II’II‘ :.""1- -- ”It“. r-II' n ZIIL I'LL 'I IL "I“I'I‘Y‘II', I'" ”1:. ‘IIL'L’II nzII‘II'I‘j: II_:.I,; L‘ . I ‘.|'~ . . .- .I II LLL ‘ ' .. I. . LII. L. “L. LL” L” ' L'L'L' LL": "' 5"" 'I. LLUI Pl LLILL I'ILLL'. ' I "LIL' I I'ILLL'I 'LIL ,lII JLLJLL' n."" IIIiITL- ' L"‘ “71"" .5» Lay/=11“ '3' II I: . . . . . ' . I :3” '1...” L . L L I LLer' Il‘lp 'L'LL' ,LLLLL’ILL -. .1 '.' " ' II . DMLLILLLQLLL ILLLLI“; ILLLLn ' LLLLII'IW‘" ILL .LLLLLLJII‘ .'L"' 'y_‘:;.& 4:35:53 -.. —-‘_ J L I“! I ILL, ‘L L - LL ' LL W. L L ILLL'LLI, II'IIILII LII IL'IILIILLLLLILLLLLLILL 'LL'LL'LLLILLI'LL" 'LLI'ILLLL .I: ILLIIL LL LLLLLLLLLL “L'Ll "'LLLLLI'JIL"LILI nLL LLLLL'L " 'LLL 'LLLL’ LLLL'LL LLLL'L'L'L'L' L 'L' L' L" ' 'LIIL L'LLILL‘ LIL'L LL‘TLLL 2‘. LLLL'L'LL' LIL. IL LL"" LILI LLLIL;‘ Ll "LLLL LLL I‘m. Ll! L‘ ,LL'L'L L I . 1’ LL .: LL. LL . ILLL'II'L'LILILL "LLLLLLLL I L LL. 9‘ ' ‘t'L-Lu . LLIL iLIIL 'I .L I Ist 'LLL'LLL L""'L'LLL III 'L' %' "II“IIIILII‘I L M. IIII‘L'LL'W I I I-IIIIII I .. IIIIIIIII‘I 'l L'. L LLL'I'I LLL I I'j l‘. .’ L'L'I' L 'XLL LII: j:L , II. JI'.'""".'LLI"I'I' "L'::" PILL"- LL. . ..II.JII|L 'LILI'" ILLILLH LLLL 'LIL LL.‘ III‘ .. ILIIIILLLI .I. . . .‘. . .I."" 'iL'n‘s'. L'nn '|:-'L' ‘ ‘ . I. L ' " .‘I ., .'i " ""‘z " In -l'-' ' L“ n..."""~"I°'VI.'.L..-'I§I'“'-J'Ll \ 'LLL'L'LLLL w. " 'I'L'L’L'L' "L“ L‘IL'L ‘ L""I' ' . .n . r L’ 3 ~""-"LI':;.I «2 I‘LL." .LLL'L'L ‘ .- L'L ‘I"‘» LL ' L " .H‘VLI" :,'L"‘ ’1} ,. "3")IIIL'I L' '4 L" J "'.'. LLL'LILI 'f- ;" . L" H’LIIIII WI. '*lx."li..r---».. .IIL-Lw“ "'L' L ‘ L.'.i'J-‘z 9"» I'LLI 'II' L'L' nL ‘ '. "L II'I LILLIL I IIIIL‘II n nI I1|LI L,I_III 04:41“ I L'L' 'IZLI'ILL' JLL" .I I%hf~ 'II'UL'I ILJ'L' L .. . '.‘;I' ‘I 'ni "" 'HL 1'! ' L' L.'I'LLn' 'L'L'L " 'LLI'L 'IL 'LL-‘L _'"L!"L' '. L . IL' ,".,'I'. I—II'nI LL'LLj'fi LILL L'L 'LLLL 'LILIIILL'L ILLLLn' ' II'LI" L MIL", I IIIIIIIIIIII IIII LI III IIII II III "'.'I [.‘L' LII 'LJLL . IIIL - II 'I'IIILLLL'L' LILL'L'L IIILLLLL'1IIL‘IIILkLIIIIfiL1LLLLI h I III I I I; IIIIIII .I II II III II II L. ILLI II IIIIILII I 'II NIL” 'LH.,II'L ILI'IILIIILIL‘JIIIIILLI L'.""|LI'L“IIILIIII‘IIIIH}:'1I;""" I LLJ' .' ' "qr-ILL 'L'M,,I "L""L"' I'LLLIL'L'LI: ILL" 'lI'LL W. L' '.I'" W L'Ln 'L""" L" ‘LL"L"LL" 'L'h' " L'L'L'" "" ' . ':l '1 L' . LL"L‘ I I. I; I L LL 1 l I. ' ‘ I I ..L'.LLLLLI« .n I..."“' ' . ILL'ULL ILI'LL I‘L. I L' n'.‘ I" unmf ”I " ILL“L""III. I'LLL."‘L "IIIL'LuLMLI 1|.LI' §I~ . ' L . l II'L LL' .. , ‘L LI‘IIL L'IL. ULIILII 'LI'IIL'L'L LLILLIL I'I I'L'IL "'1' l ' LIBRARY . l‘ W llalllfllfllllI”Mililfllljflljylflllllfljflallfllflll 293 This is to certify that the thesis entitled THE EFFECTS OF EXERCISE AND VITAMIN C SUPPLEMENTATION ON VARIOUS MORPHOLOGICAL PARAMETERS IN THE FEMUR OF MALE ALBINO RATS presented by Darlene Ulmer Jakubiak has been accepted towards fulfillment ‘ of the requirements for M.A. degreein Physical Education Major professor 0-7 639 OVERDUE FINES ARE 25¢ PER DAY ‘ PER ITEM Return to book drop to remove this checkout from your record. THE EFFECTS OF EXERCISE AND VITAMIN C SUPPLEMENTATION ON VARIOUS MORPHOLOGICAL PARAMETERS IN THE FEMUR OF MALE ALBINO RATS BY Darlene Ulmer Jakubiak 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 1979 ABSTRACT THE EFFECTS OF EXERCISE AND VITAMIN C SUPPLEMENTATION ON VARIOUS MORPHOLOGICAL PARAMETERS IN THE FEMUR OF MALE ALBINO RATS BY Darlene Ulmer Jakubiak This study was undertaken to determine the effects of eight weeks of strenuous sprint (SPT) or endurance (END) exercise and Vitamin C supplementation on various morpho- logical growth parameters of the femoral shaft of normal male albino rats. At the end of this period ten animals per treatment group were sacrificed. The left femurs were removed, wet weights and lengths determined, and then sectioned transversely at three levels along the shaft and processed for morphological studies. In absolute terms, both SPT and END animals had decreased body weights and femurs of decreased weight, length, and cross-sectional size when compared to sedentary (SED) con- trol animals. However, relative to body weight, the femurs of trained animals were actually greater in size when com- pared to those of untrained animals. Neither training performance nor long bone growth was effected by the Vitamin C supplementation. To my huaband and WW ii ACKNOWLEDGEMENTS A very special thank you is extended to Mr. A. R. Villanueva of the Calcified Tissue Research Laboratory at Henry Ford Hospital, Detroit, Michigan for his warm recep- tion, time, and expertise offered me regarding the technical aspects of my research. My sincere appreciation is given to Dr. W. D. Van Huss for the continued guidance, counseling and encouragement he provided me as my graduate advisor and committee chairman. Deep appreciation is offered to the members of my com- mittee, Dr. W. D. Van Huss, Dr. R. Echt and Dr. R. E. Carrow for making my research a worthwhile experience. A special thank you is offered to Dr. R. R. Roy for his moral support during times of frustration. iii TABLE OF CONTENTS CHAPTER Page I. THE PROBLEMOOOIO.0.O.O...COOOOOOOOOOOOOOOOOOOO [.1 Research Hypotheses........................ Research Plan.............................. Rationale.................................. Limitations................................ coma-w II. LITERATLJRE REVIEWOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 10 Normal Compact Bone Morphology and Growth.. 10 Immobilization and Disuse Effects on Long Bones................................... 15 Exercise Effects on Compact Bone........... 17 Vitamin C and Bone......................... 24 Vitamin C and Exercise as a Stressor....... 30 Recommended Dosages of Vitamin C........... 34 III. METHODS ANDMATERIALSOOOOOOOOOOOOOOOOOOOOOOOOO 36 Experimental Animals....................... 36 Exercise Groups............................ 37 Control Group........................... 37 Sprint Group............................ 37 Endurance Group......................... 38 Diet Subgroups............................. 38 Vitamin C............................... 39 Placebo Group........................... 39 Training Procedures........................ 39 Animal Care................................ 41 Sacrifice Procedures....................... 41 Bone Sectioning Procedures................. 44 Morphological Measurements................. 45 Cortical Wall Area...................... 45 Marrow Cavity Area...................... 47 Cortical Wall Area/Total Area Ratio..... 47 Greatest wall Thickness and Least Wall Thickness............................ 48 Greatest Total Diameter and Greatest of the Least Total Diameter............. 48 Analysis of Data........................... 49 iv CHAPTER Page IV. RESULTS AND DISCUSSIONOOOCOCCOOOOOOOOOOOOOOOO 51 Training Results.......................... 51 Activity Level Results.................... 55 Body Weights at Sacrifice.............. 55 Femur Weights at Sacrifice............. 60 Femur Lengths at Sacrifice............. 61 Femoral Diameter Measurements.......... 61 Femoral Area Measurements.............. 63 Vitamin C Supplementation Results......... 66 Discussion................................ 73 V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS.... 80 swam-coco...ooooooooo0.0000000000000000 80 conCIuSions I O O O O O I O O O O O O O O O O O O O O O O O O O O O O O O 82 Recomendations O C O O O O O O O O O O O O O O O O O O O O O O O O O O 84 REFERENCES...00....OOOOOOOOOOOOOOOOOOOOO00.00.0000... 85 APPENDICES A O TRAINING PROGRAMS O I O O O O O O O O O C O O O O I O O O O O O O O O O O O 9 5 B. BASIC STATISTICS FOR TRAINING DATA............ 97 LIST OF TABLES TABLE Page 1. Analysis of variance for overall training effects and Newman-Keuls tests of paired com- parisons for absolute and relative bone data.... 56 2. Analysis of variance for overall Vitamin C effects and Newman-Keuls tests of paired come parisons for absolute and relative bone data.... 67 A-l. Modified eight week spring training program for postpubertal and adult male rats in controlled- running WheelsOOOOOCOO...COCOOOOOOOOOOOCOOOOOOOO 95 A22. Modified eight week endurance training program for postpubertal and adult m ale rats in contrOlled-running WheeISOOOOOIOOOOOOOOOOOOIOIOO 96 B-l. Basic statistics for percentage of body weight loss, environmental factors and performance criteriaOOOOOOOOOOOIOOO0.0.0.000...OOOOOOOOOOOO. 97 vi LIST OF FIGURES FIGURE PAGE Diagram representing the proximal section of the femoral shaft demonstrating the Greatest Total Diameter and the Greatest of the Least Total Di- ameter.......................................... 49 Mean daily percent expected meters run for Sprint animaISOOOOOOOO0.00000000000000IOOOOOOOOO 53 Mean daily percent expected meters run for En- durance animals...OOOCOOCOOOOOOOOOOIII0.0.0.0... 54 vii CHAPTER 1 THE PROBLEM Physical activity is known to produce a variety of changes in the normal anatomical and physiological func- tion of animals and man (2, 3, 4, 7, 8, 23, 24, 25, 27, 36, 39, 63, 65, 84, 102, 108, 109, 120). More specific to the interest of this study however, are the types and degree of changes found in growing long bones due to exercise. Re— view of literature has found limited and sometimes contra- dictory reports concerning the response of growing bone when subjected to varied types of strenuous physical activity. Some data indicate that physical activity tends to increase bone weight, length, diameter, bone wall thick- ness and density (2, 12, 13, 14, 15, 24, 25, 56, 57, 58, 71, 88, 91, 96, 97, 108, 109, 114). In contrast, other reports indicate a decrease in overall stature with shorter long bone length, no change or slight increase in bone diameter and weight and accelerated maturation of long bones resulting in early closure of their epiphyseal plates (2, 3, 4, 13, 54, 56, 57, 58, 59, 60, 66, 68, 87, 108, 109, 112). With reference to such conflicting data it can therefore be stated that adaptation of bone to physical training is a function of intensity of exercise 2 (13, 64, 97). Regardless of the potentially negative effects of strenuous exercise on bone growth, implementation of rigorous conditioning programs during the rapid growth period of late childhood and early adolescence have been promoted. Not only is there a need for concrete identifica- tion of specific training effects on growing bone at var— ious levels of work intensity but also determination of the factors which might prevent the occurrence of negative training effects. The concept that exercise consists of a continuum of specific activities each of which elicits a specific re- sponse within the organism has evolved from current re- search on the topic (30, 75, 92, 115). The spectrum ranges from activities of very high intensity work of short dura- tion utilizing uniquely anaerobic metabolic pathways to those activities characterized by very low intensity work of long duration utilizing uniquely aerobic metabolic path- ways (115). Unfortunately, little information is avail- able regarding the specific response of bone when subjected to this spectrum of activity levels. An adequate supply of Vitamin C has been found to be a necessity for normal overall growth and bone formation (22, 29, 49, 50, 69, 70, 73, 86, 90). Although the specific function of Vitamin C in the body has not been adequately identified it is known to be a key factor in collagen for- mation, bone matrix deposition, prevention of scurvy and adrenal steroid release (31, 46, 47, 50, 51, S3, 61, 69, 3 70, 73, 86, 93, 111, 113, 121). In addition, Vitamin C also appears to play a role in adaptation to stress (9, 78, 79, 80, 81, 82, 83, 93). However, its specific function in bone adaptation to stress is unknown. Research Hypotheses The hypotheses to be tested in this study are as follows: 1. The body weights of sedentary animals should be signi- ficantly greater than those of exercised animals. 2. There should be a significant difference between the body weights of sprint and endurance trained animals. 3. The femoral bones of exercised animals should be signi- ficantly shorter in length but heavier in weight than the femoral bones of sedentary animals. 4. There should be a difference in femur length and weight between the sprint exercised and endurance exer- cised animals. 5. The total greatest cross-sectional diameter of femurs from exercised animals should be greater than in femoral bones of sedentary animals. ' 6. There should be a difference in the total greatest cross-sectional diameter of femurs between sprint exercised animals and endurance exercised animals. 7. Femoral bones of exercised animals should have a great- er cortical wall area than femoral bones of sedentary animals. 8. There should be a difference in the femoral cortical wall areas between sprint exercised animals and endurance exercised animals. 9. The femurs of exercised animals should have greater marrow cavity areas than femurs of sedentary animals. 10. There should be a difference in the marrow cavity area of femurs between the sprint exercised animals and endur- ance exercised animals. 11. There should be changes in the cross-sectional shape of femurs from exercised animals but not in femurs from sedentary animals. 12. There should be a difference in the cross—sectional shape of femurs between the sprint exercised animals and endurance exercised animals. 13. Exercised animals receiving Vitamin C supplementation should have significantly less long bone growth impairment than exercised animals not receiving Vitamin C supplementa- tion. Research Plan Eighty-four male albino rats (Sprague-Dawley strain) were randomly assigned to one of the following three activity groups: (a) Sedentary Control (SED), (b) Sprint Running (SPT), and (c) Endurance Running (END). One-half of the animals in each of the activity groups received 5 2 mg. of Vitamin C in .1 cc of 5% sugar water solution per 100 gm. of body weight daily. The remaining animals only received similar amounts of sugar water solutions according to their own body weight as a placebo. The SPT and END training regimens were implemented using electronically controlled running wheels (119). The two programs were more intensive than any exercise routines pre- viously used in this laboratory (see Appendix). The animals in the SPT group were subjected to an eight week 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 11 days these animals were expected to complete a 60-min. continuous run at a speed of 36 m/min. The END program has been designed to overload the aerobic capacity of the experimental animals. Ten animals in each of the six activity-diet subgroups were sacrificed at the conclusion of eight continuous weeks of treatments. Health and training performances throughout the treatment period were used as selection criteria for these animals. After removal of various tissues and organs for use in a larger activity-diet study the animals were 6 frozen and stored in a deep freezer at a temperature of 0° C. The animals were then removed from the cold and allowed to thaw at room temperature for a 24 hour period. The left femur was removed from each animal and refrigerated in an air-tight container. Each bone was then sectioned transversely at three different levels along its shaft, hand ground to approximately 50 microns in thickness, stained and mounted on glass slides. Various morphological parameters were determined with use of a Praedo micro- projector. The weight and length of the femurs were deter— mined from the right femur of each animal which was used in a bone mineral content study. Analysis of variance proce- dures were used to analyze the data. Rationale The SPT and END training regimens have been designed to simulate high intensity exercise programs for humans. It was expected that the work intensity of the two training regimens would induce growth and morphological changes in the femurs of the exercised animals. In addition, it was expected that there would be a differential response in the bone parameters under study between the SPT and END train- ing programs. Diet supplementation with Vitamin C (ascorbic acid) was included in the study as a possible preventative of ex- pected decrement in bone growth of the exercised animals. 7 Vitamin C is known to be a key factor in the normal forma- tion of bone matrix (39, 40, 49, 86, 105, 111), collagen production (10, 39, 40, 49, 51, 70, 105, 111, 113), bone growth (29, 39, 40, 49, 50, 73, 86, 90, 121), adrenal gland function (10, 31, 46, 47, 53, 61, 69, 73, 78, 79, 80, 81, 82, 83, 93, 121), and adaptation to stress (46, 47, 49, 52, 53, 55, 61, 73, 78, 79, 80, 81, 82, 83, 89, 93, 121). Although the rat synthesizes its own supply of Vitamin C, there are several reasons why it was used in this study rather than the guinea pig which, like man, does not syn- thesize the vitamin. Unlike the guinea pig, the rat is easily trained to run in a wheel. Since exercise is a major component of this study it is mandatory that trainable animal subjects be used. Also, unique to the rat is that although the animal is postpubertal at 84 days of age, which is when all experimental treatments were initiated, their skeletal growth continues until the animals are over 400 days of age (24). Thus, data concerning exercise effects on skeletal growth is obtainable. In addition, many of the earlier studies concerned with the subject of exercise effects on long bone growth have used the rat femur as their model (13, 16, 25, 59, 71, 87, 96, 97, 112). The techniques used for data collection of all morpho- logical parameters have been described by Frost (33, 34), Schock (99, 100), Villanueava (116, 117), and Weibel (118). 8 Limitations 1. Direct inferences to human beings cannot be made with results from animal studies. 2. The power of the statistical analysis may be limited due to the small size of the diet-activity subgroups. 3. Optimal durations of treatments have not been clearly established for the achievement of significant results between groups. 4. Optimal types of training regimens may not have been selected to show differential growth effects on bone. 5. Since the rat synthesizes its own supply of Vitamin C, the full effect of Vitamin C supplementation on bone growth may not be revealed in this study. 6. The results of this study are specific to the femoral bones of male albino rats. Therefore, the data apply only to the SPT and END training regimens used in this study. 7. The animals for this investigation were received in three separate shipments due to limitations of personnel and equipment. The activity treatments were not randomized across shipments. This lack of randomization could intro- duce a bias in the exercise related data. Since the animal supplier (Hormone Assay, Inc., Chicago, Illinois) had a well-controlled substrain of Sprague-Dawley rats the proba- bility of a genetic bias is small. Every possible effort was made to control unique external factors in the labora- tory. The diet treatments were randomized across all shipments. 8. Due to unavailability of personnel and feasible tech- nical procedures for preparation of bone for morphological analysis, the rat femurs were left intact with the remain- ing animal carcasses and frozen at a temperature of 0° C. This procedure may have produced some variation in the morphological data of the rat femurs. 9. The procedure followed for preparation of bone sections for microsc0pic analysis resulted in some incomplete sec- tions thus limiting the final sample size of the study. CHAPTER II LITERATURE REVIEW The literature review will cover six primary topics. Literature describing normal bone morphology and growth patterns will be covered in the first section. The follow- ing two sections will summarize literature concerning im- mobilization effects and exercise effects on bone, respec- tively. In the fourth section, the general function of Vitamin C in the body will be discussed. The relationship of Vitamin C and adaptation to stress will be covered in the fifth section while the last section will be devoted to reviewing literature on the recommended dietary allowances of Vitamin C needed to meet these various conditions of Stress . Normal Compact Bone Morphology and Growth Long bone, such as the femur, is characterized by having a hollow shaft composed of compact lamellar bone with an epiphysis at each end composed of trabecular or cancellous bone. Osteoblasts produce the matrix of compact bone in the form of concentric lamellar rings surrounding Haversian canals. Once the osteoblast ceases to produce bone matrix it resides in a lacuna in the midst of its matrix and is 10 11 now termed an osteocyte. The osteocytes of lamellar bone remain alive and in contact with each other via small nutrient transport channels called canaliculi. However, the major transportation of nerve and blood supplies is accomplished vertically via Haversian canals and horizon- tally via Volkman's canals (ll, 19, 35, 40). According to Wolff's law, as described by Koch (64), Saville and Smith (96), and Moss (77), there is a direct relationship between skeletal shape and function. In ob- servations by Currey (20), Epker (28), Frost (35), Hooper (48), Koch (64), and Moss (77), not only will the applica- tion of mechanical stresses such as weight bearing or pull by muscle attachments initiate a variety of adaptive changes in the structural aspects of the skeleton but also these changes will be specifically related to the type, intensity, and duration of the applied activity. When compressive forces are applied, such as that created by weight bearing due to physical activity, a concave surface develops on one side of the effected bone shaft. The phys- iological response is increased osteoblastic activity re- sulting in increaSed bone deposition in an attempt to fill in the hollowed out area of the bone shaft. In contrast, when tensile forces are applied to bone, such as that created by the pull of muscle attachments, a convex surface develops on the side of the effected bone shaft opposite to the concave surface. In this case, the physiological re- sponse is increased osteoclastic activity resulting in 12 increased bone resorption in an attempt to smooth out the bulging area of the shaft. The overall effect is to cause change in shape and structure in the compactum of long bones specific to the type of stress applied. The process of maintaining bone continuity and adapta- tion to stress is called bone remodelling. In a study by Takahashi et al. (110), it was found that osteoclastic activity resulting in removal of old necrotic osteocytes and bone tissue preceded invasion by active bone producing osteoblasts. Collins (19), Frost (34, 35) and Larson (68) confirmed this remodelling sequence of resorption before deposition. In addition, Frost (35) stated that the re- modelling process serves two primary functions. First, it provides a mechanism for self repair of fatigue-like damage resulting from excessive exposure to mechanical stresses. And secondly, it functions to ensure maintenance of an effective electrolyte storage depot and blood-bone buffering system. The growth process generally involves increasing long bone size in conjunction with maturation of its cellular and inorganic components. As described by Belanger (11), Collins (19), Frost (35), Ham (40) and Larson (68), long bone growth is accomplished by two key processes. The process of endochondral ossification increases long bone length via interstitial growth of fetal cartilage rudiments and maturation of epiphyseal plates while appositional growth increases long bone width. During interstitial 13 growth, chondroblasts congregate at the midpoint of the cartilage rudiment shaft to form a primary ossification center. The chondroblasts then produce large quantities of avascular cartilage matrix thus expanding the primary ossi- fication center towards each end of the shaft. As the avascular cartilage matrix increases, nutrient and waste products are no longer able to diffuse the added distance to and from the chondrocytes located at the midpoint of the shaft. The chondrocytes in the area become necrotic and die. Blood vessels then invade the area along with bone producing osteoblasts. The osteoblasts immediately begin manufacturing osteoid tissue which soon becomes calcified thus completing the replacement of cartilage with bone and increasing the length of the shaft. In addition to the interstitial growth process, long bone length is also increased with the formation of secon- dary ossification centers at the epiphyseal plates. Belanger et a1. (11), as well as Moss (77), Schenk (98), Siegling (103), and Sissons (104), described this process in great detail. The cartilage cells of the epiphyseal plate are characteristically arranged in columns like stacks of coins. Within each column of cells are four zones of chondrocytes having various shapes and functions. The uppermost zone of chondrocytes is composed of a few layers of immature cartilage cells evenly distributed throughout the intercellular matrix. The proliferative zone, which actually forms the top of the stacks, contains l4 chondrocytes which are flat and thin in appearance and function as producers of new cartilage cells. Below this zone is a region of hypertrophied chondrocytes. These cells appear rounded and swollen and function as a pro- ducer of intercellular matrix. The bottom of the epiphy- seal plate houses the zone of calcifying cartilage. It ex- tends into the bone shaft and is the area where carilage matrix is resorbed and replaced by bone. As this bone deposition front advances toward the epiphysis, the length of the bone is gradually increased. Once all of the chon- drocytes composing the various zones of an epiphyseal plate have matured and been replaced with bone, growth stops. Therefore, both interstitial growth and epiphyseal plate maturation as subprocesses of endochondral ossification result in maturation and increased length of long bone. In order to attain normal long bone growth however, there must be an increase in width commensurate with any increase in length. The process of increasing long bone width is referred to as appositional growth and has been adequately described in works by Belanger (11), Collins (19), Frost (35), and Ham (40). Surrounding the periosteal surface or exterior of the shaft of long bone is a layer of connective tissue called the periosteum. Lying underneath the periosteum are undifferentiated stem cells and osteo- blasts. Stimulation of the osteoblasts as part of the growth process results in addition of bone tissue to the periosteal surface of the shaft. However, the addition of 15 bone to the periosteal surface of the shaft by osteoblastic activity must be balanced by resorption of the interior or endosteal surface of the shaft by osteoclastic activity. The net result is an increase in the total width and mar- row cavity area in the shaft of long bones. Immobilization and Disuse Effects on Long Bones Abundant information regarding immobilization and disuse effects on the skeleton is available. However, there appears to be some variation in the observed skeletal changes reported concerning this topic. In a review article by Booth (13), data by several authors concerning immobilization effects on bone were cited. Allison and Brooks (5), in a denervation study using dogs, found that length as well as matrix and mineral contents were decreased in long bones. They also found that the marrow cavity diameter was increased while the total diameter was decreased indicating that the cortical wall thickness of the bone shaft was thinner. Along these same lines, they noted increased porosity in the bone shafts. In addition, immobilization increased the width of the epiphyseal plates while inhibiting the growth in length of the long bones. Booth (13) also referred to several other immobilization studies reporting results similar to those of Allison and Brooks (5). Klein (62) in a study also using dogs, 16 reported that the long bones showed decreased weight, ma- trix, and mineral content, and decreased cortical wall thickness. The cortical walls of the shafts of these bones were also very porous. Kittens with a denervated hindlimb were used to study immobilization effects on bone in an in- vestigation conducted by Gillespie (38). His observations confirmed those of Allison and Brooks (5) and Klein (62) as previously stated. In a study using rats, Armstrong (6) also showed decreases in the length, diameter, matrix deposition and mineral content of long bones after immobili— zation. In works by Geiser and Trueta (1958) as cited by Booth and confirmed by Heaney (42), it has been observed there is increased bone resorption with immobilization due not only to greater osteoclastic activity but also to in- creased blood flow to the effected area. Reductions in bone mineral content and density due to immobilization have been reported in studies by Donaldson (23), Manegold (72), and Abramson (l). Hattner (41), summarizing the effects of weightlessness on the skeleton, substantiates these findings and adds that enhanced vascularization may act as an adjunct to bone rarefaction. In contrast to some of the findings published in Booth's (13) article, Steinhaus (108, 109) and Malina (71) have re- ferred to studies reporting some contradictory data con- cerning immobilization effects on bone. Experimental rat data of Mfiller (1923), as referred to by Steinhaus (108, 109), showed that bones from immobilized limbs are longer 17 in length than bones from active limbs. Malina (71) and Steinhaus (108, 109) went on to report additional data of Friedlander and Thierse (1928) showing immobilized bones to have decreased diameter, increased porosity, decreased min- eral content but increased length. They also observed a reduction in the blood supply of inactive bones. Further, they found that the epiphyseal plates of immobilized bones had increased growth activity. Collins (19), in his book on bone pathology, has also reported that immobilization produced increased bone por- osity due to enhanced osteoclastic resorption as well as decreased blood flow to the effected area. Exercise Effects on Compact Bone Exercise exists as a continuum of specific activity levels having variation in type, point of application, in- tensity, and duration of activity. As a result of such variation, exercise elicits an assortment of changes in compact bone specific to each applied activity level. Authors such as Saville and Smith (96), Koch (64), Moss (77), and Kiiskinen (56) have referred to Wolff's law in an attempt to explain the diversity of exercise—related bone changes. In general, Wolff's law states that every change in the function of a bone is followed by definite changes in its internal architecture and external configuration. Booth (13), in a review article, makes application of l8 Wolff's law with reference to exercise effects on bone by stating that "...adaptation of bone to training is inextri- cably a function of the intensity of training". Thus, as reflected in the literature, inconclusive results are available regarding the specific response of bone to specific levels of activity. In an article by Beyer (12), cadets entering the 0.8. Naval Academy were used as subjects to study the effects of physical training on various growth and physiological parameters. He found that over a four year period, the cadets had favorable increases in height and weight as well as in the physiological parameters under investigation. In a strenuous low-intensity running exercise study using rats, Price—Jones (87) found that the growth rate of the exercised animals was impaired during the treatment period as indicated by decreased body weights when compared with sedentary control animals. There were also decreases in the humerus weights and lengths and femur weights and lengths of exercised animals when compared to those of con- trol animals. Price-Jones's overall conclusion was that strenuous low-intensity exercise of long duration retards growth in length and weight of long bones as well as total body length and weight. In two classic review articles, Steinhaus (108, 109) cited the work of several researchers studying the effects of physical activity on skeletal growth. Some of the re- search presented had results indicating that exercise l9 stimulates growth thus increasing arm and leg length in students participating in physical education programs. On the other hand, research involving strenuously exercised rats claimed that physical activity had a detrimental effect on growth by decreasing bone length and weight as well as body weight. At the same time it was noted that exercise of moderate intensity seemed to stimulate growth reflected by increased body weight although total body length was unaffected. In x-rays of bones and joints of highly specialized athletes such as baseball pitchers, var- ious shape and size changes in the bones of exercised limbs were observed. With reference to all of this conflicting data, Steinhaus concluded that "...pressure on the epiphy- ses of long bones from participatidn in exercise stimulates growth up to an optimal level and beyond this point retards growth". Bipedal rats were used in two studies by Saville (96) and Smith (107) to evaluate the effects of weight bearing on various characteristics of the femur. The femurs of bipedal rats showed increased bone density, volume and weight, increased breaking strength and increased mineral content. There was also an increase in the cross-sectional diameter and cortical wall thickness of the femurs from the bipedal rats although their body weights were lower than controls. It was also noted that the external shape of the femurs varied somewhat between the bipedal and control rats. 20 Rats exposed to a strenuous endurance running program described by Saville and Whyte (97), had hindlimb bones of increased weight, mineral content, density, volume and breaking strength. However, unlike bones from bipedal rats used in previous studies by Saville (96, 107), the bones from exercised rats had no change in their cortical wall thickness. King and Pengelly (60) in an investigation also using rats, subjected their animals to a high intensity sprint running program and a moderate intensity endurance running program. The tibias from the sprint runners were the only bones having a significant increase in density. However, the adrenal glands were also excised from all animals and were found to be heavier in both groups of exercised ani- mals when compared to control animals. Rats exposed to strenuous exercise programs of various intensities and durations designed by Tipton et al. (112), had femurs and tibia-fibula complexes with decreased length, width, and water content. Evaluations of the min- eral content and epiphyseal plate maturation of these bones however, showed no change between the various treatment groups. Likewise, Lamb et a1. (66), using moderately exer- cised rats also found that the animals had lower body weights and shorter tibial lengths when compared to seden- tary animals. On the other hand, Donaldson and Meeser (25) and Borer and Kuhns (l4), exposed rats and hamsters respectively, to 21 voluntary running exercise of low intensity. Results from both investigations showed decreased total body lengths and weights as well as decreased long bone lengths and weights in exercised animals when compared to their respective con- trols. In several investigations conducted by Kiiskinen et al. (56, 57, 58, 59), concerned with exercise effects on bone, mice were assigned to various endurance treatment groups and forced to run on a treadmill. Those animals subjected to the endurance program of light intensity had no change in body weight and femur length. However, the femurs of these animals did have increased weight, mineral content, density, bone deposition and an increased number of bone cells when compared to sedentary animals. On the other hand, those mice participating in the moderate and heavy endurance pro- grams when compared to sedentary animals, not only had lower body weights indicating impaired growth, but also had decreased long bone length as well as decreased vascularity. But, the bones did have increased weight, mineral content, bone deposition, density, and breaking strength. In addi- tion, Kiiskinen et a1. (56, 57) made note of the fact that the heavy endurance running program, when continued over a long duration, produced some detrimental effects in the bones of his mice. The most obvious change was a decrease in bone weight. Consequently, he concluded that exercise of excessive intensity and duration produces an overload 22 thus creating an over-training situation resulting in detrimental bone changes. Mice were also used in two studies by Heikkinen et al. (43, 44) in which the animals were forced to participate in moderate endurance prOgrams of treadmill running. The bone data from these animals paralleled the previously described data from the moderately trained animals of Kiiskinen et al. (56, 57). Several studies concerned with investigating the effects of hard physical labor and exercise on skeletal growth of humans are also prevalent in the literature. Adams (2) found that young black women engaged in hard strenuous labor were taller, heavier, and generally larger in overall size when compared to women not exposed to physical labor. In a roentgenographic examination of bones of Russian laborers, Prive (88) found that physical activity increases bone length, delays ageing and increases bone hypertrophy. He also commented that the observed skeletal changes were specific to the types of physical load placed upon the bones. In an anthropometric study of children on hand preference for work, VanDusen (114) found that the arm bone lengths and widths of preference limbs were significantly greater than those of non-preferred limbs. Buskirk et a1. (15) found similar increases in the length of arm.bones of tennis players although he did not detect any changes in the width of these bones. Adolescent boys actively involved in vari- ous physical activities of low to moderate intensity, were 23 found to have increased height and body weight when compared to inactive adolesdent boys as described in a study by Ekblom (27). With reference to ageing, research by Castillo (18), Dalen (21) and Smith et al. (106) shows that physical activity of low to moderate intensity increases the mineral content of bones and delays the onset of osteoporosis in the aged. Additional age and physical activity studies have been conducted by Reeve et a1. (94) and Lane et al. (67). They found blood flow was increased in the bones of physical- ly active mature adults over that in bones of physically in- active mature adults. Thus, the application of mechanical stressors, such as that created by participation in physical activity, results in increased bone vascularization. Kato and Ishiko (54), reporting on the results of hard physical labor on the growth of Japanese children, have found an overall detrimental effect. Consequential to carry- ing heavy loads on their shoulders, the children were found to be very small in stature, especially in leg length, the femurs and tibias showed early epiphyseal plate closure, and the bones of their feet were deformed. Correspondingly, in an investigation involving growth of adolescent boys participating in a weight training program, Kusinitz and Keeney (65) found that weight and height gains in these boys were impaired when comparisons were made to similar boys not participating in a weight training program. Likewise, in a x-ray study of elbows of pre-adolescent baseball pitchers, Adams (3) found the bones in pitching arms to have shorter 24 lengths due to early epiphyseal plate closure, increased density, fractures of various types, and damaged articular cartilage. In a review article, Malina (71) cites another x-ray report by Adams (4) in which shoulders of throwing arms of young pitchers were studied. He found the humerus of throwing arms to have a low mineral content, bone frag- mentation, and a widened proximal epiphyseal plate indicat- ing accelerated bone growth. Other supportive data indi- cating possible negative effects of strenuous physical activity on bone growth have been reported in a publication by Larson (68). Bone x-rays of physically active growing children have portrayed various types of trauma such as: the development of osteochondrosis designating derangement of the normal growth process, osteochondritis, traumatic epiphysitis, traumatic epiphyseal separation, and various bone and epiphyseal fractures. Thus, it would appear that an optimal level of physical activity stimulates and en- hances overall bone growth while excessive physical activity impairs bone growth. Vitamin C and Bone Vitamin C (ascorbic acid) is known to play a major role in the bone growth process and in the maintenance of normal bodily functions. However, its specific involvement in these processes has not been clearly identified in the lit- erature. In fact, most of the information available on the 25 function of Vitamin C in the body has been derived from studies where subjects are placed on diets deficient in the vitamin. The resultant effects of such deficiency have pro- vided the foundation for development of what is currently known about the functions of Vitamin C. According to Guyton (39), ascorbic acid, a water soluble vitamin, plays a fairly major role in regulating food intake and growth. More specifically, Vitamin C is involved in the reversible oxidation-reduction of food stuffs while also possibly acting as a cofactor in the anabolic metabolism of proteins. With reference to growth and maintenance of cellu- lar constituents, Vitamin C is necessary for the hydroxy- lation of proline to form hydroxyproline which is a requi- site for the formation of collagen. Collagen is the primary component of almost all supportive connective tissues in the body having functions ranging from maintenance of capillary wall integrity to management of normal skeletal growth. Vitamin C also affects alkaline phosphatase production by osteoblasts, thus influencing bone matrix formation. Therefore, with collagen and bone matrix production so dependent upon an adequate supply of ascorbic acid, Guyton states it is not surprising that Vitamin C deficiency results in impaired skeletal growth as well as generalized capillary and cell membrane fragility, all of which are symptomatic signs of scurvy. He concludes that an adequate intake of Vitamin C above that required to prevent the onset 26 of scurvy is necessary for normal maintenance and growth of the skeleton. Similar statements regarding the known functions of Vitamin C have been made by Ham (40). In addition, Ham notes that the vitamin is largely stored in the adrenal gland plus having some regulatory control over its function. In a biochemical study, Udenfriend (113) also indicates that ascorbic acid is involved in the overall maintenance of connective tissue and implicates that it has specific involvement in the creation of hydroxyproline which is a necessary precedent to collage formation. Jeffrey (51), in another biochemical study, reported findings corrdborating those of Udenfriend. Children receiving large pharmacological dosages of corticoids were used as subjects in a study by Liakakos et a1. (70) where large dosages of Vitamin C were also administered. The Vitamin C was given in an attempt to neutralize the inhibitory effects that corticoids have on collagen formation. His findings indicate that not only does ascorbic acid inhibit the negative effects of corti- coids on growth but also acts as a co-factor in hydroxypro- line production which is preliminary to collagen formation. In a vitamin deficiency study, Thornton (111) found scorbutic guinea pigs to have reduced osteoblastic activity resulting in decreased collagen and bone matrix formation. In addition, he found that the quality of the bone matrix was also impaired due to decreased alkaline phosphatase 27 activity. As a consequence, the scorbutic guinea pigs had bones that were incompletely calcified and very labile in nature. He concluded that an adequate intake of Vitamin C is necessary to maintain normal skeletal growth. Similar decrements in the quantity and quality of bone matrix in tibias of scorbutic guinea pigs were also re- flected in the data of Poal-Manresa et a1. (86). More generalized findings were reported by Ram (90) in that his guinea pigs had reductions in adrenal Vitamin C content, food intake, and body weight when fed on an ascorbic acid deficient diet. Evans and Hughes (29) also found that growth rate was impaired in their scorbutic guinea pigs but noted that it occurred as a result of insufficient food metabolism rather than low food intake. Literature on the topic of Vitamin C deficiency effects on skeletal growth has also been reviewed and reported on by Irving (49). The histological changes noted in such studies included increased shaft resorption with thinning of bone walls, disruption of normal metaphyseal cartilage maturation with hemorrhaging in the area, and dense calcification of pre-existing metaphyseal osteoid tissue. Chemical changes resulting from inadequate ascorbic acid intake included reduced cartilage and bone matrix production, decreased alkaline phosphatase activity consequently hindering the normal calcification of matrix, and insufficient collagen production. 28 More detailed information regarding the functions of Vitamin C in the body has been presented in another review article by Meiklejohn (73). Review of biochemical studies found ascorbic acid capable of being reversibly oxidized (accepts hydrogen) and reduced (donates hydrogen). It was also found that the majority of the vitamin in the body is present in the reduced form such that it primarily functions as an anti-oxidant in various metabolic processes. One process that Vitamin C is known to participate in is regula- tion of epiphyseal plate (metaphysis) maturation as des- cribed by Sledge (105). Its anti-oxidant function specific- ally protects chondrocyte lysosomes from breaking down when exposed to oxygen. It allows the cartilage matrix of the metaphysis to mature before being resorbed by these chondro- cyte lysosomes such that the proper environment for calcifi— cation can be maintained. Furthermore, according to Meiklehohn (73), Vitamin C has additional control over meta- physis maturation by regulating alkaline phosphatase activity and matrix calcification as well. In addition, Meiklejohn (73) goes on to say, that Vitamin C also seems to play a key role in the function of the adrenal gland which is its major storage depot. He states that ascorbic acid does not appear to be actively involved in the actual synthesis of the adrenocortical hormones but rather with inhibiting their formation and release. More detailed information on the relationship between Vitamin C and adrenal gland function has been reported in 29 the literature by several other researchers. Guinea pigs placed on a Vitamin C deficient diet were used in a study by Jones et al. (53) where it was found that adrenal ascorbic acid content decreased, body weights decreased, and plasma adrenocorticoids increased. Damage to the epiphyseal plates in the femoral and tibial bones of these animals was also Observed. In another study where Vitamin C intake was restricted, Fordyce and Kassouny (31) found hypertrophied adrenal glands with low ascorbic acid content, enhanced adrenal steroid production, and elevated plasma corticoid levels as well as decreased food intake accompanied by weight losses. In two investigations by Hodges and Hotston (46, 47), where scorbutic guinea pigs were used, similar findings of increased adrenal activity with enhanced corti- coid synthesis and release were also reported. They con- cluded that because Vitamin C seemingly has such a great impact on regulating adrenal gland activity, it may also be an important regulating factor in various metabolic adapta- tions to stress. On the other hand, many investigations have been con- ducted where large dosages of Vitamin C have been adminis— tered in an attempt to study its effect on adrenal gland function. In a study by Liakakos et a1. (69), children were given large dosages of Vitamin C to see if plasma adrenal corticoid levels would be altered. Their findings suggest that high intakes of ascorbic acid exert a braking effect on adrenal corticoid synthesis and release. Consequently, 30 large amounts of ascorbic acid may prove to have no particu- lar beneficial effect in adaptation to conditions of stress. Wilbur (121) and Kitabchi (61) reported similar findings and added that adrenal ascorbic acid stores have to be depleted before steroid synthesis and release can proceed. Further- more, Kitabchi (61) stated that adrenal cholesterol levels were exceptionally low after high intakes of Vitamin C. Vitamin C and Exercise as a Stressor As the literature indicates, Vitamin C exerts a rather strong regulatory influence over adrenal gland function and skeletal growth. It further indicates that active partici- pation in strenuous exercise or physical labor results in various functional and morphological adaptations in both of these structures. Therefore, under conditions of stress, such as that imposed by physical activity, it could be pre- sumed that additional Vitamin C may be required by the adrenal glands and skeleton in order to meet their func- tional needs. Several investigations on this topic have been reported in the literature. Numerous studies on the relationship between ascorbic acid and stress induced by participation in physical activ- ity have been cited in a review article by Irwin (50). Some data indicate a need for higher requirements of Vitamin C when doing strenuous work or exercise in either hot or cold environments. 0n the other hand, other reports 31 indicated that megadosages of Vitamin C had no effect in improving the health status of physically active subjects exposed to similar environmental conditions. Irwin speci- fically cited a study by Henschel et a1. (45) where U. S. Army soldiers walked at a pace of 3.25 miles per hour on a motor driven treadmill at a 7.5 per cent graded incline for six ten-minute alternating work and rest periods. The subjects were also given varied dosages of Vitamin C. It was found that soldiers on a high intake of ascorbic acid showed no improvement in their physiological response or their ability to do work while in a hot environment when compared to control subjects. However, Irwin also cited an in vitro frog muscle study where it was shown that Vitamin C enhanced contraction and delayed the onset of fatigue. Urinary ascorbic acid excretion and blood ascorbic acid levels were used as indicators of Vitamin C requirements by adult skiers as reported in a study by Namyslowski (80). Both parameter values were very high indicating depletion of adrenal Vitamin C stores. As a result of these findings, Namyslowski recommended that megadoses of Vitamin C be taken by athletes during training and prior to competition in their sport. Thus, the result is enhanced post-exercise recovery via adrenal ascorbic acid restoration. Other biochemical studies using athletes as subjects were also reviewed by Irwin. In general, most researchers indi- cated a need for increased Vitamin C intake by athletes partaking in various sport events. However, it should be 32 noted that this recommendation was based solely on biochemi- cal data since there was no improvement in the physiological responses or athletic performances of these subjects. In contrast to the reports supporting Vitamin C supple- mentation by athletes, Irwin also presented data showing no beneficial effects from Vitamin C supplementation. Soldiers receiving high dosages of Vitamin C were subjected to rigor- ous programs of marching on an inclined motor driven tread- mill as described in a study by Keys and Henschel (55). The response of various physiological parameters, such as heart rate, oxygen uptake and blood lactate levels, to such programs indicated that Vitamin C supplementation had no beneficial effects. Johnson et a1. (52), conducted a study where volunteers from a Civilian Public Service Camp were placed on diets either devoid of Vitamin C or supplemented with the vitamin. The data from physical fitness tests on these subjects showed that there was no significant difference in the physical efficiency of these two groups. In a study by Fox et al. (32), natives working in the gold mines of South Africa received dietary supplements of Vitamin C in an attempt to improve their physical work per- formance and overall health status. The net result was that Vitamin C supplementation had no significant beneficial effect on improving the health status or working efficiency of the natives. Two separate studies by Gey (37) and Bailey et a1. (9) also reported findings of no beneficial effects 33 from Vitamin C supplementation on the various physiological responses of their subjects to physical training. However, other studies have reported findings indicating that Vitamin C supplementation does have a positive effect on improving physical performance. In a study by Prokop (89), athletes participating in strenuous, fast running events were given dietary supplements of several vitamins, including Vitamin C. Examination of the data showed that Vitamin C supplementation improved the physiological response of the athletes by reducing their post-exercise oxygen debts, pulse rates and blood pressures. Adrenal ascorbic acid levels of rats and guinea pigs exposed to a regimen of excessive exercise were surveyed in a study by Ratsimamanga (93). His findings indicated that excessive exercise depleted adrenal ascorbic acid stores in both animal subjects. As a result of such findings, he sug- gested that ascorbic acid may be connected in some way with the function of adrenal cortical hormones. However, no specific recommendations for Vitamin C supplementation were given. Rats were also used as subjects in several investigations conducted by Namyslowski (78, 79, 81, 82). All of his results denote depletion of adrenal ascorbic acid contents following participation in various exercise programs. However, Namyslowski also pointed out that the restoration of adrenal ascorbic acid back to normal levels occurred much sooner and more completely in trained animals than in 34 control animals. Thus, he concluded that the adrenal glands of trained animals are better adapted in their ability to restore ascorbic acid levels back to normal following exer- cise. Further, he recommends that dietary Vitamin C supple- mentation be mandatory for athletes participating in strenuous physical activity. Recommended Dosages of Vitamin C As reflected in the literature, Vitamin C is a necessary dietary constituent required for normal adrenal gland func- tion and skeletal growth. However, the exact dosage level of Vitamin C needed to meet its functional roles has not been adequately identified in the literature. In fact, recommended Vitamin C supplemental dosages seem to vary according to individual growth rate and physical activity levels. In a review article by Baker (10), the League of Nations Technical Commission on Nutrition was cited as recommending that 30 mg. of Vitamin C is the daily requirement necessary to meet the functional needs of a normal human adult. However, Baker as well as Meiklejohn (73), also reported that 70 mg./day of Vitamin C is required for a normal human adult as recommended by the United States National Research Council. The League of Nations Commission interpreted this discrepancy by stating that: "... so long as there is no evidence to support the view that an intake of more than 35 30 mg. daily has beneficial effects, there is no basis for recommending an intake greater than the amount." However, under conditions of stress such as that imposed by participation in various exercise programs, some researchers have indicated the need for increased daily intakes of Vitamin C. Namyslowski (83), in a study on physical performances of adult snow skiers, has recommended supplemental Vitamin C dosages of 200-250 mg./day for athletes actively partaking in rigorous training or athletic programs. In a separate study on the physiological respon- ses of athletes participating in heavy, fast running events, Prokop (89) suggests that for a 70 kg. athlete 100-140 mg. of ascorbic acid should be ingested daily during training periods and increased to 140-200 mg. daily during competi— tion periods. In a review article completely devoted to reporting Vitamin C requirements of man, Irwin and Hutchins (50) have presented a wide variety of recommendations. Summary of the data presented on the ascorbic acid requirements necessary for a human adult under normal living conditions designated dosages of between 50 mg. to 100 mg. per day as being ade- quate. While under conditions of physical stress, the recommended daily Vitamin C requirement was increased to a range of 100 mg. to 600 mg. for an active athlete. For active, growing school-age children, a minimal dosage of 25 mg./day of Vitamin C has been recommended although the op- timal dosage level for the vitamin was closer to 50 mg./day. CHAPTER III METHODS AND MATERIALS Human metabolic responses to physical activity can be re- flected by gross measurements of total-body oxygen debt and oxygen uptake. Stressing the muscular anaerobic metabolic pathway results in an increased oxygen debt tolerance as in— duced by a program of exhaustive sprint running. Maximum workloads and short bouts of repeated exercise are character- istic of this type of training regimen. On the other hand, endurance running, characterized by light to moderate work- loads and relatively long bouts of continuous exercise stimu- lates the muscular aerobic metaboliijathway. Stimulation of this pathway results in an increased capacity to do cellu- lar work in the presence of oxygen and is directly related to oxygen uptake. This study was designed to investigate the morphological changes produced in the left femur of the male albino rat after exposure to eight weeks of sprint and endurance training with dietary supplementation of megadoses of Vitamin C. Experimental Animals Eighty-four normal male albino rats (Sprague-Dawley strain) were obtained from Hormone Assay, Inc., Chicago, Illinois. They were received at weekly intervals in three 36 37 shipments of 30, 24, and 30 animals respectively. Each ship- ment 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 of age. Exercise Groups The exercise treatment consisted of three levels of activ- ity which were administered daily between 12:30 p.m. and 5:30;»nn, Monday through Friday for a duration of eight con- tinuous weeks. The exercise treatment groups were as follows. Control Group The 24 animals in the second shipment constituted the sedentary (SED) control group. During the adjustment period and treatment period these animals were housed in individual sedentary cages (24 cm. X 18 cm. X 18 cm.) and were not forced to exercise. 5)]? in t Group The sprint (SPT) running 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) dur- ing the adjustment period and in an individual sedentary cage during the treatment period. The SPT animals were sub- jected to an interval training program of high-intensity sprint running (Appendix A-l). The workload of the SPT 38 program was increased gradually until 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. work periods alternated with four 30-sec. rest periods. During the work periods, the animals were required to run at the relatively fast speed of 108 m./min. Endurance Group The endurance (END) running group was comprised 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 (Appendix A-2). The workload was progressively 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. Diet Subgroups In addition to the three exercise treatments half of the animals in each activity group received one of two dietary supplements. The animals were given their dietary supplement by oral syringe, seven days a week, between 7:00 p.m. and 9:00 p.m. The diet treatments were administered beginning on the day before initiation of the exercise treat- ments and were terminated the day before sacrifice. 39 Vitamin C Approximately .1 cc of a 5% sugar solution with 2 mg. ascorbic acid/100 gm. of body weight was given to one-half the animals (Vit-C) in each exercise group.l Placebo Group Approximately .1 cc of a 5% sugar solution/100 gm. of body weight was given as a placebo to the remaining animals (No-C) in each activity group. Training Procedures The SPT and END groups were trained in a battery of individually controlled-running wheels (CRW). This apparat- us has been described as: . . . a unique animal-powered wheel which is capable of inducing small laboratory animals to participate in highly specific programs of controlled reproducible exercise (119). Animals learn to run in the CRW by avoidance-response operant conditioning. Motivation for the animals to run is provided by a controlled low-intensity electrical current, applied through the running surface. A light located 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 1Vitamin C crystals (30-80 mesh) were obtained from the J. T. Baker Chemical Company. 40 not reach the prescribed speed by the end of the accelera- tion time, the light remains on and shock is applied. As soon as the animal reaches the prescribed speed, the light is extinguished and shock is discontinued. The light-shock sequence is repeated if the animal fails to maintain the desired speed throughout the work period. Most animals learn to react to the light-shock stimulus after only a few days of training. A typical training session consists of alternating work and rest periods. During all rest periods the wheel is braked automatically to prevent spontaneous activity. The brake is released and the wheel is free to turn during the 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 (TEM) are used to calculate the percentage of expected meters (PEM): PEM = 100 (TMR/TEM) PEM values are the chief criterion used to evaluate and com— pare training performances. A secondary criterion is pro- vided 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) 41 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 ad libitum. 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 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 was employed to alter the normal day-night schedule for the animals so that they could be 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 SED 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 principle purposes of the study was to compare various 42 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 treatment period, ten animals were sacrificed from each of these four subgroups. They were selected for sacrifice on the basis of their health and their training performances throughout the treatment period. Those animals subjectively determined to be in good health were chosen for sacrifice. Because the training regimen was extremely vigorous, no absolute minimal performance standard was established. However, individual daily records of PEM and PSF were examined, and healthy animals that made the best adaptations to the training regimens were selected for sacrifice. In each of the sedentary subgroups, two extra animals were included originally to allow for the unlikely possibility that some of the unexercised animals may become ill during the course of the study. At the termination of the treatment period all of the animals in the sedentary subgroups were subjectively determined to be in good health and were sacrificed. 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 subgroups each day). The trained animals were killed either 72 or 96 hours 43 after cessation of their last exercise bout. This procedure was followed to eliminate any transient acute exercise affect. At sacrifice, the animals were either 140 or 141 days of age. Immediately prior to sacrifice, the final body weight of each animal was recorded. Each animal was anesthetized by an interperitoneal injection (4 mg./100 gm. body weight) of a 6.48% sodium pentabarbital (Halatal) solution. Several organs, muscles, and the tibia-fibula complex were removed and processed according to varied protocal for use in a larger diet-exercise investigation. After completion of each of the sacrifice sessions all of the dead animals were slow-frozen and stored as such for approximately nine months. The animals were then allowed to thaw for a 24 hour period. The left hindlimb was skinned and the overlying musculature removed thus exposing the femur. The femur was then detached from its articulation with the acetabulum of the pelvis and the head of the tibia at the knee. All remaining connective tissue and articular cartilage was removed and the bone weighed to the nearest milligram on a Mettler Balance. A Vernier caliper was used to measure the femur length to the nearest .1 mm. The length measurement was taken from the greater trochanter at the proximal end of the femur to the lateral femoral condyle at the distal end of the bone. Bone length is expressed in absolute terms (millimeters) and bone weight is expressed in both absolute terms (milligrams) and relative terms (percentage 44 of body weight). The bones were then placed in individual airtight vials and refrigerated until sectioned. Bone Sectioning Procedures In morphological bone studies one of the most acceptable methods of bone preparation incorporates the use of undecal- cified bone sections (33, 34, 74, 76, 99, 110, 116, 117). Morphological measurements taken from undecalcified bone sections are more representative of the actual bone size since the procedure is devoid of any chemical reagents which could distort the size and shape of the bones under study (33, 34, 115, 116). Cross-sectional samples were taken from three levels of the left femur of the rats following a method for preparation of thin undecalcified bone sections as described by Frost (33, 34). Sections were taken at approximately one-fourth the total length of the femur from both its proximal and distal ends as well as at the midpoint of the bone shaft. The distal end of the femur was cushioned between two pieces of foam rubber and clamped in a small table vice. Using a fine-toothed jeweler's saw, the bone was cut into three cross-sectional slabs approxi- mately one millimeter thick. Each slab was then hand-ground between two pieces of 400 grade abrasive waterproof carborundum sandpaper while cooled with running tap water to reduce possible heat effects due to friction produced by the grinding process. 45 The sections were ground using a circular motion to a thick— ness of approximately 50 microns. Residue from the sand- paper was cleansed from the bone sections by soaking them in a 0.01% mild soap water solution for two minutes followed by a tap water wash and distilled water rinse respectively. Each section was placed in an individual vial containing Villanueva's Bone Stain for fresh mineralized bone for a 48 hour period (116). The individual bone sections were then differentiated in a 0.01% acetic acid in 95% methanol solu- tion, dehydrated in ascending alcohols and cleared in xylene. The three sections of each femur were mounted on a glass microscope slide and permanently mounted with Permount mounting media. Morphological Measurements In addition to the femur length and absolute and relative femur weights, several other morphological parameters were studied. The additional parameters were employed as indica- tors of change in bone size and shape and were taken from sections of all three levels of each femur. The parameters under study are as follows. Cortical wall Area This parameter is commonly used among researchers as an indicator of bone wall size and structure (34, 85, 99, 100, 116). The cortical wall area (CWA) studied was that portion of the bone lying between the periosteal and endosteal 46 surfaces of each section of each bone. The CWA was deter- mined using a slightly modified point-count technique as described by Frost (33, 34), Villanueva (116), Wiebel (118), and Sedlin (101). A magnification of 21 times was used for all measurements of this type since it allowed each section to be viewed in its entirety. Using a Praedo microslide projector (Leitz wetzlar, Germany) each bone section was individually projected on a square-ruled graph paper grid having a total of 391 equi- distantly spaced intersections with a known grid area (GA) of 1 cm2 per square. The point of intersection of any two lines on the graph paper grid which fell within the cortical wall of the projected section was counted as a "hit" (H). Where a point of intersection lied tangent to either the endosteal surface or periosteal surface of a section, the count was recorded as a half hit. The sum of all total cortical hits and tangents (TCH) was recorded for each separate measurement (termed a "throw") of each bone section. Six to eight throws (T) per section were taken to accumulate approximately 150 total hits so that the calculated area for each section was equal to the true area of each section (34, 116). The cortical wall area of each bone section was calculated using the following equation: Cortical wall Area = (Total no. of cortical hits) x (Known grid area) (TotaI no. of throws) X (Total no. of intersec- tions in the whole grid) 47 and is rewritten as: TCH X KGA CWA = TT x TGI . . 2 All area measures were recorded in square centimeters (cm ). Marrow Cavity Area The marrow cavity area (MCA) studied was that portion of the bone entirely surrounded by the endosteal surface of each section of each bone. This parameter also served as an indicator of bone size and structure especially as it re- lated to the total bone area. The MCA was determined using the same method described for deriving the cortical wall area of each bone section. In the CWA equation, appropriate terms were substituted so that MCA replaced the CWA and the total marrow hits (TMH) replaced the TCH. Cortical wall Area/Total Area Ratio The ratio of cortical wall area to total section area (TSA) indicates that portion of each bone section that is actually consumed by bone tissue. This relationship was also used as an indicator of the amount of bone deposition or conversely, the amount of bone resorption that occurred on the bone surfaces as part of the remodelling process (34, 99, 100). The total section area was determined by addition of the cortical wall area to the marrow cavity area for each section (99, 100). 48 Greatest wall Thickness and Least Wall Thickness Mechanical stressors ranging from immobilization to high levels of physical activity are known to have an effect on bone morphology. The determination of the greatest cortical wall thickness (GWT) and least cortical wall thickness (LWT) of each section of each bone reflects any change in the shape and symmetry of the bone that may have occurred as a result of the experimental treatments (34, 99, 100). Those portions of each section subjectively identified as having the greatest wall thickness and the least wall thickness were measured from the appropriate endosteal surface to the appropriate periosteal surface respectively. The measure- ments were taken directly from the projected image of each section of each bone with a clear plastic ruler divided into millimeter units. All measurements of this type were re- corded in millimeter units. Greatest Total Diameter and Greatest of the Least Total Diameter Another indicator of change in shape and symmetry of bone is the greatest total diameter (GTD) and the greatest of the least total diameter (LTD) (see Figure 1). The LTD was measured from that portion of each bone section having the greatest of the least total diameter between opposing perio- steal surfaces and lying perpendicular to the GTD. The same procedure and measurement tool was used for determining these parameters as was used for the GWT and LWT. 49 ¢——9 Greatest Total Diameter ‘- — -D Greatest of the Least Total Diameter Figure 1. Diagram representing the proximal section of the femoral shaft demonstrating the Greatest Total Diameter and the Greatest of the Least Total Diameter. The morphological parameter values obtained from the sedentary-diet subgroups served as a reference standard for the various morphological parameters under study. All morphological measurements for each section of each bone were performed at the same time without knowing the identity of the treatment group. Analysis of Data This study was conducted as a two-way (3 X 2) factorial design with three levels of activity and with two levels of diet. All of the morphological parameters under study were analyzed using a two-way fixed-effects analysis of variance 50 routine on the Michigan State University Control Data 6500 Computer (CDC 6500). Newman-Keuls tests were used to evalu- ate the differences between pairs of means whenever a significant (P = .05) F-ratio was obtained. CHAPTER IV RESULTS AND DISCUSSION Four major sections of material will be covered in this chapter. The results of the Controlled-Running Wheel (CRW) training programs, which include the environmental factors that operated during training, the percentage of body weight lost during daily exercise sessions, and the performance criteria used to reflect training responses are covered in the first section. The second section deals with results of specific activity levels as indicated by the various morpho- logical parameters under study. In the following section, the results of Vitamin C supplementation on performance are reported and discussed. Finally, a general interpretation and discussion of the results are set forth in the last por- tion of this chapter. Training Resultsl The sprint (SPT) and endurance (END) Controlled-Running Wheel (CRW) training programs are presented in Appendix A. 1Some of the material in this section has been adapted, in part, from the unpublished Ph.D. dissertation of Roland R. Roy (95). 51 52 These programs are modified versions of standard regimens routinely used in the Human Energy Research Laboratory, Michigan State University, East Lansing, Michigan. The modi- fications were incorporated in an attempt to design stren- uous exercise programs which would primarily stimulate anaerobic and aerobic metabolic processes in the animals. The performance of the animals was evaluated using the per- centage of expected meters (PEM) and the percentage of shock- free time (PSF) as criterion measures. The performance data for the SPT-C and SPT-No C groups are presented in Figure 2. Progressive increases in the required running velocity were made rapidly. From the be- ginning 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 2 and Appendix A, Table Arl). 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 3. PEM values were 70% or higher each day of training in both the C and No C animals. These results indicate that the animals were able to maintain the daily requirements of the END program relatively well. 53 305:4 ezEam 8. Si 2202 3625 288$ £8 :82 .w manor. _. 9v . 8. +3 |+|om+6+-l+leml+wi mm on em cm 9 o. m [hP~n—~Ir_P_pph~P_-_-brhphnppbpp——_—_ 980 0 02 I 26.6 o I ~56)»: .15) >40 .23”: 0 ON 0 w m. N W 0? an m: a M 0w m M P m 00 u . 00. ON. 03 54 2062.4 wozo Lou uucm_..o> mo mmw>_m:< 2n 03m... 537 ..8 v oz. - - - - - ..8 v 8.8 8_.8 + 88.. + 88.8 + 88.. + 88. + 8... 8 oz. v 8.8 .88. .8... 8.... o..o. 88... ..uom ..m.o .8.. 8... o..o. 88... 8..8. ..uum .om.o ..8 v 8.8 8... H 88.8 n 88.. 8 oz. v 8.8 8.. H .8.. w .8.. 8 oz. v o.8 .88. 88... o..o. 88.8. 88... ..308 .8.: ..8 v 8.8 8.8. o... ..... 88... o8.8. ..uoo .8.: ».8 v 8.8 88.. w o... w 88.8 n 88. w 8.. 8 88.8 M oz. v 8.8 .88. 88.8. 88.8. o~.~8 88.8. ..uo8 .50.. .55. ~_8. 88. 8.... 88... ~..8. .uuom .xo.o .55. .3... ..m: .888. .3.8. ..~: .380. ».8 v oz. - . - - - - hm. v o.. 88.8 + 88.8 + 88.8 + 88.. + 88.. + 88.~ + oz. v 8.8 .88. 8..8. 88... 8...: 8...8 ..uo8 ..m.o 888. 8... .N... 88.8. .8.8. .8308 .um.o 8...: H 88.88. 88.8 8 88... N .8.... 88... u 8.8. 8... 88.88 88.88 8...8 .ouom .8.: 888. 88. 88.8. .8.8~ -.8~ .uuom .8.: oz. v 8.8 88... . o..o. 8 88.8. . .m-8. x. 88.8 n 88.. 8 88.8 u ..8 v 8.8 .88. 88.8. o~.88. 88.88. 88.88. ..uom .xo . .55. 8.8 v .88 888. .8.8 .8.88 .8..8 8..88 .ouom .xo.o .55. 43.8. ..a: .mu.o .u.z. ..u: 880.8 .a. v 8.8 88.8. H 88... w o..o. w 8.8 v ..8 8..8 « .8.8 8 8... u oz. v 8.8 .88. 88... 88.88. 8:..8. 88.88. ..308 .38.8 oz. v 5.8 .88. 88.8 .8... 88... .8.~8 .ouom .om.o ..8 v o.8 88.8. 8 88.8. H 88.8. m 88.. w .e.8 u u... w oz. v 8.8 .88. 88._8 88.88. 88.~8~ 8..~8. ..uo8 .8.: ~.8. 88. 8...8 o..oo .8.~8 .uuom .8.: .88 v 8.8 88.8. n 8.... 8 88.8. u . -8. x. 88.8 8 8..8 w o... 8 oz. v 8.8 .88. 88.88 o..oo. 88.88. 88.~8. ..uo8 .xom. .55. .8.. .8. .8.~8 .~..8 88... ..uo8 .xo.. .55. Eu... .2. u>< .53 no... o>< .88..». .88.... .88.-n. .88..». .8.. .8.<> .8.<> oz. ..8 8.8 8..o<.¢<> .8.. .8.<> .8.<> oz. ..8 o.8 8..8<_¢<> xzm . . 8z<.z .z.z.<.o. .8..<..z .z.oz...o gzm . . 8z<.z .z.:.<.o. ..8.88o< .z.oz...o .8035..8oo .. 0.88. 58 oo.~ + 8.” + 8.~ w ..3 :3 mm. + 3.. + 8.. + 2; 1.3 8... mm.» 3.3 8.3 8.3 .33 .3... e; vim .8. 2.: 8.: 8.... 8.2 .33 .3... :3 1.3 8.. u 83 u 8.. u .m. H 8.. w 8. w ea :3 .8. 8... 8.3 8.3 2.3 .33 .2: n3 vim o..o. e...” 8.: R... 2.: .33 .2: 9; 1.3 8.. « 8.~ “ 84 y . o. x. 2.. w 9... w 8. w :3 1.3 .8. 3.2 8.: 8.: 8.: .33 .xo.m-.~5. o3 vim .1... .5... 8.2 2.: 8.2 .33 .3... . .5. MUL< 0:8 HOP QUL< 0Com “OF 2: 1.3 2.. w 88 w 8.. w 83 v23 3. w mm. w .m. w e: :3 8... $3 2.2 8... E... .33 .3... as v.3 .8. 3.... $3 .m... 83 .33 .3... cm. w 8.~ M a... w .3 1.5 N... N 3. u S. N «am. a... 8.... o..o. 83 .33 .2: a3 vim So. 8; 3... mo... am... .33 .2: 2. u 8.. w 8.. w .-o. .3 a3 v2.“ 8. w R. w .3. w 8... $8 8; a...“ 8;. .33 .xEm . .5. o3 vim 2... no.“ 83 a: 3..” .33 .3... .NS. MOL< >QUNL~I QOL< >QU L0! :3 v3.9. 2.. w 8.. H o... w an v8” n... w 8. w a... w ea :3 .8. 8.2 8.2 2.2 2.... .33 .33 83 :3 :o. .63 i... 3;. N... .33 .3... :3 1.3 8.. w 88 w 8.. H on. H 8.. u S. M ea v 83 .8. 8.2 8.... 8.... 8.2 .33 .2: .8. a. S; 8.... £3 .33 .2: ea v83 2.. « 8.~ H 88 w .-o. .3 8.. w 8.. u 8.. u in 1.3 .8. 2.: 8.3 8.2 8.8 .33 irm . .5. SW :3 .3... 33 3.... an... 8.... .33 £9... . .5. QUL< __03 WLOU MUL< _—@) uWOU Gain. 1.8;». Sofa. $9.3 r3» 3...... ”.35. ea :3 93 3.2.5; 5: .35. 3.5. 8. :3 93 3.2.5... xzm L m wz:.<.._mx h2w°2mmuo xzw n. u. mz00 La: \oo.< >nu Lu: ham v oz“ .0. u no. u no. w --------- ---- ................. - ..... ham v cum .oo. o~.~_ mm. co. am. .uuom .um_o ham v cum ~o. u :o. N no. w ...... --- ---- ----- ------ ------ ------ oz“ v cum :_o. -.a mm. mm. .o. .uuom .u_z Ne. w No. u no. N ......... ---- ----- ------ ------ ------ NNM. m... gs. ox. mN. .uuum .xo.¢ A Eu. 3; .30... no: Ruck \mug< .mu_ugou \ouL< .ou_ubou Amc.uav “mo.ua. “mo.uov Amo.uav pmu» w=4<> U=J<> oz“ ham cum muao<.¢<> haw» ~24<> “34¢: oz» hum cum mu4a<_¢<> xzm a u mz_h<4w¢ pzuazuawo gzm a u mz s_o. mm.o an.mm _~.mm .uuom .u_: ca.om « om.~n w A -o_.xv o..o. n om.¢ w mum. me. on.~m~ o~.mm~ .uuum .xOma Ass. owe. m~.m mm.m~A :o.on_ .uuom .xota Assv Ana—O HOP uQOLu EQ-O uOh uflvgu oo.~ “ o... w AA. A o~. “ mas. mA. ow.o as.» Am 0. xv Asuv mmm. o... so.n mw.m Aeuv - .mg taeou .ma case; cu. w on. w _.. w c.. n cmo. .n. cm.~ MA.~ Am-o_xv Amv Am~. s~._ a... -.. Ame .u) .2450... .u) .5350... nm.~m w m~.:m w ------------ ---- ----- ------ ------ Aav u-oz A u-».> «so. mo.: mm._na c¢.Ass Amy .u: soon .u: Avon Ame..av Amo.unv u-oz u-».> Ame..o. Ame..~v u-oz u-p_> ham» mas<> u=E<> mz hawk U=E<> uss<> wz xzm a u w>.hAunAUL tea ous—Oman ecu m:OmAtmneou outmod mo «anon n.30xacuexoz van muUOumo u :AEouA> __ato>o to» oucm_to> wo m.m>_nc< .~ o_n~» (58 +n oo.a u oA.A w mo.. H N... u-A_> A u-cz soc. NN.» oo.mN oo.nN ..uum ..m.o .Ao. Am.n om.o. am.o. ..uom ....a an.. N oo.A u N... N ma. A sea. Am.m oc.AN cA.mN ..uom .u.: oAA. mo. ma... AA... ..uom .u.z o... m co.m w A -c. x. .o.. w mo.N N Am». No. o..mN oN.mN ..uom .xoma Ase. NAN. .N.. mm.N. No.m. ..uum .xota Ase. xu.;A ..m: .mao. .u.gp ..a: ammo. os.m w om.A “ m..N u .o.. w NAN. o... co.Ns an..a ..uom ....a AAA. ... AN.N. o..a. ..uom ....a as... N a..NN N mm.m N «A.m N ..A. ... oo.Am as... ..uom .v.x msN. mm.. am..N mN.AN ..uom .o.z c..oN w oo.m. w A -o. x. Ne.m A ON.m m cAm. NA. o..ms. om.om. ..uom .xoma Ase. sm.. o..N mo.no AA... ..uom .xota Ase. xu..A ..m: .mu.u Nu.;A ..m: .moto oA.m. m om.0N u mm.. H m..m m .mm. mm. cc.AN. oo.mm. ..uom ....a me. .m.. No.mA ....N ..uum ..m.a cad. ... 3.3 m m..... w .2... ... a.m. mm. oA.Am. og.mm. ..uom .u.: one. mo.m mN.ow Am.Nm ..uom .n.: om.NN w co.mN w A -o. x. Sm.m N am.. w mNN. Nm.. cm._m. om.om. ..uom .xoma Ass. gem. ms. mm..a ea.NN ..uom .xota Ass. 52o No» BE .53 no» o>< Ame..~. Ame..~. u-o2 u-A.> Amo.uo. Ame..~. u-cz u-A.> Amu» u=.<> N=.<> mz Am.» m=.<> u=.<> mz gzm a . m>_A<.u¢ Azwozuaua xzm a A uA=.oma< Azwozuauo uo:c..cou .N u.noA (59 SA.N N a... H N... N SS.. N SOA. ... oA.AN OA.AN ..uom ....o NN.. mm.N SA... mN.N. ..uom ....o oN.N w om.m “ mm.. m m... H SSA. m... SN.AN oo.SN ..uom .u.z S-oz A S-A.> Smo. SS.A .A... SS.N. ..uum .S.: om.N N oS.A w A -c. x. S... u S... w Aam. oo. oS.mN om.mN ..uum .xotm ANEu. SAN. NN.. .S.N. SN.m. ..uom .xota ANeu. MUL< 0:8 uOh ~0L< 0C8 HOP SA.. A oA.N N Na. N S... u mmm. SS. SA... oN.N. ..uum ....o Ao.. .A.N No.m Ns.m ..uom .Sm.o on.. w oS.. u AS. u .S. u SS.. mm.. om.m oa.o. ..uom .u.z 9-02 A S-A.> Sac. AN.. AN.. SS.A ..uom .u.: cm. H o... H A -o. x. An. u as. n mum. mo. oA.A OAK .Suom .xotm ANS. RS. 9. no...” MA...” .33 :6... ANS. MOL< >09 LN! QOL< >QU LOX SS.. w oA._ w NS. n ms. « NSS. an. oS.m. oS.m. ..uom ....o AAA. Am. NA.S SS.S ..uom ..m.a om.. w oo.m H mA. H mm. H omS. S.. om.A. oS.A. ..uum .u.: AS.. Sm.. SA.A .S.A ..uom .u.: om.N u SA.N w A o. x. m... H .m. w a.m. .o. oA.NN SA.NN ..uuS .xotm-A 56. SSN. AS.. AA.m Ao.o. ..uum .xota A 56. mot< ..m: «Lou mot< ..u: «tau AS..... .A...... S-.... 3; . .8.... AS..... IE .1; ASS» S=.<> S=.<> Sz ASSA S=.<> S=S<> Sz xzm a . S>.A<.S¢ AzSazSASa Sam A A SA=.oSS< Azuazuauo uo==.S=ou .N u.S~A 70 +I :o. + co. ............ ---- ----- ------ ------ .Nuom .ASAQ «AA. Am.A Na. :4. .Suvm .NSAQ :o. u So. u ----- ....... ---: ..... ------ ------ .uuom .v.t «on. wo._ on. Am. .uuom .u_: ..o. N. No. ... ------ ..... . ---- ........... ------ .uuom .xota A so. OAS. ”A. AN. :N. .uuom .xotm A so. mot< Amu0h not< .mu0h Anot< >~u to: Anot< >mu to: ..o. w ..o. 4” ------ ...... ---- ................. .Nuum .SSAG mAA. Am.A mm. mm. .uuum .uSAa co. m co. w ..... ------- ---- I--- ------ ------ doom .3... can. A3.— ..S. MS. .38. ...... ..o. N N... u ......u...‘ I... ..---- nus--- 2.-..-- .33 $8.... A Eu. NS. 3. on. AK. .uuum £95 A Eu. NOL< —flu0h NUL< —“u0h \mo..< AmuAuLOu \mo..< Ana—atom. Amo.nm. Amo.umv o-oz uuh_> Amo.umv Amo.u~v uuoz u-h.> hmmh m=A<> m3A<> mz emu» u=A<> man<> mz 22m A. .A u>.._.<._u¢ bzuozmauo xzm A. ... whaomm< hzwozmmmo vo::_uc0u .N uASMA 71 animals not receiving Vitamin C. On the other hand, 31% of the parameters indicated the Vit-C group had larger bones than the No-C group while only 9% of the parameters had equivalent values. When the tabulated data was analyzed further, some inter- esting trends between absolute and relative data were also noted. Forty-three percent of the parameters, consisting of just absolute values, demonstrated the Vit-C animals actual- ly had femurs of smaller size than those animals not receiv- ing a Vitamin C supplement while only 17% of the relative parameters exhibited such a trend. In contrast, the rela- tive data shows that 22% of the parameters had values indi- cating the Vit-C animals had femurs of increased size although only 9% of these animals demonstrated this trend when absolute values were examined. However, it must be emphasized that none of these results represent a statis- tically significant difference between the Vit-C and No-C groups. Thus, it would appear that diet supplementation with Vitamin C had little or no effect on enhancing bone growth in rats placed on strenuous running exercise programs. Because of the great diversity of information on the functional roles of Vitamin C in bone growth and adaptation to stress, it is very difficult to interpret the diet supplementation findings reported in this study. It was hypothesized that high dosages of Vitamin C would enhance long bone growth, especially in the trained animals, by increasing their ability to adapt to the physical stress 72 imposed by the exercise programs. However, it is evident from the data that the findings do not support this hypothe- sis. In fact, it would appear that high intakes of Vitamin C may actually be detrimental to long bone growth. A possi- ble explanation of such results may lie in the regulatory role that Vitamin C plays in controlling functions of the adrenal gland. Various hormones synthesized in the adrenal gland are known to be released into the blood stream upon subject exposure to stress. However, adrenal Vitamin C stores must be depleted prior to synthesis and release of adrenal hormones. Under ordinary circumstances, physical exertion is known to deplete adrenal Vitamin C stores thus allowing the normal sequence of adrenal hormone synthesis and release to occur. However, under the conditions imposed by this study, large dosages of Vitamin C were administered daily throughout the eight week training period. Although adrenal Vitamin C stores may have been depleted following each exercise session, it may be possible that the daily Vitamin C intake was excessive enough to continuously replenish the adrenal gland storage depot, prior to body excretion, thus inhibiting normal responses of the gland to stress. Furthermore, since the response of the adrenal gland to stress is systemic in nature, it is also possible that the lack of a positive Vitamin C effect on bone growth is an indirect result of the atypical adrenal gland function previously proposed. 73 Discussion Accurate interpretation of the effect exercise has on bone growth is dependent upon the results of both absolute and relative bone data. Evaluation of either absolute data or relative data alone would lead to misinterpretation of the exercise results as indicated by the opposite femoral growth trends previously reported in this chapter. It is obvious from the body weight data that both groups of exercised animals, especially those on the sprint program, are significantly smaller in overall size when compared to the sedentary animals. This diversity in body weights be- tween the three exercise treatment groups could be explained in several ways. One possibility is that the great increase in average body weight of the sedentary group could be attributed to excessive food intake. On the other hand, it could also be possible that the exercise programs could have decreased the appetites and thus lowered the food intake of the sprint and endurance animals resulting in their dimin- ished overall body size. However, these are merely supposi- tions since food intake was not considered to be an integral part of the study and therefore was not monitored. The most probable explanation for the body weight varia- tions corresponds to the caloric expenditures characterized by the activity levels of each of the respective exercise treatment groups. Since the sedentary control animals were innately inactive throughout the 8-week treatment period, 74 it is not surprising that these animals may have merely taken in more calories than they were able to expend during their daily activities. As a result, these animals had higher body weights and increased overall size when compared to their exercised counterparts. Furthermore, it also follows that the animals placed on the relatively low intens- ity endurance running program would expend fewer calories than those animals on the relatively high intensity sprint running program. Thus, based on this body weight data, it can be concluded that overall body size is a function of the intensity of exercise. Because of these significant between group body weight differences, it should be kept in mind that the interpreta- tions of all subsequent findings will be greatly effected by this data. In particular, absolute data will especially be effected. Therefore, interpretations and conclusions of all subsequent exercise results will be based on the rela— tive bone data which takes into account these body weight differences. General bone growth changes are indicated by femur weight and length measurements. Both sets of relative data show the femurs of exercised animals to be significantly heavier in weight and longer in length proportional to their overall size than the femurs of sedentary animals. It is known from the findings of studies concerned with immobilization effects on bone growth, that optimal levels of both compres- sive and tensile forces are needed for normal, healthy long 75 bone growth. Adequate exposure to both of these types of forces results in balanced osteoblastic and osteoclastic activity. It is also known that running types of exercise exert both tensile and compressive types of forces at the proximal and distal ends of the femoral shaft, respectively. More specifically, it would appear that these exercise stressors had a positive effect on bone growth by enhancing epiphyseal plate maturation. This would account for the increased long bone length of the trained animals. However, exactly how cells of the epiphyses are effected by the vari- ous types of stressors imposed by running exercise is unknown. Since maturation of the epiphyseal plates depends, to some degree, on an adequate oxygen supply, it is possible that bone vascularity may also play a major role in the stress adaptability of long bones. However, vascular quanti- fication and epiphyseal plate maturation was not determined in this investigation thus leaving these explanations, as to how exercise effects long bone growth, to pure supposition. Evaluation of the various relative diameter measures also shows there was a positive exercise effect on growth in width of the femoral shaft of trained animals. Consistently, the relative data shows both trained groups to have femurs significantly larger in breadth than those of the unexer- cised group. In addition, although the relative diameter data was not statistically significant, the SPT animals also had femurs that were larger in width than those of END animals. Of further interest, is that in two of the three 76 relative diameter measures under study, the SPT animals showed a greater width increase at the proximal level of the femoral shaft than found at the same level in the END animals. Conversely, at the distal level there was less width increase in the femurs of SPT animals when compared to similar measurements on the END animals. Interestingly, these findings indicate that the bone response to running exercise varies along the length of the femoral shaft accord- ing to the type, intensity, and point of application of stress. Since the adductor muscles attach along the medial aspect of the proximal level of the femoral shaft, it is probable that a tensile type of force is created by contrac- tions of these muscles. The result is an increase in bone width as noted in the SPT animals. Evidently, the higher intensity of the sprint program over that of the endurance program was great enough to stimulate additional osteo- blastic activity at this particular area of bone. This bone increase would then act as a means of increasing the resist- ance of the bone to potential fatigue created by the tensile stress of the adductor muscles. Since exercise is known to increase the number, size, and strength of muscle fibers(95), it follows that bone tissue should show similar adaptations to exercise stress by also increasing in size. Therefore, it seems reasonable to assume that the bone size increase should at least be proportional to the increased strength of its attached muscles. 77 At the distal level of the femur, a compressive rather than tensile type of force seems to have been applied as a result of both sprint and endurance exercise programs. However, this time the greatest diameter increase occurred in END animals rather than SPT animals. It would appear that a great deal of compressive force is applied to the distal portion of the femur as an inherent part of any type of running exercise. In this study, the findings would indicate that the SPT animals have been exposed to an exces- sive amount of compressive force due to the high intensity of their exercise program. As a result, their femurs were not as readily adaptable to the compressive type of stress as they were to the tensile type of stress applied at the proximal portion of their femurs. A more optimal level of exercise intensity, allowing adequate adaptability to the compressive type of stress, appears to have been provided by the endurance program. Based on these findings, it can be concluded that compressive types of stress, such as that produced by running exercise, also enhance the appositional growth of long bones. However, it should be noted that unlike the tensile forces created at the proximal level of the femur, compressive forces acting at distal sites are very sensitive to exercise regimens of high intensity. Evaluation of the various relative cross-sectional area determinations further indicates the positive effect that exercise has on appositional growth of the femur. Consistent with relative diameter data, a significant 78 increase in relative total cross-sectional area of the femurs of both SPT and END animals was also found. Again, this increase varied along the length of the femoral shaft. Also, consistent with the relative diameter data, is the finding that these size variations are reflective of the type, intensity, and point of application of the exercise stressors imposed by this study. It is interesting to speculate that the tensile force applied at the proximal level of the femur seems to have had a greater stimulatory effect on the osteoblastic activity on the periosteal sur- face of the femoral shaft than it did on the osteoclastic activity of the endosteal surface of this bone. However, at the distal level of the femur where compressive forces have their greatest effect, stimulation of both osteoblastic and osteoclastic activity was more balanced. These state- ments are based on the fact that at the proximal level of the femur approximately 76% of the total bone area was found to be composed of actual bone tissue with the remain- ing 24% of the total area consumed by the marrow cavity while at the distal portion of the femur only 58% of the relative total cross-sectional area was found to be composed of actual bone tissue leaving the marrow cavity to assume the remaining 42% of the total area. Thus, with respect to relative cross-sectional area determinations, it is apparent that appositional growth is enhanced in femoral bones exposed to both tensile and compressive forces as a result 79 of participation in either strenuous sprint or endurance running exercise programs. CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary This investigation was undertaken to study the effects of two strenuous running exercise regimens and Vitamin C supple— mentation on various morphological growth measurements of the femur. Eighty-four normal male rats (Sprague-Dawley strain) were used as subjects and randomly assigned to one of three exercise programs. The exercise programs consisted of high intensity sprint (SPT) and endurance (END) running regimens which were used in an attempt to selectively tax the anaerobic and aerobic metabolic pathways of the experi- mental animals, respectively. So that the specific intens- ity and duration of these exercise regimens could be regulated and the performance of each animal monitored, the experimental animals were trained on electronically con- trolled running wheels (CRW). The animals assigned to the sedentary (SED) group received no exercise and were used as controls. In addition, one-half of the animals in each training group received a dietary supplement of Vitamin C (Vit-C) while the remaining animals received a sugar water placebo (No-C). Both dietary supplements were administered daily 80 81 by oral syringe. The Vitamin C dosage consisted of 2 mg of ascorbic acid in a .1 cc 5% sugar water solution per 100 gm of body weight. At the start of the eight week treatment period all animals were 84 days of age. At the cessation of this treat- ment period, selected rats were sacrificed 72 to 96 hours after their last training session. The body weight of each animal was determined just prior to sacrifice. After being in frozen storage for approximately nine months, the animals were then thawed and the left femur removed. wet weights and lengths were determined on the femurs prior to their processing for morphological studies. As a result of performance criteria and sampling error, all morphological data analyses were based on an equal cell size of ten animals. All of these parameters were evaluated in terms of both absolute and relative values. In general terms, the absolute data showed the exercised animals, especially those on the sprint program, to be significantly smaller in overall size including all femoral size measure- ments, when compared to sedentary animals. However, when body size was taken into consideration, the relative data showed the exercised animals actually had femurs of larger magnitude proportional to their overall size than the unexercised animals. Thus, strenuous sprint and endurance running was found to enhance both the longitudinal and appositional growth of the femur. 82 The Vitamin C data was also analyzed in terms of absolute and relative values. However, no significant differences in femoral growth trends were noted between the Vit-C and No-C groups . Conclusions l) The average body weights of both SPT and END exercise groups are significantly less than those of the SED group with the SPT animals also significantly smaller in overall size than the END animals. 2) The effects of tensile and compressive forces, created by strenuous SPT and END running exercise regimens, vary along the length of the femoral shaft. 3) The greatest size and shape alterations, resulting from participation in strenuous SPT and END running programs, occur at the distal and proximal levels of the femoral shaft, respectively. 4) The middle portion of the femoral shaft is least affected by size and shape changes resulting from partici- pation in strenuous SPT and END running programs. 5) The femurs of both SPT and END exercised animals are significantly longer in length and heavier in weight, rela- tive to each group's average body weight, than the femurs of unexercised sedentary animals. 6) There were no significant differences between the relative femur weights and lengths of SPT and END animals. 83 7) The femurs of SPT and END exercised animals had sig- nificantly larger cross-sectional diameters, relative to body weight, than the femurs of SED animals. 8) There were no significant differences between the relative total cross-sectional diameters of femurs of SPT and END animals. 9) Foci of greater cross-sectional shape alterations were noted in the femurs of animals trained on SPT and END exerCise programs when compared to the femurs of unexer- cised, sedentary control animals. 10) There were no significant bone shape alterations noted in the femurs of either SPT or END animals except at the distal portion of the bone where SPT animals had signifi- cantly greater shape alterations compared to END animals. 11) The relative femoral cortical wall areas of exercised animals were significantly larger than those of sedentary animals. 12) There were no significant differences between the relative femoral cortical wall areas of SPT and END exer- cised animals. 13) There were no significant differences between the relative femoral marrow cavity areas of any of the three exercise treatment groups except at the distal level of the femur where the relative marrow cavity area of the END group was significantly increased. 84 14) Dietary Vitamin C supplementation had no effect on either the longitudinal or appositional growth of femurs of any of the three exercise treatment groups. Recommendations 1) Due to the significant body weight differences between the three exercise treatment groups, food intake should be monitored. 2) Due to the altered longitudinal growth patterns ob- served in the exercised animals, the processes of epiphyseal plate maturation should be studied. 3) Bone vascularity quantification should be determined due to the altered longitudinal as well as appositional growth patterns observed in SPT and END animals. 4) Femoral strength and deformation characteristics should be determined in further investigations to establish the true training effects of various strenuous running exercise regimens on bone growth. 5) Urinary Vitamin C excretion should be monitored in order to better determine the functional requirements of the vitamin in animals placed on strenuous running exercise programs. 6) Further investigations are needed to determine whether the sugar water placebo had any effect on altering the femoral growth patterns of trained animals. REFERENCES 10. ll. 12. REFERENCES Abramson, A. S., and E. F. Delagi. Influence of weight bearing and muscle contraction on disuse osteoporosis. Arch. Phys. Med. 42:47, 1961. Adams, E. H. A comparative anthropometric study of hard labor during youth as a stimulator of physical growth of young colored women. Res. Quart. 9:102-108, 1938. Adams, J. E. Injury to the throwing arm. Calif. Medi- cine. 102:127, 1965. Adams, J. E. Little league shoulder. Calif. Medicine. 105:22-25, 1966. Allison, N., and B. Brooks. Bone atrophy: An experi- mental study of the changes in bone which result of nonuse. Surg. Gynec. Obstet. 33:250-260, 1921. Armstrong, W. D. Bone growth in paralyzed limbs. Proc. Soc. Exp. Biol. Med. 61:358-362, 1946. Asmussen, E., and K. Heebill-Nielson. A dimensional analysis of physical performance and growth in boyd. J. Applied Physiol. 7:593-603, 1955. Astrand, P. 0., and K. Rodahl. Textbook of WOrk Physiol- ogy. New York: McGraw-Hill, 1970, pp. 375-411. Bailey, D. A., A. V. Carron, R. G. Teece and H. Wehner. Effect of Vitamin C supplementation upon the physiologi- cal response to exercise in trained and untrained sub- jects. Int. 2. Vitaminforsch. 40:435-441, 1970. Baker, E. M. Vitamin C requirements in stress. Amer. J. Clin. Nutrition. 20:583-590, 1967. Belanger, L. F. "The skeletal tissues," Histology. L. Weiss and R. O. Greep (Eds.). Toronto: McGraw-Hill, 1977, pp. 205-249. Beyer, H. G. The influence of exercise on growth. J. Exp. Med. 1:546-558, 1896. 85 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 86 Booth, F. W., and E. W. Gould. "Effects of training and disuse on connective tissue," Exercise and Sport Science Reviews. J. F. Keogh (Ed.). New York: Academic Press, 1975, pp. 83-112. Borer, K. T., and L. R. Kuhns. Radiographic evidence for acceleration of skeletal growth in adult hamsters by exercise. Growth. 41, 1:1-13, 1977. Buskirk, E. R., K. Lande Andeesen, and J. Brozek. Unilateral activity and bone muscle development in the forearm. Res. Quart. Amer. Assoc. Health Phys. Educ. 27:127-131, 1956. Byrd, R. J. The effect of controlled, mild exercise on the rate of physiological ageing of rats. J. Sports Med. and Phys. Fitness. 13:1-3, 1973. Carter, D. R., and W. C. Hayes. Compact bone fatigue damage 1. Residual strength and stiffness. J. of Biomechanics. 10, 5-6:325-338, 1977. Castillo, B. A., R. A. El Sallab, and J. T. Scott. Physical activity, cystic erosions and osteoporosis in rheumatoid arthritis. Annals Rheumatoid Disease. 24:522-527, 1965. Collins, D. H. Pathology of Bone. London: Butter- worths, 1966. Currey, J. D. The adaptations of bone to stress. J. Theor. Biol. 20:91, 1968. Dalen, N., and K. E. Olsson. Bone mineral content and physical activity. Acta Orthop. Scand. 45:170-174, 1974. Davies, J. E. W., J. Pulsinelli, and R. E. Hughs. Ascorbic acid saturation levels in young and old guinea pigs. The Proc. of the Nutr. Soc. 35, 3:117A-118A, 1976. Donaldson, C. L., S. B. Hulley, J. M. Vogel, R. S. Hattner, J. H. Bayers, and D. E. McMillan. Effect of prolonged bed rest on bone mineral. Metab. Clin. Exp. 19:1071-1084, 1970. Donaldson, H. H., and S. B. Conrow. Quantitative studies on the growth of the skeleton of the albino rat. Amer. J. Anat. 26:237, 1919. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 87 Donaldson, H. H., and R. E. Meeser. Effect of prolonged rest following exercise; on the weights of organs of albino rats. Amer. J. Anat. 56:45, 1935. Doyle, F., J. Brown, and C. Lachance. Relation between bone mass and muscle weight. Lancet. 1:391-393, 1970. Ekblom, B. Effect of physical training in adolescent boys. J. Applied Physiol. 27:350-355, 1969. Epker, B. N., and H. M. Frost. Correlation of patterns of bone resorption and formation with physical behavior of loaded bone. J. of Dental Res. 44:33-41, 1965. Evans, J. R., and R. E. Hughes. The growth maintaining activity of ascorbic acid. Br. J. Nutr. 17:251-255, 1963. Faulkner, J. A. New perspectives in training for maxi- mum performance. J. Amer. Med. Assoc. 205:741, 1968. Fordyce, M. K., and M. E. Kassouny. Influence of Vitamin C restriction on guines pig adrenal calcium and plasm corticosteroids. J. of Nutr. 107, 10:1846-1851, 1977. Fox, F. W., L. F. Dangerfield, S. F. Gottlich, and E. Jokl. Vitamin C requirements of native mine workers. Br. Med. J. 2:143-147, 1940. Frost, H. M. Preparation of thin undecalcified bone sections by rapid manual method. Stain Tech. 33:273- 277, 1958. Frost, H. M. Tetracycline based histological analysis of bone remodelling. Calcified Tis. Res. 3:211-237, 1969. Frost, H. M. Orthopaedic Biomechanics. Vol. 5, Springfield, 111.: Charles C. Thomas, 1973. Galbo, H., E. A. Richter, J. J. Holst, and N. J. Christensen. Diminished hormonal responses to exercise in trained rats. J. of A lied Ph siol.: Res ir., Environ. and Exer. Physiol. 43, 6:553-958, 1977. Gey, G. 0., K. H. Cooper, and R. A. Bottenberg. Effect of ascorbic acid on endurance performance and athletic injury. J. Amer. Med. Assoc. 211:105, 1970. Gillespie, J. A. The nature of the bone changes associated with nerve injuries and disuse. J. Bone and Joint Surg. 36-B:464, 1954. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 88 Guyton, A. C. Teprook of Medical Physiology. 4th ed. Philadelphia: W. B. Saunders Co., 1971. ' Ham, A. W. Histology. 7th ed. Philadelphia: J. B. Lippincott Co., 1974, pp. 378—447. Hattner, R. S., D. E. McMillan. Influence of weight- lessness upon the skeleton: A review. Aerospace Medi- Cineo 393849-855, 1968. Heaney, R. P. Radiocalcium metabolism in disuse osteo- porosis in man. Amer. J. of Med. 33:188-200, 1962. Keikkinen, E., H. Suominen, T. Vihersaari, I. Vuori, and A. Kiiskinen. Effect of physical training on enzyme activities of bones, tendons, and skeletal muscle in mice. Proc. Int. Symp. Exer. Biochem. 2nd., 1973, Abstract p. 53. Heikkinen, E., and I. Vuori. Effect of physical activ- ity on the metabolism of collagen in aged mice. Acta Physiol. Scand. 84:543-549, 1972. Henschel, A., H. L. Taylor, J. Brozek, O. Mickelson, and A. Keys. Vitamin C and ability to work in hot environ- ments. Amer. J. Trop. Med. 24:259-265, 1944. Hodges, J. R., and R. T. Horston. Ascorbic acid defi— ciency and pituitary adrenocortical activity in the guinea pig. Br. J. Pharmaco. 40:740—746, 1970. Hodges, J. R., and R. T. Horston. Supression of adreno- corticotropic activity in the ascorbic acid deficient guinea pig. Br. J. Pharmaco. 42:595-602, 1971. Hooper, A. C. B. Effects of divergent selection for body weight on bone length and diameter in mice. Animal Production. 24, 1:77-82, 1977. Irving, J. T. A comparison of the influence of hormones, vitamins and other dietary factors upon the formation of bone, dentine and enamel. Vitamins and Hormones. 15: 291-323, 1957. Irwin, M. I., B. K. Hutchins. Research on Vitamin C requirements of man. J. Nutr. 106:823-879, 1976. Jeffrey, J. J., G. R. Martin. The role of ascorbic acid in the biosynthesis of collagen. II. Site and nature of ascorbic acid participation. Biochemica Et Bio— physica Acta. 121:281-291, 1966. 52. 53. 54. 55. 56. 57. 58. S9. 60. 61. 62. 63. 89 Johnson, R. E., R. C. Darling, F. Sargent, P. Robinson, M. Bartlett, and A. Kibler. Effects of variations in dietary Vitamin C on the physical well-being of manual workers. J. Nutr. 29:155-165, 1945. Jones R. S.. L. Peric-Golia, and K. Eik—Nes. Ascorbic acid deficiency and adrenocortical function in the guinea pig. Endocrinology. 63:659, 1958. Kato, S., and T. Ishiko. "Obstructed growth of chil- dren's bones due to excessive labor in remote corners," Proceedings of the International Congress of Sports Sciences. K. Kato, ed. Tokoyo: Japinese Union of Sport Sciences, 1966, p. 476. Keys, A., and A. F. Henschel. Vitamin supplementation of U. S. army rations in relation to fatigue and the ability to do muscular work. J. Nutr. 23:259-269, 1942. Kiiskinen, A. Physical training and connective tissues in young mice--Physical properties of achilles tendons and long bones. Growth. 41, 2:123-138, 1977. Kiiskinen, A., and E. Heikkinen. Effects of physical training on development and strength of tendons and bones in growing mice. Scand. J. Clin. Lab. Inves. 29 Suppl. 123:20, 1973. Kiiskinen, A., and E. Heikkinen. Effect of prolonged physical training on the development of connective tissues in growing mice. Proc. Int. Symp. Exer. Bio- chem. 2nd. Abstracts p. 25, 1973. Kiiskinen, A., and H. Suominen. Blood circulation of long bones in trained growing rats and mice. European J. of Applied Physiology. 34:303-309, 1975. King, D. W., and R. G. Pengelly. Effect of running exercise on the density of rat tibias. Med. and Science in Sports. 5:68-69, 1973. Kitabchi, A. E. Ascorbic acid in steroidogenesis. Nature (London) 215:1385-1386, 1967. Klein, L., D. G. Kanefield, and K. G. Heiple. Effect of disuse osteoporosis on bone composition: The fate of bone matric. Calcified Tissue Research. 2:20-29, 1968. Knehr, C. A., D. B. Dill, and W. Neufeld. Training and its effect on man at rest and work. Amer. J. of Physiol. 136:148-156, 1942. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 90 Koch, J. C. The laws of bone architecture. Amer. J. Anat. 21:177-298, 1917. Kusinitz, I., and C. E. Keeney. Effects of progressive weight training on health and physical fitness of adolescent boys. Res. Quart. 29:294, 1958. Lamb, D. R., W. D. Van Huss, R. E. Carrow, W. W. Heusner, J. C. Weber, and R. Kerzler. Effects of pre- pubertal physical training on growth, voluntary exercise, cholesterol, and basal metabolism in rats. Res.4Quart. Amer. Assoc. Health and Phys. Educ. 40:123-133, 1969. Lane, L. B., A. Villacin, and P. G. Bullough. The vascu- larity and remodelling of subchondral bone and calcified cartilage in adult human femoral and humeral heads. J. of Bone and Joint Surg. 59, 3:272-278, 1977. Larson, R. L. "Physical activity and the growth and development of bone and joint structures," Physical Activity: Human Growth and Development. G. L. Rarick, edT’ New York: Academic Press,’l973, pp. 32-59. Liakakos, D., N. L. Doulas, D. Ikkos, C. Amussakis, P. Vlachos, and G. Jouranani. .Inhibitory effect of ascorbic acid (Vit. C) on cortisol secretion following adrenal stimulation in children. Clinica Chimica Acta. 65:251-255, 1975. Liakakos, D., D. G. Ikkos, P. Vlachos, K. Ntalles, and C. Coulouris. Effect of ascorbic acid on urinary hydroxyproline of children receiving corticosteroids. Archives of Disease in Childhood. 49:400-403, 1974. Malina, R. M. Exercise as an influence upon growth. Review and critique of current concepts. Clinical Pediatrics (Phila). 8:16-26, 1969. Manegold, C., C. Krempien, D. Baumann, H. wesch, and G. Geiger. Skeletal homeostasis and ageing. Studies in human femora. Calcified Tissue Research. 22 Suppl.: 389-392, 1977. Meiklejohn, A. P. The physiology and biochemistry of ascorbic acid. Vitamins and Hormones. 11:61-96, 1953. Melsen, F., and L. Mosekilde. Morphometric and dynamic studies of bone changes in hyperthyroidism. Acta Path. Microbiol. Scand. Sect. A. 85:141-150, 1977. Moffroid, M. T., and R. H. Whipple. Specificity of speed of exercise. Physical Therapy. 50:1692, 1970. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 91 Mosekilde, L., F. Melsen, J. P. Batger, O. Myhre-Jensen, and N. Serensen. Bone changes in hyperthyroidism; inter- relationships between bone morphometry, thyroid function and calcium-phosphorus metabolism. Acta Endocrinologica. 85, 3:515-525, 1977. Moss, M. L. "The regulation of skeletal growth," n.d., nope, pp. 127-1420 Namyslowski, L. Ascorbic acid content of the supra- renals of rats during physical effort. Rocaniki Panstwo- we 0 Zakladu Hi . 8:79, 1957. From: Chem. Abstr. 51: 10695, I956. Namyslowski, L. Influence of stress on ascorbic acid content of rat adrenals. Roszniki Panstwowe o Zaklad Hig. 7:425, 1956. From: Chem. Abstr. 51:3789, 1957. Namyslowski, L. Vitamin C requirements in sportsmen depending on physical exertion. Rgczniki Panstwowegp_ Zakladu H;g. 7:97, 1956. From: Chem. Abstr. 51:4518, 1957. Namyslowski, L. Course of return to normal of ascorbic acid content in rat adrenals after exhaustive exercise. Roczniki Panstwowego Zakladu Hig. 8:265, 1957. From: Chem. Abstr. 52:1418, 1958. Namyslowski, L. Effect of training on the adaptation of rat adrenals to efforts. Endokrynologia Polska. 9:223, 1958. From: Chem. Abstr. 53:8350, 1959. Namyslowski, L. Estimation of ascorbic acid require- ments of athletes in relation to physical exertion. Roszniki Panstwowego Zakluda Hi . 7:97, 1956. From: Chem. Abstr. 54:13303, I959. Parizkova, J. Longitudinal study of the development of body composition and body build in boys of various physical activity. Human Biology. 40:212-225, 1968. Park, E. Cortical bone measurements in Turner's syn- drome. Amer. J. Phys. Anthrop. 46, 3:455-461, 1977. Poal-Manresa, J., K. Little, and J. Trueta. Some observations of the effects of Vitamin C deficiency on bone. Br. J. Exp. Path. 51:372-378, 1970. Price-Jones, C. The effect of exercise on the growth of white rats. Quart. J. Exp. Physiol. 16:61-67, 1926. Prives, M. G. Influence of labor and sport upon skele- ton structure in man. Anat. Record. 136:261, 1960. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 92 Prokop, L. Vitamins and athletic performance. Med. u. Ernahrung. 2:174-199, 1961. From: Nutr. Abstr. 33:180. Ram, M. M. Growth rate and protein utilization in Vitamin C deficiency. Indian J. Med. Res. 54:964-970, 1966. Rarick, G. L. "Exercise and growth," Science and Medi- cine of Exercise and Sports. W. R. Johnson, ed. New York: Harper and Row Publishers, 1960, pp. 440-465. Rasmussen, H., P. Bordier. The Physiolpgical and Cellu- lar Basis of Metabolic Bone Disease. Baltimore: Williams and Wilkins Co., 1974, p. 41. Ratsimamanga, R. Variations in the ascorbic acid con- tent of the suprarenal glands during work. C. R. Soc. Biol. 131:863-865, 1939. From: Nutr. Abstr. 9:338. Reeve, J., C. P. Swain, and R. Wootton. The acute ef- fect of moderate exercise on blood flow to the skeleton in man. J. of Physiol. 267, 1:41p-32p, 1977. Roy, R. R. Specific changes in a histochemical profile of rat hindlimb muscle induced by two exercise regimens. Unpublished Ph.D. Thesis, Department of Health, Physical Education and Recreation, Michigan State University, East Lansing, Michigan, 1976. Saville, P. D., and R. Smith. Bone density, breaking force and leg muscle mass as function of weight in bipedal rats. Amer. J. Phys. Anthrop. 25:35-39, 1966. Saville, P. D., and M. P. Whyte. Muscle and bone hyper- trophy. Positive effects of running exercise in rat. Clin. Orthop. 65:81-88, 1969. Schenk, R. K., D. Spiro, and J. Wiener. Cartilage resorption in the tibial epiphyseal plate of growing rats. J. Cell Biology. 34:275, 1967. Schock, C. C., F. R. Noyes, and A. R. Villanueva. Measurement of haversian bone remodelling by means of tetracycline labelling in rib of Rhesus monkeys. Henry Ford Hospital Medical Journal 20, 3:131-144, 1972. Schock, C. C., F. R. Noyes, M. M. Crouch, and C. H. E. Mathews. The effects of immobility on long bone remodelling in the Rhesus monkey. Henry Ford Hospital Medical JOurnal.23, 3:107-116, 1975. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110 . 111. 112. 113. 93 Sedlin, E. D., and C. Hirsch. Factors affecting the determination of the physical properties of femoral cortical bone. Acta Orthop. Scand. 1966. Severson, J. A., R. D. Fell, J. G. vander Tiug, and D. R. Griffith. Adrenocorticoid function in ageing exercise-trained rats. J. of Applied Physiol: Respir., Environ. and Exer. Physiol. 43, 5:839-843, 1977. Siegling, J. A. Growth of the epiphysis. J. of Bone and Joint Surg. 23:23-36, 1941. Sissons, H. A. Experimental determination of rate of longitudinal bone growth. J. of Anat. 87:228-237, 1953. Sledge, C. B. Biochemical events in the epiphyseal plate and their physiologic control. Clin- Orthop. 61:37-47, 1968. Smith, E. L., and S. W. Babcock. Effects of physical activity on bone loss in the aged. Med. and Sci. in Sports. 5:68, 1973. Smith, R. E., P. D. Saville. Bone breaking stress as a function of weight bearing in bipedal rats. Amer. J. of Phys. Anthrop. 25:159-164, 1966. Steinhaus, A. H. Chronic effects of exercise. Physiol. Rev. 13:103-147, 1933. Steinhaus, A. H. Toward an Understandipg of Health and Physical Education. Iowa: wm. C. Brown Co., 1963, pp. 196-197. Takahashi H., B. N. Epker, R. Hattner, and H. M. Frost. Evidence that bone resorption precedes formation at the cellular level. Henrprord Hospital Medical Bulletin. 12:359-364, 1964. Thornton, P. A. Effect of ascorbic acid deprivation in guinea pigs on skeletal metabolism. J. Nutr. 95:388-392, 1968. Tipton, C. M., R. D. Matthes, and J. A. Maynard. Influence of chronic exercise on rat bones. Med. Sci. Sports. 4:55, 1972. Udenfriend, S. Formation of hydroxyproline in colla- gen. Proline is incorporated into peptides before it is hydroxylated. Science 152, 3727:1335-1340, 1966. 114. 115. 116 . 117. 118. 119. 120. 121. 94 Van Dusen, C. R. An anthropometric study of the upper extremities of children. Human Biology. 11:277-284, 1939. Van Huss, W. D., W. W. Heusner, and O. Mickelson. "Effects of prepubertal exercise on body composition," Exercise and Fitness. D. Franks, ed. Chicago: Athletic Institute, 1970, p. 201. Villanueva, A. R. Method of sectioning, staining and application of mineralized sections of bone. Presented at: First International Workshop on Bone Morphometry. University of Ottawa, Ontario, Canada. March, 1973. Villanueva, A. R. A bone stain for osteoid seams in fresh, unembedded, mineralized bone. Stain Tech. 49, 1:1-8, 1974. Weibel, E. R., C. Fisher, J. Gahm, and A. Schaeffer. Current capabilities and limitations of available stereological techniques. J. Micros. 95:367-392, 1972. Wells, R. L., and W. W. Heusner. A controlled running wheel for small animals. Lab. Animal Sci. 21:904- 910, 1971. Wells, J. B., J. Parizkova, J. Bohanan, and E. Jokle. Growth, body composition and physical efficiency. J. Assoc. Phys. Ment. Rehab. 17:37-40, 1963. Wilbur, V. A., B. L. walker. Dietary ascorbic acid and time of response of guinea pig to ACTH administra- tion. Nutr. Reports International 16, 6:789-794, 1977. APPENDICES 95 APPENDIX A TRAINING PROGRAMS Table L-l. Modified Eight Heek Sprint Training Program for Postpubertal and Adult Hale Rats in Controlled-Running wheels Total Ac- Time Time Total celer- Hork Repeti- Be- of Total Hort 0a, 0a, ation Time Rest tions No. tween Run Prog. Ext. Time cf of Time (R10: Time per of BOuts Shock Speed (min: Meters (52:) 9.. HR. Tr. (sec) sec) (sec) Baut Bouts (min) (ma) (m/min) sec) TEP THT 0 4-T -2 3.0 40:00 l0 1 l 5.0 0.0 27 40:00 --- --- S-F -l 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --- l l=M l 2.0 00 l0 10 lo 8 2.5 l.2 36 42:50 480 800 2:7 2 2.0 00.10 lo 10 8 2.5 l.2 36 42:50 480 800 3=u 3 l.5 00:10 15 lo 8 2.5 l.2 54 49:50 720 800 4:7 4 1.5 00 10 15 l0 8 2.5 1.2 54 49:50 720 800 5:? 5 1.5 00:l0 15 lo 8 2.5 l.2 54 49:50 720 803 2 ls“ 6 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 =' 7 1.5 02.10 l5 10 8 2.5 l.2 54 49:50 720 800 3=n 8 1.5 00:15 30 6 7 2.5 l.2 72 43:00 756 63: =7 9 1.5 00:l5 30 6 7 2.5 l.2 72 43 00 756 630 5=F l0 l.5 00 15 30 6 7 2.5 l.2 72 43:00 756 630 3 l-H l l.5 00 l5 30 6 7 2.5 l.2 72 43:00 756 630 2=T l2 1.5 00 15 30 6 6 2.5 l.2 Bl 36:30 729 540 3=H l3 1.5 00 15 30 6 6 2.5 l.2 8l 36:30 729 540 4:7 14 1.5 00 l5 30 6 6 2.5 l.2 81 36:30 729 540 5=F 15 1.5 00 15 30 6 6 2.5 l.2 81 36:30 729 540 4 l=H l6 1.5 00 l5 30 6 6 2.5 1.2 81 36 30 729 540 2-T l7 2.0 00 l5 C 5 6 2.5 l.2 90 32:00 675 450 B'H l8 2.0 00:l5 30 5 6 2.5 l.2 90 32:00 675 450 4:7 19 2.0 00 15 30 5 6 2.5 l.2 90 32:00 675 450 5sF 20 2.0 00 l5 30 5 6 2.5 l.2 90 32 00 675 453 5 l-H 2l 2.0 00 l5 30 5 6 2.5 1.2 90 32:00 675 450 2:7 22 2.0 00 l5 30 5 6 2.5 1.2 99 32:00 743 450 3-H 23 2.0 00 15 30 5 6 2.5 l.2 99 32:00 743 450 4sT 24 2.0 00 l5 30 5 6 2.5 l.2 99 32:00 743 450 S-F 25 2.0 00 l5 30 5 6 2.5 1.2 99 32:00 743 450 6 l-M 26 2.0 00 l5 30 5 6 2.5 l.2 99 32:00 743 450 2=T 27 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 810 450 3=H 28 2.0 00 l5 30 5 6 2.5 l.2 108 32:00 810 450 4=T 29 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 8l0 450 5-F 30 2.0 00 15 30 5 6 2.5 l.2 l08 32:00 8l0 450 7 l-H 31 2.0 00 15 30 5 6 2.5 1.2 l08 32:00 Bl 450 2=T 32 2.0 00 l5 30 5 6 2.5 l.2 108 32:00 8l0 450 38H 33 2.0 00 15 30 5 6 2.5 1.2 l08 32:00 8l0 450 4sT 34 2.0 00 l5 30 5 6 2.5 l.2 108 32 00 810 450 S-F 35 2.0 00 15 30 5 6 2.5 l.2 108 32:00 8l0 450 8 l-H 36 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 8l0 450 2-7 37 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 8l0 450 3-H 38 2.0 00 l5 30 5 6 2.5 l.2 l08 32 00 8l0 450 4-T 39 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 8l0 450 5-F 40 2.0 00 l5 30 5 6 2.5 l.2 l08 32:00 810 450 This training program is a modified version of a standard program designed using male rats of the Sprague-Dewley strain(23,49). 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 on the animals and will seriously impair the effectiveness of the training program. 96 APPENDIX A--continued Table A-2. Hodified Eight week Endurance Training Pragram for Postpubertal and Adult Hale Rats in ' Controlled-Running Wheels Total Ac- No. Par- Time Time Total celer- work Repeti- of tial Be- of Total Nork 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) Uh. Uh. Tr. (sec) sec) (sec) Bout Bouts sec) (min) (ma) (m/min) sec) TEN TNT 0 4=T -2 3.0 40:00 10 1 5.0 0.0 27 40:00 --- --- 58F -1 3.0 40 00 10 1 1 5.0 0.0 27 40:00 --- -.- 1 18M 1 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 2'1 2 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 3'H 3 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 4:1 4 1.5 05:00 0 1 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'H 6 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 2'1 7 1.0 07:30 0 1 2 5.0 1.2 36 20:00 540 900 3'H 8 1.0 07:30 0 1 2 2.5 1.2 36 17:30 540 900 4=T 9 1.0 07:30 0 1 2 1.0 1.2 36 16:00 540 900 5=F 10 1.0 15:00 0 1 1 0.0 1.2 36 15:00 540 900 3 1=H 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 1 1 07:30 1.0 1.0 36 23:30 810 1350 3=H 13 1.0 15:00 0 1 1 10:00 1.0 1.0 36 26:00 900 1500 4:1 14 1.0 15:00 0 1 1 12:30 1.0 1.0 36 28:30 990 1650 5=F 15 1.0 15:00 0 1 2 1.0 1.0 36 31:00 1080 1800 4 1-H 18 1.0 15:00 0 1 2 05:00 1.0 1.0 36 37:00 1260 2100 2=1 17 1.0 15:00 0 1 2 07:30 1.0 1.0 36 39:30 1350 2250 3=H 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 0 1 2 12:30 1.0 1.0 36 44:30 1530 2550 58F 20 1.0 15:00 0 1 3 1.0 1.0 36 47:00 1620 2700 5 1tM 21 1.0 15:00 0 1 3 05:00 1.0 1.0 36 52 00 1800 3000 2'1 22' 1.0 15:00 0 1 3 07:30 1.0 1.0 36 54:30 1890 3150 38H 23 1.0 15:00 0 1 3 10:00 1.0 1.0 36 57:00 1980 3300 4‘1 24 1.0 15:00 0 1 3 12:30 1.0 1.0 36 59:30 2070 3450 5'5 25 1.0 15:00 0 1 4 1.0 1.0 36 63:00 2160 3600 6 1'” 26 1.0 15:00 0 1 4 1.0 1.0 36 64:00 2160 3600 2‘1 27 1.0 30:00 0 1 2 5.0 1.0 36 65:00 2160 3600 338 28 1.0 30:00 0 1 2 2.5 1.0 36 62:30 2160 3600 4-1 29 1.0 30:00 0 1 2 1.0 1.0 36 61:00 2160 3600 53F 30 1.0 60:00 O 1 1 0.0 1.0 36 60:00 2160 3600 7 1'H 31 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 231 32 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 38H 33 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 4'1 34 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 5-F 35 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 8 13H 36 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 2:1 37 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 3'" 38 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 4'1 39 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 S‘F 40 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 This training program is a modified version of a standard program designed using male rats of the Sprague-Dawley strain (23.49). 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. BASIC STATISTICS FOR TRAINING DATA APPENDIX B 9'7 Basic statistics for Percentage of Body Height Loss. Environmental Factors and Performance Criteria Simple Correlations a Standard Air Per Bar Per Body Variable N Mean Deviation Temp humid Press Ht Loss PEN so; Air Temp (F) 367 72.9 4.8 Per Humid 367 39.0 12.1 .110 Bar Press (nnflg) 367 740.7 4.3 -.276 -.713 Per Body wt loss 367 1.7 .5 -.066 ..205 .044 PEH 367 55.4 20.2 -.199 -.359 .121 .258 PSF 367 56.8 19.5 0.398 -.312 .164 .196 .872 EEILOE Air Temp (r) 375 73.1 4.6 Per Humid 376 38.5 12.3 .125 Bar Press (nuMg) 376 740.8 4.2 -.263 -.715 Per Body wt less 376 1.7 .6 .000 -.200 .046 PER 376 61.6 25.9 -.165 -.383 .210 .115 PSF 376 62.6 23.4 -.260 -.271 .129 .032 .868 .952: Air temp (F) 340 73.9 4.0 Per Humid 340 47.1 10.7 .134 Bar Press (nmflg) 340 739.5 3.8 -.288 -.675 Per Bocy wt Toss 340 2.5 1.0 .374 .139 -.240 PEM 340 82.8 24.7 -.279 -.173 .232 -.069 PSF 340 70.7 18.6 -.403 -.083 .159 -.118 .685 21212;. Air Temp (F) 348 73.9 4.0 Per Humid 348 47.0 10.6 .151 Bar Press (nnMg) 348 739.5 3.8 -.294 -.677 Per Body wt loss 348 2.6 1.0 .439 .114 -.159 PEH 348 82.2 18.7 -.231 -.253 .286 -.003 PSF 348 69.6 19.2 -.254 -.102 .153 .021 .748 “a - total days training. all animals nrcwran STATE UNIV. LIBRARIES WWWW”IWill”IIIWIWIIHIIWWWHI 31293100633878