f 7’ , , if! : : 7v — 7 7 ; ’ é ,, __ i 7* ,_ 7’17“ 77, . PROGRESSWE “EFFECTS OF PHYSECAL AC’i‘WiTY’ ANS .tMX?ETY-?RGUUC!NG STRESS on THE EASAL METABOUC HA'i'E OF YOUNG mun“ mu 4L0 ms Thesis for the Degree of M A“ memm STA‘!‘E UN‘E‘JERSH'Y PAUL D. ROMESW 1956 'Ix@nmyggmnmnflnflnnmnjggnm , ’ gm R; ABSTRACT PROGRESSIVE EFFECTS OF PHYSICAL ACTIVITY AND ANXIETY-PRODUCING STRESS ON THE BASAL METABOLIC RATE OF YOUNG ADULT MALE ALBINO RATS by Paul D. Robinson The purpose of this study was to investigate the progressive effects of physical activity and anxiety- producing stress on the basal metabolic rate of young male albino rats. One hundred and sixty animals were randomly assigned to ten equal treatment groups, two of which were control groups. Five groups lived in sedentary cages and five in voluntary activity cages. Treatments comprised forced activity in the form of swimming for 30 minutes with two per cent of body weight attached to the tail, or anxiety- producing stress in the forms of an electrical shock received every 15 seconds for 30 minutes. Four groups, two voluntary and two sedentary, received both treatments but in a different sequence. Treatments were administered five days a week for ten weeks. Eight animals from each group were chosen at random, from which weekly serial blood samples of 3cc were taken throughout the study. The B. M. R. of each rat was tested during a two—day rest period when treatments were not administered. Oxygen uptake in mls per gram of body weight per 2“ hours was the B. M. R. measurement and it was taken in a closed circuit apparatus. Data were examined by analysis of variance with alpha set at the 0.05 level and little significance was found. Longitudinal effects were demonstrated graphically, It was shown that B. M. R. decreases with increasing age and that this decrease may be affected by the treatments. PROGRESSIVE EFFECTS OF PHYSICAL ACTIVITY AND ANXIETY—PRODUCING STRESS ON THE BASAL METABOLIC RATE OF YOUNG ADULT MALE ALBINO RATS By Paul D. Robinson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Health, Physical Education and Recreation 1966 To Jessica ACKNOWLEDGMENTS The author wishes to thank Dr. William Heusner for his advice and guidance; also Ken Coutts, David Anderson, Cynthia Gillespie and Carol Kowalewski for their cheerful assistance and encouragement throughout this study. PDR iii TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES Chapter I. INTRODUCTION Statement of Problem Need for the Study Definition of Terms . . Limitation of the Study II. REVIEW OF LITERATURE Factors Affecting the Basal Metabolic Rate Basal Metabolic Rate Increase with Training Basal Metabolic Rate Decrease with Training The Relationship Between Training and Basal Metabolic Rate . . . . III. METHOD Testing Procedure Analysis of Data IV. RESULTS AND PRESENTATION OF DATA Introduction Treatment and Presentation of Data Vertical Analysis . . . . . . . . Discussion . . . . . . . . . . . . Longitudinal Analysis Discussion . . . iv Page iii vi vii viii NOON ON rtww H HFJ t4 H U] U) [UH I—‘CD 22 22 23 27 28 28 3A Page V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS . . . 35 Summary . . . . . . . . . . . . . . . . . . . 35 Conclusions . . . . . . . . . . . . . . . . . 36 Recommendations . . . . . . . . . . . . . . . 37 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 38 APPENDICES . . . . . . . . . . . . . . . . . . . . . . A3 LIST OF TABLES Table 1. Mortality Rate of Animals During Experimental Period . . 2. Equal Three-Way Analysis of Variance Test on Cage, Blood and Treatment Effects 3. One-Way Analysis of Variance Tests on Cage, Blood and Two Treatment Effects . vi Page 2” 25 26 LIST OF FIGURES Figure Page 1. Apparatus for Measuring Oxygen Consumption in Small Animals by Closed Circuit Method; Single Unit . . . . . . . . . . . . . 19 2. Sedentary Group . . . . . . . . . . . . . . .3. 29 Voluntary Group . . . . . . . . . . . . . . . . 29 Sedentary Forced Group . . . . . . . . . . . . . 3O 5. Sedentary Anxiety Group . . . . . . . . . . . . 3O 6. Voluntary Forced Group . . . . . . . . . . . . . 31 7. Voluntary Anxiety Group . . . . . . . . . . . . 3l 8. Sedentary Forced Anxiety Group . . . . . . . . . 32 9. Sedentary Anxiety Forced Group . . . . . . . . . 32 10. Voluntary Forced Anxiety Group . . . . . . . . . 33 ll. Voluntary Anxiety Forced Group . . . . . . . . . 33 vii LIST OF APPENDICES Appendix A. Analysis of Variance with Equal Cells-- All Categories B. One-Way Analysis of Cells--Categories C. One-Way Analysis of Cells--Categories D. One-Way Analysis of Cells--Categories E. One-Way Analysis of Cells--Categories Variance with Equal Sedentary v. Voluntary Variance with Equal Non-Blood v Blood Variance with Unequal Non-Forced v Forced Variance with Unequal Non—Anxiety v Anxiety F. Treatment Group Means . . . . viii Page AA 50 53 56 59 62 CHAPTER I INTRODUCTION The changes brought about by automation have signif- icantly reduced the necessity for man to do much physical activity in daily living. This decrease in daily physical activity and the change in type of anxiety—producing situations encountered by modern man have both been pro- posed as factors involved in the development of degenera- tive cardiovascular disease. To evaluate the relative roles of these factors, more complete information of a longitudinal nature is required. How these factors affect the basal metabolic rate (B. M. R.) over a period of time is a small but important piece of this required information. There are many factors which affect the basal meta- bolic rate. When measuring the B. M. R. of any animal it is vital that the animal be in the post-absorptive condition, not having eaten for at least twelve hours, for the diges- tion of food increases the B. M. R. (l, A, 10, Al). The age of the animal is also important; a very young animal has a higher B. M. R. than one which is more mature (A, 12, 13). One must take into account the animal's state of activity and the time of day measurements are to be taken, for though man is diurnal, rats are nocturnal (A, 8, 10, 12). Seasonal_variation also occurs (19, 23). Sex differences must be considered for during menstruation the female's B. M. R. is lower (19, 20, 23). Basal metabolism has been shown to be higher in men than in women and this is probably due to body composition (3, A). Controllinthhe environ- mental temperature while measuring is vital, for extreme variations_cause an increased B. M. R. (10). There is a definite relationship between emotional state and the B. M. R. in man (5, 7, 8, 22, 23). It has also been pointed out that there can be after effects from strenuous exercise which may last for more than 24 hours (A3, 44). Stress, whether from exercise or mental, stimulates the thyroid which releases thyroxine causing an increase in the meta- bolic rate (U7). How long this increase persists is not, as yet, known. Evidence supporting increased B. M. R. as a result of training is opposed by equivalent evidence showing a decreased B. M. R. Supporters of increased metabolism during training state that it may be due to the development of more muscle tissue. The supporters of decreased B. M. R. suggest that it is caused by functional processes becoming increasingly efficient. A lower B. M. R. may be exhibited in an athlete because he learns to relax at will. However, the majority of investigators believe that the B. M. R. is unaffected by training. In this case, the high degree of relaxation which is acquired by the trained animal may explain why it is possible for muscles to hypertrophy, without an increase in B. M. R. (40). Although the results of animal studies cannot be translated directly to human subjects, experiments conducted on animals do provide clues as to what may take place in man. Statement of Problem The purpose of this investigation was to study the progressive effects of physical activity and anxiety— producing stress on the B. M. R. of young adult male albino rats. In particular, the effects of sedentary versus voluntary or spontaneous exercise cage existence, forced exercise, and electrical shock were evaluated singly and in various combinations. Hypothesis: That the B. M. R. of young male albino rats is affected by physical activity and anxiety-producing stress. Need for the Study Very few studies relating the B. M. R. to physical activity or to anxiety have been longitudinal in nature. There is a need for a longitudinal investigation which will provide serial determination of the influence of physical activity and anxiety-producing stress, imposed singly and simultaneously, on the B. M. R. Definition of Terms Basal Metabolic Rate.—-This is the energy output of an individual under standardised resting conditions, i.e., complete bodily and psychical rest in a post-absorptive period and in an equable environmental temperature. Anxiety-Producing Stress or "Anxiety" Group.--Each animal in this group was placed in individual electrical- stress cages for 30 minutes five days per week. During the electrical-stress period each animal received a .4 sec. D. C. electrical shock of -A to -5 milliampers every 15 seconds. This intensity of stimulation was found to be disturbing but non-injurious (21). Forced Activity or "Forced" Group.--Each animal in this group was forced to swim for 30 minutes five days a week in an individual metal cylindrical tank. A weight equal to two per cent of the animal's body weight was attached to the base of his tail during the swimming period. Sedentary Group.--This refers to the housing of animals in standard individual cages 2Acm long by 18cm wide by 18cm tall. Spontaneous Exercise--Voluntary Group.--This refers to the housing of animals in spontaneous exercise cages. Limitation of the Study 1. It is impossible to accurately assess the resting state of the animal within the apparatus used for measuring the B. M. R. It varies from active movement to the sleeping condition. It was found that if an animal is observed he remains more restless. The animals were therefore left in complete silence under bright illumination between deter- minations. 2. The taking of blood samples from half the animals weekly might have affected the B. M. R. If this occurred it would reduce the effective number of animals in the ten treatment groups posed by half, forming 20 different treat— ment groups containing 8 animals each. 3. Murine pneumonia may have developed as a result of forced swimming. A. It was impossible to stress each animal to the same degree without separate stimulation cages, for each animal will provide a different resistance. CHAPTER II REVIEW OF LITERATURE Factors Affecting the Basal Metabolic‘Rate In every individual there are periodicities in the rate of metabolism during the day (l). The factors affecting the basal metabolic rate are many and varied and need to be taken into account when designing a study such as this. According to Benedict gt_al. (l) the injestion of a mixed diet produces an increase in metabolism. An increase in the B. M. R. is created by a carbohydrate- free diet which causes acidosis(b). Williams et_al. (Al) found that heat production in a dog was doubled during the first three hours after feeding and was maintained for seven hours after which it began to fall. The increase in heat production was prOportional to the amount of food injested. It was also proportional to the amount of protein injested except during the first two or three hours after feeding. "The prerequisite of all experiments is that the subject should not have eaten for twelve hours; he should be in the post-absorptive condition." (1, 5, 10) It has been shown that age affects the B. M. R. According to Benedict (A) younger men have a higher metabolic rate than older men. Davis (l2, 13), working with rats, found that the average oxygen consumption rate showed a continual decrease throughout life. The decrease was rapid during the first four months and more gradual thereafter. He found a close relationship between the heat production of rats and of men, both in its per-unit amount and in its rate of decrease with advancing age. "Sleep favours complete relaxation." Benedict (8) found that B. M. R. determinations were affected by varia- tions in the length and depth of sleep. The possibility of tension is obviously less and the deeper the sleep the lower the B. M. R., although the possibility of muscular movements cannot be ruled out entirely (A, 10). Davis (12) observed three states of quietness in measuring the B. M. R. of rats: (a) where the rat is wide awake; (b) where the rat seems to be sound asleep; (c) where the rat is in an intermediate state. Davis cautions that the state of quietness, age and sex should be carefully taken into account in metabolism work on rats. Diurnal variations in the B. M. R. have been found to occur in men and in rats. In man the lowest rate is at night, 14 per cent higher in the morning and 22 per cent higher in the afternoon (A). Rats are nocturnal animals and have been found to be quietest during the day, especially when they are brightly illuminated (10). Seasonal variations have been found in man. Gustafson and Benedict (19) making determinations on 20 women showed that the average oxygen consumption strongly suggested that metabolism tends to be at a lower level in the winter and rises to a higher level in the spring and summer. Hitchoek and Wardwell (23) working with four subjects also found a decrease in the rate during the winter and spring with a sharp rise in the summer maintained into the fall. The B. M. R. during menstruation in the same subjects was also recorded and found to be lower (19, 20, 23). "Under certain conditions animals will produce only the amount of heat they need for the maintenance of life processes, but when subjected to extreme variations in environmental temperature they produce additional heat." (10) There is a relationship between heat production and body composition, for Benedict (14) found that heat produc- tion was greater with increased amounts of active protoplasm tissue such as muscle. Benedict and Emmes (3) showed that metabolism was 5 per cent higher in men than that in women of similar height and body weight. Their explanation for this fact was the disproportion between body weight and body surface, and the large proportion of subcutaneous fat found in women and not in men, the latter possessing more muscle. Martin and Fuhrman (32) have shown that in the average dog, summated tissue respiration will acccount for 70 per cent of the resting metabolism. This was also found by Field §£_al. (15) working with rats. Work by Bertalonffy and Pirozynski (11) shows that the decrease of B. M. R. with increasing size is not due to cellular factors alone. The cause must be sought in the organism as a whole. According to Gephart and Du Bois (17) the average metabolism of fat and thin subjects is the same if determined in relation to surface area. There is a definite relationship between basal meta- bolic processes and emotional states in man. Henry (22) has listed the corresponding metabolic rates with emotional states as follows: 1. Elated, overactive states accompanied by accel- eration of B. M. R. 2. Depressed, underactive states accompanied by retardation of B. M. R. 3. Apprehensive, tense, agitated states cause an acceleration of B. M. R. but this may be counter- acted by a feeling of depression. A. Apathetic states cause low or retarded B. M. R. "It is probable that normal variation in the metabolic rate is due to different emotional states presented by the tested individual." Benedict (5, 8) found that "any emotional disturbance caused a marked increase in the metabolism which did not subside for several days." The mental state of Hitchcock's subjects (23) also affected the B. M. R. Yet individuals who appeared to be rejuvenated after a summer vacation had unaltered B. M. R.'s according to Benedict and Finn (7). 10 It has been pointed out by Wishart (A3, AA) that there is a possibility of after effects from strenuous exercise on the B. M. R. Very strenuous work may produce changes which result in a raised metabolism longer than 2A hours. He also points out that spontaneous changes can occur in the B. M. R. which are incorporated in a range of 15 per cent of average figures for the individual. Gephart and Du Bois (17) have obtained similar results. The effect of stress on basal metabolism has been investigated by Selye through the effect of stress on the thyroid gland (A7). "This organ can influence all the organs of the body through the release of thyroxine which intensely stimulates the metabolism of every tissue." Factors such as muscular activity, digestion, sleep and emotional disturbance influence basal metabolism thus its interpretation is not a simple procedure. B. M. R. Increase with Training Benedict and Smith (2) compared the metabolism of normal individuals with that of athletes and found the athletes had a much greater metabolic rate. "With a low basal metabolism there is seemingly a lowered physical fitness compared with the vitality when a higher metabolism is noted with the same individual." (6) "The greatly increased proportion of active protOplasmic tissue, muscle, in the trained individual is alone sufficient to account 11 for the increased metabolism." (2) Speck (37) also found that muscular individuals had a greater basal respiratory exchange than non-athletes. Monro (33) compared the B. M. R. of British physical training instructors with that of clerks and found a higher rate in the former. Leilich (28) compared college men in groups composed of sedentary individuals, physical educa- tion majors, varsity and highly trained athletes. The three latter groups all showed higher average B. M. R.'s than the sedentary group. It was also determined that with training the B. M. R. was increased. Lusk and Du Bois (29) stated that "the influence of sedentary life in reducing basal metabolism in the dog has a probable counterpart in lack of exercise and indoor confinement in man." The dog they studied had a higher B. M. R. when it returend to the laboratory after an active summer. "The B. M. R. of dogs on a high caloric diet is decreased by restrictions of physical activity and increased by exercise" according to Kunde and Nordlund (26). The increased metabolism during the period of training may be due to the development of muscle tissue, or more possibly to the difference in the metabolism of a muscle which is made physiologically fit by exercise from one reduced to a state of fatigue by inactivity. B. M. R. Decrease with Training Benedict, in a later study with Sherman (9) on basal metabolism in relation to old age and exercise during old age in rats has stated that in exercised animals there is a distinct trend to a lower B. M. R. and that exercise prevents the rise of basal metabolism shown in rats of advancing age. Morehouse (3A) found a small difference between the B. M. R. of athletes out of training compared with those in training. However the average B. M. R. of athletes was below the average normal standards. Schneider e£_al. (35) compared athletes and non-athletes, the results of which lend no support to the idea that taking daily exercise increases the B. M. R. On the whole they indicate that the B. M. R. is reduced during training. Schneider and Foster (36) studied 9 athletes and 3 non-athletes. Of the athletes in the training program, the B. M. R. was lowered in 7, unchanged in one and elevated in another. Of the non-athletes, two were raised and one lowered. The explanation of these results was that "if cells are stim- ulated to increased activity they will use more oxygen; but if the life processes become increasingly efficient they will use less oxygen." Lowered B. M. R. may also be exhibited in the athlete because he learns to relax at will (36). Jung gt_al. (2A) tested students and found lower B. M. R.'s tended to occur in those who gave high haemato- crite readings and lower lymphocyte percentages. These 13 students also tended to be heavier than the average. The lowered B. M. R. did not appear to be an indication of unfitness. The Relationship Between Training and B. M. R. According to Krehi et_al. (25) the basal metabolism of man was not significantly altered by track training three times weekly. Griffith et_al. (18) also noted that the B. M. R. was not affected by training followed by a ten-day canoe trip. With the daily repetition of the same amount of light work Gemmill et_al. (16) found no signif- icant change in B. M. R. values. Magnus and Falk (31) found that three muscular individuals had the same or slightly lower B. M. R.'s than untrained persons. Steinhaus (38) obtained data on trained dogs which indi- cated that the B. M. R. is not increased by a period of extensive physical activity. Later, with the assistance of Jenkins, Steinhaus (A0) confirmed his previous work. He proposed "that the high degree of relaxation which is acquired by the trained animal may explain why it is possible to have muscle hypertrophy without increase in B. M. R." Lee and Van Buskirk (27) working with rats found that the B. M. R. shows no close variation with the amount of spontaneous activity manifested. The evidence is also 1A against the idea that the amount of spontaneous activity manifested is dependent on the B. M. R. "The data presents no direct evidence that exercise increases basal metabolism by increasing the bulk of the musculature. Dogs and Man in training relax more readily when required to than those not in training." (39) "Most of the basal metabolic values are normally distributed." (1A) Evidence has been furnished which supports an increase, or a decrease, or no change in B. M. R. with training. The evidence which supports an increase in B. M. R. with training is counteracted by an equivalent quantity and quality of evidence supporting a decrease in B. M. R. with training. It is hoped that this study will shed further light upon the effect of exercise and anxiety-producing stress on the B. M. R. CHAPTER III METHOD The purpose of the study was to investigate the progressive effects of physical activity and anxiety- producing stress on the basal metabolic rate in young adult male albino rats. The study was designed with two housing treatments, two blood treatmetns and five activity and anxiety treatments. B. M. R. was measured weekly. The number of animals chosen for the experiment was based solely upon the accommodation and handling facilities of the laboratory. For statistical signif- icance, alpha was set at the 0.05 level. No attempt was made to control the probability of making a type II error. Two hundred young adult (77-day-old) male rats were housed in spontaneous-exercise cages for two weeks to permit adjustment to the laboratory and stabilization of volun- tary-activity patterns. Activity records were kept during the final three days of this period. To obtain a rela- tively homogeneous sample, those animals with activity records in the highest and lowest deciles were eliminated from the study. The remaining 160 animals were assigned randomly to the ten housing and treatment groups at 98 15 16 days of age. Half of each group was assigned at random for blood sampling. Sedentary animals were housed in standard individual sedentary cages 2Acm long by 18cm wide by l8cm tall. Voluntary animals were housed in individual spon— taneous-exercise cages. Each of these cages has a standard individual sedentary cage for living quarters plus a freely revolving activity wheel 130m in width and 350m in diameter which allows the animal to rest or exercise at will. An automatic counter records each revolution of the wheel. Forced-exercise animals were forced to swim for 30 minutes five days per week in an individual metal cylin- drical tank having a diameter of 28 cm and a height of 75cm (water depth 70cm). A weight equal to two per cent of the animal's body weight was attached to the base of his tail during the swimming period. Anxiety animals were placed in individual electrical- stress cages for 30 mintues on five days per week. Each electrical stress cage was constructed of k-inch clear plastic and had a stainless steel rod grid serving as a floor. These cages were 17cm long by 17cm wide and 16cm tall. During the electrical-stress period, each animal received a .A-second D. 0. electrical shock of .A to .5 milliampers every 15 seconds. This intensity of stimulation was chosen so as to be disturbing but non- injurious. 17 Initial samples of blood, approximately 3cc, were taken from every animal in the first five days. Thereafter weekly serial samples were taken only from the eight animals randomly selected for blood sampling from each group. Each rat was anesthetised, using ether vapour, and the blood sample was drawn from the orbital sinus using a one-millimeter bore, heparinized, capillary tubing. Weekly serial measurements of B. M. R. were made on all animals throughout the experimental period. During the study every animal was handled daily, all were fed a standard ground feed, ag_libatum, and had access to water at all times. The following ten treatment groups of animals were used in this investigation. 1. A sedentary control group received no Special treatment during the experimental period and was housed in standard individual sedentary cages for small animals. 2. A sedentary forced-exercise group was also confined in sedentary cages. 3. A sedentary anxiety group was housed in seden— tary cages and was placed in individual electrical-stress cages for 30 minutes five days per week. A. A sedentary forced-exercise plus anxiety group was housed in sedentary cages and received both the swimming and the electrical-stress treatments five days per week. 5. A sedentary anxiety and forced-exercise group was housed in sedentary cages. 18 6. A voluntary control group received no special treatment during the experimental period but was housed in individual spontaneous-exercise cages. 7. A voluntary forced-exercise group was housed in spontaneous-exercise cages and received the swimming treatment. 8. A voluntary anxiety group was also housed in spontaneous—exercise cages but received the electrical- stress treatment. 9. A voluntary forced-exercise,plus anxiety group was housed in spontaneous-exercise cages and received both the swimming and electrical-stress treatment. 10. A voluntary anxiety and forced-exercise group was housed in spontaneous-exercise cages. TestingiProcedure The B. M. R. was determined by measuring the oxygen consumption of the animal by closed-circuit method (30). The apparatus used for each animal is shown in Figure 1. These were set up in series so that 16 animals could be independently measured at the same time. Oxygen uptake was determined using the following formula: P 273 60 _ (V-Va) —7€5— x (273+t) x CD — Oxygen in ml/hr (30) l9 /— Thermometer :- Plostic Tubing Deeiccator—\ 3 _ way Stopcock-—\ / . Oxygen To Vacuum to replace Pump vacuum (-200 mm Hg) ~ Mercury SOdO Lil“. MOflOMCIOI' (ZOOgm = l44ml) Figure l. Apparatus for measuring oxygen consumption in small animals by closed circuit meihod. Single unit. 20 Where: V = net volume of unit in ml. Va = volume of animal (assume Igm = Iml). P = pressure difference in mm. Hg. t = temperature in desiccator. T = time-—1ength of determination in minutes. Each animal was weighed to the nearest gram and then placed in the desiccator. The lid was replaced, a firm seal being obtained with a vaseline lubricant. The stopcock was closed for a period of fifteen minutes to check for leaks in the apparatus, then air was removed to minus 200m.m. mercury by use of a vacuum pump. This was then replaced with pure oxygen. The animals were left for a further fifteen minutes to acclimatize further, after which the initial pressure reading was made. Since all the carbon dioxide in the desiccator is absorbed by the soda lime, any change in pressure as shown by the mercury manometer was due to oxygen uptake by the animal. After exactly fifteen minutes, the second reading was taken and the difference recorded. A third reading was taken fifteen minutes later, again the difference was recorded. Since a closed-circuit method was used, the humidity within the apparatus was constant for all animals. At each reading the temperature within the desiccator was recorded and used in the correction factor shown in the formula. 21 B. M. R. determinations took place during week ends when the laboratory was at its quietest and were begun at approximately 9:00 a.m. The rats were randomly assigned to the order to testing, half the determination being made in five groups, each of 16 rats,on Saturday and half in another five groups on Sunday. The procedure for measuring the B. M. R. was tested prior to the experimental period and it was found that the lowest reading was obtained 30 to 60 minutes after the animals wereplaced in the desiccator. It was during this period that the measurements were taken in this study. Since two values were obtained, the lowest was accepted as that which would approach the true B. M. R. of the animal. The oxygen uptake of the animal in mls. per hour was converted to oxygen uptake in mls. per gram weight per 2A hours, and it is in the latter form that the B. M. R. is expressed. Analysis of Data The analysis of variance for a three-factor, fixed- effects model, completely randomized design, the one-way analysis of variance with equal and unequal cells, and a single Tukey test were used to analyze the data (A5). The analysis of variance tests was conducted with the use of the M. S. U. Computer. The means from each treatment group, non-blood group, and blood group are presented graphically to show longitudinal variations in the B. M. R. CHAPTER IV RESULTS AND PRESENTATION OF DATA Introduction The purpose of this investigation was to study the progressive effects of physical activity and anxiety- producing stress on the B. M. R. of young adult male albino rats. The 160 animals were randomly assigned to ten different housing-treatment groups. The rats received treatments five days per week, 30 or 60 minutes each day, for a period of ten weeks. B. M. R. was measured, in the form of oxygen uptake in mls. per gram body weight per 2A hours, during the two days between treatments. Thus a total of 11 measurements were made throughout the ten-week period of the study. Unfor- tunately, B. M. R. measurements taken between the sixth and seventh weeks of treatments and between the seventh and eighth weeks of treatments were much higher than would have been expected for true B. M. R. values. On these two occasions, the laboratory temperature was approximately 290 C and the temeprature within the desic- cators ranged from 30° C to 3A° C. These high temperatures and the 100 per cent humidity within the apparatus made the animals more restless and caused them to pant in an attempt 22 23 to lower the body temperature. It was decided to omit B. M. R. measurements obtained during these two testing periods since an extraneous factor, temperature, appeared to have affected the results and certainly disturbed the animals at the time of testing. During the study twelve animals died, six during blood sampling, three were found dead in their cages, two drowned and one animal had to be sacrificed for it was suffering from paralysis of the lower extremities, the cause of which was unknown. The treatment groups and time of death of these animals is shown in Table l. The missing data caused by the death of animals was replaced by the mean score of each specific group for that particular week. Treatment and Presentation of Data Analysis of variance and Tukey test were used to analyze the data, the results of which are shown in Tables 2 and 3. The means from each treatment group are illustrated graphically to show longitudinal variations. The presenta- tion is divided into two parts: (a) vertical analysis; (b) longitudinal analysis, each of which are discussed separately. 2A 11.... i . 5.. c l» .. . . v... M . .. ....._ Ti] 3.. 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Cmo:.; unm:.m Cams.~ cflwm.m cmmm.n Cumm.~ Comm.o :mmfl.fi meH.o pooan uoouum .> oooanlcoz poon Amaamo Amadov mmwm.n anwo.m seam.m coco.o :Hom.o maoo.o amm:.o amHH.w swam.m humpczao> vacuum .> mumpcovom ammo m m m a m m m m m m m Ha ma d m r m m l m m H mocmflum> uo mousom xooz .muoouum pnmfiumoha 038 Ucm pooflm .zmm; so mumme moCMHum> uo mwmmflmn< mmzlmCOIl.m mqm<8 27 Vertical Analysis The variance each week due to difference in treatments was analyzed using the "F" test and was found not to be significant at the 5 per cent level. When the cage effect, blood effect and treatment effect were analyzed as a whole, significance was found once in the cage effect and once in the treatment effect from the nine tests made (see Table 2). Significance was also only found once in the combined cage-blood effect. In this study a large number of comparisons have been made and many probabilities were computed for each source of variance. Since a large number of probabilities were computed some of these might appear significant by chance, as is evident from the data shown in Tables 2 and 3 (A2). Where one-way analysis of variance tests was made on the cage effect, blood effect and the two major treatment effects, significance was found only in the final week in the cage effect and in the fifth week of the non-forced versus forced treatment effect. During the other seven weeks differences were not significant in these two groups. The remaining sources of variance were not significantly different in any of the nine weeks of testing, as shown in Table 3. As stated previously, the significance found is probably due to chance since many porbabilities were cal- culated. 28 Discussion These results show that within any one week variation of B. M. R. between any of the ten treatment groups was not significant. If B. M. R. differences were present in the study they were not perceptible. This may have been due to the limited number of animals used and to the large variability, from week to week, of each animal tested. Longitudinal Analysis The variations of each treatment group from week to week are represented graphically. In every group with the exception of the sedentary anxiety forced, the B. M. R. increased during the first week of treatments. The increase may be attributed to the change from a voluntary cage exis- tence to the eXperimental treatments. However the increase shown by the voluntary control group, since its treatment was unchanged, cannot be explained. After the first week of treatment the voluntary and sedentary control groups show the tendency for the B. M. R. to decrease with in- creasing age; this is especially evident in the sedentary control group (Figure 2). This "ageing" curve can also be seen in the sedentary anxiety group (Figure 5) and in the sedentary anxiety forced group (Figure 9). The effect of treatments on all other voluntary groups and on the seden- tary forced anxiety group has altered the B. M. R. suf- ficiently such that the effect of age is not evident (see Figures 6, 7, 8, 9, 10 and 11). Only the sedentary forced 02 Uptake in ml/Gm/24 hrs. 02 Uptake in ml/gm/24hrs. Sedentary Group 29 KEY Whole Group Non - Blood — -- Blood —-—- \ \ ’——-— _-—‘- .0- Time in Weeks Voluniary Group 30- Figure 2 Time in Weeks Figure 3 T T l 9 IO \ \ \ \\\ ,. \\ T I 1 8 9 IO 02 Uptake in ml/gm/24hrs. 02 Uptake in ml/gm/24hrs. 30 £112.! Whole Group ----- Sedentary Forced Group Non-Blood --— —--- 30 “ Blood _._,-. -- l““-~ l \ \ \\\\.-/’ \;,/ ‘L lbw 0 T r l 1 ‘ "i """'“l"’""‘" “7‘ r 1 1 O I 2 3 4 5 £5 7 B 9 IO Time in Weeks Figure 4 Sedentary Anxiety (Srcup 30 - 28 -4 \\\ ,r’dfi-k: I 1 l T l 6 7 B 9 IO Time in Weeks 02 Uptake in willow/24 hrs. 02 Uptake in ml/gm/24 hrs. 31 5.6.! Whole Group Voluntary Forced Group Non - Blood —- — Blood --—- cut —1 —I - q .1 d d I I O I 2 3 4 5 6 7 8 9 l0 Time in Weeks Figure 6 Voluntary Anxiety Group ‘ /~—-_ ”.2 I 1 I i 1 6 7 8 9 l0 Time In Weeks Figure 7 32 5.5.! Whole Group Sedentary Forced Anxiety Group Nonealood --—— 30 _ Blood ——- — V ’E \ \\ .5 3 2 22 ~ 3 .. ON ~ 0 I T I I I I I I I I 0 l 2 3 4 5 6 7 8 9 IO Tlme in Weeks Figure 8 Sedentary Anxiety Forced Group 30 - . 28 - V Q 2e -l é E 24 d \ // .5 x. _ . / \\// g 22 '1 \ J 6" I o L I I I I I I I I I n O I 2 3 4 5 6 7 8 9 l0 Time In Weeks Figure 9 KEY Whole Group Voluntary Forced Anxiety Group Non~8lood —— 28 -l m‘ z v Q 26 /‘\ //\ \ ‘~ _ E 24—1 ’_,.J;/‘ \// /— \\ ‘____._:/ .c P / \// \ / - / \ / .3 \/ g 22 - 3 T 8‘ a o I I T I I I I I I 1 O l 2 , 3 4 5 6 7 8 9 IO Time in Weeks Figure IO Voluntary Anxiety Forced Group 30 - , 28 d a z e i 26 - i _q\ .s \ . \ / 3 \ / 2 22 - \ / 3 ‘r 8‘ 0 I I I I I I I I 1 O l 2 3 4 5 7 8 9 l0 Time in Weeks Figure II “* 3A group showed a continuous increase in the B. M. R. during several successive weeks, after which there was a continuous decrease (Figure A). Discussion These results on th whole point to the fact that the treatments may, over a period of time, affect the B. M. R. by either maintaining it at a relatively constant level and not allowing the usual decrease with increasing age, or even cause it to increase slightly. However it is not possible in this study to draw any definite conclusion about the longitudinal effect of the treatments on the B0 M. R. since so few animals were used in each group in the study and since the variability of each animal was so high from week to week. The study confirmed work by Davis (l2, 13) who found that the average oxygen consumption rate shows a continual decrease throughout life. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary The study investigated the progressive effects of physical activity and anxiety—producing stress on the basal metabolic rate (B. M. R.) of young adult male albino rats. The physical activity was forced swimming for thirty minutes with two per cent of the body weight attached to the base ofthe tail. The anxiety-producing stress was non- injurious electrical shock received every 15 seconds for 30 minutes. Treatments were administered five days a week for ten weeks. One hundred and sixty rats were randomly assigned to ten treatment groups. Half the animals were housed in sedentary cages and half in spontaneous activity cages (voluntary). Eight animals from each group were chosen at random and blood samples of 3cc were removed from each of these animals once a week throughout the study. The B. M. R. was measured in the form of oxygen uptake in mls. per gram i of body weight per 2A hours. Oxygen uptake was measured in a closed-circuit apparatus (30). 35 36 Analysis of variance and Tukey test was used to analyze the daba,and.longitudinal effects are demonstrated graphically. It was found that treatments were not signif- icantly different at the 5 per cent level; however, the graphs did show that B. M. R. decreases with increasing age and that treatments may stop or inhibit the decrease in B. M. R. due to ageing. Conclusions The results show that for this study physical activity and anxiety-producing stress may, if applied over a long period of time, affect the decrease of B. M. R. due to ageing. There is no significant difference between the forced activity treatments and the anxiety treatments, or in fact between any combination of treatment. Whether the stress is from physical activity or from anxiety, produced by an electrical shock, it has the same effect on the B. M. R. of the adult male albino rat. More specific conclusions cannotlxedrawn from this study since so few animals were used in each group and the variability of each animals was so high. 37 Recommendations Further study should be confined to fewer groups with larger numbers of animals in each group. B. M. R. measurements should be made in a temperature- controlled environment. Another method of forced exercise, other than swimming, needs to be developed. r. “(bl ’n -. 10. ll. BIBLIOGRAPHY Periodicals Benedict, Francis G., L. E. Emmes, P. Roth, H. Monmouth Smith. The basal, gaseous metabolism of normal men and women. Journal of Biological Chemistry l8zl39. 191A. Benedict, F. G., H. Monmouth Smith. The metabolism of athletes as compared with normal individuals of similar height and weight. Journal of Biological Chemistry 38:2A3. 1915. Benedict, F. G., L. E. Emmes. A comparison of the basal metabolism of normal men and women. Journal of Biological Chemistry 28:253. 1915. Benedict, F. G. Factors affecting basal metabolism. Journal of Biological Chemistry 88:263. 1915. Benedict, F. G. The measurement and standards of basal metabolism. Journal of American Medical Asso— ciation 11:2A7-250. 1921. Benedict, F. G. Age and basal metabolism of adults. American Journal of Physiology 88:651. 1928. Benedict, F. G. and M. D. Finn. 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American Journal of Physiology 88:595-601. 1931. Speck, C. Physiologie des Mendschlichen Athmens, Leipsic, p. 22A, 1892. From Schneider, Ring, Clark, The effect of physical training on basal respiratory exchange, pulse rate and arterial blood pressure. American Journal of Physiology 88:256. 1927. Steinhaus, A. H. Studies on the influence of physical work on the basal metabolism. American Journal of Physiology 18:18A. 1926. Steinhaus, A. H. Studies in the physiology of exercise 1. Exercise and basal metabolism in dogs. American Journal of Physiology 88:658. 1928. Steinhaus, A. H., and Jenkins, T. A. Studies in physiology of exercise IV. Further concerning exer- cise and basal metabolism in dogs. American Journal of Physiology 88:202. 1930. Williams, H. B., Riche, J. A., and Lusk, G. Animal calorimetry second paper. Metabolism of the dog following ingestion of meat in large quantity. Journal of Biological Chemistry 88:3A9. 1912. Wilkinson, B. A statistical consideration in psycho- logical research. Psychological Bulletin 88;156- 158. 1951. Wishart, G. M. The variability of the basal metabolic rate. Quarterly Journal of Medicine 88:193. 1927. Wishart, G. M. The influence of previous muscular activity and other on the basal metabolic rate. Quarterly Journal of Medicine 88:199. 1927. A5. A6. A7. A2 Books Guenther, W. C. Analysis of Variance, pp. 126-128. Prentice Hall, Inc., Englewood Cliffs, J. J. 196A. Selye, Hans. The Story of the Adaptation Syndrome. ACTA, Inc. Medical Publishers, Montreal, Canada. 1952. Selye, Hans. The Stress of Life, pp. 111, 182. McGraw-Hill Book Co., Inc., New York. 1956. APPENDICES A3 APPENDIX A Analysis of Variance with Equal Cells—- All Categories. AA Source of Sum of Degrees Mean F variance squares freedom square Statistic Week 1 Cage effect AA.0160 1 AA.0160 3.767 Blood effect 2.2231 1 2.2231 0.190 Cage-blood effect A1.6976 l A1.6976 3.569 Treatment effect 35.3778 A 8.8AAA 0.757 Cage-treatment effect 106.89AA A 26.7236 2.287 Blood-treatment effect 8.2572 A 2.06A3 0.176 Cage—blood— treatment effect A0.7980 A 10.1995 0.837 Remaining error 1635.6503 10A 11.6832 TOTAL 191A.91A7 159 Week 2 Cage effect 1.2691 1 1.2691 0.119 Blood effect 12.8879 1 12.8879 1.215 Cage-blood effect 18.5981 1 18.5981 1.75A Treatment effect 65.5991 A 16.3997 1.5A7 Cage-treatment effect 106.A959 A 26.6239 2.511 Blood-treatment effect 32.9073 A 8.2268 0.776 Cage—blood— treatment effect 12.2176 A 3.05AA 0.288 Remaining error 1A83.8590 1A0 10.5989 TOTAL 1733.83uu 159 A6 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 3 Cage effect 5.917A 1 5.917A 0.AA2 Blood effect 11.29A3 l 11.29A3 0.8A5 Cage-blood effect 1.AOA3 1.AOA3 0.105 Treatment effect 35.A897 A 8.872A 0.66A Cage-treatment effect 15.0947 u 3.7736 0.282 Blood-treatment effect 96.7668 A 2A.1917 1.810 Cage-blood- treatment effect 99.8951 A 2A.9737 1.869 Remaining error 1870.3135 1A0 13.3593 TOTAL 2136.1761 159 Week A Cage effect 0.020A l 0.020A 0.002 Blood effect 12.6731 1 12.6731 1.253 Cage-blood effect 12.5832 1 12.5832 1.2AA Treatment effect 61.9951 A 15.A987 1.532 Cage-treatment effect A2.2226 A 10.5556 1.0AA Blood-treatment effect A0.053A A 10.0133 0.990 Cage-blood— treatment effect A5.3605 A 11.3A01 1.121 Remaining error 1A15.5126 1A0 10.1108 TOTAL 1630.A213 159 A7 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 5 Cage effect 2.2633 1 2.2633 0.223 Blood effect A.3l32 l A.3l32 0.A26 Cage—blood effect 101.8088 1 101.8088 10.063 Treatment effect 115.1013 A 28.7753 2.8AA Cage-treatment effect 89.9A38 A 22.A859 2.222 Blood-treatment effect 22.9161 A 5.7290 0.566 Cage-blood- treatment effect 27.178A A 6.79A6 0.671 Remaining error 1A16.3335 1A0 10.1166 TOTAL 1779.8587 159 Week 6--No results Week 7--No results Source of Sum of Degrees Mean F variance squares freedom square statistic Week 8 Cage effect 0.0052 1 0.0052 0.000 Blood effect A5.7318 1 A5.7318 3.8A8 Cage-blood effect 1A.18A8 l 1A.18A8 1.193 Treatment effect A2.0218 A 10.505A 0.88A Cage-treatment effect 79.6337 A 19.908A 1.675 Blood-treatment effect A.1669 A 1.0Al7 0.087 Cage-blood- treatment effect 58.0366 A lA.509l 1.221 Remaining error 1663.A95A 1A0 11.8821 TOTAL 1907.2766 159 Week 9 Cage effect A0.6627 1 A0.6627 3.701 Blood effect 18.5232 1 18.5232 1.686 Cage-blood effect 18.7A16 l 18.7A16 1.706 Treatment effect 3A.3660 A 8.5915 0.782 Cage-treatment effect 17.0190 A A 25A7 0.387 Blood-treatment effect 10.1776 A 2 5AAA 0.231 Cage—blood- treatment effect 36.1176 A 9.029A 0.821 Remaining error 1537.96Al 1A0 10 985A TOTAL 1713.5721 159 A9 __‘,i 7* Source of Sum of Degrees Mean F variance squares freedom square statistic Week 10 II I Cage effect 25.3AA6 l 25.3AA6 2.01A Blood effect 30.15A3 l 30.15A3 2.397 Cage-blood effect A.733A l A.733A 0.376 Treatment effect 7.8009 A 1.9502 0.155 Cage-treatment effect 67.5367 A 16.88Al 1.3A2 Blood-treatment effect 35.672A A 8.9181 0.709 Cage-blood- treatment effect 1A.3887 A 3.5971 0.285 Remaining error 1760.9511 1A0 12.5782 TOTAL 19A6.582A 159 Week 11 Cage effect 6A.22A2 1 6A.22A2 5.A89 Blood effect 0.0273 1 0.0273 0.002 Cage-blood effect 2.1275 1 2.1275 0.181 Treatment effect l6.A03A A A.1008 0.350 Cage-treatment effect 25.70A7 A 6.A26l 0.5A9 Blood—treatment effect 2.6087 A 0.6521 0.055 Cage-blood- treatment effect 13.9633 A 3.A908 0.298 Remaining error 1637.7996 1A0 11 6985 TOTAL 1762.8590 159 __r._,_ APPENDIX B One-Way Analysis of Variance with Equal Cells Categories Sedentary v Voluntary 50 51 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 1 Between categories AA.0160 1 AA.0160 3.717 Within categories 1870.8986 158 11.8All Total 191A.91A7 159 Week 2 Between categories 1.2691 1 1.2691 0.115 Within categories 1732.5652 158 10.9656 Total 1733.83AA 159 Week 3 2 Between categories 5.917A l 5.917A 0.A38 Within categories 2130.2587 158 13.A826 Total 2136.1761 159 Week A Between categories 0.020A 1 0.020A 0.001 Within categories 1630.A009 158 10.3189 Total 1630.A213 159 Week 5 Between categories 2.2633 1 2.2633 0.201 Within categories 1777.5953 158 11.2506 Total 1779.8587 159 52 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 6--No results Week 7—-No results Week 8 Between categories 0.0052 1 0.0052 0.000 Within categories 1907.2713 158 12.1713 Total 1907.2766 159 Week 9 Between categorie A0.6627 l A0.6627 3.8A0 Within categories 1627.909A 158 10.5880 Total 1713.5721 159 Week 10 Between categories 25.3AA6 1 25.3AA6 2.08A I Within categories 1921.2378 158 12.1597 Total 19u6.582u 159 Week 11 Between categories 6A.22A2 l 6A.22A2 5.973 Within categories 1698.63A8 158 10.7508 Total 1762.8590 159 APPENDIX C One-Way Analysis of Variance with Equal Cells Categories Non-Blood v Blood 53 5A Source of Sum of Degrees Mean F variance squares freedom square statistic Week 1 I Between categories 2.2231 1 2.2231 0.183 Within categories 1912.6916 158 12.1056 Total 191A.91A7 159 Week 2 Between categories 12.8879 1 12.8879 1.183 Within categories 1720.9A6A 158 10.8920 Total 1733.83AA 159 Week 3 Between categories 11.29A3 1 11.29A3 0.839 Within categories 212A.8818 158 13.AA86 Total 2136.1761 159 Week A Between categories 12.6731 1 12.6731 1.237 Within categories 1617.7A82 158 10.2389 Total 1630.A213 159 Week 5 Between categories A.3l32 1 A.3l32 0.383 Within categories 1775.5A55 158 11.2376 Total 1779.8587 159 55 Source of Sum of Degrees Mean F variance .squares freedom square statistic Week 6--No results Week 7—-No results Week 8 Y Between categories A5.7318 1 A5.7318 3.881 Within categories 1861.5AA8 158 11.7819 Total 1907.2766 159 Week 9 I Between categories 18.5232 1 18.5232 1.726 Within categories 1695.0A89 158 10.7281 Total 1713.5721 159 Week 10 3 SI Between categories 30.15A3 1 30.15A3 2.A86 Within categories 1916.A281 158 12.1292 Total 19A6.582A 159 Week 11 Between categories 0.0273 1 0.0273 0.002 Within categories 1762.8317 158 11.1571 Total 1762.8590 159 APPENDIX D 1ll‘u- -_e 3 Rd. One-Way Analysis of Variance with Unequal Cells Categories Non-Forced v Forced 56 57 Source of Sum of Degrees Mean F variance .squares freedom square statistic Week 1 Between categories 6.A026 l 6.A026 0.530 Within categories 1908.5120 158 12.0791 Total 191A.91A7 159 Week 2 Iv Between categories A0.6A38 l A0.6A38 3.792 Within categories 1693.83A3 158 10.7163 Total 1733.83A3 159 Week 3 Between categories 17.7860 1 17.7860 1.326 Within categories 2118.3901 158 13.AO75 Total 2136.1761 159 Week A Between categories 20.859A l 20.859A ‘2.0A7 Within categories 1609.5619 158 10.1871 Total 1630.A213 159 Week 5 Between categories 63.8AA6 l 63.8AA6 5.878 Within categories 1716.01A0 158 10.8608 Total 1779.8587 159 58 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 6--No results Week 7—-No results Week 8 Between categories 30.3170 1 30.3170 2.552 Within categories 1876.9595 158 11.879A Total 1907.2766 159 Week 9 Between categories 7.88A3 l 7.88A3 0.730 Within categories 1705.6877 158 10.795A Total 1713.5721 159 Week 10 Between categories 0.0620 1 0.0620 0.005 Within categories 19A6.5203 158 12.3197 Total 19A6.582A 159 Week 11 Between categories 5.A828 1 5.A828 0.A92 Within categories 1757.3762 158 11.1226 Total 1762.8590 159 _,f .12.. . 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I‘ APPENDIX E One-Way Analysis of Variance with Unequal Cells Categories Non-Anxiety v Anxiety 59 60 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 1 Between categories 0.5367 1 0.5367 0.0AA Within categories 191A.3779 158 12.1163 Total 191A.91A7 159 Week 2 Between categories 29.AA55 l 29.AA55 2.729 Within categories 170A.3888 158 10.7872 Total 1733.83AA 159 Week 3 If Between categories 23.A125 1 23.A125 1.750 Within categories 2112.7636 158 13.3719 Total 2136.1761 159 Week A I Between categories 22.1707 1 22.1707 2.178 Within categories 1608.2506 158 10.1788 Total 1630.A213 159 Week 5 Between categories 21.1909 1 21.1909 1.903 Within categories 1758.6678 158 11.1308 Total 1779.8587 159 61 Source of Sum of Degrees Mean F variance squares freedom square statistic Week 6--No results Week 7--No results Week 8 7 Between categories 2.8A38 1 2.8A38 0.235 Within categories 190A.2766 158 12.0533 Total 1907.2766 159 Week 9 Between categories 2.5575 1 2.5575 0.236 Within categories 1711.01A6 158 10.8292 Total 1713.5721 159 Week 10 Between categories 0.6651 1 0.6651 0.05A Within categories 19A5.9l72 158 12.3159 Total 19A6.582A 159 Week 11 Between categories 0.2056 1 0.2056 0.018 Within categories 1762.653A 158 11.1560 Total 1762.8590 159 APPENDIX F Treatment Group Means 62 63 ——'——— Group Week Total mean Non-blood mean Blood Mean Sedentary 0 2A.62 2A.A9 2A.75 1 26.31 26.22 26.A0 2 26.0A 27.01 25.07 3 2A.72 25.09 2A.35 A 2A.65 25.33 23.97 g 23.56 2A.32 22.79 7 8 23.13 2A.55 21.70 9 22.A9 23.00 21.97 10 22.06 22.11 22.01 Voluntary 0 2A.A3 2A.53 2A.33 1 26.AA 27.07 25.81 2 26.6A 27.72 25.55 3 2A.90 25.19 2A.61 A 26.23 25.72 26.75 2 25.62 26.25 2A.99 7 8 25.23 2A.69 25.77 9 2A.20 2A.0A 2A.35 10 23.80 2A.19 23.Al Sedentary forced 0 23.57 2A.76 22.AA l 23.67 23.21 2A.13 2 25.09 2A.85 25.32~ 3 25.35 26.32- 2A.37 A 27.60 28.69 26.15 2 26.19 26.11 26.26 7 8 2A.51 25.25 23.76 9 22.62 22.93 22.30 10 23.3A 22.30 23.37 6A fir Group Week Total mean Non-blood mean Blood Mean Sedentary anxiety 0 2A.20 2A.Al 23.99 1 26.07 25.88 26.25 2 25.69 25.A7 25.90 3 22.85 2A.A9 21.21. A 23.63 2A.7l 22.55 2 23.7A 25.61 21.86 7 8 23.65 2A.78 22.52 9 22.75 22.60 22.91 10 22.37 22.71 22.02 Voluntary forced 0 2A.97 23.85 26.08 1 26.39 27.22 25.56 2 26.A9 28.97 2A.01, 3 25.A6 26.3A 2A.58 A 2A.95 25.33 2A.56 2 2A.16 25.18 23.13 7 8 25.09 2A.83 25.35 9 2A.33 25.08 23.57 10 2A.20 25.18 23.82 Voluntary anxiety 0 23.13 23.2A 23.02 1 2A.35 23.95 2A.75 2 25.65 2A.07 27.22 3 2A.37 23.33 25;A0 A 23.99 22.12 25.8A 2 23.81 23.08 2A.55 7 8 2A.2A 2A.56 23.92 9 2A.5A 2A.33 2A.7A 10 2A.AA 2A.28 2A.59 65 Week Total Group mean Non-blood mean Blood mean Sedentary forced anxiety 0 25.09 25.A2 2A.75 l 25.52 2A.12 26.91 2 25.30 25.37 25.22 3 25.22 2A.52 25.92 A 26.53 27.97 25.09 2 25.38 25.85 2A.90 7 8 2A.78 25.A2 2A.1A 9 2A.32 2A.63 2A.00 10 23.91 23.67 2A.15 Sedentary anxiety forced 0 26.8A 28.39 25.28 1 2A.78 23.79 25.77 2 2A.87 26.11 23.62 3 25.18 25.71~ 2A.6A A 25.29 25.62 2A.95 2 2A.60 25.82 23.57 7 8 23.09 22.56 23.62 9 23.1A 23.8A 22.AA 10 22.90- 22.1A 23.66 Voluntary forced anxiety 0 23.18 22.63 23.73 1 23.76 23.56 23.95 2 2A.93 2A.25 25.61 3 23.51 22.99 2A.03 A 25.29 2A.58 26.00 2 2A.A6 2A.A9 2A.A2 7 8 25.1A 25.17 25.11 9 23.12 2A.05 22.18 10 2A.23 2A.3A 2A.11 66 Group Week Total mean Non-blood mean Blood mean Voluntary anxiety forced 0 23.35 22.87 23.83 1 2A.A9 23.92 25.05 2 25.2A 2A.78 25.69 3 2A.97 25.36 2A.57 A 26.20 25.69 26.71 2 25.56 25.79 25.33 7 8 2A.50 2A.93 2A.06 9 23.2A 2A.93 21.55 10 23.63 2A.0A 23.22 "lllllllllilll