A STUEY 0F THE EFFECTS OF EXERCISE OE VARIGUS FREQUENCEES AND ENTENSETIES UPON THE SWIMMENG ABELETY AND PHYSEOLOGICAL WELLBEING 0F SPECIFIC PATHOGEN FREE MALE ALBTNO RATS um Them for degree of Ph. D. Michigan State University Wynn Franklin Updyke 1962 This is to certify that the thesis entitled A STUDY OF THE EFFECTS OF EXERCISE OF VARIOUS FREQUENCIES AND INTEV‘TSITIES UFO“.T THE SWIMMING ABILITY AND PHYSIOLOGICAL HELL-BEING OF SPECIFIC PATHOGEN FREE MALE ALBINO RATS presented by Wynn F. Updyke has been accepted towards fulfillment of the requirements for Ph. D. degree in Physical Education /' // {/41 Egg/{Ll / §w\, MAIL/’1 / Major professor Date Get. 5’, 1962 0-169 u LIBRAR Y1 Michigan State University ABSTRACT A STUDY OF THE EFFECTS OF EXERCISE OF VARIOUS FREQUENCIES AND INTENSITIES UPON THE SWIMMING ABILITY AND PHYSIOLOGICAL WELL-BEING OF SPECIFIC PATHOGEN FREE MALE ALBINO RATS by Wynn F. Updyke The purpose of the study was to investigate the effects of exercise of various frequencies and intensities upon the swimming ability and physiological well-being of specific pathogen free male albino rats. One hundred and twenty mature male albino rats (Carworth Farms (CFE), free of specific pathogens, were trained by swimming in water maintained at a temperature of thirty five to thirty seven degrees centigrade. All animals, except twenty controls, were re- quired to swim daily for gradually increasing periods of time (up to 30 minutes) with a small weight in the form of fishing sinkers attached to the base of the tail. The size of this weight was gradually increased as the animals were able to manage it. Following a training period of four weeks (which was inter- rupted by a respiratory illness) all animals were subjected to an all-out swim test with two percent of body weight attached. The results of this test indicated that the swimming ability of the con— trol animals was so vastly superior to that of the experimental animals that it was decided to abandon the original objective of the experiment and study instead the reasons underlying the poor Wynn F. Updyke performance of the experimental animals, and to determine the best training combination for recovery of swimming ability. Following a second all-out swim test with three and one half percent of body weight attached, all animals were assigned to groups so that the mean swimming times of groups were approximately equal. The experiment was designed so that the rats of Group IA were required to swim daily for thirty minutes with as much weight as they could carry while those in group IB, swimming daily, were required to carry a much greater percentage and to swim until they became exhausted. The weight was to be great enough to prevent swim times in excess of five minutes. A similar pattern was set up for groups IIA and IIB except that they swam every other day. Groups IIIA and IIIB were made to exercise by swimming under the same set of conditions but only every fifth day, while on every tenth day groups IVA and IVB were forced to swim. Finally group V was allowed to re- main sedentary, serving as a second control group differing from the overall controls only in the reSpect of having undergone the pre- liminary training with the other eXperimental animals. After training according to this schedule for five weeks, a .second all-out swim test with three and one half percent of body weight attached was administered. Following this was another five weeks of training and then a final all-out swim test. Two days after the final swim all animals were sacrificed and subjected to complete post-mortem examination. Statistical analysis showed that the controls were signifi- cantly better swimmers than all other animals on Test No. 1 only. There were no differences between the mean swimming abilities of the various groups on either of the other two all-out swim tests. Wynn F. Updyke There appeared to be slight, although significant improvement for the animals generally during the last half of the experimental period. Only four of the individual groups, however, (Control, III A, III B, and IV B) exhibited significant changes in this regard. The most striking factor observed was that of the abrupt dr0p in swim times of the control animals after all-out swim test No. l, a drOp which was regained only in part during the last five weeks of the experiment. The post-mortem examination revealed that virtually all animals were suffering from mild to severe forms of chronic murine pneumonia upon which was superimposed an acute reSpiratory disease. Bacteriological cultures, however, failed to clarify the etiology of either disease. Moreover, there was no apparent or statistically positive relationship between the severity of lung lesions and dura- tion of swim time. There was a statistically significant indication (P = .99) that animals with the most severe lung involvement swam the longest during the final all-out test. Analysis of the results of this study led to the following conclusions: 1. The trapping of air in the fur of the swimming rats was the dominant factor in the experiment, and was responsible for distorting swim times to the extent that other relationships were obscured. 2. The fact that animals, supposedly free of the pathogens responsible for chronic murine pneumonia, were found to be afflicted with that disease indicates that such animals must be housed under Special conditions if their health is to be maintained. 3. The great lack of consistency of swimming performance Wynn F. Updyke indicates that the methods used in this eXperiment are inadequate as a means of differentiating between various degrees of swimming potential. 4. The generally poor swimming performance of the animals involved must be at least partially attributed to the reSpiratory diseases discovered in the animals. As to whether the chronic or acute form of the illness was more debilitating no conclusions are drawn. A STUDY OF THE EFFECTS OF EXERCISE OF VARIOUS FREQUENCIES AND INTENSITIES UPON THE SWIMMING ABILITY AND PHYSIOLOGICAL WELL-BEING OF SPECIFIC PATHOGEN FREE MALE ALBINO RATS By Wynn F1 Updyke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health, Physical Education and Recreation 1962 ACKNOWLEDGEMENTS The writer is indebted to Dr. William Field, Mr. Paul Yevich and Dr. Robert Schirmer for their expert advice, assistance and willing cooperation, without which this study could not have been undertaken. ii TABLE OF CONTENTS Chapter I. THE ‘PROBLEM AND DEFINITION OF TERMS USED The Problems . . Nature of the problem . Statement of the problem Limitations of the study Definitions of Terms . Specific Pathogen Free Chronic Murine Pneumonia Initial Test . . Test No. 1 Test No. 2 Test No. 3 II. REVIEW OF RELATED LITERATURE The Nature of Training . Biochemical Adaptation . Cardio-Respiratory Adaptation Training Methods . . . Variability of Swim Times Training and Body Weight . . Training and Resistance to Disease . Respiratory Disease In the Rat . III. EXPERIMENTAL METHOD . Material and Equipment . Design of the Experiment . The Original Design . . . Interruption of the Study . Alteration of the Design IV. RESULTS AND DISCUSSION Results Swimming Times . . Pathological Findings . Discussion . . . Discussion of Results . Discussion of Pathological Findings . V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary Conclusions Recommendations BIBLIOGRAPHY . iii "U m 00 (D O‘O‘O‘O‘O‘O‘O‘U‘WHHH TABLE OF CONTENTS-—Continued Page APPENDIXES S4 55 57 59 6O 61 64 65 66 HHL‘EQMMUOW» iv Table II. III. IV. VI. VII. VIII. LIST OF TABLES Pre-Experimental Training Schedule December 26 to March 18 . The Basic Design of the Experiment H Values For Rank Sum Test of Homogeneity of Swim Time Rank Sum Test of Differences Between All-Out Swim Tests . Rank Correlation of Swim Times (Reliability Coefficients) . . . . . . . . . . Correlation of Body Weight and All-Out Swim Time Chart-Summary of Pathological Findings Rank Sum Test Comparison of Gross Lung Involvement and All-Out Swim Times Page 20 24 31 33 34 35 37 42 LIST OF FIGURES Figure Page 1. Mean and Median All-Out Swim Times in Minutes . . . . . . 29 2. Means and Ranges of All-Out Swim Times in Minutes . . . . 30 vi CHAPTER I THE PROBLEM AND DEFINITIONS OF TERMS USED The Problem Nature g£_£h§ problem. The problem of controlling undesirable variables is such a universal one, in any type of experimental work in which human subjects are involved, that frequently animal studies are undertaken in order to attempt to establish general principles which may be applicable to the animals and human beings alike. There is no question that such studies are often fruitful and occasionally even indispensable. In fields such as physical education there are areas in which knowledge is so limited that it is essential to obtain general information as quickly and as accurately as possible. It is often impractical or even impossible to obtain the necessary information through direct study of human subjects, because of the strict controls required, the numbers involved, the necessity of studying changes in organs, or for similar reasons. Because of such limitations, animal research in physical education offers great promise as a worthwhile endeavor. Such a vieWpoint is tenable only, however, if the assumed advantages actually exist. This is particularly true in the case of experimental variables. If variables can be more effectively re- duced in working with animals than with humans, then perhaps animal work can be justified. If not, perhaps such effort is worse than useless. 2 In recent years considerable effort has been directed toward producing laboratory animals which would provide reliable and valid data. The albino rat, being one of the most commonly used experi- mental animals, has undergone a particularly intensive program of refinement. A number of highly specialized strains has been deve10ped in an attempt to provide experimental material for research workers in widely divergent fields of study. Despite great strides which have been made in this direction, albino rats and mice have continued to exhibit a strong tendency to develOp respiratory diseases under cer- tain circumstances, notably when subjected to physiological stressors such as toxicity, trauma, and forced exercise. The term "chronic murine pneumonia" or "endemic pneumonia" is often used in describing such ailments. One of the most disturbing factors involved in this problem is the fact that the chronic murine pneumonia is clinically silent and can be diagnosed, both as to existence and extent of in- volvement, only by means of post mortem examination. By implication then, the results of all rat training studies involving even moderate exercise or other stressors are subject to question, unless such ex- aminations are performed. If, however, it could be clearly shown that respiratory involvement does not inevitably result in such cases, the material and methodology involved would be of considerable interest. Several breeding laboratories have developed colonies of rats which are free of specific pathogens. Such colonies are derived from a nucleus of animals which are taken by caesarean section and cross ‘suckled on another strain of germ-free stock in order to avoid the milk-borne pathogens from the mother. Such animals are advertised as being remarkably free of respiratory illnesses such as chronic 3 murine pneumonia. They are, however, bacteria susceptible. The present study developed out of an attempt to utilize Specific Pathogen Free rats (Carworth Farms) in studying the mainte- nance of cardio-respiratory endurance. Because of previous diffi- culties encountered in using standard laboratory strains in work of this general nature, it was decided to employ the SPF rats. Statement g£_£hg_problem. I The original problem: The original purpose of this study was to ascertain the minimum amounts of exercise necessary for the main- tenance of a pre-established level of cardio-respiratory endurance in specific pathogen free albino rats. Importance of the problem: Because of the difficulty with respiratory diseases encountered in previous experiments involving heavy training of rats by means of swimming, it was decided to use SPF animals because of their greater freedom from such diseases. Since the SPF animals in question were advertised as free of the pathogens commonly associated with chronic murine pneumonia, it was believed that the use of such animals would yield data unclouded by the post mortem discovery of pathological lesions of the kind which had been encountered in some previous work. The fact that chronic murine pneumonia cannot be detected by any known clinical diagnostic technique, and that its existence and/or the extent of involvement can be determined only by post mortem ex- amination, has caused considerable concern among researchers who experiment with rats under conditions involving moderate to severe stress. Such stressors, regardless of their specific nature (cold, drugs, trauma, exercise, etc.) frequently have been found to trigger the onset of chronic pneumonia in otherwise apparently healthy animals. 4 Thus, in an experiment in which data is collected in terms of swimming times, although the presence of chronic murine pneumonia may be re- flected in terms of performance, sacrifice of suspected animals is the only sure method of determining the existence of the disease. It is conceivable, then, that a training study of some type could be con- ducted, analysed, and reported upon without any consideration of the presence of respiratory disease, if sacrifice of the animals (in- -cluding histological examination of lung sections) were not provided for. It thus becomes important to determine whether there are strains of rats which can be successfully utilized in studies of exercise physiology. In accordance with the original purpose of this investigation, 125 male SPF albino rats (Carworth Farms CFE) reportedly free of salmonella, bartonella, and endemic pneumonia were obtained at the age of eight weeks (maturity). These animals were all trained until they could swim for 20 minutes with 2 percent of their body weight attached to the base of their tail. The training was accomplished by gradually increasing load and swim time over a period of four days. Twenty of these rats were randomly selected as the control group. The rats in this group were not permitted to swim again ex- cept at the three all-out test periods. Training of the remaining animals was continued with gradual vincreases of weight along with an increase in swimming time up to 30 minutes. (Table No. I shows the entire training schedule for the group). Following the training period of approximately seven weeks (not including a five week interruption due to an acute illness to be 5 discussed in Chapter III), the animals were subjected to an all-out endurance swim test. Two percent of each animal's body weight was attached to its tail, and the length of time it was able to swim, be- fore drowning became imminent, was recorded. The revised problem: The results of this initial test were such that it was impossible to pursue the original design_further. As has been stated above, it was necessary to deve10p a given level of endurance in the experimental animals before beginning the formal study of the factors affecting the maintenance of such a character- istic. The test results revealed, however, that not only had the ‘experimental animals failed to develOp the desired endurance, but had actually regressed to the point that the nonaexercised control animals outperformed them in average swim time by a ratio of about seven to one. Because of the extreme unexpectedness of this result it was deemed important to attempt to determine the cause of this phenomenon. Of secondary interest was the attempt to determine which of several training methods might be most effective in returning the animals to the normal levels of swimming ability. The altered objective of this study was to study the effects of exercise of various frequencies and intensities upon the swimming ability and physiological well-being of Specific Pathogen Free male albino rats. Limitations 9_f_ the £31511. 1. No attempt was made to control humidity of the animal ‘ room. 2. No attempt was made to control or measure food intake. All animals were fed ad libitum. 3. Results of this study are assumed to apply only to albino rats of similar strain under similar conditions. 6 4. The attempts to determine causes for the various per- formance patterns of individual animals have been limited to an analysis of routine pathological findings reported by Dr. William Field and Mr. Paul Yevitch. 5. No attempt was made to study any learning or con- ditioning effects (in the Pavlovian sense) in this experiment. Definitions of Terms Specific Pathogennggg, Strain of rats (Carworth Farms) started by aseptic caesarian section from mother rats belonging to the original Carworth-Wistar colony, free of salmonella, bartonella and endemic pneumonia. Chronic mg£in§_pneumonia. Also called endemic pneumonia; a chronic respiratory disease of rodents which is attributed to a virus- like etiological agent. This disease, which may involve two independ- ent diseases (i.e. infectious catarrh and chronic bronchiectasis) is clinically silent and can be identified only by post-mortem examina- tion. Initial £235. The first all-out swim test; conducted under the assumptions upon which the original design was based, namely that training would increase swimming ability. Igg£.Ng;_l, The second all-out test conducted three days after the initial test above. This was the first of three tests set up under the assumptions of the altered design. Ig§£.Ng;.§, The mid-point or fifth week test under the altered design. Test No. 3, The final test of the ten week eXperimental period. CHAPTER II REVIEW OF RELATED LITERATURE In making a study of the effects of exercise on any organism it is helpful, if not essential, to have some knowledge of mechanisms underlying the adaptation processes. This is particularly true in a case such as this one, in which the expected adaptation apparently was prevented from occurring. Any attempt to explain the failure and/or to undertake remedial action should be based upon a basic understanding of the physiology of the experimental subjects. An awareness of common pathological findings, with respect to specific experimental condi- tions, is also of considerable value. Ihg'nature 2: training. "Even if we know pretty well the re- sult of training on different bodily functions we know nothing of the essence of training." (19) This statement, made by P. O. Astrand sums up the situation well with regard to the true nature of the effects of vigorous exercise. In his review of the various effects of training he makes special note of the fact that little is known concerning which forms of training produce the desired results in the shortest possible time. His general conclusion is that the work load should be gradually increased so that the organism will have sufficient time to adapt itself to the work. Biochemical Adaptation. The site at which the adaptation pro- cess occurs has undergone considerable scrutiny. Energy sources for muscular contraction have been studied with some consistency of findings. 7 8 Rawlinson and Gould (9) reported that the adenosine triphis- photase and creatine phosphokinase levels, assayed in muscles of animals trained by swimming thirty minutes daily for six 5 day weeks, were unaffected. In a further study, (27) in which 8, 11, and 15 week old rats were required to swim for thirty minutes daily for six weeks no dif- ferences in muscle assays of lactic dehydrogenase, molic dehydrogenase and phosphorylase were detected. Their conclusion was that the adaptive reaponses appeared to involve regulatory mechanisms rather than any change in enzyme levels. Hearn and Wainio (11) studied the succinic dehydrogenase activity of the heart and skeletal muscle of rats exercised by swimming for 1/2 hour daily at 320 C. Although exercise did not significantly alter the unit and total activities of skeletal muscles, it was found to increase the relative total activities of the heart ventricles. Aldolase activity of both skeletal muscles and heart ventricles was found to be increased in the exercised animals. (12) In contrast to their findings, Hearn and Wainio reported that Chepinoga found an increase of succinic dehydrogenase activity of 50 to 100 percent with exercise. Yakovlev was quoted as reporting that enzymes of aerobic metabolism were first to increase during training and last to decrease upon the cessation of training. Gollnick and Hearn (8) concluded from their work that enzyme activities could be altered as a result of exercise. Their study in- dicated that the exercise used by them (swimming) was a greater stress on the heart than on the skeletal muscle. In studying the mechanisms of muscular fatigue in adrenalec- tomized rats, Ramey, Goldstein and Levine (26) found that the normal 9 controls swam up to three hours without suffering any ill effects, while the adrenalectomized animals appeared to be fatigued after ten to twenty minutes and had become exhausted by thirty minutes. No differences in glycogen levels were found between groups before or after the swimming. The conclusion drawn from this work was that part, if not all, of the difficulty encountered by the adrenalectomized animal stemmed from failure to make proper neuro-circulatory adjust- ments. In utilizing rats which had undergone adrenal enucleation, Harris and Ingle (10) were unable to show any differences between normal and enucleated animals in terms of all-out swim times. In another approach to study of specific adaptive mechanisms, Milder and Darrow (22) demonstrated that within wide limits, the amount of potassium in the muscle cells did not limit the capacity of rats to swim continuously for sixty minutes. Cardio-respirggggy.adaptation. The limiting factors for work capacity (i.e. tidal volume, red cell volume, heart rate, stroke volume, maximal 02 uptake, etc.) are fairly well known and it is not intended that this chapter should contain an exhaustive review of re- search in this area. Most of these factors are directly concerned with the ability of the blood to deliver the required amount of oxygen to the body tissues. (1)(3)(4)(6)(7)(13)(25)(28)(30) That training increases this capacity via several avenues can easily be demonstrated. Reasons for the failure of this capacity to increase with training are not so easily determined, and studies dealing with this particular problem are rare. The type of training itself might well play some part in 10 failure of an endurance factor to be develOped. Vanderhoof, Imig and Hines (35) found that "significant changes in blood flow response to exercise stress are associated with improvement in endurance rather than strength." They suggested that the vascular bed was capable of opening up to a greater extent during and following exercise as endurance training progressed. The implication is that if a training method which was designed to develop strength was employed, a con- current develOpment of endurance should not be expected. The concept of stress physiology as developed by Selye sug- gests the possibility that the intensity of the exercise itself may be detrimental to the normal adaptation process, particularly if the »stressor (exercise) is applied unremittingly over too long a period. (l7)(32) Training methods. A major problem encountered in any attempt to deve10p a specific characteristic (such as endurance) through training is the selection of method to be used. Scheer, Efihél- (29) approached the problem of assigning equiv- alent weights to swimming rats on the basis of specific gravity. If the specific gravity of an animal was found to be less than 1.04, brass weights were attached to the thorax with elastic bands to adjust it. The physical capacity of the rats was determined by requiring animals to swim in water 360-38o C. while weights were added in 2 gram increments every three minutes. Failure to reach the surface during a ten second period constituted the termination criterion. In support of this procedure the following statement was made: "It would perhaps be possible to devise a schedule of loads for rats of different sizes, but there is no evident theoretical basis for such a schedule." (6) 10 failure of an endurance factor to be develOped. Vanderhoof, Imig and Hines (35) found that "significant changes in blood flow response to exercise stress are associated with improvement in endurance rather than strength." They suggested that the vascular bed was capable of opening up to a greater extent during and following exercise as endurance training progressed. The implication is that if a training method which was designed to deve10p strength was employed, a con- current development of endurance should not be expected. The concept of stress physiology as developed by Selye sug- gests the possibility that the intensity of the exercise itself may be detrimental to the normal adaptation process, particularly if the istressor (exercise) is applied unremittingly over too long a period. (l7)(32) Training methods. A major problem encountered in any attempt to develop a specific characteristic (such as endurance) through training is the selection of method to be used. Scheer, 25 El. (29) approached the problem of assigning equiv- alent weights to swimming rats on the basis of specific gravity. If the specific gravity of an animal was found to be less than 1.04, brass weights were attached to the thorax with elastic bands to adjust it. The physical capacity of the rats was determined by requiring animals to swim in water 360-38o C. while weights were added in 2 gram increments every three minutes. Failure to reach the surface during a ten second period constituted the termination criterion. In support of this procedure the following statement was made: "It would perhaps be possible to devise a schedule of loads for rats of different sizes, but there is no evident theoretical basis for such a schedule." (6) ll Kimeldorf and Jones (13)(19) attempted to standardize their swimming procedure to the extent that when rats, with ten gram weights clipped to chest fur, were unable to rise above a line eighteen inches below the surface of the water, the test was terminated. In their work with rats, Miller and Darrow (22) attached five grams to the base of the animals' tails regardless of size (250- 425 grams). Harris and Ingle (10) used a similar system except that the weight used was either ten or twenty grams, depending upon the -segment of the experiment involved. As a technique for testing swimming endurance Montoye g£_al. (23) attached six percent of each rat's body weight about the chest by means of an elastic harness. Under these conditions no correlation was found between body weight and swim time (r = 0.15). Several studies in which swimming was used as a training medium for rats reported no added weight at all. (8)(9)(11)(22)(26) In his conclusions regarding the use of swimming as an experi- mental technique with small mammals, Wilbur (36) suggests that animals be swum.singly rather than in groups in order to obtain pre- cise results. Variability 2f swim times. Eng Meng Tan, Hanson and Richter (33) showed that temperature of the water bath exerts a great effect on swim times of rats. In water at or near body temperature, rats swam 350 times longer than in water 630 F., and 130 times longer than in water at 1050 F. At 980 F. one rat swam for 72 hours while 50 hours was average for this temperature. It was reported the bath was twelve inches deep and the container 8-3/4 inches in diameter. In contrast to the previous study, Hearn and Wainio (ll) 12 reported that two hours was considered maximum swimming time for their rats (Wistar, 250 grams average weight). Temperature of the bath was 32° c. In their experiments, Kimeldorf and Jones (19) used a 24 gallon cylindrical tank filled to a constant level with water at a temperature of 15 to 210 C. They reported that after two weeks of conditioning "most animals" could swim for fifteen to thirty minutes with a 10 gram weight attached, before becoming exhausted. Great variability in swim time was reported by Miller and Darrow (22) in swimming rats (250—425 grams) with five grams attached to the base of the tail. At 370 C. and with water 22 inches deep, "normal" rats ranged in time from exhaustion in ten minutes up to 110 minutes with no apparent fatigue. They concluded that "It seems justified to assume that sixty minutes of continuous swimming indicates a satisfactory performance." Harris and Engle (25) reported that rats, if not weighted, were capable of swimming for several hours before exhaustion under their conditions (i.e., 30 degrees Centigrade). Training and body weight. That training reduced the weight of animals involved is almost always noted. (9)(23)(34) Gollnick and Hearn (8) observed that with rats which swam for thirty minutes daily for 35 consecutive days the exercised animals gained 38.7 percent less weight than the controls. It should be noted that in this case the animals were pair-fed with the exercised rat being the limiting animal. Kimeldorf and Baum (18) collected similar data in that animals which swam 50 to 30 minutes per day (with 10 grams attached) for six 13 weeks, lost weight steadily with respect to the controls. Weight dif- ferences at 3, 9, 18 and 30 days were 6 grams, 7 grams, 27 grams and 39 grams respectively. Training and resistance Eg_disease. Exercise and its effect upon the organism's ability to resist disease has undergone consider- able investigation. In an early study Merrill and Howe (20) concluded that training greatly increased the resistance of the albino rat to infection with pseudomonas aeruginosa. It was found, however, that fatigue favored susceptibility to the infection and that fatigue after infection was more disastrous than fatigue before. Zimkin (37) reviewed the literature and reported that results of a series of studies in which training was seen to have a positive effect upon resistance to various unfavorable factors. He concluded that training could lead to a non-specific increase of physiological resistance to many factors including infections and other illnesses, provided the duration of the training period was sufficient and the intensity of the exercise not excessive. He further observed that this increased resistance persisted for a limited time after training ended. Respiratory disease in_£hg_g§£, The problem of reapiratory diseases in rat and mouse colonies has been reviewed by Joshi, Blackwood and Dale. (15) WideSpread chronic reapiratory diseases generally grouped under the term "chronic murine pneumonia", include enzootic pneumonia, endemic pneumonia, infectious catarrh, broncho- pneumonia, lung abcesses, enzootic bronchiectasis and rodent bronchiectasis. It was reported that incidence in rats obtained from established colonies reaches 75 percent in some cases. The importance 14 of endemic pneumonia and infectious catarrh was stressed, not because they are the only respiratory diseases affecting rats, but because of their wide distribution. Despite this wide distribution, however, Innes 25.31. (14) have pointed out that it is rare for an author to admit the presence of disease in his experimental animals. Part of this failure may be due to the wide experience in pathology of laboratory animals neces- sary for the detection of such disease. Innes g£_§l, (l4) quoted Nelson's studies as indicating that chronic murine pneumonia involves two independent diseases, namely, infectious catarrh with occasional middle and inner ear infection, and the condition referred to as chronic bronchiectasis. Innes and Nelson agreed that most diseased rats exhibit few, if any, clinical signs of respiratory disease although in advanced stages rough hair coat and loss of weight occurs, followed by labored and audible breathing. As quoted by Josi g£_§l,,(15) Nelson stated that the animal finally loses weight rapidly and finally dies about the tenth to twelfth month following infection. Nelson (24) reported that the involvement of the middle ear, resulting in labyrinthitis and twisting, is fairly common in rats. Innes and co-workers, (14) in discussing the etiology of the disease indicated that the pathogenic organisms commonly isolated have been streptobacillus moniliformis, pleurOpneumonia-like organisms and brucella bronchiseptica. They pointed out, however, that there are no published data to prove successful experimental reproduction of the chronic pneumonia using pure cultures of any identifiable microbe. They further state that "murine penumonia is another example of 15 disease in which the 'cause' is not a single etiologic entity." (14) Nelson (15) reported that nursing rats acquire the virus of enzootic bronchiectasis from their mothers, whereas infectious catarrh is usually transmitted by direct contact in both rats and mice. Attempts to produce rats free of the respiratory diseases were reported by Innes §£_al. (14) Their study showed that a disease free rat colony can be established without Special environmental pre- cautions, and that it "costs no more in terms of time, labor, food, and money than any other colony". CHAPTER III EXPERIMENTAL METHOD Material and equipment. The experimental animals used in this study were male albino rats reported to be Specific Pathogen Free (free of salmonella, bartonella, and endemic pneumonia) and obtained at the age of eight weeks, from Carworth Farms, New City, New York. Of the original number delivered (125) complete data was collected on a total of 88 animals. Two rats died of causes unrelated to the ex— periment while 27 animals drowned sometime during the training period. Eight other rats were utilized in various pilot studies relating directly to the experimental procedures and were thus deemed unqualified for inclusion in the experiment prOper. All animals were fed a pathogen-free baked diet produced by Dietrich and Gambrill eSpecially for laboratory rats and mice. This diet was prepared in the form of 1/2 inch X 1/2 inch X 1/8 inch biscuits which were placed on the floor of the cages. Care was taken to keep an adequate supply of feed and water available to the animals at all times. No effort was made to measure feed intake. All animals were housed in individual "self-cleaning” cages of quarter inch wire mesh which were suSpended over litter pans con- taining a sterilized animal bedding. (DeOdor Grade Animal Bedding, Paxton Processing Co., Paxton, Illinois). These cages, measuring 10 X 8 X L were arranged in wheeled racks so that five horizontal rows of six cages each were available on each side of the rack making a 16 17 total of 60 individual cages per rack. All cages were thoroughly cleaned and soaked in quanternary ammonium compound before the experiment began. A specially constructed swimming tank was utilized for the training and testing of animals. The tank, measuring 8 feet long by 3 feet wide and 30 inches deep, was divided into twenty-four cubicles (each 1 foot square by 30 inches deep) by means of 1/8 inch thick transparent plastic dividers. This arrangement made it possible for each animal to swim without interference from others both during training and testing, thus providing greater control of the intensity of the exercise involved. Water temperature for all swimming was maintained between 35-37 degrees centigrade. Each day the tank was thoroughly rinsed after use and the entire tank was sterilized with quanternary ammonium compound at irregular intervals. Upon completion of each swim, the rats were removed from the water and placed in rectangular cages of 4 by 4 hardware cloth, measuring 10 by 4 by 4 inches, which were located approximately 24 inches below a bank of four 250 watt infra red heat lamps. The rats were allowed to remain in these cages for up to thirty minutes or until found to be sufficiently dry to be replaced in their permanent cages. Only on rare occasions were any animals toweled off before being returned to the cage rack. The temperature of the room was maintained at 80 degrees Fahrenheit. Upon one occasion, at the conclusion of the experiment, the temperature exceeded this level for a period of about two days. This occurred during the month of May when the outside temperature exceeded ninety degrees. The temperature in the animal room at this time rose to about 83 degrees Fahrenheit. 18 With the lack of air conditioning no control of humidity was possible and no record of humidity was maintained. Special care was taken to handle all rats daily, regardless of whether or not they were -schedu1ed to swim. All rats, without exception became extremely docile and easy to handle with no apparent differences in temperament or ex- citability being noted. All animals were weighed twice weekly at approximately the same time each Wednesday and Saturday. Continuous plots of average weight fluctuations for the control group and the experimental group were maintained. (See appendix E). Design g£_the experiment. The original design: As mentioned in Chapter I, this study was initiated when the unexpected performance of the rats involved forced the abandonment of the original experiment. The original study called for all animals to be eXposed briefly to the swimming method employed before control animals were selected. It was hOped that some of the learning effect involved in swimming might be eliminated by this procedure. Following this, the control animals were to be selected randomly and the remaining animals trained until they could swim for thirty minutes with four percent of their body weight attached to the base of the tail. Once this objective had been attained, the rats were to have been divided into nine groups (in addition to the control group) each of which was to swim under different conditions of frequency and duration. At the conclusion of the experiment it was hoped that it would be possible to determine (within certain limits) the minimum amount of training which would serve to maintain the level of endurance to 19 which the rats had been originally trained. As previously described, the initial all-out swim test revealed, that instead of deve10ping greater endurance, the trained animals apparently regressed in swimming ability to the extent that the non- trained control group swam, on the average, longer than did the ex- perimentals. Since it was then impossible to study maintenance of the endurance factor (such a factor having failed to deve10p) it was decided to determine, if possible, the reasons for the failure of these rats to respond as expected, and to investigate the relative effectiveness of various systems of training in improving the swimming ability of the experimental animals. Following is a description of the complete experiment as it was actually conducted. One hundred and twenty Specific Pathogen Free male albino rats (Carworth CFE) were obtained at the age of eight weeks (maturity). After two days of acclimation to the laboratory, (during which time they were handled twice daily) all rats were placed in the exercise tank and allowed to swim for five minutes. On the fourth day they swam for ten minutes, once in the morning and again in the afternoon. Two a day sessions continued for eight days with the animals being required to swim for various lengths of time as weights were attached. (See Table 1). On the second day of training approximately two percent of each animal's body weight was attached to the base of the tail by means of adhesive tape. Small fishing sinkers of various sizes strung on a bit of wire served to provide the necessary burden. On the third day of training two percent of body weight was carried for fifteen minutes and on the fourth day the percentage was increased 20 PRE-EXPERIMENTAL TRAINING SCHE Y"? $94.23 TABLE I DECEMBER 26 TO MARCH 18 Swim Time % Swim Time I Date (Minutes) Carried Date (Minutes) Carried A.M. P.M. A.M. P.M. Dec. 26 5 0 Feb. 24 3O 0 Dec. 27 10 2 Feb. 25 Dec. 28 15 15 2 Feb. 26 30 1 Dec. 29 20 20 4 Feb. 27 Dec. 30 20 20 4 Feb. 28 30 2 Dec. 31 O 0 March 1 Jan. 1 3O 4 Marsh 2 20 2 5 Jan. 2 10 10 4.5 March 3 20 2.5 Jan. 3 15 15 4.5 March 4 Jan. 4 20 20 3-4 March 5 30 2.5 Jan. 5 20 2.5-4 March 6 Jan. 6 20 2.5-4 March 7 30 2.0 Jan. 7 25 2-4 March 8 Jan. 8 30 2.5-5 March 9 30 Variable Jan. 9 10 10 2.5-5 March 10 Jan. 10 15 2.5-5 March 11 30 Variable Jan. 11 20 2.5-5 March 12 Jan. 12 20 2.5-5 March 13 30 2 Jan. 13 3O 2.5-5 March 14 30 2 Jan. 14 3O 2.5-5 March 15 Jan. 15 30 2.5-5 March 16 Jan. 16 3O 2.5-5 March 17 Jan. 17 30 2.0-5 March 18 All Out Swim Jan 18 to Feb. 23 Rest March 22 - June 3 See Table II 21 to four percent to be carried for twenty minutes. This procedure of gradually increasing the percentage carried, as well as increasing the swimming time (up to 30 minutes), was followed for the entire period of time preceeding the initial all-out swim test. Table I summarizes the training schedule for this part of the experiment. Following the fourth day, twenty rats were selected, by means of a random numbers table, to serve as a control group. These animals did not swim again except at the all-out swim tests. Interruption of the study: During the third week of the con- ditioning period, abrupt weight losses were noted in several of the animals, accompanied by bloody serrous nasal discharge and snuffling, of moderate to severe nature. Upon consultation with Dr. Robert Schirmer of the School of Veterinary Medicine of Michigan State Univer- sity, (See report in Appendix A), it was confirmed that most of the animals were afflicted with upper respiratory congestion, and although gross pulmonary congestion was not noted, subsequent necrOpsy reports on two animals indicated a viral or influenza-type bronchOpneumonia in lungs of both. (See Appendix B). Cultures of nasal secretions re- vealed two different micrococci which were sensitive on the plate-test only to the nitrofurans. Although the causative factor was not deter- mined, nitrofurantain (Dantafur, Eaton Labs) was administered in the drinking water to the extent that each rat should have received 4 to 6 mg. daily. In keeping with the prescribed treatment, the training of the animals was interrupted until the symptoms appeared to have been alleviated and the weight loss restored. This resulted in a lay—off from January 17 to February 24; a total of five and one-half weeks. The 22 nitrofurantain treatment was continued, however, until March 27. I By February 20, all rats were adjudged by Dr. Schirmer to be recovered and training was resumed with animals swimming every other day only. On the 24th all animals swam for thirty minutes with no weight attached. On the 26th the animals were swum with approximately one percent of body weight attached for thirty minutes. This percent- ‘age was increased to two percent on the 28th of February and to 2.5 percent on March 2. On March 2, however, swimming time was reduced to twenty minutes. Following this, the swimming time was held constant at thirty minutes and weights adjusted individually for each animal to permit him to successfully complete the thirty minute swim if pos— sible. In any case in which a rat was unable to continue for thirty minutes the weight was removed and the rat returned to the tank to complete the swimming time unencumbered. During this phase of the study it was observed that swimming ability was generally poor. In several cases it was not only impos- sible to increase the weight carried, but it was necessary to decrease it. Because of the large number of animals which were unable to swim for thirty minutes with at least three percent of their body weight attached, it was necessary to reduce the required percentage for the all-out swim to two percent. The results obtained under these conditions showed such great variance between animals, and indicated that the swimming ability of the experimental animals was so vastly inferior to that of the control animals, as was eXplained above, that the original design had to be abandoned. The results of the initial test can be summarized as follows: Ten of the twenty control animals swam for at least seven hours and 23 appeared to be capable of swimming indefinitely when they were removed from the water. Only three of this group were unable to swim for as long as thirty minutes. Of the eighty nine eXperimental animals, ten were removed from the water after seven hours while still able to swim easily and eighteen more were removed after four hours under the same conditions. Fifty-six of the experimentals failed to swim for even thirty minutes. The median swim times for this truncated distribution were 240 minutes for the control group and 19 minutes for the rats which had been trained. Appendix G contains the swim times for all animals. Alteration of the design: The decision to attempt to deter- mine the reasons underlying the failure of the experimental animals to respond to the training as expected was made almost immediately. When clinical observations and hematocrit tests showed no apparent differences between groups it was decided to divide the experimental animals into groups undergoing training of various intensities in order to determine whether the intensity of exercise was a causative factor in the poor performance of these animals. A secondary purpose of this design was to observe whether swimming at various intervals for short periods with a heavy overload, or with less of an overload for periods of greater duration would be more effective in improving the swimming ability of the animals. Table II shows the basic design. The length of the experi- mental period was to be ten weeks. Three all-out swimming ability tests were scheduled; one at the beginning of the experimental period, the second at the midpoint, and the third at the end of the tenth week. 24 TABLE II THE BASIC DESIGN OF THE EXPERIMENT _ Y ‘— - ‘7. S w i m I n t e n s i t y Swim Frequency Moderate Overload Extreme Overload 30 Minutes * All-out ** Daily Group I A Group I B Every 2 days Group II A Group II B Every 5 days Group III A Group III B Every 10 days Group IV A Group IV B Test periods Group V only Control Group * Animals in this column are required to swim for 30 minutes with the heaviest possible weights attached. Weights adjusted daily. ** Animals in this column are required to swim for as long as possible with approximately 5% of body weight attached. In any case, weight must be sufficient to in- sure animal's inability to swim for more than five minutes. Three days after the initial all-out swim, a second all-out swim test (to be referred to throughout as Test No. l) was performed. Since there was now no absolute time which it was desired for the animals to swim, the primary consideration was to attach an overload great enough to prevent extremely long swim times but which, on the other hand, would allow all animals to swim for a measurable period. After limited experimentation with four randomly chosen rats, it was decided that each animal should carry 3-1/2 percent of his body weight for all three all-out swim tests. At this time all animals were given identification markings corresponding to the group to which they had been assigned. Five colors of indelible ink were used for this purpose, and a code was 25 developed which necessitated the placing of only two marks on each rat's tail. These markings had the advantage of being easily and quickly applied, with no discomfort to the animals, and could also be easily read when the rats were being handled. Data for all—out swimming were collected to the nearest five seconds and in all cases termination of swimming time was dependent upon the subjective judgment of the experimentor. In general, an animal was considered to have reached the all-out point when it was obvious that he could no longer regain the surface, and was judged to be in imminent danger of drowning. During the initial swim test it was observed that those rats which were able to continue swimming for at least thirty minutes had, by this time, trapped so many small bubbles in their fur that their bouyancy was greatly increased. In some cases animals which found it necessary to struggle vigorously to remain at the surface early in the test were, after two or three hours, able to float motionless with the nose protruding from the water. Because of the fact that the intensity of the effort was found to decrease as the duration increased, it was decided that in sub- sequent tests all animals reaching sixty minutes should have the fur- trapped bubbles removed by gentle underwater massage. If they con- tinued to swim, the bubbles were to be removed again at precise fifteen minute intervals thereafter. This truncation of the distribution of swim times was considered justified because of the fact that with the alteration in bouyancy the factor being measured also changed. The choice of time for massage of the animals was arbitrary, based partially upon practical consider- ations involved in swimming a large number of rats. 26 If, for any reason, data were not complete for any given animal, that animal was excluded from consideration in the calculations made for this study. After Test No. 1, all rats were trained according to the sched- ule in Table II. Group V served as a sort of second control group, differing only in the respect that all animals in this group had undergone the entire period of preliminary training, whereas the control group had not. At the midpoint of the experimental period (five weeks) the all-out Test No. 2 was conducted. Following this, training was continued for another five weeks before the final all-out test was administered. Before each test, training of all animals was suspended for two days during which time the rats were permitted to rest. Two days after Test No. 3 half of the animals were sacrificed for post-mortem examination. 0n the third day following Test No. 3 the remaining animals were also sacrificed. Because of the importance of the information being sought in these necropsies, Dr. William Field, National Institutes of Health Research Fellow, School of Veterinary Medicine, Michigan State Univer- 'sity, and Mr. Paul Yevich, Histopathologist from the U.S. Department of Health Occupational Rehabilitation and Training Facility, Cincinnati, Ohio, were retained to perform the necessary Operations. Gross examina- tion was performed immediately upon sacrifice while tissues were taken for routine bacteriological analysis and histOpathological study from every fifth animal as well as from all rats exhibiting gross abnor- malities. Included in the routine gross examination was careful observa- tion of the following: 27 Skin and hair Lymph nodes Stomach Small intestine, cecum, colon Spleen; weight of spleen Liver; weight of liver Adrenals; weight of adrenals Kidneys; weight of kidneys Heart; weight of heart 10. Lungs and pleura; weight of lungs ll. Mesentery 12. Uro-genital systems 13. Nasal passages 14. Internal and external musculature 15. Bones and joints l6. Tongue, mouth, eSOphagus 17. Primary arteries and veins \DmNO‘U'I-l-‘UJNH Gross findings, histopathological and bacteriological results were reviewed independently (from coded data) by both Dr. Field and Mr. Yevich and a final joint report submitted. (Appendix C). CHAPTER IV RESULTS AND DISCUSSION Results Swimming times. In each all-out swim test there were several animals which were able to swim for long periods of time without difficulty. As the initial (pre-experimental) swim showed, these rats found the task easier and easier as time elapsed because of the accumu- lation of air bubbles in the fur. In order to reduce the distortion of the data resulting from this phenomenon, the air bubbles were re- moved by gentle underwater massage at precisely sixty minutes. In most cases the animal involved lost all ability to continue almost immediately, but in those few instances in which they continued to swim, massage was again applied at fifteen minute intervals. Because of the two foregoing factors (i.e., swim times of extraordinary duration and termination of swim), the distribution of swim times was observed to lack normality. (See appendix G). For this reason, non-parametric statistical methods were employed in analysis of the data. The results of the three all—out swim tests are shown in Figures I and II. Both mean and median for each group are graphed in order to indicate the size of differences between these two measures. In order to determine whether differences in swim time between 28 65 55 45' 35 29 FIGURBI mmmmsnpourswmrmsmmms RR Tess Is. 1 Test Is. I fidisns nuns Q I O C] \U IB rm No. 3 30 FIGURE 2 MEANS AND RANGES OF ALL-OUT SWIM TIMES IN MINUTES 1.0 - 100 - ‘ 1‘0 _ . 120 - ‘ Mbsns Hill! 100 - Runs- [:1 90 - Test No. 1 Test No. 2 Test No. 3 I] Illf/[l‘ u- I o .— ‘ I - . .:E;:‘ nnnnnnhMI r7 /’3- 31 groups were significant a Kruskal-Wallis rank sum test was employed. The statistic H is distributed approximately as Chi Square with K-l degrees of freedom, and the hypothesis that all values come from identical pOpulations is rejected for large values of H. As shown in Table III, there was significant difference be- tween grOUps demonstrated for Test 1 only. The hypothesis could not be rejected for either Test 2 or Test 3. TABLE III H VALUES FOR RANK SUM TEST OF HOMDGENEITY OF SWIM TIMES N Test No. 1 Test No. 2 Test No. 3 88 66.3* 9.9 4.5 74 9.0 - - * Significant with probability of .99 (X‘s 20.09) Control Rats removed Since the data suggested that the significant difference ob- served in Test 1 was probably due to the fact that swim times for the controls were so much greater than any other group, this possibility was tested. A second rank swim test was performed with all control values eliminated from consideration. Thus, if the controls were entirely responsible for the rejection of the hypothesis of equality, exclusion of these values from the test would be eXpected to result in an H statistic smaller than 20.1. As shown in Table III the re- ‘sultant value was found to be 9.0, thus confirming the assumption. In analyzing the swimming performance of rats from one all- out test to the others two types of computation were made. First a 32 Mann-Whitney test was performed to determine whether the swim times for each group were significantly different from one test to another. The performance of all experimental animals taken together was also examined in this manner. The second test utilized was that of rank correlation, which was used to ascertain the consistency of swimming performance of the rats from test to test. In Table IV are seen the rank sums T'. The boundaries of the critical region for each comparison are listed in Appendix I. In order to reject the hypothesis of random samples down from identically distributed papulations, T' may not fall between these two numbers. For a sample size greater than ten, the chance that the statistic T' will exceed a given value can be calculated on the basis of the normal curve. Thus, for the control group and the combined experimental groups significance was judged on the basis of the Z statistic. The values in Table IV indicate that in the comparison of Test 1 with Test 2, only the control values changes significantly. A comparison of Test 2 and Test 3 revealed that significant differences occurred for groups III A, III B, IV B and the controls. When all eXperimental animals were lumped together there appeared to be a significant change. An examination of the results of Test 1 and Test 3 showed that there was a significant difference in performance at these two periods for groups II B and IV B only, although once again the ex- perimentals taken as a single group, showed a significant change. A study of the consistency of performance of individual animals, both with respect to others within their own groups and to 33 others regardless of group, provided the results shown in Table V. Appendix J lists the critical values for r in this study. TABLE IV RANK SUM.TEST OF DIFFERENCES BETWEEN ALL-OUT SWIM TESTS Test Comparison Group N 1 versus 2 1 versus 3 2 versus 3 Control 14 Z = 2.33 Z = .87 Z = 2.60** I A 7 425-57.5 47-58 50.5-54.5 I B 9 76-95 69.5-101.5 80.5-90.5 II A 7 51.5-53.5 47-58 51.0-54 II B 8 51-85 48-88* 61-75 III A 10 109.5-99.5 79-131 75.5-134.5* III B 9 77-94 65-106 60-99* IV A 7 57.5-47.5 43-62 39-66 IV B 9 73.5-97.5 61-110* 56.5-114.5** V 8 65.5-70.5 60-76 58-78 Overall 74 Z = 1.24 Z = -4.44** Z = -4.08** (minus controls) * Significant with a probability of .95 ** Significant with a probability of .99 34 TABLE V RANK CORRELATION OF SWIM TIMES (RELIABILITY COEFFICIENTS) — i_. Test Comparison Group N 1 versus 2 1 versus 3 2 versus 3 Control 14 .5539 .5154 .5877 I A 7 .5358 -.0714 .5715 I B 9 -.1333 .3917 .7917** II A 7 .2858 .0358 .8572* II B 8 -.4642 .0239 .5000 III A 10 .2576 .3455 .0334 III B 9 .5292 .6959* .3084 IV A 7 .7500* .5715 .2500 IV B 9 .2917 -.0708 -.0250 V 8 .4762 .6905* .7381* Overall .2686* .2955* .3947* * Significant with probability of .95 ** Significant with probability of .99 It is readily seen that significant correlations for performance of animals in the group taken as a whole were obtained for each of the three comparisons (i.e., Test 1 vs Test 2, Test 2 vs Test 3 and Test 1 vs Test 3). When the coefficients for each of the groups in the com- parison of Test 1 with Test 2 are examined, however, it is seen that only group IV A showed consistency of performance significantly greater than could be accounted for by chance. The results of Test 2 vs Test 3 indicate that only the animals of groups I B, II A and V 35 exhibited any significant consistency in terms of their swim time rank. A study of the swim time data suggested that the relationship between animals size and duration of swim might be of interest. Since all animals carried a standard 3-1/2 percent of their body weight during each all-out test, the correlation between body weight and swim time.is identical to the correlation between weight carried and swim time. As shown in Table VI, the coefficients of correlation for the three all-out tests were found to be 0.008, -0.21 and -O.l4 respec- tively, none of which indicates the existence of mutual relationship. TABLE VI CORRELATION OF BODY WEIGHT AND ALL-OUT SWIM TIME 1 I N Test No. 1 Test No. 2 Test No. 3 88 r = 0.008 r = 0.21 r = -.14 Pathological findingg. .As explained in Chapter III all animals in the study were subjected to post-mortem examination in an attempt to determine the cause of their poor performance. A random sample of 16 rats was drawn for the purpose of histOpathological examination of sections from the lungs, spleen, kidney, liver and gut. Tissues were taken from these same organs as well as from those of five additional animals for routine bacteriological cultures. Two professional pathologists performed the autopsies and made the subsequent histo- pathological study. Bacteriological cultures were prepared and re- ported on by the laboratories of the Michigan State University School of Veterinary Medicine. 36 The report of findings as submitted by Dr..Field after con- sultation with Mr. Yevich, is included in Appendix C. Their findings indicated that only the respiratory system was found to be abnormal in any way, but that the lesions noted there were frequently of a severe nature. While gross examination revealed lung involvement in all but 31 of the rats, the microscOpic study revealed that even these animals suffered at least mild lymphoid collaring of the bronchioles. Most of the rats examined microsc0pically suffered more severe lesions, with presence of atelectasis, bronchiectasis and hyperplasia of the bronchiolar epitehlium, with various gradations of interstitial pneumonitis. Mucuous and neutrOphils filled many of the dilated bronchioles and the alveoli. The changes seen in the rats of this study were consistent with those commonly reported for chronic murine pneumonia except for the abcesses which were frequently noted. This was considered to be an acute suppurative process superimposed on the chronic condition. Table VII contains a chart-summary of the pathological findings in this eXperiment. It should be noted that there is no apparent consistency of relationship between the organisms noted and the type or severity of lesions reported. All of the organisms re- ported in the intestinal cultures are considered normal, except per- haps the diptheroid, and the effect of their presence in the rats was not known. In animals such as IV B2, III A8, I A2, C 10, and IV A5 where lesions of moderate to severe nature were apparent and no growth was reported in the bacteriological statement, it was believed that some organisms must have been present but the laboratory procedures utilized failed to disclose them. 37 TABLE VII CHART-SUMMARY OF PATHOLOGICAL FINDINGS* Body Swim Lobes Weight Times Involved Bronchi- Atelec- Hyper- Suppura- # (gms.) (min.) Grossly ectasis tasis plasia tion Mucin IV BZ 488.0 23.9 Azygos Severe 4+ Severe 4+ 3+ 3-4+ 2+ 3.5 Cardiac 8.5 IV B7 524 0 2.25 Azygos 4+ 3-4+ 2+ 3+ 2+ 4.00 4.50 II A4 491 O 4.33 No visible 1+ 1+ 2+ 2+ 1+ 8.75 Lesions 7.17 III A2 478.0 7.60 Apical 2-3+ 2+ 2+ Not seen 2+ 4.00 Azygos 6.08 III A8 461.0 2.83 Apical Not seen 2+ 2+ Not seen Not seen 6.00 Azygos 5.00 II 32 483.0 13.42 Apical 4+ 4+ 3+ 4+ 3+ 3.25 Rt. Diaph. 62.75 II B9 475.5 2.60 Azygos 1+ 1+ 2+ l+ 2+ 7.75 Apical 7.00 Rt.Diaph. I A2 481.0 8.00 Apical 2-3+ 2+ 2+ 2-3+ 2+ 4.50 Rt.Cardiac 3.50 Lt.Diaph. IA7 473.0 4.12 Rt.Apica1 -- -- -- -- -- 5.50 5.50 C-1 620.0 -- No visible 4.50 Lesions -- -- -- -- -- * Severity of pathology is coded l to 4 with 1 being mild and 4 being severe. TABLE VII--Continued Bacteriological Findings Spleen Kidney Remarks Organs Neg. Intestine: Coli- form & proteus Hemisiderosis 2+ White pulp ex- haustion 2+ Organs- Veridans Strep. Bacillus Species Gut- Pseudomonas Hemisiderosis 1+ 1+ Organs: micro- coccus Sp. & Coliform Gut- Negative Hemisid. 1+ 1+ Casts Emphysema & Edema with peribronchiolar hyalinization Organs: Neg. Gut: Pseudomonas & proteus Hemisid. 1+ 1+ Casts No growth Hemisid. 2+ 1+ Fibrosis 2+ Organs: Coliform enterococcus Gut: Pseudomonas Hemisid. 1+ 1+ Casts Emphysema Fibrosis 1+ Organs: Micro. Sp. coliform PSEudomonas Gut: Proteus Hemisid. 1+ 1+ Emphysema Organs: No growth Gut: Pseudomonas Hemisid. 1+ Organs: No growth Gut: Pseudomonas Only mild lymphoid collaring seen Organs Pseudomonas Gut Coliform Hemisid. 2+ 2+ 39 TABLE VII-—Continued Body Swim Lobes Weight Times Involved Bronchi- Atelec- Hyper- Suppura- # (gms.) (min.) Grossly ectasis tasis plasia tion Mucin C-6 519.0 3.75 Cardiac 1+ 2+ 1+ 2+ -- 11.00 63.67 C-lO 455.5 114.73 Azygos 2-3+ 1+ 4+ 4+ 2-3+ 63.00 Apical 41.00 Cardiac Left Lobe D-18 589.5 3.10 Rt.Diaph. 2+ 4+ l-2+ 3+ 1-2+ 2.50 tip 4.25 III B3 503.5 5.50 -- 1+ 1+ l-2+ -- —- 29.00 8.33 III B8 582.0 2.58 Cardiac 1+ 3+ 3+ 1+ 1+ 3.75 2.50 IV A5 540.0 3.97 -- 1+ 2+ 1+ -- -- 4.25 4.08 C-5 500.0 Bacteriological Only C-7 507.0 Bacteriological Only C-l7 558.5 Bacteriological Only C-8 560.0 Bacteriological Only C-ll 587.0 40 TABLE VII--Continued Bacteriological Findings Spleen Kidney Remarks Organs: pseudomonas Hemisid. 1+ 2+ Edema 2+ enterococcus & coliform Gut: pseudomonas No Culture -- Hemorrhage 1+ Fibrosis 1+ Hyalinization 1+ Emphysema 2+ Organs: micro. Sp. diptherold Gut: Pseudomonas Hemisid.'1-2+ 1-2+ Edema 2+ Organs: Veridans Strep. Pseudomonas Gut: Pseudomonas Proteus, Coliform Hemisid. 1+ Organs: Veridans Strep., diptheroid Gut: Neg. Organs: No Growth Gut: Proteus 1+ Casts & -- Calcification Pseudomonas Bacillus Species Negative Negative Negative Gut: Proteus 41 A complete lack of positive relationship between severity of lesions and swimming ability was also noted. Some of the best swimmers (C 10, II 82) were observed to be most severely afflicted. On the other hand, animals such as I A7, C l and III B3 which appeared to be relatively free of lesions were found to have swim times ranging from only poor to fair. As a result of this apparent lack of relationship between swim time and severity of pathology, a test was performed to determine whether statistical procedures would verify this situation. As a general indicator of the degree of lung involvement, the gross report of the number of lung lobes showing pathological changes was used. A Kruskal-Wallis rank sum test was applied to four groups set up on the basis of (1) no lobes involved, (2) one lobe, (3) two lobes, and (4) three or more lobes involved. As Table VIII shows, there was no statistical support for rejection of the hypothesis that the swim times of individual animals in all four groups were randomly drawn from identical distributions, insofar as all-out swim Test 1 and Test 2 were concerned. For the data of all-out Test 3, however, a highly significant difference was obtained (p = .99). The direction of this difference, as indicated by examination of the data, was, however, in direct constrast to the expected result. That is, the mean (or median) swim times of the animals with two, or three or more lobes involved was significantly greater than that of the rats which showed no gross lesions or those with involvement reported in only one lobe. 42 TABLE VIII RANK SUM TEST COMPARISON OF GROSS LUNG INVOLVEMENT AND ALL-OUT SWIM.TIMES W S w i m T i m e s Number of Lobes Showing Test 1 Test 2 Test 3 Gross Lesions Mean Median Mean Median Mean Median None 17.6 4.3 5.6 4.5 11.7 7.2 One 12.47 4.0 5.4 4.0 12.4 5.2 Two 37.2 7.6 12.1 4.0 24.7 14.5 Three or More 8.8 4.2 9.0 5.4 27.8 16.75 Statistic Test 1 Test 2 Test 3 H ' 6.16 1.84 28.05* fi n *— H _ *Significant with P - .99 “£3.01“ --. 8 11034) Discussion Discussion.2£Presults. Swim times: While some statistical significance has been demonstrated with regard to differences in swimming abilities of rats in various comparisons, the practical significance of such results is questionable. At least the reasons for the differences which have been shown are not at all clear. There seems to be no clear pattern of performance on which to base any assumptions. The underlying cause of the difference between the control group and the experimental animals on the first test can only be guessed at. That the bouyancy of the animals was a factor there is no doubt. It might be conjectured that the control animals, having 43 not been exposed to the water, possessed more oil in their fur which accounted for the formation of more air bubbles than in the experi— mental groups. If this was the predominate factor, however, it would seem that their performance should not have been so drastically changed in Tests 2 and 3, since they were not exposed to the water for five weeks between each of these tests. A.second possibility exists in the realm of stress physiology. As has been previously noted, the entire colony was afflicted with an acute respiratory ailment which necessitated interruption of the study for some five weeks. It seems, entirely possible that the exercise.to which the experimental animals were subjected, following so closely after the illness, caused a general depression of the physical efficiency of the trained animals. If this hypothesis is accepted, however, it must be conceded that one all-out swimming test, lasting perhaps only four minutes, could cause the same reaction in the control animals. It seems doubtful, however, that such a depres- sion of ability, if it occurred at all, would not be overcome by five weeks of rest. A third factor which should be considered is that of the withdrawal of medication (nitrofurantain) from the animals on March 27. .It will be recalled from Chapter III that the nitrofurantain was administered via the drinking water for a short time after the animals had apparently fully recovered. It is conceivable that this medication was sufficient to maintain a prOphylactic effect in the non-exercising controls but was not able to do so in the experimental animals. Thus, in all-out swim Test 1 the control animals might have been healthier than their eXperimental counterparts. Following the 44 withdrawal of the medication, however, the acute respiratory condition might have become sufficiently debilitating to result in the swim times obtained for the second and third tests. In attempting to interpret the differences in swim times from Test 2 to Test 3 it is necessary to examine both the mean and median swim times. (Figure I). Groups III A, III B, IV B and the controls were the only groups exhibiting significant changes. That the changes resulted from a specific type of training is doubtful. There may be some indication, however, that swimming every five days or even less frequently may have contributed to better swim times than swimming more often. It is possible that performance was better because swimming on the less frequent schedule was less harmful rather than more beneficial. If this logic is extended, however, group IV A and especially group V would also be expected to perform better. Since this was not the case, it is almost impossible to determine the true facts of the matter. The practical significance of the statistically significant correlations shown in Table V is questionable. In any training study one would normally expect to have a high rank correlation within groups. If correlation is absent it would indicate that the swim-time variability overshadowed any effect of the treatment involved. The fact that so few significant within group correlations were obtained indicates that the variability of swim times of individual rats in this study was great. This, of course, merely confirms the intuitive impressions given by casual examination of the data. This problem of the extreme variability in swim times from one test to another was a serious one. In studying the data the 45 possibility of a relationship between body size and/or fatness was repeatedly suggested. The correlations reported in Table VI for swim time and body weight were, however, adequate evidence for dismissal of the body size possibility. It might have been enlightening to have data on the carcass specific gravities of these animals, but such information was not obtained. Pathological findiggg. As previously mentioned, there is no doubt that the animals were suffering from chronic pneumonia upon which was superimposed a secondary disease of an acute nature. The problem of the poor performance of the animals in general would cer- tainly seem to be related to this condition. The difficulty of interpretation lies in the fact that there appeared to be no positive relationship between swimming ability and severity of disease. The only logical-explanation would seem to be that the test criterion (swimming, with 3-1/2% attached) was inadequate as a discriminatory test of cardio-respiratory capacity. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary The purpose of the study was to investigate the effects of exercise of various frequencies and intensities upon the swimming ability and physiological well-being of Specific pathogen free male albino rats. One hundred and twenty mature male albino rats (Carworth Farms CFE), free of specific pathogens, were trained by swimming in individual compartments measuring 12 x 12 x 30 inches. The water temperature was maintained between 35 and 37 degrees centigrade. All animals, except twenty controls, were required to swim daily for gradually increasing periods of time (up to 30 minutes) with a small weight.in the form of fishing sinkers attached to the base of the tail. The size of this weight was gradually increased as the animals were able to manage it. During the third week of the training period an acute respira- tory condition, resulting in weight loss, nasal congestion and bloody nasal discharge, caused the study to be interrupted for approximately five weeks. Although the causative factor of the illness was not determined, 4 to 6 mg. of nitrofurantain (Dantafur, Eaton Labs) was administered daily in the drinking water. This treatment was continued for approximately two weeks after the symptoms disappeared, and then discontinued. 46 47 After the illness, training was resumed with the animals swimming every two days only. Following a training period of four weeks all animals were subjected to an all-out swim test with two percent of body weight attached. The results of this test indicated that the swimming ability of the control animals was so greatly superior to that of the eXperimental animals that it was decided to abandon the original objective of the eXperiment and study instead the reasons underlying the poor performance of the eXperimental animals, and to determine the best training combination for recovery of swimming ability. Following a second all—out swim test with 3-1/2 percent of body weight attached, all animals were assigned to groups so that the mean-swimming times of groups were approximately equal. The experiment was designed so that the rats of Group I A were required to swim daily for thirty minutes with as much weight as they could carry while those group I B, swimming daily, were re- quired to carry a much greater percentage and to swim until they be- came exhausted. The weight was to be great enough to prevent swim times in excess of five minutes. A similar pattern was set up for groups II A and II B except that they swam every other day. Groups III A and III B were made to exercise by swimming under the same set of conditions but only every fifth day, while on every tenth day groups IV A and IV B were forced to swim. Finally group V was allowed to remain sedentary, serving as a second control group differing from the overall controls only in the respect of having undergone the preliminary training with the other experimental animals. After training according to this schedule for five weeks, a 48 second all-out swim test with 3-1/2 percent of body weight attached was administered. Following this was another five weeks of training and then a final all-out swim test. Two days after the final swim all animals were sacrificed and subjected to complete post-mortem examination. Statistical analysis (Kruskal-Wallis rank sum test) showed that the controls were significantly better swimmers than all other animals on Test No. 1 only. There were no differences between the mean swimming abilities of the various groups on either of the other two all-out swim tests. There appeared to be slight, although significant improvement for the animals generally during the last half of the experimental period. Only four of the individual groups, however, (Control, III A, III B, and IV B) exhibited significant changes in this regard. The most striking factor observed was that of the abrupt drop in swim times of the control animals after all-out swim test No. 1, a drop which was regained only in part during the last five weeks of the experiment. The post-mortem examination revealed that virtually all animals were suffering from mild to severe forms of chronic murine pneumonia upon which was superimposed an acute reSpiratory disease. Bacteriological cultures, however, failed to clarify the etiology of either disease. Moreover, there was no apparent or statistically positive relationship between the severity of lung lesions and duration of swim time. There was a statistically significant indication (P = .99) that animals with the most severe lung involvement swam the longest during the final all—out test. 49 Conclusions .Analysis of the results of this study has led to the following conclusions: 1. The trapping of air in the fur of the swimming rats was the dominant factor in the experiment, and was responsible for dis- torting swim times to the extent that other relationships were obscured. 2. The fact that animals, sUpposedly free of the pathogens responsible for chronic murine pneumonia, were found to be afflicted with that disease indicates that such animals must be housed under special conditions if their health is to be maintained. 3. The great lack of consistency of swimming performance indicates that the methods used in this experiment are inadequate as a means of differentiating between various degrees of swimming potential. 4. Despite several notable exceptions, it is concluded that the generally poor swimming performance of the animals involved must be at least partially attributed to the reSpiratory diseases dis- covered in the animals. As to whether the chronic or acute form of the illness was more debilitating no conclusions are drawn. Recommendations Findings related to this study have prompted the following recommendations: 1. A study should be made to determine whether or not it is possible to remove the bouyancy effect of prdgressive air entrapment in the fur of swimming rats. It is suggested that the effects of steps such as clipping the fur, rinsing thoroughly in detergent and 50 utilizing detergent in the swimming tank might be studied. 2. It is recommended that control of temperature and humidity be a prerequisite for any further animal studies. 3. A random sampling of any experimental animals should be subjected to post-mortem examination as soon as they are obtained from the breeders in order to determine their status with regard to respir- atory disease. 4. Some control animals should be maintained completely free from contact with swimming in order to determine whether it is this Specific factor which has contributed to the difficulties encountered. 5. Animal quarters should be kept as clean as possible with every effort made to sterilize cages and other equipment frequently. It is eSpecially important that only those persons who must handle the animals be permitted to enter the animal quarters. 6. A comparative study utilizing SPF rats as well as other strains should be made in order to determine whether there are some animals which are superior for work of this type. 7. A study should be made of the effects of swimming as opposed to heavy exercise of another type in order to determine whether the respiratory difficulties encountered in this study were specifically linked to swimming. 10. ll. 12. BIBLIOGRAPHY Astrand, Irma, P. O. Astrand, E. H. Christensen and Rune Hedman, "Intermittent Muscular Work," Acta Physiologica Scandinavica, 48:448—53, April, 1960. Astrand, P. 0., "Human Physical Fitness--With Special Reference to Age and Sex," Physiological Reviews, 36:307-335, 1956. Bannister, R. G., D. J. C. Cunningham, and C. J. Douglas, "The Carbon Dioxide Stimulus to Breathing in Severe Exercise," Journal 2£_Physiolggy, 125:90, 1954. Beckner, G. L. and T. Winsor, "Cardiovascular Adaptations to Prolonged Physical Effort," Circulation, 9:835-846, June, 1954. Birren, J. E., and Harry Kay, "Swimming Speed of the Albino Rat," Journal g£_Gerontology, 13:374, October, 1958. Bruce, R. A., F. W. Lovejoy, R. Pearson, P. N. G. Yu, G. B. Brothers, and T. Velasquez, "Normal Respiratory and Circulatory Pathways of Adaptation in Exercise," Journal gf Clinical Investigation, 28:1423-1430, November, 1949. Gasser, H. 8., and A. V. Hill, "The Dynamics of Muscular Con- traction," Proceeding 22 the Royal Societngf London, 96: 398, 1924. . Gollnick, P. D. and G. R. Hearn, "Lactic Dehydrogenase Activities of Heart and Skeletal Muscle of Exercised Rats," American Journal gf Physiology, 201:694-696, October, 1961. Gould, M. K. and W. A. Rawlinson, "Biochemical Adaptation as a Reaponse to Exercise," The Biochemical Journal, 73:41-44, 1959. Harris, R. E. and Dwight J. Ingle, "The Capacity for Vigorous Muscular Activity of Normal Rats and of Rats after Removal of the Adrenal Medulla," American Journal gf Physiology, 130:151, 1940. Hearn, G. W. and W. W. Wainio, "Succinic Dehydrogenase Activity of the Heart and Skeletal Muscle of Exercised Rats," American Journal of Physiology, 185:348, 1956. Hearn, G. W. and W. W. Wainio, "Aldolase Activity of the Heart and Skeletal Muscle of Exercised Rats," American Journal of Physiology, 190:206, 1957. 51 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 52 Hill, A. V., C. N. H. Long and H. Lupton, "Muscular Exercise, Lactic Acid and the Supply and Utilization of Oxygen," Proceeding§_2£_the Royal Society 2E London, B, 438:455, 1923-24. Innes, J. R. Ms, E. J. Donati, M. A. Ross, R. M. Stoufer, P. P. Yevich, C. E. Wilson, J. F. Farber, J. A. Pankevicius and T. 0. Downing, "Establishment of a Rat Colony Free From Chronic Murine Pneumonia," The Cornell Veterinarian, 47:260-280, April, 1957. Joshi, N. N., A. C. Blackwood and D. G. Dale, "Chronic Murine Pneumonia--A Review," Canadian Journal 2£_Comparative Medicine and Veteranary Science, 25:267-273, November, 1961. Kay, Harry and James Birren, "Swimming Speed of the Albino Rat-- II Fatigue, Practice, Drug Effects on Age ans Sex Differences," Journal 2; Gerontology, 13:378, October, 1958. Korobkov, A. V., D. A. Goloracheva and V. A. Shkurdoda, "Effect of Muscular Training and Toning Substances on Non-Specific Endurance and Work Capacity In Rats," Sechenov, Physiological Journal 9; the U.S.S.R., 47:30-37, 1961. Kimeldorf, D. J. and S. J. Baum, "Alterations of Organ and Body Growth of Rats Following Daily Exhaustive Exercise, X- Irradiation and Post Irradiation Exercise," Growth, 18:79-96, June, 1954. Kimeldorf, D. J. and D. C. Jones, "The Relationship of Radiation Dose to Lethality Among Exercised Animals Exposed to Roentgen Rays," American Journal g£_Physiology, 167:626, 1951. Merrill, Ida B. and Eugene C. Howe, "The Effect of Exercise and Fatigue Upon Resistance to Infection," American Physical Education Review, 33:68-74, February, 1928. Michael, E. D., K. E. Hutton and S. M. Horvath, "Cardio-Respiratory Responses During Prolonged Exercise," Journal of Applied PhysiOlogy, 16:997-1000, November, 1961. Miller, H. C. and D. C. Darrow, "Relation of Serum and Muscle Electrolyte, Particularly Potassium, to Voluntary Exercise," American Journal 9; Physiology 132:801, 1941. Montoye, H. J., R. MtNab, R. Nelson and P. Johnson, "Effects of Exercise on Swimming Endurance and Organ Weights in Mature Rats," Research Quarterly, 31:434-439, October, 1960. Nelson, J. B., "Studies on Endemic Pneumonia of the Albino Rat," Journal 2: Experimental Medicine, 84:15-23, 1946. Pickford, Mary, "Respiration Adaptation," Lancet 2:1225-1226, December, 1954. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 53 Ramey, E., M. S. Goldstein and R. Levine, "Mechanisms of Muscular Fatigue in Adrenalectomized Animals," American Journal of Physiology, 162:10, 1950. Rawlinson, W. A., and M. K. Gould, "Biochemical Adaptation as a Response to Exercise," The Biochemical Journal, 73:45, 1959. Ruosteenaja, R., "Circulatory, Respiratory and Thermal Adaptation During Heavy Exercise," Acta Physiologica Scandinavica, 31: 248-262, 1954. Scheer, Bradley T., S. Dorst, J. F. Codie and D. F. Soule, "Physical Capacity of Rats in Relation to Energy and Fat Content of the Diet," American Journal of Physiology, 149: 194, 1947. Sjostrand, Torgny, "Volume and Distribution of Blood and Their Significance in Regulating the Circulation," Physiological Reviews, 33:202, 1953. Smith, F. and W. Smith, "Exercise Following Whole-Body Irradiation of Mice," Federation Proceedings, 9:117, 1950. Selye, Hans, The Stress g£_Life. New York: McGraw-Hill Book Co., 1956, pp. 1-326. Tan, E. M., M. E. Hanson and C. P. Richter, "Swimming Time of Rats with Relation to Water Temperature," Federation Pro- ceedings, 13:150, 1954. Thomas, B. M. and A. T. Miller Jr., "Adaptation to Forced Exercise in the Rat," American Journal of Physiology, 193:350-354, May, 1958. Vanderhoof, E. R., C. J. Imig and H. M. Hines, "Effect of Muscle Strength and Endurance Deve10pment on Blood Flow," Journal gf Applied Physiology, 16:873-877, September, 1961. Wilber, Charles G., "Some Factors Which Are Correlated with Swimming Capacity in Guinea Pigs," Journal 2£_Applied Physiology, 14:199, 1959. Zimkin, N. V., "Stress During Muscular Exercises and the State of Non-Specifically Increased Resistance," Sechenov, Physiological Journal of the U.S.S.R., 47:6, 741-751, 1961. APPENDIX A Clinical Report Experimental SPF Rats Department of Physical Education & Recreation History: Three male rats were examined on January 23, 1962. The colony had generally shown a rapid drop in average weight for the three days previous to this. Many individuals in the colony were snuffling and sneezing. Some showed bloody nasal discharge. Physical Examination: All three rats showed upper respiratory congestion. Bloody mucoid nasal discharge was evident. Very slight increased bronchial rales noted on auscultation. Gross pulmonary congestion was not noted. Cultures revealed two different micrococci which were sensitive on plate test only to the nitrofurans. Slight inhibition was noted to several other antibiotics. Blood counts were within normal ranges. An effort to demonstrate PPLO from these and subsequent specimens failed. Treatment: Nitrofurantain* was administered in the drinking water to the extent that each rat should have received 4 to 6 mg of the drug daily. This was continued for approximately two months. Necropsy report on two live rats presented on February 13, 1962 is included. Conclusions: These rats were showing a generalized pulmonary involvement which could probably be classified in the chronic murine pneumonia com- plex. The finding of micrococcus sp. in the respiratory tracts of those rats examined was consistent. One may only speculate as to whether or not this might be the organism of cause. Limited attempts to infect normal rats by nasal secretion transfer failed. * Dantafur, Eaton Laboratories 54 APPENDIX B REPORT OF LABORATORY EXAMINATION History. The rats have been on stress experiments and show signs of respiratory infection. Q5225 Lesions. The carcasses are in good nutritional condition. Both rats show a rhinitis with a mucopurulent exudate present in the external nares. A viral or influenza type bronchOpneumonia is seen in both lungs with consolidation of primarily the anterior lobes. A catarrhal gastroenteritis is present in the intestinal tract. Laboratory Findings. HistOpathological examination: peribronchitis, interstitial and perivascular lymphocytic infiltration, lobular gray hepatization of the lung. Fibrinonecrotic enteritisin the intestine. Bacteriological examination: coliforms from the lung, the kidney and liver were negative and pseudomonas from the intestine. Conclusions. An influenza type bronchopneumonia was the outstanding finding in this case. Our bacteriological examination did not yield any pertinent findings. Supplemental Report Bacteriological examination: Micrococcus species was isolated from the lung; pseudomonas and proteus isolated from the intestine. R. G. Schirmer, Veterinarian H. J. Rothenbacher, Pathologist 55 APPENDIX C PATHOLOGISTS' REPORT £5232 Pathology. All of the gross changes were limited to the lungs. The lesions ranged from small focal areas of sunken plum colored consolidation to disseminated areas. In many cases whole lobes were involved. Some areas were nodular in appearance, and on cut section were filled with a yellowish to yellowish-green viscous material, resembling abcesses. MiscroscOpic Pathology. Microsc0pically there was a wide variation in~severity but generally the pathology was similar. The lesions ranged from mild lymphoid collaring of the bronchioles to extremely severe. When the severity was increased one saw atelectasis, bronchiectasis, hyperplasia. of the bronchiolar epithelium, with mild to complete interstitial pneumonitis. There was a considerable amount of mucous in the dilated bronchioles and many, along with alveoli, were packed with neutrOphils. The hyperplasia, at times, was severe enough to close off the airway. The nature of the cellular infiltrate changed from the mild lymphoid collaring to a more severe generalized infiltration with a mixed granulomatous character. The hemisiderosis observed in the spleen and the mild changes found in the kidney were not considered as contributory and may even be within the normal range of variation. The changes seen.were considered consistent with those reported for chronic murine pneumonia with the exception of the purulent nature. The abcessing was considered an acute suppurative process superimposed on the chronic. W. Field 56 APPENDIX D SWIM TIMES OF RATS AS CLASSIFIED BY LOBES INVOLVED L o b e s I n v o 1 v e d 1 2 Test Test Test #1 #2 #3 #1 #2 #3 #1 #2 #3 4.28 4.50 4.33 3.25 4.00 32.75 62.75 4.00 15.75 14.50 7.25 8.00 '2.25 4.00 4.50 23.93 3.50 8.50 4.33 8.75 7.17 6.13 13.25 6.92 3.63 5.25 4.75 3.00 6.75 4.83 5.17 10.00 67.33 3.43 16.00 62.42 3.33 3.50 3.75 2.83 6.00 5.00 66.12 3.00 2.42 5.75 4.25 11.42 2.55 4.50 4.00 63.67 4.00 40.08 4.08 3.00 6.25 9.83 4.00 5.25 7.60 4.00 6.08 4.45 4.50 6.00 4.00 3.75 3.85 13.42 3.25 62.75 3.00 2.00 67.17 4.17 7.00 5.50 2.67 3.00 5.75 3.17 3.50 3.33 3.33 6.00 5.33 2.33 64.75 14.50 4.62 7.25 7.25 4.75 4.08 5.17 6.00 9.42 13.50 2.67 2.50 2.50 3.75 2.75 3.42 5.25 5.22 32.67 2.33 5.25 7.50 1.75 3.80 2.58 6.00 3.75 17.00 65.00 4.75 8.75 4.00 3.17 4.50 205.5 62.5 72.58 4.75 8.00 6.75 4.12 5.50 5.50 94.42 3.25 5.67 ,1.22 4.50 4.00 4.00 4.00 43.17 63.00 7.00 44.33 P 4.50 2.33 3.33 3.75 3.75 3.33 3.00 11.75 P 4.00 7.33 3.00 3.00 3.08 P 2.50 2.42 3.75 11.00 63.67 144.93 3.25 43.17 93.95 7.00 5.67 2.55 3.50 4.92 71.83 3.25 3.83 163.33 7.00 5.42 3.10 2.50 4.25 5.88 3.25 62.58 4.42 6.00 10.50 13.25 4.75 11.33 2.58 2.75 3.50 5.50 9.00 8.33 62.67 8.50 15.75 5.08 7.00 17.75 9.87 6.25 5.17 2.75 6.25 6.33 10.00 4.00 12.33 3.97 4.25 4.08 3.75 3.75 4.17 4.30 7.25 10.75 57 58 APPENDIX D--Continued L o b e s I n v o 1 v e d 3 4 5 Test Test Test #1 #2 #3 #1 #2 #3 #1 #2 #3 2.25 2.50 16.75 4.00 9.50 24.67 2.50 4.00 4.75 1.22 2.00 4.42 2.60 7.75 7.00 4.67 5.25 5.00 4.83 7.75 67.75 6.25 9.00 31.92 P 6.50 56.58 3.53 9.00 63.50 3.67 3.75 11.33 69.75 4.75 51.00 8.00 4.50 3.50 3.38 3.00 5.00 114.73 63.00 41.00 4.32 5.75 64.83 4.75 5.00 13.50 APPENDIX E NM Hm om mm mm nm em mN «N mm mm HN ON a.” ma 2” 0H nH a: m." NH .3 OH m J 4 q A q . q a q a a j . u _ _ . _ 1 q _ _ J . AV gob—“:30 0 House “.396— WW..\.<\ \LUIIIhYl _\\hW\\\hY\ \Ol0\ \<\4\\< CFIIJQ\1\hV\\\ "VJW\\\X\\ gm NET “.50sz Amxmm» ow.‘ oom oem own owe 00¢ com can “use” .2 APPENDIX F DIAGRAM.OF RAT LUNG Apical Cardiac Diaphramatic Azygos Left Lobe 6O APPENDIX G ALL OUT SWIM TIME IN MINUTES Test No. G r o u p Animal Control IA 13 IIA IIB IIIA IIIB IVA IVB V 1 -- -- 65.00 66.12 -- 63.67 63.08 62.67 62.75 69.75 2 -- 8.0 9.83 14.50 13.42 7.60 13.25 10.00 23.93 9.42 3 205.50 6.25 4.75 -- 4.62 5.75 5.50 9.87 4.28 5.22 4 -- 4.67 4.83 3.92 -- 6.13 5.08 -- 3.63 3.75 5 3.00 4.33 4.00 4.33 2.55 5.17 4.75 3.97 .3.25 -- 6 3.75 -- 3.53 -- 2.67 4.08 4.42 3.75 3.43 4.08 7 3.38 4.12 4.17 4.00 2.67 4.45 3.67 4.30 2.25 2.75 8 {144.93 4.00 4.45 3.00 -- 2.83 2.58 3.33 2.25 3.80 9 -- 3.33 2.58 3.33 2.60 3.00 2.75 -- 2.50 3.17 10 114.73 -- -- -- 2.33 3.17 11 2.55 1.22 2.33 12 163.33 13 93.95 14 -- 15 4.32 16 94.42 17 71.83 '18 3.10 19 -- 20 5.88 Mean 65.33 4.96 11.46 13.78 4.15 10.58 11.68 13.98 12.03 12.74 Median 38.85 4.33 4.45 4.00 5.27 4.81 4.75 4.30 3.43 3.94 62 APPENDIX G--Continued ALL OUT SWIM TIME IN MINUTES Test No. 2 G r o u p Animal IIIA IIIB IVA IVB IIA IIB IB Control IA <.n.fi.nv <.<.s.nu 7.n.9.n. 7.7.7ln. .4,b.5,b ".4.J.1.4 n.n.n.s.n.n.nvn.n. n.<.:.9.n.n.nvs.nu 43454m424 nvnvs. :.<.R.nv s.nv9. 9.7.9.nu .8.4,b ".4.J.I.J n.<.nvnvn.nvs.t.<. nvvlnunvnvnu7.7.9. .I,4Mw_l.5,o.302,o nvnv<.<.nunvnunvn. nonvozoznvnv:.n.n. 444303462 11. <.s. nunvnv R. 9.9. <.<.nv 1: . 1.1: .,4n2.3 ._l . - . nus. a. n.<.n. nuoz .I .5_/.5 1.7." a." Q.A.1." 500550000 vlnvnv7.7,n.nvnvs. 448739764 nunvq.<. nvnva. s.nu9.7. s.nu1: ".4.9.5.4 ".5.4xJ n. n.n.nvs. . . 1.. n.n.n.7.. . . 9.. 1.1.1.1.. ,b .1 1.9.1.A.§.A.7.R.o. 2.00 64.75 3.50 5.25 3.50 7.00 7.00 63.00 10 11 12 13 14 15 5.75 3.25 3.25 2.50 16 17 18 19 20 3.25 5.25 6.08 5.82 12.28 5.45 7.94 5.50 5.30 4.53 13.0 Mean 4.75 6.00 6.75 4.87 4.62 6.00 4.25 4.00 4.75 3.37 Median 63 APPENDIX G--Continued ALL OUT SWIM TIME IN MINUTES Test No. 3 m G r o u p Animal Control IA IB IIA IIB IIIA IIIB IVA IVB V 1 -- -- 8.75 2.42 -- 40.08 44.33 15.75 15.75 51.00 2 -- 3.50 5.25 8.00 62.75 6.08 11.33 12.33 8.50 13.50 3 72.58 31.92 6.75 -- 7.25 11.42 8.33 5.17 4.33 32.67 4 -- 5.00 67.75 7.17 -- 6.92 17.75 -- 4.75 17.00 5 3.08 4.50 3.85 24.67 4.00 67.33 13.50 4.08 32.75 -- 6 63.67 -- 63.50 4.83 2.50 6.25 10.50 4.17 62.42 5.17 7 5.00 5.50 5.50 3.75 5.75 6.00 11.33 10.75 4.50 3.42 8 43.17 21.25 5.33 -- -- 5.00 3.50 11.75 16.75 2.58 9 -- 3.75 4.00 4.42 7.00 67.17 6.33 -- 4.75 4.50 10 41.17 -- 14.50 3.33 11 4.92 7.50 12 5.42 13 ’5.67 14 -- 15 64.83 16 5.67 .17 3.83 18 4.25 19 -- 20 62.58 Mean 25.26 10.77 18.96 7.89 13.91 21.96 14.10 9.14 17.17 16.23 Median 5.67 .5.00 ‘5.50 4.83 7.62 6.58 11.33.10.75 8.50 9;33 ‘ J Animal APPENDIX H Hematocrit (Z) Animal HEMATOCRIT VALUES FOR SELECTED RATS MARCH 24 Hematocrit (Z) I I l I a I l I l I I I I I I HHmO\NHHNI—I\O\IO\NHHHH\IO‘UIo-IHI—Ii—Io—Ikomw \oooun-I \IO‘UJNO lllllll 00‘on hizgzghihizghi '< '<-< <:Pihihihlrih4C3C3C3C)C3C3C3CDCDC3CDCDCDCDCE ON I 51 54 49 49 49 51 58 48 49 50 52 51 51 49 54 45 42 47 49 54 53 51 48 48 49 52 51 51 III-15 III-21 III-24 50 51 52 50 50 53 52 50 54 49 50 52 43 53 48 45 47 48 53 48 64 APPENDIX I CRITICAL REGIONS FOR RANK SUM TEST OF TABLE Iv, pg. 33. Critical Regions Sample Size 5 percent level 1 percent level 7,7 37 to 68 33 to 72 8,8 49 to 87 44 to 92 9,9 63 to 108 57 to 114 10,10 79 to 131 72 to 138 14,14 Z = $1.96 Z = £2.57 74,74 2 = 111.96 2 = 152.57 65 APPENDIX J CRITICAL VALUES OF 1', FOR RANK CORRELATION TEST OF TABLE V, pg. 34 N = .05 = .01 7 .714 .893 8 .643 .833 9 .600 .783 10 .564 .746 14 .456 .645 30 .306 .432 88 .177 .249 ER "11111171111111fifllfltfllfllfiflflflfflfifllfl“