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UBC Theses and Dissertations

Study on the effects of exercise and confinement on the calcium metabolism of the rat. Ross, Bruce H. 1968

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A STUDY ON THE EFFECTS OF EXERCISE AND CONFINEMENT ON THE CALCIUM METABOLISM OF THE RAT by BRUCE H. ROSS Dip. Phys. Ed., U n i v e r s i t y o f Otago, 1964 M.A., U n i v e r s i t y o f Iowa, 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Physiology  We accept t h i s t h e s i s as conforming required  standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1968  t o the  In  presenting  an  advanced  the I  Library  further  for  this degree shall  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make that  i t freely  permission  purposes  may  representatives. thesis  for  be It  financial  available for  by  the  is understood gain  Department Columbia  for  extensive  granted  permission.  The U n i v e r s i t y of B r i t i s h V a n c o u v e r 8, Canada  British  shall  Head  be  requirements  reference copying  that  not  the  of  and  of my  I agree  for that  Study.  this  thesis  Department  copying  or  allowed  without  or  publication my  ABSTRACT  The  e x p e r i m e n t was  designed t o demonstrate  the  effects  o f e x e r c i s e and c o n f i n e m e n t on t h e c a l c i u m m e t a b o l i s m o f rats.  A s u b s i d i a r y p u r p o s e was  t o d e t e r m i n e i f any  e f f e c t s a r e m e d i a t e d by a h u m o r a l Twenty-four male Sprague i n t o three groups. a c t i v i t y cages g r o u p was  One  such  agent.  Dawley r a t s were d i v i d e d  g r o u p was  exercised i n motorized  f o r one h o u r p e r d a y  f i v e d a y s p e r week;  one  c o n f i n e d i n s m a l l i n d i v i d u a l metabolism cages f o r  26 d a y s and i n b o d y h o l d e r s f o r 12 d a y s ; one g r o u p was i n i n d i v i d u a l m e t a b o l i s m cages and s e r v e d  housed  as c o n t r o l s .  Half  of t h e r a t s o f each o f t h e above t h r e e groups r e c e i v e d 2 ml whole b l o o d  from r a t s s i m i l a r l y t r e a t e d ( e x e r c i s e d , c o n f i n e d  o r c o n t r o l ) , on each o f t h e l a s t f i v e days o f t h e e x p e r i m e n t . Thus t h e e x e r c i s e d r a t s r e c e i v e d b l o o d the c o n f i n e d r a t s r e c e i v e d blood control r a t s received blood  from c o n f i n e d  rats,  f r o m e x e r c i s e d r a t s and  the  from c o n t r o l r a t s .  A c a l c i u m b a l a n c e s t u d y was  c a r r i e d out f o r the  last  26 d a y s o f t h e 38 d a y e x p e r i m e n t and a c a l c i u m - 4 5 b a l a n c e s t u d y was ment.  c a r r i e d out f o r the l a s t s i x hours of the e x p e r i -  Thus t h e f o l l o w i n g s p e c i f i c p a r a m e t e r s o f  m e t a b o l i s m were measured: utilization,  calcium balance,  calcium  per cent calcium  food i n t a k e , f a e c a l calcium, u r i n a r y calcium,  t o t a l c a r c a s s c a l c i u m , femur  calcium, calcium-45 absorption  iii and femur calcium-45.  The e n t i r e experiment was performed  twice and the data analyzed u s i n g a randomized complete block design a n a l y s i s o f v a r i a n c e . The e x e r c i s e or confinement d i d not s i g n i f i c a n t l y  alter  the c a l c i u m metabolism o f the male Sprague Dawley r a t under the c o n d i t i o n s of t h i s study. discussed.  Two p o s s i b l e c o n c l u s i o n s were  E i t h e r t h i s experiment c a s t s some doubt on the  c u r r e n t concepts  r e g a r d i n g i m m o b i l i z a t i o n and o s t e o p o r o s i s ,  or t h a t the humane c o n d i t i o n s adopted i n t h i s study d i d not s u c c e s s f u l l y immobilize a l s o suggests  the r a t s .  The present experiment  t h a t r e g u l a r vigorous e x e r c i s e may not a f f e c t  the c a l c i u m metabolism of the r a t t o a g r e a t e r extent normal a c t i v i t y or m i l d e x e r c i s e .  than  No c o n c l u s i o n s c o u l d be  drawn about the r o l e o f the blood i n r e g u l a t i n g o r cont r o l l i n g the e f f e c t s of e x e r c i s e or confinement on c a l c i u m metabolism.  iv TABLE OF CONTENTS Page LIST OF TABLES  v  i  LIST OF FIGURES  x  ACKNOWLEDGEMENT.  x  i  INTRODUCTION  1  Statement o f the Problem . . REVIEW OF LITERATURE  2 . .'  4  Summary  8  EXPERIMENTAL DESIGN Subjects  9 • •  9  i? Parameters  10  Treatments  14  Summary. . .  17  METHODS  1  8  Grouping o f Rats  IS  Weighing o f Rats  18  Diet  18  Housing o f the Rats  19  Method o f E x e r c i s e  24  Stool Collection  27  Calcium A n a l y s i s  • • 27  Blood T r a n s f u s i o n s  28  Radiocalcium Methods  29  Summary  34  V  TABLE OF CONTENTS  (Continued) Page  ANALYSIS OF DATA  35  RESULTS  39  DISCUSSION  75  Summary  82  Conclusions.  82  LITERATURE CITED  •  84  vi LIST OF TABLES TABLE I. II.  III.  Page S p e c i f i c a t i o n s o f Metabolism Cages Used to House Rats.  20  Computations o f Sums o f Squares and F R a t i o s as w e l l as the V a r i a n c e estimated by the Mean Square f o r a Randomized Complete Block Design  36  A n a l y s i s o f V a r i a n c e - Body Weight (g) Day 0  41  IIIA. Ranked Treatment Means - Body Weight (g) IV.  Day 0  41  A n a l y s i s o f Variance - Body Weight (g) Day 12. .  42  IVA. Ranked Treatment Means - Body Weight (g) Day 12.  42  V.  A n a l y s i s o f V a r i a n c e - Body Weight (g) Day 19. .  43  VA. Ranked Treatment Means - Body Weight (g) VI.  Day 19  43  A n a l y s i s o f V a r i a n c e - Body Weight (g) Day 26. .  44  VIA. Ranked Treatment Means - Body Weight (g) VII.  Day 26  .  A n a l y s i s o f V a r i a n c e - Body Weight (g) Day 33. .  44 45  VIIA. Ranked Treatment Means - Body Weight (g) VIII.  Day 33  A n a l y s i s o f V a r i a n c e - Body Weight (g) Day 38. .  VIIIA. Ranked Treatment Means - Body Weight (g) Day 38 IX. A n a l y s i s of V a r i a n c e - Calcium Balance Data (Mg) Days 12-19 IXA. Ranked Treatment Means - Calcium Balance (Mg) Days 12-19  45 46 46 47 47  vii LIST OF TABLES  (Continued)  TABLE X.  PAGE A n a l y s i s of V a r i a n c e - Calcium Balance (Mg) Days 19-26  XA. Ranked Treatment Means - Calcium Balance (Mg) Days 19-26 . XI.  A n a l y s i s o f V a r i a n c e - Calcium Balance (Mg) Days 26-33  XIA. Ranked Treatment Means - Calcium Balance (Mg) Days 26-33 XII. XIII.  XVII. XVIII.  49  A n a l y s i s of V a r i a n c e of Per Cent Calcium Utilization Days 12-19  51  A n a l y s i s of V a r i a n c e - Per Cent Calcium Utilization Days 19-26.  A n a l y s i s of V a r i a n c e - Per Cent Calcium Utilization Days 26-33.  XVA. Ranked Treatment Means - Per Cent Calcium Utilization Days 26-33. XVI.  49  50  XIVA. Ranked Treatment Means - Per Cent Calcium Utilization Days 19-26 XV.  48  A n a l y s i s of V a r i a n c e - Calcium Balance (Mg) Days 33-38  XIIIA. Ranked Treatment Means - Per Cent Calcium Utilization Days 12-19 XIV.  48  51 52 52 53 53  A n a l y s i s of V a r i a n c e - Per Cent Calcium Utilization Days 33-38  54  A n a l y s i s of V a r i a n c e - Food Intake (g.) Days 12-19  55  A n a l y s i s of V a r i a n c e - Food Intake (g) Days 19-26  56  XVIIIA. Ranked Treatment Means - Food Intake (g) Days 19-26  56  viii LIST OF TABLES  (Continued)  TABLE XIX.  Page A n a l y s i s of V a r i a n c e - Food Intage Days 26-33  (g) 57  XIXA. Ranked Treatment Means - Food Intake (g) Days 26-33 . . . XX.  57  A n a l y s i s of V a r i a n c e - Food Intake (g) Days 33-38 .  ,  XXA. Ranked Treatment Means - Food Intake (g) Days 33-38 . XXI. XXII.  (Mg)  A n a l y s i s of V a r i a n c e - F a e c a l Calcium Days 19-26  (Mg)  59 60  A n a l y s i s of V a r i a n c e - F a e c a l Calcium Days 26-33 . .  A n a l y s i s of V a r i a n c e - F a e c a l Calcium Days 33-38  XXVI.  A n a l y s i s of V a r i a n c e - U r i n e Calcium Days 33-38  (Mg) 61  (Mg) 62 (Mg) 62  (Mg) 63  A n a l y s i s o f V a r i a n c e - T o t a l Carcass Calcium  XXVII.  60 61  XXIVA. Ranked Treatment Means - F a e c a l Calcium Days 33-38 XXV.  (Mg)  (Mg)  XXIIIA. Ranked Treatment Means - F a e c a l Calcium Days 26-33 XXIV.  58  A n a l y s i s o f V a r i a n c e - F a e c a l Calcium Days 12-19  XXIIA. Ranked Treatment Means - F a e c a l Calcium Days 19-26 XXIII.  58  (Mg)  A n a l y s i s of V a r i a n c e - Femur Calcium  A n a l y s i s o f V a r i a n c e - Calcium-45 A b s o r p t i o n (% Administered Dose) XXVIIIA. Ranked Treatment Means - Calcium-45 A b s o r p t i o n (% Administered Dose)  64 (Mg). . . .  65  XXVIII.  66 66  XX  LIST OF TABLES  (Continued) . Page  TABLE XXIX. XXX.  A n a l y s i s o f V a r i a n c e - Femur (% A d m i n i s t e r e d Dose)  Calcium-45  A n a l y s i s o f V a r i a n c e - R e l a t i v e Heart Weight (Mg/g) ,  XXXA. Ranked Treatment Means - R e l a t i v e Heart Weight (Mg/g)v  67 68 68  X  L I S T OF  FIGURES  FIGURE 1. 2. 3.  4.  5. 6.  7.  8.  9.  Page C o n f i n i n g cage d e s i g n e d movement b y t h e r a t Wahman a c t i v i t y run r a t s  t o minimize 23  cage m o d i f i e d t o f o r c e 25  Quench c a l i b r a t i o n c u r v e . Plotted points represent the e f f i c i e n c y of counting a known s t a n d a r d w i t h known q u a n t i t i e s o f a q u e n c h i n g agent added  32  Calcium-45 s p e c t r a l curve showing spectrometer window s e t t i n g s f o r c o u n t i n g c a l c i u m - 4 5 u s i n g t h e c h a n n e l s r a t i o method o f quench correction  33  Body w e i g h t g r o w t h c u r v e s f o r t h e e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s  40  Mean w e e k l y c a l c i u m b a l a n c e o f t h e e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . Significant i n t e r a c t i o n F r a t i o f o r calcium balance data (days 33-38) p r e v e n t e d t h e v a l u e s f o r d a y s 33-38 b e i n g p l o t t e d  70  Mean w e e k l y p e r c e n t c a l c i u m u t i l i z a t i o n o f the e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . S i g n i f i c a n t i n t e r a c t i o n F r a t i o f o r per c e n t c a l c i u m u t i l i z a t i o n d a t a (days 33-38) p r e v e n t e d t h e v a l u e s f o r d a y s 33-3 8 b e i n g plotted  71  Mean w e e k l y f o o d i n t a k e o f t h e e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . Significant i n t e r a c t i o n f o r f o o d i n t a k e d a t a (days 12-19) p r e v e n t e d t h e v a l u e s f o r d a y s 12-19 b e i n g p l o t t e d  72  Mean w e e k l y f a e c a l c a l c i u m o f t h e e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . Significant i n t e r a c t i o n f o r f a e c a l c a l c i u m d a t a (days 12-19) p r e v e n t e d t h e v a l u e s f o r d a y s 12-19 b e i n g p l o t t e d  73  ACKNOWLEDGEMENT  T h i s w r i t e r would l i k e t o thank Dr. C.F. Cramer f o r h i s confidence and encouragement, Dr. M. F r a n c i s f o r her p a t i e n t c o u n s e l l i n g i n s t a t i s t i c s and Mrs. M. N i c h o l s o n f o r her i n v a l u a b l e technical  assistance.  INTRODUCTION Calcium  i s one o f the most important  i n v o l v e d i n the l i f e p r o c e s s .  Calcium  bivalent cations  i s i n v o l v e d i n blood  c l o t t i n g , neuromuscular t r a n s m i s s i o n , muscle c o n t r a c t i o n and it  i s the main s t r u c t u r a l component o f bone.  The l i t e r a t u r e  on c a l c i u m metabolism i n the l i v i n g organism i s e x t e n s i v e . I n t e s t i n a l c a l c i u m a b s o r p t i o n was reviewed by Wiseman (196 4) and P f i e f f e r  (1968a), plasma c a l c i u m r e g u l a t i o n by p a r a -  thormone and c a l c i t o n i n was c l a r i f i e d by Copp (1962) and i t s f u n c t i o n i n bone was d i s c u s s e d by many authors, i n c l u d i n g Rodahl e t a l . (1960).  However, t h e r e i s a notable  of r e s e a r c h r e p o r t s concerning  scarcity  the e f f e c t s o f e x e r c i s e on  the r e g u l a t i o n , a b s o r p t i o n and bone k i n e t i c s o f c a l c i u m . This lack of i n t e r e s t i s s u r p r i s i n g since osteoporosis caused by i m m o b i l i z a t i o n i s o f s e r i o u s c l i n i c a l (Whedon, 1960).  Several studies  concern  ( A l l i s o n and Brooks, 1921;  D e i t r i c k e t a l . , 1948; Abramson, 1948; Whedon and Shorr, 1957;  Vogt e t a l . , 1965; and Birkhead,  1966) document the  f a c t t h a t i m m o b i l i z a t i o n and d i s u s e r e s u l t i n i n c r e a s e d u r i n a r y e x c r e t i o n o f c a l c i u m and atrophy bone.  The s t u d i e s o f Abramson  (1954), G i l l e s p i e  of the immobilized  (1948), Wyse and P a t l e e  (1954) and Whedon and Shorr  p r o v i d e i n d i r e c t evidence  (1957) a l l  t h a t normal muscle f u n c t i o n i s  r e q u i r e d t o prevent undue c a l c i u m l o s s from bone.  2 The  only d i r e c t evidence  t h a t e x e r c i s e may  effect  c a l c i u m metabolism i s p r o v i d e d by Vogt e t a l . (1965), demonstrated t h a t i s o m e t r i c e x e r c i s e i n h i b i t e d  who  immobili-  z a t i o n o s t e o p o r o s i s , and by L u i and McCay (1953),  who  suggest t h a t e x e r c i s e promoted c a l c i u m r e t e n t i o n i n o l d dogs. Although, as p r e v i o u s l y c i t e d , there i s a c o n s i d e r a b l e body of evidence  i n d i c t i n g i n a c t i v i t y or i m m o b i l i z a t i o n as a  cause of h y p e r c a l c i u r i a and bone atrophy, (Burkhart  and Jowsey, 1967)  only one  study  attempted t o determine the  mechanism whereby i m m o b i l i z a t i o n causes o s t e o p o r o s i s . Since the e f f e c t s of e x e r c i s e on calcium metabolism do not appear to have been thoroughly  i n v e s t i g a t e d , and  s i n c e the mechanism causing i m m o b i l i z a t i o n o s t e o p o r o s i s not been completely  has  e l u c i d a t e d , t h i s study proposed to i n -  v e s t i g a t e the e f f e c t s of e x e r c i s e on e x p l i c i t parameters of c a l c i u m metabolism, and  to determine i f b l o o d p l a y s any  i n causing the e f f e c t s of e x e r c i s e or i m m o b i l i z a t i o n  role  (con-  finement) on c a l c i u m metabolism. Statement of the Problem  The  f i r s t purpose of t h i s study was  any p h y s i o l o g i c a l e f f e c t s of e x e r c i s e and the c a l c i u m metabolism of r a t s .  t o demonstrate confinement on  3 The second purpose of t h i s study was  t o determine i f  the b l o o d c a r r i e s a humoral agent which mediates any of the p h y s i o l o g i c a l e f f e c t s of e x e r c i s e and confinement on the c a l c i u m metabolism of r a t s .  REVIEW OF LITERATURE The  only d i r e c t evidence t h a t e x e r c i s e has an e f f e c t  on c a l c i u m metabolism i s p r o v i d e d and by Vogt e t a l . (1965).  by L u i and McCay (1953)  L u i and.McCay  (1953), i n v e s t i -  g a t i n g c a l c i u m metabolism i n dogs, r e p o r t e d t h a t e x e r c i s e caused an i n c r e a s e o f c a l c i u m r e t e n t i o n i n three o l d dogs. Vogt e t a l . (1965) r e p o r t e d t h a t i s o m e t r i c e x e r c i s e apparently i n h i b i t e d calcium they were b e i n g  l o s s i n seven h e a l t h y young men while  subjected  t o 14 days o f bed r e s t .  S e v e r a l s t u d i e s i n d i r e c t l y i m p l i c a t e e x e r c i s e as a f a c t o r which prevents e x c e s s i v e m o b i l i z a t i o n o f c a l c i u m bone.  from  D e i t r i c k e t a l . (1948) conducted a study i n which  f o u r h e a l t h y young men were c o n f i n e d t o bed i n a c a s t extending  from the w a i s t  seven weeks.  t o the t o e s , f o r a p e r i o d o f s i x t o  D i e t a r y c a l c i u m was c o n t r o l l e d and c a l c i u m  e x c r e t i o n measured.  These i n v e s t i g a t o r s found t h a t immo-  b i l i z a t i o n brought about a prompt and g r a d u a l l y i n c r e a s i n g l o s s o f c a l c i u m which reached a maximum r a t e a t about f o u r to f i v e weeks.  The u r i n a r y c a l c i u m e x c r e t i o n r a t e appeared  to i n c r e a s e two t o three f o l d . recovered  A f t e r the f o u r  from the i n i t i a l s i x t o seven week  p e r i o d , the i m m o b i l i z a t i o n was repeated, were rocked  subjects  immobilization  but the p a t i e n t s  on a bed which o s c i l l a t e d from h o r i z o n t a l t o 20°  5 and back every two minutes.  T h i s treatment decreased the  s e v e r i t y o f c a l c i u m l o s s due t o i m m o b i l i z a t i o n , but d i d n o t prevent the development o f a n e g a t i v e c a l c i u m b a l a n c e . a f o l l o w up study, Whedon and Shorr  In  (1957) found t h a t the  r o c k i n g bed treatment had no e f f e c t on the severe o s t e o p o r o s i s s u f f e r e d by p a r a l y z e d p o l i o v i c t i m s . and Shorr  Thus Whedon  (1957) concluded t h a t f u n c t i o n i n g muscles are  important f o r the maintenance o f a p o s i t i v e c a l c i u m b a l a n c e . Another  study which r e l a t e d c a l c i u m l o s s d u r i n g  i m m o b i l i z a t i o n d i r e c t l y t o the d i s u s e o f the i n v o l v e d limb or l i m b s , was a c l i n i c a l study performed  by Abramson  He d i v i d e d p a r a p l e g i c s i n t o two groups.  One group of 30  p a t i e n t s perambulated  (1948).  i n wheel c h a i r s , w h i l e a group o f  e i g h t p a t i e n t s walked w i t h braces and c r u t c h e s f o r a t l e a s t one hour per day.  Over a three year p e r i o d 25 o f the 30  wheel c h a i r o p e r a t o r s had marked t o severe o s t e o p o r o s i s o f the p e l v i s , as assessed by X-ray, w h i l e o n l y one o f the e i g h t walkers showed s l i g h t p e l v i c bone r a r e f a c t i o n . Wyse and P a t l e e (1954) extended Abramson  (1948) by demonstrating  the f i n d i n g s o f  t h a t bed r o c k i n g or weight  b e a r i n g by the i n v o l v e d limbs o f p a r a p l e g i c s d i d n o t change t h e i r negative calcium balance.  Thus i t appears  unlikely  t h a t bone t e n s i o n alone can prevent c a l c i u m l o s s from bone. Gillespie  (1954), by s e c t i o n i n g the a n t e r i o r o r  p o s t e r i o r nerve r o o t s s u p p l y i n g the h i n d limbs o f k i t t e n s ,  6 found t h a t the l o s s i n bone weight c o r r e l a t e d  highly  with the decrease i n muscle weight of the p a r a l y z e d and d i d not c o r r e l a t e with Therefore  (0.79)  limbs,  the degree of nerve d e s t r u c t i o n .  he concluded t h a t bone changes due  to  immobili-  z a t i o n r e s u l t from l o s s of muscular a c t i v i t y and  not  from  the l o s s of a t r o p h i c i n f l u e n c e of the nerves. C o n t r a d i c t i n g the c o n c l u s i o n s the f i n d i n g s of Dunning and Plum  of G i l l e s p i e  (1957).  (1954) are  These i n v e s t i g a t o r s  found t h a t h y p e r c a l c i u r i a developed i n p a t i e n t s who  contracted  p o l i o m y e l i t i s r e g a r d l e s s of the degree of p a r a l y s i s . those p a t i e n t s w i t h  s l i g h t bulbar  ceased t o e x c r e t e e x c e s s i v e  However,  and high s p i n a l p a r a l y s i s  calcium  i n the u r i n e by  the  f o u r t e e n t h week a f t e r the onset of p a r a l y s i s , whereas the severely paralyzed  continued  Thus Dunning and Plum  to s u f f e r from h y p e r c a l c i u r i a .  (1957) concluded t h a t there i s a n e u r a l  or neurochemical f a c t o r i n v o l v e d i n bone d e m i n e r a l i z a t i o n . More r e c e n t s t u d i e s have documented the e f f e c t s of immobilization  on c a l c i u m e x c r e t i o n .  Vogt e t a l . (1965)  performed a c a l c i u m balance study on seven h e a l t h y young d u r i n g a seven day p e r i o d of bed  rest.  They found t h a t  i m m o b i l i z a t i o n promoted a n o t i c e a b l e l o s s i n bone w i t h i n three days of the bed  rest period.  calcium  They a l s o found  n o t i c e a b l e changes i n bone d e n s i t y as measured by scanning.  men  However, as p o i n t e d out by Hegsted  radiographic  (1967) i t  appears t h a t the r a d i o g r a p h i c d e n s i t y measurement  techniques  7 are somewhat s u s p e c t , s i n c e the r e p o r t e d changes i n bone density  (3% - 9%)  i s f a r above the calcium, e x c r e t i o n l e v e l  r e p o r t e d i n balance s t u d i e s . By i m m o b i l i z i n g Macaca Trus monkeys i n c a s t s , or by t r a n q u i l l i z e r , or by p a r a l y s i s , Gross e t a l . (1966) f a i l e d f i n d any  to  change i n the c a l c i u m balance i n Macaca Irus monkeys  as a response  to immobilization.  I t appears from the consensus of l i t e r a t u r e r e p o r t s to date, t h a t r e s o r p t i o n of bone c a l c i u m d e f i n i t e l y occurs i n response  to t o t a l or p a r t i a l i m m o b i l i z a t i o n .  The  physio-  l o g i c mechanism governing  t h i s e f f e c t has not been exten-  sively investigated.  c a r e f u l study by Burkhart  (1967) suggests  One  and Jowsey  t h a t both the t h y r o i d s and p a r a t h y r o i d s are  i n v o l v e d i n the i m m o b i l i z a t i o n bone r e s o r p t i o n mechanisms. These r e s e a r c h e r s immobilized  i n c a s t s the r i g h t l e g s of  a d u l t dogs, f o r a p e r i o d of three to 12 weeks. were d i v i d e d i n t o f o u r groups:  intact,  The dogs  parathyroidectomized,  t h y r o i d e c t o m i z e d , and t h y r o p a r a t h y r o i d e c t o m i z e d .  The  and one m e t a t a r s a l were removed from the immobilized limbs of each animal.  By performing  tibia and  t h a t bone r e s o r p t i o n i n the immobilized  o n l y i n the i n t a c t dogs.  free  q u a n t i t a t i v e micro-  r a d i o g r a p h i c a n a l y s i s of each bone, Jowsey and Burkhart found  30  (1967)  limb o c c u r r e d  T h e r e f o r e , these i n v e s t i g a t o r s con-  clude t h a t both the t h y r o i d and p a r a t h y r o i d glands r e q u i r e d f o r i m m o b i l i z a t i o n o s t e o p o r o s i s to occur.  are  8 Summary . The r e s e a r c h which suggests: t h a t e x e r c i s e calcium metabolism by p r e v e n t i n g  affects  undue bone r e s o r p t i o n , i s  meagre, and f o r the most p a r t i n d i r e c t .  Although i t i s  w e l l documented t h a t t o t a l or p a r t i a l i m m o b i l i z a t i o n osteoporosis,  causes  the mechanism c o n t r o l l i n g t h i s e f f e c t has not  been e l u c i d a t e d . and p a r a t h y r o i d  The mechanism may i n v o l v e both the t h y r o i d glands.  EXPERIMENTAL DESIGN  Once the r e s e a r c h problem had been formulated, an experiment was  designed.which might d e t e c t some p h y s i o l o g i c  e f f e c t s o f e x e r c i s e and confinement on c a l c i u m metabolism. The experiment was  a l s o designed t o s e a r c h f o r a humoral  agent which might mediate any e f f e c t s of e x e r c i s e and confinement on c a l c i u m metabolism.  F a c t o r s considered i n the  d e s i g n of t h i s experiment were:  the s u b j e c t s t o be used,  the  parameters t o be measured, the treatments or e x p e r i -  mental v a r i a b l e s t o be i n v e s t i g a t e d , the d u r a t i o n o f the experiment, and the s t a t i s t i c a l p r i n c i p l e s of e x p e r i m e n t a l design. Subjects Rats were chosen as the e x p e r i m e n t a l animals because they adapt r e a d i l y t o e x e r c i s e  ( T i p t o n , 1965)  and are  e a s i l y housed i n metabolism and r e s t r i c t i v e cages. Forty-two male Sprague-Dawley r a t s , 43 days o l d , were used i n each experiment. was  A second and s i m i l a r  used when the experiment was repeated.  group  10  P a r a m e t e r s  T h e C a l c i u m  d i e t .  f o l l o w i n g  p a r a m e t e r s  w e r e  m e a s u r e d  o n  e a c h  r a t .  B a l a n c e C a l c i u m  b a l a n c e  i s  t h e  C a l c i u m  b a l a n c e  w a s  n e t  c a l c i u m  c a l c u l a t e d  r e t a i n e d  u s i n g  t h e  f r o m  t h e  f o l l o w i n g  f o r m u l a : C a ^  =  C a ^  —  C a p  —  C a ^  w h e r e : C a  r e p r e s e n t s  c a l c i u m  C a j _  r e p r e s e n t s  t h e  c a l c i u m  C a  F  r e p r e s e n t s  t h e  f a e c a l  C a y  r e p r e s e n t s  t h e  u r i n e  B  T h e  c a l c i u m  P e r  C e n t  b a l a n c e  C a l c i u m  P e r  c e n t a  w a s  c o m p u t e d  i n  m i l l i g r a m s ,  i n g e s t e d c a l c i u m c a l c i u m e a c h  i n i n  m i l l i g r a m s m i l l i g r a m s  i n  m i l l i g r a m s .  w e e k .  U t i l i z a t i o n c a l c i u m  e x p r e s s e d  a s  p a r a m e t e r  p r o v i d e s  d e p e n d e n t  o f  u t i l i z a t i o n  p e r c e n t a g e  t h e  b a l a n c e  a n  o f  i n d e x  v a r y i n g  t h e o f  a m o u n t  i s  c a l c i u m  t h e  c a l c i u m  b a l a n c e  i n g e s t e d .  n e t  c a l c i u m  o f  c a l c i u m  r e t e n t i o n  T h i s i n -  i n g e s t e d .  i . e . % C a u t i l i z a t i o n = C a j _ i s c a l c i u m  d e r i v e d : i n  f o o d ,  g r a m s  o f  e x p r e s s e d  f o o d a s  C a ^ 7  B  x 1 0 0  i n g e s t e d m u l t i p l i e d b y m i l l i g r a m s  o f  c a l c i u m .  %  11 Weekly Food  Intake  Food i n t a k e i s the weight i n grams o f the t o t a l i n g e s t e d p e r week.  food  I t was measured t o d e t e c t the e f f e c t of  e x e r c i s e and confinement on a p p e t i t e . Weekly F a e c a l  Calcium  F a e c a l c a l c i u m i s the m i l l i g r a m s o f calcium i n the faeces c o l l e c t e d over a p e r i o d o f one week.  The f a e c a l  cium was used t o c a l c u l a t e the c a l c i u m balance used t o assess whether the treatments f a e c a l e x c r e t i o n of calcium.  cal-  and was a l s o  were a f f e c t i n g the  T h i s i s an i n d i r e c t  reflection  of "endogenous f a e c a l calcium e x c r e t i o n . " Weekly Urine  Calcium  U r i n e c a l c i u m i s the m i l l i g r a m s of calcium i n the u r i n e pooled  from t r i - w e e k l y c o l l e c t i o n s .  T h i s parameter was  measured as i t was c o n s i d e r e d t o be the most probable  route  of c a l c i u m e x c r e t i o n i f body calcium l o s s was i n c r e a s e d d u r i n g the experiment, e s p e c i a l l y i n the event o f i n c r e a s e d bone, r e s o r p t i o n . Carcass  Calcium Carcass  c a l c i u m i s the m i l l i g r a m s o f c a l c i u m i n the  ashed r a t c a r c a s s .  The c a r c a s s calcium r e f l e c t s the p h y s i o -  l o g i c a l s t a t u s o f the bone, s i n c e 9 9.5 c a l c i u m i s found  i n the s k e l e t o n .  per cent o f body  Any change i n t h i s  para-  12 meter over a p e r i o d might be a s e n s i t i v e i n d i c a t o r of f a c t o r s which a l t e r bone calcium s t o r a g e .  This i s distinguished  from r a d i o - c a l c i u m s k e l e t a l r e t e n t i o n which measures the non-cumulative p h y s i o l o g i c a l s t a t u s of bone a t a g i v e n i n t h i s case at the end of the t h i r t y - e i g h t day training period.  Femur  exercise  However, s i n c e s o f t t i s s u e calcium i s  p r e s e n t , a secondary more s p e c i f i c i n d i c a t o r of bone calcium was  time;  also chosen—femur  cumulative  calcium.  Calcium Femur c a l c i u m i s the net calcium p r e s e n t i n a femur  removed a t autopsy and  ashed.  The  femur c a l c i u m p r o v i d e s  index of the p h y s i o l o g i c s t a t e of a s p e c i f i c bone, w i t h  an  no  s o f t t i s s u e p r e s e n t , a t a g i v e n time, i . e . , the end of the 38 day experimental  period.  During  t h i s p e r i o d an e x p e r i -  mental v a r i a b l e which might a l t e r bone r e t e n t i o n was introduced. Body Weight. The body weight of each r a t at the end of each week was  used as an index of g e n e r a l h e a l t h , n u t r i t i o n , and  growth.  R e l a t i v e Heart Weight R e l a t i v e h e a r t weight i s the r a t i o of the h e a r t weight compared to the body weight.  Since T i p t o n  (1965) has shown  t h a t the r e l a t i v e h e a r t weights of t r a i n e d r a t s are g r e a t e r  13 than the r e l a t i v e h e a r t weights of non t r a i n e d r a t s ,  relative  h e a r t weight was used i n t h i s study as a measure of the e f f e c t i v e n e s s of the e x e r c i s e used i n t h i s study. Calcium-45 A b s o r p t i o n Calcium-45  a b s o r p t i o n i s d e f i n e d here as the per  cent of the o r a l l y a d m i n i s t e r e d dose of calcium-45 absorbed i n s i x hours. the  T h i s was c a l c u l a t e d by two methods t o i n c r e a s e  c e r t a i n t y of i t s measurement... The f o l l o w i n g two formulae  were' used a f t e r the r a d i o a c t i v e counts had been converted t o per  cent dose: 1.  % Dose Ca  2.  % Dose Ca  45 A  45 =  100% Ca  =  % Dose C a  45 Dose - % Dose C a  45  45 A  c  p  45 + % Dose Ca^  where: 45" Ca  A  r e p r e s e n t s n e t a b s o r p t i o n of calcium-45,  45 Ca  r e p r e s e n t s the calcium-45 i n the i n t e s t i n a l  p  contents p l u s the f a e c e s , 45 Ca  r e p r e s e n t s the calcium-45 i n the ashed  c  c a r c a s s minus the i n t e s t i n a l t r a c t , and 45 Ca^ Femur  r e p r e s e n t s the calcium-45 i n the u r i n e .  Calcium-45 Femur calcium-45 i s d e f i n e d here as the per cent dose  of calcium-45 which was r e t a i n e d i n the r i g h t femur o f each  14 r a t s i x hours a f t e r the a d m i n i s t r a t i o n of the o r a l dose. T h i s parameter r e f l e c t s the n e t bone uptake o f the calcium-45 o r a l dose; i t r e f l e c t s the n e t e f f e c t of a c c r e t i o n and almost n e g l i g i b l e r e s o r b t i o n o f c a l c i u m i n a s p e c i f i c bone d u r i n g the f i n a l s i x hour i n t e r v a l o f the 38 day experimental p e r i o d .  :  Treatments  Three main groups were employed were: group.  i n t h i s study.  They  an e x e r c i s e d group, a c o n f i n e d group, and a c o n t r o l A subgroup of each o f the above three groups was  i n j e c t e d w i t h whole b l o o d over the f i n a l f i v e days o f the experiment t o g i v e a t o t a l o f s i x treatments.  The three  main groups were used t o i n v e s t i g a t e the e f f e c t s o f e x e r c i s e and confinement on c a l c i u m metabolism.  The three  subgroups  were used t o i n v e s t i g a t e the r o l e of the b l o o d i n m e d i a t i n g or c o n t r o l l i n g any e f f e c t s of e x e r c i s e and confinement on c a l c i u m metabolism. The e x e r c i s e d r a t s were r e q u i r e d t o run twice a day, f i v e days per week f o r the d u r a t i o n o f the experiment (38 days) . The c o n f i n e d r a t s were housed i n s m a l l metabolism cages f o r the f i r s t  26 days of the experiment and were housed  i n s m a l l e r cages, s p e c i a l l y designed t o r e s t r i c t movement, f o r the l a s t 12 days of the experiment.  15 T h e c a g e s f o r  c o n t r o l  d a y  d a y s  t h e  s u b g r o u p f i n e d ; b l o o d  s u b g r o u p  c r o s s  o f  t h e f r o m  d o n o r i n  d o n o r  w h i c h  b l o o d  h a d  t r a n s f u s i o n ,  o f a n d  r a t s  r a t s  b e e n  s u b g r o u p  m i l l i l i t e r s  t h e  i n  w h i c h  f i n a l  t h e h a d  e x e r c i s e d ;  r e c e i v e d  f i v e  e x e r c i s e d b e e n  s u b g r o u p  c o n -  " r e c e i v e d a n d  b l o o d  t h e f r o m  c o n t r o l s .  t h e t h e  c o n f i n i n g m e t h o d  o f  c a g e s ,  t h e  e x e r c i s e  m e t h o d  c a n  b e  o f  f o u n d  s e c t i o n , p p . 18-34.  : D u r a t i o n o f t h e ' E x p e r i m e n t  E a c h  m e t a b o l i s m  t w o  f o r  c o n f i n e m e n t h a d  b e e n  r e c e i v e d  b l o o d ,  d o n o r  t h e  c o n t r o l  d e s c r i p t i o n  i n t h e M e t h o d s  i n  i n d i v i d u a l  r e c i p i e n t  f r o m  r a t s . w h i c h  t h e  w h o l e  T h e  b l o o d  i n  r e c i p i e n t s o f  e x p e r i m e n t .  r e c i p i e n t s r a t s  b l o o d  r e c i p i e n t s  h o u s e d  t h e e x p e r i m e n t ( 3 8 d a y s ) .  t r a n s f u s i o n  r e c e i v e d  A  w e r e  t h e d u r a t i o n o f  T h e p e r  r a t s  r e p e t i t i o n  o f  t h e  e x p e r i m e n t  l a s t e d  :  a  t o t a l  o f  3 8 d a y s . T h e f i r s t 1 2 d a y so f e a c h3 8 d a y e x p e r i m e n t a l p e r i o d c a g e s ,  w e r e  u s e d  t o  a c c l i m a t i z e  a n d  i n  t h e  c a s e  o f  i n t r o d u c e d  t o  t h e  f o r c e d  t h e  t h e  e x e r c i s e d  r u n n i n g  i d e d T h e  i n t o d a t a  p e r i o d .  f o u r w e r e  w e e k s c o m p i l e d  l a s t 2 6  d a t a  c o l l e c t i o n  ( t h e  l a s t  w e e k  t h e  e n d  o f  w a s  t h e  r a t s ,  T h e  a t  t o  p r o c e d u r e .  c o l l e c t e d f r o m e a c h r a t d u r i n g t h e e x p e r i m e n t a l  r a t s  t h e y D a t a  p e r i o d  e a c h  w a s  d a y s  d a t a  w e r e w e r e  d a y s o f  f i v e  e a c h  m e t a b o l i s m  d i v -  l o n g ) .  c o l l e c t i o n  16 week  ( s e e R e s u l t s , p p . 39-74) . S i n c e o n l y f o u r r a t s c o u l d be h a n d l e d i n e a c h  ment g r o u p a t one t i m e  r  provide a s t a t i s t i c a l l y  t h e e x p e r i m e n t was r e p e a t e d acceptable  sample  size.  treatto  Thus b y  r e p e a t i n g t h e e x p e r i m e n t w i t h s i m i l a r r a t s , t h e sample o f e a c h g r o u p was i n c r e a s e d f r o m f o u r t o e i g h t  size  rats.  Statistics I t was e s t a b l i s h e d t h a t t h e m o s t e f f i c i e n t  statistical  f o r m a t f o r t h i s e x p e r i m e n t was a n a l y s i s o f v a r i a n c e , , e m p l o y i n g a randomized complete block design. i z e d c o m p l e t e b l o c k d e s i g n demanded 1.  t h e r a t s were randomly  The u s e o f t h e randomthat:  assigned  to the s i x  treatments, 2.  t h e number o f r a t s i n e a c h t r e a t m e n t was  3.  a l l t r e a t m e n t s were a p p l i e d d u r i n g each  equal, repetition  of the experiment so t h a t each r e p e t i t i o n o f the experiment provided  a complete block of data.  C o m p a r i s o n b e t w e e n t h e i n d i v i d u a l t r e a t m e n t means was made u s i n g D u n c a n ' s new m u l t i p l e r a n g e t e s t Torrie,  1960).  ( S t e e l and  Summary  The e x p l a n a t i o n o f - t h e experimental d e s i g n u t i l i z e d i n t h i s study i n v o l v e d a d e s c r i p t i o n of the model, the subjects,: the t r e a t m e n t s , t h e d u r a t i o n of the  experiment.  statistical  parameters and  the  METHODS  T h i s s e c t i o n d e s c r i b e s the equipment in this  and methods used  experiment.  : Grouping of Rats To assure random sampling 42 r a t s were assigned t o the s i x treatment and t o the t h r e e e q u a l donor groups a t a b l e of random numbers  using  ( S t e e l and T o r r i e , 1960) .  Weighing o f Rats A l l the r a t s were weighed  t o the n e a r e s t gram on  a r r i v a l , 1 2 days a f t e r the s t a r t of the experiment, and a t the end of each subsequent week, u n t i l the end o f the experiment. 1  Diet :  A powdered form o f B u c k e r f i e l d s chow was used as the r a t food f o r the e n t i r e experiment.  Weekly a n a l y s i s of the  food i n d i c a t e d t h a t i t averaged 0.23 ±0.03 per cent magnesium,  0.72 ±0.05 per cent phosphate  and 0.66 ±0.06 per cent  c a l c i u m , and was n u t r i t i o n a l l y adequate i n a l l r e s p e c t s .  19 Housing of the Rats Each r a t was housed i n an i n d i v i d u a l metabolism cage throughout the experiment.  The e x e r c i s e d and c o n t r o l r a t s  were housed i n s i m i l a r metabolism cages  (see Table I ) .  The  c o n f i n e d r a t s were housed i n metabolism cages which were s m a l l enough t o r e s t r i c t movement.  The dimensions of a l l  the metabolism cages are p r e s e n t e d i n Table I . A l l cages were designed t o minimize contamination of the f a e c e s and u r i n e by s p i l l e d food.  To f u r t h e r minimize  food s p i l l a g e the food was p r e s e n t e d t o the r a t s i n e i g h t ounce g l a s s j a r s . was  P l a c e d on top of the food i n these j a r s  a s t a i n l e s s s t e e l d i s c p e r f o r a t e d w i t h 12 1 cm diameter  h o l e s , through which the r a t s o b t a i n e d g r a n u l a r food. During the f i r s t run of the experiment, the l a r g e c y l i n d r i c a l metabolism cages were used t o house the exerc i s e d and c o n t r o l r a t s .  Each of these cages was  p l a c e d on  a c i r c u l a r sheet of open wire mesh which i n t u r n covered an aluminium tray.. . The wire s c r e e n r e t a i n e d the faeces w h i l e the aluminium t r a y c o l l e c t e d the u r i n e . For the second run of the experiment, new  and more  convenient s t a i n l e s s s t e e l cages were o b t a i n e d and these were used t o house the e x e r c i s e d and c o n t r o l r a t s . t u n a t e l y , the use of new  Unfor-  cages c o n s t i t u t e s a v a r i a b l e change  d u r i n g the experiment and may  be r e f l e c t e d by the l a r g e  b l o c k and i n t e r a c t i o n v a r i a n c e shown i n the r e s u l t s .  TABLE I S p e c i f i c a t i o n s of Metabolism Cages Used t o House Rats  CAGE  SHAPE  DIMENSIONS Cm  Large Metabolism*  Upright Cylinder  18 12  Diameter High  Acme Metabolism**  Rectangular prism  12 20 11  Small Metabolism  Rectangular prism  80 25 8  Restrictive Metabolism  Horizontal Cylinder  5.5 - 7.5 Diameter 25 Long  FLOOR AREA Cm2  DAYS HOUSED OR CONFINED BY GROUP EXERCISE CONFINED CONTROLS  254  38  38  Wide Long High  240  38  38  Wide Long High  200  26  *125  Used i n f i r s t experiment Used i n second experiment  -  12  (Block A) (Block B)  o  21 The new  s t a i n l e s s s t e e l metabolism cages manufactured  by ACME C o r p o r a t i o n are r e c t a n g u l a r prisms and have f e e d i n g tunnels which are s i m i l a r t o those on the c y l i n d r i c a l cages. These cages have e f f e c t i v e u r i n e and f a e c a l s e p a r a t o r s attached. The r a t s of the group which were r e s t r i c t e d i n a c t i v i t y d u r i n g t h i s experiment were housed i n s m a l l metabol i s m cages f o r the f i r s t  26 days of the experiment and were  housed i n s p e c i a l l y " d e s i g n e d " c o n f i n e m e n t cages f o r ' t h e 12 days of the experiment.  last  Since the s p e c i a l l y designed  confinement cages were h i g h l y r e s t r i c t i v e , the c o n f i n e d r a t s were not housed i n these cages f o r the e n t i r e experiment. Twelve days was  c o n s i d e r e d t o be a s u f f i c i e n t p e r i o d i n the  h i g h l y r e s t r i c t i v e confinement cages.  Vogt e t a l . (1965)  found changes i n human c a l c i u m balance a f t e r three days of bed  rest. The s m a l l metabolism cages used t o house the c o n f i n e d  r a t s d u r i n g the f i r s t  26 days of the experiment are con-  s t r u c t e d of s t a i n l e s s s t e e l and two of these cages are c o n s t r u c t e d as a u n i t . are  Funnels w i t h u r i n o - f a e c a l s e p a r a t o r s  attached under each cage u n i t .  A g l a s s j a r w i t h a per-  f o r a t e d s t a i n l e s s s t e e l d i s c food cover, s i m i l a r t o those d i s c s used i n the c y l i n d r i c a l metabolism cages, was wired i n t o each cage as a food c o n t a i n e r .  22 The  r e s t r i c t i v e confinement cages were s p e c i a l l y  designed by t h i s i n v e s t i g a t o r t o prevent, as much as p o s s i b l e , any undue movement by the r a t s .  I t was necessary  t h a t the  r a t s , w h i l e i n the cages would remain h e a l t h y and comfortable but were unable were unable breathe  t o move t o and f r o more than two inches and  t o r e v e r s e t h e i r body p o s i t i o n .  f r e e l y and c o u l d r o t a t e along t h e i r  They c o u l d longitudinal  axes.  F i g u r e I i n d i c a t e s the c o n s t r u c t i o n of t h i s type of  cage.  E s s e n t i a l l y the s p e c i a l l y designed  cages c o n s i s t o f  a r o l l e d c y l i n d e r of s t a i n l e s s s t e e l mesh. 25 cm long and has a diameter 5.5 cm t o 7.5 cm.  t h a t can be a d j u s t e d from  Adjustments i n the diameter  i n d e r are made by means of two expansive Attached  The c y l i n d e r i s  pipe  box.  clips.  t o the head end o f the t r a y i s a s t a i n l e s s  food box, 12 cm h i g h , 5 cm long and 5 cm wide. diameter  o f the c y l -  steel  A 4.5 cm  hole p r o v i d e s access of the r a t ' s head t o the feed  A removable food hopper i s attached t o the food box  by means o f a f l a n g e on the food box which s l i d e s i n t o a groove on the food hopper.  The food hopper c o n s i s t s of a  wedge shaped box which allows powdered food t o become a v a i l able as needed.  The back o f the food hopper c o n s i s t s o f a  sheet of s t a i n l e s s s t e e l bent t o an angle o f 140° t o p r o v i d e a p l a c e o f attachment f o r the water b o t t l e , the spout o f which i s a v a i l a b l e through  a hole c u t i n the s i d e o f the  c y l i n d e r near the head end.  The t a i l end o f the cage con-  23  Figure Confining  1.  cage d e s i g n e d t o movement by  minimize  the r a t .  24 s i s t s of a sheet of s t a i n l e s s s t e e l 11 cm high and 7 cm wide. A 2 cm diameter h o l e 3 cm above the base p r o v i d e s an o u t l e t f o r the t a i l . During use, each o f the confinement cages was mounted on an aluminium t r a y .  Under t h e . t a i l two t h i r d s o f the cage  a cupped sheet of p o l y e t h y l e n e served t o c a t c h the u r i n e . The faeces were trapped by a n y l o n f i n e mesh s c r e e n mounted s l i g h t l y above the p o l y e t h y l e n e sheet.  Method o f E x e r c i s e  :  S i x t e e n r a t s were e x e r c i s e d d u r i n g each experiment; f o u r r a t s from the e x e r c i s e treatment group, f o u r r a t s from the e x e r c i s e sub-group, s i x r a t s from the b l o o d donor group, and two spare r a t s .  Four Wahmann a c t i v i t y cages were modi-  f i e d t o f o r c e run the r a t s , s i m i l a r t o the m o d i f i c a t i o n s o f Tipton  (personal communication).  The f o u r cages were  mounted on a s i n g l e s h a f t which was  r e v o l v e d by a motor  coupled t o a Graham v a r i a b l e r e d u c t i o n gear box  (Fig. 2).  The Graham gear box allowed a c c u r a t e c o n t r o l o f the speed of the e x e r c i s e cage.  To f o r c e the r a t s t o run r a t h e r than  r i d e the r e v o l v i n g cage a padded b a f f l e was mounted i n s i d e the cage on the s t a t i o n a r y cage support.  To minimize the  trauma t o the r a t s f e e t caused by the e x e r c i s e , the i n s i d e of each e x e r c i s e cage was  l i n e d w i t h f i n e nylon mesh.  No  25  Figure  2.  Wahman a c t i v i t y cage m o d i f i e d f o r c e run  rats.  to  26 •torn or damaged f e e t were ever found on the r a t s used i n t h i s experiment; these symptoms have been found by p r e v i o u s workers  (Tipton,  1965).  Each o f the e x e r c i s e d r a t s was  run f i v e days a week  (Monday t o F r i d a y ) from the f i r s t day of the 12 day a c c l i m a t i z a t i o n p e r i o d t o the l a s t day of the experiment. For  the f i r s t  26 days o f the experiment, the i n t e n -  s i t y and d u r a t i o n of the e x e r c i s e was  increased.  During the  l a s t 12 days of the experiment the r a t s ran a t the r a t e of 0.5 m i l e s per hour f o r two h a l f - h o u r p e r i o d s .  The two  hour p e r i o d s were separated by a two hour r e s t p e r i o d . h a l f - h o u r e x e r c i s e schedule was first, the  halfA  chosen f o r two reasons:  t o p r o t e c t the f e e t of the r a t , and second, t o reduce  change of any r a t r e f u s i n g ' t o run because of f a t i g u e . T h i s e x e r c i s e regimen was  c a r r i e d out f o r a p e r i o d o f  38 days; the r a t s were e x e r c i s e d on 28 of the 38 days. A maximum running r a t e of 0.5 m i l e s per hour chosen because T i p t o n  (1965) r e p o r t e d t h a t a t speeds above  t h i s , r a t s develop f o o t t r o u b l e . study was moderate  was  S i n c e the purpose of t h i s  to i n v e s t i g a t e e f f e c t s on calcium metabolism o f e x e r c i s e , not strenuous t r a i n i n g , i t was  running a t 0.5 m i l e s per hour was  assumed t h a t  s u f f i c i e n t l y i n t e n s e exer-  c i s e t o demonstrate these p h y s i o l o g i c  effects.  27 Stool Collection T o t a l faeces and u r i n e were c o l l e c t e d twice weekly. Chemical c a l c i u m was determined and the r e s u l t s  presented  as a weekly u r i n e calcium and f a e c a l calcium e x c r e t i o n r a t e . In the f i r s t experiment the u r i n e and faeces were c o l l e c t e d from a l l the 42 r a t s , but i n the second run c o l l e c t i o n of the s t o o l from the donor r a t s was not deemed  necessary.  To prevent p r e c i p i t a t i o n o f c a l c i u m from the u r i n e w h i l e i t was being c o l l e c t e d , three drops of g l a c i a l a c i d were added t o each u r i n e sample as suggested Horner  acetic  by  (1955). The  covered  faeces e x c r e t e d by the e x e r c i s i n g r a t s was r e -  from the e x e r c i s e wheels a t the end of the e x e r c i s e  period.  Calcium A n a l y s i s A l l t i s s u e s were d r i e d and then ashed i n a m u f f l e furnace a t 600° C f o r 12 hours. Calcium  a n a l y s i s o f the u r i n e and faeces r e q u i r e d the  p r e l i m i n a r y p r e c i p i t a t i o n of the phosphate as d e s c r i b e d by Horner  (1955), f o l l o w e d by EDTA t i t r a t i o n of c a l c i u m as des-  c r i b e d by Copp The  (1963).  u r i n e was analyzed without  ashing.  28 Blood  Transfusions  The r a t s which r e c e i v e d blood  c o n s t i t u t e d h a l f of  the c o n t r o l group, h a l f of the c o n f i n e d group and h a l f of the e x e r c i s e d group. rats.  Each such sub-group c o n s i s t e d of f o u r  The e x e r c i s e d sub-group r e c e i v e d blood  from  donor r a t s , the c o n f i n e d sub-group r e c e i v e d blood  confined from exer-  c i s e d donor r a t s and the c o n t r o l sub-group r e c e i v e d from c o n t r o l donor  blood  rats.  Two m i l l i l i t e r s of whole blood  from a given  donor,  were i n j e c t e d i n t o the t a i l v e i n of each r e c i p i e n t r a t once a day f o r a p e r i o d o f f i v e days. s u f f i c i e n t blood  One donor r a t s u p p l i e d  t o i n j e c t one treatment group each day.  Thus three donor r a t s  (an e x e r c i s e d , a c o n f i n e d and a con-  t r o l ) , were s a c r i f i c e d as donors on each day of the f i v e day injection period. equipped with the blood  A 2 ml g l a s s t i p t u b e r c u l i n s y r i n g e  a h a l f i n c h 26 gauge needle was used t o i n j e c t  i n t o the t a i l v e i n of the r e c i p i e n t s .  p i e n t r a t s were l i g h t l y a n a e s t h e t i z e d  The  reci-  w i t h ether t o f a c i l i -  t a t e the i n j e c t i o n procedure. Exsanguination  of the donor r a t s was performed by  a n a e s t h e t i z i n g the r a t and drawing blood aorta with  a h e p a r i n i z e d s y r i n g e and a 20 gauge needle.  Once the blood was was  from the abdominal  e x t r a c t e d from the donor r a t , i t  t r a n s f e r r e d from the s y r i n g e t o a s i l i c o n e coated  test  29 tube kept a t 37° C.  The blood was immediately  to the f o u r r e s p e c t i v e r e c i p i e n t  :  The using  Radiocalcium  administered  rats.  Methods  a b s o r p t i o n and r e t e n t i o n of calcium was measured  calcium-45.  Calcium-45 S p e c i f i c a t i o n s The  calcium-45 stock used f o r t h i s experiment was  obtained from the New England Nuclear  Corporation,  Massachussets, U.S.A., d i s s o l v e d i n 0.1 ml o f 5N HC1. I t s s p e c i f i c a c t i v i t y was 15.6 m i l l i c u r r i e s / m g o f c a l c i u m and the r a d i o m e t r i c p u r i t y o f the i s o t o p e was 99 per cent. The dose c o n s i s t e d o f f i f t e e n m i c r o c u r r i e s o f c a l c i u m 45 c o n t a i n i n g 10 micrograms o f calcium i n 0.25 ml o f water. Each dose was administered  by stomach tube i n t o the conscious  rats. Radioisotope  Assay  Six hours a f t e r the a d m i n i s t r a t i o n o f the o r a l dose of calcium-45,  each r a t was s a c r i f i c e d with 0.3 ml s o l u t i o n  of 6gm/100ml sodium p e n t o b a r b i t a l d i l u t e d i n e t h a n o l .  A  blood sample, the h e a r t , the r i g h t femur, and the i n t e s t i n a l tract  (excluding the oesophagus) were removed from each r a t  and p l a c e d i n separate Pyrex beakers.  The i n t e s t i n a l  tract,  30 the femur, and the c a r c a s s of each r a t were assayed s e p a r a t e l y for  calcium-45. Sample P r e p a r a t i o n : The  i n t e s t i n a l t r a c t , the femur and the c a r c a s s of  each r a t were ashed s e p a r a t e l y i n a m u f f l e furnace a t 600° C for of  twelve hours. 3N HC1  The c a r c a s s ash was  d i s s o l v e d i n 20  and d i l u t e d w i t h 20 ml of 0.01N  t r a c t ash was  d i s s o l v e d i n 5 ml of 3N HC1  5 ml of 0.01N  HC1;  3N HC1  the femur ash was  the  and d i l u t e d w i t h  HC1.  d i l u t e d w i t h an equal volume of 0.01N  Each sample  p i p e t t e d from each of  cm x 4.3  cm f i l t e r paper  which had been p l e a t e d by making f o u r f o l d s 1.5  cm apart  A f t e r d r y i n g , the f i l t e r papers were  i n v e r t e d and p l a c e d i n t o 20 ml g l a s s counting v i a l s . t i l l a t i o n solution to  was  HC1.  A volume of 100 m i c r o l i t e r s was  along t h e i r long a x i s .  intestinal  d i s s o l v e d i n 3 ml of  and d i l u t e d w i t h 3 ml of 0.01N  the above samples and p l a c e d on 7.5  HC1;  ml  (10 ml) was 1  added t o each v i a l j u s t  Scinprior  counting. S c i n t i l l a t i o n Solution: The mixture used i n the c o u n t i n g v i a l s i s d e s c r i b e d  by Humphreys PPO  (1965).  I t was  (2,5-diphenyloxazole)  prepared by adding 4 grams of  and 0.2  grams of POPOP  ( l , 4 - D i [ 2 - ( 5 p h e n y l o z o l y l ) ] b e n z e n e ) t o one The  s o l u t i o n was  cupboard.  l i t e r , of. t o l u e n e .  s t o r e d i n an amber j a r and kept i n a dark  31 Liquid S c i n t i l l a t i o n  Counting  A P i c k e r Nuclear Liquimat 3., y l i q u i d was  scintillator  used t o measure r a d i o a c t i v i t y i n the samples.  f o r quenching, a l l sample c o u n t s - r a t e s (CPM)  To c o r r e c t  were converted  to a b s o l u t e counts or d i s i n t e g r a t i o n s per minute  (DPM)  means of a channels r a t i o quench c a l i b r a t i o n curve. quench c a l i b r a t i o n curve set  The  prepared by c o u n t i n g a  of s t a n d a r d s , a f t e r adding a known amount of quenching  agent ,, the  ( F i g . 3) was  by  ( n o n r a d i o a c t i v e a c i d d i s s o l v e d c a r c a s s a s h ) , and p l o t t i n g  , . .• channels r a t i o  , CPM channel B . , ,, . . ( channel A a g a i n s t the c o u n t i n g C  p  M  efficiency. The c o u n t i n g e f f i c i e n c y of unquenched standards of calcium-45 u s i n g the method d e s c r i b e d above was cent.  85 ±1.31 per  T h i s e f f i c i e n c y i s comparable t o t h a t r e p o r t e d by  H a r d c a s t l e e t a l . (1966) and c o n s i d e r a b l y b e t t e r than the e f f i c i e n c y r e p o r t e d f o r calcium-45 by Lutwak (1959), C a r r and Parsons  (1962), Sarnat and J e f f a y To adapt the l i q u i d  i t was  (1965).  s c i n t i l l a t o r t o count calcium-45,  f i r s t necessary t o analyze the 3 s p e c t r a l curve f o r  calcium-45 for  (1962), and Humphreys  ( F i g . 4 ) , then determine the d i s c r i m i n a t o r  settings  the two spectrometer windows (channels) f o r use when  c o u n t i n g calcium-45 by the.channels r a t i o method. allows quench c o r r e c t i o n s t o be made.  The s e t t i n g s  T h i s method chosen  were 100 d i v i s i o n s and 800 d i v i s i o n s * on the lower and  *  arbitrary  units  upper  Quench  Calibration Curve  % Counting Etticiency 90  80  60  —i  1  0.1  1  1  0.2  1  0.3  Channels  Ratio  1  1  0.4  B/A  F i g u r e 3. Quench c a l i b r a t i o n curve.  Plotted points  r e p r e s e n t the e f f i c i e n c y of c o u n t i n g a known standard w i t h known q u a n t i t i e s o f a quenching  agent  added.  Spectral Curve Calcium - 45 Radio Activity Counts/Min. 12 K 10 K  -  8 K -  6 K-  4 K  2 K  -  8 0 0  Discriminator Divisions (Arbitrary Units)  Figure Calcium-45  4.  s p e c t r a l curve showing  spectrometer window s e t t i n g s  f o r counting  calcium-45 u s i n g the channels r a t i o method of quench  correction.  34 d i s c r i m i n a t o r s of channel A and 575 d i v i s i o n s and 800 d i v i s i o n s of the lower, and upper d i s c r i m i n a t o r s e t t i n g s on channel B ( F i g . 4 ) .  Summary The methods of grouping, f e e d i n g , housing and exerc i s i n g of the r a t s have been d e s c r i b e d described  i n this section.  Also  are the methods i n v o l v e d i n the sample c o l l e c t i o n ,  the c a l c i u m  a n a l y s i s , the blood  radiocalcium.  t r a n s f u s i o n s and the use of  ANALYSIS OF DATA  The data were analyzed by computing  the F r a t i o s f o r  treatment v a r i a n c e , the b l o c k v a r i a n c e and the i n t e r a c t i o n v a r i a n c e , u t i l i z i n g a randomized analysis of variance.  block design  The computational model f o r t h i s  a n a l y s i s i s g i v e n i n Table I I . performed  complete  A l l the computations  were  on an Olivetti-Underwood Programma 101 desk top  computer. I f the i n t e r a c t i o n F r a t i o was not s i g n i f i c a n t (p > 0.05) then a comparison between treatment means was made u s i n g Duncan's new m u l t i p l e range t e s t 1960,  ( S t e e l and T o r r i e ,  p. 107). I f the i n t e r a c t i o n F r a t i o was s i g n i f i c a n t  (p < 0.05) the treatment means c o u l d not be compared u s i n g Duncan's new m u l t i p l e range  test.  The treatment means o f those parameters which were measured more than once d u r i n g . t h e experiment were graphed t o assess the changes i n each parameter  from week t o week.  treatment means f o r these data c o u l d be p l o t t e d  The  o n l y when  the i n t e r a c t i o n was not s i g n i f i c a n t . Definitions A s i g n i f i c a n t F r a t i o f o r the treatment v a r i a n c e means t h a t the e f f e c t s o f the treatments are not the same. I t does n o t s p e c i f y which treatments d i f f e r s i g n i f i c a n t l y nor  TABLE II Computations of Sums of Squares and  F Ratios as well as the Variance  estimated by the Mean Square  for a Randomized Complete Block Design.  •  1  SOURCE TREATMENT BLOCKS  t-i  bs.-  t s  INTERACTION (t-l)(b-l) ERROR TOTAL  MS TREATS MS INTERACTION  bst  Y?..  b-1  u  s  bs  rri  bst  ij  ij  e  .  ts£\  e  TB  s... 'J  I*  tbs-1  Yj.. represents it II  Y  j i  block total treatment j ' block total (cell total)  , h  th  t h  k  it  s  II it  TB  treatment total  t n  grand total  t  2  bst  II  b  2  2  it  tl  tsc7\ B sc7\  Y. ..  i  tn  value in i  , h  treatment and j  t h  number of blocks »/ " treatments "  "  replications in each cell  error variance  It  block variance  It  interaction variance  2  B  + sr?v.  C\  bst  tSy  tb(s-l)  F RATIO  MEAN SQUARE (MS) ESTIMATES  SUMS OF SQUARES (SS) COMPUTATIONS  df  block  MS BLOCKS ERROR  MS  MS INTERACTION MS ERROR  37 does i t g i v e the d i r e c t i o n these can be i n f e r r e d  o f the d i f f e r e n c e .  either  However,  by i n s p e c t i o n o f the treatment  means o r by comparing them u s i n g a t e s t such as Duncan's new m u l t i p l e range t e s t .  Thus i n t h i s study a s i g n i f i c a n t  treatment F r a t i o means t h a t the e f f e c t s the  o f a t l e a s t two o f  s i x treatments are not the same. Duncan's new m u l t i p l e range t e s t may be used even  when the treatment F ' r a t i o i s not s i g n i f i c a n t because i t sometimes r e v e a l s a s i g n i f i c a n t d i f f e r e n c e between any two of a group treatments even though the treatment F r a t i o i s not  significant. A s i g n i f i c a n t F r a t i o f o r the b l o c k v a r i a n c e means  t h a t the e f f e c t o f the b l o c k s i s not the same.  Since i n  t h i s study a b l o c k c o n s t i t u t e s a i l the data obtained d u r i n g each experiment a s i g n i f i c a n t b l o c k F r a t i o means t h a t the data o b t a i n e d from the f i r s t experiment i s not the same as the  data o b t a i n e d from the second experiment.  The b l o c k  v a r i a n c e i s an estimate o f the e r r o r t h a t would have o c c u r r e d i f t h i s method o f a n a l y s i s had not been used. A significant interaction  F r a t i o means t h a t the  e f f e c t o f the b l o c k s i s not the same f o r each treatment. Thus the treatments and b l o c k s i n t e r a c t . action invalidates  Significant  inter-  the treatment F r a t i o and makes comparison  between the treatment means meaningless. study s i g n i f i c a n t i n t e r a c t i o n  Therefore i n t h i s  i m p l i e s t h a t the r e p e t i t i o n o f  the experiment caused a t l e a s t one of the treatments, t o d i f f e r i n a manner d i s i m i l a r t o the other treatments.  RESULTS  A n a l y s i s of the body weight data showed t h a t c o n f i n e ment d i d not s i g n i f i c a n t l y a l t e r body weight u n t i l the r a t s were p l a c e d i n t o the r e s t r i c t i v e metabolism cages III - VIII).  (Tables  F i g u r e 5 shows t h a t the e x e r c i s e d r a t s a p p a r e n t l y  grew slower than the c o n t r o l r a t s although only on day  26  d i d the e x e r c i s e d r a t s weigh s i g n i f i c a n t l y l e s s than the c o n t r o l r a t s (Table V I ) . The  e x e r c i s e or confinement used i n t h i s experiment  d i d not s i g n i f i c a n t l y a l t e r the calcium metabolism of r a t s as measured by the s p e c i f i c parameters of c a l c i u m balance (Tables IX - X I I ) , per cent calcium u t i l i z a t i o n XVI), XXI  food i n t a k e  (Tables XVII - XX),  - XXIV), u r i n a r y calcium  calcium  absorption femur  (Table XXVIII) and  (Table XXIX).  f i n d i n g can be found.  total  (Tables  carcass  (Table XXVII), calcium-45  calcium-45 d e p o s i t i o n i n the  Four exceptions The  f a e c a l calcium  (Table XXV),  (Table XXVI), femur calcium  (Tables X I I I -  to the above g e n e r a l  confined rats u t i l i z e d a  signifi-  c a n t l y g r e a t e r per cent of t h e i r d i e t a r y c a l c i u m than d i d the e x e r c i s e d and  c o n t r o l r a t s d u r i n g days 19 - 26  XIV), and the c o n f i n e d r a t s e x c r e t e d s i g n i f i c a n t l y  (Table less  f a e c a l calcium than the e x e r c i s e d or c o n t r o l r a t s d u r i n g same p e r i o d  (Table X X I I ) .  The  exercised rats u t i l i z e d  a  the  Growth Curve  Time in Days  F i g u r e 5. Body weight growth curves f o r the e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s .  41  TABLE I I I A n a l y s i s o f V a r i a n c e - Body Weight Source  df  (g)  Day 0  Sums o f Squares  Mean Square  Treatments  5  125.854  25.171  Blocks  1  172.521  172.521  Interaction  5  144.854  28.971  Error  35  Total  46  1281.25  *  0.869 4.814* 0.814  35.59  S i g n i f i c a n t , p < 0.05  TABLE I I I A Ranked Treatment Means - Body Weight  (g)  Day 0  C  XB  R  CB  RB  X  124.9  122.9  122  121  120.5  120.2  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  CB  -  c o n t r o l blood  X  -  e x e r c i s e d , XB  -  e x e r c i s e d blood  R  -  confined,  -  c o n f i n e d blood  RB  recipients, recipients, recipients.  42 TABLE IV A n a l y s i s o f V a r i a n c e - Body Weight df  Source  (g) Day 12  Sums o f Squares  Mean Square  Treatment  5  814.42  162.884  1.206  Blocks  1  768.003  768.003  6.149**  Interaction  5  675.497  135.009  1.082  Error  36  4496.0  Total  47  6753.92 **  124.888  S i g n i f i c a n t , p < 0.025  TABLE IVA Ranked Treatment Means - Body Weight CB 218.9  RB  C  217.3  216.4  (g) Day 12 R .  215.9  XB 214.3  X 206.1  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  CB  -  c o n t r o l blood  recipients,  X  -  exercised,  XB  -  e x e r c i s e d blood  R  -  confined,  RB  -  c o n f i n e d blood  recipients, recipients.  43, TABLE V A n a l y s i s of V a r i a n c e - Body Weight Source  df  (g) Day 19  Sums of Squares  Mean Square  Treatments  2  362.64  Blocks  1  Interaction  2  315.37  157.6 8  Error  42  9256.26  220.38  Total  47  16371.92  725.29 6075.  ***,  F 1.64  6075.  27.56*** 0.71  H i g h l y s i g n i f i c a n t , p < 0.01  TABLE VA Ranked Treatment Means - Body Weight  (g) Day 19  C 255.3  R  X  251.7  245.9  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  44 TABLE VI A n a l y s i s of V a r i a n c e - Body Weight df  Source  (g) Day 26  Sums of Squares  Treatments  2  1845.88  922.24  Blocks  1  5720.33  5720.33  Interaction  2  775.79  387.90  Error  42  17334.  Total  47  25676. *.*•*  F  Mean Square  2.236 13.86*** 0.94  412.71  H i g h l y s i g n i f i c a n t , p < 0.01  TABLE VIA Ranked Treatment Means - Body Weight  (g) Day 26  C  R  287.1  X  275.6  272.8  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  45  TABLE V I I A n a l y s i s o f V a r i a n c e - Body Body Weight  Source  df  (g) Day 33  Sums o f Squares  Mean Square  F  Treatments  2  9398.29  4699.14  6.36  Blocks  1  7252.0 8  7552.08  9.82***  Interaction  2  209.28  104.64  Error  42  30998.02  738.04  Total  47  47857.67 .***.  0.14  H i g h l y s i g n i f i c a n t , p < 0.01  TABLE VIIA Ranked Treatment Means - Body Weight  (g.) Day 33  C  X  R  310.9  2 89.8  277  Any two means not u n d e r l i n e by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  46  TABLE V I I I A n a l y s i s of V a r i a n c e - Body Weight Source  df  (g) Day 38  Sums o f Squares  Mean Square  Treatments  5  16374.85  3274.97  Blocks  1  9718.52  9718.52  Interaction  5  1494.86  298.97  Error  36  26920.25  747.78  Total  47  54508.48 **•*•  F 4.37 12.99*** 0.39  H i g h l y s i g n i f i c a n t , p < 0.01.  TABLE VI11A Ranked Treatment Means - Body  C  Weight  (g) Day 38  X  XB  CB  313.8  311.3  305.5  281.4  RB  R  273  269.1  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's m u l t i p l e range t e s t . C  -  control,  CB  -  c o n t r o l blood  X  -  exercised,  XB  -  e x e r c i s e d blood  R  -  confined,  RB  c o n f i n e d blood  recipients, recipients, recipients.  47  TABLE IX A n a l y s i s o f V a r i a n c e - Calcium Balance Data Source  (Mg) Days 12-19  Sums o f Squares  df  Mean Square  F  Treatments  2  22442.17  11221.08  2.6  Blocks  1  16875  16875.00  3.91  Interaction  2  21348.48  10674.24  2.47  Error  42  180891.02  4306.92  Total  47  241556.67  TABLE IXA Ranked Treatment Means - Calcium Balance  (Mg) Days 12-19  C  X  R  320.3  327.6  283.1  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  -  exercised.  48  TABLE X A n a l y s i s o f V a r i a n c e - Calcium Balance df  Source  (Mg)  Days 19 - 26  Sums o f Squares  Mean Square  F  Treatments  2  36141.79  18070.89  2.0  Blocks  1  2508.52  2508.52  0.27  Interaction  2  2639.29  1319.64  0.14  Error  42  379194.88  9028.44  Total  47  420484.48  TABLE XA Ranked Treatment Balance  (Mg)  Means - Calcium  Days 19 - 26  R  C  X  340.9  292.6  276.3  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p. < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  49  TABLE XI Analysis of Variance Balance df  Source  (Mg)  - Calcium  Days 26 - 33  Sums o f Squares  Mean Squares 1981.58  Treatments  2  3963.17  Blocks  1  Interaction  2  24737.9 8  12368.99  Error  42  309748.52  7374.96  Total  47 .*.*•*  0.26  299568.  299568.  F  40.61*** 1.67  H i g h l y s i g n i f i c a n t , p < 0.01  TABLE XIA Ranked Treatment Means - Calcium Balance  (Mg)  Days 26 - 33  R  X  C  230.4  218.8  208.1  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  TABLE XII A n a l y s i s o f V a r i a n c e - Calcium Balance Source  df  (Mg)  Days 33 - 38  Sums o f Squares  Mean Square  F 0 .734  Treatments  5  80466.765  16093.353  Blocks  1  41139.720  41139.720  12 73***  Interaction  5  109562.971  21912.594  6 . 78***  Error  35  113110.417  3231.726  Total  46  344279.873  *.**  H i g h l y s i g n i f i c a n t , p < 0.01  51  TABLE X I I I A n a l y s i s o f V a r i a n c e o f Per Cent Calcium df  Source  Utilization  Sums o f Squares  Days 12-19  Treatments  2  75.59  37.798  Blocks  1  2676.65  2676.651  Interaction  2  55.41  27.703  Error  42  854.66  20.349  Total  47  3662.32 .*.**.  F  Mean Square  Highly s i g n i f i c a n t ,  1. 86 131. 54*** 1.36  p < 0.01  TABLE XIIIA Ranked Treatment Means - Per Cent Calcium  Utilization  X  Days 12-19 R  C 28.7  29.8  26. 7  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  -  exercised.  52  TABLE XIV A n a l y s i s o f V a r i a n c e - Per Cent Calcium U t i l i z a t i o n Source  df  Days 19-26  Sums o f Squares  Mean Square  Treatments  2  292.809  146.404  Blocks  1  1443.213  1443.213  Interaction  2  2.008  1.00.4  Error  42  2434.443  57.963  Total  47  4172.423 .*.**• H i g h l y s i g n i f i c a n t ,  F 2.53 24.90*** 0.02  p < 0.01  TABLE XIVA Ranked Treatment Means - Per Cent Calcium U t i l i z a t i o n R  Days 19-26  C  30.9  X  25.9  25.5  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t C  -  control,  R  -  confined,  X  -  exercised.  53  TABLE XV A n a l y s i s of V a r i a n c e - Per Cent Calcium U t i l i z a t i o n df  Source  Days 26-33  Sums o f Squares  Mean Square  Treatments  2  307.726  153.863  Blocks  1  5203.335  5203.335  Interaction  2  86.554  43.277  Error  42  4334.726  103.208  Total  47  9932.341 *.**•  Highly s i g n i f i c a n t ,  F 1.49 50'. 42*** 0.42  p < 0.01  TABLE XVA Ranked Treatment Means - Per Cent Calcium U t i l i z a t i o n R  Days 26-33  X  27.9  C  23.5  22.0  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  54  TABLE XVI A n a l y s i s of V a r i a n c e - Per Cent Calcium U t i l i z a t i o n Source  Sums o f Squares  df  Days 33-38 Mean Square  Treatment  5  4847.614  Blocks  1  1775.  Interaction  5  1630.815  826.63 115.08  Error  35  4027.92  Total  46  12281.049 *  .*•**  969.523 1775.  S i g n i f i c a n t , p. < 0.05 H i g h l y s i g n i f i c a n t , p < 0.01  F 2.97 15.42*** 2.83*  55  TABLE XVII A n a l y s i s o f V a r i a n c e - Food Intake Source  df  (g)  Days 12-19  Sums o f Squares  Mean Square  Treatments  2  662.79  331.39  Blocks  1  2929.68  2929.68  Interaction  2  1267.13  633.56  Error  42  6240.88  148.59  Total  47  11100.48  .**  S i g n i f i c a n t , p < 0.025  .*.**• H i g h l y s i g n i f i c a n t , p < 0.01  F 2.23 19.71*** 4.26**  56  T A B L E A n a l y s i so f  V a r i a n c e- F o o d  I n t a k e ( g )  d f  S o u r c e  S u m s  X V I I I  o f  D a y s 19-26  S q u a r e s  M e a n  S q u a r e  T r e a t m e n t s  3  620.66  310.33  B l o c k s  1  2227.68  2227.68  I n t e r a c t i o n  2  1003.5  501.75 262.49  E r r o r  42  11024.64  T o t a l  47  14876.48 .*.**  Highly  significant,  T A B L E  F 1.18 8.48*** 1.91  p < 0.01  X V I I I A  R a n k e d T r e a t m e n t M e a n s I n t a k e (g)  - F o o d  D a y s 19-26  C  X  R  140.7  138.4  132.2  A n y t w o m e a n s n o t u n d e r l i n e d b y t h e s a m e l i n e d i f f e r s i g n i f i c a n t l y ( p < 0.05) a c c o r d i n g t o D u n c a n ' s n e w m u l t i p l e r a n g e t e s t . C  -  control,  R  -  confined,  X  e x e r c i s e d .  57  TABLE XIX A n a l y s i s of V a r i a n c e - Food Intake Source  df  (g)  Days 26-33  Sums o f Squares  Mean Square  Treatments  2  12038.37  Blocks  1  363.  363.  1.3  Interaction  2  1429.61  714.8  2.56  Error  42  11712.02  Total  47  25543. *  6019.18  21.58*  278.19  S i g n i f i c a n t , p < 0.05  TABLE XIXA Ranked Treatment Means - Food Intake  (g)  Days 26-33  C  X  R  147.1  139.3  110.3  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  58  T A B L E A n a l y s i so f  V a r i a n c e- F o o d  I n t a k e (g)  S o u r c e  S u m s  d f  o f  X X  D a y s 33-38  S q u a r e s  M e a n  F  S q u a r e  T r e a t m e n t s  5  1399.722  279.944  4.096  B l o c k s  1  130.355  130.355  0.77  I n t e r a c t i o n  5  341.693  68.339  E r r o r  35  5923.167  169.233  T o t a l  46  7794.937  T A B L E  X X A  R a n k e d T r e a t m e n t M e a n s I n t a k e C B  C  80.9  79.0  (g)  X  - F o o d  D a y s 3 3-38 R  75.6  0.404  B  74.8  R 72.4  X  B 64.1.  A n y t w o m e a n s n o t u n d e r l i n e d b y t h e s a m e l i n e d i f f e r s i g n i f i c a n t l y ( p < 0.05) a c c o r d i n g t o D u n c a n ' s n e w m u l t i p l e r a n g e t e s t . C  -  c o n t r o l ,  C B  R  -  c o n f i n e d ,  R  X  -  e x e r c i s e d ,  X B  B  c o n t r o l b l o o d r e c i p i e n t s , -  -  c o n f i n e d b l o o dr e c i p i e n t s , e x e r c i s e db l o o d r e c i p i e n t s .  59  TABLE XXI A n a l y s i s of V a r i a n c e - F a e c a l Calcium Source  df  (Mg)  Days 12-19  Sums o f Squares  Mean Square  Treatments  2  123706.63  61853.31  Blocks  1  2624077.69  2624077.69  Interaction  2  129927.12  64963.56  Error  42  279081.38  6644.79  Total  47  3156792.82 ***  H i g h l y s i g n i f i c a n t , p < 0.01  F 9.3 394.9*** 9.77***  60  TABLE XXII Analysis of Variance - Faecal Calcium Source  (Mg)  Days 19-26  Sums o f Squares  df  Mean Square 73517.52  147035.4  Treatments  2  Blocks  1  2881200.  Interaction  2  24071.  12035.68 11733.27  Error  42  492797.52  Total  47  3545103.92 .*.**  F 6.26 245.55***  2881200.  1.02  Highly s i g n i f i c a n t .  TABLE XXIIA Ranked Treatment Means Calcium  (Mg)  C 891  Faecal  Days 19-26  X  R  875.7  766.7  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g to Duncan's new m u l t i p l e range t e s t . C  -  control,  R  -  confined,  X  exercised.  61  T A B L E A n a l y s i s o f  X X I I I  V a r i a n c e -F a e c a l  C a l c i u m(Mg)  S o u r c e  d  fS u m s  o f  D a y s 26-33  S q u a r e s  M e a n  S q u a r e  T r e a t m e n t s  2  92995..88  46497..94  B l o c k s  1  1805364..19  1805364..19  I n t e r a c t i o n  2  15444..87  7722..43  E r r o r  42  901399..88  T o t a l  47  2815204..82  .*.**  Highly  2,.16 84. 0..35  21461..9  significant,  T A B L E  F  p < 0.01  X X I I I A  R a n k e d T r e a t m e n t M e a n s C a l c i u m(Mg)  -F a e c a l  D a y s 26-33  C  X  R  810.8  747.6  703.5  A n y t w o m e a n s n o t u n d e r l i n e d b y t h e s a m e l i n e d i f f e r s i g n i f i c a n t l y ( p < 0.05) a c c o r d i n g t o D u n c a n ' s n e w m u l t i p l e r a n g e t e s t .  C  -  control,  R  -  confined,  X  e x e r c i s e d .  62  TABLE XXIV A n a l y s i s of V a r i a n c e - F a e c a l Calcium  (Mg)  Days 33-38  df  Sums o f Squares  Treatments  5  44876.726  8975.345  Block  1  797884.033  797884.033  Interaction  5  28058.907  5611.781  Error  35  118737.821  3392.509  Total  46  989557.487  Source  F  Mean Square  1.59 235.189*** 1.654  .*.**• H i g h l y s i g n i f i c a n t , p. < 0.01  TABLE XXIVA Ranked Treatment Means - F a e c a l Calcium CB  RB  521.7  481.3  (Mg)  C  Days 33-3 8 X  470.6  454.6  R  XB  451.6  421.1  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  CB  -  c o n t r o l blood  recipients,  X  -  exercised,  XB  -  e x e r c i s e d blood  R  -  confined,  RB  -  c o n f i n e d blood  recipeints, recipients.  63  TABLE XXV A n a l y s i s o f V a r i a n c e - Urine Calcium Source Treatments  df  (Mg)  Days 33-38  Sums o f Squares 11.5258  5  Mean Squares 2.3051  F 0.29  *  49.3976  26.64***  39.507  7.9014  4. 26***  1.854  Blocks  1  49.. 3976  Interaction  5  Error  35  63.0392  Total  46  163.4683 *.**•  H i g h l y s i g n i f i c a n t , p < 0.01  64  TABLE XXVI A n a l y s i s of V a r i a n c e - T o t a l Calcium  Source  df  Carcass  (Mg)  Sums of Squares  Mean Square 19367..782  Treatments  5  96838.,912  Blocks  1  3593493,.547  Interaction  5  48766..547  9757..309  Error  35  116452,.549  3327,.215  Total  46  3855571..555 * ***  F 1..98  3593493,.547 1080..03***  S i g n i f i c a n t , p < 0.05 H i g h l y s i g n i f i c a n t , p < 0.01  2..93*  65  TABLE XXVII A n a l y s i s o f V a r i a n c e - Femur Calcium Source  df  (Mg)  Sums of Squares  Mean Square  Treatment  5  601.611  120.322  Blocks  1  1369.242  1369.242  Interaction  5  874.139  174.827  Error  35  2640.93  Total  46  5485.922 *  75.45  S i g n i f i c a n t , p < 0.05  .*.**. H i g h l y s i g n i f i c a n t , p < 0.01  F 0.688 18.146**.* 2.316*  TABLE XXVIII A n a l y s i s o f V a r i a n c e - Calcium-45 Absorption Source  df  (% Administered  Sums o f Squares  Dose)  Mean Square  F  Treatments  5  447.654  89.531  0. 369  Blocks  1  202.824  202.824  1.216  Interaction  5  1211.953  242.391  1. 453  Error  35  5938.255  166.807  Total  46  7800.686  TABLE XXVIIIA Ranked Treatment Means - Calcium-45 . Absorption C  RB  52.8  48.6  R 46.6  (% Administered  Dose)  XB  CB  45.1  42.9  X 42.2  Any two means not u n d e r l i n e d by the same l i n e d i f f e r s i g n i f i c a n t l y (p < 0.05) a c c o r d i n g t o Duncan's new m u l t i p l e range t e s t . C  -  control,  CB  -  c o n t r o l blood  X  -  e x e r c i s e d , XB  -  e x e r c i s e d blood  R  -  confined,  -  c o n f i n e d blood  RB  recipients, recipients, recipients.  67  TABLE XXIX A n a l y s i s o f V a r i a n c e - Femur Calcium-45 Source  df  (% Administered Dose)  Sums o f Squares  Mean Square  F  Treatments  5  1.1425  0.2285  0.267  Blocks  1  0.0102  0.0102  0.302  Interaction  5  4.2749  0.8549  2.532*  Error  35  11.8171  0.3376  Total  46  17.2447  *  S i g n i f i c a n t , p < 0.05  68  T A B L E A n a l y s i s o f H e a r t  S o u r c e  d  f S u m s  X X X  V a r i a n c e - R e l a t i v e  W e i g h t  o f  ( M g / g )  S q u a r e s  M e a n  S q u a r e  T r e a t m e n t s  2  0.72  0 .144  B l o c k s  1  2.7509  2 .7509  I n t e r a c t i o n  2  1.2759  0 .2551  E r r o r  42  6.6395  0 .1952  T o t a l  47 * * *  H i g h l y  s i g n i f i c a n t , p  T A B L E R a n k e d  T r e a t m e n t H e a r t  X B  2.986  2.982  M e a n s  2.903  A n y t w o m e a n s n o t u n d e r l i n e d s i g n i f i c a n t l y ( p < 0.05) m u l t i p l e r a n g e t e s t .  14. .093** 1..307  < 0.01  -  R el a t i v e  ( M g / g ) R B  2.956  0..564  X X X A  W e i g h t  C B  X  F  C  2.875  R  2.622  b y t h e s a m e l i n e d i f f e r a c c o r d i n g t o D u n c a n ' s n e w  C  -  c o n t r o l ,  C  B-  c o n t r o l b l o o d r e c i p i e n t s ,  X  -  e x e r c i s e d , X  B-  e x e r c i s e db l o o d r e c i p i e n t s ,  R  -  c o n f i n e d ,  B-  c o n f i n e d b l o o dr e c i p i e n t s .  R  69 s i g n i f i c a n t l y g r e a t e r percentage of t h e i r d i e t a r y c a l c i u m than d i d the c o n f i n e d r a t s d u r i n g days 12 - 19 The c o n f i n e d r a t s ate s i g n i f i c a n t l y  (Table X I I I ) .  l e s s food than the  e x e r c i s e d or c o n t r o l r a t s d u r i n g days 26 - 33  (Table XIX).  The b l o o d t r a n s f u s i o n s used i n t h i s experiment  failed  t o s i g n i f i c a n t l y a l t e r any of the parameters measured on the e x e r c i s e d , c o n t r o l or c o n f i n e d r a t s (Tables V I I I , X I I , XVI, XX, XXIV, XXV,  XXVI, XXVII, XXVIII, XXIX, and  XXX).  Calcium balance over the l a s t 26 days of the e x p e r i ment i s graphed i n F i g u r e 6.  T h i s f i g u r e suggests t h a t  d i e t a r y c a l c i u m r e t a i n e d by a l l r a t s d e c l i n e d d u r i n g days 19 - 38.  However, the graph of the per cent calcium  z a t i o n over the l a s t 26 days of the experiment  utili-  ( F i g . 7) does  not show an obvious decrease i n the per cent d i e t a r y calcium u t i l i z e d d u r i n g days 19 - 38. The graphs of food i n t a k e and f a e c a l c a l c i u m over the l a s t 26 days of the experiment  ( F i g s . 8 and 9) show a n o t i c e -  able decrease i n the food i n t a k e and f a e c a l calcium  excreted  d u r i n g the l a s t f i v e days of the experiment. The e x e r c i s e or confinement used i n t h i s study d i d not s i g n i f i c a n t l y a l t e r the r e l a t i v e h e a r t weights of the rats  (Table XXX). A s i g n i f i c a n t i n t e r a c t i o n F r a t i o was  found t o occur  i n seven of the 28 measurements made d u r i n g t h i s experiment.  70  Weekly  Calcium  Balance  Mg  ^  350T  Control Exercised Confined  J  Mean standard error  3005  250  c o  u o  <D c  200  J  flllllft  Days 12-19  n  Days 19-26  Days 26-33  Days 33-38  E i g u r e 6. Mean weekly c a l c i u m balance o f the e x e r c i s e d , c o n f i n e d and c o n t r o l  rats.  Significant interaction F ratio for  c a l c i u m balance data  (days 33-38) prevented the values  f o r days 33-38 being  plotted.  71  Weekly percent Calcium  Utilization.  % 35  \yyy\ Control  -t  ||[ 111 Exercised fcs$$| Confined J  Mean standard error  30-  25-  c o o o  w  +0c  20  ZZZA  Days 12-19  Days 19-26  Days 26-33  L  J  Days 33-38  F i g u r e 7. Mean weekly per cent c a l c i u m u t i l i z a t i o n o f the e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . S i g n i f i c a n t i n t e r a c t i o n F r a t i o f o r p e r cent c a l c i u m u t i l i z a t i o n data  (days 33-38) prevented  the v a l u e s f o r days 33-38 being p l o t t e d .  Weekly  Food  Intake  Grams  s  Days 12-19  Days 19-26  Days 26-33  Days 33-38  F i g u r e 8. Mean weekly food i n t a k e o f the e x e r c i s e d , c o n f i n e d control rats. i n t a k e data  S i g n i f i c a n t i n t e r a c t i o n f o r food  (days 12-19) prevented  days 12-19 being  the values f o r  plotted.  * Values f o r days 33-38 e x t r a p o l a t e d t o g i v e i n t a k e f o r seven days.  food  73  Weekly  Faecal  Calcium  Mg  Y//X Control  9 0 0 ~t  HI HI Exercised  fcs$$) Confined J  Mean standard error  800 -  o o 700-  CD C m  600  MA  J  Days 12-19  Days 19-26  Days 26-33  Days 33-38  F i g u r e 9. Mean weekly f a e c a l calcium o f the e x e r c i s e d , c o n f i n e d and c o n t r o l r a t s . calcium data  Significant interaction for faecal  (days 12-19) prevented  the values f o r  days 12-19 being p l o t t e d . *  Values f o r days 33-38 e x t r a p o l a t e d t o g i v e f a e c a l c a l c i u m f o r seven days.  o  74 They were:  c a l c i u m b a l a n c e , days 33-38 (Table X I I ) ; per cent  c a l c i u m u t i l i z a t i o n , days 33-38 (Table XVI); food i n t a k e , days 12-19  (Table XVII); f a e c a l c a l c i u m , days 12-19  XXI); u r i n e c a l c i u m , days 33-38 (Table XXV); c a r c a s s (Table XXVI); and femur calcium-45 (Table XXIX). f i c a n t i n t e r a c t i o n v a r i a n c e prevented the above parameters being  (Table calcium  The s i g n i -  the treatment  means of  compared.  A s i g n i f i c a n t b l o c k F r a t i o was not found the 28 measurements made d u r i n g t h i s experiment. calcium balance, days 12-19  and days 19-26  i n f i v e of They were:  (Tables IX and X ) ,  food i n t a k e , days 26-33 and days 33-38 (Tables XIX and XX), and calcium-45 a b s o r p t i o n  (Table  XXVIII).  DISCUSSION  The  r e s u l t s obtained d u r i n g t h i s experiment  no c o n c l u s i v e evidence  provide  t h a t e x e r c i s e or confinement a f f e c t s  the c a l c i u m metabolism of r a t s .  T h i s i s somewhat s u r p r i s i n g  i n the l i g h t of the l i t e r a t u r e which suggests t h a t i s l o s t from bone d u r i n g p a r t i a l or t o t a l In the present  study  method of confinement may  immobilization.  the c o n f i n e d r a t s  f a i l e d to l o s e calcium f o r any  calcium  probably  of three reasons.  not have s u f f i c i e n t l y  First,  the  immobilized  the r a t s t o b r i n g about i m m o b i l i z a t i o n o s t e o p o r o s i s .  In  f a c t , when the c o n f i n e d r a t s were p l a c e d i n the h i g h l y r e s t r i c t i v e metabolism cages f o r the l a s t 12 days of the e x p e r i ment, s e v e r a l of them v i g o r o u s l y gnawed a t the cage.  This  gnawing and body t e n s i n g may  exer-  c i s e to prevent  have p r o v i d e d  sufficient  immobilization osteoporosis.  However, i t  i s d o u b t f u l t h a t r a t s c o u l d remain h e a l t h y under c o n d i t i o n s of g r e a t e r  restriction.  Second, the p e r i o d of i m m o b i l i z a t i o n may insufficient  have been  t o cause any d e t e c t a b l e changes i n the r a t ' s  c a l c i u m metabolism. T h i r d , the calcium metabolism of the r a t may from t h a t of c a t s , dogs or man.  Hegsted  t h a t the r a t i s probably  beast"  an "odd  metabolism i s concerned and  differ  (1967) suggests as f a r as i t s bone  t h e r e f o r e care should be  taken  76 i n i n t e r p r e t i n g c a l c i u m s t u d i e s performed on r a t s .  Gross  e t a l . (1965) f a i l e d t o demonstrate any changes i n the c a l cium balance of monkeys when they were immobilized, so perhaps both the r a t and the monkey m e t a b o l i s e c a l c i u m d i f f e r e n t l y than does  man.  The apparent f a i l u r e of e x e r c i s e t o a l t e r the r a t s ' c a l c i u m metabolism i n t h i s study suggests e i t h e r t h a t e x e r c i s e had no e f f e c t on c a l c i u m metabolism or t h a t v i g o r o u s r e g u l a r e x e r c i s e d i d not a f f e c t c a l c i u m metabolism t o a g r e a t e r degree than d i d m i l d e x e r c i s e .  In other words, the minimal e x e r c i s e  performed by the c o n t r o l r a t s had as much of an e f f e c t on c a l c i u m metabolism as d i d the r e g u l a r f o r c e d e x e r c i s e performed by the e x e r c i s e d  rats.  S i n c e t h i s study r e v e a l e d no c o n c l u s i v e e f f e c t s of e x e r c i s e or confinement on c a l c i u m metabolism, the d a t a obt a i n e d from the b l o o d r e c i p i e n t r a t s i s meaningless.  This  p a r t of the study u n f o r t u n a t e l y does not p r o v i d e enough evidence t o even s p e c u l a t e about the r o l e of b l o o d i n media t i n g the e f f e c t s of e x e r c i s e or confinement on c a l c i u m metabolism. The body weight d a t a suggests t h a t the e x e r c i s e d and c o n f i n e d r a t s were s u b j e c t e d t o s t r e s s s i n c e both the exerc i s e d and c o n f i n e d r a t s a p p a r e n t l y grew slower than the control rats  (Fig. 5).  77 The f a c t t h a t the e x e r c i s e d r a t s appeared t o g a i n body weight a t a slower r a t e than the c o n t r o l r a t s p o r t s the r e p o r t by Mayer provides  ( F i g . 5) sup-  (1960), t h a t r e g u l a r  a mild continuing  exercise  s t r e s s t o which r a t s adapt by  u t i l i z i n g some of t h e i r m e t a b o l i c energy f o r muscular work, r a t h e r than body weight growth. Figure  5 a l s o shows t h a t the c o n f i n e d  growing and even l o s t weight when p l a c e d t r i c t i v e cages  (days 26-38).  r a t s stopped  i n the h i g h l y r e s -  T h i s suggests t h a t the method  of confinement used i n t h i s study d i d provide  physiologic  s t r e s s and i t agrees w i t h the f i n d i n g s r e p o r t e d (1968b).  by P f i e f f e r  Some of the slowed growth was probably normal  s i n c e the c o n t r o l r a t s showed a p l a t e a u about 30 days  i n growth r a t e a t  ( F i g . 5).  The c e s s a t i o n of growth and the l o s s of body weight demonstrated by the c o n f i n e d diminished  food i n t a k e .  of the c o n f i n e d  r a t s most l i k e l y r e f l e c t  Figure  8 shows t h a t the food  r a t s d e c l i n e d when they were p l a c e d  h i g h l y r e s t r i c t i v e cages  (days 26-38).  The c o n f i n e d  their intake  i n the rats  ate s i g n i f i c a n t l y l e s s food than the e x e r c i s e d or c o n t r o l r a t s i n the f i r s t  seven days a f t e r being p l a c e d  t r i c t i v e cages.  T h i s suggests t h a t severe confinement i n h i -  b i t e d the a p p e t i t e o f these r a t s . should  i n the h i g h l y r e s -  However, t h i s  conclusion  be q u a l i f i e d , because the c o n t r o l r a t s , the e x e r c i s e d  78 r a t s and the c o n f i n e d r a t s a l l ate l e s s food d u r i n g the f i v e days of t h i s experiment  ( F i g . 8).  The  reason  last  for this  d e c l i n e i n food i n t a k e i s unknown. The  graph showing the changes i n f a e c a l calcium  the l a s t 12 days of the experiment  ( F i g . 9) c l o s e l y resembles  the graph of the food i n t a k e over the same p e r i o d T h i s supports  over  ( F i g . 8).  the f i n d i n g s r e p o r t e d by Wiseman (1964) t h a t  the c a l c i u m content of faeces i s l a r g e l y determined.by the dietary  calcium. The measurements which showed s i g n i f i c a n t d i f f e r e n c e s  between the e x e r c i s e d and c o n f i n e d r a t s (Tables X I I I , XIX,  and XXII) are d i f f i c u l t to understand.  XIV,  The e x e r c i s e d  r a t s u t i l i z e d a s i g n i f i c a n t l y g r e a t e r percentage of t h e i r d i e t a r y c a l c i u m than d i d the c o n f i n e d r a t s d u r i n g days but t h i s was  r e v e r s e d d u r i n g days 19-26.  The  12-19  f a c t t h a t the  c o n f i n e d r a t s u t i l i z e d a s i g n i f i c a n t l y g r e a t e r percentage of t h e i r d i e t a r y calcium d u r i n g the days 19-26, i s r e f l e c t e d the f a c t t h a t the c o n f i n e d r a t s e x c r e t e d s i g n i f i c a n t l y  by  less  f a e c a l c a l c i u m than d i d the e x e r c i s e d r a t s although  the  food  i n t a k e i s q u a n t i t a t i v e l y comparable between the two  groups  during t h i s period. There were no s i g n i f i c a n t d i f f e r e n c e s between the r e l a t i v e h e a r t weights of the e x e r c i s e d , c o n f i n e d or c o n t r o l rats.  T h i s may  have r e s u l t e d from the l o s s of body weight  79 suffered  by  the  confined r a t s .  would probably i n c r e a s e the actual may  f a c t the  A sudden l o s s of body weight  r e l a t i v e h e a r t weight.  r e l a t i v e h e a r t weights of the  Thus i n  confined  rats  r e f l e c t a body weight change r a t h e r than a h e a r t weight  change. Since the d i d not of the  r e l a t i v e h e a r t weights of the  d i f f e r s i g n i f i c a n t l y from the control  rats  i t can  be  regimen used i n t h i s study was t r a i n the  rats.  But  exercised  r e l a t i v e h e a r t weights  concluded t h a t the not  exercise  s u f f i c i e n t to  physically  the body weight data i n d i c a t e  that  e x e r c i s e used i n t h i s study d i d cause a p h y s i o l o g i c T h e r e f o r e the the  e x e r c i s e regimen was  A n a l y s i s of the  not  t r a i n the  o c c u r r e d f o r some of the  It i s unlikely  caused by  metabolism cages to house the  block  the  e x e r c i s e d and  because t h i s cage change d i d not  aim  investigation  and  parameters.  an  rats.  data compiled d u r i n g t h i s  l a r g e b l o c k v a r i a n c e was  stress.  necessarily  revealed that s i g n i f i c a n t i n t e r a c t i o n  the  the  considered s u f f i c i e n t f o r  purposes of t h i s study, as i t was  of t h i s study to p h y s i c a l l y  rats  use  variation  of  different  control  a f f e c t a l l the  that  rats,  experimental  animals. The  cage change probably c o n t r i b u t e d to the  v a r i a n c e but interaction  i t is unlikely because the  v a r i o u s parameters.  interaction  t h a t i t i s the main cause of  interaction  Probably the  i s inconsistent  significant  for  the the  interaction  80 r e f l e c t s the f a c t t h a t the day t o day, or week t o week, or month t o month v a r i a t i o n i n c a l c i u m metabolism o f the r a t , i s g r e a t e r than the v a r i a t i o n r e s u l t i n g from e x e r c i s e o r confinement. It  i s p o s s i b l e t h a t the l a r g e i n t e r a c t i o n v a r i a n c e  o b t a i n e d f o r many o f the parameters may r e s u l t from an unknown v a r i a b l e such as d i s e a s e .  T h i s p o s s i b i l i t y i s suggested  because a u t o p s i e s o f spare r a t s from the f i r s t run o f the experiment showed, a t the end o f the e n t i r e experiment, Salmonella i n f e c t i o n which was p a r t i c u l a r l y n o t i c e a b l e as c y s t s i n the lungs. B a n k i e r , D.V.M.) run  (This d i a g n o s i s was confirmed by J .  None of the r a t s used d u r i n g the second  o f the experiment were found t o be i n f e c t e d .  There i s no  c e r t a i n t y t h a t the i n f e c t i o n was p r e s e n t i n the r a t s used i n the  f i r s t run o f the experiment.  In f a c t most o f the e v i -  dence suggests otherwise as the growth curve f o r the c o n t r o l animals f o l l o w s a normal p a t t e r n  ( F i g . 5 ) . Body weight was  chosen as a c r i t e r i a o f g e n e r a l h e a l t h and metabolism, and i f a d i s e a s e d i d i n f e c t some of the r a t s t h i s should be r e f l e c t e d i n the body weight d a t a .  T h i s was not the case.  A l s o s i g n i f i c a n t i n t e r a c t i o n occurred only seven times out of  of a p o s s i b l e 28.  I f a d i s e a s e had s t r i c k e n some o r a l l  the r a t s used d u r i n g the f i r s t run o f the experiment  81 s i g n i f i c a n t i n t e r a c t i o n probably would have o c c u r r e d i n n e a r l y even s t a t i s t i c a l comparison. bility  t h a t d i s e a s e caused  Therefore the  proba-  the l a r g e i n t e r a c t i o n v a r i a n c e  o b t a i n e d f o r some of the parameters i s c o n s i d e r e d to be small. Since the r e s u l t s obtained from t h i s experiment were i n c o n c l u s i v e , a r e p e t i t i o n of the experiment u s i n g a d i f f e r e n t s t r a i n of r a t s , and c o n f i n i n g the one group of r a t s f o r a longer p e r i o d , would be a d v i s a b l e .  I d e a l l y , the con-  f i n e d r a t s should be t o t a l l y immobilized by p l a c i n g them i n a p l a s t e r c a s t or s i m i l a r d e v i c e .  However, t h i s o b v i o u s l y  becomes a p r a c t i c a l problem of perhaps insurmountable portions.  I t may  a l s o be a d v i s a b l e to use a d u l t r a t h e r  than growing r a t s , i f as Hegsted  (1967) s t a t e s "bone l o s s i s  not the r e v e r s e of bone f o r m a t i o n . " due  to growth may  pro-  T h e r e f o r e , bone formation  be a unique process and the c a l c i u m k i n e -  t i c s i n bone r e s u l t i n g from e x e r c i s e or confinement may obscured  be  i n the growing rat..  A l s o , a more accurate and r e l i a b l e method f o r d e t e r mining  r a t u r i n e c a l c i u m should be d e v i s e d b e f o r e  experiment i s r e p e a t e d .  the  82 1  Summary  The r e s u l t s of t h i s experiment suggest t h a t e i t h e r the p r e v i o u s l i t e r a t u r e i s i n e r r o r i n c o n c l u d i n g t h a t i m m o b i l i z a t i o n causes bone breakdown and body c a l c i u m  loss,  or t h a t t h i s study d i d not t o t a l l y immobilize the r a t s . F u r t h e r study i s probably r e q u i r e d t o e s t a b l i s h which conc l u s i o n i s t r u e , although t h i s i n v e s t i g a t o r favours the latter. On the b a s i s of these experiments i t i s suggested t h a t e x e r c i s e , i n excess of normal a c t i v i t y , does not a p p r e c i a b l y a l t e r the r a t ' s c a l c i u m metabolism.  Conclusions From the data o b t a i n e d d u r i n g t h i s experiment i t was concluded t h a t : 1.  Moderate  e x e r c i s e a p p a r e n t l y d i d not a f f e c t  c a l c i u m metabolism i n the growing male Sprague Dawley r a t . 2.  Confinement a p p a r e n t l y d i d not a f f e c t  calcium  metabolism i n the growing male Sprague Dawley r a t . the method o f confinement used i n t h i s study may  However,  not have  s u c c e s s f u l l y immobilized the r a t , although i t d i d p r o v i d e a physiologic  stress.  83 3.  Since the e x e r c i s e and confinement used i n t h i s  study d i d not a l t e r the r a t ' s c a l c i u m metabolism no cont r o l l i n g o r r e g u l a t i n g mechanism i n v o l v i n g blood c o u l d be established.  84 LITERATURE CITED  Abramson, A.S. (1948) Bone d i s t u r b a n c e s i n i n j u r i e s t o s p i n a l cord and cauda equina; t h e i r p r e v e n t i o n by ambulation. J . Bone and J o i n t Surg. 3OA:982. A l l i s o n , N. and B. Brooks. (1921) Bone atophy. An experimental and c l i n i c a l study of the changes i n bone which r e s u l t from non use. Sur. Gynec. and Obstet. 33:250. Birkhead, N.C., J . J . B l i z z a r d , B. I s s e k u t z , R. Myers and K. e x e r c i s e , standing, negative p r e s s u r e on bed r e s t induced U.S. A i r Force Aerospace Med. AMRL-TR-66-6.  J.W. Daly, G.J. Haypt, Rodahl. (1966) E f f e c t of trunk and p o s i t i v e s k e l e t a l o r t h o s t a s i s and h y p e r c a l c i u r i a . Res. Lab. Rept. Number  Burkhart, J.M. and J . Jowsey. (1967) P a r a t h y r o i d and T h y r o i d hormones i n the development of i m m o b i l i z a t i o n o s t e o p o r o s i s . E n d o c r i n o l o g y 81:1053. C a r r , T.E.F. and B.J. Parsons. (1962) A method f o r the assay of calcium-45 by l i q u i d s c i n t i l l a t i o n c o u n t i n g . I n t . J . A p p l . Rad. and Isotopes. 13:57. Copp, D.H. (1962) Evidence f o r c a l c i t o n i n - a new hormone from the p a r a t h y r o i d s t h a t lowers blood c a l c i u m . E n d o c r i n o l o g y 70:638. Copp, D.H. (1963) Simple and p r e c i s e micromethod f o r EDTA t i t r a t i o n of c a l c i u m . J . Lab. C l i n . Med. 61:1029. D e i t r i c k , J.E., G.D. Whedon and E. Shorr. (1948) E f f e c t s of i m m o b i l i z a t i o n upon v a r i o u s m e t a b o l i c and p h y s i o l o g i c f u n c t i o n s of normal man. Amer. J . Med. 4:3. Dunning, M.F. and F. Plum. (1957) H y p e r c a l c i u r i a f o l l o w i n g poliomyelitis: I t s r e l a t i o n s h i p to s i t e and degree of p a r a l y s i s . A r c h . I n t . Med. 99:716. G i l l e s p i e , J.A. (1954) Nature of bone changes a s s o c i a t e d w i t h nerve i n j u r i e s and d i s u s e . J . Bone and J o i n t Surg. 36B:464.  85 Gross, A.L., K.T. Robertson, L.H. Krough and J.W. Meisse. (1966) A study of c a l c i u m , phosphorous, and n i t r o g e n m o b i l i z a t i o n r e s u l t i n g from c o n d i t i o n s of i n a c t i v i t y i n Macaca Irus monkeys. U.S. A i r Force School of Aerospace Med. Rept. Number SAM-TR-66-94. H a r d c a s t l e , J . E . , R.J. Hannapel and W.H. F u l l e r . (1966) A l i q u i d s c i n t i l l a t i o n technique f o r the r a d i o a s s a y of calcium-45. I n t . J . A p p l . Rad. and Isotopes. 18:193 Hegsted, D.M. (1967) Proc. 26:1747. Horner, W.H. material.  M i n e r a l i n t a k e and bone l o s s .  Fed.  (1955) Determination of c a l c i u m i n b i o l o g i c a l J . C l i n . Lab. Med. 45:951.  Humphreys, E.R. (1965) An improved method f o r determining calcium-45 i n b i o l o g i c a l m a t e r i a l . I n t . J . A p p l . Rad. and Isotopes 16:345. L i u , C H . and C M . b o l i s m i n dogs.  McCay. (1953) S t u d i e s of c a l c i u m metaJ . G e r o n t o l . 8:264.  Lutwak, L. (1959) E s t i m a t i o n of r a d i o a c t i v e calcium-45 l i q u i d s c i n t i l l a t i o n c o u n t i n g . A n a l . Chem. 31:340.  by  Mayer, J . (1960) E x e r c i s e and weight c o n t r o l , p. 110-121 i n E x e r c i s e and f i t n e s s . A c o l l e c t i o n of papers presented a t the c o l l o g i u m on e x e r c i s e and f i t n e s s . U n i v e r s i t y of Illinois. P f e i f f e r , C.J. Med. 43:199.  (1968a)  A b s o r p t i o n of c a l c i u m .  Postgrad.  P f e i f f e r , C.J. (1968b) The p h y s i o l o g i c e f f e c t s of r e s t r i c t e d a c t i v i t y i n the r a t : s t r e s s e f f e c t s of c h r o n i c r e s t r a i n t . Ex. Med. Surg. 25:201. Rodahl, K., J.T. N i c h o l s o n and E.M. Brown (ed.). (1960) Bone as a t i s s u e ; proceedings of a conference Oct. 30-31, 1958. B l a k i s t o n D i v i s i o n , McGraw-Hill, New York. Sarnat, M. and H. J e f f a y . (1962) Determination of r a d i o a c t i v e c a l c i u m by l i q u i d s c i n t i l l a t i o n . A n a l . Chem. 34:643.  86 S t e e l , R.G.D. and J.H. T o r r i e . (1960) P r i n c i p l e s and procedures of s t a t i s t i c s . McGraw-Hill, New York. Tipton, CM. (1965) T r a i n i n g and b r a d i c a r d i a i n r a t s . Amer. J . P h y s i o l . 209:1089. Vogt, F.B., P.B. Mack, W.G. B e a s l e y , W.A. Spencer, D. Cardus and C. Valbonna. (1965) The e f f e c t of b e d r e s t on v a r i o u s parameters of p h y s i o l o g i c a l f u n c t i o n : P a r t X I I . The e f f e c t of b e d r e s t on bone mass and c a l c i u m b a l a n c e . Texas I n s t . Rehab. Res. Rept. N a t l . Aeron. U.S. NASA Cr 182. Whedon, G.D. and E. S h o r r . (1957) M e t a b o l i c s t u d i e s i n p a r a l y t i c acute a n t e r i o r p o l i o m y e l i t i s , I . A l t e r a t i o n s i n n i t r o g e n and c r e a t i n e metabolism. J . C l i n . I n v e s t . 36:941. Whedon, G.D. (1960) I n a c t i v i t y o s t e o p o r o s i s , p.67 i n K. Rodahl, J.T. N i c h o l s o n and E.M. Brown (ed.). Bone as a t i s s u e ; proceedings of a conference Oct. 30-31, 1958. B l a k i s t o n D i v i s i o n , . McGraw-Hill, New York. Wiseman, G. (1964) P r e s s , London.  A b s o r p t i o n from the i n t e s t i n e .  Academic  Wyse, D.M. and C.J. P a t l e e . (1954) E f f e c t of the o s c i l l a t i n g bed and t i l t t a b l e on c a l c i u m , phosphorous and n i t r o g e n metabolism i n p a r a p l e g i a . Amer. J . Med. 17:645.  

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