nu. mazu 9...? Rub a.» a In h. AH...- .~aw.. .. .- nuh .nmufi axav m.un “mug ‘hhe ‘mhfibb ’n.?~w ”53. w::. ‘W... on. I»; #6.. 92:. I. OVWPV “DA .. or. ..h.l ul- .. axdo ”nab“: fihmw Hme o» .3». t otl.‘ «H of: .2. vAOOD. h- arc muuw ’hounuo .3 ‘0‘. .33 Pm;a RWt¢ «\Jm I I.~@I-. n '6' . u it. ‘nh.’ a! ‘0‘ HI. I fi‘.‘vt Du . .0 qr\ OH'L TL Ito-w...‘ I. T‘I‘ .4 Aw. 00.4 1.. h! .wwu. av s; '0' I. T. I‘ npflsfln CG . nan-W. A V hfl.ptl .01 i O.- 7‘ Q oi . a I c’Q “U '11-? .0 has VM I at rams...“ nanw 7.... av,» if; :5 LIBRARY ABSTRACT SPECIFIC CHANGES IN RAT FEMURS INDUCED BY TWO EXERCISE REGIMENS AND VITAMIN C SUPPLEMENTATION By Margaret Anne Kolka This investigation was undertaken to determine the effects of eight weeks of sprint (SPT) or endurance (END) training and/or vita- min C supplementation on the wet weight, dry weight, length and ash content of the femur in the male albino rat (Sprague-Dawley Strain). Net weights were recorded using a Mettler Balance. Dry weights were obtained fOllowing 24 hours in a 110 degree (C) drying oven by the same method. The dried samples were ashed for 24 hours at 600 de— grees centigrade to determine the mineral content. All statistical analyses were run using a two-way fixed effects AOV with Neuman- Keuls tests run on significant F values. The SPT and END exercise regimens both produced a number of alterations in the femurs of the animals. The CON animals had the greater absolute wet weights and dry weights when compared to the SPT and END animals. The END and CON groups had longer bones (abso- lute) than the SPT group and the CON group had a greater ash content than the SPT group (absolute). In all relative neasurements, the SPT and END animals had great- er values than the CON group. Margaret Anne Kolka The vitamin C supplementation did not appear to have any benefi- cial or other effect on training performance or long bone growth with the high intensities of training in this investigation. SPECIFIC CHANGES IN RAT FEMURS INDUCED BY TWO EXERCISE REGIMENS AND VITAMIN C SUPPLEMENTATION By Margaret Anne Kolka 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 1976 DEDICATION To my gamifly and finienda. ii ACKNOWLEDGEMENTS My sincerest appreciation belongs to my family, for their un- selfishness, patience, understanding and encouragement throughout my graduate program. A very special thank you is extended to Dr. W. D. VanHuss 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. D. VanHuss, Dr. R. R. Roy and Dr. K. W. Ho for making the writing of this thesis a worthwhile experience. A special thank you is extended to Dr. R. R. Roy for his assis- tance in statistical procedures and editing of early drafts, to David Anderson for his assistance in laboratory techniques, to Dr. G. Mikles for his continued support and to Ann Snyder, Dee Ulmer and Charlene Troyano for their assistance during the processing of the tissues. A very special thank you is offered to Nancy McNames, Jennifer Parks, Bonnie Smoak and Crystal Fountain for their friendship and concern. Gratitude is due to Dr. 0. Mickelsen for the extensive use of the facilities of his laboratory, Department of Food Science and Human Nutrition. CHAPTER II. III. IV. TABLE OF CONTENTS Page LIST OF TABLES .......................................... vi LIST OF FIGURES ......................................... vii THE PROBLEM ............................................. 1 Statement of the Problem ............................. 3 Significance of the Problem .......................... 3 Limitations of the Study ............................. 4 REVIEW OF RELATED LITERATURE ............................ 5 Composition of Bone .................................. 5 Mechanical Stress and Bone Growth .................... 5 Mechanism of Vitamin C ............................... 8 Vitamin C and Athletic Performance ................... 8 Specificity of Training .............................. 9 RESEARCH METHODS ........................................ 10 Experimental Animals ................................. 10 Research Design ...................................... 10 Training Procedures .................................. 12 Animal Care .......................................... I4 Sacrifice Procedures ................................. 14 Tissue Removal and Analysis .......................... 15 Statistical Analysis ................................. 16 RESULTS AND DISCUSSION .................................. 17 Training Results ..................................... 17 Body Weights at Sacrifice ............................ 21 Vitamin C Effects .................................... 23 Femur Results ........................................ 23 Discussion ........................................... 27 iv TABLE OF CONTENTS--continued CHAPTER Page V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS ................ 28 Summary .............................................. 28 Conclusions .......................................... 29 Recommendations ...................................... 29 BIBLIOGRAPHY .................................................... 31 APPENDICES A. Training Programs ....................................... 36 B. Basic Statistics for Training Data ...................... 38 LIST OF TABLES TABLE Page 1. Analysis of Variance for overall vitamin C effects for body weights at sacrifice and absolute femur results.... 24 2. Analysis of Variance for overall vitamin C effects for relative femur results .................................. 24 vi LIST OF FIGURES FIGURE Page 1. Mean Daily Percent Expected Meters Run for SPRINT Animals .................................................. 19 2. Mean Daily Percent Expected Meters Run for ENDURANCE , Animals .................................................. 20 3. Means and S. E. for Body Weight and Percent Femur Ash.... 22 4. Means and S. E. for Absolute and Relative Femur Wet Weight and Dry Weight .................................... 25 5. Means and S. E. for Absolute and Relative Femur Length and Ash Content .......................................... 26 vii CHAPTER ONE THE PROBLEM The stress and strain of muscular activity and weight bearing is thought to provide a stimulation for the growth and development of bone. However, research results on the effects of exercise on bone growth in young animals are not clear at this point. It is believed that there is a resultant increase in the rate of calcium deposition to the bone with exercise and conversely, a decrease in the rate of calcium resorption from the bone (4,6). Current literature indicates that exercise induced changes in bone may be related to the intensity of the training program. High intensity and relatively short duration repetitive running, 60-80 m/min for 30 sec, apparently stimulates the growth of new bone around the diaphyses of rat tibias (51). Training at slow speeds and low in- tensities has not produced any noticable alterations in the bones of rats (40,44). However, swimming of moderate intensity has been shown to retard the long bone growth in young animals (30,51,52). The response of bone to vitamin C supplementation during high in- tensity training should be positive since vitamin C is involved in the fOrmation of the fibrous structures of the body. The effects of vitamin C on performance has been studied in both man and animals (7,22,25,53). Animal data presented by Demarais, tend to confirm the need for larger than normal quantities of vitamin C with training (8). However, no studies have been found which evaluate the effects of vitamin C supplementation as a means of preventing the retardation of long bone growth which has been observed in endurance training. Current literature has indicated that exercise consists of a continuum of specific levels, each of which elicits a somewhat differ- ent response within the organism. However, little is known about the specific effects of the different exercise on long bone growth. The two training programs used in this study were designed to provide func- tional overloads at distinctly different points in the metabolic contin- uum. The intensities of the two programs were designed to provide very strenuous physical stress which stimulates overtraining in short- duration high-intensity work and in long-duration low-intensity work. The femur of the rat is a long bone which provides a workable tool for bone analysis, is easily accessible and directly involved in the flexion and extension of the hip. Use of a long bone from the hind- limb of the rat provides comparable data with previous and current in- vestigations. Previous findings in the rat indicate a depletion of the intrin- sic supply of vitamin C in the adrenals during heavy stress (1,54). These data indicate the supply of the vitamin is not readily replaced even though the rat is known to synthesize this vitamin. Normal growth of the fibrous tissues, including bone, is dependent on an adequate supply of vitamin C within the animal (7,19,47). It was reasoned that supplementation might prevent the vitamin C depletion, thus have a fa- vorable effect upon bone growth. Statement gf_the Problem To determine the effects of high intensity training of short and long duration and/or vitamin C supplementation on the wet weight, dry weight, length and ash content in the right femur of the rat. It was hypothesized that the high intensity training regimens in this inves- tigation and the vitamin C supplementation should result in a greater relative weight, relative length and relative mineral content of the femur in the trained animals when compared with control animals. Significance of the Problem Exercise related alterations in the long bones, although contro- versial, are well documented (4,6,9,11,21,26,28,3D,40,41,44,46,51). In particular, confusion exists in attempting to determine the work inten- sity in the various programs. The need for further assessment of bone response to various controlled, reproducible levels of training is needed, preferable with high levels or intensity. Changes in the femur of the highly trained animals in this investigation should provide fur- ther insight into the relationship between the intensity of training and long bone growth. The effect of vitamin C on bone during high intensity exercise is not clear in the literature. The inclusion of vitamin C in this inves— tigation should lead to a better understanding of the relationship be- tween vitamin C and high levels of training on bone growth. Limitations of the Study The results are limited to the right femur of adult male rats. The rat synthesizes its own supply of vitamin C, therefore, the significance of the supplemental vitamin C effect may not be ful- ly revealed in this study. The training programs were not of equal intensity, thus may not have been truly comparable regarding the type of exercise used. CHAPTER TWO REVIEW OF RELATED LITERATURE This review focuses on the composition of bone, stress and bone growth, the mechanism of vitamin C, vitamin C and its relationship to athletic performance and specificity of training. Composition-9f.Bone Bone consists of an organic and an inorganic component (5,6,11, 12,14,19). The organic element consists of collagenous fibers embed- ded in a ground substance. This organic component determines the den- sity of the tissue. An appatite crystal of calcium and phosphate con- stitutes the inorganic component which provides rigidity to the bone. The two constituents are consolidated in a matrix, resulting in great strength and adaptability to the tissue. The inorganic matrix, usually comprising one third to one half of whole bone, is primarily calcium phosphate (60-80%), calcium car- bonate (10%), magnesium phosphate (1-2%) and magnesium carbonate (1- 2%) (11,13,19). The inorganic matrix, or mineral content of the bone, can be represented by the ash content of the bone. Ash content is a desirable gravimetric reference state for analysis of bone samples (31). Mechanical Stress and Bone Growth The first suggestion of an intensity of training-bone growth relationship was cited by Steinhaus (46), who hypothesized that the pressure produced by exercise upon bone epiphyses stimulated bone growth until an optimal length had been achieved. If the pressure continued beyond this point bone growth would be retarded. Rarick, in support of this contention, concluded that an undefined minimum of phy- sical activity is essential for normal bone growth (41). Donaldson (9) has presented evidence in support of the stress involvement in the growth of long bones in rats and dogs; and increases in bone growth have been reported with low-intensity exercise programs (41). However, im- paired growth following training is also supported in well-controlled investigations (40,46,52). A number of investigators have trained growing animals and record— ed lengths and weights of bones. Training has been reported to both in- crease bone length and decrease bone length; to increase bone weight and decrease bone weight. Current literature has indicated that the exer- cise induced changes in bone may be related to the intensity of the training program (6,46). Experiments with animals have demonstrated that the bones of animals receiving stressful programs are short and dense (44) and can tolerate large lateral forces before breaking (43). Growing animals (rats) trained by running up to six hours a day at a low intensity showed no effect on bone growth as indicated by length and weight when compared to body weight (40,44). Alteration in bone growth in mice occured in a training program of 18 m/min for 80 min a day for 12 weeks with longer and heavier femurs appearing (28). However, when the training regimen was altered to 80 min/day for 21 weeks or 120 min/day for 12 weeks, shorter and lighter bones appeared. This is in support of the premise that higher duration will lead to in- hibition. In a training regimen involving speeds of 26-30 m/min fOr 6O , minutes a day, Tipton found shorter and lighter bones in rats when com- pared to body weight (51). High intensity training has been shown to inhibit the growth in length of the long bones in laboratory animals, and furthermore to in- crease the mineral content of the specific bone (6). King found that with high intensity repetitive exercise, 30 sec at 60-80 m/min, density of the bone in rats increased (29). However, there was no apparent change in the density of the bone fbllowing 26-30 m/min one hour a day for ten weeks (29). Work by Tipton (51) and Saville (44) showed no changes in the mineral content of the long bones in rats. An increase in the relative mineral content (calcium) of mice trained in a high- intensity program was shown in work by Kiiskinen and Heikkinen (21,28). Removal of the local physiological environmental factors (mechani- cal stress, weight-bearing and muscle action) results in metabolic al- terations in the bone tissue (4,6,11,19). Cortical thinning, loss of trabeculation (supporting structures), increased fragility and loss of mineral are apparent. The loss of mineral appears to occur without sig- nificant alteration in the chemical composition or histology of the re- maining bone. 0n the basis of the literature review it was expected that the two high intensity training programs would result in heavier, denser bones relative to body weight. However, it was expected that the overall growth of the trained animals would be less, therefore the absolute bone lengths and weights would be less. Mechanism 9: Vitamin_C All animals with exception of the guinea pig, one species of bat, monkey and man synthesize vitamin C (ascorbic acid) (7,19,47). Vitamin C is involved in the formation of intracellular substances to include the collagen of fibrous structures, and the matrices of bone, cartilage and dentin. In the absence of this vitamin, the c0ndition known as scurvy develops. The pathology of scurvy induces the cessation of bone forma- tion and a breakdown in collagen (47). Resorption of mineral depositions occurs and a resultant atrophy and bone fragility is apparent. Supple- mentation of vitamin C is shown histologically to reverse this trend within a few hours through tissue regeneration. Normal bone fOrmation has been found to be dependent on an adequate supply of vitamin C (39,50). The need for this vitamin appears to be in- creased during exercise in children involved in strenuous and/or pro- longed training programs (55). A search of the literature has revealed no evidence available which evaluates the effect of supplementary vita- min C intake as a measure of preventing the bone growth retardation which accompanies heavy exercise. Vitamin C and Athletic Performance Previous investigations (25,53) have shown that a slight increase in ascorbic acid with training is desirable. Doses of up to one gram daily have shown a slight, but not significant, alteration in performance (25). Beneficial effects in both cardiovascular, as measured by PWC170, and metabolic responses have been reported in exercise studies using vi- tamin C supplementation. However, very high doses of vitamin C may in- crease 02 utilization by metabolically active systems and thus have a negative effect on performance by disturbing the equilibrium between 02 transport and O2 utilization (25). Ascorbic acid appears to improve the biosynthesis of epinephrine and norepinephrine (25). The increased secretion of norepinephrine sub- sequently enhances stroke volume by increasing cardiac contractility. The heart can reach the same cardiac output by working at a lower heart rate and therefore working more efficiently. The addition of vitamin C during training also enhances the utilization of the plasma free-fatty- acids as an energy source in the working muscle (25). This shift in metabolism is a result of the glycogen-sparing effect on muscle tissue and the liver. Beneficial effects would be most prominent during endur- ance exercise. Specificity of Training Gross measurements of total-body oxygen uptake and debt have been used to reflect human metabolic responses to physical activity. Repeat- ed bouts of exhaustive sprint running lead to an increased tolerance of oxygen debt which presumably reflects a greater compacity for the gen- eration of muscular energy via anaerobic metabolism. Training regimens based on this type of running are characterized by maximal workloads and relatively short bouts of repeated exercise. In contrast, distance run- ning is thought to be dependent chiefly upon oxidative muscle metabolism and tends to increase total-body oxygen uptake compacity. Moderate or light workloads and relatively long bouts of continuous exercise are typical of endurance training programs (23,42,49). The two training pro- grams used in this study were developed to simulate in animals, the sprint and endurance programs of man. CHAPTER THREE RESEARCH METHODS This investigation was designed to determine possible exercise and/or vitamin C induced changes in the wet weight, dry weight, length and ash content in the right femur of the rat. Experimental Animals Eighty-four normal male albino rats (Sprague-Dawley Strain) were obtained from Hormone Assay Inc., Chicago, Illinois. They were re- ceived at weekly intervals in three shipments of 30, 24 and 30 animals respectively. Each shipment was designated as a separate activity group. A standard period of 12 days was allowed for adjustment to the laboratory conditions. Activity was initiated when the animals were 84 days old. Research Design This study was organized as a two-way design with three activity groups and two dietary supplement groups. The supplement (vitamin C) was crossed throughout the activity groups. gasp; ED In 10 10 IO 10 10 IO l2 0 In 10 11 The duration of the experiment was eight weeks, a period which has been sufficient to produce marked changes in body weight, body ash and muscle responses (52). The three activity groups are as follows: Sedentary Control Group The 24 animals in the second shipment constituted the sedentary control (CON) group. These animals received no exercise and were forced to remain relatively inactive throughout the experiment. The animals were housed in individual sedentary cages (24 cm by 18 cm by 18 cm) during both the adjustment and activity periods. Sprint Group The Sprint running (SPT) group was comprised of the 30 animals in the first shipment. Each of these animals was housed in individual vol- untary activity cages (sedentary cages with access to a freely revolving wheel) during the adjustment period and in individual sedentary cages during the experimental period. The SPT animals were subjected to an interval training program of high-intensity sprint running. The work- load of the SPT group was gradually increased until on the 27th day of training and thereafter, the animals were expected to complete six bouts of exercise with 2.5 minutes of inactivity between bouts. Each bout in- cluded five 15 second work periods with four 30 second rest periods. During the work periods, the animals were expected to run at the extreme- ly fast speed of 108 m/min. Endurance Group The endurance running (END) group was composed of the 30 animals of the third shipment. These animals were housed the same as the SPT 12 animals. The END animals were subjected to a demanding program of dis- tance running. The workload was gradually increased so that on the 30th day of training and thereafter, the animals were expected to complete 60 minutes of continuous running at 36 m/min. The two dietary supplement groups are as follows: Vitamin C QEQUB One half of the animals in each activity group received vitamin C supplementation (C group). Vitamin C supplementation was administered orally by syringe with a dosage of 2.4 mg vitamin C (Merck) in a .1 ml 5% sugar solution per 100 g body weight between 7 and 9 pm daily. The administration of the supplement was begun the day prior to the initi- ation of activity and terminated the day prior to sacrifice. The dos- age used was equivalent to 1680 mg daily for a 70 kg man. Placebo Group The remaining animals in each of the activity groups (no C group) received an identical quantity of the sugar solution per unit of body weight. Training Procedures The SPT and END groups were trained in a battery of individual controlled running wheels (CRW). The apparatus has been described as: .. a unique animal-powered wheel which is capable of induc- ing small laboratory animals to participate in highly speci- fic programs of controlled, reproducible exercise. (58) Animals learn to run in the CRW by avoidance-response Operant con- ditioning. A low intensity controlled shock current, applied through 13 alternating grids comprising the running surface, provides the motivation for the animal to run. A light above the wheel signals the start of each work period. The animal is given a predetermined amount of time (accel- eration time) to attain a prescribed running speed. If the animal does not reach the prescribed speed by the end of the acceleration period, the light remains on and shock is administered. As soon as the animal reaches the desired speed, the light is immediately extinguished 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 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 the work periods. Perfbrmance data are prescribed for each animal in terms of the total meters run (TMR) and the cumulative duration of shock (C05). The TMR and the total expected meters (TEM) are used to calculate the percen- tage of expected meters (PEM): PEM‘= 100(TMR/TEM) PEM values are the chief criteria used to evaluate and compare training performance. A secondary criteria is provided by the percentage of shock- free time (PSF) which is calculated from the C05 and the total work time (TWT): PSF = 100 - lOO(CDS/TWT) 14 In this study all exercise treatments were administered once a day, Monday through Friday, between 12:30 and 5:30. Animal Care All housing cages were steam-cleaned every two weeks. Standard procedures for CRW cleaning and maintenance were observed. The animals received fOOd (Wayne Lab Blox) and water ad_libitim. A relatively constant environment was maintained for the animals by daily handling as well as by temperature and humidity control. The animals were exposed to an automatically regulated daily se- quence of 12 hours of light followed by 12 hours without light. Since the rat is normally a nocturnal animal, the light sequence was estab- lished so that the lights were off between 1:00 pm and 1:00 am and on between 1:00 am and 1:00 pm. 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 Twelve animals were sacrificed from each of the six subgroups. Since one of the inherent purposes of the study was to compare various parameters in two groups of highly trained animals, three extra rats originally were included in the SPT and END groups. Twelve animals were selected for sacrifice from each of the six groups on the basis of their 15 health and training perfbrmance throughout the treatment period. Only animals subjectively determined to be in good health were chosen. Be- cause the training requirements were extremely vigorous, no minimal per- formance criteria were established. However, individual daily records of PEM and PSF values were examined and those animals making the best adaptations to the training regimens were selected for sacrifice. All 24 CON animals were judged to be healthy and were sacrificed. Three sacrifice periods of two days duration (Monday and Tuesday) were established. All animals within a treatment group were killed dur- ing a single sacrifice period (i.e., 12 animals each day). The trained animals were killed either 72 or 96 hours after their last bouts of ex- ercise were completed. This procedure was followed to eliminate any transient effects of acute 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 ml/100 g body weight) of a 6.48% sodium pentobarbital (Halatal) solution. After selected tissues were removed for analysis by other investigators, the animals were eviscerated, placed in plastic bags and frozen at 20 degrees Fahrenheit. Tissue Removal and Analysis_ The frozen carcasses were removed after 9 months of storage and thawed overnight. The right femur was excised and all extraneous tissue removed. A wet weight was determined to the nearest .001 9 using a Met- tler Balance. Bone length as measured from the greater trochanter to the 16 lateral condyle was recorded to the nearest .1 mm using a vernier cali- per (37). The tissues were dried at 110 degrees centigrade for 24 hours, placed in a dessicator, and the dry weight determined using the same pro- cedure as followed for the wet weight. The dried samples were crushed with a mortar and pestle, to allow fOr complete ashing. The samples were then placed in vycor crucibles in a 600 degree centigrade oven for ashing, a method for removing or destroying organic matter associated with bone (2,15,16,17,18,27,31,33,34,44,56). After 24 hours the samples were removed and returned to room temperature in a dessicator and ash measured to the nearest .001 g (2,16,17,27). The ash samples were then placed in capped one dram bottles to await further analysis. Statistical Analysis Body weight, absolute and relative wet weight, dry weight, length and ash content were analyzed using a two-way fixed effects (activity and vitamin C) analysis of variance routine on the Michigan State Uni- versity Control Data 6500 Computer (CDC 6500). Neuman-Keuls tests were used whenever a significant overall F ratio (<.05) was obtained. CHAPTER FOUR RESULTS AND DISCUSSION The material in this chapter is organized into four main sections. The first part deals with the training results from the Controlled- Running Wheel (CRW) programs. It includes the percentage of body weight lost during the daily exercise periods, the environmental conditions during the training period and the training response as reflected by the two performance criteria. Secondly, body weight results are given. Thirdly, the femur data and analyses are presented. Finally, a discus- sion is offered that attempts to relate the present findings to those of other investigations reported in the literature. Training Results The sprint (SPT) and endurance (END) Controlled-Running Wheel (CRW) training programs are presented in Appendix A. These programs are modi- fied versions of standard regimens routinely used in the Human Energy Re- search Laboratory, Michigan State University, East Lansing, Michigan. The modifications were incorporated in an attempt to design strenuous ex- ercise programs which would primarily stimulate anaerobic or aerobic me- tabolic processes in the animals. The performances of the animals were evaluated using the percentage of expected meters (PEM) and the percen- tage of shock-free time (PSF) as criterion measures. The performance data for the SPT-C and the SPT-No C groups are pre- sented in Figure 1. Progressive increases in the required running velo- city were made rapidly. From the beginning of the fourth week to the end 17 18 of the program, the animals were expected to run at velocities ranging from 90 to 108 m/min. No comparable exercise program for small animals has been found in the literature. The results indicate the animals could not maintain the program requirements. PEM values fell to ap- proximately 45 during the last three weeks of training. Several possible explanations could account for these relatively poor perfOrmance data. The required running velocities may have been too fast, however observations during the training sessions revealed that the animals were capable of sprinting at the desired speeds. Low PEM values might suggest that the animals responded to the unconditioned shock stimulus rather than to the conditioned light stimulus. Improper initial training and defects in the CRW equipment could lead to such a learning problem, but the END animals learned to run under the same con- ditions and had no such difficulties (see Figure 2). A lack of control of environmental factors affecting training performance might have ac- counted for these results. This is particularly true for air tempera— ture and percent humidity, but again the END data make this explanation improbable (See Appendix B). The most likely cause of the low PEM values is that the SPT regimen may have produced a state of overtraining. The data presented in Figure 1 supports the view that the SPT regimen pro- duced a state of overtraining. Increases in the required velocity were expected repeatedly from the SPT animals before they were fully adapted to the previous velocity. The animals clearly could not handle the training program. The constant additional stress could have resulted in overtraining. 19 T mo. VTImmliTlomI'TIlSIl'TINhII'TIvnIIVTona— O¢ mm on mm ON 0. o. m F__.____c____._____F__________r_________ m_oE_c< FzEam .8 any. 2222 02896 283a 360 cows. . _ .9“. 9.90 0 02 I 990 o 010 275,5 i_m> >40 2.4m: 0 ON O? 00 00 00. ON. 03 ”slaw pepedxg Io sx, pemdwoo 20 225.2 moz o¢ mm on 0N cu m. . 0. >40 .235. E._P__H_r__._F.~L._.._.b»..».r..____b_p.10 L ION” w 1 N w 10¢” % 1 m. .omn. d 9 - w 9 D. camera .oow 9 - a u .00. aaeoooz I 18. 9.200010 1 L o! 21 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 group and the No C group. These results indicate that the animals were able to maintain the daily requirements of the END program relative- ly well. The END animals ran at the relatively slow speed of 36 m/min. Periods of continuous running were progressively increased to 60 minutes at the end of five weeks of training and were maintained at this level for the remainder of the eight week program (Appendix A). The single bout of exercise was determined subjectively to result in daily physical exhaustion of the animals. Repeated exposure to this level of stress could have resulted in a mild state of overtraining. On the average, the END animals lost 2.55% of their body weight during each training session. 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 days of the program, but the results were not recorded due to a technician error. Body Weights 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 training groups of animals was not statistically significant. Both trained groups were approxi- mately 20% lighter than the CON group. These results are in agreement with those of previous studies using the CRW (23,49) and support the general observation that strenuous exercise slows the usual gain in 22 Body Weight (om) 5” —I— .500): ' 475 - 450 t- 425 r- #1 375 350 00L SPT CON END CON > SPT CON > END Percent Femur Ash .72 " .70- .ee- ‘ ’ seb- 1L I .62- .501.“ .001. SPT CON END END >CON SPT >CON Fig. 3 Means and S.E.(n-ZO) with significant (p<0.05) Newmon— Keuls Contrasts 23 body weight seen in the male rat over time (42). The slower rate of weight gain is usually attributed to an increase in caloric expenditure associated with exercise. In some instances the growth impairment has been attributed to a significant reduction in food intake (38). Vitamin £_Effects The training responses of the animals supplemented with vitamin C as compared to those performances of the No C groups are shown in Fig— ures 1 and 2. The vitamin C supplementation did not appear to affect the training performance of the animals. In the ten analyses run on vitamin C effects (Tables 1 and 2), on— ly two showed significant P values. Absolute femur length indicated that the No C group had longer bones. The relative ash measurement showed greater values for the C groups. However, post hoc tests (Neuman Keuls) failed to show significance in any comparisons. Femur Results The femur absolute wet weight and absolute dry weight results fol- lowed the same pattern as was found for the body weight (Figure 4). The CON group had significantly larger mean absolute wet and dry weights than did either of the trained groups. The absolute wet and dry weights of the SPT and END groups were not significantly different. Absolute femur lengths were significantly shorter in the SPT group when compared to the END and CON groups (Figure 5). There was no dif- ference however, between the END and CON groups. The CON group was Table 1. Analysis of Variance for overall vitamin C effects for body weights at sacrifice and absolute femur results. Dependent Treatment Means F p Variable C No C Value Value Body weight at sacrifice 451.533 445.500 3.162 .077 (9) ESTUI "eight 1.183 1.215 1.637 .204 05’ femur 790 811 1 725 192 weight (g) ' ’ ' ' Femur Length 3.698 3.857 7.946 .007* (cm) AZ? we‘ght .489 .499 .987 .999 *Significant overall vitamin C effect at the 0.05 level. revealed no significant differences between the means. Post Hoc test Table 2. Analysis of Variance f0r overall vitamin C effects for rela- tive femur results. Dependent Treatment Means F p Variable C No C Value Value E;?”r "e‘ght 2.773 2.748 .213 .999 Dry femur weight (g) 1.854 1.837 .255 .999 f3?”” length 8.703 8.756 .091 .999 Relative Ash 1.575 1.492 5.133 .026* Percent Ash .664 .654 1.952 .165 *Significant overall vitamin C effect at the 0.05 level. revealed no significant differences between the means. Post Hoc test 25 Femur Weight (gm) Femur th Body VIMIIO’) L361- 3.001- 1.50~ I 250- L} Ff) 1.25 b I 280- IZOI- 2.70)— I.l5"’ ,1. 2508- no“- 250- TI“ 1.05 I- 2. 1 .oor 22:1 - L °°°L' SPT cow (END 9° SPT cow END cow > END END > cow cow > SPT SPT >cow Dry Femur Weight (gm) 0 Dry Femur Wt/Body Vlt. (“03) l" .90 (- 20 + .85 b «+1 195 - F} .30 *- ('1‘ .1... 130 r .75 - 1.70 - ‘I— .70 - L60 - .65 - 1.50 - 301- L401:- , 'L. - .001-1 - 00 SPT CON END SPT CON END CON > END END > CON CON > SPT SPT > cow Fig. 4. Means and S. E. (0:20) with significant (p<0.05) Newman—Keuls Contracts 26 Femur Length (mm) Femur Longth/Body Wt. (xl03) 4.00 1.000)- I a- 3901" 9.50 ._ FI— 3.80 F- F‘I‘} 9.00 ~ ff 370L 950— 3.60 - F} 800 - 3.5o - 7.50 - F!- 5.40 r 7.00 _. asor ssqf ooh» .mfi4- SPT CON END SPT CON END END > SPT END > CON CON > SPT SPT > CON Femur Ash Weight (gm) Ash Wt./ Body Wt. (x103) .60 r- 220 55 " 2.00; .5o& . . 160—- + 2- + I .45 - [.60- Aob 1.40H .35- 120- .30 LOOK- xxfla. 0&5: ~ , SPT CON END SPT CON END CON > SPT END > CON SPT > CON Fig.5 Means and S.E. (n=20) with significant (p<0.05) Newman-Keuls Contrasts 27 significantly greater in absolute ash content than the SPT group. No significant differences appeared between the CON and END or the SPT and END groups (Figure 5). The relative wet and dry femur weights for both of the trained groups were significantly larger than those of the CON group (Figure 4). Relative femur lengths, percent ash and relative ash content all fol- lowed the same pattern as the relative weights (Figures 3,4,5). In all cases the SPT and END animals had significantly larger relative values than the CON animals. There was no significant difference in any group between the SPT and END animals. Discussion Exercise related alterations in long bone growth are a result of the intensity of the training program (6,46). The increase in relative density as a result of an increase in the mineral content as measured by relative ash weight, agrees with previous investigations (6,29,44). Kiiskinen and Heikkinen reported that increased mineral content with stressful exercise is probably a result of an increase in the calcium and hydroxyproline concentrations (6,21,28). The greater relative weight of the SPT and END bones is in agree- ment with Wolff's Law that bone will develop the structure most suited to resist the forces acting upon it (10,11). Longer bones have been ob- served in mice following intense training (21,28). The vast amount of the data detailing the effects of strenuous exercise on long bone growth is consistent with the findings of this investigation (40,44,46,51,52). The consensus among these experiments is that there is a retardation of long bone growth with high intensity training. CHAPTER FIVE SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary This study was undertaken to determine the effects of two high- intensity exercise programs and/or vitamin C sopplementation on various femur measurements. Normal male adult rats (Sprague-Dawley strain were used as subjects). The two training regimens were modifications of Controlled-Running Wheel routines previously reported from this labora- tory (58). The modified programs, an endurance running routine (END) and a sprint (SPT) running routine, represented attempts to stimulate selectively either aerobic or anaerobic metabolic processes in the ex- perimental animals. Eighty-four animals were brought into the laboratory and randomly assigned to CON, SPT and END activity groups. One half of the animals in each activity group received vitamin C supplementation (C group) and the remaining animals received a placebo (No C group). Vitamin C supple- mentation was administered orally by syringe with a dosage of 2.4 mg vi- tamin C (Merck) in a .1 ml 5% sugar solution per 100 g body weight. The No C group received an identical quantity of the sugar solution per unit of body weight. An eight week treatment period began when the animals were 84 days of age. Selected animals were sacrificed 72 to 96 hours after their last training session. Selection criteria developed for training performance resulted in a final cell frequency of ten animals per treatment group. Wet weight, dry weight, length and ash content were determined for the right femur 28 29 of each animal. Relative weights were determined for each of the mea- surements to account for the size of the animal. Absolute weights indicated significantly (P<.05) heavier bones in the CON group. The END and CON groups had significantly greater absolute lengths than did the SPT animals. Absolute ash weight in the CON group was also significantly larger than the SPT group. In all relative measurements, the SPT and END groups had signifi- cantly larger values than did the CON animals (P<.05). Analysis of variance of the C group and the No C group indicated significant differences in absolute length and relative ash, however post hoc tests revealed no significant differences between the C and the No C groups on these measurements. Conclusions 1. Both the SPT and END training regimens resulted in greater relative weight, relative length and relative mineral content in the femur of the trained animals when compared to the control animals. 2. The inclusion of vitamin C in this investigation did not appear to serve as a preventive to long growth retardation in the trained ani— mals. 3. 0n the basis of this investigation, it appears that judgment must be withheld on the effect of vitamin C on long bone growth. Recomnen dati ons 1. The present study should be repeated with further analysis of the bone ash as to specific mineral content. 3O Power-type events for animals must be included to add to the present knowledge of bone adaptations to intense training. High jumping and weight lifting programs should be developed for this purpose.. Further investigation into the mechanism of vitamin C in the bone during high intensities of training should be investigated. 10. 11. 12. 13. 14. BIBLIOGRAPHY Altman, P. and D. S. Dittmer. Biological Data Book, Philadelphia: Saunders, 1964. Anderson, D. Personal Communication, 1976. Bacchus, H. 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Effects of parathyroid hormone on bone of thy- roparathyroidectomized rats. Am. J. Path., 75:529, 1974. Wells, R. L. and W. W. Heusner. A Controlled running wheel for small laboratory animals. Lab. Animal Sci., 21:094, 1971. Yevich, P. P., W. D. VanHuss, R. E. Carrow, H. J. Montoye and J. Weber. Pulmonary pathology to be considered in exercise research in the rat. Res. Quart., 40:251, 1969. 36 APPENDIX A TRAINING PROGRAMS Table A-1. Modified Eight Week Sprint Training Program fo Controlled-Running Wheels r Postpubertal and Adult Male Rats in Total Ac- Time Time Total celer- Work Repeti- Be- of Total Work Day Day ation Time Rest tions No. tween Run Prog. Exp. Time of of Time (min: Time per of Bouts Shock Speed (min: Meters (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEM TWT 0 4-T -2 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --- 5=F -l 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --- l 1=M 1 2.0 00:10 10 10 8 2.5 1.2 36 42:50 480 800 2=T 2 2.0 00:10 10 10 8 2.5 1.2 36 42:50 480 800 3=W 3 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 4=T 4 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 5=F 5 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 2 1=M 6 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 2=T 7 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 3=W 8 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 4=T 9 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 5=F 10 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 3 1=M 11 1.5 00 15 30 6 7 2.5 1.2 72 43:00 756 630 2=T 12 1.5 00 15 30 6 6 2.5 1.2 81 36:30 729 - 540 3=W 13 1.5 00 15 30 6 6 2.5 1.2 81 36:30 729 540 4=T 14 1.5 00 15 30 6 6 2.5 1.2 81 36:30 729 540 5=F 15 1.5 00 15 30 6 6 2.5 1.2 81 36:30 729 540 4 1=M 16 1.5 00 15 30 6 6 2.5 1.2 81 36:30 729 540 2=T 17 2.0 00 15 30 5 6 2.5 1.2 90 32:00 675 450 3=W 18 2.0 00 15 3O 5 6 2.5 1.2 90 32:00 675 450 4=T 19 2.0 00 15 30 5 6 2.5 1.2 90 32:00 675 450 5=F 20 2.0 00 15 3O 5 6 2.5 1.2 90 32:00 675 450 5 1=M 21 2.0 00 15 30 5 6 2.5 1.2 90 32:00 675 450 2=T 22 2.0 00 15 30 5 6 2.5 1.2 99 32:00 743 450 3=W 23 2.0 00 15 30 5 6 2.5 1.2 99 32:00 743 450 4=T 24 2.0 00 15 30 5 6 2.5 1.2 99 32:00 743 450 5=F 25 2.0 00 15 30 5 6 2.5 1.2 99 32:00 743 450 6 1=M 26 2.0 00 15 30 5 6 2.5 1.2 99 32 00 743 450 2=T 27 2.0 00 15 3O 5 6 2.5 1.2 108 32:00 810 450 3=W 28 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 4=T 29 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 30 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 7 1=M 31 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 2=T 32 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 3=W 33 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 4=T 34 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 35 2.0 00 15 3O 5 6 2.5 1.2 108 32:00 810 450 8 1=M 36 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 2=T 37 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 3=W 38 2.0 00 15 30 5 6 2.5 1.2 108 32 00 810 450 4=T 39 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 40 2.0 00 15 30 5 6 2.5 1.2 108 32:00 810 450 This training program is a modified version of a standard Sprague-Dawley strain(23,49). All animals should be exposed to a minimum of one week of start of the program. Failure to provide this adjustment program designed using male rats of the voluntary running in a wheel prior to the period will impose a double learning situation on the animals and will seriously impair the effectiveness of the training program. APPENDIX A—-continued 37 Table A-2. Modified Eight Week Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels Total Ac- No. Par- Time Time Total celer- Work Repeti- of tial Be- of Total Work Day Day ation Time Rest tions Com- Bouts tween Run Prog. Exp. Time of of Time (min: Time per plete (min: Bouts Shock Speed (min: Meters (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts sec) (min) (ma) (m/min) sec) TEM TWT O 4=T -2 3.0 40:00 10 1 l 5.0 0.0 27 40:00 --- --- 5=F -1 3.0 40:00 10 l 5.0 0.0 27 40:00 --- --- 1 1=M 1 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 2=T 2 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 3=W 3 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 4=T 4 1.5 05:00 0 l 3 5.0 1.2 36 25:00 540 900 5=F 5 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 2 1=M 6 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 2=T 7 1.0 07:30 0 1 2 5.0 1.2 36 20:00 540 900 3=W 8 1.0 07:30 0 l 2 2.5 1.2 36 17:30 540 900 4=T 9 1.0 07:30 0 l 2 1.0 1.2 36 16:00 540 900 5=F 10 1.0 15:00 0 1 l 0.0 1.2 36 15:00 540 900 3 1=M 11 1.0 15:00 0 1 1 05:00 1.0 1.2 36 21:00 720 1200 2=T 12 1.0 15:00 0 l 1 07:30 1.0 1.0 36 23:30 810 1350 3=W 13 1.0 15:00 O 1 l 10:00 1.0 1.0 36 26:00 900 1500 4=T 14 1.0 15:00 0 l 1 12:30 1.0 1.0 36 28:30 990 1650 5=F 15 1.0 15:00 O l 2 1.0 1.0 36 31:00 1080 1800 4 1=M 16 1.0 15:00 0 l 2 05:00 1.0 1.0 36 37:00 1260 2100 2=T 17 1.0 15:00 0 1 2 07:30 1.0 1.0 36 39:30 1350 2250 3=W 18 1.0 15:00 0 1 2 10:00 1.0 1.0 36 42:00 1440 2400 4=T 19 1.0 15:00 O 1 2 12:30 1.0 1.0 36 44:30 1530 2550 5=F 20 1.0 15:00 0 l 3 1.0 1.0 36 47:00 1620 2700 5 1=M 21 1.0 15:00 0 1 3 05:00 1.0 1.0 36 52:00 1800 3000 2=T 22 1.0 15:00 0 l 3 07:30 1.0 1.0 36 54:30 1890 3150 3=W 23 1.0 15:00 0 1 3 10:00 1.0 1.0 36 57200 1980 3300 4=T 24 1.0 15:00 0 1 3 12:30 1.0 1.0 36 59:30 2070 3450 5=F 25 1.0 15:00 0 1 4 1.0 1.0 36 63:00 2160 3600 6 1=M 26 1.0 15:00 0 1 4 1.0 1.0 36 64:00 2160 3600 2=T 27 1.0 30:00 0 l 2 5.0 1.0 36 65:00 2160 3600 3=W 28 1.0 30:00 0 1 2 2.5 1.0 36 62:30 2160 3600 4=T 29 1.0 30:00 0 1 2 1.0 1.0 36 61:00 2160 3600 5=F 30 1.0 60:00 0 l l 0.0 1.0 36 60:00 2160 3600 7 1=M 31 1.0 60:00 0 1 l 0.0 1.0 36 60:00 2160 3600 2=T 32 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 3=W 33 1.0 60:00 0 1 l 0.0 1.0 36 60:00 2160 3600 4=T 34 1.0 60:00 0 l 1 0.0 1.0 36 60:00 2160 3600 5=F 35 1.0 60:00 O 1 l 0.0 1.0 36 60:00 2160 3600 8 1=M 36 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 2=T 37 1.0 60:00 0 l 1 0.0 1.0 36 60:00 2160 3600 3=W 38 1.0 60:00 0 1 l 0.0 1.0 36 60:00 2160 3600 4=T 39 1.0 60:00 O 1 1 0.0 1.0 36 60:00 2160 3600 5=F 40 1.0 60:00 O 1 1 0.0 1.0 36 60:00 7160 3600 This training program is a modified version of a standard program Sprague-Dawley strain (23,49). designed using male rats of the All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation in the animals and will seriously impair the effectiveness of the training program. 323 APPENDIX 8 BASIC STATISTICS FOR TRAINING DATA Basic statistics for Percentage of Body Weight Loss, Environmental Factors and Performance Criteria Simple Correlations Standard Air Per Bar Per Body Variable Na Mean Deviation Temp Humid Press Wt Loss PEM at Air Temp (F) 367 72.9 4.8 Per Humid 367 39.0 12.1 ..110 Bar Press (mmHg) 367 740.7 4.3 .276 -.713 Per Body wt loss 367 1.7 .5 .066 —.206 .044 PEM 367 55.4 20.2 .199 -.359 .121 .258 PSF 367 56.8 19.5 .398 -.312 .164 -196 .872 §P_TM£ Air Temp (F) 376 73.1 4.6 Per Humid 376 38.5 12.3 .125 Bar Press (man) 376 740.8 4.2 .263 -.715 Per Body wt loss 376 1.7 .6 .000 -.200 .046 PEM 376 61.6 25.9 .165 -.383 .210 .115 PSF 376 62.6 23.4 .260 -.271 .129 .032 .868 010. 9. Air temp (F) 340 73.9 4.0 Per Humid 340 47.1 10.7 .134 Bar Press (ang) 340 739.5 3.8 .288 -.675 Per Bocy wt loss 340 2.5 1.0 .374 .139 -.240 PEM 340 82.8 24.7 .279 -.173 .232 -.O69 PSF 340 70.7 18.6 -.403 —.O83 .159 -.118 .685 99 u_ 9 Air Temp (F) 348 73.9 4.0 Per Humid 348 47.0 10.6 .151 Bar Press (ang) 348 739.5 3.8 .294 -.677 Per Body wt loss 348 2.6 1.0 .439 .114 -.159 PEM 348 82.2 18.7 .231 -.253 .286 -.003 PSF 348 69.6 19.2 .254 -.102 .153 .021 .748 N8 = total days training, all animals