THE EFFECTS OF DIANABOL AND ANAEROBIC ENDURANCE EXERCISE 0N SELECTED ANATOMICAL AND HISTCCHEMICAL PARAMETERS IN THE ADULT MALE ALBINO RAT A Thais for the Degree of M. A. MICHIGAN STATE UNIVERSITY ROBERT CHARLES HICKSON 1972 - O-"~..”.~-qv“~v-'-D“O' ° - .w .m S n .mo ”m M .w: m .m U ABSTRACT THE EFFECTS OF DIANABOL AND ANAEROBIC ENDURANCE EXERCISE ON SELECTED ANATOMICAL AND HISTOCHEMICAL PARAMETERS IN THE ADULT MALE ALBINO RAT BY Robert Charles Hickson The purpose of this study was to determine the separate and combined effects of an anabolic steroid and an anaerobic program of endurance running on selected anatomical and histochemical parameters in the adult male albino rat. Dianabol, a product of the CIBA Pharmaceutical Co., was the anabolic steroid used. The training program was the high-intensity, short-duration Controlled Running Wheel program developed in this laboratory. Body compo- sition and various organ weights were investigated. Histochemical determinations were made of glycogen storage and phosphorylase activity in ten locations of the gastrocnemous-plantaris-soleus muscle group. The cross- sectional areas of thirty muscle fibers were measured in these same locations. Forty-two, normal, male albino rats (Sprague- Dawley strain) of three different age levels, were brought Robert Charles Hickson into the laboratory in one shipment. The differences in age were required to accommodate staggered treatment periods set up in conjunction with other concurrent studies using the same facilities. Initiation of treatments began for all animals at 100 days of age. Fifteen animals were 90 days old (Age-Level 1), twelve animals were 76 days old (Level 2), and fifteen animals were 62 days old (Level 3) at the time of arrival. Each animal was randomly assigned to training-drug treatments within his own age group. All animals were allowed a minimum of 10 days to become acclimated to laboratory conditions before the study began. Since all animals began their training at 100 days of age, the Level 1 animals began the program first and the Level 2 and 3 animals followed at succeeding two-week intervals. Dianabol was administered subcutaneously at a l-mg/day dose. Treatments were administered Monday through Friday for eight weeks. All animals were supplied with food and water ad libitum. The exercised animals were selected for sacrifice on the basis of having the highest percent of expected revolutions (PER) within their own drug groups. The final sample consisted of 36 animals (six per cell). At sacrifice, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbitol. Selected organ weights were immediately removed, trimmed, Robert Charles Hickson and weighed while wet. The gastrocnemous, plantaris, and soleus muscles were removed as a unit and frozen in a isopentane-liquid nitrogen system. Fresh-frozen, cross sections were cut at 10 microns using a rotary microtome in a cryostat. Relative localization of glycogen and phosphorylase were quantitatively determined by a histo- chemical photometer for a sample of thirty fibers in each of ten areas of the muscle unit. Absolute fiber size (micronsz), as measured with a polar plainimeter, was also determined for thirty fibers in the same ten muscle areas. The remaining carcass was saved for subsequent body composition analysis. The results indicated that anaerobic training for eight weeks produced smaller body weights and smaller absolute weights of the liver, spleen, kidneys, and muscle in the exercise group than in the sedentary group. The relative weights of the adrenals, heart, liver, testes, kidneys, and muscle in the exercise group were larger than those in the sedentary group. Relative spleen weight was smaller in the exercise group. Body weights of the Dianabol and placebo groups were both greater than that of the control group but not different from each other. Both absolute and relative liver weights were higher in the Dianabol group than in the control group, and the relative liver weights were also higher in the Dianabol group than in the placebo group. 9'1 Robert Charles Hickson Relative spleen weights were less in the Dianabol and placebo groups than in the control group. Phosphorylase was depleted in all 10 muscle areas as a result of exercise. The Dianabol and placebo groups had less phosphorylase activity than the control group in area 3. An increase of glycogen in areas 4, 5, 6, 7, and 8 occurred with the training program. The Dianabol and placebo groups had less glycogen in area 5 than the control group. Absolute fiber size showed no changes with either the training or drug treatments. Carcass weight and the percentage of fat were lower while the percentages of water, protein, and ash were higher in the exercise group than in the sedentary group. All of the absolute carcass components in the exercise group were lower than those in the sedentary group. THE EFFECTS OF DIANABOL AND ANAEROBIC ENDURANCE EXERCISE ON SELECTED ANATOMICAL AND HISTOCHEMICAL PARAMETERS IN THE ADULT MALE ALBINO RAT BY Robert Charles Hickson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Health, Physical Education and Recreation 1972 DEDICATION To my Mom, Dad, and Aunt Doris for their confidence and faith in me. ii ACKNOWLEDGMENTS This study was made possible with the support and guidance of Dr. William W. Heusner. The assistance and intellectual setting created by Dr. Wayne D. Van Huss is also greatly appreciated. Thanks are extended to Mr. David J. Anderson and Dr. James F. Taylor for their mechanical and technical help. Special thanks are extended to Dean Jackson for his aid with the animal training program. Further thanks are extended to Dr. Rexford E. Carrow, Mrs. Barbara Wheaton, Mr. Kwok-Wai Ho, Mr. Arthur Psaledas, Miss Linda Frome, Miss Trudy Van Russ, and Mrs. JoAnn LaFay. Lastly, thank you Susie Jones! iii TABLE OF CONTENTS Page LI ST OF TABLES O O O O O 0 O O O O O O 0 Vi i LIST OF FIGURES. . . . . . . . . . . . . ix Chapter I. INTRODUCTION . . . . . . . . . . . 1 Statement of the Problem. . . . . . . 2 Rationale. . . . . . . . . . 3 Significance of the Problem. . . . . . 4 Limitations of This Study . . . . . . 4 Definition of Terms . . . . . . . . 6 II. REVIEW OF RELATED LITERATURE . . . . . . 8 Effects of Dianabol and Other Anabolic SterOidS O O O O O O O O O O O 8 Measurement of Myotropic Activity . . . 8 Dianabol as an Anabolic Agent . . . 13 Role of Anabolic Steroids on Skeletal Muscle Glycogen Concentration. . . . 17 Effects of Exercise . . . . . . . . 20 Response of Skeletal Muscle Glycogen and Phosphorylase. . . . 20 Body Composition and Organ Weights. . . 26 Effects of Exercise and Anabolic Steroids . . . . . . . . . . . 26 Human Studies. . . . . . . . . . 26 Animal Studies . . . . . . . . . 32 iv Chapter III. RESEARCH METHODS . . . Sampling Procedures . Research Design. . . Training Groups. . . Exercise (E) . . . Sedentary (S). . . Drug Groups . . . . Dianabol (D) . . . Placebo (P) . . . Control (C) . . . Experimental Procedures Animal Care . . . . Sacrifice Procedures . Organ Weights and Body Composition Procedures. . . . Histochemical Procedures. Histochemical Methods. Histochemical Analysis Morphological Analysis Statistical Procedures IV. RESULTS AND DISCUSSION . Training Results . . Histochemical Results. Phosphorylase. . . Glycogen . . . . Morphological Results. Organ Weight Results . Body Composition Results. Discussion . . . . V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. Summary . . . . . Conclusions . . . . Recommendations. . . REFERENCES . . . . . . . Page 34 34 35 36 36 38 38 38 39 39 39 4O 4O 42 42 43 45 47 47 50 50 50 56 56 56 59 64 68 77 77 80 82 84 Chapter APPENDI Appendi A. CES x Training Program . . . Histochemical Raw Data . Morphological Raw Data . Organ Weight Raw Data . Body Composition Raw Data vi Page 96 97 99 100 101 LIST OF TABLES Table Page 1. Effects of Exercise on the Absolute (gm) and Relative (Percent) Body Composition Components . . . . . . . . . . . . 27 2. Previous Studies of Effects of Forced and Spontaneous Exercise on Organ Weights From Montoye, et a1. . . . . . . . . 28 3. Effects of Exercise on the Absolute (gm) and Relative (Percent) Organ Weights Since 1960 O O O O I O O O O O O O O O 29 4. Random Assignment of Animals to Treatment Groups Within Age Levels . . . . . . . 35 5. Experimental Design With Final Cell Frequency . . . . . . . . . . . . 36 6. Analysis of Variance and Tukey Test Results for Phosphorylase Activity in the Ten Selected Areas of Gastrocnemous, Plantaris, and Soleus Muscles . . . . . . . . . 52 7. Analysis of Variance and Tukey Test Results for Glycogen Storage in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles . . . . . . . . . 54 8. Analysis of Variance and Tukey Test Results for Absolute Fiber Size (Square Microns) in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles. . . . . . 57 9. Analysis of Variance and Tukey Test Results for Body Weight and Absolute and Relative Organ Weights . . . . . . . . . . . 61 vii Table 10. 11. 12. Page Analysis of Variance and Tukey Test Results for Carcass Weight and the Relative (Percent) and Absolute (gm) Carcass Components . . . . . . . . . . . . 66 Effects of Training and Anabolic Steroid Use on Phosphorylase, Glycogen, and Absolute Fiber Size in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles . . . . . . . . . . . . . 72 Effects of Training and Anabolic Steroid Use on Body Weight and on Absolute and Relative Organ Weights . . . . . . . . . . . 73 Effects of Training and Anabolic Steroid Use on Carcass Weight and on Body Composition. . 76 Standard Eight-Week, Short-Duration, High- Intensity Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels . . . . . . . 96 Mean Phosphorylase Activity Per Animal of 30 Muscle Fibers Per Area in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles (Percent Light Absorbed = 100 - Percent Light Transmitted). . . . . 97 Mean Glycogen Storage Per Animal of 30 Muscle Fibers Per Area in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles (Percent Light Absorbed = 100 - Percent Light Transmitted). . . . . 98 Mean Absolute Fiber Size (cmz) Per Animal of 30 Muscle Fibers Per Area in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles. . . . . . . . . . . 99 Body Weight and Organ Weight Results (gm) Presented by Animal Number, Training and Drug Treatments . . . . . . . . . . 100 Body Composition Results Presented by Animal Number Training and Drug Treatments. . . . 101 viii Figure 1. 2. 3. LIST OF FIGURES Histochemical Photometer, Front View . . Close Up of Control Panel. . . . . . Diagram of a Typical Cross Section of a "Sandwich" Black of the Gastrocnemius, Plantaris and Soleus Muscles . . . . Mean Daily Percent Expected Revolutions (PER) for CRW SHORT Program . . . . . . Significant Interaction Effect: Absolute Fiber Size--Area 4 . . . . . . . Significant Interaction Effect: Liver weight 0 I O O O O O O O O 0 Significant Interaction Effect: Percent Water. . . . . . . . . . . . ix Page 46 46 48 51 6O 65 69 CHAPTER I INTRODUCTION Early investigations led to the conclusion that the use of steroid hormones produced both an androgenic effect and the retention of nitrogen, an anabolic effect (66, 93, 114). Since then, anabolic steroids have been synthesized which stimulate positive protein metabolism as their main function. That is, chemical modifications of testosterone have led to the development of synthetic compounds with largely dichtomized anabolic and androgenic activities. Krfiskemper (73) has summarized the effects of anabolic steroids on protein synthesis. The proposed mechanism of action on protein assimilation is related to an increase in the ribonucleic acid content of cells, and an increase in the activities of enzymes which activate amino acids. Initially, practical applications of anabolic steroids were limited to clinical medicine where they were used to counteract muscular atrophy, osteoporosis, and the effects of corticoids. In recent years, steroid application has shifted from the pathological to the exercise end of the pathological-normal-exercise continuum with widespread usage among athletes. With the current emphasis on winning at all costs in sports, steroid use has risen markedly. The type of sports most affected appear to be those which include anaerobic-type events emphasizing strength. As a result of this trend, anabolic steroid usage has become quite common with some athletes consuming the steroids in dosages for surpassing what is recommended clinically. Anabolic steroid usage by athletes has not had the prior experimentation necessary to determine whether it is useful and without accompanying pathological significance. Information is needed regarding the effects of anabolic steroids on cellular alterations, particularly in skeletal muscle which is most affected by these drugs. Statement of the Problem The purpose of this study was to determine the separate and combined effects of an anabolic steroid and an anaerobic program of endurance running on selected anatomical and histochemical parameters in the adult male albino rat. Dianabol, a product of the CIBA Pharmaceutical Co., was the anabolic steroid used. The training regimen was the high-intensity, short-duration Controlled Running Wheel program previously reported from this laboratory (115). Body composition and various organ weights were investigated. Histochemical determinations were made of glycogen storage and phosphorylase activity in ten locations of the gastrocnemous-plantaris-soleus muscle group. The cross-sectional areas of thirty muscle fibers were measured in these same locations. Rationale It was hypothesized that anatomical and histo- chemical changes are mediated through the metabolic requirements of the animal. Both exercise and anabolic steroids are known to alter metabolic requirements. Thus, it was assumed that the effects of these imposed treatments should be reflected in the parameters selected for investigation. It was further postulated that cellular responses in skeletal muscle are not only specific to the metabolic requirements of the total muscle but are identifiable by specific muscle areas. Ten pre-selected muscle locations were used to test this hypothesis. Dianabol has been reported to have moderate or low anabolic activity as compared to other commercially available steroids. It was chosen for this study because it has been the steroid most widely used by athletes. The rat was selected since it has been shown to provide a reasonably valid biological model for skeletal muscle. Furthermore, the effects of exercise or organ weights and body composition have been studied chiefly in the rat. Significance of the Problem Specific knowledge of the anatomical and histo- chemical effects of exercise, steroids, and of the exercise- steroid combination are needed. The results of this study can add to that knowledge. Of course, the ultimate questions of whether steroid usage can increase performance and whether steroids can be used in athletes without corresponding pathological changes must await further investigation. Limitations of This Study 1. The steroid dose of 1 mg/rat/day was chosen after consultation with Dr. J. J. Chart of the department of Endocrinology at CIBA Pharmaceutical Co. This dosage was selected to maximize the anabolic effects of Dianabol. However, there were no known quantitative data to support this judgment. In fact, recent evidence indicates the dosage may have been too low for maximum effects in the rat (l4). 2. The exercise program selected for this study represents only one form of "anaerobic" exercise. It was the program judged to be most appropriate for this study from those currently available at the Human Energy Research Laboratory, Michigan State University. Another program, especially one emphasizing a higher expenditure of strength, might have yielded entirely different results. Due to laboratory facilities and the number of Controlled Running Wheels (CRW) available, the number of animals was limited to forty-two. The experimental period was limited to eight weeks. There was no way of knowing whether this duration was optimal for maximizing the exercise and drug effects. The Cornfield-Tukey argument for statistical inference was applied to the population of all rats similar to those chosen for this study. The results of animal studies cannot be translated directly to humans. However, such studies do provide clues as to the structural and functional changes which may take place in the human. Shock provided the stimulus for the animals to run. However, no control for the shock itself was included in the study. Definition of Terms Steroid Hormones.--Those hormones possessing the cyclopentanoperhydrophenanthrene ring system (steroid nucleus) in their molecules. They include the androgens, estrogens, and corticoids. Androgen.--A generic term for an agent (usually_a hormone, e.g., testosterone) that stimulates the activity of the accessory sex organs of the male, encourages the development of the male sex characteristics, or in special cases prevents the latter. Anabolic Steroid.--A compound which relates to or promotes the process of assimilation of nutritive matter and its conversion into living substance. This includes synthetic processes and requires energy. Testosterone.--A male steroid hormone with both androgenic and anabolic effects. It is produced by the Leydig cells of the testes under normal conditions. Dianabol.--A synthetic anabolic steroid derivative of testosterone, produced by CIBA Pharmaceutical Co. The pharmacological name of Dianabol is methandrostenolone while the structural name is 17-methyl-l7-hydroxyandrosta- 1, 4-dien-3-one. Myotropic Effect.--The property of an anabolic steroid to increase muscle mass. This term was used, interchangeably with "anabolic effect" in this study. CHAPTER II REVIEW OF RELATED LITERATURE In order to provide a better understanding of the separate effects of exercise and anabolic steroids, these two topics are first reviewed individually. With this background, the work done with exercise plus anabolic steroids is then reviewed. Effects of Dianabol and Other Anabolic Steroids Measurement ongyotropic Activity Pioneer work in this area began with Kochakian (66) who found an increase in nitrogen retention in three castrated dogs which had received a male sex hormone sub- cutaneously. The positive nitrogen balance was due to changes in urinary urea. Nitrogen retention was greater after the administration of repeated doses (2X daily) than after single large doses. There was a point beyond which increasing the amount of hormone did not increase the amount of nitrogen retained. The weights of the dogs showed significant but not large increases during the injection period but returned to preinjection levels after cessation of the treatments. Wainman and Shipounoff (114) initiated research for determining the myotropic effect of steroids. They observed that the perineal muscles (levator ani, bulbocavernosus and ischiocavernosus) were more responsive to castration and treatment with testosterone propionate than were other striated muscles. In normal rats, the administration of testosterone propionate caused an increase in the bulk of these muscles as manifested by an increase in the width of the muscle fibers. This line of investigation eventually led to the development of an appropriate method for determining an anabolic-androgenic index. Eisenberg and Gordan (73) asked whether the effect on the perineal musculature was due to the anabolic or androgenic component of testosterone. Working with castrated rats, they concluded that any steroid-induced gain in weight of the levator ani muscle was exclusively an expression of the anabolic property of the steroid. Their experimental procedure began with castration of three-week-old rats. Twenty-three days after castration, a steroid was injected daily. The reference compound was testosterone or testosterone propionate. The effects of the reference compound on the levator ani, seminal vesicles, and prostate were compared quantitatively with 10 those produced by the tested steroid. The ratio of the activities of the two compounds was then calculated as an anabolic-androgenic index. Papanioclaou and Falk (93) found hypertrophy of the temporal muscles in castrated immature male guinea pigs when treated with testosterone propionate. No quanti- tative data were given. In a study examining the same muscle group, Kochakian, Humm, and Bartlett (70) found that castration decreased the weight of the temporal muscles in immature male albino guinea pigs to less than one-third that of normal animals. Subsequent subcutaneous implantation of various steroid pellets increased the weight of the temporal muscles. However, the increase was only to about half of that of normal animals. The myotropic-androgenic ratio (increase in temporal muscle mass divided by the increase in accessory sex organs--seminal vesicles and prostate) was determined for various steroids. The castrated animals' body weight was less than that of the normals. The steroids restored the body weight to normal; but when a maximal response was attained, a further increase in dose either had no further effect or was less effective. The method of Eisenberg and Gordan was later modified by Hershberger, Shipley, and Meyer (54). They eliminated the twenty-three-day post-castration rest 11 period for the animals, thus reducing the total assay time from thirty-one to eight days. In evaluating compounds for myotropic effects, the ratio of the response of the levator ani (LA) to the response of the ventral prostate (VP) was employed. The ratio was calculated as follows: Levator ani weight Levator ani weight LA = (Experimental) - (Control) VP Ventral prostate Ventral prostate (Experimental) - (Control) Their preliminary screening with young male cas- trated rats showed l9-nortestosterone and other l9-nor analogs of androgens to be effective anabolic and rela- tively weak androgenic agents. At the same dose levels, l9-nortestosterone showed weak androgenic activity while testosterone showed strong androgenic activity. Metcalf and Broich (84) measured the anabolic potential of steroids using C14 alpha amino-isobutyric acid (AIB). Male rats (150 gm) were castrated and imme- diately given steroids intramuscularly while under ether anesthesia. Thirty hours later 1.0 m1 of AIB solution was administered subcutaneously. Nine hours after AIB injection, the animals were exsanguinated under ether anesthesia by direct cardiac puncture. The sensitivity of the AIB test Was five to seven- fold that of the levator ani weight increment test on the basis of the ratio between percentage increase in uptake 12 to percentage increase in weight. There was a reduction in urinary AIB excretion even before any nitrogen retention became established. The authors suggested the possibility of using this reduction as an additional or alternate indicator of the anabolic activity of steroids. The major problem was to determine what proportion of the uptake of AIB was attributable to the anabolic effect and what proportion was attributable to the androgenic effect of these steroids. Overbeck and de Visser (91) compared (a) the phenyl propionates (PP) of testosterone (T) and nandrolone (N), and (b) the decanoates (D) of testosterone and nandrolone after the subcutaneous injection of a single threshold dose into young rats (50-60 gm). The rats were castrated the day before the experiment, and the levator ani test was used as described by Hershberger, Shipley, and Meyer (54). The anabolic-androgenic ratios were calculated in the following way: = §Anab. (NPP/TPP) Andr. (NPP/TPP) Q (NPP/TPP) fAnab. (ND/TD) Q (ND/TD) RAndr. (N67TBT 13 where: RAnab. potency ratio based on effects on levator ani muscle, R Andr. potency ratio based on effects on seminal vesicle. Thus, the principle of this calculation was to establish potency ratios for each activity and then to calculate ratios of these activities. Their results showed that the nandrolone esters were relatively more anabolic and less androgenic than the corresponding testosterone esters. The phenylpropionates were more active than the decanoates but their duration of action was much shorter, especially with regard to the levator ani muscle. Linearity of the dosage-activity curve which is the essential premise of the Overbeck and de Visser calculation was not met according to Krfiskemper (73). He also points out that the reference steroids of the Hershberger method (testosterone propionate, 17 alpha- methyltestosterone and others) are more androgenic than myotropic. Dianabol as an Anabolic Agent At a high steroid dose level, the uptake of Dianabol (methandrostenolone), as measured by the C14 labeled AIB method (84), was lower than the mean uptake 14 value of testosterone propionate which served as the standard. 1220 and Glasser (59) also recorded low anabolic behavior of Dianabol, in that it did not inhibit the course of protein catabolism in fasting rats. Kochakian (68) observed that testosterone propionate (unlike Dianabol) hastened the replenishment of protein in starved rats, although after body weight was restored the nitrogen retention decreased below that of the controls. It also was observed by Lloyd and Anthony (82) that nitrogen retention did not increase in pigs during a six-week period of feeding methandrostenolone at a level of 1 mg/kg of feed starting when the animals were three weeks old. The animals taking methandrostenolone had a larger number of muscle fibers of small diameter than did the controls. Almqvist, Ikkos, and Luft (3) used graded doses of methandrostenolone (5, 10, and 25 mg/day) and testosterone propionate (25 mg/day) on three metabolically stable subjects all of whom had received steroid therapy before. The results indicated 5 mg/day of methandrostenolone induced nitrogen and calcium retention, while the effects observed with the larger doses were not quantitatively different from the S-mg/day dose. The nitrogen and calcium retention with the S-mg/day dose was as great or greater than that induced by testosterone propionate (25 mg/day). Methandrostenolone induced creatinurea but 15 had no effect on sodium and chloride balances and urinary excretion of l7-ketosteroids. Arnold, Potts, and Beyler (5) using castrated male rats (200 gm) determined methandrostenolone to be 1.2 i 0.14 times as effective for nitrogen retention as methyl- testosterone as measured by urinary nitrogen. When andro- genic activity was measured by the ventral prostate weight method of Hershberger, methandrostenolone was found to be 0.35 :_0.045 times as androgenic as methyltestosterone. The resultant relative anabolic-androgenic ratio was 3.4. However, these values were the lowest in nitrogen retention, highest in androgenic activity, and lowest in nitrogen retention-androgenic ratio of the five steroids studied, disregarding methyltestosterone which was the standard. Other investigators have reported low anabolic effects of Dianabol. Dorfman and Kincl (31) observed decreased seminal vesicles in young castrated rats (21—23 days) treated with Dianabol as compared to those found in similar animals given 17 alpha-methyltestosterone. Levator ani weights were not different. Saarne, Bjerstaf, and Erman (96) administered Dianabol to hospitalized patients and recorded nitrogen retention to be only 1 gm/day. This value corresponds to the generally accepted figure for possible losses through the skin. However, a positive effect was observed by Sloper and Pegrum (99) who injected 0.2 mg of Dianabol daily in mice (25-40 gm) whose right gastrocnemous was crushed. 23‘- -q 16 The treated mice had an accelration both in phagocytosis and in muscular regrowth. They postulated that the acceleration in myogenesis reflected either an increase in RNA and DNA or the special susceptibility of regenerating tissue to the steroid. The effect on myogenesis was probably maximal on the second and third days after injury. Further attempts to quantitate the beneficial effects of anabolic steroids on protein metabolism were made by Albanese (2). He developed the Steroid Protein Activity Index (SPAI). The formula is: _ NSBP _ NBCP SPAI ’ HIE? ’ NICP x 100 where: NBSP = nitrogen balance in steroid period NISP = nitrogen intake in steroid period NBCP = nitrogen balance in control period NICP = nitrogen intake in control period Anabolic agents have a positive SPAI and the magnitude of the value is directly proportional to the metabolic effect. Dianabol's SPAI was determined to be +16 which was the median value of the nine anabolic steroids studied. Recently Boris, Stevenson, and Trmal (l4) injected doses of Dianabol and eleven other steroids for ten consecutive days in rats (60-70 gm) which were 24 to 25 days old at the start of the experiment. Testes weight 17 decreased significantly at 100 mcg/rat/day. Seminal vesicle weight and ventral prostate weight increased at 1,000 mcg/rat/day. These results showed Dianabol to be neither as active anabolically nor as active androgenically as most of the other steroids. The androgenic values are in disagreement with the high values recorded by Arnold (5). In a subsequent study Boris, Stevenson, and Trmal (15) administered Dianabol and eleven other steroids for seven consecutive days using the same experimental con- ditions as before. Potencies were evaluated in terms of the dosages required to double the weights of target organs. Dianabol ranked last in potency rank for the levator ani, ventral prostate, and seminal vesicles. Role of Anabolictgteroids on Skeletal Muscle Glycogen Concentration Lewis and McCullagh (81) examined fasted adult male rabbits (2.7-3.5 kg) with administration of methyltes- tosterone (MT), MT and a high carbohydrate diet, and MT and testosterone propionate TP. The differences observed in the gastrocnemous were not significant although the mean glycogen values (gm %) were: .311 for controls, .338 for MT, .360 for MT and diet, and .351 for MT and TP. The conclusions drawn by Lewis and McCullagh appear to be in the minority. Leonard (79) observed an increase in the glycogen content of the perineal muscles in normal, 18 castrated, and hypohysectomized fasting rats that were injected with 1 mg of testosterone propionate for three days. He postulated that the increase in glycogen con- centration was an index of renewed growth in these muscles. Supporting this view is Kochakian (68) who reviewed the biochemical evidence on the anabolic property of testo- sterone, and suggested that the increase in muscle glycogen was indicative of the mechanism by which the hormone exerts its effect. In order to pursue his theories further, Leonard (80) observed an increase in the glycogen content of the rectus femoris, abdominal, and cremaster muscles, using a 1 mg dose for six days beginning three days after castration in fasting male and female rats. These muscles were used to show that skeletal muscle can be a part of the mechanism by which the hormone exerts an anabolic effect. The duration of steroid use was studied by Meyer and Hershberger (85) who examined the effects of testo- sterone propionate (TP) on the glycogen content of the levator and muscle following administration for one, three, five, and seven days in 21- and S4-day-old castrated rats. There was an initial increase in TCA soluble glycogen; however, with continued use of TP (0.1 mg daily) a decrease in glycogen concentration associated with rapid growth of the levator ani was observed. The authors speculated that the early rise in glycogen represented a storage of 19 potential energy which became depleted in the course of protein synthesis and rapid muscular growth. Supporting this initial rise in glycogen were Adolfson and Ahren (1) who also found glycogen to increase in the levator ani both 10 and 24 hours after injection of testosterone propionate (100 mg/kg) in immature male rats. The effects of various doses were studied by Talaat and Habib (107), who reported an increase in thigh muscle glycogen in male rabbits with a dose of 5 mg/kg of testo- sterone propionate for ten days. Twelve days after cessation of treatment with a 10 mg/kg dose an increase in muscle glycogen was recorded, while no residual change was noted with a 5 mg/kg dose. In a later study, Talaat and Habib (108) castrated male rabbits (1 kg) and administered single injections of testosterone propionate at doses of either 1 mg/kg or 5 mg/kg. In animals castrated for 14 days, muscle glycogen levels were elevated both after 12 hours and after 3 days for both doses. In those animals castrated for 30 days, a 1 mg dose increased glycogen levels after 12 hours; however, then values returned to castrated levels after 3 days. The animals receiving 5 mg showed no changes. The effects of repeated injections of 1 mg for 7 days in the rabbits castrated 14 days earlier resulted in elevated muscle glycogen levels both 12 hours and 3 days after the last injection. With repeated injections of 20 5 mg doses, glycogen was only temporarily increased after 12 hours. Both the present findings and the results of Almqvist, Ikkos, and Luft (3) indicate that the anabolic activity potential of the steroids is not directly related to increases in dosage. Effects of Exercise Response of Skeletal Muscle Glycogen and Phosphorylase Various studies (10, 57, 58) have shown that a high carbohydrate diet increases the resynthesis of muscle glycogen after exercise to levels far above normal values. It has also been shown that a reciprocal relationship exists between phosphorylase and oxidative enzyme activity (33). However, the specific metabolic relationships between glycogen and phosphorylase in the various skeletal muscle fiber types has not been elucidated conclusively. Beatty, Peterson, and Bocek (9) determined that glycogen concentrations were higher in the white fibers than in the red fibers of the adductor muscles of rats immediately after sacrifice. After a two-hour incubation period, glycogen concentration in the red fibers was higher. In a later study, the same group of coworkers (13) again found initial glycogen concentrations lower in the red fibers of the rat adductor group. After a two-hour incubation in litre, the decrease of glycogen in red muscle was one-half as great as in white muscle. Ten 21 times more labeled glucose C14 was incorporated into red muscle during sixty minutes of incubation and three times as much after two hours. Studies also have shown the red area of muscle to have higher glycogen content. Jeffress, Peter, and Lamb (60) found the increase in total glycogen synthetase activity to be greater in the red area than in the white area of the vastus lateralis muscle of guinea pigs. The trained group (treadmill running for 30 minutes at 1.9 km/hr for three weeks) had the highest values, while the exercised group which ran only once had the lowest. Gillespie, Simpson, and Edgerton (45) showed biochemically that the guinea pig vastus lateralis contained more glycogen in the red region (9.7 mg/g) than in the white region (7.4 mg/g). Histochemically, the red fibers showed more intense staining with PAS than either the white or intermediate fibers. They concluded that the metabolic characteristics of muscles can best be described in terms of their histochemically determined fiber populations. Other studies have shown no differences in glycogen content by fiber areas. Short, g£_§l. (97) trained rats for eight weeks by submaximal running (13.7 m/min) on a motor-driven drum. The animals exercised a total of four hours daily, with five-minute rest periods between each thirty-minute running period, six days a week. Examination of the adductor magnus muscle in vivo showed the glycogen 22 concentration to be higher in red muscle than in white muscle only for the trained animals. The in vitro incorporation of glucose C14 into glycogen was greatly accelerated in red as compared to white muscle, and glycogen specific activity at the end of the incubation period was higher in the red fibers. However, these results were present in both the control and experimental groups and were not affected by training. The authors concluded that the differences in glycogen concentrations in red and white fibers may be exaggerations of their normal relationships and not unequivocally attributable to training. Lamb, EE_El° (76) exercised guinea pigs on a motor- driven treadmill (1.9 km/hr). Glycogen concentration was increased 48 hours after exercise; however, both red and white sections of the vastus lateralis muscle exhibited similar patterns of glycogen changes immediately and 48 hours after exercise. Glycogen values of the trained and untrained groups immediately after exercise, when compared with those before exercise, suggested that approximately the same amounts of muscle glycogen were used for the first thirty minutes of exercise by both groups. However, the trained animals with greater glycogen stores could continue to exercise for a longer time. This finding was in agreement with Bergstrom, et a1. (11) who showed a positive 23 relationship between muscle glycogen concentration and exercise tolerance in man. Phosphorylase activity was studied by Rawlinson and Gould (94) who swam rats of three different age groups, for one and two thirty-minute periods daily, for eight weeks. They concluded from biceps femoris homogenates that the total activity of phosphorylase was not affected by either swimming program in any of the groups. Edgerton, g£_gl. (38) exercised guinea pigs on a treadmill at 1.6 km/hr for 5 minutes, 10 minutes, or to exhaustion. Phosphorylase-negative fibers in the plantaris muscle were found with increasing durations of exercise. A higher percentage of fibers in the red region than in the white region became phosphorylase-negative. After exhaustive exercise, white fibers were the most resistent to becoming phosphorylase-negative. No consistent changes were observed in the soleus muscle. This possibly was due to its homogeneous composition of intermediate fibers. The simultaneous investigation of glycogen and phosphorylase has led to divergent results. Stubbs and Blanchaer (105) demonstrated histochemically in guinea pigs that phosphorylase is more active in white muscle fibers (quadriceps femoris) than in red muscle fibers (adductors), while glycogen synthetase showed no differ- ences. However, quantitative determinations showed glycogen synthetase to be higher in red fibers and total 24 phosphorylase (A+B) to be higher in white fibers. Stimu- lation (30 sec. with 1 volt impulses of 20 miliseconds duration at a rate of 20 pulses per sec.) produced a significant conversion of phosphorylase B to A only in white muscle. Stimulation did not alter the synthetase level of red muscle but increased it significantly in white muscle. The quantitative evaluation of glycogen synthetase compared favorably with the results of Jeffress, Peter, and Lamb (60). Kugelberg and Edstrom (74) induced muscular con— tration in the anterior tibial muscle of the rat with low frequency shock (S/sec, and lO/sec) and found phosphorylase and glycogen to become negative fastest in A fibers, next in B fibers, and slowest in C fibers. No changes were observed in the soleus. After one and two hours of stimu- lation (5/sec), glycogen negative fibers were identified but phosphorylase negative fibers were absent. The phosphorylase results were in direct contrast to those of Edgerton, g£_gl. (38), however the methods of inducing changes were not the same. Kugelberg and Edstom concluded that the histochemical method reflects the active form of phosphorylase rather than total phosphorylase, and that histochemical changes in phosphorylase are secondary to changes in glycogen. In order to investigate the differences further, Edgerton, et al. (39) exercised guinea pigs with wind 25 sprints and endurance running on a treadmill for twenty weeks. Following the training program, muscular contraction was induced by electrical stimulation (S/sec for 1 hour) of the medial gastrocnemous. Total phosphorylase activity, when examined histochemically, was selectively depleted in the white region. Phosphorylase depletion was paralleled by glycogen depletion when measured histochemically and biochemically. Every phosphorylase-negative fiber was negative for glycogen as determined by the PAS stain. The histochemical depletion of phosphorylase and glycogen was greater in the nontrained than in the trained animals. Phosphorylase activity did not return to prestimulation levels as was reported by Kugelberg and Edstrom. Quantitative determinations with a hiStochemical photometer have been done at Michigan State University (unpublished data). Using the same ten selected muscle areas as in the present study (see Figure 3, p. 48) and the same exercise regimen (SHORT), with a duration of eight weeks, phosphorylase was found to decrease in areas 9 and 10 and to increase in areas 4, S, 6, and 7. Glycogen as measured by the PAS stain showed increased in areas 1, 2, 3, 5, 7, 8, and 9. A slight decrease was observed in area 4. 26 Body Composition and grggn Weights The known effects of various types of exercise on the absolute and/or relative values of water, fat, protein, and ash are presented in Table l. VanHuss, Heusner, and Mickelson (111) have presented data on the residual effects of exercise. No significant differences were observed. Studies of the effects of various exercise programs on organ weights have been reviewed by Montoye, 2E_2l' (87) up to 1960. The results are presented in Table 2. A continuation of the literature since 1960 is presented in Table 3. It has been shown that the residual effects of exercise are not reflected by differences in organ weights (111). Effects of Exercise and AnabOIic Steroids Human Studies Johnson and O'Shea (61) recorded significant increases in dynamic strength (bench press and squat) and static strength (cable tensiometry) in twelve matched pairs of subjects, aged 19 to 39, who were on a six-week weight training program. All subjects were fed a high-protein diet throughout the program with the experimental group receiving Dianabol (5 mg) twice daily during the final three weeks of the program. 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I + I manor ucwsadzm mason ovum .nm. 0 o o o o o o ensue: unausauo> nuance: o>auaaum ouaaound o>auaaa¢ sundown: o>wudaum «usaoand o>wuaaom ousHOand o>wuaaom dungeon: abwuoaox ouaaoon¢ vague: «wounnsm oawuuoxm ousuuouom mean no onus naocvax nounoa cooamw uo>wa undo: «account .oooa oucam manage: cacao .ucoouom. o>wuoaox can Asa. ouaaoan¢ oz» no oaaouoxm no auoouunII.n names 30 uptake test increased in the treated group. The increase in oxygen uptake was not expected. In an earlier study, Fowler, Gardner, and Egstrom (44) administered l-methyl-A'-androstenolone acetate (Nibal), at a dose of 20 mg/day, alone or in conjunction with a sixteen-week physical condition program. No differences were observed in strength, oxygen uptake ability, serum enzymes, anthropometric measurements, or performance in either the trained or untrained college men who were used as subjects. However, the intensity of the exercise program may have been too low to cause strength increases. In addition, the subjects in the study were not placed on a high-protein diet. O'Shea and Winkler (90) administered a 10 mg/day dose of oxandrolone (Anavar) to eight competitive swimmers and three weight lifters for six weeks during an eleven- week study. Swimming performance was not improved, however the weight lifters showed considerable improvement in strength during the steroid period. Body weight increased for all subjects during the treatment period. An increase in protein utilization, as measured by Albanese' SPAI index, was found in eight of the eleven subjects. SPAI and body weight of the individuals showed a high corre- lation. In a subsequent study, Johnson, gE_gl. (62) selected subjects from college physical education activity classes and randomly assigned them to treatment and 31 placebo groups after four weeks of weight training. A double blind method was used to administer a 10 mg/day dose of Dianabol and a protein supplement for 21 days. There were no significant changes in maximal oxygen uptake, sperm count, or deposition of subcutaneous adipose tissue. Dynamic and static strength and body weight increased in the treatment groups. Similar results were recorded by Bowers and Reardon (17). They administered Dianabol, 10 mg daily for the last 21 days of training, and a protein supplement throughout a six-week weight training program to eighteen experienced weight trainers. The experimental group showed increases in bench press, squat, body weight, and biceps and forearm girths. No changes were recorded in aerobic capacity. Contrasting results were obtained by Fahey and Broun (43). Young men aged 19 to 32 were matched and the experimental group was given nandrolone decanoate (deca- Durabolin R) intramuscularly at a dosage of 1.0 mg/kg of body weight. The subjects were placed on a ten-week weight training program with steroid injections adminis- tered at weeks two, five, and eight of thegprogram. Body weight and oxygen uptake remained unchanged while dynamic and isokinetic strength increased in both groups. It has been observed that Dianabol increases motor time and decreases latency time in the knee jerk reflex. A greater contractile force, as measured by 32 maximal weight lifting, also has been attributed to the steroid (4). Animal Studies The results of studies of the effects of exercise and anabolic steroids on organ weights are conflicting. Murphy and Eagan (89) administered a "steroid cocktail" of Winstrol (9.3 mg/day), Dianabol (0.3 mg/day), and Durabolin (2.5 mg/wk) and an exercise program of S mi/wk of treadmill running to adult male rats for two weeks. Four weeks later, the results of an endurance run were: exercised-steroid group (ES) > exercised (E) > steroid > (S) > control (C). The order of body weights was: C > S = E > ES. Three weeks later, the mean ES weight still remained below that of the other groups. Heart weights did not differ among the groups, while pituitary and testes weights were less for the S and ES groups. Liver weight was 17 percent lower in the ES group than in the C, E, or S groups. Brown and Pilch (21) administered a low dose (0.5 mg/kg) and a high dose (5 mg/kg) of Dianabol and trained rats both by running (1 ft/sec, 5 bouts, 10 min. duration) and by a progressive high jumping program for six weeks. Adrenals, brain, and heart weights were increased in the high jumpers. Testes, kidney, and levator ani weights were significantly increased in rats injected with low doses of Dianabol. High doses only produced a testes 33 weight increase, while performance was not affected by either dose. Measurements of glycogen concentrations have yielded conflicting results. Gillespie and Edgerton (46) determined the role of testosterone in exercise-induced glycogen supercompensation on normal and castrasted guinea pigs. Testosterone propionate (0.833 mg/day) was adminis- tered to the castrated guinea pigs. The exercise program consisted of treadmill running (31 m/min) every other day for ten trials. Trials one through five and trials six through ten were 30 and 40 minutes in duration, re- spectively. The experimental period was twenty-five days and the guinea pigs were sacrificed 48 hours after the final exercise trial. Glycogen values in the vastus teteralis muscle (mg/g) were greater in the normal-trained and castrated-replacement-trained animals than in the castrated—trained and sedentary treatment animals. Exercise was not considered as an independent variable. Taylor and Murray (110) injected Dianabol (.02 mg/kg) daily and exercised rats at 1 mph., 1 hr/day, five days a week. Exercise produced a mobilization of free fatty acids (FFA) from plasma and adipose tissue and glycogen from the biceps and gastrocnemous muscles. Dianabol had no effect upon the storage and utilization of glycogen or upon mobilization of FFA. CHAPTER III RESEARCH METHODS Sampling Procedures Forty-two, normal, male albino rats (Sprague- Dawley strain) of three different age levels, were brought into the laboratory in one shipment. The differences in age were required to accommodate staggered treatment periods set up in conjunction with other concurrent studies using the same facilities. Initiation of treatments began for all animals at 100 days of age. Fifteen animals were 90 days old (Age-Level l); twelve animals were 76 days old (Level 2); and fifteen animals were 62 days old (Level 3) at the time of their arrival. Each animal was randomly assigned to a training- drug treatment group within his own age level. The 90- day-old animals were allowed 10 days to become acclimated to laboratory conditions before the study began. Since all animals began their training at 100 days of age, the Level 1 animals began the program first and the Level 2 and 3 animals followed at succeeding two-week intervals. 34 35 See Table 4 for a complete assignment of animals to treatment groups. TABLE 4.--Random Assignment of Animals To Treatment Groups Within Age Levels. Factor A: Training Level 1 (n=15) Level 2 (n=12) Level 3 (n=15) Exer- Seden- Exer- Seden- Exer- Seden- cise tary cise tary cise tary Factor B: Dru D1anabol 4 l 0 4 4 1 Placebo 4 l 0 4 4 1 Control 4 l 0 4 4 l Research Design The study was organized into a 2 x 3 factorial design. Factor A, Training, consisted of two treatment groups: (A1) an exercise group which was subjected to an anaerobic endurance training program, and (A2) a sedentary group. Factor B, Drug, consisted of three treatment groups: (B1) a Dianabol group, (82) a placebo group, and (B3) a control group. For the trained animals, percent of expected revolutions (PER) served as the performance criterion to eliminate one of the four animals within each of the Dianabol, placebo, and control groups of Age Levels 1 and 3. However, due to a possible infectious leg injury, 36 one animal in the exercise-control group of Level 1 was automatically eliminated from the study. A representation of the experimental design with final cell size can be seen in Table 5. TABLE 5.--Experimental Design With Final Cell Frequency. Factor A: Training Exercise Sedentary Factor B: Drug Dianabol n = 6 n = 6 Placebo n = 6 n = 6 Control n = 6 n = 6 Traininngroups The two training groups in the study were as follows: Exercise (E) The exercise treatment that these animals were subjected to was the SHORT program, which is a high- intensity, short-duration controlled running wheel (CRW) program developed at the Human Energy Research Laboratory, Michigan State University. The CRW apparatus can be described as " . . . a unique animal-powered wheel which is capable of inducing small laboratory animals to par- ticipate in highly specific programs of controlled, reproductible exercise" (115). The animals learned to 37 run by avoidance-response operant conditioning. A low- intensity controlled shock current provided motivation for the animals to run. Following body weight recordings and drug injections at the start of each treatment period, the animals were placed in individually braked running wheels. A light above the running wheel signaled the start of each work interval. If the animal responded to the light by running at or faster than a preset speed, the light was extin- guished and shock was avoided. The time during which the light was on was termed the "acceleration period." If the animal was not running at a predetermined speed by the end of the acceleration period, the light was turned off and a current was applied to the grid running surface of the wheel to induce the animal to run at the prescribed speed. If the animal attained the prescribed speed while being shocked, the shock was immediately discontinued. If the animal slowed down below the prescribed speed, the light and shock sequence was repeated. A typical running program consisted of alternate work and rest periods. During the work periods, the wheel was free to turn; while during the rest periods, the wheel was braked automatically to prevent spontaneous activity. A specified number of alternate work and rest periods (repetitions) constituted one bout of exercise. A single training period would 38 include several such bouts separated by a relatively long time between bouts. The exercise program was progessive in nature. That is, the intensity of the program was gradually increased until on the thirty-seventh day of training, and thereafter, the animals are expected to complete eight bouts of exercise with 2.5 minutes of inactivity between bouts. Each bout consisted of six repetitions of 10 seconds of work alternated with 40 seconds of rest. During the work intervals, these animals were required to run at the relatively fast speed of 5.5 ft/sec. For a complete day-by-day description of the training program see Appendix A. Sedentaryq(S) These animals did not receive any type of forced exercise. To compensate for the handling of the exercised animals, the sedentary animals were weighed during each treatment period. DrggfiGroups The three drug groups used in this study were as follows: Dianabol (D) The animals were given Dianabol five times a week, prior to each exercise period, throughout the eight-week program. The concentration level was 10 miligrams (mg) 39 per cubic centimeter (cc) and the dosage level was 1 mg/rat/day or 0.1 cc/day. The Dianabol was dissolved in Mazzola corn oil (the solvent was chosen following a personal communication with Dr. J. J. Chart, CIBA Pharmaceutical Co.). Placebo (P) These animals were given Mazzola corn oil, 0.1 cc/day, prior to each exercise period throughout the eight-week program. The corn oil corresponds to the solvent system that was used with the Dianabol group. The placebo was also given to counteract any effects which the injection procedure might have had on the Dianabol rats. Control(C) These animals did not receive an injection of any kind. Experimental Procedures The animals were given the training and drug treatments once a day, between 6:30 A.M. and 9:30 A.M., Monday through Friday, for eight weeks in the Human Energy Research Laboratory at Michigan State University. Body weights of the trained animals were recorded before and after each exercise period. Dianabol and the placebo, corn oil, (0.1 cc/day) were injected subcutaneously into the animals following 40 initial body weight recordings. The area of drug adminis- tration was in the lower back (Lumbar) region of the rat. The performance data for each trained animal was recorded daily. Total revolutions run (TRR) and total expected revolutions (TER) were used to calculate percent of expected revolutions (PER). PER = TRR/TER x 100. Animal Care All of the animals were housed in standard, individual, sedentary cages (24 cm. x 18 cm. x 18 cm.) throughout the entire investigation. Since rats are normally more active at night than during daylight hours, the light sequence in the animal quarters was automatically timed to reverse the rat's active period by having the lights off between 1:00 P.M. and 1:00 A.M. A relatively constant environment was maintained for the animals by daily handling, temperature and humidity control, and regular cage cleaning. Throughout the experi- ment, all animals had access to food (Wayne Laboratory Blox) and water fig libitum. Sacrifice Procedures Three sacrifices of twelve animals each were conducted forty-eight to seventy-two hours following the fortieth day of exercise for each age level. After the last treatment period, the animals were placed in metabolism Cages. The housing was changed from the sedentary cages 41 in order that urine volume could be collected for another concurrent study. On the sacrifice day, final body weights were recorded and then each animal was anesthetized by an intraperitoneal injection, 6 cc of a 6.48 percent Halatal solution (sodium pentobarbitol). Selected organs were immediately removed, trimmed, and weighed wet. The right hind limb was skinned and the superficial posterior crural muscles were exposed by reflecting the overlying tissues. The right gastrocnemous, soleus and plantaris muscles were removed as a unit. The unit was held by forceps and quick frozen immediately in 2-methy1butane (isopentane). The isopentane had been previously cooled to a viscous fluid (-l40 to -185°C) by liquid nitrogen. The frozen muscles were put in aluminum 35 mm film containers and placed in a cryostat at -20°C. Frozen muscle weights were obtained before further processing. A block of tissue was cut approximately 10 mm long from the bellies of the muscle unit. The "sandwich" blocks were then mounted onto cryostat chucks using 5 percent gum tragacanth. The use of a "sandwich" block was to insure identical freezing, cutting, incubation, fixation, and mounting of tissues from the three muscles of each animal. Fresh-frozen, serial cross sections, 10 micra thick, were cut using a rotary microtome in a 42 cryostat. Sections were mounted on cover glasses and air dried for at least one hour. The remaining carcass was placed in a plastic bag and frozen for subsequent body composition analysis as described by Mickelson and Anderson (86). Organ Weighpg and Body Composition Procedures Absolute organ weights (gm) were recorded for the adrenals, heart, liver, spleen, testes, and kidneys. Relative organ weights (percent) also were obtained for each animal by dividing the absolute organ weights by body weight. The body composition parameters studied were the percentages of water, fat, protein, and ash. Absolute values in grams were obtained by multiplying the percentage values by the animal's carcass weight and dividing by 100. Additional dependent variables in the study included body weight (gm), carcass weight (gm), and absolute (gm) and relative muscle weights (percent). Histochemical Procedures Glycogen localization was studied by using the periodic acid-Schiff reaction (PAS) (101). Phosphorylase (Phos) activity was demonstrated by the method of Takeuchi (106). 43 Histochemical Methods The metabolic properties of skeletal muscle do not appear to be uniform thorughout a given muscle. Different classifications of skeletal muscle fibers have been developed which are based on cellular constituents and metabolism. In 1962, Stein and Padykula defined A, B, and C fiber types. In 1967, Padykula and Gauthier recommended a change of classification of red, white, and intermediate fibers. Mitochondrial content determined by electron microscopy, was the basis for the change in nomenclature. However, various researchers have continued to use the first method (42, 48, 53, 77, 117). Recent studies have been conducted to type fibers as red, white, or intermediate, but these have not been entirely consistent with each other (8, 9, 13, 26, 36, 37, 38, 45, 88). Other authors have classified only two types of fibers (I and II), some with subclasses of type II (18, 19, 20, 32, 33, 34, 40, 41). A standard nomenclature has not been developed due to differences in fiber typing techniques based upon cellular characterisitcs of fibers. Regardless of the system of classification, fiber types and metabolic characteristics have been rated subjectively, usually by visual microscopy. These subjective ratings may have led to the existing differences in fiber typing. 44 As a result of the fiber typing inconsistencies and a need for a more objective and quantitative method to evaluate histochemically stained tissues, a Histo- chemical Photometer (HCP) was developed by Wells and Heusner (116) at Michigan State University. The HCP provides an accurate and objective method of determining the percentage of light passing through very small areas of tissue (less than one cell). The HCP consists of a Prado microprojector, a photocell with associated circuits to measure light intensity, and a digital voltmeter readout. The projecting microscope is mounted so that light passing through a tissue section is projected upwards at an angle, reflected off a flat front-reflecting mirror, and directed onto a white plastic projection surface mounted on a horizontal table top. The intensity of light passing through a l/l6-inch hole in the center of the circle is measured by the photocell and is displaced as a number between 00.0 and 100.0 percent. The readings are independent of inci- dent light level. Zero percentage transmission corresponds to no light received by the photocell. One hundred per- cent transmission is arbitrarily defined as the amount passing through the glass slide, mounting medium, and cover slip with no intervening tissue. Calibration is required only once for each slide and consists of setting the photometer at zero percent 45 transmission and then at 100 percent transmission as described above. Recalibration is required when the tissue section is changed because variations in the optical density of glass slides at 100 percent trans- mission may cause errors of several percent. The operation of the HCP consists of inserting a slide in the mechanical stage of the microprojector at a magnification of X200. The image of the tissue (muscle cell) to be examined is positioned over the center hole, and following initial calibration, the percentage of light transmission is displayed automatically by the digital voltmeter readout. A model of the histochemical photo- meter can be seen in Figures 1 and 2. Histochemical Analysis The formation of glycogen represents the end product of the synthesis and bonding of glucose units. Phosphorylase is an enzyme responsible for the breakdown of glycogen to glucose-l-phosphate which can then enter the glycolytic sequence of intermediary metabolism. Glycogenn+x + xPiz===2-Glycogenn + xGlucose-l- Phosphate Thus, the substrate glycogen and the enzyme phos- phorylase are important constituents in anaerobic metabo- lism although they do not represent the rate limiting steps of metabolic pathways (78). 46 Fig. I. HISTOCHEMICAL PHOTOMETER, FRONT VIEW Fig.2. CLOSE UP OF CONTROL PANEL 47 To determine glycogen localization and phos- phorylase activity, thirty muscle fibers were randomly selected from each of ten areas in the gastrocnemous, plantaris, and soleus muscles. Thirty fibers were selected as being sufficient to represent the metabolic characteristics of an area of muscle. Figure 3 shows the ten areas of the sandwich block selected for study. Values were recorded as percentage of light transmission but analyzed as percentage of light absorption by subtracting the recorded values from 100. Morphological Analysis Thirty muscle fibers were randomly selected in each of the same ten areas. These fibers were projected by means of a Prado microporjector X200 on a sheet of white paper and carefully traced. Absolute muscle fiber size was measured with a polar plainimeter for each muscle fiber in all ten areas. The units of area represented square centimeters (cmz). These values were transformed to square microns and analyzed as such. Statistical Procedures Mean values for the thirty fibers per area represented the units of analysis for glycogen, phos- phorylase, and absolute fiber size. An arc sine trans- formation (angular transformation) was applied to normalize 48 CD Gastrocnemius CD Plantaris <9 @ Lateral Head Medial Head Fig. 3. Diagram of a Typical Cross Section of a "Sandwich” Block of the Gastrocnemius, Plantaris and Soleus Muscles. The Ten Areas Chosen for Study Are Shown as Numbered Circles. 49 the glycogen and phosphorylase data. This was required to meet the assumption of symmetry neccessary for analysis of variance (100). As a result of freezing artifacts, the glycogen, phosphorylase, and muscle fiber size values for animal number 25, area 1, were lost. Adjusted cell means, which represented the mean value of the other animals in each respective cell, were substituted for the missing data. The data were analyzed using the FACREP routine on the Michigan State University Control Data 3600 Computer (CDC 3600). The model for the data represents a two-way, fixed effects ANOVA. The Tukey Test was used to determine the significance of differences between means following significant analysis of variance results for Factor B; Drug, and the Training-Drug interaction. Egg; ggg procedures were not necessary for Factor A; Training, as it contained only two levels. Statistical significance was set at the .05 level for the two-way ANOVA and at the .10 level for the Tukey post hoc procedures. CHAPTER IV RESULTS AND DISCUSSION Training Results The performance of the three drug groups on the CRW SHORT program was determined by the mean daily percent of expected revolutions (PER). Figure 4 shows that the Dianabol and placebo groups responded similarly during the eight-week program, while the control group had slightly lower PER values. This was particularly evident during the last fourteen days of training when the expected running velocity was increased to 5.0 and 5.5 ft/sec. Histochemical Results THe raw phosphorylase and glycogen values1 for the thirty muscle fibers per area are tabulated by muscle area, animal number, training and drug treatments in Tables B-1 and B-2, Appendix B. The mean values, the analysis of variance results, and the appropriate Tukey Test comparisons are presented in Tables 6 and 7. 1Phosphorylase and glycogen values are given in terms of the percent of light absorbed. 50 51 E800... .EOIm >>mo to. Ema. 225651 888$ 28th 260 822:4 .3... _? no .0T_ll on .._lle_ he I'T|_ o... IiT|_ an lvT|_ on liwmf.n7v_ 63:... HS m A m n c n m _ . _ m 23.2.5: ow on on on. 8 n. :5 .225 IPP-__b-_——-___-_npb-b__—b—_—b-____b_bnH $406 a.» JFK»? deAdr no» r. Ir» M. x). 9 A a .1. XI... 10m 10m I09 102 ION. 30528 oII.o Ommo S-U B-P < s-P Table 8.5. Absolute Fiber Sise--Area 5 Drag Dianabol 4375 3792 4083 F=1.0l Placebo 4083 3958 4021 P=0.377 Control 4208 4583 4396 Column Means 4222 4111 4167' ANOVA Results !-0.23 by Columns P-O.634 Interaction P-l.43; P-0.254 rants 8.--Csntinued. 58 Training ANOVE Tukey Row Results Results Exercise Sedentary Means by Rows by Rows Table 8.6. Absolute Fiber size--Area 6 D Dianabol 4750 4750 4750 P-1.04 Placebo 4917 5125 5021 P-0.366 Control 5250 5125 5188 Column Means 4972 5000 4986' AmVA Results F-0.0l by Columns P-0.912 Interaction F-0.151 P-O.86O Table 8.7. Absolute Fiber Sizeo-Area 7 2’22 Dianabol 4458 3667 4063 F-0.07 Placebo 4125 4292 4208 P-0.934 Control 3750 4542 4l46 Column Means 4111 4167 4139* ANOVA Results P-0.03 by Columns P-0.864 Interaction F-2.051 P-0.147 Table 8.8. Absolute Fiber size--Area 8 Dru Dianabol 3750 3458 3604 F-0.18 Placebo 3667 3542 3604 P-0.837 Control 3625 3833 3729 Column Means 3681 3611 3646' ANOVA Results F-0.l2 by Columns P-o.727 Interaction F-0.S6: P-0.579 Table 8.9. Absolute Fiber Sise--Area 9 Drug Dianabol 3375 3375 3375 P-o.71 Placebo 3333 3917 3625 P-0.501 Control 3708 3250 3479 Column Means 3472 3514 3493' ANOVA Results F-0.06 by Columns P-0.811 Interaction F-3.05; P-0.062 Table 8.10. Absolute Fiber Size--Area 10 Dr! Dianabol 4625 4667 4646 F-0.33 Placebo 4625 5000 4813 P-O.723 Control 4583 4417 4500 Column Means 4611 4694 4653* ANOVA Results F-0.07 by Columns P-0.793 Interaction F-0.25; P-O.780 'Grand Mean. 59 Neither the training nor the drug treatments used in this study resulted in significant changes in mean fiber size. A significant interaction did occur in area 4. Both the training and drug factors were disordinal. The disordinal drug interaction indicated that the sedentary- Dianabol animals had a smaller mean fiber size than the sedentary-control animals. In the disordinal training interaction, the exercise-Dianabol group showed an increase in mean fiber size over the sedentary-Dianabol group; however, the exercise-placebo group had a smaller mean fiber size than the sedentary-placebo group. Figure 5 illustrates the absolute fiber size interaction effect for area 4. Organ Weight Results Body weight and the absolute organ weights are tabulated by animal number, training, and drug treatments in Appendix C. The analysis of variance results and appropriate Tukey Test comparisons are presented in Table 9. I Body weight and the absolute weights of the liver, spleen, kidneys, and muscle were smaller in the exercise group than in the sedentary group. However, the relative weights of the adrenals, heart, liver, testes, kidneys, and muscle in the exercise group were larger than those in the sedentary group. Relative spleen weight was smaller E‘j ‘ 60 WE 6500,. Disordinal Interaction with Drug 6000 l. / Contr0| Placebo 5500 - 5000 - 4500 >- 6; Dianabol § 4000 - .2 J. g Qt l 1 v Exercise Sedentary < TRAINING m I: <1 I w a e e e l e g 6500,. DISOl'dInOI Interaction wuth Tl'CIll'lll'lg tr 3 6000 - Sedentary E Exerclu m 5500'- I'- D g 5000 p 4 4500 L 4000 - J. 01 l 1 l Olanabol Placebo Control DRUG Fig- S-Significant Interaction Effect: Absolute Fiber Size - Area 4 61 TABLE 9.--Analysis of Variance and Tukey Test Results for Body Weight and Absolute and Relative Organ Weights. Training ANOVA Tukey . Row Results Results Exercise Sedentary Means by Rows by Rows Table 9.1. Body Weight Drag Dianabol 436 493 454 F-5.69 D>C Placebo 430 507 468 P-0.008 P>C Control 411 483 447 - Column Means 426 494 450. ANOVA Results F-155.38 by Columns P<0.000S 7 L' Interaction F-l.l3: P-0.336 f Table 9.2. Absolute Adrenals Height (no-2) - Drug “‘4 Dianabol 5.90 5.35 5.52 9-1.22 . “i Placebo 5.45 5.08 5.27 P-0.309 Control 5.49 4.64 5.06 . Column Means 5.61 5.02 5.32' ANOVA Results F-3.92 by Columns P-0.057 Interaction F-0.22; P-0.800 Table 9.3. Absolute Heart Weight Drag Dianabol 1.35 1.35 1.35 P-2.27 Placebo 1.38 1.40 1.39 P-0.121 Control 1.28 1.38 1.33 Column Means 1.34 1.38 1.36' ANOVA Results F=3.20 by Columns P-0.084 Interaction F-1.83t P-0.178 Table 9.4. Absolute Liver Weight Drug Dianabol 13.70 13.67 13.68 F-6.56 h‘c Placebo 12.62 13.78 13.20 P-0.004 Control 11.80 13.33 12.57 Column Means 12.71 13.60 13.15. ANOVA Results F-12.51 by Columns P-0.001 Interaction Ps3.48: P-0.044 Disordinal Interaction with Drug, E-D > E-P 8-0 > E-C Disordinal Interaction with Training, a-v > n—P E-C > S-C Table 9.5. Absolute Testes Height Drug Dianabol 3.58 3.73 3.66 P-0.44 Placebo 3.75 3.80 3.78 v-0.647 Control 3.68 3.78 3.73 Column Means 3.67 3.77 3.72'I ANOVA Results F-0.92 by Columns P-0.345 Interaction F-0.07; p-o.929 TABLE 9.--Continued. 62 Training ANOVA Tukey Row Results Results Exercise Sedentary Means by Rows by Rows Table 9.6. Absolute Spleen Weight (x1071) 2522 - Dianabol 7.92 10.32 9.12 F=l.60 " Placebo 7.99 9.90 8.90 P-0.218 :. I Control 7.87 11.66 9.76 F . Colunn Means 7.93 10.60 9.26' ANOVA Results F-42.44 by Columns P<0.0005 Interaction F-2.04; P-0.l48 ,1 Table 9.7. Absolute Kidneys Height {1” Drgg “' Dianabol 2.73 2.92 2.82 F-0.23 Placebo 2.80 2.93 2.86 P-0.797 Control 2.82 2.84 2.83 Column Means 2.79 2.89 2.84. ANOVA Results P-4.l9 by Columns P-0.049 Interaction F-0.92; P-0.408 Table 9.8. Absolute Muscle Height Dr Dianabol 3.20 3.33 3.26 F=1.31 Placebo 3.27 3.57 3.42 P=0.284 Control 3.21 3.47 3.34 Column Means 3.23 3.46 3.34. ANOVA Results F-8.92 by Columns P-0.006 Interaction F-0.503 P-0.613 Table 9.9. Relative Adrenals Height (x10'4) Drug Dianabol 1.4 1.1 1.2 F-0.70 Placebo 1.3 1.0 1.1 P-0.505 Control 1.3 1.0 1.2 Column Means 1.3 1.0 1.2' ANOVA Results F-21.l9 by Columns P<0.0005 Interaction F-0.32: P-0.728 Table 9.10. Relative Heart Height (X10'3) Drug Dianabol 3.10 2.74 2.92 P-1.06 Placebo 3.21 2.77 2.99 P-0.359 Control 3.12 2.87 3.00 Column Means 3.15 2.79 2.97. ANOVA Results F-6l.41 by Columns P<0.0005 Interaction P-l.51: P=0.236 TABLE 9.--Continued. Training ANOVA Tukey Row Results Results Exercise Sedentary Means by Rows by Rows Table 9.11. Relative Liver Weight (X10'2) Dr Dianabol 3.14 2.78 2.96 F-3.48 D>P Placebo 2.94 2.72 2.83 P-0.044 D>C Control 2.87 2.76 2.82 Column Means 2.98 2.75 2.87. ANOVA Results F-22.27 by Columns P<0.0005 Interaction F-2.4l: P-0.107 Table 9.12. Relative Testes Height (x10’3) D Dianabol 8.20 7.57 7.89 F-2.40 Placebo 8.70 7.51 8.11 P-0.108 Control 8.97 7.82 8.40 Column Means 8.63 7.63 8.13* ANOVA Results F=27.19 by Columns P<0.0005 Interaction F-0.90; P-0.417 Table 9.13. Relative Spleen Height (x10'3) DIE Dianabol 1.82 2.09 1.96 P-4.38 DP Placebo 8.43 12.49 10.46 P-0.028 D>C Control 8.38 12.28 10.33 Column Means 8.48 13.50 10.99' ANOVA Results F-73.28 by Columns P<0.0005 Interaction F-3.15: P-0.057 Table 10.4. Percent Protein Drug Dianabol 21.87 21.17 21.52 F-3.02 Placebo 22.54 21.33 21.93 P-0.064 Control 22.58 21.85 22.22 Column Means 22.33 21.45 21.89. ANOVA Results P-l4.16 by Columns P-0.001 Interaction P-0.513 P-0.604 Table 10.5 Percent Ash Drug Dianabol 3.92 3.73 3.83 P-1.27 Placebo 4.32 3.83 4.08 P-0.296 Control 4.21 3.68 3.94 Column Means 4.15 3.75 3.95* ANOVA Results F-9.92 by Columns P-0.004 Interaction r-o.73x P-O.489 TABLE lO.--Continued. 67 Training ANOVA Tukey Row Results Results Exercise Sedentary Means by Rows by Rows Table 10.6. Absolute Water Dru Dianabol 225 239 232 F-2.56 Placebo 224 256 240 P-0.094 Control 215 247 231 Column Means 221 247 234' ANOVA Results F=49.85 by Colmns P<0.0005 Interaction F-2.82; P-0.076 Table 10.7. Absolute Pat Dru Dianabol 29.94 63.64 46.79 F-4.36 D>C Placebo 27.16 52.00 40.58 P-0.022 Control 27.97 49.04 38.50 Column Means 29.02 54.89 41.96. ANOVA Results F-117.75 by Columns P<0.0005 Interaction F-2.74: P-0.081 Table 10.8. Absolute Protein Drug Dianabol 75.88 85.77 80.83 P-1.53 Placebo 78.05 88.50 83.27 Ps0.233 Control 75.24 87.33 81.28 Column Means 76.39 87.20 81.80. ANOVA Results F-79.26 by Columns P<0.0005 Interaction F-0.30: P-0.746 Table 10.9. Absolute Ash Drug Dianabol 13.58 15.12 14.35 F-2.60 Placebo 14.98 15.89 15.43 P-0.09l Control 13.99 14.67 14.33 Column Means 14.18 15.23 14.71. ANOVA Results F-5.37 by Columns P-0.028 Interaction F-0.321 P-0.726 'Grand Mean. 68 Carcass weight and the percentage of fat were lower in the exercise group than in the sedentary group. The exercise group, however, had higher percentages of water, protein, and ash than the sedentary group. On an absolute basis, all of the carcass components in the exercise group were lower than those in the sedentary group. The per— ‘”“1 percentage and the absolute values of fat were both higher treatment. The percentage of fat was also greater in the in the Dianabol group than in the control group for the drug g t E. Dianabol group than in the placebo group. Data on the percentage of water yielded a signifi- cant interaction and was disordinal with the drug treatment. The sedentary-Dianabol group had a lower percentage of water than either the sedentary-placebo or the sedentary- control groups. Figure 7 illustrates the percent water interaction effects. Discussion The effectiveness of Dianabol as a performance stimulant was not evident when measured by PER values obtained on the CRW SHORT program. The placebo group had values similar to the Dianabol group during the entire study. The control group had the lowest PER values of all groups during the most vigorous parts of the training program. The exercise-induced histochemical depletion of phosphorylase in all ten muscle areas, and the accompanying 69 Disordinal Interaction with Drug 65 F 64 r- 63 i- 62 L- Control Placebo 6| P 60 - 59 - Dianabol JL OT l 1 ES Banks gummy, I; TRAINING 3: I- 65 OrdInoI Interaction wIth Training 2 F' u: 8 Exercise w 54 " 0.. 63 - 62 I. —— Sedentary 6| - 60 - 59 Ii 01 . . . Dianabol Placebo Control DRUG rig. 7- Significant Interaction Effect: Percent Water 70 glycogen supercompensation in five muscle areas, lend support to the hypothesis that there are different mechanisms of glycogen breakdown and subsequent resynthesis. Phosphorylase enzymatically degrades glycogen into glucose-l-phosphate which is then acted upon by phosphoglucomutase to form glucose-6-phosphate which ‘h‘n enters the glycolytic sequence of anaerobic metabolism. . . The resynthesis of glycogen, however, is not the reverse of this pathway, and phosphorylase is not a contributing : H enzyme. The enzyme responsible for the synthesis of glycogen from glucose phosphate units is glycogen synthetase. The presence or absence of phosphorylase does not preclude the presence or absence of glycogen synthetase. Areas 4, 5, 6, 7, and 8 were the regions that showed an increase in the localization of glycogen. Besides considering the metabolic mechanisms involved, it is important to recognize the possibility that certain muscle areas participated more in the exercise program than other muscle areas. Thus, specific muscle area participation might have influenced the sites of glycogen resynthesis. Referring to Figure 3, it can be seen that the central and medial portions of the gastrocnemous and plantaris had the greatest increases in localization of glycogen. The training and drug treatments exhibited a reciprocal relationship on glycogen localization in 71 area 5. Exercise produced an increase in glycogen content while the Dianabol and placebo groups both had lower relative concentrations of glycogen than the control group. Various studies (17, 61, 62) have shown increases in the total muscle girth of humans as a result of anabolic steroid use with weight training. The present results showed no indication of a change in mean absolute fiber size of rats with anaerobic running exercise or with steroid use. Muscle weights did change as a result of training. Relative values were larger and absolute values were smaller in the exercise group than in the sedentary group. However, neither the relative nor the absolute muscle weight differences between the groups are attributable to alterations in absolute fiber size. The changes in phosphorylase, glycogen, and absolute fiber size with training and steroid use in the ten selected muscle areas are summarized in Table 11. The smaller body weights of the exerise group were very evident and confirms the results of previous research (see Table 12). It was difficult to understand why the mean body weights of the Dianabol and placebo groups were both greater than that of the control group but not differ- ent from each other. The Dianabol was dissolved in a 72 .mpcofiumouu mean can moflcfimuu soda pommmm scavooumucfl Honouomflo omnoooum m mmuéIIaufim “when muSHOmnd «ouoz .oa>uomno osmoHMHcmHm no: u z z z z z z z z z z z ouem Hones audaomnd Uvm z z z z z Uvo z z z z camoomaw Uvm % m z z z z z z z Uvo z 2 wood no: mo m mono z z z z z z z z z z ouflm Honflm ouoHOmnm z z mAm mAm mAm mAm men 2 z z comoomao mvm mvm mvm mvm mvm mvm mvm mvm mvm mvm mmmHmoonamonm mcflsflmne OH m m b . o m e m m a usasunoua wand odomoz .mmHomsz msoaom use .mflumusmHm .moosmooouummo an» no means oouooaom cos one ow anew Hones ouoHOmn< can .oomoomao .omoamuonmmosm so amp owoumum oaaonmsd one mowowmue mo muoommmII.HH momma 73 .nucesusouu mono one newsman» new: uuouuo sawuuouuucH Hauduuomfiu a voodooum unmwoa uo>wa sundown: "ouoz .ueeoeueemee yo: I z ova vo one 2 z z z ova z z 2 one qu z z z 2 can some men mvm mxw mvm mvm mvm mAu z mAu mvu mew a men 2 mvm meanness 0 u I I >«ueaeu condoned e>wueuax ousaomnd ebuueaum condemns o>wueaum ausaomA< a>wueaam sundown: use n.0w ousaonnd eawueuam sundownd unoaos usefiueoua moon eaves: sheen“: cooHom moumoa ue>wq uuaom oneseuot .eunmaoz eeueo o>aueflem one condone: :0 one prose: soon ea men neoueum essences one meanness no euouuquI.NH memes 74 corn oil solvent and the placebo contained only corn oil. Whether or not the higher body weights in these two groups can be attributed to the corn oil is not known at this time. While the absolute weights of the liver and kidneys were smaller in the exercise group than in the sedentary group, all of the relative organ weights in the exercise group were larger than those in the sedentary group with the exception of that of the spleen.. The spleen showed both smaller absolute and relative weights in the exercise group than in the sedentary group. The exercise program used in this investigation was of a highly anaerobic nature. All of the previous research reviewed on organ weights involved only voluntary or aerobic-type exercise. The relative changes in organ weights resulting from those exercise regimens were considerably less than were observed in the present study. Thus, it can be postulated that the marked changes in relative organ weights are a direct result of the high intensity of the CRW SHORT exercise program. The relative spleen weight results presented an enigma since this was the only relative weight to be smaller in the exercise group than in the sedentary group. The ability of the spleen to store and release blood is offered as an explanation. The smaller weight may have resulted from the donation of blood by the spleen to help 75 fulfill an acute or chronic circulatory need in the exercised animals. In man the spleen has been known to decrease in size to release as much as 150 ml of blood to the general circulation (49). Larger absolute and relative liver weights in the Dianabol group than in the control group and larger F. relative liver weights in the Dianabol group than in the placebo group suggest a possible functional role of that organ with anabolic steroids. Furthermore, the Dianabol . group had higher relative and absolute fat values than the 53 control group, and higher relative fat values than the placebo group. Additional investigation is needed to determine if liver lipids are the cause of the higher liver weights. The lower carcass weight and absolute and relative fat content as a result of training agree with previous findings. The higher percentages of water, protein, and ash that occurred with training also concur with the literature. The changes in body composition brought about by the exercise regimen and by anabolic steroid use are summarized in Table 13. 76 .ucoEuoouu mono ecu new: vacuum :oHuoouauow Hosanuomao e couscous nous: ucaouom ”auoz .ucaofiuwcmwm was a z er z z z 2 a8 08 7. z z 965 who mvm mAm mvm mvm mvm .uxw mvm mvm 65539 0>H d 0 0 D On 0>H M 0 0 D Om h H .u H m u a .2 .o A m u a be 9.323. 33022 e a 30m 338.2 ”236: ucosueoee see someone one seems emeoeeo .coHuwwanou moom so can uan03 mmflUHMU CO 0mD chuwum UfiHOflMG‘ 0G0 OCHCAGHB NO muowummllemd qu48 CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of this study was to determine the separate and combined effects of an anabolic steroid and an anaerobic program of endurance running on selected anatomical and histochemical parameters in the adult male albino rat. Dianabol, a product of the CIBA Pharmaceutical Co., was the anabolic steroid used. The training program was the high-intensity, short-duration Controlled Running Wheel program developed in this laboratory. Body compo- sition and various organ weights were investigated. Histochemical determinations were made of glycogen storage and phosphorylase activity in ten locations of the gastrocnemous-plantaris-soleus muscle group. The cross— sectional areas of thirty muscle fibers were measured in these same locations. Forty-two, normal, male albino rats (Sprague— Dawley strain) of three different age levels, were brought into the laboratory in one shipment. The differences in age were required to accommodate staggered treatment 77 78 periods set up in conjunction with other concurrent studies using the same facilities. Initiation of treatments began for all animals at 100 days of age. Fifteen animals were 90 days old (Age-Level l), twelve animals were 76 days old (Level 2), and fifteen animals were 62 days old (Level 3) at the time of arrival. ”a Each animal was randomly assigned to training-drug 'n treatments within his own age group. All animals were ‘ allowed a minimum of 10 days to become acclimated to q laboratory conditions before the study began. Since all if animals began their training at 100 days of age, the Level 1 animals began the program first and the Level 2 and 3 animals followed at succeeding two-week intervals. Dianabol was administered subcutaneously at a l-mg/day dose. Treatments were administered Monday through Friday for eight weeks. All animals were supplied with food and water ad libitum. The exercised animals were selected for sacrifice on the basis of having the highest percent of expected revolutions (PER) within their own drug groups. The final sample consisted of 36 animals (six per cell). At sacrifice, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbitol. Selected organ weights were immediately removed, trimmed, and weighed while wet. The gastrocnemous, plantaris, and 79 soleus muscles were removed as a unit and frozen in a isopentane-liquid nitrogen system. Fresh-frozen, cross sections were cut at 10 microns using a rotary microtome in a cryostat. Relative localization of glycogen and phosphorylase were quantitatively determined by a histo- chemical photometer for a sample of thirty fibers in each of ten areas of the muscle unit. Absolute fiber size (micronsz), as measured with a polar plainimeter, was also determined for thirty fibers in the same ten muscle areas. The remaining carcass was saved for subsequent body composition analysis. The results indicated that anaerobic training for eight weeks produced smaller body weights and smaller absolute weights of the liver, spleen, kidneys, and muscle in the exercise group than in the sedentary group. The relative weights of the adrenals, heart, liver, testes, kidneys, and muscle in the exercise group were larger than those in the sedentary group. Relative spleen weight was smaller in the exercise group. Body weights of the Dianabol and placebo groups were both greater than that of the control group but not different from each other. Both absolute and relative liver weights were higher in the Dianabol group than in the control group, and the relative liver weights were also higher in the Dianabol group than in the placebo group. 80 Relative spleen weights were less in the Dianabol and placebo groups than in the control group. Phosphorylase was depleted in all 10 muscle areas as a result of exercise. The Dianabol and placebo groups had less phosphorylase activity than the control group in area 3. An increase of glycogen in areas 4, 5, 6, 7, and 8 occurred with the training program. The Dianabol and placebo groups had less glycogen in area 5 than the control group. Absolute fiber size showed no changes with either the training or drug treatments. Carcass weight and the percentage of fat were lower while the percentages of water, protein, and ash were higher in the exercise group than in the sedentary group. All of the absolute carcass components in the exercise group were lower than those in the sedentary group. Conclusions The results of the study have led to the following conclusions with regard to the albino rat, to the steroid dosage used, to the CRW SHORT program, and to the duration of the experimental period: 1. Anaerobic endurance running produces a non- selective depletion of phosphorylase in the gastrocnemous, plantaris, and soleus muscles, while glycogen localization is selectively increased in the central and medial portions of these same muscles. 81 Dianabol does not produce as marked change in phosphorylase activity and glycogen localization as does the training program. There are no significant training-drug interaction effects with regard to phosphoylase activity or glycogen storage in any of the muscle areas studied. Absolute fiber size is not affected by either exercise or Dianabol usage. Body weight and the absolute weights of the liver, spleen, kidneys, and muscle are all lower with exercise; however, on a relative basis the adrenals, heart, liver, testes, kidneys, and muscle weights are all higher. Relative spleen weight is lower with exercise. Dianabol increases both absolute and relative liver weights over those found in control animals and also increases relative liver weights over those found in animals injected with a corn oil placebo. Body weights are not different in the Dianabol and corn-oil placebo groups; however, both have greater body weights than control animals. 82 Exercise decreases carcass weight and the per- centage of fat while increasing the percentages of water, protein, and ash. Dianabol produces higher percentage and absolute fat values than are found in control animals and a greater percentage of fat than is found in animals receiving a corn oil placebo. Recommendations A wider variety of enzymes and substrates should be studied. The typing of skeletal muscle fibers is needed. The effects of Dianabol should be studied in conjunction with specific anaerobic and aerobic training programs. The effects of different doses of Dianabol should be studied. 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Total eler- Work ti- Bet- Run of Revo- Work Day Day ation Time Rest tions Mo. ween Speed Prog. lu- Time of of Time (min: Time per of Bouts Shock (ft/ (min: tions (sec) Mk. Mk. Tr. (sec) sec) (sec) Bout Bouts (min) (me) see) sec) TBR TNT 0 4-T -2 3.0 40:00 10 l 5.0 0.0 1.5 40:00 . . . . S-P -1 3.0 40:00 10 1 1 5.0 0.0 1.5 40:00 . . . . 1 l-M 1 3.0 00:10 10 4O 3 5.0 1.2 1.5 49:30 450 1200 2-T 2 3.0 00:10 10 4O 3 5.0 1.2 1.5 49:30 450 1200 3." 3 3.0 00:10 10 40 3 5.0 1.2 1.5 49:30 450 1200 4-T 4 2.5 00:10 10 40 3 5.0 1.2 2.0 49:30 600 1200 SI! 5 2.0 00:10 10 40 3 5.0 1.2 2.0 49:30 600 1200 2 1-M 6 1.5 00:10 10 28 4 5.0 1.2 2.5 51:40 700 1120 2-T 7 1.5 00:10 15 27 4 5.0 1.2 3.0 59:00 810 1080 3-M 8 1.5 00:10 15 27 4 5.0 1.2 3.0 59:00 810 1080 4-T 9 1.5 00:10 15 27 4 5.0 1.2 3.0 59:00 810 1080 S-F 10 1.5 00:10 15 27 4 5.0 1.2 3.0 59:00 810 1080 3 l-M 11 1.5 00:10 15 27 4 5.0 1.2 3.0 59:00 810 1080 2-T 12 1.5 00:10 20 23 4 5.0 1.2 3.5 59:40 805 920 3dW 13 1.5 00:10 20 23 4 5.0 1.2 3.5 59:40 805 920 4-T 14 1.5 00:10 20 23 4 5.0 1.2 3.5 59:40 805 920 s-r 15 1.5 00:10 20 23 4 5.0 1.2 3.5 59:40 805 920 4 l-M 16 1.5 00:10 20 23 4 5.0 1.2 3.5 59:40 805 920 2-T 17 1.5 00:10 25 20 4 5.0 1.0 4.0 60:00 800 800 3-M 18 1.5 00:10 25 20 4 5.0 1.0 4.0 60:00 800 800 4-T 19 1.5 00:10 25 2O 4 5.0 1.0 4.0 60:00 800 800 SC! 20 1.5 00:10 25 20 4 5.0 1.0 4.0 60:00 800 800 5 l-M 21 1.5 00:10 25 20 4 5.0 1.0 4.0 60:00 900 800 2-T 22 1.5 00:10 30 16 4 5.0 1.0 4.5 55:40 720 640 3-M 23 1.5 00:10 30 16 4 5.0 1.0 4.5 55:40 720 640 4-T 24 1.5 00:10 30 16 4 5.0 1.0 4.5 55:40 720 640 S-F 25 1.5 00:10 30 16 4 5.0 1.0 4.5 55:40 720 640 6 1-M 26 1.5 00:10 30 16 4 5.0 1.0 4.5 55:40 720 h40 2-T 27 2.0 00:10 35 10 5 5.0 1.0 5.0 54:35 625 600 3-M 28 2.0 00:10 35 10 5 5.0 1.0 5.0 54:35 625 500 4-T 29 2.0 00:10 35 10 5 5.0 1.0 5.0 54:34 625 500 s-e 30 2.0 00:10 35 10 5 5.0 1.0 5.0 54:35 625 500 7 l-M 31 2.0 00:10 35 10 5 5.0 1.0 5.0 54:35 625 500 2-T 32 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560 3-W 33 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560 4-T 34 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560 S-P 35 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560 8 1-M 36 2.0 00:10 35 7 8 2.5 1.0 5.0 54:50 700 560 2-T 37 2.0 00:10 40 6 8 2.5 1.0 5.5 52.10 660 480 3" 38 2.0 00:10 40 6 8 2.5 1.0 5.5 52:10 660 480 4-T 39 2.0 00:10 40 6 8 2.5 1.0 5.5 52:10 660 480 5'! 40 2.0 00:10 40 6 8 2.5 1.0 5.5 52:10 660 480 96 44. APPENDIX B HISTOCHEMICAL RAW DATA en we on on em no no no me me o m a. on an no on on 46 me no mm mm a m so as me we we no em no «a om no a m 64 an on no em no on on on en ea 0 u an as He no no an an an em on no u m an we no on he an an no on an on o u en en no on ca no nm em no om no a m mm on we us on pm me no no he so a u em a. on on no ~a on an ms en en a u ~n on as me on no me no no «a on o u an pm we ~o no on do no as an en a m an on no as an no he as am on no a u on on no mm on «a va us an no am 0 m RN me me we no va me as em on mm o m e~ an en mo «a we on he ~a ca no u m «N on an no on on do me em on we 0 m «w an on on em cm on me no no ~a a m aw me we ~m me an no «a no em am a m «a en ~n on we «a na mm an we «a a m Ha en me ea em mm mm on do me on a m ca me on He no Ha am me as do on o m an an no no no no we as on mm as n m as an en mm ea on we ea an ea ~a a m nu on me me we me nm em as me me o m no me .6 on an on on on an we ~o o m me an we no no no no no en «a on a 6 ea an am an so me on .6 an ~o on o m ma no me an em me he an mm an no u m as no pm we on no he no an no me o m ca on an as on «a am up on «a as o u no me me an we on me on no on he a a no He an on up we we no me on mm o u no em mm «a en en en 46 em me an o u no mm mm Na No mm on no we no as o u so am me me on an on an me no en a n no me me we we on we me an «a en o u do as a a n e m c n w a mono unannnns eonssz smut sauna: nusofiueoua defiant ..oouuannneus ununo unmouee I one : seasoned anode unouuoe. mesons: eneaom one .ennnuneam .asoeosoouuaeo one no neau< vauoaaom sea on» a“ mesa use mucosa muons: on uo Haawsa Ham aua>wuo¢ onaahuonmaonm seQZII.AIm mamas 9'7 98 H. HH HH HH H. HH HH HH HH H. o H H. H. H. HH HH H. HH HH H. HH o. H H H. H. H. HH HH H. HH .. H. 0. HH o H o. .. H. H. HH H. HH HH H. HH H. u m HH .H HH HH HH .. HH .H HH HH 0. u H HH HH H. H. HH H. H. HH .. HH .. u m HH HH H. H. HH H. HH HH .. HH H. H H HH o. HH H. .H H. H. HH HH HH H. H m .H HH HH HH HH H. .H HH HH HH H. H m HH HH oH H. HH HH HH H. H. HH 0. a m HH H. HH .. HH H. HH HH HH HH o. a H HH .H HH HH HH H. OH HH HH HH H. o H HH H. H. HH HH HH HH HH HH HH 0. u H HH HH H. o. HH H. HH HH H. HH H. o H HH HH HH HH HH HH HH oH HH HH H. o H HH HH HH HH HH HH HH HH HH HH H. u H .H HH H. HH HH .H HH HH H. HH HH H H HH HH H. HH HH .H HH HH HH HH HH H H HH H. H. HH HH HH HH HH HH HH H. H H HH H. H. HH HH H. HH HH HH HH H. H H OH .. HH HH HH HH HH HH HH HH H. a H HH H. H. HH HH H. HH HH H. HH HH a H HH HH HH HH .H HH HH HH .H HH HH a H HH .H .. .H HH a. HH .H HH HH 0. o H HH H. .. HH HH H. HH HH HH HH H. u H HH OH H. HH HH HH HH HH H. .H .. H H .H H. o. HH HH H. HH HH HH HH H. a H HH H. H. HH HH o. o. H. HH HH HH 0 m HH HH H. .. H. H. H. H. H. HH HH u H OH H. oH HH o. H. HH H. HH HH HH 0 a HH .. H. HH H. 0. HH HH HH HH 0. H m Ho HH HH HH .H H. H. H. .. H. HH H m Ho .H HH HH HH H. HH HH HH HH HH H H Ho HH H. HH H. HH H. H. HH .H HH 9 m .0 HH HH HH HH .. HH HH .H HH .H a m HO OH H. HH HH H. HH HH HH HH HH 0 m HH OH H H H H H . H H H Haun HchHHua Honeaz Hoquc c0: Onomfl: nun—03900“? .vauuwewcnua unqu unmouwm I OOH I cmnuomat unawq unmoummv noaomaz mamaom can .uwuoucoam .nsoemcoouumoo as» no nomad vouooHom :09 0:» ca aunt uwm muonfih «Hons: on no Hmewcc umm ooououm cmuooaao amozln.~um Handy APPENDIX C MORPHOLOGICAL RAW DATA TABLE C-1.-Mean Absolute Fiber Size (cmz) Per Animal of 30 Muscle Fibers Per Area in the Ten Selected Areas of the Gastrocnemous, Plantaris, and Soleus Muscles. Muscle Area Treatments Animal 10 Drug Training Number 99 HNNHOOOWWOHM‘DMNMHHFQFONFIN"'QwommO‘ONm 00000000000000coo-000.000.000.000... NNNNF‘NNHHNNHHHMHNNHHHNNHHHHHHHNHNHNH WMWMNmV'mel‘HHH.NO§DMI‘mmmHMMH'IflMOle‘Mu-C Flo-lHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH IhhIDIDNM'ID"M‘OHHOHMWH‘MFOM'WMOmvmv-‘mIDFM HHHHHHHHHHHHHHNHHHHHHHHHHHHHHHHHHHHH F'Q‘DU‘OVNO'VFM'WWQFMQOOMGMH'MmCVVFI‘MID FINFlu-4HNHHHHHHHHHHHHHHHNHNHNHHHHHHHHNH OOOOFGHONMMQOOOU‘GFOOH‘DOHQNOOWF‘DMMG'U‘! HNNHHHNHNNNHHNHHHHNNNNNNHNHHNHNHNHNH \DGFMMUF\Dl‘VOID"!FWM‘O'OHMOOOHMtflm‘ONOWtflW HHHHHHHHP‘HHHHHHHHHHHNHNHNNHHNHNHHHHH IDMMI"l‘HmHWOQ'FNWOmNNQHMU-INOMDFVWI‘QHQ‘ NNMHHHNNNHNMHHNHNNNNHNNNNHHHHNNHNNNN fiOIDMO‘MMH'HMNNONGIO‘DONP‘MIDHNNH'MU‘I‘O'NU‘H HHHHOHHHHHHHHHHOHHHHHHHHHHHHHHHHHHHH I"CGOI‘WOMOOWNVNF‘OQNV’MNQFMOOVI‘QQHO‘DI‘I‘O‘ NNHHHHHNHHNNHNHHHNHNNHHNHNHHHHNNHHHH HOMIfiFOOFGI‘OMW‘DGMH'FMHN'WDOQFHNHMMGONH NNNNHNHHHHNNHNHHNNHNNNNNNHHNNNNNHNNN nannmaouoomooanommmmuooooonmmmuooomu n:alumnaramalmanac:mmmmmmmmmmmwmmmmmummmmmm APPENDIX D ORGAN WEIGHT RAW DATA 100 TABLE D-1.--Body Weight and Organ Height Results (gm) Presented by Animal Number, Training and Drug Treatments. Animal Treatments Number BOdY Training Drug Height Adrenals Heart Liver Testes Spleen Kidneys Muscle 01 E D 435 0.0774 1.3154 14.9227 3.9494 0.7992 2.7664 2.9742 03 E D 453 0.0496 1.2804 13.7146 3.7946 0.8101 2.7571 3.2956 04 E D 446 0.0576 1.3970 13.7973 3.5026 0.8289 2.7175 3.0954 05 E P 426 0.0413 1.3334 12.2499 3.6749 0.6918 2.6670 3.1535 06 E P 414 0.0334 1.3007 12.5351 3.3929 0.8090 2.9472 2.8682 07 E P 456 0.0797 1.4260 13.5044 3.9198 0.8629 2.9375 3.4621 09 E C 415 0.0646 1.3189 13.2426 3.9931 0.7276 3.1373 2.9913 10 E C 398 0.0592 1.2246 11.8115 3.6359 0.7312 2.6746 3.1042 11 E C 410 0.0496 1.2759 12.0839 3.3544 0.7800 2.6113 2.9937 13 s D 485 0.0526 1.3914 14.2140 3.6710 1.0171 3.0307 3.3397 14 s P 508 0.0500 1.5547 14.6358 3.5619 1.1100 2.9462 3.5358 15 s C 502 0.0475 1.5257 13.4519 4.2062 1.1809 2.8254 3.3868 16 s D 488 0.0510 1.3124 13.8906 3.2496 0.8593 3.1193 3.1889 17 S D 490 0.0575 1.3023 11.9455 3.6099 0.9166 2.9169 3.6033 18 s D 500 0.0599 1.4270 14.0523 3.6838 1.0034 3.1048 3.4016 19 s D 487 0.0459 1.3202 13.5571 3.8265 1.1025 2.5021 3.2030 20 s P 514 0.0578 1.3750 13.6198 3.8173 1.0557 2.9516 3.4564 21 s P 483 0.0480 1.2760 13.3880 3.6385 0.7778 2.9183 3.7287 22 s P 502 0.0500 1.4273 13.2878 4.1472 0.9716 2.9953 3.4493 23 s P 528 0.0523 1.3931 13.7386 3.8738 1.0239 2.9352 3.7961 24 S C 482 0.0438 1.3073 13.2789 3.4885 1.1696 2.8341 3.4404 25 s C 494 0.0484 1.4141 13.7450 3.8381 1.4645 2.8286 3.3758 26 S C 467 0.0555 1.3106 13.3838 3.4420 0.9390 2.7073 3.3548 27 s C 500 0.0359 1.3410 13.4236 3.9663 1.1207 2.8917 4.0844 28 z D 419 0.0542 1.3766 12.9060 3.4391 0.8411 2.5778 3.3314 29 s D 451 0.0586 1.4094 14.6118 3.9617 0.7534 2.9678 3.5409 31 E D 409 0.0565 1.3213 12.2279 2.8280 0.7196 2.6014 2.9787 32 B P 451 0.0608 1.4813 12.7851 4.1782 0.7945 3.0099 3.5551 34 E P 433 0.0559 1.4628 12.5734 3.9467 0.9079 2.5777 3.5015 35 E P 401 0.0557 1.2879 12.0442 3.3775 0.7302 2.6568 3.0524 36 E C 435 0.0476 1.3057 12.4489 3.5389 1.0356 2.8642 3.3643 38 E C 409 0.0572 1.2774 10.5546 3.8675 0.7541 2.8552 3.4768 39 s C 398 0.0509 1.2915 10.6853 3.7050 0.6947 2.8047 3.3241 40 s D 506 0.0539 1.3539 14.3664 4.3504 1.2971 2.8163 3.2235 41 s P 505 0.0469 1.3849 14.0388 3.7759 0.9438 2.8269 3.4487 42 s C 453 0.0472 1.4100 12.9939 3.7266 1.1194 2.9295 3.1828 APPENDIX E BODY COMPOSITION RAW DATA TABLE E-l.--Body Composition Results Presented by Animal Number 101 Training and Drug Treatments. Treatments Percent Animal Carcass Number Training Drug Weight(gm) Water Fat Protein Ash 01 E D 342 65.04 10.68 20.38 3.21 03 E D 366 65.66 8.23 21.94 3.49 04 E D 351 63.52 9.25 21.50 4.41 05 E P 343 65.54 7.56 21.94 4.19 06 E P 333 62.93 10.23 21.88 4.39 07 E P 364 63.49 9.71 21.81 5.27 09 E C 332 64.01 9.36 22.19 4.07 10 E C 317 64.74 7.36 22.31 4.56 11 E C 330 63.64 8.81 22.31 4.50 13 S D 398 61.43 12.68 21.44 3.68 14 S P 420 62.88 12.58 20.50 4.06 15 s C 408 62.72 11.45 21.50 3.68 16 S D 401 58.52 16.86 21.00 3.70 17 s D 407 62.26 11.60 22.12 3.62 18 S D 410 57.74 17.66 20.75 3.58 19 S D 399 55.24 20.12 20.19 4.05 20 S P 429 58.43 15.93 20.75 3.99 21 S P 389 62.29 10.72 22.06 3.96 22 S P 406 61.61 12.40 21.38 3.86 23 S P 434 62.71 11.56 21.88 3.75 24 S C 399 61.33 12.83 21.62 3.75 25 S C 409 62.56 12.73 21.12 3.17 26 S C 384 60.78 12.80 22.19 4.08 27 S C 416 62.82 10.90 22.00 3.39 28 E D 337 64.40 7.20 23.44 4.56 29 E D 363 65.14 8.92 21.69 3.82 31 E D 324 65.89 7.38 22.25 4.02 32 E P 364 65.44 7.85 23.50 3.69 34 E P 348 66.25 6.47 23.31 4.26 35 E P 325 64.63 8.78 22.81 4.13 36 E C 350 64.76 9.12 22.25 3.67 38 E C 340 65.23 7.56 23.12 4.01 39 E C 330 63.98 8.09 23.31 4.44 40 S D 416 58.92 15.35 21.50 3.77 41 S P 413 62.75 11.77 21.38 3.35 42 S C 383 59.89 12.99 22.69 4.00 "”'l’fliii'iifllflmifl 11111111171153