THE EFFECTS OF TWO EXERCISE REGIMENS AND SUPPLEMENTAL VITAMIN C INTAKE UPON BONE GROWTH IN ALBINO RATS BY Lesley Ann Sive A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Health, Physical Education and Recreation 1978 A: OLA ABSTRACT THE EFFECTS OF TWO EXERCISE REGIMENS AND SUPPLEMENTAL VITAMIN C INTAKE UPON BONE GROWTH IN ALBINO RATS BY Lesley Ann Sive This investigation was undertaken to determine the effects of eight weeks of sprint (SPT) or endurance (END) training and vitamin C supplementation on the weight of the tibia-fibula complex and the length of the tibia in normal male albino rats. Animals from each training group were sacrificed 72 to 96 hours after their last exercise sessions. Selection criteria developed for training performance resulted in a final cell frequency of ten animals per treatment group. Absolute tibia lengths of the END and SPT animals were significantly shorter than those of the sedentary control (CON) group. No differences in bone length were found between the two training groups. The SPT group had significantly lower absolute and relative tibia-fibula weights than did the END and CON animals. The vitamin C supplementation did not appear to have any effect on training performance or long bone growth under the high-intensity training conditions imposed in this investigation. To my parents ACKNOWLEDGEMENTS My sincerest appreciation belongs to my family, for their patience, understanding and encouragement throughout my graduate program. A very special thank you is extended to Dr. W. W. Heusner for the continued guidance, counseling and assistance he provided me as my graduate advisor and committee chairman. Deep appreciation is given to the members of my committee, Dr. W. W. Heusner, Dr. W. D. Van Russ and Dr. K. W. Ho for making the writing of this thesis a worthwhile experience. A special thank you is extended to Dr. W. D. Van Huss for his continued support and guidance during my years as a graduate student. A very special thank you is offered to my friends for their concern and understanding, and for helping to maintain my morale in times of difficulty and frustration. iii TABLE OF CONTENTS CHAPTER II. III. IV. LIST OF TABLES . . . . . . . . LIST OF FIGURES . . . . . . . THE PROBLEM . . . . . . . . . Need for the Study . . . . Statement of the Problem . Research Hypotheses . . . . Research Plan . . . . . . . Rationale . . . . . . . . . Limitations . . . . . . . . REVIEW OF LITERATURE . . . . . Exercise and Growth . . . . Vitamin C and Bone Growth . Vitamin C and Stress . . . Requirements of Vitamin C . METHODS AND MATERIALS . . . . Sample . . . . . . . . . . Activity Groups . . . . . Diet Subgroups . . . . . Training Procedures . . . Animal Care . . . . . . . Sacrifice Procedures . . Analysis of Data . . . . RESULTS AND DISCUSSION . . . . Training Results . . . . . Body Weights at Sacrifice . Effects of Vitamin C Supplementation Activity Level Results . . Discussion . . . . . . . . iv Page vi vii mmnwww H 00 10 12 14 18 18 19 20 21 22 23 25 26 26 30 33 33 34 TABLE OF CONTENTS--continued CHAPTER Page V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS . . . . 38 Summary . . . . . . . . . . . . . . . . . . . 38 Conclusions . . . . . . . . . . . . . . . . . 39 Recommendations . . . . . . . . . . . . . . . 40 REFERENCES 0 o o o o o o o o o o o o o o o o o o o o o 4 l APPENDICES A. Training Programs . . . . . . . . . . . . . . . 49 B. Basic Statistics for Training Data . . . . . . . 51 TABLE 1. LIST OF TABLES Analysis of variance for effects and Newman-Keuls comparisons for absolute data . . . . . . . . . . Analysis of variance for effects for body weights absolute and relative bone data . . . . . . . . . Page overall training tests of paired and relative bone 0 O O I O O O O O O 0 O O 32 overall vitamin C at sacrifice and vi FIGURE 1. 2. Mean Daily Percent Expected Meters Run for SPRINT Animals Mean Daily Percent Expected Meters Run for ENDURANCE Animals Means and S.E. for Body Weight and Tibia Length . Means and S.E. Weight . for Absolute and Relative Tibia LIST OF FIGURES , vii Page 28 29 31 35 CHAPTER I THE PROBLEM Many studies have been conducted to determine the effects of exercise on various measures of body growth and development. However, limited and sometimes contradictory evidence has been presented concerning the growth response of bone to physical activity. Some data have indicated that the pressure effect of low-intensity exercise on the epiphyses increases both the length and the weight of long bones (14, 40). Other data have shown that retardation of linear long bone growth occurs in response to strenuous exercise programs during the developmental period of an organism (19, 36, 40, 65, 78, 80). The adaptation of bone to physical training has been hypothesized to be a function of the intensity of training (7). Despite the serious implications of the observed negative effects of exercise on bone growth for the well-being of children involved in competitive athletics at the Elementary and Junior High school levels, little attempt has been made to determine the possible physiological factors involved in growth impair- ment or the steps which might be taken to prevent its occurrence. Current literature has indicated that exercise consists of a continuum of specific activities each of which elicits a specific response within the organism (17, 54, 68). However, other than a small amount of data on intensity of exercise, there is a dearth of information regarding the effects of different types of exercise programs on long bone growth. Overall body growth and normal bone formation have been found to be dependent on an adequate supply of vitamin C (16, 64, 67, 77). In particular, the vitamin may play a role in the synthesis and/or metabolism of the adrenal steroid hormones (29, 42, 74) which are related to growth. Heavy stress is known to deplete the supply of vitamin C in the adrenals (57, 69). This information has resulted in recommendations of increased vitamin C intake for athletes who participate in strenuous and/or prolonged physical activity (56, 66). A search of the literature, however, revealed no study which has assessed the effect of supplementary vitamin C intake as a means of preventing the bone-growth retardation that is induced by stressful exercise. Need for the Study Data were needed to determine the effects of high- intensity sprint and endurance exercise regimens on long bone growth. In addition, an investigation was required to identify the role that vitamin C may play in altering bone-growth changes induced by exercise. Expectations were that a study of bone growth, using specific exercise routines with and without the administration of supplementary vitamin C, would furnish useful information about the relationships under consideration. Statement of the Problem The purpose of this study was to determine the effects of two different types of prolonged physical activity, plus daily vitamin C supplementation, on long bone growth in the male albino rat. The length of the left tibia and the weight of the left tibia-fibula complex were used as criterion measures of bone growth to facilitate comparisons between treatment groups. The exercise programs that were used were designed with the expectation that one would produce primarily aerobic and the other more anaerobic metabolic adaptations in the animals. Research Hypotheses The hypotheses that were tested in this study are as follows: 1. The tibial bones of exercised animals should be signifi- cantly shorter and heavier than the tibial bones of sedentary animals. 2. The two exercise regimens should produce differential long bone growth in the exercised animals. 3. Significantly less impairment of long bone growth should be found in exercised animals that receive vitamin C supplementation than in exercised animals that do not receive vitamin C supplementation. Research Plan Eighty four male albino rats (Sprague-Dawley strain) were randomly assigned to one of the following three activity groups: (a) Sedentary Control (CON), (b) Sprint Running (SPT), and (c) Endurance Running (END). One-half of the animals in each of the activity groups received 2 mg of vitamin C in a .1 cc 5% sugar solution per 100 gm of body weight daily. The remaining animals received similar amounts of a sugar solution as a placebo according to body weight. The SPT and END training regimens were implemented using electronically controlled running wheels (82). The two programs were more intensive than any exercise routines previously used in this laboratory (see Appendix A). The animals in the SPT group were subjected to an interval training program of high-intensity sprint running. During the final 14 days, they were expected to run at speeds of 108 m/min. Six bouts of exercise were used with 2.5 min between bouts. Each bout consisted of five lS-sec work periods alternated with four 30-sec rest periods. The SPT program was expected to tax the anaerobic capacity of the experimental animals. The END animals were subjected to a rigorous program of distance running. During the final eleven days of the training period, these animals were expected to complete a 60-min continuous run at a speed of 36 m/min. The END program was designed to overload aerobic metabolic capacity. 5 Ten animals in each of the six activity-diet subgroups were sacrificed at the conclusion of eight weeks of treat- ments. Health and training performances throughout the treatment period were used as selection criteria for these animals. The left tibia-fibula complex was removed from each animal. The wet weight of the complex and the length of the tibia were determined. Analysis of variance procedures were used to analyze the data. Rationale The SPT and END training regimens were designed to simulate high-intensity exercise programs for humans. The intensity of the programs was expected to induce bone growth changes in the exercised animals. Differential long bone growth in the experimental animals was anticipated in response to the differences between the SPT and END training programs. Vitamin C (ascorbic acid) supplementation was included for study as a possible preventative of the expected decrement in bone growth of the exercised animals. Vitamin C is important in the normal formation of bone matrix (64, 77). The rat synthesizes its own supply of vitamin C. However, there are several reasons why the rat was used in this study rather than the guinea pig which, like man, does not synthesize the vitamin. The rat is easily trained to run in a wheel whereas the guinea pig is not. In addition, the rat tibia had been used in earlier studies concerned with the effects of exercise on long bone growth. Treat- ments were initiated when the animals were 84 days old. Although the animals were postpubertal at this age, rat skeletal growth continues until the animals are over 400 days of age (15). The techniques that were used for data collection were based on those of a previous bone-growth study completed at the Human Energy Research Laboratory (80). Limitations l. The results of animal studies cannot be used to make direct inferences to human beings. 2. The small subgroup size may limit the power of the statistical analyses. 3. Optimal durations of treatments for the achievement of significant results between groups have not been clearly established. 4. Optimal types of training to show differential growth effects on bones may not have been selected. 5. The rat synthesizes its own supply of vitamin C. Therefore, the significance of the supplemental vitamin C effect on bone growth may not be fully revealed in this study. 6. The results of this study are specific to the tibial bones of male albino rats. The data apply only to the SPT and END training regimens used in the investigation. Due to limitations of personnel and facilities, the animals for this investigation had to be received in three separate shipments. The activity treatments were not randomized across shipments. This lack of randomization could have introduced a bias in the exercise-related data. The probability of a genetic bias was small because the supplier (Hormone Assay, Inc., Chicago, Illinois) had a well-controlled substrain of Sprague-Dawley rats. Every possible effort was made to control unique external factors in the laboratory. The diet treatments were randomized across shipments. CHAPTER II REVIEW OF LITERATURE The following review of literature is divided into four sections. The first section will focus on the nature of the effects of exercise on bone growth. The role that vitamin C plays in the formation and maintenance of bone will be covered in the second section. The third section will be devoted to the relationship between ascorbic acid and stress. Theories on the function of ascorbic acid in the adrenal cortex also will be presented. The final section will deal with requirements of vitamin C particularly under conditions of stress. Exercise and Growth Mechanical stress is believed to play an important role in the structure and growth of bone (10, 44, 84). However, a review of literature indicates there is little agreement among researchers concerning the nature of the effects of exercise on bone growth. Steinhaus (75), in his review of the effects of chronic exercise, reported evidence suggesting that the pressure effect of exercise on the epiphyses stimulates bone growth. Donaldson (14) found that voluntary running for a three-month period or longer produces gains in weight 8 9 and length of rat leg bones. A comparison of dominant and non-dominant forearms of tennis players, carried out by Buskirk gt a1. (9), indicated that tennis produces increased length of the radius and ulna in the forearm used to swing the racket. Recently, Saville and Whyte (71) ran a group of thirty- day-old rats 2000 m/day, five days a week, for periods of up to 10 weeks. They noted increased wet weight and volume of the long bones in the exercised animals. In another study by Saville and Smith on male rats (72), bone responded to isometric exercise by increases in density and breaking strength. In contrast to these findings, other evidence indicates that excessive and prolonged pressure on the epiphyses may retard bone growth. In 1926 Price-Jones (65) subjected newly-weaned rats to a forced-exercise running program and reported that control animals had longer, heavier bones than did the exercised animals. Working with two-week-old mice, Kiiskinen and Heikkinen (40) found slight increases in femoral length and weight under low-intensity training conditions (80 m/day at 18 m/min for 12 weeks). However, when the duration of running was progressively increased to 180 m/day, the mean femur length was significantly shorter in the exercised group than in a control group. In a study by Van Huss et 31. (80), a group of 30—day- old albino rats was forced to swim 30 min daily for 35 days. 10 An impairment of body size was indicated by the finding of significantly lower body weights and shorter tibias in the animals forced to exercise during prepuberty than in control animals. These observations were reinforced in an investigation conducted by Tipton gt gt. (78). Statistically significant differences in length, width and mineral percentage of long bones were found between a group of growing rats subjected to strenuous exercise and a group of normal controls. The mechanisms of these growth changes are yet to be determined. Weinmann and Sicher (81) believe that an "increase of pressure or tension beyond the limits of tolerance leads to destruction of bone by resorption." Evans and Hughes (16) cited Gelbke's study (19) which was designed to determine the effect of tension and pressure on the growth of endochondral bone in young dogs. Continuous prolonged tension and compression were found to result in the replacement of epiphyseal material by a spongy bone resistant to mechanical forces. The conclusion, therefore, was that bone may sacrifice its growth zone to retain its configuration and stability. Vitamin C and Bone Growth Vitamins play a major role in the normal metabolic processes of the body. Although some of the specific physiological functions of vitamin C (ascorbic acid) have not been ascertained as yet, the fact that a deficiency manifests itself in numerous symptoms of defective body ll functioning is well known. One of the most important roles of ascorbic acid involves the formation of collagen, a fibrous protein which constitutes the major component of bone matrix. The primary effect of ascorbic acid recently was shown to be related to the hydroxylation of proline and lysine after the formation of protocollagen by peptide binding (5, 12, 33, 79). Hydroxyproline and hydroxylysine are rare amino acids and an integral constituent of collagen. Evidence indicates that the formation of hydroxyproline and hydroxylysine takes place by means of analOgous reactions (5). However, studies it ytttg suggest that lysine hydroxylation may be less readily affected by ascorbate deficiency than proline hydroxylation (6). The precise mode of action of the vitamin in the hydroxylation reactions has yet to be determined. Ascorbic acid may function as an essential cofactor participating directly in the hydroxylation mechanism (5, 31, 46) or as an enzyme activator responsible for the conversion of an inactive precursor to an active enzyme (5). However, the two activities of ascorbic acid may really be two aspects of a single phenomenon (5). Jeffrey and Martin (33) demonstrated that ascorbic acid can neutralize the inhibitory actions of puromycin on new collagen formation. Likewise, Liakakos gt gt. (47) found that large doses of ascorbic acid can supress the inhibitory actions of the corticosteroids on new collagen formation. 12 Some of the changes that occur in bone as a result of varying degrees of vitamin C deficiency were determined by Poal-Manresa gt gt. (64). They found that six-week-old guinea pigs, when placed on a scorbutogenic diet for three weeks, had thinner than normal epiphyseal bone plates in the tibia, decreased trabeculae, and a scanty matrix. Administration of 5 mg/day of ascorbic acid to the scorbutic animals resulted in improved quality of the matrix and the formation of new bone in areas which were not severely damaged. Thornton (77) demonstrated that ascorbic acid deficiency also affects bone salt deposition and stability. Scorbutic guinea pigs were found to have decreased bone salt depositions which paralleled the length of time of the deficiency. Decreased stability of bone salts was significantly greater in ascorbic acid deficient animals than in control animals. Lability of bone salts increased as deposition of the salts decreased which suggested that the two factors were inversely related. Osteoblastic activity and the quantity and quality of the bone matrix appeared to have been influenced by lack of vitamin C. Vitamin C and Stress High concentrations of ascorbic acid are present in most metabolically active tissues in the body. It has been well established that the adrenal cortex contains great amounts of this vitamin. During stressful situations, the release of adrenocorticotropin (ACTH) from the 13 adenohypOphysis stimulates the adrenals and results in marked depletion of the gland's ascorbic acid content. Namyslowski (57) found that the adrenals of a group of white rats subjected to prolonged exhaustive swimming contained an average of 242 mg % of ascorbic acid which was less than half the value measured in control animals. In addition, it appears that adrenal ascorbic acid levels in both the guinea pig, which cannot synthesize vitamin C, and the rat, which does synthesize the vitamin, are not fully restored for some hours after cessation of adrenal stimulation (50, 59, 69). A more economical utilization of adrenal ascorbic acid in trained animals has been indicated by Namyslowski (58). Following exhaustive exercise, the decrease of ascorbic acid in trained rats was less than in untrained rats; furthermore, normal adrenal levels were restored much faster in the trained animals after exercise (59, 60). Namyslowski also noted an increased concentration of adrenal ascorbic acid in trained animals that were rested (60). These findings suggest that training results in the adaptation of the adrenal gland to strenuous exercise. The body is able to resist different types of stresses with the help of the corticosteroid hormones, in particular the glucocorticoids, which are released from the adrenal cortex. The high concentration of ascorbic acid in the adrenals has led to the concept that this vitamin is related to steroid biosynthesis. A number of conflicting hypotheses 14 have arisen to attempt to explain the role it plays. Enhancement of tg ztttg steroid formation in the presence of ascorbic acid has been reported by some investigators (21, 37, 38). However, no such direct tg zttg evidence appears to be available at this time. In fact, observations have pointed to increased pituitary- adrenocorticotrophic activity with increased corticosteroid production in guinea pigs on a diet which is ascorbic 'acid deficient (ll, 28). More recently, the high concen- tration of ascorbate in the adrenals has been hypothesized to prevent steroidogenesis (24, 35, 42, 43). Experimentation has shown that ascorbic acid release precedes corticoid out- put (49). Kitabchi (42) believes that this ascorbate release reverses hydroxylase inhibition in the adrenal with the resulting facilitation of steroid production and release. Liakakos gt gt. (48) have demonstrated that an excess of ingested ascorbic acid may exert an inhibitory effect on the secretion of the major glucocorticoid, cortisol, following adrenal stimulation with ACTH. Finally, the results of studies by Hodges and Hotston (28, 29) have led to doubts that adrenal ascorbic acid has any influence on steroidogenesis. However, these authors do suggest that the vitamin may be important in the metabolism of corticosteroids. Requirements of Vitamin C A review of the literature on ascorbic acid shows that there are wide variations from one study to another in the 15 estimated vitamin C requirements for most population groups. This difference of opinion appears to be the result of insufficient knowledge concerning the metabolic role of ascorbic acid and differences in methods by which ascorbic acid status is evaluated (32). Evidence does suggest that the minimum daily intake of vitamin C needed to insure prevention of scurvy is 10 mg (4, 22, 53), although amounts of less than 10 mg/day may provide adequate protection for some peOple (4, 27, 30). In 1938 the League of Nations Technical Commission on Nutrition (45) recommended an allowance of 30 mg/day for the human adult. This value appears to be sufficient to replenish the quantity of ascorbic acid metabolized daily (3). In the U.S., the Food and Nutrition Board of the National Research Council (61) has advocated dietary allowances of 40 mg/day for children up to 11 years of age and 45 mg/day for adults. The Recommended Dietary Allowances (RDA) have been described as: ... the levels of intake of essential nutrients considered, in the judgement of the Food and Nutrition Board on the basis of available scientific knowledge, to be adequate to meet the known nutritional needs of practically all healthy persons (61). In order to cover the needs of most people, the RDA are estimated to exceed average requirements. The increased metabolic demands placed on the body during exercise would seem to suggest that the need may exist for an additional daily intake of vitamins, particularly vitamin C. Physiological parameters such as l6 muscular endurance, pulse rate, vital capacity, respiratory quotient, blood pressure, and recovery time have been used to evaluate the need for increased amounts of vitamin C during physical exertion. The results of these studies have indicated both negative and positive effects of vitamin C administration. No beneficial effects of vitamin C supplementation on physiological performance were shown by Keys and Henschel (39), Fox gt gt. (18), Johnson gt gt. (34), Bailey gt gt. (2), Gey gt gt. (20) and Kirchhoff (41). Studies indicating positive effects of additional vitamin C ingestion have been conducted by Brunner (8), Matthes (52), Wiebel (83), Yakovlev (85) and Harper gt gt. (23). When biochemical indices such as urinary ascorbic acid and blood ascorbic acid levels are used, researchers usually interpret the data as being indicative of an increased requirement for vitamin C during vigorous physical activity (32). Namyslowski (55) studied healthy young athletes while they were engaged in normal activities at a physical culture academy and during physical training at ski camp. In both instances he noted decreased urine and serum ascorbic acid levels when the athletes had a vitamin C intake of 100 mg/day. A rise in intake to 300 mg/day produced increases in both urine and blood ascorbic acid levels. Based on these observations Namyslowski recommended daily vitamin C intakes of 100 to 150 mg during normal activities, 200 to 250 mg during ski-training camps, and 200 to 400 mg for long ski runs (56). Bachinsky (1) found 17 that physically active peOple have lower amounts of ascorbic acid in the urine than do less active subjects. He inter- preted this observation to be evidence of a need for vitamin C supplements in athletes. Maksjutinskaya gt gt. (51) studied 20 school boys at summer camp who swam from 1,000 to 5,000 m daily. Ten of the boys were given 75 mg of ascorbic acid per day along with supplements of vitamin A and thiamin. The control subjects received only the samp diet. The group on vitamin supplements excreted slightly higher amounts of ascorbic acid in their urine than did the control group. The results were assumed to be support for an increased intake of vitamins by active subjects. CHAPTER III METHODS AND MATERIALS Gross measurements of total-body oxygen debt and oxygen uptake have been used to reflect human metabolic responses to physical activity. Exhaustive sprint running leads to an increased tolerance of oxygen debt which presumably reflects a greater capacity for the generation of muscular energy via anaerobic metabolism. Training regimens based on this type of running are characterized by maximal work— loads and relatively short bouts of repeated exercise. In contrast, distance running is thought to be dependent chiefly upon aerobic muscle metabolism and tends to increase total-body oxygen uptake capacity. Moderate or light workloads and relatively long bouts of continuous exercise are typical of endurance training programs. This study was designed to investigate the effects of eight weeks of sprint and endurance training on the weight of the tibia- fibula complex and the length of the tibia in the male albino rat. In addition, the effects of daily vitamin C supplementation on tibial growth were determined. Sample Eighty four normal male albino rats (Sprague-Dawley strain) were obtained from Hormone Assay, Inc., Chicago, 18 19 Illinois. They were received at weekly intervals in three shipments of 30, 24 and 30 animals respectively. Each shipment was designated as a separate activity group which was then divided into two diet subgroups. A standard period of 12 days was allowed for adjustment to laboratory conditions. The treatments were initiated when the animals were 84 days old. Activity Groups The duration of the experimental period was eight weeks. The three activity treatments were as follows: SedentarypGroup The 24 animals in the second shipment constituted the sedentary control (CON) group. These animals were not forced to exercise and were housed in individual sedentary cages (24 cm x 18 cm x 18 cm) during both the adjustment period and the treatment period. Sprint Group The sprint running (SPT) group was comprised of the 30 animals in the first shipment. Each of these animals was housed in an individual voluntary-activity cage (sedentary cage with access to a freely revolving activity wheel) during the treatment period. The SPT animals were subjected to an interval training program of high-intensity sprint running. The workload of the SPT program was increased gradually until on the 27th day of training, and thereafter, the animals were expected to complete six bouts of exercise with 2.5 min of inactivity between bouts. Each bout included five lS-sec 20 work periods alternated with four 30-sec rest periods. During the work periods, the animals were required to run at a speed of 108 m/min. Endurance Group The endurance running (END) group was composed of the 30 animals in the third shipment. These animals were housed under the same conditions as the SPT animals. The END animals were subjected to a demanding program of distance running. The workload was progressively increased so that on the 30th day of training, and thereafter, the animals were expected to complete 60 min of continuous running at 36 m/min. Diet Subgrogps In addition to the three activity regimens, two diet supplements were administered. Both supplements were given by oral syringe, seven days a week, between 7 p.m. and 9 p.m. Administration of the diet treatments was begun on the day prior to the initiation of the activity treatments and was terminated on the day prior to sacrifice. Vitamin C Group One half of the animals in each activity group were given approximately .1 cc of a 5% sugar solution with 2 mg ascorbic acid/100 gm of body weight.1 1Vitamin C crystals (30-80 mesh) were obtained from the J. T. Baker Chemical Co. 21 Placebo Group The remaining animals in the three activity groups received approximately .1 cc of a 5% sugar solution/100 gm of body weight as a placebo. Trainigg Procedures The animals were trained in a battery of individual controlled-running wheels (CRW). This apparatus has been described as: ... a unique animal-powered wheel which is capable of inducing small laboratory animals to partici- pate in highly specific programs of controlled reproducible exercise. (82) Animals learn to run in the CRW by avoidance-response Operant conditioning. A controlled low-intensity electrical current, applied through the running surface, provides motivation for the animals to run. A light above the wheel signals the start of each work period. The animal is given a predetermined amount of time (acceleration time) to attain a prescribed running speed. If the animal does not reach the prescribed speed by the end of the acceleration time, the light remains on and shock is applied. As soon as the animal reaches the prescribed speed, the light is extinguished and the shock is discontinued. When the animal responds to the light and attains the prescribed running speed during the acceleration time, the light is extinguished immediately and shock is avoided. If the animal fails to maintain the prescribed speed throughout the work period, the light-shock sequence is repeated. Most animals learn to 22 react to the light stimulus after only a few days of training. A typical training session consists of alternated work and rest periods. The wheel is braked automatically during all rest periods to prevent spontaneous activity. The brake is released and the wheel is free to turn during work periods. Performance data are displayed for each animal in terms of the total meters run (TMR) and the cumulative duration of shock (CDS). The TMR and the total expected meters UHMM are used to calculate the percentage of expected meters (PEM): PEM = 100 (TMR/TEM) PEM values are the chief criterion used to evaluate and compare training performances. A secondary criterion is provided by the percentage of shock-free time (PSF) which is calculated from the CD8 and the total work time (TWT): PSF = 100 - 100 (CDS/TWT) Animal Care All housing cages were steam-cleaned every two weeks. Standard procedures for daily CRW cleaning and maintenance were observed. The animals received food (Wayne Laboratory Blox) and water gg libitum. A relatively constant environment was maintained for the animals by daily handling as well as by temperature and humidity control. 23 The animals were exposed to an automatically regulated sequence of twelve hours of light (1:00 a.m. to 1:00 p.m.) followed by twelve hours without light (1:00 p.m. to 1:00 a.m.). Since the rat normally is a nocturnal animal, this lighting pattern altered the normal day-night schedule for the animals so that they were trained during the active phase of their diurnal cycle. Body weights of the SPT and END animals were recorded before and after each training session. The CON animals were weighed weekly. Sacrifice Procedures Anticipated limitations of time and personnel restricted the number of animals that could be handled at sacrifice to 10 in each activity-diet subgroup. Since one of the inherent purposes of the study was to compare various parameters in two groups of highly trained animals and a group of untrained animals, five extra rats originally were included in each of the four subgroups that were subjected to regimens of forced exercise. At the end of the investigation, the ten animals to be sacrificed from each of these four subgroups were selected on the basis of their health and their training performance throughout the treatment period. Only animals subjectively determined to be in good health were chosen. Because the training requirements were extremely vigorous, no absolute minimal performance criteria were established. However, individual daily records of PEM and PSF were examined, and healthy animals making the best adaptations 24 to the training regimens were selected for sacrifice. Two extra animals were included originally in each of the two sedentary subgroups to allow for the unlikely possibility that some of the unexercised animals might have become ill during the course of the study. Again, only animals subjectively determined to be in good health were chosen for sacrifice. If there were more than 10 healthy animals in either subgroup, the animals to be sacrificed were chosen by lot. Three sacrifice periods of two-days duration (Monday and Tuesday) were established. All 20 animals within an activity group were killed during a single sacrifice period (i.e., five animals from each of the two diet sub- groups each day). The trained animals were killed either 72 or 96 hours after their last exercise bouts were completed. This procedure was followed to eliminate any transient acute effects of exercise. The animals were either 140 or 141 days old at sacrifice. Final body weights were recorded immediately prior to sacrifice. Each animal was anesthetized by an interperitoneal injection (4 mg/100 gm body weight) of a 6.48% sodium pentobarbital (Halatal) solution. The lower part of the left hind limb was stripped of superficial muscles. The tibia-fibula complex was removed and cleaned of all remaining muscle and epiphyseal cartilage. The bones were placed in Ringer's solution for several minutes to allow additional cleansing. After removal from the solution, they were 25 blotted dry and weighed to the nearest milligram. Vernier calipers (Craftsman, West Germany) were used to measure the length of the tibia to the learest .1 mm. The length measurement was taken from the most lateral point of the lateral condyle of the proximal tibial epiphysis to the most lateral point of the inferior malleolus. Bone length was expressed in absolute terms (millimeters) and bone weight was expressed in both absolute terms (milligrams) and relative terms (percentage of body weight). AnalysiS'of‘Data This study was conducted as a two-way (3 x 2) factorial design with three levels of activity and two levels of diet. The bone lengths and the absolute and relative bone weights were analyzed using a fixed-effects analysis of variance routine on the Michigan State University Control Data 6500 Computer. Newman-Keuls tests were used to evaluate differences between pairs of means whenever a significant (p g .05) F-ratio was obtained. CHAPTER IV RESULTS AND DISCUSSION The material in this chapter is organized into five main sections. The first part covers the training results from the Controlled-Running Wheel (CRW) programs and includes the percentage of body weight lost during the daily exercise periods, the environmental factors that Operated during training and the training response as reflected by the performance criteria. Body weight results at sacrifice are given in the second section. The effects of vitamin C supplementation on performance and on tibial length and weight are presented next. The fourth major section deals with the tibia data for each training group. Finally, a discussion is offered that attempts to relate the present findings to those of other investigations reported in the literature. Training Resultsl The sprint (SPT) and endurance (END) Controlled- Running Wheel (CRW) training programs are presented in 1Some of the material in this section has been adapted, in part, from the unpublished Ph. D. dissertation of Roland R. Roy (70). 26 27 Appendix A. These programs are modified versions of standard regimens routinely used in the Human Energy Research Laboratory, Michigan State University, East Lansing, Michigan. The modifications were incorporated in an attempt to design strenuous exercise programs which would primarily stimulate anaerobic or aerobic metabolic processes in the animals. The performances of the animals were evaluated using the percentage of expected meters (PEM) and the percentage of shock-free time (PSF) as criterion measures. The performance data for the SPT-C and the SPT-No C groups are presented in Figure 1. Progressive increases in the required running velocity were made rapidly. From the beginning of the fourth week of training to the end of the program, the animals were expected to run at velocities ranging from 90 to 108 m/min (see Figure l and Appendix A, Table A-l). No comparable exercise programs for small animals have been found in the literature. The results indicate that the animals could not maintain the program requirements. PEM values fell to approximately 45% during the last three weeks of training as contrasted with the usual criteria of 75% for satisfactory completion of an exercise regimen. The training data for the END-C and END-No C groups are shown in Figure 2. PEM values were 70 or higher each day of training in both the C and the No C animals. These results indicate that the animals were able to maintain the daily requirements of the END program relatively well. 28 22.34 5:35 .8 5m 2222 8.83. 2851 2:5 :85. ._ .91 T 8. . +lmm lleomlleu 6 IlemslaTl filo—$1 Es)... Jw> 9. on on mm 8 n. o. n >3 .225 p _ P — P p P — P P p P P p b L P b b P — P F P b P P P — PL P P — P — — b — P I. o .‘. Low 1 ”new posoodxa :0 95 pomdunoo 990002 I 9.9.001 ION. L 03 29 2252 moz<§ozm .3 5m 2.2: 8.82m. .82.... 2:5 :82 as: T - 3 ATM.— ..séé. d> o¢ mm on 0N ON 9 . 0. >40 .23": _pb_Pr__r.__~P__p._-.Ppppbrbprprp+rrkpp O “new puaadxg 100/.pomdmoa - cm L g 1 .. om .. oo. 93.5 002 I 1 ON. 9.20 0 Old .. 30 The END animals ran at the relatively slow speed of 36 m/min. Periods of continuous running were progressively increased to 60 min at the end of five weeks of training and were maintained at this level for the remainder of the eight week program (see Figure 2 and Appendix A, Table A-2). The single bout of exercise was determined subjectively to result in daily physical exhaustion of the animals. On the average, the rats lost 2.55% of their body weight during each training session (see Appendix B). Body weight data were used to award an unplanned recovery day on Wednesday of each of the last three weeks of training. The animals were run on the 39th and 40th day of the program, but the results were not recorded due to a technician error. Bodngeights at Sacrifice At the end of eight weeks of exercise, the trained animals were significantly lighter than the sedentary control animals (Figure 3). The differences in body weight between the SPT and END groups of animals were not statistically significant (Table 1). Both trained groups were approximately 20% lighter than the CON group. These results are in agreement with those of previous studies using the CRW (26, 76) and support the general observation that strenuous exercise slows the usual gain in body weight seen in the male rat over time (13). The slower rate of weight gain usually is attributed to an increase in caloric expenditure associated with exercise. In some instances the growth impairment has been ascribed to a £8.28 Mamxucoezmz EQOVE EoozEQm 2:3 8m "5 mm oco .885. .m .9... ozm A 200 02w A 200 ...aw A 200 Pam A 200 ozu .Em zoo . azm Km 28 Hoe o . w a i. / .\ MM / .\ n W m M“ W & HM...“ n._.o // .. ow... - owe - 8... J owe , 1 on... .. one [H1 1 ems? .. 000 .. 9.1V iHL .. our. Es. £83 0:5 2.9 2925 In8m. 32 Table 1. Analysis of variance for overall training effects and Newman-Keuls tests of paired comparisons for absolute and relative bone data. Dependent Treatment Means F P fizzTgn- Variable CON SPT END Value Value T ** est Body weight at sacrifice 515.1 397.3 408.3 96.7311<0.0005* SPT