body mass, with no significant seasonal dif- ference or difference between control and growth hormone-treated toads. The composition of the organic matter was about the same in controls and growth hormone-treated toads in the spring experiment, whereas the protein and glycogen percentages were significantly higher and fat lower in the controls than in the growth hor- mone-treated toads in the autumn experiment (Table 1). This higher percentage of protein in the controls can be explained by the relatively small increase in body mass in the controls com- pared with the growth hormone-stimulated

Allocation of Matter 59

ii I . N 09-\4 09 000 3 z $ 1 :: +I t l tl

Q1 103 3 .z tl 7 ti +I '" tl

-zI. G . " Z w vl

C 3 c M

a v)

I .- B

vl 09 0

+I 0 3

00

z 0

toads. In the controls the composition of the organic matter at autopsy was thus largely deter- mined by the composition prior to the exper- iment, whereas the composition of the growth hormone-treated toads largely reflected the allo- cation of ingested organic matter during the feed- ing period. The composition thus indicates that treatment with growth hormone resulted in a relative increase in deposition of fat. This is sup- ported by the significantly larger fat bodies in the growth hormone-treated toads. It is note- worthy that there was no fat depositing effect of exogenous growth hormone outside the fat bod- ies (Table 1).

Mass of Parts of the Body

The relative masses of the fat bodies and liver, as representatives of storage organs, and of the digestive tract and 'blood' are shown in Table 2. It may be seen that the mass of the fat bodies tended to be larger in the growth hormone- treated toads than in the controls, in accordance with the data on fat content (Table 1). However, the differences were not statistically significant. The relative masses of liver, digestive tract and 'blood' were about the same in all groups.

Standard Growth and Condition

Treatment with growth hormone significantly enhanced growth in mass, AW. The effect was particularly drastic in the autumn experiment, and it increased with feeding level (Table 3). Growth hormone treatment also significantly increased the relative proportion of mass allo- cated to standard growth.

In order to see whether this reallocating effect of treatment with growth hormone was specific to the exogenous hormone or rather was an effect of the enhanced growth, standard growth rates, AW,/Wo, were plotted against total growth rates, AWIWO. Figure 1 shows that standard growth rate was closely correlated with overall growth rate, with no obvious specific effect of the hor- mone treatment or of sex and season. The eq- uation AWJW, = 0.28 (AW/Wo)1.36 closely fitted the plot, the correlation coefficient being r = 0.93 (n = 47). The proportion of increase in mass apportioned to standard growth thus increased with increasing growth rate.

Relations Between Condition and Constituents

As mentioned, the condition index tends to increase in toads growing in the laboratory on a

60 C. B. Jorgensen and H . Wind-Larsen

Table 2. Masses of parts of the body per 100 g body mass

n Fat bodies Liver Digestive tract 'Blood' Season Treatment (Yo mass) (% mass) (Yo mass) (Yo mass)

15 5.1 f 1 . 1 5.1 f 0.6 4.7 f 0.9 16.1 f 4.0 Autumn Controls GH 15 1.7 2 2.8 4.2 f 0.6 4.6 f 1.0 19.9 f 4.0

Spring Controls 8 5.3 f 1.7 5.3 f 1.5 5.2 f 1.6 11.5 f 5.9 GH 8 6.1 f 1.5 5 .6 f 0.9 6.6 f 1.3 12.8 * 4.3

Table 3. Increase in body mass, AW, and partitioning between standard growth and increase in bulk AW,,

Meals Per

Season week Treatment Sex n AW AW, (YoAw)

Autumn 1 Controls GH

2 Controls GH

3 Controls GH

Spring Controls + T, GH+GT,

juv. 5 9 .0 f 1.9 9 9 4 20.4 f 1.2 juv. 5 1.8 f 1.4

juv. 5 12.5 f 3.4 9 9 4 51.0 f 3.4

I 43.9 f 4.3 9 76.4 f 3.1

9 9 4 43.4 t 4.9

9 9 d d

139( 11 1-191) 78 f 4.6

59 f 2.5

94(0-166) 62 t 2.3

161( 117-305)

70 f 4.1 59 f 1.8

2 Controls+T, $? $? 6 13.2 f 0.9 71 f 2.1 GH+GT, d d 4 33.0 f 3.9 50 2 1.3

~

Statistical significances. Autumn-GH-treated, 1 meal vs. 2 meals; AW, P<O.Ol; AW,,, P<O.Wl-GH-treated, 1 meal vs. 3 meals; AW, P<O.OOl; AW,,, K 0 . 0 2 . Spring- controls vs. GH-treated; $? $? AW, P<O.OOl; AW,,, K 0 . 0 5 ; 0" d A W , P = 0.001, AWb, P<O.OOl.

diet of meal-worms. The effects of the deviations from standard growth on the constituents of the body are shown in Table 4 for water, organic matter, fat and protein, as reflected in the allo- metric relations between condition indices and constituent indices. In most groups condition index was highly correlated with constituent index, especially in the case of the most precisely determined constituents, water and organic matter, with no obvious difference in the relationships between controls and growth hor- mone-treated toads. These were therefore pooled. Water was the predominant constituent of the growth in bulk, amounting to about 70% of increases in condition. Organic matter increased relative to water with increasing con- dition. This increase was entirely due to the steep increase in the fat index. The relative increase in fat with condition was faster in the autumn

experiment than in the spring experiment, the value of 3.20 for the exponent p in the autumn experiment being significantly higher than the value of 2.16 in the spring experiment, controls and growth hormone-treated toads being pooled (P < 0.05).

It can be calculated from the allometric relations listed in Table 4 that at an early phase in the development of bulkiness fat and protein contributed about equaUy to the increases in con- dition index, about 10%. At an advanced stage of bulkiness, corresponding to a condition index of about 200, protein had declined in importance, whereas the contribution of fat to further increase in condition approached 20-30%.

Presumably, fat is deposited with very little water, in contrast to protein. The ratios protein/ water obtaining in the condition increases are low, ranging from 0.12 to 0.10 with increasing

Allocation of Matter 61

A W W 0

0 2.0

f - 0 z

r0 cn

C 0 - cn V

e,

.-

a - 3

0

- 0 0 0

0 0 0 e e g

a *o "0% A - A A O e

a eo 0

, A ? I I 1 I

Fig. 1. Relationship between massspecificstandardgrowthAWJW,andactual increase in mass AW/W,, in young growing toads in the spring experiment. 0 Femalecontrols,OfemalesGH-treated, A malecontrols, A malesGH-treated.

Table 4. Allometric relations between condition indices and constituent indices*

Constituent Season Treatment n a P r

Water Autumn Controls 15 0.99 0.95 0.90 G H 15 1.65 0.85 0.91 Controls + GH 30 1.47 0.87 0.91

Spring Controls 16 1.14 0.92 0.93 G H 16 1.15 0.92 0.97 Controls + G H 32 1 .oo 0.95 0.95

Organic matter Autumn Controls 15 0.048 1.27 0.86 G H 15 0.008 1.61 0.88 Controls + G H 30 0.032 1.36 0.86

Spring Controls 16 0.069 1.22 0.80 G H 16 0.036 1.34 0.93 Controls + G H 32 0.068 1.22 0.86

Fat Autumn Controls 15 6.9 x 2.79 0.75 G H 15 0.8 x lo-" 3.22 0.94 Controls + GH 30 0.9 X 3.20 0.87

Spring Controls 8 3.6 x 2.52 0.82 GH 8 4.2 x 2.02 0.88 Controls + G H 16 2.1 x 2.16 0.85

Protein Autumn Controls 15 0.71 0.65 0.80 G H 15 0.25 0.83 0.83 Controls + G H 30 1.22 0.53 0.63

Spring Controls 8 2.39 0.41 0.73 G H 8 2.89 0.37 0.35 Controls + G H 16 3.47 0.33 0.41

*General equation: constituent index a, = a(condition index a)@. In the spring experiment the condition indices of sexually mature female toads were corrected for vitellogenic ovarian mass.

62 C. B. J0rgensen and H . Wind-Larsen

Table 5. Allometric relations between condition indices and indices of body parts, component indices *

Component Season Treatment n (Y P r

Fat bodies Autumn Controls GH Controls + GH

Spring Controls GH Controls + GH

Liver Autumn Controls GH Controls + GH

Spring Controls GH Controls + GH

Digestive tract Autumn Controls GH Controls + GH

GH Controls + GH

Spring Controls

'Blood'

Carcass

Autumn Controls GH Controls + GH

GH Controls + GH

Spring Controls

Autumn Controls GH Controls + GH

GH Controls + GH

Spring Controls

15 15 30

16 16 32

15 15 30

16 16 32

15 15 30

16 16 32

15 15 30

16 16 32

15 15 30

16 16 32

1.1 x 10-8 1.2 x 10-12 1.1 x lo-"'

1.9 x 10-5 5.1 x 10-5 2.8 x 10-5

9.0 x 10-4

6.2 x 10-4

1.5 x 10-3

1.2 x 10-2

3.3 x 10-2

1.6 x

3.6 x 0.4 x 1.8 x lo-'

1.4 2.4 0.2

7.9 x 10-4 2.5 x 10-3 5.5 x 10-4

9.8 x 10-l" 2.2 x 10-2 3.3 x 10-6

7.1 9.3

11.5

31.2 7.5

14.8

3.99 5.77 4.90

2.58 2.39 2.50

1.29 1.74 1.06

1.90 1.70 1.72

1.05 1.47 1.18

0.34 0.29 0.72

2.04 1.83 2.12

4.66 1.33 3.05

0.54 0.48 0.44

0.23 0.51 0.38

0.72 0.78 0.78

0.73 0.86 0.81

0.73 0.85 0.59

0.84 0.84 0.85

0.48 0.62 0.58

0.15 0.24 0.33

0.69 0.70 0.75

0.54 0.45 0.50

0.76 0.63 0.66

0.25 0.66 0.47

*General equation: component index a, = &(condition index a)@.

condition index. It is thus indicated that during the developing bulkiness water was retained in excess of that normally associated with protein synthesis and cell growth. This indication was supported by the relations observed between increases in condition and 'blood' (Table 5).

Relations between Condition and Body Parts

The relations between condition index and indi- ces of organs and parts of the body, including component indices of fat bodies, liver, digestive tract, 'blood' and carcass are shown in Table 5.

There were no obvious specific effects of treat- ment with growth hormone, so controls and growth hormone-treated toads were pooled. There was no significant effect of season, except for the fat bodies. As might be expected from the relationship between condition and total fat indices, the relative increase in fat body mass with condition was larger in the autumn exper- iment than in the spring experiment (P < 0.01).

Besides the fat body mass, the liver also increased relatively faster in mass than did the overall increase in bulk, as expressed in the condition index. The digestive tract increased in mass in the same proportions as the overall

Allocation of Matter 63

Table 6. Percentage contributions of organs and structures to increases in bulk

Increase Season Percentage contribution in CI

Fat bodies Liver Digestive tract ‘Blood’ Carcass

100-110 Autumn 4.1 4.6 4.9 21.4 37.3 Spring 7.5 7.4 3.9 14.1 31.4

190-200 Autumn 46.0 4.8 5.5 42.8 26.5 Spring 19.1 11.5 3.3 50.1 21.4

increase in condition, whereas carcass accounted for decreasing fractions with increasing con- dition.

Interestingly, the amounts of fluid lost from the body during autopsy also increased relative to increases in condition, the exponents p in the allometric relations between condition indices and fluid indices amounting to about 2 in the autumn experiment and about 3 in the spring experiment (Table 5). Presumably, the fluid con- stituting blood drained from the cut vessels and lymph from the lymph sacs and body cavity.

From the allometric relations between con- dition and component indices the apportioning of condition indices on carcass and organs were calculated. Table 6 shows that the contributions of the fat bodies increased from less than 10% early in the phase of increasing condition index to about 20% or more late in the phase. Fluid that drained from the body during autopsy con- tributed 10-20% early in the phase, increasing to 40-50%.

Discussion

J@rgensen (1983) assumed that increases in con- dition index, or bulkiness, in growing toads, Bufo viridis, reflected deposited fat. The present study showed that this assumption was wrong and that fat presumably constituted only about 1&20% of the increase in bulk. The caloric value of this increase derived from fat deposition thus amounted to about 1-2 kcal g-l, instead of 9 kcal g-’ as used in the previous calculations on partitioning of energy equivalents between lean growth and fattening.

The increase in bulkiness observed in toads growing in the laboratory on a diet of meal- worms thus covers other growth phenomena than deposition of fat. These include synthesis of pro- tein, usually associated with lean growth, and expansion of extracellular fluid compartments. It

is noteworthy that protein synthesis and expan- sion of fluid compartments contributed equally to bulkiness in the non-growing and growing toads of the autumn experiment. The non-grow- ing control toads even tended to reallocate stan- dard body mass to an increase in bulkiness.

Treatment with growth hormone has been observed to stimulate growth in length at the expense of deposition in amphibians and teleosts fed constant rations. In the present study the effects of growth hormone treatment were pre- sumably largely indirect, the allocation of matter to growth in length and to increases in bulk being determined by rates of growth, whether ‘spontaneous’ or stimulated by treatment with growth hormone. In other studies suggesting a specific effect of growth hormone in stimulating growth in length the effect may also at least partly be indirect, correlated with the growth rates, for instance in studies on the effects of growth hormone, thyroxin and androgen on growth in young coho salmon (Higgs et al. 1975, 1976, 1977, Markert et al. 1977). These authors observed that androgen stimulated growth according to isometric proportions, whereas growth hormone-treated fish increased in slen- derness, with no difference from the allometric proportions of the controls in the thyroxin- treated fish. However, the values of the exponents b in the allometric growth equations could be shown to be negatively correlated with growth rate, with a correlation coefficient r = 0.66. It therefore remains to be assessed to what extent the effects observed are specific to the hormones and to what extent they are indirect effects correlated with growth rate.

Relations between condition index and constituent and component indices. The sum of the con- tributions of the constituents water, fat and pro- tein to increases in condition amounted to 98% at the early phase of increase above the standard condition and to 96% at the late phase. The few per cent not accounted for include glycogen and

64 C. B. Jergensen and H. Wind-Larsen

inorganic constituents. The allometric equations thus served as satisfactory models of the relations between condition index and indices of con- stituents. The allometric equations described the relations between condition and organ and tissue indices less satisfactorily. They tended to under- estimate contributions early in the phase of growth in bulk and to overestimate contributions late in the phase. The mean of the sum of the contributions was 99% in the autumn experi- ment, i.e. the theoretical prediction. In the spring experiment the mean value was 85%. The remaining 15% include the ovaries. The mean gonadal index for the group amounted to 9%, leaving only 6% unaccounted for.

Acknowledgements

The gift of bovine growth hormone from the National Hormone and Pituitary Program, Bal- timore, MD, U.S.A., is gratefully acknowl- edged. Drs Lis Olesen Larsen and Per Rosen- kilde rendered valuable comments and suggestions for improvement of the manuscript.

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