UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on animal growth Bailey, Charles Basil Mansfield 1956

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STUDIES. ON ANIMAL GROWTH by Charles B a s i l M. Bailey A Thesis Submitted i n P a r t i a l F u l f i l l m e h t of The Requirementa For The Degree of MASTER OF SCIMGE IN AGRICULTURE i n The Division of Animal Science We accept this-thesis.as conforming t o the standard.required from candidates f o r the degree of Master of Science i n Agriculture THE UNIVERSITY OF BRITISH COLUMBIA October, 1956. ABSTRACT In order to c l a r i f y some of the problems connected with a study of the growth of meat-producing animals an invest* igation of certain aspects of growth has been undertaken. Because of the d i f f i c u l t y of conducting large scale experiments with the common meat producing animals, the mouse was chosen for the present study. As a pi l o t to future work with larger animals, average age changes i n body composition and metabolic rate of mice from birth to maturity have been determined* In addition, the effect of plane of nutrition on these two variables and on growth rate and feed efficiency have been investigated. The composition of the body varies i n a regular manner with increasing body weight especially on a fat-free basis. Changes i n the major constituents apparently exhibit d i f f e r e n t i a l growth. The protein to water ratio i s suggested as a v a l i d index of physiological age i n a l l mammals. Metabolic rate does not bear a constant relationship to body weight, age, surface area, fat-free mass or protein mass i n growing animals* i It i s , however, directly proportional to the fat-free mass divided by the protein to water ratio and to total body-water. The former expression is considered a valid index of physiologically active mass because i t is a function of that fraction of the body which is metabolizing and of changes in i t s composition. It is the amount of metabolizing tissue corrected to a common physiological age. ELane of nutrition has an effect on growth rate, feed efficiency, body composition and metabolic rate. Restricted nutrition slows up the growth process such that the rate of physio-logical aging decreases. Thus, most of the changes in the growth complex resulting from restricted nutrition may be due to changes in the relative growth rates of the various body tissues. i i TABLE OF CONTENTS Page I Intoduction 1 II Experimental Methods • • • • • . * 5 A* Body Composition • 5 B. Metabolic rate . . . o . <>•..•.•<> 6 C. The feeding experiment* . . 7 III Results and Discussion • • • • • • • • • • • • 19 A* Body composition • • • • • • • • • • • • 19 Weight changes: i n protein, water, fat and ash 20 (2) The composition of the fat-free body 21 (3) The formulation of an index of physiological age • . . • • 23 (4) The calculation of body composition i n vivo 26 B<> Metabolic rate 31 C. The feeding experiment . . . « * . . . * 47 (1) Growth rate 47 (2) Body composition 51 (3) Efficiency of gain 54 (4) Metabolic rate » • . . . . 59 IV Summary and Conclusions 60 V Appendix • • • • • • 65 VI Bibliography i i i LIST OF TABLES TABLE PAGE 1 . Body composition and metabolic rate for 1 1 6 white mice . . . . . . . 9 2 . Composition of diet . . . . . 1 7 3 . High and low plane feeding standard . 18 4 . Composition data for the rat from Mitchell and Carmen . . . . . . 2 7 5 . Composition data for the cow from Murray 28 6 . Slopes of the log protein-log water relation-ship for the mouse, rat and cow . . 4 6 7 . Analytical data for the high plane mice 4 8 8 . Analytical data for the low plane mice 4 9 9 . Analytical data for the 1 5 gram control mice 5 0 1 0 . Average composition of gain and efficiency of gain for the high and low plane mice 5 7 1 1 . The relationship between environmental temperature and heat production for Swiss Albino mice . . . . . .. 7 2 1 2 . Oxygen consumption and basal metabolism of four representative male mice . . 7 3 iv LIST OF FIGURES Page 1. Age equivalence of the mouse, cow, pig and sheep 3a 2. Average growth rate of male mice 7a 3 . Feed consumption of male mice 7b 4. Changes in protein weight with changes in body weight 20a 5 . Changes in water weight with changes in body weight 20b 6. Changes in ash weight with changes in body weight 20c 7. Changes in fat weight with changes in body weight 20d 8. Changes in water weight with changes in fat-free body weight 22a 9 . Changes in protein weight with changes in fat-free body weight 22b 10. Changes in ash weight with changes in fat-free body weight 22 c 11. Changes in protein weight with changes in water weight 23 a 12. Changes in ash weight with changes in protein weight 23b 13. Changes of the protein to water ratio with weight 24a 14. Weight changes in daily basal metabolic rate 34a 15. Weight changes in basal metabolism per unit of surface area . . . . . . . . 34b 16. The relationship between daily metabolic rate and physiologically active mass 38a 17. The relationship between daily metabolic rate and body water 38b 18. Diagramme of the respirometer apparatus . . . . 65a 19. Changes of water bath temperature with time . . 71a 20. Changes of metabolic rate with temperature . . 71b v Acknowledgement I wish to thank Dr. B. A. Eagles, Dean of the Facility of Agriculture and Chairman of the Division of Animal Science, for his permission to undertake this project and for the use of departmental faci l i t i e s . For his guidance, interest and enthusiasm throughout the execution of this project I express my sincere appreciation to Dr. A. J. Wood, Professor of Animal Science. My thanks are due also to Dr. W. D. Kitts for his criticism and many helpful suggestions. The financial assistance provided by a National Research Council of Canada Studentship is gratefully acknowledged. " the scientific account of living things does already, in its f i r s t stages, completely exhaust the science of chemistry. That unit of living organisms, the cell, both in the substances that compose i t and in the arrangement of those substances, presents problems which extend beyond the frontier of present chemical and physical knowledge." J. N. W. Sullivan The Limitations of Science v i i INTRODUCTION INTRODUCTION The importance of the meat producing industry to the economy and welfare of the country is well recognized. The production of meat exploits one of the most fundamental activities of l i f e : the phenomenon of growth. Any intelligent attempts to improve the efficiency of meat production presuppose a thorough and basic understanding of the nature of animal growth. That is, an answer to the question, *what is growth?* must be attempted. Growth cannot involve only a consideration of the kinetics or time rate of mass increase but must also involve a consideration of such other variables as feed efficiency, body composition and metabolic rate. This is so because changes in each of these variables parallel changes in the others as growth proceeds. It is important, therefore, to assess the character of these changes and the nature of their environmental interrelationships. Then, by a judicious manoeuvering of the environment, i t will be possible to take advantage of such knowledge in the production of animals for meat. Growth studies may proceed on several levels, the molecular, the cellular or the organismic. The present study was designed to elucidate some of the problems connected with - 1 -- 2 -growth at the level of the organism. Its particular purpose was to attempt an answer as to why variations exist in the rate and efficiency with which animals grow. That variations do exist is well recognized, vast differences appearing between individuals of the same species and even between individuals from the same l i t t e r (40) . Two possibilities present themselves to explain individual growth rate and efficiency differences. Either they arise as a result of prevailing and/or previous environmental circumstances or they are genetic effects. It seems logical, however, that whichever is the ultimate cause, its influence must be mediated through a differential disposition of ingested nutrients between the maintenace expense and the energy of the gain. It is unreasonable that the differences could be due to variations in the net energetic efficiency. If the distribution of calories between maintenance and gain may differ, then not only the absolute amount of gain but also the composition of. the gain my differ. This is so because the composition of gain at various stages of growth depends on the priority of the various tissues for available nutrients. Insofar as basal metabolic rate i s a function of the composition of the body i t , too, may be expected to vary with growth and feed efficiency. Before beginning a study of growth rate and efficiency, i t was considered desirable to delineate the average changes in metabolic rate and body composition as growth proceeds. Because of the difficulty of undertaking large scale experiments on the common meat producing animals and the relative ease of working with small experimental animals such as the mouse, the latter animal was chosen for the present study. Thus, as a pilot to future work with larger animals, changes in basal metabolic rate and body composition of male Swiss albino mice ranging in age from birth to 118 days were investigated. In order to show that this age range parallels a significantly long physiological period in the l i f e of the major meat producing animals, a chart (9) showing the weight-growth equivalence of several animals is here partially reproduced (Figure l ) . It may be seen that an 118 day mouse is the approximate equivalent of a 13 month sheep, a 2+0 month pig or a 45 month cow. These ages represent, especially in the case of the pig, relatively mature states. By comparing a mouse day to the physiologically equivalent time in the l i f e of the larger animals i t is obvious that physiological events are happening at a tremendous pace in the former as compared with the latter. In order to measure the basal metabolic rate of the mouse a new respirometer for small animals has been developed. It is similar in principle to the familiar Warburg respirometer. Finally, a short experiment was conducted in which male l i t t e r mate groups of three mice each having the same weight for age were separated into three groups at a body weight of fifteen grams. One member from each group was MOUSE C B AGE OF ANIMAL IN MONTHS 2 4 6 J 8 COW i i i i i i i • i i I I C B 10 20 30 40 50 60 70 80 90 SWINE I I I I I I I I I C B 10 20 30 40 50 60 70 SHEEP 1 ' 1 ' 1 1 1 C B 5 10 15 20 25 B • BIRTH C • CONCEPTION - 4 -killed and analysed for protein, water, ash and fat; the other two were placed on different planes of nutrition and grown to 25 grams. At this weight their basal metabolism was measured and then they were killed and analysed as the 15 gram controls had been. Average growth rates, feed efficiencies, body compositions and metabolic rates were compared between the two groups. EXPERIMENTAL METHODS - 5 -EXPERIMENTAL METHODS 1. Body Composition* The whole carcasses of one hundred and sixteen male UBC Swiss Albino mice were analysed for fat, protein, water and ash. They ranged in age from birth to 118 days and in weight from 1.34 to 27.42 grams. In a l l cases except for very young mice (below six grams) each analysis represents a single animal. For the smaller mice several l i t t e r mate * males were analysed as a group at each weight. The analytical data are presented in Table 1. The analytical methods used for determing the four main body constituents were as follows. After killing the animal by a sharp blow on the head, i t s three body cavities (abdominal, thoracic and cranial) were opened and the whole carcass was dried to a constant weight in an oven at 100 to 105 C. The weight lost during the interval is reported as body water. The dried carcass was then ground in a mortar and the residue transferred quantitatively to a small flask. The percentage of ether extract (reported as percentage fat) was obtained by repeated extraction of this residue by shaking with several aliquots of ethyl ether each followed by decantation through f i l t e r paper. The amount of extract - 6 -was determined as an average between the amount of residue left after evaporation of the ether and the weight loss of the original dried carcass. In order to ascertain whether this method permitted complete extraction, the residue was re-extracted in several cases and no detectable amount of fat was found. The fat and moisture free substance was analysed for ash and protein according to the A.O.A.C. (l) methods. 2. Metabolic rate. Three hundred individual metabolic rate determina-tions were performed on male white mice ranging in age from birth to 118 days and in weight from 1.34 to 27.42 grams. An indirect method especially designed for this purpose was employed. A detailed description of the design and operation of this apparatus is given in appendix I. In order to demonstrate the relationships between body composition and metabolic rate, those animals which were analysed for their protein, water, fat and ash were subjected to a preliminary determination of metabolic rate. Strict basal conditions were only possible of attain-ment in animals above about 10 grams because of the impossibility of limiting the movement of the very young mice suffering from inanition. However, i f , as is generally believed true, resting (non-fasting) metabolism represents a constant increment above basal metabolism, the pattern of age changes will be the same for resting and basal conditions. - 7 -3 . The Feeding Experiment. In order that an attempt might be made to discover why large differences exist in the rate and efficiency with which animals grow a feeding experiment was set up which could supple-ment the findings of and be interpreted in the light of the body composition and basal metabolism data. Nine lots of three male white mice were divided one into each of three groups. To minimize genetic variability each trio of mice was selected from a single l i t t e r and each member of the trio was required to reach 1 5 grams at the same age. At 15 grams one randomly selected member from each lot was killed and analysed for protein, water, fat and ash, one member was placed on a high plane of nutrition and the third was placed on a low plane of nutrition (70% of the high plane). The composition of the diet i s given in Table 2 . The metabolic rate of each animal was determined at 1 5 grams. The feeding schedule for the high plane animals was determined from the feed intake of a li t t e r of mice from 1 5 to 2 5 grams. The growth curve and feed consumption data of these mice are given in Figures 2 and 3 * Table 3 shows the feeding standard for the high and low plane animals. The level of feed intake of the high plane animals was restricted to 90% of the measured intake of the preliminary mice to ensure complete daily consumption. The animals were weighed every second day and the feed offered was adjusted every second day in accordance with their weights. The low plane animals were maintained on the low plane of nutrition and AGE IN DAYS Figure 2. Average growth curve of male white mice. - 8 -their intake adjusted each time the intake of their high plane controls was adjusted. On the average they just maintained their weights or lost slightly until the high plane animals reached about twenty grams at which time their feed intake was sufficient to permit them to grow very slowly. At this point, they were switched to the high plane feeding standard and maintained on this basis, according to their weight, exactly as the high plane animals had been. Both groups of animals were fasted for twenty-four hours when they reached 25 grams, their basal metabolism was measured and they were killed and analysed for the four main body constituents. A comparison between body composition, the composition of the gain, the rate and efficiency of growth and the basal metabolism of the average for the two groups was,conducted. The composition of the average gain from 15 to 25 grams was calculated as the difference between the composition of the 15 gram mice and the 25 gram mice. TABEE 1. BODY COMPOSITION AND METABOLIC RATE FOR 116 WHITE MICE Body Body Composition Protein/ BMR-KCALS FFBM Weight FFBM Age Fat Water Protein Ash /H20 Per Day Per Unit Surface Protein/H20 Gms. Gms. Days Gins. Gms. Cms. Gms. Gms. 1.37 1.34 0 0.03 1.14 0.16 0.03 0.142 0.19 17.3 9.4 N 2.59 2.45 4 0.14 2.05 0.32 0.05 0.156 0.66 39.2 15.7 N 4.23 4.05 10 0.18 3.28 0.64 0.10 0.195 1.10 47.0 20.8 N 4.36 4.08 8 0.28 3.34 0.62 0.09 0.187 1.29 53.5 21.8 N 4.46 3.95 7 0.51 3.23 0.64 0.10 0.184 1.11 45.1 21.5 N 6.02 5.45 12 0.57 3.28 0.91 0.13 0.209 1.49 50.2 26.1 N 5.37 5.24 50 0.13 3.84 1.03 0.24 0.268 - — 19.6 N 7.30 6.87 21 0.43 5.35 1.19 0.21 0.222 2.48 73.4 31.0 N 7.36 6.97 20 0.39 5.41 1.22 0.21 0.226 2.56 75.0 30.8 N 7.43 7.15 22 0.28 5.56 1.23 0.18 0.220 2.24 65.7 32 .5 N 7.46 6.68 14 0.78 5.26 1.15 0.18 0.219 2.35 68.3 30.5 N 7.49 6.97 17 0.52 5.42 1.26 0.20 0.233 2.54 73.8 29-9 N 8.58 8.00 19 0.58 6.24 1.37 0.21 0.220 3.24 85.9 36 .4 N 8.64 8.09 22 0.55 6.32 1.38 0.29 0.219 3.59 95.2 36 .9 . N 8.95 8.55 19 0.40 6.69 1.45 0.28 0.219 2.94 76.2 39.0 N 9.33 8.88 20 0.45 6.95 1.48 0.28 0.214 3.79 95.4 41.5 N Body Body Composition Protein/ BMR-KCALS FFBM Weight FFBM Age Fat Water Protein Ash /H20 Per Day Per Unit Protein/H20 Gms. Gms. Days Gms. Gms. Gms. Gms. Surface Gms. # 9.42 9.24 — 0.18 6.99 1.77 0.37 0.253 2.50 62.5 36.5 N 9.45 8.98 25 0.47 6.96 1.53 0.26 0.220 3.12 78.0 40.8 N 10.26 10.08 31 0.18 7.61 1.90 0.42 0.249 2.87 67.5 40.5 11.26 11.05 - 0.21 8.09 2.31 0.46 0.286 2.78 61.4 38.6 11.56 10.88 26 0.68 8.48 1.90 0.39 0.224 3.60 78.2 48.6 N 11.73 10.41 - 1.32 8.07 1.73 0.36 0.215 4.03 87.0 48.4 12.04 11.51 30 0.53 8.90 1.97 0.38 0.222 - - 51.6 N 12.34 11.31 29 1.03 8.48 2.12 0.43 0.250 4.47 88.2 45.2 N 12.70 12.28 30 0.42 9.33 2.30 0.42 0.247 3.65 74.6 49-7 N 12.84 12.38 35 0.46 9.33 2.39 0.45 0.256 3.37 68.5 48.4 12.85 12.09 22 0.76 9.27 2.21 0.39 0.238 3.92 77.3 50.8 N 12.88 12.13 22 0.75 9.18 2.31 0.44 0.252 - - 48.1 N 13.23 12.34 30 0.89 9.48 2.21 0.37 0.233 - — 52.9 N 13.81 12.90 37 0.91 9.79 2.36 0.47 0.242 4.59 88.8 53.3 N 13.82 12.91 30 0.91 10.06 2.36 0.41 0.235 4.31 83.4 54.94 N 13.82 12.95 25 0.87 10.04 2.31 0.43 0.230 4.45 86.1 56.30 N 13.88 13.01 22 0.87 10.06 2.30 0.41 0.229 4.12 79.3 56.8 N Body- Body Composition Protein/ BMR-KCALS FFBM Weight FFBM Age Fat Water Protein Ash /H20 Per Day Per Unit Protein/H20 Gms. Gms. Days Gms. _ Gms. Gm3. Gms. Surface Gms. •H-13.94 13.28 - 0 . 66 10 .37 2.28 0 .44 0.220 4.11 79.1 60 .4 N 14.11 13.85 31 0 .26 10 .32 2 .78 0.58 0.269 3.95 75.3 51.5 14.13 13.40 23 0 . 73 10 .09 2 .54 0 . 4 7 0 .252 - - 53.2 N 14.15 12 .92 - 1 .23 9.42 2 . 74 0 .49 0.291 3.72 70.9 47.8 14.18 13.36 35 0.82 10 .05 2 .67 0 . 50 0.265 3.43 65 .1 50 .4 14.18 13.30 - 0.88 10 .30 2 .34 0 .43 0 .227 - — 58 .6 N 14.34 13.81 29 0 . 53 10.38 2 .71 0 . 51 0.261 4 .00 75.5 52 .9 14.52 14.10 30 0 .42 10 .61 2 .76 0.48 0.260 4.50 75 .8 54.2 14.57 13.71 29 0.86 10 .65 2.36 0 . 42 0.222 .- - 61 .7 N 14.68 14.40 62 0.28 10 .29 3 .25 0 .62 0.316 2.82 52.3 45 .6 14.75 13 .84 32 0 . 91 10.80 2 .33 0.48 0.216 3 .48 64.5 64.1 N 15.08 14.19 24 0.89 11 .07 2 .46 0 . 43 0.222 4 .34 78 .4 63 .9 N 15.08 13.98 31 1.10 10 .53 2 .75 0 . 50 0.261 4.35 79.2 53 .6 N 15.18 14.19 28 0 . 99 10 .91 2 .57 0 . 44 0.235 4 .86 88.1 6 0 . 4 N 15.38 14.43 45 0 . 95 10 .55 2 .78 0 . 60 0.264 3 .21 57 .7 54 .7 15.70 14.77 - 0 .93 11.38 2 .69 0.48 0.236 3.83 68.0 62 .6 15.76 14.68 45 1.08 10 .73 2 . 94 0 . 61 0.271 3 .98 70 .3 54.2 Body-Weight FFBM Age Body Composition Protein/ /H20 BMR-KCALS FFBM Fat Water Protein Ash . Per Day Per Unit Surface Protein/H20 Gms. Gms. Days Gms. Gms. Gms. Gms. Gms. 16.31 15.37 45 0.94 11.38 3.11 0.60 0.274 3.73 64.6 56.1 16.41 15.78 31 0.63 11.77 3.17 0.57 0.269 4.08 70.3 58.7 16.82 16.35" 98 0.47 11.70 3.82 0.78 0.326 3.27 55.5 50.2 16.88 15.63 38 1.25 11.38 3.29 0.62 0.289 4.07 68.8 54.1 16.96 15.98 - 0.98 11.90 3.37 0.55 0.283 4.35 73.2 56.5 17.03 16.41 . 33 0.62 12.22 3.38 0.60 0.276 4.66 78.4 59.5 17.55 15.77 44 1.78 11.55 3.36 0.61 0.291 3.96 65-4 54.2 18.10 17.28 33 0.82 12.82 3.60 0.56 0.280 3.94 63.6 61.7 18.39 17.11 - 1.28 12.44 3.68 0.58 0.296 4.10 65.5 57.8 18.55 17.23 118 1.32 12.54 3.69 0.75 0.294 - - 58.6 18.61 17.01 61 1.60 12.39 3.69 0.70 0.298 4.01 63.6 57.08 18.83 17.53 — 1.30 13.10 3.63 0.52 0.277 4.73 74.4 63.3 19.00 18.38 44 0.62 13.14 - 0.80 - 4.12 64.4 -19.03 18.01 - 1.02 13.04 3.98 0.70 0.305 5.09 79.5 59.1 19.16 16.53 46 2.63 12.31 3.33 0.67 0.270 4.50 69.8 61.2 19.66 18.78 48 0.88 12.61 4.07 0.72 0.299 4.46 67.1 62.8 Body Weight FFBM Age Body Composition Protein/ /H20 BMR-KCALS FFBM Fat Water Protein Ash Per Day Per Unit Protein/H20 Gms Gms. Days Gms. Gms. Gms. Gms. Surface Gms. 19.66 18.24 46 1.42 13.62 3.46 0.78 0.254 4.28 65.3 71.8 19.71 18.35 46 1.36 13.37 3.89 0.68 0.291 5.09 77.6 63.1 If. 81 18.92 43 0.89 13.57 - 0.73 - 4.33 65.8 -19.88 19.10 56 0.78 14.13 3.76 0.73 0.266 4.51 68.3 71.8 19.93 19.22 48 0.71 14.25 3.98 0.72 0.279 4.96 73.7 68.8 19.97 18.85 37 1.12 13.93 3.93 0.72 0.282 4.44 67.1 66.8 19.98 18.84 42 1.14 13.80 3.94 0.67 0.285 4.19 63.3 66.1 20.07 18.96 72 1.11 13.77 4.11 0.81 0.299 4.38 65.9 63.4 20.21 18.44 57 1.77 13.79 3.66 0.73 0.266 4.95 72.3 69.3 20.25 18.50 60 1.75 13.62 3.90 0.74 0.286 4.82 72.1 64.7 20.26 18.49 71 1.77 13.38 4.00 0.79 0.299 4.95 74.0 61.8 20.30 19.33 45 0.97 13.94 4.22 0.82 0.302 4.94 73.9 64.0 20.38 19.48 71 0.90 14.45 4.05 0.74 0.280 4.75 70.8 69.6 20.41 18.65 41 1.76 13.40 4.02 0.71 0.300 5.16 76.9 62.2 20.42 18.42 43 2.00 13.12 - 0.69 - 5.17 77.1 -20.42 19.60 45 0.82 14.17 4.43 0.84 0.312 5.25 78.2 62.8 Body-Weight FFBM Age Body Composition Protein/ /H20 BMR-KCALS FFBM Fat Water Protein Ash Per Day Per Unit Surface Protein/H20 Gms. Gms. Days Gms. Gms. Gms. Gms. Gms. 20.43 18.37 71 2.06 13.46 3.82 0.77 0.284 - - 64.7 20.47 17.93 59 2.54 13.12 3.76 0.81 0.286 4.72 70.1 62.7 20.53 17.56 69 2.97 12.87 3.72 0.75 0.289 4.35 64.6 60.8 20.61 19.09 58 1.52 13.77 3.88 0.80 0.282 4.69 69.4 67.7 20.74 18.66 35 2.08 13.69 3.99 0.70 0.292 - - 63.9 20.76 18.97 58 1.79 14.01 3.98 0.76 0.284 4.74 69.7 66.8 20.89 19.70 114 1.19 14-41 4.19 0.77 0.290 4.66 68.2 67-9 21.01 16.83 60 4.18 12.15 3 .64 0.69 0.291 4 .64 67.8 57.8 21.13 17.78 61 3.35 13.07 3.71 0.70 0.284 4.69 68.3 62.6 21.30 . 18.77 54 2.53 14.02 3.65 0.69 0.250 5.35 77.5 75.1 21.34 19.11 70 2.23 13.94 3.89 0.67 0.279 - - 68.5 21.41 20.09 73 1.32 14.69 4.28 0.86 0.292 4.35 62.8 68.8 21.58 19.49 48 2.09 14.74 3.81 0.70 0.258 5.39 77.3 75.5 21.77 19.69 42 2.08 14.32 4.23 0.71 0.295 4.49 64.0 66.8 21.91 19.83 — 2.08 14.48 4.22 0.78 0.291 4.65 66.1 68.1 £ Body-Weight FFBM Age Body Composition Protein/ /H20 BMR-KCALS FFBM Fat Water Protein Ash Per Day Per Unit Surface Protein/H20 Gms Gms. Days Gms. Gms. Gms. Gms. Gms. 21.94 17.43 57 4.51 12.94 3.51 0.61 0.271 4.56 64.8 64.3 22.15 19.84 97 2.31 14.53 4.24 0.78 0.291 5.06 71.3 68.2 22.20 18.11 51 4.09 13.08 3.85 0.72 0.313 5.04 71.0 57.9 22.26 19.25 55 3.01 13.98 4.19 0.78 0.299 4.31 60.5 64.4 22.65 21.72 - 0.93 15.99 4.59 0.85 0.287 4.86 67.6 75.7 23.02 21.22 39 1.80 15.68 4.35 0.74 0.277 5.02 69.1 76.6 23.24 19.90 52 3.34 14.40 4.30 0.83 0.298 4.85 66.3 66.7 23.42 . 22.00 58 1.42 16.00 4.70 0.89 0.294 4.87 66.2 74.8 23.96 20.52 50 3.44 15.63 3.68 0.76 0.275 5.70 76.2 74.6 24.34 22.43 59 1.91 16.50 4.70 0.80 0.285 5.19 68.8 78.7 24.36 22.56 - 1.80 17.18 5.10 0.96 0.297 5.35 70.8 76.0 24.52 20.99 56 3.53 15.60 4.24 0.76 0.272 - 77.2 • 24.70 22.71 51 1.99 16.46 4.98 0.86 0.303 5.01 65.7' 75.0 25.53 24.00 66 1.53 17.56 5.00 0.89 0.285 5.51 70.8 84.2 26.36 23.25 3.11 16.90 4.96 0.85 0.294 5.59 70.1 79.1 Body Body Composition Protein/ BMR-KCALS FFBM Weight FFBM Age Fat Water Protein Ash /H20 Per Day Per Unit Proteln/H20 Gms. Gms. Days Gms. Gms. Gms. Gms. \ Surface Gms. 26.94 24.38 45 2.56 17.83 5.24 0.88 0.294 6.26 77.6 82.9 27.42 23.36 - 4.06 16.98 5.14 1.03 0.303 5.93 72.6 77.1 * Non-fasted animals H ON 1 -17-TABLE 2. COMPOSITION OF DIET Ingredient Weight - lbs. Ground Wheat 750 Wheat Bran 100 Soya Bean Meal 1 150 Fish Meal (70#) 300 Powdered Skim Milk 100 Dried Yeast 25 Liver Meal 50 Bone Meal 10 Fish Oil 5 Molasses 200 Ground Yellow Corn 300 Iodized Salt 10 Total 2000 TABLE 3. HIGH AND LOW PLANE FEEDING STANDARD Body Weight Feed Standard High Low 15 3.6 2.5 16 3.7 2.6 17 3.9 2.7 18 4.1 2.9 19 4.3 3.0 20 4.5 3.2 21 4.8 3.4 22 5.0 3.5 23 5.1 3.6 24 5.2 3.6 25 5.2 3.6 RESULTS AND DISCUSSION - 19 -RESULTS AND DISCUSSION 1 . Body Composition For years there has been sporadic interest in changes in the gross composition of animals. The concept that changes in body composition result from, or are the cause of variations in metabolic rate, growth rate and growth efficiency is relatively new. There is increasing evidence, however, that compositional changes are important determinants in the growth complex. Two examples will suffice to make this clear. First, since the original formulation of the "surface law" in 1837 (50 ) , metabolic rate has commonly been expressed as a function of surface area or what amounts to the same thing, as a fractional power of body weight ( 9 , 2 6 ) . The newer concept of a physiologically active fraction of the body (generally taken to be the fat-free portion), however, has made the surface concept open to question. It is now held by some that metabolic rate changes are linked to changes in body composition ( 6 ,17 ,24 ,35 )• Second, differences in growth rate and efficiency may logically be connected with differences in body composition and differences in the nature of the gain. This is so because of the known variations in the energy cost and the density of the constituents of gain. It was a recognition of the importance of changes in body composition which prompted the present study of age changes in the body composition of mice. - 20 -(a) Weight changes in protein, water, fat and ash. In accordance with the concept of differential growth elaborately discussed by Huxley (23) and shown to apply to changes in body composition in the pig by McMeekan (34) and in sheep by Hammond (19) and Palsson and Verges (46) the present data has been graphically represented by a log-log relationship. It should be mentioned that many size relationships assumed, a priori, to be best described by the differential or heterogonic growth equation are, in fact, best described by a linear relation-ship. Scholl (54) has pointed out the wisdom of caution in using this type of growth formula. However, the present results appear to be best described in this manner and the results are considered to have physiological significance. The relationships between the four main body constituents are given in Figures 4 to 7. From an inspection of the correlation coefficients and the standard errors of estimate of the lines for protein, water and ash i t is seen that, as might be expected, these components of gained tissue bear a very definite relationship to body weight. Although obviously correlated with body weight as well, there is considerably more variation in the relation between fat and body weight. The slopes of the lines for protein, fat and ash are greater than unity while that for water is less than unity. This indicates that, per unit mass increase, there is a greater than unit increase in protein, fat and ash and a less than unit increase in water. From the above data i t is seen that the increase in 5 10 B O D Y W E I G H T IN G R A M S Figure 4. Changes in protein weight with changes in body weight. o SmUQ NI H 3 1 V M J O 1 H 9 I 3 M Figure 5. Changes i n water weight with changes i n body weight. • I 1 I 1 I I I I 5 10 2 0 B O D Y W E I G H T IN G R A M S Figure 6. Changes i n ash weight with changes' i n body weight. I 5.01-< o X S2 UJ U_ 1.0 0.5 0.1 J I M i l l 10 3 0 B O D Y W E I G H T IN G R A M S Figure 7. Changes in fat weight with changes,, in body weight. - 21 -in mass of the four main body constituents, especially protein water and ash, is an orderly and directed process. Thus, for the population as a whole, older, heavier animals will tend to have more protein, ash, and fat but less water percentagewise. This cannot be construed to mean, however, that two animals of the same weight or age will have the same composition, for considerable variation can exist within any age or weight group. (b) The composition of the fat free body. As noted above, the most striking variations in body composition occur in the percentage of fat. These fluctuations may be largely a response to variations in nutritive state and are presumably a reflection of the differential demand for nutrients by the various tissues, under conditions of restriction ( 1 0 , 3 4 , 4 5 ) . The considerable variation in fat content was recognized by Murray (43) and Moulton (42) who proposed that the chemical composition of animal bodies be compared on a non-fat basis. In this connection i t has been stated by Brozek ( 10 ) : "The separation of the body weight into the fat component and the lean body mass represents the f i r s t and fundamental step in the quantitative description of the compartments of the body." Moulton has presented evidence for many species that the composition of the fat-free body mass changes in a regular manner with age ( 42 ) . Beyond a certain point, however, l i t t l e or no further change was noted and the age at which this Occured was called the point of "chemical maturity". After chemical - 22 -maturity, the composition of the fat-free body was stated to be unaffected by age, degree of fatness and nutritive state. More recently, Spray and Widdowson (57) have cautioned that, though valuable, the concept of chemical maturity requires further elaboration and definition i f i t is to be used for detail. This conclusion, based on their work with the rat, rabbit, dog and cat, arose from the discovery that the four gross body constituents as well as many of the minor ones did not reach their "chemically mature" concentration at the same time. It may logically be argued that age is a poor baseline against which to relate changes in body composition because the fat-free body weight may vary considerably at any given age depending upon the nutritive status of the animal. With the differential growth concept in mind i t seemed reasonable to represent changes in the composition of the fat-free body on this basis. Figures 8, 9 and 10 show log-log plots of fat-free body weight against water, protein and ash respectively. It is obvious that, with respect to the fat-free body, the percentage of these three constituents continues to change over the age range studied. The variation of the data about the smoothed line is less than when plotted against body weight. This tends to confirm the value of considering the animal body as composed of a fat-free portion accompanied by a variable amount of fat. In view of the above facts, the changing relation-ships between the non-fat body constituents themselves is of o io SVWelO NI U31VM J O J.H9I3M Figure 8, Changes in water weight with changes i n fat-free weight. EQUATION Y = 0,147 X I 1 I I I • l l i 5 10 W E I G H T O F F A T - F R E E B O D Y IN G R A M S Figufee 9. Changes in protein weight with changes in fat-free weight. 5 10 W E I G H T O F F A T - F R E E B O D Y IN G R A M S Figure 10. Changes in ash weight with changes in fat-free weight. - 23 -is of considerable interest. Figures 11 and 12 show the log-log relationship between body water and body protein and between body protein and body ash respectively. Here again the two variables are seen to be closely correlated in each case. The slope of the log body protein-log body a&relation-ship is nearly unity (1.07) suggesting a nearly constant ratio of protein to ash. This tends to confirm a similar conclusion presented elsewhere by Murray (43). The slope of the log body water-log body protein relationship is greater than unity suggesting that a fundamental property of aging or growth is a progressive dehydration of the tissues. This increasing de-hydration, however, proceeds at a diminishing rate, approaching a maximum limiting value. In conclusion i t may be said that as aging progresses the composition of the dry, non-fat portion of the body remains relatively constant, the hydration of this fraction uniformly decreases and the amount of fat present shows great variation and is largely dependant on nutritive condition. (c) The formulation of an index of physiological age. The inadequacy of body weight or chronological age as indices of physiological age is immediately apparent. From the dat4 i t may be seen that composition differences between animals of the same weight or age may be very great so that a mouse which is young according to its weight or age may be old in terms of its composition, and vice versa. In other words, because one fundamental property of physiological aging is change in body composition any index of physiologic age in Figure 11. Changes in protein weight with changes i n water weight. SWVU9 NI HSV dO 1H9I3M Figure 12. Changes in ash weight with changes in protein weight. - 24 -which such change is not implicit cannot be taken as valid. For this reason any single component of, or any combination of the components of, or the whole of the fat-free body itself are a l l inadequate indices of physiological age. Presumably the protein and water components of body weight comprise the bulk of the physiologically "active" portion of the body. The uniformity of the relationship between water and protein has already been noted. Herein lies a due to the discovery of a suitable index of physiological age. It is apparent from the slope of the log protein-log water plot that the ratio of protein to water steadily increases but at a dininishing rate. This protein to water ratio immediately recommends itself as a useful index of physiological age for i t satisfies the requirements which such an index should meet. That i s : (l) i t increases on the average with body weight and age at a decreasing rate and approaches a maximum (see Figure 13); (2) within limits i t i s independent of both weight and age as shown by the fact that i t is a dimensionless number; (3) i t takes into consideration changes in the composition of the fat-free body and (4) i t is a function only of the "active" components of the body. Further support for the use of the protein to water ratio as an index of physiological age may be found in a review by Lansing (28). This author has cited work which demonstrates U31VM / / NI310Ud Figure 13, Changes in the protein to water ratio with changes in weight. - 25 -that rats placed on a restricted plane of nutrition during the growing phase live longer than control rats on a high plane of nutrition during the same time interval. From results to be presented in a later section of this study i t is shown that mice placed on a low plane of nutrition during the second half of the growth period have a lower protein to water ratio than high plane l i t t e r mate controls of the same weight even though the former are chronologically older than the latter. In addition, Tauber (59) has noted that the apparent age of a group of 496 undernourished children was well below their actual age. On the basis of this informa-tion i t is tempting to suggest that caloric restriction retards physiologic aging and hence prolongs the time of approach to maturity. In other words, caloric restriction decreases the rate of change of physiologic age. The control exercised by the level of caloric intake over physiological aging may be indirectly mediated through a retardation of the output of growth promoting hormones such as the anterior pituitary growth hormone as well as directly through a deficit of the substrate necessary for growth (12). That pituitary activity may be depressed by under nutrition has been indicated by Samuels (49). The range of values of the protein to water ratio appears to be very similar for species of greatly divergent - 26 -size. Recalculated data for the rat (36,37) and the cow (43) demonstrate this point. These data appear in tables 4 & 5. The reason that the very low values noted for young mice are not duplicated for the cow and the rat is because the former are physiologically more mature at birth and the latter are not represented in this comparison by very young animals. On the basis of their protein to water ratio animals of very different size may be subject to a valid comparison. (d) The calculation of body composition in vivo. The body composition data presented here may be used to extend the usefulness of the present in vivo methods of computing body composition. Present in vivo methods rely on an indirect estimation of total body water or extracellular water using antipyriaie (8,56) or deuterium oxide (51) and sodium thiocyanate (13) or sodium thiosulphate (ll) respectively. Such methods rely on the tacit assumption that body water is a constant percentage of the fat-free body (44). From the foregoing discussion, however, i t is obvious that this assumption is invalid for the growing animal and only approximately valid for mature animals. The formula of Pace and Rathbun (44) used to calculate percentage fat from percentage water i s : %Y - 100- gtf 0.732 where: F = fat W =: water 0.732 = water as a fraction of the fat-free body - 27 -TABLE 4 COMPOSITION DATA FOR THE RAT FROM MITCHELL AND CARMEN (36,37) Body Weight Age Days Protein Gms. Water Gms. Protein Water KCALS Water 58 32 10.3 38.4 0.269 -64 32 10.0 45.0 0.223 -68 32 11.2 48.0 0.234 -75 32 11.6 52.8 0.219 -109 97 19-7 81.2 0.242 -154 97 31.7 103.1 0.308 0.167 158 97 31.4 106.3 0.296 0.169 223 188 42.4 131.7 0.322 0.154 225 165 38.8 150.4 0.258 0.179 240 165 45.3 134.2 0.337 0.162 241 188 46.8 144.7 0.323 0.193 264 169 49.4 146.3 0.338 0.173 266 165 48.9 161.9 0.302 0.129 264 173 39.2 172.2 0.228 0.173 267 173 50.6 160.3 0.316 0.228 272 169 50.4 156.4 0.332 0.162 280 188 53.8 178.4 0.302 0.138 290 169 49.1 159.8 0.307 0.172 Average 0.167 - 28 -TABLE 5. COMPOSITION DATA FOR THE COW  FROM MURRAY (43). Body Weight Protein Water Protein KCALS Kilograms Water Water 40.8 8.1 29.3 0.276 0.0444 74.0 14.2 52.3 0.272 0.0421 111.6 21.0 73.3 0.286 0.0416 154.0 29.8 101.4 0.294 0.0365 189.7 36.3 119.3 0.304 0.0357 223.9 43.5 138.8 0.313 0.0328 226.6 49.6 160.8 0.308 0.0312 313.9 59.0 183.3 0.322 0.0300 351.0 62.0 190.1 0.326 0.0305 400.0 68.4 207.8 0.329 0.0298 443.0 72.7 211.7 0.343 0.0312 489.0 78.2 234.4 0.334 0.0299 522.0 82.4 250.1 0.329 0.0288 561.0 90.6 267.9 0.338 0.0280 614.0 96.4 266.8 0.361 0.0304 Average 0.0335 - 29 -In the light of the present study a different formula can be derived by replacing 0.732 with a new expression for water as a fraction of the fat-free body. Thus, from figure 8: ¥ = 0.8677 FFBM 0* 9^ 6 where: FFBM = fat-free body mass And water as a fraction of the fat-free body i s : 0.8677 FFBM 0' 9^ 6 _ 0.8677 FFBM 0.0554 FFBM Now, since FFMB = BW - F (where BW is body weight), the above expression can be changed to: 0.8677 , ,0.0554 (BW-F) Substituting this in the Pace formula gives: % F =" 100 - 0.8677 ~ (BW-F) 0" 0" 4 or % F = 100 - % W (BW-F)°*°554-0.8677 Multiplying by B.W. and dividing by 100 gives: F =• BW - W (BW-F) 0' 0 5 5 4 0.8677 To calculate the protein content i t is merely necessary to use the expression relating body protein to body water, thus: Protein ^ 0.1456 W1*2^ The ash content may be similarly calculated from the - 30 -protein content (see figure 12). It must be remembered that i t is necessary to establish the various numerical relationships between the body compartments before an in vivo method of analysis based on a determination of water alone can be effective. - 31 -2. Metabolic Rate "Growth i s inseparable from metabolism". Samuel Brody. The metabolic rate of any organism i s the heat energy released incidental to a l l the biochemical processes associated with l i f e . It must be equivalent, i n terms of calories, to that portion of ingested feed energy which i s not recovered i n the faeces or urine and which i s not incorporated into the tissues. It represents the energy cost of voluntary movement, of a l l involuntary a c t i v i t i e s v i t a l to l i f e such as digestion, res-piration, circulation, secretion and excretion etc., and, f i n a l l y , the cost of maintaining intact the thermodynamically unstable l i v i n g state ( 9 ) . This l a t t e r cost of maintaining the integrity of complex body constituents requires large amounts of energy because the equilibrium state i n synthetic processes i s far removed i n the direction of hydrolytic breakdown ( 7 ) . Basal metabolic rate i s defined as that energy elaborated during complete rest i n a metabolically indifferent (thermoneutal) environment and i n the post-absorptive condition. It differs from total metabolism i n being uncomplicated by the heat increment of feeding or by unnecessary physical ac t i v i t y . It theoretically represents the irreducible minimum of energy necessary to maintain the organism intact. Since a l l the - 32 -energy so expended must be recovered as heat, the heat loss of an animal is a direct index of basal metabolic rate when measured under basal conditions. In comparing the basal heat production of mature animals of several different mammalian species i t was discovered many years ago that as weight increased the basal energy production per unit of weight decreased. (See Kleiber (26) for a review of this subject). Thus, each weight unit in a mouse is about 25 times as active metabolically as the same unit in a cow. By an application of the principles of heat loss, Sarrus and Rameaux formulated for the first time the so-called "surface law" which stated, in effect, that since heat loss must balance heat production and heat loss is proportional to surface area, heat production is proportional to the surface area of an animal or to the square of its linear dimensions (cited by Brody, page 354, (9)« If this be true, and i f it.be assumed that the density of all mammalian tissue is the same, i t can then be shown that basal metabolic rate is proportional to the 2/3 power of body weight, or: BMR = KW2/3 where BMR = basal metabolism K = a constant W =- body weight. In actual fact, although the surface concept may s t i l l be held valid, the exponent of W has empirically been found to be closer to 3 / 4 than 2 / 3 . The value of K is very close to 70 which indicates that for a l l mammalian species every kilogram of weight raised to the 3 /4 power produces 70 Calories of heat ( 5 , 9 , 2 7 ) . ' This relationship appears valid intra- and inter-specifically. For growing animals the constancy of the ratio of basal metabolic rate to a power function of body weight does not hold. The reasons for this have been listed by Brody ( 9, ) as follows: 1 . There may be a work energy cost of growth which changes with age and growth rate. 2 . Early l i f e i s essentially poikilothermic and thermoregulatory mechanisms are not developed. 3 . The neuro-endocrine system does not attain maximum functional level until relatively late in l i f e . 4 . Muscular mass increases as a percentage of body weight from birth to maturity. 5 . There is a difference in rate of change of shape with age. In order to establish the age changes in metabolic rate for the mouse, 300 individual basal metabolic rate deter-minations have been performed on animals ranging in weight from 1.34 to 27.42 grams. Figure 14 shows a graphical representation of this data on log-log coordinates. There appear to be three distinct phases of activity for the non-- 34 -fasted mice which cover the greater part of the growth period. During the first two phases the metabolism is increasing at a greater rate than would be predicted from the line for mature animals of a l l species (BMR =• 70.5 W0,7-3(9) ). The slopes are 0.858 and 1.071 respectively as against 0.73• The third phase indicates a decline (K = 0.621) relative to the same baseline. For the fasted animals there does not appear to be more than one phase although only about two-thirds of the total growth period is represented by these animals. When the same data are calculated per unit of surface area and plotted against weight (Figure 15) there is an i n i t i a l rise to a maximum at about ten grams and a subsequent decline such that a constant low value is approached. For this graph body surface was calculated by the formula of Benedict (5) (Surface =^ 9 W2/3). The results reported here are strikingly similar to those reported for the rat by Kibler, and Brody (25). Brody (page 402) (9) points out the parallel between changes in growth rate and metabolic rate and declares that the rate of heat production parallels growth rate because of the work cost of growth but mainly because rapidly growing tissue is more youthful than non-growing or slow-growing tissue. This and other evidence throws serious doubt oil the surface concept as i t applies to growing and mature animals within a single species or breed. Thus, i f surface were the only factor limiting metabolic level, then even mature animals Figure l i * . Weight changes i n daily metabolic rate Figure 15. Weight changes In daily metabolic rate per unit of Surface area. - 35 -in any species should be capable of an energy expenditure at least as great as the animal at the maximum of the metabolism/ surface area curve. Further, i t should be possible to demonstrate very real maximum lijnits to activity imposed by the extent of surface area. Therefore, surface area cannot regulate heat production although i t may set a maximum limit to the potential rate of heat transfer. The following quote from Wedgewood et al (6l) is pertinent to the present discussion: "Computed and empiric relationships between basal metabolic rates and such factors as age, size, sex and similar functions used over the past century have been found useful tools but are subject to criticism because they imply either physiologic concepts of causation or regulation that may not exist, or else are empirical formulations without physiologic rationale." These authors go on to say that i t appears unlikely that there is any simple relationship between metabolic rate and any single standard of reference. Nevertheless, Best (6) states that the total evidence strongly suggests a close relation between basal metabolic rate and active protoplasmic mass regardless of other parameters of size. Thus, although the independence of surface area and heat production within a species is reasonably certain, i t may yet be possible to discover some standard of reference against which to relate basal metabolic rate. Such a standard must be a function of that fraction of the body which is involved in the production of heat. Furthermore, under basal conditions - 36 -metabolism must be directly proportional to the size of this "chemically reactive system" (61) or active protoplasmic mass or active tissue mass or physiologically active mass. In this connection i t has been stated as probable that in the basal state each unit of active mass is reacting at a rate independent of body size (6). In practice, any such estimate of active protoplasmic mass must consider the amounts of the metabolically active gross body components together with a correction for changes in their relative concentrations. This is so because in practice, and particularly in vivo, the gross components are the most readily estimated quantit-atively. Many attempts have been made to associate certain gross body compartments with metabolic rate. The successful use of the fat-free body mass as a reference base for the human has been demonstrated (4,6,35)• This approach testifies to the metabolic inertness of fat per se (as opposed to adipose tissue) and to the recognition of the necessity for considering only "active" body components. In fact, i t has been shown that normal and hereditarily obese mice have comparable basal metabolic rates based on the non-fat portion of the body (17). In addition, Best (6) indicates that the apparently lower metabolic rate of females becomes the same as for males when based on metabolically active mass. Other workers have demonstrated relationships between basal metabolic rate and - 37 -various body water compartments in humans.For example, on the basis of the in vivo measurement of body water in adult humans, Shock (53) noted a correlation between intracellular body water and metabolic rate. Wedgewood et al (6l) have demonstrated correlations between basal metabolic weight and intracellular, extracellular and interstitial fluid volumes in men. In general, however, these researches have been concerned with relatively mature subjects and have not attempted to correlate metabolically active mass with metabolic rate over the complete growth phase. In the present study the body composition of 116 white mice has been determined together with their basal metabolic rates. Fasting (basal) metabolism was obtained for mice above 10 grams and non-fasting (resting) metabolism for mice from 1.3 to 15.2 grams. Ih the above discussion i t was pointed out that the relation between basal metabolic rate and weight or surface was relatively inconstant. As a f i r s t step in the discovery of an estimate of physiologically active mass metabolism has been calculated per unit of fat-free body mass and per unit of protein. The number of Calories per day per respective unit of reference was seen to decline with increasing weight (see Table l ) . However, the character of this decline appeared to parallel, in an inverse sense, the increase in the protein to water ratio: that is, the physiologic age. Inasmuch as the two trends exhibit this - 38 -inverse parallel, the products of basal metabolic rate/fat-free body mass and basal metabolic rate/protein respectively and physiologic age were computed. In each case a very nearly constant value for fasted animals and a slightly higher but also constant value for non-fasted mice above six grams was obtained. The increment of non-fasted above fasted animals i s , of course, due to the heat increment of feeding as previously explained. In effect, then, the products, BMR x FROT. and BMR x PROT FFBM ¥ PROT ¥ are constant for a l l male mice above about six grams i n weight. Rearranging these expressions gives: f l \ BMR , v BMR K ( 1 ) FFBM K l a n d W ¥ K 2 PROT ¥ The expressions say, i n effect, that when basal metabolic rate i s plotted against fat-free body mass divided by the protein to water ratio or against weight of body water on arithmetic coordinates a straight line results which inter-sects the origin. Figures 16 and 17 show two such plots. The best straight li n e through the points representing the fasted mice i s seen not to intersect the origin. This means that the apparently constant values are, i n fact, only approximately constant. In other words, there i s a constant metabolism per unit of reference employed indicated by the value of the slope plus an increment represented by the value of the Y-intercept. Figure 16. The relationship between daily metabolic rate and physiologically active mass. t0 C M AVO a3d S3iaoivo - wsnoavi3w nvsva Figure 17. The relationship between d a i l y metabolic rate and body water. - 39 -This, however, should not be unexpected. A large part of the energy expended under basal conditions results from a l l those energy producing reactions in the body necessary to maintain the equilibrium state between breakdown and synthesis far in the direction of the latter. During growth, when anabolic processes are proceeding at a maximum rate (i.e. when equilibrium is as far removed as possible towards synthesis (2) ) energy expenditure will be at a maximum and will decline as the rate of mass increase declines. The excess energy above true maintenance which is produced during growth will be partially present even under conditions of severe inanition and hence i t is impossible to realize a true basal state in growing animals. If a line be drawn roughly parallel to the lowest basal metabolism values (presumably the slowest growing animals) in Figures 16 and 17, i t intersects the origin and converges with the calculated line. This line represents the approximate relation between true basal metabolism and the reference bases employed, and the difference between the two lines represents the "work" cost of growth. Thus the number of basal calories per unit of fat-free body maaB divided by the protein to water ratio is about 0.067 and per unit of water is about 0.30. The expression fat-free body mass divided by the protein to water ratio appears to have a definite physiological significance. As previously mentioned, basal metabolic rate is highly correlated with fat-free body mass for the mature human. r - 40 -In the above discussion i t was stated that any practical index of physiologically active mass must be a function of that portion of the body directly involved in metabolism together with a correction for changes in i t s composition. The fat-free body mass (or the fat - and ash - free body mass although the two are l i t t l e different) is an index of the active fraction of the animal body. "Therefore, i t is not surprising that mature humans have basal metabolic rates of a magnitude proportionate only to the size of their fat free body masses. This is true because physiologic age (and hence the composition of the fat free body mass) is almost constant in mature mice and presumably also in mature humans. In growing animals, however, the protein to water ratio is changing at a very rapid rate and hence the physiologic significance of a unit of the fat-free body also changes. Dividing the fat-free mass by the protein to water ratio corrects the former for changes in its own composition. Herein lies the significance of the fi r s t expression. It may be taken as an index of the amount of physiologically active mass because i t represents the total amount of metabolizing tissue corrected to the same physiological age or, in other words, i t is the amount of tissue responsible for the production of heat per unit of physiological age. The foregoing interpretation of the results, i f valid provides a nice confirmation of the conclusion of Brody (9) that the higher metabolic rate during growth is due to the - 41 -"work" of growth and to the fact that growing tissue is more youthful than non-growing tissue. It is shown here that the more youthful (in the present physiologic sense) the higher the metabolism and also that there is an apparent work cost of growth. The second expression relating basal metabolic rate to total body water indicates that water may be the sole agent responsible for controlling the metabolic level of an animal. In view of the changing relationship between protein and water with growth, i t seems much more reasonable that the basal metabolism - total water relationship is a reflection of a change in the significance of a unit of protein as i t relates to the water fraction. In this connection, Wedgewood et al (61) have shown for the human male that there are constant relationships between intra- and extra-cellular body water and basal metabolism but point out that any relation between total water and metabolic rate is dependent on a function of at least these two compartments. Furthermore, Friis-Hansen (16) has demonstrated growth differences in the relative amounts of the body water compartments in young humans of both sexes. Although some of the conclusions of Mayer et al (32) do not seem valid on the basis of the results of the present study, yet these authors do recognize the importance of compositional changes when they state that h water, amongst other things, varies so that the termochemical efficiency of growth is constant from weaning to puberty. - 42 -Because of the ease of its determination in vivo, however, total water represents a very useful index of basal metabolic rate and of metabolically active mass. On the basis of the present experiments i t may be tentatively concluded that every unit of physiologically active mass or approximately every unit of water has the same metabolic rate for mice of a l l weights above about six grams. Below six grams the basal metabolic rate appears to be too low according to this relationship. However, the number of animals investigated below this weight are few and no con-clusion can be drawn until more analytical data has been accumulated in this region. It is interesting to speculate as to whether this same relationship holds for many or a l l mammalian species and, i f so, i f they have the same basal metabolic rate per reference unit. The fact that physio-logical age varies over roughly the same range of values for such different species as the mouse, the rate and the cow might seem to affirm this question. However, when basal metabolic rate (actual in the case of the rat, average literature values in the case of the cow) is computed per unit of water or physiologic mass, i t is seen to be relatively constant within species but declines as the mature size of the species declines. (Tables 4 & 5)« The average values per unit of water are 0.167 and 0.0335 respectively for the rat and the cow. The apparent tendency - 43 -for the values to increase with decreasing weight for very young cows may be a reflection of the fact that the basal metabolic rate values were not collected from the same animals on which body composition was determined. A further tentative conclusion, therefore, is that the range of physio-logic age for a l l mammalian species is the same but that metabolic activity of the physiologically active mass uniformly declines with increasing size. An explanation for the differences in the metabolic level of active tissues of different animal species is needed. The following discussion, though largely speculative as yet, allows one to make conclusions that have some basis in fact. Kleiber (26) has stated that the apparent validity of the surface law is due to the fact that the total potential heat transfer of an animal and the total transport capacity of its circulatory system are both proportional to the 2/3 power of body weight. The primary factor limiting heat production would appear to be the former because no matter what the potential capacity for nutrient transport, the maximum rate of heat production is governed by the maximum rate of heat transfer which i s definitely a surface phenomenon. Thus, though any correlation between surface area and basal metabolism for animals of different size within a species is probably spurious, the same correlation between different sized animals of different species is not. If there is - 44 -survival value in homeothermy, "then the surface law may be understood as the result of natural selection" ( 2 6 ) and the potential metabolic level of the tissues of any animal will be genetically fixed. In homeotherms the upper and lower limits of heat production are imposed by the necessity for maintaining a constant body temperature. Therefore, the smaller the animal and the greater the extent of i t s surface relative to i t s mass, the higher will be i t s relative heat production. Inasmuch as growth is an energetic process, those animals possessing a high inherent metabolic rate would also be expected to possess a high potential growth rate. This is generally known to be the case. For example, the mouse may reach relatively mature size in three months 'whereas i t takes the cow some three years. It is a corollary of the above discussion that high metabolic rate and hence high growth rate always accompany small mature size and vice versa. It is a second corollary that the slower the growth rate and the lower the metabolic rate the longer the time taken to reach mature size. A third corollary is that a l l animals must, very roughly, attain the same percentage of their birth weight at maturity. These points may be demonstrated in Figure i where i t is seen that, for the mouse, physiologic aging proceeds much more rapidly and mature size is reached much soon than for the cow. - 45 -In other words, the cow experiences the same passage of physiologic events as the mouse but these events are occuring much more slowly in the former than in the latter. Factual support for this contention i s found in a comparison of the actual rates of physiologic aging of these animals. If physiologic aging, in the new sense developed above, is faster in smaller animals than in larger, then the rate of change of protein with respect to water should decrease as size of species increases. That i s , the slope of the log protein-log body water relationship should decrease with increasing size. This is seen to be the case for the mouse, rat and cow. (Table 6 ) . It would be of great practical interest to know exactly what genetically controlled physiologic mechanisms govern the metabolic level of the tissues of different species. Although l i t t l e is actually known about this subject KLeiber ( 2 6 ) presents evidence that tissues from any animal species respire at a similar rate in vivo and in vitro. He denies the presence of an inherent metabolic level in the tissues themselves but states that the animal as a whole responds to changes in somatic conditions and that reactions to such conditions are transmitted to the tissues by centralized metabolic regulators. Examples of such regulators may be electrical potentials, ion concentrations, structural relations, and the concentrations of oxygen, metabolites and enzymes. - 46 -TABLE 6. SLOPES OF THE LOG PROTEIN- LOG WATER RELATIONSHIP FOR  THE MOUSE. RAT AND COW. Animal Slope Mouse 1.25 Rat 1.19 Cow 1.14 - 47 -3 . The Feeding Experiment. Having established the average growth changes of body composition and metabolic rate for the mouse, i t was considered desirable to investigate the nature of the inter-relationships between these two variables on the one hand and growth rate and food efficiency on the other. Some progress towards discovering why differences exist between animals in theirrate and efficiency of gain was hoped to accrue from such an investigation. A presentation and discussion of the results of the present feeding t r i a l are given here. Tables 7 and 8 present the results of the measurement of growth rate, growth efficiency, body composition and metabolic rate for nine animals each on the high and low planes of nutrition. Table 9 presents similar results for the 15 gram control mice. These results will be discussed under four separate headings as follows: (a) Growth rate. As might be expected, the low plane animals grew at a considerably slower rate than their high plane pairs. The average time required to reach 25 grams from 15 grams is 30.9 days for the former and 22.6 days for the latter. These times represent average growth rates of 0.33 and O.46 grams per day respectively. The difference is highly significant (P<0.01). When, however, the i n i t i a l stationary growth period of the low plane group is excluded from consideration, the low plane animals are seen to reach 25 grams in less time than the high plane animals. Under these conditions - 48 -TABLE 7. ANALYTICAL DATA FOR THE HIGH PLANE MICE. Body Weight Age % of Body Weight BMR . Per Day Protein Water Fflt Protein Ash Water Cms. Days KCALS 20.47 59 64.1 12.4 18.3 4.0 4.72 0.286 19.03 - 68.6 5.4 20.9 3.7 5.09 0.305 20.42 45 69.4 4.0 21.7 4.1 5.25 0.312 19.93 58 71.5 3.6 20.0 3.6 4.96 0.279 20.30 45 69.6 4.8 20.8 4.0 4.94 0.302 19.71 46 67.8 6.9 19.8 3.5 5.09 0.291 19.88 56 71.2 4.0 18.9 3.7 4.51 0.266 19.97 37 69-9 5.6 19-7 3.6 4.44 0.282 22.26 55 62.7 13.5 18.8 3.5 4.31 0.299. Mean 20.22 48.9 68.3 6.7 19.9 3.7 4.81 0.291 Time on Growth Active Feed % of FFBM Test Rate Tissue Consumed Protein Water Ash Days Gms/Day Gms. Gms. 29 0.35 62.7 120.1 20.4 73.3 4.5 21 0.48 59.1 56.4 22.1 72.5 3.9 22 0.45 62.8 73.1 22.6 72.3 4.3 21 0.48 68.9 71.6 20.7 74.2 3.7 23 0.43 64.0 87.8 21.8 73.2 4.2 17 0.59 63.1 73.8 21.2 72.9 3.7 26 0.38 71.8 108.1 19.7 74.2 3.8 15 0.67 66.8 56.3 20.9 74.0 3.8 29 0.35 64.4 120.4 21.7 72.6 4.1 L 22.6 0.46 64.8 85.3 21.2 73.2 4.0 - 49 -TABLE 8. ANALYTICAL DATA FOR LOW PLANE MICE  Body Age" % of Body Weight BMR Protein Weight Water . Fat Protein Ash Per Day Water Grams Days ; KCALS  21.13 61 61.9 15.9 17-4 3.3 4.69 0.284 21.91 - 66.1 9.5 19.2 3.5 4.65 0.291 20.61 58 66.8 7.4 18.8 3.9 4.69 0.282 20.21 57 68.3 8.8 18.1 3.6 4.95 0.266 20.76 58 67.4 8.6 19.2 3.6 4-74 0.284 21.30 54 65.8 11.9 17.2 3.2 5.35 0 .250 20 .25 60 67.2 8.6 19.2 3.8 4.82 0.286 21.58 48 68.3 9.7 17-6 3.3 5.39 0.258 21.94 57 59.1 20.6 16.0 2.8 4.56 0.271 Mean 21.08 56.6 65.7 11.2 18.1 3.4 4-87 0.275 Feed Consumed % of FFBM Time on Growth Active Growing Test Rate Tissue Total Phase Protein Water Ash  Days Gms/Days Gms Gms Gms 31 0.32 62.6 107.5 62.5 20.3 73-3 3.9 32 0.31 68.1 99.2 83.5 21.3 73.0 3.9 35 0 .29 67-7 123.3 96.5 20.3 72.1 4.2 30 0.33 69.3 95.1 51.5 • 19.8 74-8 4.0 36 0.28 66.8 132.1 80.8 21.0 73.9 4.0 25 0.40 75.1 89.8 51.5 19-5 74.7 3.7 30 0.33 64.7 99.7 56.0 21.1 73.7 4.0 28 0.36 76.3 78.2 52.7 19.6 75.5 3.6 31 0 .32 64.3 103.6 79.7 20.1 74.3 3.5 Mean30.9 0.33 68.3 103.2 68.3 20.3 73.9 3.9 - 50 -TABLE 9. ANALYTICAL DATA FOR 15 GRAM CONTROL MICE. Body Weight % of Body Weight Protein Water Fat Protein Ash Water Gms. 12.70 73.5 3.3 18.1 3.3 0.247 13.94 74.3 4.7 16.3 3.2 0.220 14.13 71.3 5.2 18.0 3.3 0.252 13.81 70.9 6.7 17.1 3.4 0.242 12.85 72.2 5.9 17.2 3.1 0.238 12.34 68.7 8.4 17.2 3.5 0.250 13.82 72.9 6.6 17.1 3.0 0.235 13.89 72.5 6.3 16.6 3.0 0.229 15.18 72.0 6.5 16.8 2.9 0.235 l?-63 72.0 6.0 17.2 3.2 - 51 -the low plane group grew at 0.59 grams per day although the difference between this and the growth rate of the high plane group was not significant. This result demonstrates the remarkable ability of the mouse to recover from periods of inanition. Similar findings have also been reported by Thompson (60) for the mouse by Williams (64) for the rat and by Winchester and Howe (65) for the steer. (b) Body Composition. Rather striking and unexpected differences occurred in the average composition of the two groups of mice. The low plane animals had very similar ash contents but less water and less protein than the high plane animals. The greatest but least expected difference occurred in the fat content of the animals. The low plane group had nearly twice as much fat (11.2$) as the high plane group (6.7%)* This difference is statistically significant. On the basis of the rapid growth recovery of the low plane animals after realimentation, i t might be expected that they would make up for lost time, as i t were, and would gain largely protein (i.e. muscle tissue). The fact that this does not occur, however, indicates that their recovery does not represent a simple time delay in growth rate. There must have been a fundamental shift in the overall growth process in order that relatively passive storage tissue could preferentially accumulate instead of the metabolically more active - 52 -proteinaceous tissue. Pickens et al (47) have demonstrated an essentially similar finding for rats after realimentation following nutritional inadequacy. Considering the difference in final carcass composition between the high and low plane animals on a fat- free basis illustrates the great physiological difference between these two groups of animals. The low plane animals had more water but less protein as a percentage of the fat-free body than did the high plane animals. This variation is reflected in their significantly lower (P<0.05) physio-logical age (0.291 for the high and 0.275 for the low plane animals). Thus, the low plane animals are physiologically much younger, but chronologically much older, than the high plane animals even though they both weigh the same. The same result has been reported elsewhere for the rat although the physiological significance of this finding was not recognized (47). 3h addition, recalculation of some of the data of Wellington et al (62) demonstrates the same situation for the-"steer:.-3. Reference to the literature provides information which may explain the above result i f the findings for one animal species may be extrapolated to another. McMeekan has demonstrated for the pig (34) and Hammond (19) and Palsson and Verges (46) for the sheep that the main body constituents (fat, muscle and bone) exhibit differences in their relative - 53 -growth rates. Furthermore, the different tissues attain their maximum rates of growth (expressed as gain in tissue weight per unit of time) at different times. In other words, the tissues mature at different rates and the order of maturation parallels their growth rates. The order of increasing maturation rate of the three tissues is fat, muscle and bone. From this i t follows that there are definite age changes in the proportions of the tissues. The proportions of the various tissues, moreover, may be changed at will by altering the plane of nutrition at different stages during growth. The reason for this is two fold. First, tissues which are at the stage of their highest growth intensity will be most affected by an energy deficit. Second, because nutrient priority is higher at any age for the tissue having the higher growth rate, the earliest maturing parts are least, and the late maturing parts most affected by undernutrition (45). Although tissues have a considerable capacity to recover from a nutrient deficit, i f such a deficit is too severe and persists for too long, recovery may never occur (IS). The previous demonstration of heterogenic growth for the four body constituents fat, water, protein and ash render the results of the feeding experiment subject to an interpretation based on the above discussion. During the period of restricted feeding, the protein and fat fractions - 54 -suffered the greatest retardation and the ash fraction the least. Subsequent return to an optimum plane of nutrition found the protein fraction past its period of most rapid growth. Because the protein fraction could then not increase it s size at a rate sufficient to utilize a l l the provided nutrients, the excess could not but be stored as fat. The imposed nutritional setbakc also caused a failure of complete recovery of the physiological age of the low plane animals, when transferred to the high plane. It is conceivable that the relative proportions of the body constituents of the low plane animals may have reached the mature state had they been maintained on a high plane for a prolonged period. In terms of the meat producing animals, however, the animals would have been slaughtered long before this point had been reached. (c) Efficiency of gain. Variations in the relative rates of gain of the tissues imposed by variations in the level of nutrient intake should be accompanied by changes in the gross feed efficiency and the gross caloric efficiency. This i s true for the following reasons. First, the efficiency of growth will depend on the relative distribu-tion of available nutrients between maintainance and other waste energy costs on the one hand and the energy cost of the gain itself on the other. Thus, the greater the fraction of the feed going for gain, the greater the efficiency. Dickerson (15) has produced evidence in work with hereditary obese mice - 55 -and normal mice that differences in feed efficiency between them (favouring the former) are due to an increase in food intake and a reduction in energy expended, especially for activity. Because the animals stored only 2-11% of their total energy intake, small maintenance cost reductions caused large increases in food storage. Second, the energy content of a unit of gain differs greatly depending on whether i t i s largely fat or protein because the energy content of a unit of fat gain is five to six times as great as that of a unit of proteinaceous gain. The reason for this is not only because fat contains more gross energy per unit weight than protein but also because a unit of protein gain is associated with two to three units of water whereas a unit of fat is associated with only about one tenth of a unit of water. In this regard, Winters et al (66) have computed on the basis of their work with swine, that, excluding maintenance, less nutrients are required to produce a pound of lean meat than a pound of fat. Third, the maintenance cost of protein tissue is considerable whereas that of fat tissue is very low. Thus, a greater and greater proportion of the body of an animal laying on fat will become metabolically inert decreasing thereby the total maintenance cost and allowing a greater fraction of the total feed intake to be used for gain. Growth efficiency i s therefore a complex of many inter-related factors. A l l are basically dependent on the relative - 56 -growth rates of the various body constituents which, in turn, are controlled by both genetic and environmental influences. The feed consumption and feed efficiency values for the animals are listed in table 10. The low plane animals used more feed per gram of gain than the high plane animals when both are considered over the complete experimental period. This result is not unexpected, however, because of the large amount of feed required for maintainance over the static phase of the experimental period. When the efficiency for the low plane group is calculated over the non-static phase, the amount of feed required to reach 25 grams is less than for the high plane group. Thompson (60) reported a similar result many years ago for the albino mouse. She found, in addition, that some animals subjected to a period of growth suppression actually grew more efficiently even when the period of restricted food intake was included in the calculations. The caloric content of the gain of the two groups of mice may be estimated from the change in average composition between they and the group of l i t t e r mate mice killed at 15 grams (Table 10). Despite the long period of weight growth stasis, the gross energetic efficiency of the low plane group exceeded that of the high. Again considering only the growing phase of the low plane animals the difference is even wider. The apparently anomalous situation where feed efficiency is lower but energetic efficiency is higher for the low plane animals over the complete experimental period may be explained as follows. On a weight - 57 -TABLE 10. AVERAGE COMPOSITION OF GAIN AND EFFICIENCY  OF GAIN FOR THE HIGH AND LOW PLANE MICE. High Plane Low Plane  Total Growing Phase Gms. Gain Protein 1.68 1.48 Water 4.00 3.96 Fat 0.53 1.54 Ash 0^ 3.1 0.28 Energy of Gain Protein 6.72 KCALS. 5.92 Fat 4«98 14.48 TOTAL 11.70 20.40 Efficiency Gms. Gain/Gm. Feed 0.117 0.097 0.146 KCALS Gain/ Gm. Feed 0.137 0.198 0.300 - 58 -basis proteinaceous tissue is more efficiently laid down than fatty tissue because of its large water content. The energy content of fatty tissue i s , however, five to six times greater than that of proteinaceous tissue and, per unit weight gain, represents more stored energy. An explanation as to why the low plane group should have exhibited a greater feed efficiency and a greater energetic efficiency over their growing period than the high plane group may be found in the theoretical discussions of body composition and efficiency. Because of the differential nature of the growth of body constituents, there is a consequent differential effect of super- or sub-maintenance diets on the proportions of these constituents. It may be concluded that, at the point where the low plane animals were returned to optimum nutritional conditions, the rate of growth of the proteinaceous fraction of their bodies had severely declined. Therefore, less of the total caloric intake was used for the growth of this fraction in the low than in the high plane group and the excess was stored as fat. More-over, as the amount of fat relative to the total weight increased, the maintenance cost per unit of weight decreased permitting more of the total calories to be used for fat gain. Despite the greater energy cost of fat gain, therefore, the low plane animals gained more fat but at a lower overall feed cost. - 59 -(c) Metabolic rate. On the basis of the previous results whereby basal metabolic rate was considered to be a function of the total amount of metabolizing tissue as well as a function of its composition, i t is of interest to see what effect nutritionally imposed changes in body composition may have on basal metabolic rate. The average metabolic rates of the two groups are the same. (Tables ^  and 8). The interpreta-tion of this result is simple in the light of the second part of the discussion. The high plane group of mice contained a greater percentage of metabolizing tissue than the low plane group but its physiological age was greater. These two effects, one tending to increase and the other to decrease metabolic rate, cancel each other. The net effect is an equality of total heat production by the two groups. As this result implies, the total average amount of physiologically active tissue is the same for each group (Tables 7 and 8). SUMMARY AMD CONCLUSIONS -60-SUMMARY AND CONCLUSIONS Age changes in the body composition and metabolic rate of male BBC Swiss Albino, mice ranging in age from birth to 118 days and in weight from 1*34 to 27*42 grams have been investigated. In addition, the growth of mice on a high plane of nutrition has been compared with that of mice fed the same diet af 7G# of the high plane intake for a portion of their post-weaning growth period. The average effects of differences in the plane, of nutrition on body composition and metabolic rate as well as on growth rate and feed efficiency have studied* The high, andthe low plane groups of mice were composed of nine l i t t e r mate pairs, one from each pair being in each group. A new apparatus herein described was designed to measure,the basal meatbolio rate of the mice* Cto the average, body composition changes in a regular manner with age. The ratio of protein to ash remains nearly constant, the hydration of the protein-ash fraction uniformly decreases and the amount of associated fat varies depending on the nutritive state. The ratio of protein to water increases with age but at a decreasing rate. It is suggested that this ratio i s a. valid index of physiological age. Metabolic rate does not bear a constant relationship to body mass, age, surface area, fat-free body mass or protein mass. It i s however, almost, directly proportional to the total water and to the weight of the fat-free body divided by the protein to water ratio. The latter expression is considered to be physiol-ogically meaningful and is defined as the weight of themetabol--61-i c a l l y active fraction of the body corrected to a common physiologic age* It i s concluded that this expression i s a suitable index of the size of the physiologically active mass. Total body water represents a useful and easily measured index of metabolically active mass and, hence, of basal metabolic rate. Alterations i n the plane of nutrition during growth were seen to have a significant affect on the proportions of the body constituents, on growth rate and feed efficiency. Nutrient restriction retards physiological aging as measured by the protein to water ratio. It also slows the growth rate but may not affect the efficiency of growth. These results are interpreted as resulting directly from the influence of nutritive l e v e l on the d i f f e r e n t i a l growth rates of the various body constituents. Diffenences i n the genetic growth rates and i n the proportions of the various parts of animals may be masked or accentuated i f , at any time from the foetal stage onward, nutrientrestriction i s Imposed. As Montague has pointed out(38), an animal i s the product of both i t s environmentsand i t s heredity and environment and heredity are inseparable parts of the same coordinate system. Any comparison of animals on a genetic basis must, therefore, be tempered with a consideration of the nutritional history of the animals prior to the period during which the comparison i s to be made. —62— Recognition of the above findings i s of great importance when comparing two or more animals on the basis of any single criterion such as weight or age. It i s recommended here, that where body compostion data are available, any such comparison must include a consideration of the magnitude of both the physiological age and the size of the fat-free body* The unit of comparison would then be the dividend of these two factors or the size of the physiologically active mass. APPENDIX I A SIMPLE RESPIROMETER FOR SMALL ANIMALS A great number of techniques have been described for the indirect measurement of basal metabolic rate. They range from manually operated, short term devices to those which operate and record the results automatically for periods up to 24 hours and more (14, 22 , 29, 30 , 31, 33, 41, 48, 55, 63). The closed circuit devices which directly measure volume or pressure changes in a closed system appear to be the most convenient for the small animals. Brody (9) and Swift and French (58) have reivewed the various techniques. lh this study i t became desirable to measure the metabolic rate of large numbers of individual animals, and the need for a simple, rapid and accurate apparatus soon became apparent. Since the familiar techniques did not appear to f u l f i l l a l l of the above requirements, a respirometer of an original design was constructed. A description of the design and operation of the respirometer together with some experi-mental results obtained with i t are presented here. 1. Description of the Apparatus The design adopted is a modified version of a device described elsewhere for studying the kinetics of certain oxida-tion reactions (67)• It is similar in principle to the familiar Warburg respirometer with the exception that measurements are made at constant pressure rather than at constant volume. A diagrammatic representation of the apparatus is given in Figure 18. -65-AMMAL V E S S E L N-BLANK V E 3 S E L K GLASS BOTTOM _WATER BATH - 6 6 -The apparatus consists of four separate parts; a U-tube manometer (B); a modified glass burette (A) and two identical glass vessels (J and K). The manometer i s constructed of 5 mm inside diameter glass tubing and is 3 0 cms. long. Each arm is equipped with a glass stopcock (C and D) and a short side-arm (G and H). (A) is a 5 0 cc burette modified to the extent that an outlet tube (E) has been added below the lowest scale reading and the top has been pinched off to form a sidearm just above the 2 0 cc mark (Q). The vessels (J and K) are cylindrical and are constructed of pyrex glass of 8 cms. inside diameter and 1 6 cms. deep. The cage containing the animal rests on three glass projections on the inside of the vessels 7 cms. from the bottom. The animal cage (M) is a cylinder of £ inch mesh hard-ware cloth, 4 cms. deep and 6 cms. in diameter equipped with a metal dropping pan. The vessels are fitted with lids having greased ground glass seals. Each l i d has two outlet tubes attached. In operation, the parts are assembled as shown in Figure 18. The animal is placed in a wire cage which, in turn, is placed within one of the vessels (j). Both the •animal1 and the •blank* vessel are immersed in a constant temperature water bath up to the greased seal and then attached to the manometer as shown. Stopcocks C and D and outlets L and M are open. Fifteen or twenty minutes are allowed for temperature equilibra-tion, during which time the bottom of the burette (at F) is - 67 -connected to a resevoir of water with sufficient head to f i l l the burette. Water is allowed to enter to the lowest scale reading. . The pinch clamp at E is closed. When the temperature has equilibrated, an aliquot of a potassium hydroxide solution is pipetted into each vessel. The vessels are then flushed with oxygen through C and D, after which a l l vents in the system are closed to the outside. Now, as the expired carbon dioxide i s absorbed by the potasium hydroxide and the oxygen is consumed by the animal, there is a net pressure drop in the system. This causes the water levels in the manometer to be displaced from the rest position. Periodic restoration of pressure in the system to atmospheric conditions by allowing water from the resevoir to enter at F permits a direct reading from the burette of the volume of O2 used by the animal in the given time interval. Readings may be taken as often as desired. When the effective range of the burette has been exhausted, the water in the burette may be restored to its original level by quickly opening stop-cocks C and D, allowing the water to escape through the exit at E and again closing C and D. The whole procedure is then repeated. In order to assure complete carbon dioxide absorp-tion, the potassium hydroxide solution is constantly agitated. This is accomplished by placing a magnetic stirring bar in the potassium hydroxide solution in the animal chamber. Beneath the water is placed a magnetic stirring motor which induces - 68 -rotation of the magnetic bar through the glass bottom of the water bath. The blank vessel was included in the design of this apparatus to prevent fluctuations of temperature and barometric pressure from influencing the results. Thus, i t serves the same function as the tthermobarometert familiar in the use of the Warburg apparatus. The volume of the Carbon dioxide produced by the animal is determined by a double titration of the potassium hydroxide solution in the animal vessel. Standard hydrocBoric acid is run in to the phenolphthalein end point and then to the methyl red end point. Twice the difference between these two titrations multiplied by the normality of the acid gives the number of milliequivalents of produced at standard temperature and pressure. From this value and the total oxygen consumed, the respiratory quotient may be calculated. The heat produced may then be estimated by reference to standard tables showing the caloric equivalent per l i t r e of oxygen or carbon dioxide. It is obvious that the respiratory quotient may be calculated for the total test period only. Therefore, in order to compute heat production for any interval of time within the total period, the overall respiratory quotient must be used. Over short time intervals this introduces no errors of signif-icance because variations in the state of activity of the animal cannot be expected to effect the respiratory quotient. - 69 -The major advantage of this apparatus is its simplicity of operation. It takes but a few minutes to place the animal within the chamber and then to assemble the parts. Furthermore, by the simple expedient of subtracting the burette reading at any instant from the previous reading, the volume of oxygen used in any given time interval is obtained directly. It is thus possible, by observing the animal, to eliminate from consideration the oxygen consumption during periods of activity. The only required observations are the burette readings, the hydrochloric acid titre for the potassium hydroxide solution, the water bath temperature and the barometric pressure. By varying the sizes of the burette and vessels and the concentration of the potassium hydroxide solution, animals of various sizes may be accommodated in the apparatus. It may also be necessary to vary either the size of the manometer or the density of the manometer fluid in order that the fluid levels in the manometer be not displaced to an extreme degree and thus sp i l l over into the burette or the animal vessel. Finally, by having a battery of respirometer units, i t is possible to measure the gas exchange of several animals simultaneously. 2. Experimental Procedure Using the above described apparatus, the oxygen con-sumption and heat production have been determined for normal Swiss Albino mice at different environmental temperatures from 22 to 35 C. - 70 -and at different states of activity at constant temperature. Three activity phases have been defined for the purpose of this discussion. 'Awake* (A) designates that state where physical activity is readily apparent; * quiet* (Q) designates that state where the animal exhibits l i t t l e movement but is not asleep; and * sleeping* (S) designates that state where the animal shows no movement and where its eyes are closed. Since a l l readings on the burette were taken at six minute intervals, the state of the animal in doubtful cases could be assigned according to the value of the reading. In a l l cases, except where the activity increment was to be determined, metabolic rate was calculated for the sleeping state only. Thus the values of the metabolic rate so calculated are the closest possible approach to an estimate of true basal metabolism. Furthermore, since this appears to be the most reproducible state, the metabolic rates of animals in this state should be directly comparable. In order to investigate the relationship between the ambient temperature of the mouse in the respirometer vessel and the temperature of the water bath, the temperature difference between the air surrounding the animal and the water in the bath was measured with a copper-eonstantine therm-ocouple for periods of one and a half hours. After temperature equilibration, (20 minutes) fluctuations of ambient temperature from bath temperature never exceeded three tenths of a centigrade degree (always in the positive sense). This difference falls - 71 -within the range of accuracy of the thermostatic temperature control of the water bath. (See Figure 19). 3. Heat Production of Swiss Albino Mice at Various Environmental Temperatures The basal metabolic rate was determined at nine different temperatures between 22.7 C. and 35 C. The basal metabolic rate per unit of body weight declined from 0.270 Kcals/day at 22.7 C. to 0.168 Kcals/day at 30 C. and thereafter rose to a high of 0.268 at 35 C. at which temperature the animal exhibited signs of acute heat stress. The results are shown in Table H and Figure 20. It is apparent that the thermo-neutral temperature for these animals is about 30 C. The very narrow range (about 29 to 31 C.) of minimum metabolic rate is very striking. These values corroborate results reported elsewhere for the same species (21). It must be remembered, however, that the thermo-neutral point is a function of the previous environmental temperature and, as a result, is subject to some fluctuations (52). 4. Heat Production of Swiss Albino Mice at Different States of Activity Table 12 shows the results of the determination of oxygen consumption for different mice of various ages. The readings during the quiet and active states vary considerably, whereas, when the mouse was asleep, the readings were relatively more constant. The variation coefficients of the means for each activity state demonstrate this point. The basal metabolism values calculated from this data are very close to values reported elsewhere for the white mouse (14,20). Furthermore, Figure 19. Changes of water bath temperature with time 'igure 20. Changes of metabolic rate with temperature„ - 72 -TABLE 11 The Relationship Between Environmental Temperature and Heat  Production for Swiss Albino Mice  Temperature R. Q. Heat Production Kcals/day Kcals/gm B.W./day 22.7 C. 0.71 6.92 0.272 25.0 0.81 5.62 0.219 27.0 0.78 5.17 0.212 29.0 0.85 4.76 0.187 30.0 0.80 3.29 0.168 31.0 0.73 4.18 0.193 32.2 0.84 5.03 0.201 32.5 0.78 4.67 0.184 35.0 0.77 5.63 0.268 - 73 -TABLE 12 Oxygen consumption and basal metabolism of four  representative male mice. Animal Number 1 2 3 4 Body Weight 7.4 gms. 15.1 21.3 27 .4 R.Q. 0.78 0.79 0.73 0.81 Awake 7.37 0 .70 9.29 1.28 10.96 0 .52 ( 9 . 5 ) * (13.8) (4 .8) Means Quiet 3.37 0 .37 5.19 0.52 6.34 0 .51 7.08 0 .21 (11 .0) (10.0) (8 .0 ) (3 .0 ) Sleeping 2.53 0 .22 4 .2 0 .21 5.28 0 .34 5.76 0 .22 (8 .7 ) (5 .0 ) (6 .4 ) (3 .8 ) Basal Heat Production Kcals per day 2.64 4.35 5.35 5.93 According to the equation BM 70.5 W°*73 1.96 3.36 4.25 5.10 Kcals per gm. body weight per day 0.357 0.288 0.251 0.216 Kcals per sq. meter body surface per day 773.7 792.2 774.6 725.8 * coefficients of variation - 74 -as the weight of the animal increases to the mature weight, the heat production approaches that value calculated from the expression relating basal metabolic rate to body weight for mature animals of a l l species (9 ) *« The reproducibility of the replicate readings and the closeness with which these results parallel values reported elsewhere tend to confirm the validity of the apparatus. * BMR = 70.5 W°* BIBLIOGRAPHY BIBLIOGRAPHY 1 . 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