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Body composition studies on the growing pig. Groves, Tom David Douglas 1960

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BODY C O M P O S I T I O N S T U D I E S  ON T H E G R O W I N G P I G  by Tom D a v i d D o u g l a s  A Thesis of  Submitted the  i n  Partial  Requirements Degree  Groves  Fulfillment  for  the  of  MASTER OF S C I E N C E I N A G R I C U L T U R E in Division  of  the Animal  Science  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o the standard required from candidates for the degree of Master of Science i n A g r i c u l t u r e  THE U N I V E R S I T Y OF B R I T I S H O c t o b e r , i960.  COLUMBIA  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the r e q u i r e m e n t s f o r an advanced degree a t the  University  o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make it  freely  a v a i l a b l e f o r r e f e r e n c e and  agree t h a t p e r m i s s i o n f o r e x t e n s i v e f o r s c h o l a r l y purposes may  study.  I further  c o p y i n g of t h i s  be g r a n t e d by the Head o f  Department o r by h i s r e p r e s e n t a t i v e s .  be a l l o w e d w i t h o u t my w r i t t e n  Department The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Date  ^To^  ,  f^4Q  my  I t i s understood  t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r g a i n s h a l l not  thesis  financial  permission.  A B S T R A C T  The  present  been  undertaken  meat  producing animals.  i t  is  range  of  The in-vivo  was  closely  with  at  to  used  The  p i g was  age.  deuterium oxide  intervals  total  relating  water  the  ash,  relationships  determinations,  total  as  in-vitro  determinations  was  analysed  for  in-vivo  analysis.  the  growth of  were  intimately  growth  phase,  slowly  increased  rates  of  The  the  pigs  related  the  to  relative to  gain.  total  results  i n  of  consisted  of  process  rates  of  relative  a  of  one  i  the  the  above the  of  protein  gain,  agreed  pigs  distinct  however,  body  indicated  In  and  been  phases  body  abrupt  estimated  in-vitro  obtained  of  determina-  in-vivo  which had  and of  days  body  was  the  for  litters  calculated  in-vivo  of  was  equations  were  piglets  results  of  water  in-vitro  physiological aging.  fat  through  from four  Prediction  f o l l o w e d by an of  because  and s i x t y - f i v e  sacrificed  series  gain of  rate  animal  i n  The m e t h o d  body water  the  case  being  has  growth  which  piglets  body composition data  a maximum w h i c h was The  the  p r i o r to  serial  results  dessication.  body composition of  obtained  the  give  methods.  to  early  determining body  weekly for  Good agreement between  body water  that  both  grew.  and f a t  and the  serially  composition  they  the  classical  of  rapidly, passing  between  killed  growing p i g  experimental  thirty-three  were  the  time.  carcass  i n  was  short  found to  animals  l i t t e r  an  method f o r  i n - v i t r o by  body p r o t e i n ,  these  grows  i n a  of  aspects  and so  body water  from each  the as  a n d was  when the  of  chosen  dilution  body composition by  total  and u s i n g  obtained  determine  One p i g l e t of  birth  p h y s i o l o g i c a l events  those  to  body composition  c l a r i f y some  thoroughly tested,  frequent  tion  order  on the  p h y s i o l o g i c a l l y young at  a wide  then  i n  study  which  each  weight  decrease followed  i n an  inverse  pattern  of  growth phase,  each  each  phase  to  to  that  support  above,  and w i t h the  with  fat  body f a t  accelerated  being energy  rate  i i  of  gained being protein  r a p i d l y at used  at  gain.  the  the end  beginning of  TABLE OF CONTENTS Page 1  I . The In-vivo Determination of Total Body Water i n the Suckling Pig.... A. Introduction........  1  B. Methods and Materials.  7  1. Analysis of Deuterium Oxide i n Water..  7  a) Mass spectrometer.......... * b) Densitomometric methods  7 8  2. Determination of Body Water i n the Pig................. a) b) c) d)  14  Animals Rations and management Analytical in-vivo..... Analytical in-vivo  14 14 15 17  3. Procedure Checks and Evaluations  17  a) Physical b) B i o l o g i c a l  17 18  C. Experimental Results.  '  19  1. Evaluation of the P u r i f i c a t i o n Technique  19  2. Standard Curves*.*  19  3. Evaluation of the Precision of the In-vivo a n a l y t i c a l Methods  20  4. Equilibration of Deuterium Oxide i n the Pig.....  21  5. Comparison of In-vitro and In-vivo Body Water Values...  21  6. Extent of Urination by Animals During Injection and Sampling Period. •  22  •  7. The Effects of the Techniques Used for the Determination of Body Water In-vivo on the Subsequent Growth and Development of the Pig.................  22  D. Discussion 1. Densitometric Method f o r Deuterium Oxide Determination.. a) b) c) d)  Linearity of calibration curves.. Micro-dropping pipette.•........................ Water bath S e n s i t i v i t y of the densitometric method iii  37 37 38 38 40  2. The Equilibration of Deuterium Oxide i n the P i g  Page 41  a) Equilibration time 41 b) Distribution of heavy water i n the body after injection ........••••..42 c) Equilibration and loss of weight during the sampling schedule ••••.43 E. Summary and Conclusions..,.  ..........44  I I . Changes i n Body Composition i n the Suckling P i g  45  A* Introduction.  45  B. Methods and Materials.  58  1* Animals.......  •  2. Rations  58 58  3 • Management.  .58  4* Analytical  .59  a) In-vitro  ................................59  b) In-vivo  61  C. Experimental Results  ,.62  1. Changes i n Body Composition, Determined In-vitro........62 a) Body Water • .62 b) Body nitrogen ••.•..•••••.••••••62 c) Body ash 63 d) Body fat 63 e) Fat-free dry mass. 63 2. Relationship of Body Nitrogen to Body Water 64 a) The nitrogen-to-water r a t i o . . . . . . . . . . . . b) The regression of body water against body nitrogen. 3« The Relationship of Body Ash to Body Water  .....64 .65 66  a) The ash-to-water r a t i o . . . . .......66 b) The regression of body ash against body water. ,66 4. Relationships Involving the Fat-Free Dry Body Mass  67  a) The relationship of fat-free dry mass to body water •••••••67 b) The relationship of percent fat-free dry mass to body weight,......,..,....,., , 67 iv  Page 68  5. The Calculation of Body Composition In-vivo  a) The prediction equations..., ..68 b) The in-vivo body composition by weight..........68 6. The Estimation of the Chemical and Energetic Composition of Gain i n Growing Pigs...........  70  D. Discussion 1. The Changes i n the Relationships of Nitrogen, Ash, and Water i n Relation to Physiological Aging...........106 2. The Constancy of Proportion of Fat-Free Dry Mass Relative to Body Weight  ...109  3. The Composition of Gain i n Relation to and Growth i n Piglets •  110  4. The Effects of Restricted Feed Intake  123  •  5. The Effects of the Analytical Methods on the Growth Rates of the Piglets  124  6. The Practical Significance of the Present Findings.....125 E. Summary and Conclusions... Appendix. •  ....127 130  Index of Tables Table No. 1.  2.  3.  Page  Summary of Analysis of Variance to Detect Differences i n Dropping Times Between Samples of D i s t i l l e d Water ( i ) , Unpurified Water.... Recovered from Pigs not Injected with DoO (II), and Purified Water from the Same Biological Sources ( H i ) •  24  Summary of analysis of variance to detect differences i n dropping times of purified and unpurified aliquots of standard 0»1% D2O resulting from change i n concentration caused by fractional d i s t i l l a t i o n i n the purification train...  25  Summary of analysis of variance to detect differences i n dropping times of purified and unpurified aliquots of standard 0.5$ D2O resulting from change i n concentration caused by fractional, d i s t i l l a t i o n i n the purifying train  26  4. Standard Curve No. 1  27  5.  Standard Curve No. 2..  28  6.  Comparison of the slopes of typical standard curves  7.  Calculation of the precision and sensitivity of the methos f o r D 0 determination.  ..»•• 29 30  2  8. The effects of analytical sensitivity and of weighing and injecting errors on the precision of in-vivo body water measurement........• 31 9.  Summary of equilibration analyses  •  *  •  10.  Equilibration times for intraperitoneal D 0 injections  11.  Comparison of body water values determined in-vitro by dessication (X) and in-vivo on the same animals just prior to sacrifice by the deuterium oxide dilution technique. The in-vivo values are calculated on both an i n i t i a l weight (Y^) and f i n a l weight (Y ) basis  32 34  2  2  12  Linear regressions of Y-^ and Y  2  35  against X from Table No.11  36  13-A Litter No.l - Percentage body composition - based on in-vitro analysis  72  13-B L i t t e r No.l analysis. •  73  Body composition by weight - based on in-vitro  14-A Litter No.2 - Percentage body composition - based on in-vitro analysis. 14-B L i t t e r No.2 - Body composition by weight - based on in-vitro analysis vi  74 ....  75  Page 15-A  L i t t e r s 3 and 4 Percentage body composition - based on i n - v i t r o analysis  76  15-B  L i t t e r s 3 and 4 - Body composition by weight - based on i n - v i t r o analysis • .....*....*. 78  16.  Relationship between nitrogen to water r a t i o (kg/kg) and kilograms of f a t - f r e e dry mass (X) •  17.  Differences i n body composition and p h y s i o l o g i c a l age between new born p i g l e t s i n the same l i t t e r * .  IB*  20. 21. 22.  23.  •  .....*• 81  Differences between the nitrogen to water r a t i o s of pigs of the same chronological ages  19«  30  .............•..*........•. 82  The r e l a t i o n s h i p of grams t o t a l body nitrogen t o kilograms t o t a l body water.«...  33  Relationship between the ash t o water r a t i o (kg/kg) (Y) and Kg. of f a t - f r e e dry mass (X)«..«.  35  Relationship between Gms. t o t a l body ash (Y) and Kgs. t o t a l body water (X)  36  A. Relationship of Kg. f a t - f r e e dry mass (Y) to Kg. body water (X)« B. Relationship of percent f a t - f r e e dry mass (Y). to Kg. l i v e weight (X)  87  Summary of the prediction equations used i n c a l c u l a t i n g body composition i n - v i v o . ..*••  88  24.  Body composition of growing p i g l e t s estimated i n - v i v o . . . . . . . . . . . . . . . 89  25.  Composition of gain  •  98  Index of Tables i n the Appendix  1.  Farrowing data  130  2.  Percentage proximate compositions of the rations....  3.  Growth data  4*  Blood haemoglobin and i r o n i n j e c t i o n data..........  5.  Sow weights  6.  D a i l y feed intake of sows........  ....131 132 ••••••••139 142  vii  ••••143  LIST OF FIGURES, Figure number*  Page 9a  1.  Schematic diagram of the water p u r i f i c a t i o n tran........••••••••  2.  The dropping tube  3.  Schematic diagram of the water bath...................  4.  Schematic diagram of the pipette used to deliver a constant sized drop.  12a  Two t y p i c a l standard curves showing the relationship between the reciprocal dropping time and the concentration of deuterium oxide i n water. ••  19a  6.  The changes i n concentration of deuterium oxide i n blood water following the i n t r a c a r d i a l injection of deuterium oxide....•••••  21a  7»  The changes i n percent body water with age (in-vitro data)......  62a  8.  The changes i n percent body nitrogen with age ( i n - v i t r o data)...  62a  9.  The changes i n percent body ash with age ( i n - v i t r o data)........  63a  10.  The changes i n percent body f a t with age (in-vitro data)........  63a  11.  The relationship of the nitrogen to water ratio (Y) to fat-free dry matter (X) (arithmetic grid) •  64a  5.  •••«  ...  10a  ••  11a  12.  Relationship between nitrogen to water r a t i o and fat-free dry body mass, (logarithmic grid)................. •••••• 64a  13•  The relationship of body nitrogen to t o t a l body water...........  65a  14.  The relationship of the ash to water r a t i o (Y) to fat-free dry mass (X) (arithmetic grid).....  66a  15.  The relationship of the ash to water ratio (kg/kg) (Y) to f a t free dry mass (kg) (X) (logarithmic grid)  66a  16.  The relationship of body ash to t o t a l body water................  66a  17.  The relationship of fat-free dry mass to t o t a l body water.......  67a  18.  Percent fat-free dry matter vs. body weight....  67a  19-22. Cumulative changes i n the body compartments with age............ 23-31* Changes i n protein, fat and body weight with age......... viii  70a 110a  Acknowledgement  I of  wish  to  thank  Dr.  B.A. Eagles,  A g r i c u l t u r e and Chairman of  for  his  use  of  kind  permission to  departmental  the  Division  undertake  this  his  grateful  continued  duration Kitts  of  and D r .  for  his  interest  the  project.  S.W.  of  the  Animal  project  Faculty Science  and f o r  the  facilities.  To D r . A . J . W o o d , P r o f e s s o r sincerely  Dean o f  of  encouragement  and constructive I  Nash f o r  Animal  and guidance advice  would also  like  their  interest  kind  Science,  to  I  am  and  for  throughout  the  thank  D r . W.  D.  and h e l p f u l  criticisms.  I vided by  am a l s o  grateful  a National Research  Fellowship  given by  the  for  the  Council  financial Student  assistance  Bursary  B r i t i s h Columbia E l e c t r i c  IX  pro-  and by  Company.  a  I.  THE IN-VIVO DETERMINATION OF TOTAL BODY WATER IN THE SUCKLING PIG  A.  INTRODUCTION  The composition of the animal body may be regarded as the r e s u l tant of a l l of the biochemical and physiological l i f e p r o c e s s e s , and, as such, i t s measurement has been the task of many who have sought a more complete understanding of the phenomenon of animal growth and development. I t has been demonstrated that the gross compartments of water, ash, prot e i n , and f a t i n the animal body bear a d e f i n i t e r e l a t i o n s h i p to each other, and that the r e l a t i v e proportions of these compartments tend to change i n a d e f i n i t e manner as the i n d i v i d u a l grows to maturity and/or i s subjected to exogenous stresses.  Much of our present knowledge of body  composition i s necessarily based on the d i r e c t physical and chemical analysis of carcasses (l£, 2 7 , 3 2 , 3 7 , 59, 7 2 , 7 7 , 1 0 2 ) . In order to apply t h i s knowledge i t has become increasingly important to be able t o measure accurately the body composition of the l i v i n g animal, and to t h i s end, many i n d i r e c t methods of variable usefulness have been devised ( 1 9 , SO)*  In these i n d i r e c t methods, one or more body compartments are measured,  and the others are estimated on the basis of the relationships established by i n - v i t r o analysis.  Body water i s the most frequently used parameter  f o r the in-vivo estimation of body composition, since i t i s conveniently measured, using blood water as a representative sample of the t o t a l water compartment.  - 2 The in-vivo measurement of body water may be approached i n two ways.  F i r s t l y , s p e c i f i c gravity may  be measured and body water calculated  on the basis of an assumed constant composition of the lean body mass (68, 85).  This method has two main disadvantages, namely that accurate  s p e c i f i c gravity measurement i s d i f f i c u l t to obtain and that the assumpt i o n of constant lean body composition i s not s a t i s f a c t o r y i n the l i g h t of present knowledge of animal growth (k, 8, 75, 102), although f o r pract i c a l calculations such an assumption may not produce large errors.  A more widely used technique f o r the determination of body water has involved the i n j e c t i o n of a suitable solute with subsequent determinat i o n of the extent of i t s d i l u t i o n and excretion by the body.  Soberman  and Associates (100) and Cowgill i n h i s review (19) have outlined the desired properties of such a solute as follows:  1.  I t should dissolve r e a d i l y i n water, and should become evenly and r a p i d l y d i s t r i b u t e d i n a l l of the body water.  2.  I t should not be s e l e c t i v e l y bound by the tissues, should be only slowly metabolized, and should be e l i m i nated from the body at a s a t i s f a c t o r y measurable r a t e .  3.  I t should be e a s i l y and accurately determined i n the blood water.  il.  I t should not be toxic to the animal at the required l e v e l of dosage, nor should i t have any cumulative t o x i c i t y when administered sequentially i n a time s e r i e s .  - 3 Many such solutes have been proposed, including ethyl alcohol (50),  g l y c e r o l (50),  8U, 93), (88),  potassium (50),  antipyrine (10,  19,  sulphanilamide (50),  water (2J4., 79).  3h,  100,  thiourea (50), 112,  50),  deuterium oxide (19,  urea (19,  50, 5U,  58,  N-acetyl U-amino antipyrine U0, 50, 9k),  and  tritiated  A l l of these compounds f a l l short i n some measure of the  above requirements f o r an i d e a l solute, and most have been discarded as body water reference substances, i n some cases perhaps without a f a i r t r i a l (50).  Of the above compounds antipyrine (2,3-dimethyl,  5-oxo-l-  phenyl-3-pyrazoline) and deuterium oxide have been used most extensively i n the determination of t o t a l body water. Soberman et a l . (100)  and Wellington et a l . (112), using a n t i -  pyrine, have obtained body water values which are i n good agreement with data obtained from the same individuals by deuterium oxide d i l u t i o n , s p e c i f i c gravity determination and i n - v i t r o analysis.  Garret et a l .  (3I4.)  observed that antipyrine used to determine body water at monthly i n t e r v a l s i n c a t t l e gave results which were more variable than could p h y s i o l o g i c a l l y be expected.  Hardy and Drabkin (1.0) have c r i t i c i s e d the use of antipyrine  on the basis of slow e q u i l i b r a t i o n with abnormal f l u i d deposits, s e l e c t i v e binding by proteins and a variable rate of metabolism. N-acetyl l|.-amino derrivative used by Brodie (10a)  In t h i s respect the  and recently by Reid  et a l . ( 8 8 ) , may constitute an improvement i n that this compound i s not metabolized to any appreciable extent. Deuterium oxide, the solute of choice i n the present study, has been discussed as an i d e a l reference substance (Uo).  I t i s not metabolised  - h and becomes r a p i d l y equilibrated i n the tissues (26,  9k), and although  k i n e t i c considerations would lead one to expect some differences i n reaction rates of exchanges of D and H i n the body, heavy water i n low concentrations apparently behave e s s e n t i a l l y as ordinary water, as evidenced by the f a c t that the b i o l o g i c a l h a l f l i f e of deuterium oxide i s only very s l i g h t l y l e s s than that f o r ordinary water (9k).  Explanation  of t h i s phenomenon i s probably that at body temperatures, the equilibrium constant f o r the reaction H2O + D20^-2 HDO  i s about 3.8  (lOj?,  109).  Thus, very shortly a f t e r i n j e c t i o n of a n a l y t i c a l amounts of deuterium oxide i n t o the body, almost none remains as D2O, and i t i s probable that the k i n e t i c s of the r e s u l t i n g HDO  are not s u f f i c i e n t l y d i f f e r e n t from  those of H2O f o r the two molecules to be p h y s i o l o g i c a l l y distinguishable. Deuterium oxide administered i n high concentrations over extended  time  i n t e r v a l s does however have some a n t i b i o l o g i c a l e f f e c t s , including the production of s t e r i l i t y (hh), the i n h i b i t i o n of c e l l d i v i s i o n (kS,  111),  and the reduction of glomerular f i l t r a t i o n and renal plasma flow (106).  One weakness of deuterium oxide as a reference solute i s that deuterium atoms exchange with l a b i l e hydrogen atoms i n the various organic compounds of the body (55,  107).  The extent of t h i s exchange has been  estimated as leading to a one h a l f to four per cent over-estimation of body water (50).  I t has been found that the ether extracts of carcasses  contained about one t h i r d as much exchangeable hydrogen as the amount found i n the p r o t e i n - r i c h residue, and also that a stable f i x a t i o n of deuterium oxide i n the body may occur  (97).  - 5 T r i t i a t e d vrater, the beta ray emitting radioactive form of water, has also been used as a reference solute (2U,  79),  are e s s e n t i a l l y the same as f o r deuterium oxide.  giving r e s u l t s which This substance has  the  advantage of requiring a smaller administered dose, but i s more l i k e l y to d i f f e r from water on a k i n e t i c basis ( i l l ) , and may,  as a r e s u l t of the  r e l a t i v e l y long h a l f l i f e of t r i t i u m , not be desirable f o r animal or human use  (50).  Various methods f o r the determination of deuterium oxide i n water have been described including the measurement of r i s e i n freezing point (86), emission of photo-neutrons a f t e r bombardment of the sample with high energy gamma r a d i a t i o n (35), mass spectrometry (51).  (51,  101,  105,  109),  i n f r a - r e d spectrophotometry (96), and various densitometric methods  Of these methods, the mass spectrograph  and the densitometric  method of Keston, Rittenberg and Schoenheimer (U9),  have found the most  frequent use i n studies i n which body water i s determined by the deuterium oxide d i l u t i o n  technique.  In addition to or instead of the measurement of t o t a l body water, some workers have found i t desirable to measure the " a v a i l a b l e " and " i n t e r s t i t i a l " water compartments (20,  26, 31,  58,  67).  Such measurement  i s s i m i l a r to that of the determination of t o t a l body water by the d i l u t i o n technique, except that the solutes used are only d i s t r i b u t e d i n the e x t r a c e l l u l a r compartment of the water.  Suitable solutes have been  sodium thiosulphate (33), and sodium thiocyanate (20).  Available water  refers to the compartment measured by thiocyanate or thiosulphate d i l u t i o n , while i n t e r s t i t i a l water i s defined by Forbes et a l . (31)  as being the  -  sodium thioeyanate the  blood  seventy  cell  per  determining does  not  analysed  to  be  of  the  the  from the  present  ash,  i n  for  the  i n Section used  i n  relation  in-vitro.  the to  blood  the  II.  the  the  dye  per  blood  cent  cells  measured  Evan's  body composition to  l i t t e r body  results The  Blue  the  age  values  was  of  eight  sacrificed  the  the  dilution.  compartments of  of  of  are  by  (31)  which  body  of  for  the  obtained  suckling  weeks. In  total  in-vivo from the  Body  carcass fat,  composition is  pig  addition,  and the  following discussion  experiment the  of  that  conveniently  deuterium oxide  major The  is  and seventy  stream.  from b i r t h  weekly by  and water.  presented  dessication  study,  animal from each  chemically  methods  the  blood  sequentially  one  body water  assumption  by  dilution  determined  (protein)  on the  the  was m e a s u r e d  each week,  plasma volume  Plasma volume  dialyse  was  (57a),  volume  the  -  cent water.  In  water  space minus  6  an  nitrogen  study  are  evaluation  determination  of  same a n i m a l s  by  B.  1.  METHODS AND MATERIALS  Analysis of Deuterium Oxide i n Water a)  Mass spectrometer  Some preliminary work was done with the U.B.C. Chemistry Department's a n a l y t i c a l mass spectrometer.  The instrument was evacuated f o r  three quarters of an hour, f i l l e d with the sample, and then re-evacuated before measurement of the deuterium oxide concentration.  The p a r t i c u l a r  instrument was not designed to record accurately the mass 2 and 3 p a r t i c l e s normally used i n such an analysis (92, 101) making i t necessary to use a comparison of the peak heights i n the mass range of 17 to 20 to determine deuterium concentration.  Thomas (105) and Washburn et a l . (109) describe  methods of mass spectrometry i n which the r a t i o of mass 19 to mass 18 has been d i r e c t l y proportional to the deuterium oxide concentration of the sample i n the d i r e c t mass spectrometric analysis of water vapour.  In the  preliminary work c a r r i e d out i n the present study using the mass spectrometer i t was found to be more convenient to measure deuterium oxide concentration by the Mass 19  r a t i o , on the assumption that Mass 19 was corn-  Mass 17 posed c h i e f l y of HDO  p a r t i c l e s and that Mass 17, i n the d i l u t e heavy water  solutions used would e s s e n t i a l l y represent OH* p a r t i c l e s from H2O. work indicated l i n e a r i t y i n the r e l a t i o n s h i p of Mass 19 Mass 17 of deuterium oxide i n water.  Initial  to the concentration  The method was, however, not pursued further,  since i t was f e l t that the amount of time necessary to evacuate and f l u s h the  appaid-us between samples to reduce memory effects r e s u l t i n g from a  - 8r e s i d u a l f i l m of water on the walls of the glass tubing (101) might render the method impractical f o r the rapid analysis of a large number of samples.  Also, because of the large number of samples envisioned,  and because the apparatus was at the time being used by several other workers, i t was f e l t that almost continuous use of the mass spectrometer by the author might be an imposition on the generosity of the Chemistry Department.  b)  Densitomometric methods i)  Balancing drop method Linderstrom-Lang et a l . (576) have described a method f o r the  determination of heavy water i n which drops of water containing d i f f e r e n t amounts of deuterium oxide came to r e s t at d i f f e r e n t l e v e l s i n a density gradient between kerosene having a density of about  0.703  and bromoben-  zene, with a density of 1.1*991. In the preliminary work done with t h i s method, i t was found that a mixture of chlorobenzene, having a density of  1.1066  produced a much more sensitive density gradient f o r separating  heavy water samples i n the concentration range of 0 to 0.8 atoms % of added deuterium oxide. A d i r e c t r e l a t i o n s h i p was found between the depth of the drop below the l i q u i d meniscus and the concentration of heavy water i n the sample.  The p o s i t i o n of the sample drops may be determined with a s e n s i -  t i v e cathetometer, using a point-source of l i g h t behind the solvent container to illuminate the centers of the suspended drops.  The method has  the one great advantage that the s i z e of the drops of sample need not be controlled.  Reproducibility of r e s u l t s i s , however, dependent on the same  thermostating problems as encountered i n the f a l l i n g drop method discussed  - 9 below, as w e l l as on the f a c t that f r a c t i o n a l evaporation from surfaces of the mixture changes the composition of the density gradient.  of the l a t t e r and a l t e r s the p o s i t i o n  A further d i f f i c u l t y i s that experienced i n  t r y i n g to i d e n t i f y d i f f e r e n t bubbles of sample through the eyepiece of the cathetometer i f several drops are placed i n the solvent mixture a t once.  This method was not followed past the preliminary stages i n the  present study, however, i t should be mentioned that, i n view of wide spac i a l separation i n the chlorobenzene-kerosene density gradient of samples d i f f e r i n g only s l i g h t l y i n deuterium oxide content, i t i s possible that modification of t h i s method could lead to a very simple and accurate means of measuring deuterium oxide i n water.  ii)  F a l l i n g drop method  Keston, Rittenberg and Schoenheimer (U9) describe a technique f o r the determination of the density of heavy water samples based on the method proposed e a r l i e r by Barbour and Hamilton (5)»  A modification of  t h i s method has been used extensively i n the present  study.  One h a l f c c . samples of water recovered from blood, urine, muscle or f a t were p u r i f i e d by four successive in-vacuo d i s t i l l a t i o n s i n an apparatus e s s e n t i a l l y the same as that described by Keston et a l . (i+9). Figure 1 i s a diagram of the p u r i f i c a t i o n t r a i n used.  These workers  i g n i t e d deuterated organic material i n a quartz combustion chamber and collected the r e s u l t i n g water f o r analysis.  Dissolved oxides of nitrogen  produced by the combustion were removed by treatment of the water sample with BaCDj.  This trap was omitted from the present analysis since i t was  f e l t that oxides of nitrogen would not be present i n water recovered  F/GOftE  /  SCHEMATIC WATER  D / A G K A M  PURIFICATION  orr«c TRAIN  40_  30 . S UCT/O/V  20 _  0  J  JO _  0  _  6 THREE  T  -  WAN  STOPCOCK  F  - F/AJE T I P T O C O N T R O L  RATE O F A I R F L O W T H R O U G H  T3RVNC, T U B E  TJ  - 10 d i r e c t l y as such from blood or t i s s u e .  The f i r s t trap i n the p u r i f i c a t i o n  t r a i n used contained a few crystals of r e c r y s t a l l i z e d I^C^O^ to oxidize organic material.  The second trap contained KMnO^ as a further o x i d i z i n g  agent as w e l l as a small amount of dry NaOH, which prevented ammonia from d i s s o l v i n g i n the water condensing i n trap 2 from trap 1. were kept blank.  Traps 3 and k  The p u r i f i e d sample was f i n a l l y c o l l e c t e d i n trap 5 from  which i t was removed with a f i n e pipette and placed i n a t i g h t l y stoppered glass storage v i a l .  Vacuum i n the apparatus was obtained using a water  aspirator, and a mercury manometer was mounted on the main suction hose to detect pressure leaks.  D i s t i l l a t i o n was aided by heating with a small  (550 Watt) hot plate controlled by a rheostat and placed about 2 inches below the bare trap. the  Vapour was condensed i n the next trap by submerging  l a t t e r i n a beaker of c o l d tap water a t 10°C.  A second c o l d bath was  applied to the next trap, and a t h i r d cold bath was placed under trap 6 i n order to minimize the d i f f u s i o n of water vapour to the apparatus from the  water tap.  wooden frame. the  The apparatus was mounted on an aluminum f o i l - c o a t e d A hood of heavy aluminum f o i l was placed over the top of  t r a i n during operation i n order to r e f l e c t heat onto the top of the  traps, reducing condensation i n the upper bends of the tubing. A i r entering the system through the 3-way stopcock was dried with CaCl2. The glass tubing used f o r the apparatus was eight millimeter inside diameter pyrex f o r the f i r s t four traps, fourteen millimeter i n s i d e diameter pyrex for  trap 5 and s i x millimeter pyrex f o r the tubing leading to the vacuum  line.  F / C U R E  THE  2.  D f i o m N G TUBE  70  0  B / O  TJROPPlNG  GLASS  TJROPP/NG  -I'O Cn  PIPETTE  HtDIUM  INSIOE  LtVEL  UIAWE1ER  PYHHX  . UPPE«  CALIBRATION  MARK  40  30  .  20 LQWCRCAUBR/NTJON  10  HARK  .  50  o.  SLEEVE  STANDARD  _ ^  50  I / V S / O C Ul*nETER  T O GUIDE  CMS.  60  MM  C C . C ~ A P A C I T ? TZ>UL£>  TUBE  - 11  -  A l l glassware coming i n contact with the water samples i n c l u d i n g storage bottles was  scrupulously cleaned with f r e s h potassium dichromate-  concentrated sulphuric a c i d cleaning s o l u t i o n followed by many rinsings with d i s t i l l e d water from a glass s t i l l a f t e r each series of p u r i f i c a t i o n s . In addition, trap 5 "was washed with b o i l i n g d i s t i l l e d water a f t e r the p u r i f i c a t i o n of each sample. the apparatus was  P r i o r to the introduction of the sample,  dried completely by heat under vacuum.  Great care  was  taken when the suction was being turned on, or when outside a i r was l e t i n a f t e r p u r i f i c a t i o n , that the a i r turbulence d i d not sweep a small part i c l e of e i t h e r I^C^O^, KMno^, KOH,  or Mn02 (which was produced when the  KMhO^ was heated) i n t o the f i n a l trap to contaminate the sample.  To  check that this had not happened, the p u r i f i e d sample i n the f i n a l trap was held over a white card i n a strong l i g h t and viewed from the top of the tube i n order to detect colour traces.  I f colour was  evident i n the  water, the sample was r e p u r i f i e d by again passing i t through the  two  blank traps 3 and k a f t e r c a r e f u l re-washing and drying of trap 5«  The f a l l i n g drop apparatus consisted of a one centimeter inside diameter pyrex tube s i x t y four centimeters long, f i t t e d with a Qufck f i t B-10  ground glass outside j o i n t at the top and attached at the bottom to  a $0 cc pyrex glass bulb.  (Figure 2)  Two rings were scratched on the tube,  one twenty-five centimeters from the top and the other f i f t y - f i v e c e n t i meters from the top.  The dropping tube was  suspended r i g i d l y i n a water  bath (see Figure 3) having a f i v e cubic foot capacity and i n s u l a t e d with two inches of spun f i b r e g l a s s wool i n s i d e a one quarter-inch plywood easing.  A s l i t was  cut i n the side of the casing so that the  dropping  z  -  J  >J  —  a  CO H  QC o h <£. or O Ul  cQ  faC <  C/5  o  0  <  z  i—  03  if  ar  0  Z  u  c cO o O -J 0 cr  cr X  a U.  < <J R  U.  f-  2 u  Oi <j CQ I  <j  x.  < ^  r-  ^  u * I 1/1  <c  QC  3  o  Ul  2 ar  <  a:  U M  D O  x  o CL  o o O  a. 2 o; OC  7  j  Z  o  -j -=>  -  3  <C  <  P  z < >  i  UJ  (=>  cr Uj UJ  Z Ui or ^ *•  h O tu  x  u > u -J. cc u  <  2  * ? S  f-  m m  CC  z  z  QC  c  QC O  cj  3  o <  <  o  5  - 12 -  tube could be observed, and a small hole was cut i n the opposite side so that a l i g h t could be shone into the tank.  The bath was f i l l e d with  water to within four centimeters of the top of the dropping tube.  Tem-  perature control was maintained with a "Colora Ultrathermostat"* equipped with a one thousand Watt heating c o i l , a very e f f i c i e n t s t i r r i n g blade, a c o i l through which cold water could be c i r c u l a t e d and which was  speci-  f i e d by the manufacturer as being able to maintain a constant temperature i n a f o r t y - f i v e gallon water bath with a f l u c t u a t i o n of t l 0.01°C.  A  very small flow of cold tap water was allowed to pass through the cooling c o i l to compensate f o r the small heating e f f e c t of the s t i r r i n g motor.  Ortho-fluorotoluene* having a density of 0.9996 a t 26.8°C. was used as the dropping medium. man  The dropping tube was f i r s t coated with Beck-  "Desicote" 18772 an organic-silicone compound which forms a monomolecular  f i l m on the tube and prevents the sample drops from adhering to the walls of the tube, but does not a f f e c t the properties of the dropping medium. The tube was then f i l l e d with o-fluorotoluene to within four centimeters of the surface of the water i n the bath.  A dropping pipette was made which would d e l i v e r a constant drop s i z e (Figure U ) .  I t s i n t e r i o r was also coated with Desicote to ensure a  complete d e l i v e r y of contents.  In the course of analysis, the dropper  was f i l l e d with the sample, the t i p of the pipette placed beneath the surface of the dropping medium and the drop expelled a f t e r which i t was detached from the top of the pipette by drawing the l a t t e r slowly through the solvent meniscus.  Great care was taken not to draw any solvent up i n t o  ft U.S.P. produced by the Eastman-Kodak Co. Colora S c i e n t i f i c Instruments, Lorch/Wurttemberg, Germany  FIGURE  4  D/AGftAM  OF  USED  PlPClTE  A CONSTANT  TJELIVER  To  SI^ED U R O P  ' /(VtLV T H R t A b t D S C R t W /1£7AL S Y R I N G E HOL/DER  SVRiMGtSTEEi.  BARREL  SPRING  2.0 cc. G L A S S  SYRINGE  TVGON CONNECTING  TUBE  HEAVY fW&RCfi TO INT  1USINC  CALIBRATION  HARK  CONSTRICTION T O AT  0.5  CALIBRATION  mm  TU6/A/C  To S o P P O « r  TJETCRCASE  B O R f OF T U B E  MARK  INSlOEDIAMETER  PV R E X C A P l L L l A R V  pipette.  The pipette was r i n s e d twice with the sample before each r e p l i -  cate series of drops to eliminate any memory effects from the previous sample.  Care was also taken that no extraneous water adhered to the  outside of the t i p of the pipette, as t h i s would be added to the drop as the  pipette was withdrawn from the dropping tube.  The i n t e r i o r of the  pipette was cleaned a t frequent i n t e r v a l s with potassium dichromate cleaning s o l u t i o n followed by r i n s i n g with d i s t i l l e d water. The time of the f a l l of the drops between the two etched rings on the dropping tube was measured using a two-pointered stop watch which recorded sixths of a second.  The s i z e of drop used was  and s i x drops of a single sample constituted an a n a l y s i s .  2^.68  cu. mm.,  A f t e r each s i x  r e p l i c a t e drops, the l e v e l of the o-fluorotoluene i n the dropping tube was adjusted back to i t s o r i g i n a l l e v e l by withdrawing with a pipette a volume of medium equal to that of the drops added to the tube.  The same  dropping medium was used f o r a succession of analyses u n t i l the reservoir at the bottom of the tube became f u l l a t which time the l a t t e r was cleaned and r e f i l l e d with fresh o-fluorotoluene.  Used o-fluorotoluene  was dried with anhydrous sodium sulphate and was then stored over a small amount of d i s t i l l e d water u n t i l i t was re-used.  Standard water samples were made up gravimetrically i n 100 ml. volumetric f l a s k s using commercial 99.7h per cent pure dueterium oxide, and made up to 100 ml. with f r e s h l y d i s t i l l e d water.  The concentration  was expressed as per cent deuterium oxide by weight.  No correction was  made f o r the density of p u r i t y of the deuterium oxide, since i t was f e l t that such differences would be n e g l i g i b l e .  In addition, no allowance  - lU was made f o r the f a c t that pure water normally contains 0.015  atoms per  cent deuterium oxide (9k) since the f i n a l analysis was based on the difference of two samples and the e f f e c t of the r e s i d u a l deuterium oxide i n water was thus eliminated. Standard curves with a p o s i t i v e slope were established by regressing the r e c i p r o c a l of the dropping times of s i x standard samples ranging from 0.07 per cent to 0.k5 per cent deuterium oxide against the respective concentrations. When samples within the deuterium oxide concentration range of the standards were run, pure water was not included i n the standard curve, whereas, i f samples of very low suspected deuterium content were analysed, pure water was substituted f o r the highest reference concentration i n the standard curve.  Standard samples were run i n each  analysis to check the position and slope of the standard curve, and i f these samples and the curve d i d not agree closely, a new curve was calculated.  2.  Determination of Body Water i n the P i g  A)  Animals  Four l i t t e r s of Landrace-Iorkshire p i g l e t s were available f o r the experiment.  A l l l i t t e r s were by the same registered Landrace boar,  and were out of Yorkshire sows, which were a l l h a l f s i s t e r s .  The l i t t e r  s i z e was adjusted to eight p i g l e t s a t one week post partum, and when possible such adjustment provided equal numbers of males and females. b)  Rations and management  The sows and t h e i r l i t t e r s were f e d on standard U.B.C. swine  -15 rations and were subjected to the management procedures described more f u l l y i n Section I I . c)  A n a l y t i c a l in-vivo The p i g l e t s were t r a n q u i l i z e d (see appendix) with acepromazine  maleate (0.5 mg/lb body weight, given intramuscularly) and were then given an intramuscular i n j e c t i o n of p e n i c i l l i n ( D i c r y s t i c i n , containing  150,000  I.U. of p e n i c i l l i n G procaine,  potassium, 0.125  Gm.  50,000  I.U. of p e n i c i l l i n G  of dihydrostreptomycin sulphate and 0.125  streptomycin sulphate per cc.) at the rate of  20,000  Gm.  of  I.U. of p e n i c i l l i n  G. per kilogram of body weight to o f f s e t any possible i n f e c t i o n introduced while blood was being withdrawn and the deuterium oxide i n j e c t e d . was then weighed as accurately as possible and 5*0  The p i g  cc. sample of blood  was withdrawn by heart puncture, using s t e r i l e vacutainer equipment. Deuterium oxide (99.7k% pure) was  then i n j e c t e d i n t r a p e r i t o n e a l l y at the  rate of 2.0 cc. per Kg. of predicted t o t a l body water, giving a f i n a l in-vivo concentration of about 0.2 body water.  per cent of deuterium oxide i n the  A f t e r s u f f i c i e n t time had elapsed f o r the deuterium oxide  to become completely d i s t r i b u t e d throughout the tissues (2k, 36, a second blood sample was f o r the second time.  79,  9k)  taken, a f t e r which the animals were weighed  Urine was not generally c o l l e c t e d , since i t was  observed that the t r a n q u i l i z e d animals d i d not urinate u n t i l a f t e r the excitement of the second heart puncture, a t which point the heavy water would be e q u i l i b r a t e d i n the tissues (though not necessarily with the urine i t s e l f ) , and any u r i n a r y loss could not greatly change the concent r a t i o n of heavy water i n the body f l u i d s .  The animals were allowed  - 16 access to food and water up u n t i l the time of analysis, but were not allowed anything to eat or drink during the time of the i n j e c t i o n and sampling schedule.  A f t e r each weekly determination one animal was  selected a t random from each l i t t e r and k i l l e d f o r t o t a l chemical analysis in-vitro.  P r i o r to s a c r i f i c e , these animals were subjected to a t h i r d  heart puncture, i n order that the extent of deuterium oxide e q u i l i b r a t i o n could be checked.  In addition, urine, muscle and f a t samples were taken  to check on the d i s t r i b u t i o n of the reference solute i n the t i s s u e s . Water was recovered from the blood, urine and tissue samples by placing the l a t t e r i n t i g h t l y stoppered 250 cc. Erlenmeyer  flasks,  having pointed cold fingers projecting through the tops and 5*0 cc. c o l l e c t i n g v i a l s placed beneath the cold f i n g e r s .  The f l a s k s were then  placed i n a water bath at 80°C. and cold water a t 10°C. was c i r c u l a t e d through the cold f i n g e r s .  Water was thus removed almost completely (to  the vapour pressure of water a t 10°C.) water samples were then p u r i f i e d and analysed by the method of Keston e t a l . (U9) as described above. F i n a l i n - v i v o body water calculations are based on the following formula:  Per cent t o t a l body water ~  (cc. deuterium oxide injected) x (10) (concentration o f D2O i n ) (blood water a t T2 minus) (concentration of D2O i n ) (blood water a t T ) x  ^  D °in  x  2  AC x f ,  1  0  x  (Body Weight) ( at T ) 9  2  - 17 d)  Analytical in-vitro  The saw and were Body w a t e r after  then  was  and  weight  i n a  on weights  small tunnel  taken  on  slices  with  band  75>°C.  dryer at the  a  carcasses  just  then  subjected  to  further  analyses  as  Evaluations  effects  and e f f e c t i v e n e s s  was  actually  compared  densitometrically with  necessary.  t h e r e was  of  same s t a n d a r d  i i )  estimated. expected  The  used  also  deuterium oxide samples  i n  made  distillation i n  the  precision  On t h e  determine  was  the  p u r i f i c a t i o n method  basis  v a r i a t i o n i n the  whether  same  animal  d i s t i l l e d water  or not  i n  purification  to  determine  whether  the  purifying  t r a i n by  or  i n p u r i f i e d and u n p u r i f i e d  was  not  comparing aliquots  of  the  r e p r o d u c i b i l i t y and l i n e a r i t y  of  the  analysis.  of  of  freshly  from the  were  densitometrically.  A s t u d y was made  curves  i i i )  to  A test  any f r a c t i o n a l  concentrations  standard  of  P u r i f i e d and u n p u r i f i e d b i o l o g i c a l water  f a l l i n g drop apparatus,  the  up i n t o  II.  Checks  The  evaluated.  of  based  cut  Physical i)  the  constant  were  dry carcass  i n Section  Procedure a)  the  frozen,  sacrifice.  described  source  were  d r i e d to  calculations  The  3.  carcasses  the  the  dropping and p u r i f y i n g  pooled variance  measurement  of  total  for  the  operation  was  dropping runs,  body water  resulting  the  from  - 18 a n a l y t i c a l errors and v a r i a b i l i t y was calculated.  Consideration was also  given to v a r i a t i o n i n t o t a l body water measurement due to errors i n i n j e c t i n g the deuterium oxide and i n weighing the animals. b)  Biological i)  The e q u i l i b r a t i o n of heavy water i n the p i g was studied.  The  e q u i l i b r a t i o n curves f o r two small pigs which were i n j e c t e d i n t r a c a r d i a l l y with deuterium oxide are presented.  In addition a s t a t i s t i c a l analysis  was c a r r i e d out to test f o r s i g n i f i c a n t differences i n deuterium oxide content of water taken from the two f i n a l blood samples and from muscle, f a t and urine of the animals s a c r i f i c e d f o r i n - v i t r o analysis.  ii)  The e f f e c t of i n i t i a l and f i n a l weights on the c a l c u l a t i o n  of body water was investigated from a t h e o r e t i c a l and s t a t i s t i c a l point of view i n order to determine which of the two would provide the most v a l i d calculated r e s u l t s .  iii)  A study was made of the agreement between body water  determined by in-vivo and i n - v i t r o methods i n the same animals. iv)  An i n v e s t i g a t i o n was carried out to determine the amounts of  urine excreted by the animals during the time of the i n j e c t i n g and sampling schedule.  v)  Consideration was given to the possible e f f e c t s of the body  water determination techniques on the normal growth and development of the piglets.  - 19 -  C.  1.  EXPERIMENTAL RESULTS  Evaluation of the P u r i f i c a t i o n  Technique  Table 1. summarizes an experiment inhich shows that whereas i t could be expected that water recovered from the blood of normal pigs which had not been injected with deuterium oxide would be the same as d i s t i l l e d water, the unpurified b i o l o g i c a l water evaporated d i r e c t l y from blood was densitometrically very d i f f e r e n t from pure water.  On the other hand,  p u r i f i e d b i o l o g i c a l water was not s i g n i f i c a n t l y d i f f e r e n t from d i s t i l l e d water.  I t i s therefore evident that p u r i f i c a t i o n of the b i o l o g i c a l water  samples p r i o r to densitometric analysis i s j u s t i f i e d and necessary.  The  s l i g h t l y s i g n i f i c a n t F t e s t f o r w i t h i n and between aliquot variances does not change the v a l i d i t y of the h i g h l y s i g n i f i c a n t F test comparing between and within treatment variances. Tables 2. and 3. present evidence to show that no measureable f r a c t i o n a l d i s t i l l a t i o n of H^O and HDO occurs during p u r i f i c a t i o n of heavy water solutions containing 0.1 and 0.5% deuterium oxide. 2.  Standard Curves Tables 1; and 5 present descriptions of two t y p i c a l standard  l e a s t squares l i n e a r regressions of reciprocal dropping times against the concentrations of deuterium oxide i n standard solutions.  Both curves  exhibit very small standard errors of estimate, and both showed no deviat i o n from l i n e a r i t y over the range of concentrations included i n the regression  (0.08$  to  0.36$  DO).  Figure 5» shows how very s l i g h t (not  f/GUfifc.  'hncfsi  /wo 73f /  5  WELN  S /A/VDA8U  TW/-. 8 c c / r K O C H L  CONCENTRATION  2.0  21  CuRvt  j  SHOWING  IHL  / /nr AMU  F\OPPING  O l j j f U l i f i / O f l OX'OiT iftj  WA  RLLK  7  i £ R,  I . x /O  T T -- 77 Mt' SIXTHS  Or A  C U R V E A/o, 1  /  ^  0-OII2  6/*w  /VO.  2  Y = /«5/24 X  - oooq I  1-2.  OOT_ OO  0<2  O'l PERCENT  BI  0-3  WC/GHT o r 9 9 74 % 7J 0 w STANDARD 2  SOLUTIONS  H  f  HONSH  measurable) changes i n the adjustment of the water bath thermostat affects the slopes of the standard curves.  A comparison of the slopes  of the two curves shown (Table 6) indicated that the difference i n slope was not s i g n i f i c a n t .  However, s l i g h t l y greater changes i n ther-  mostat s e t t i n g could be expected to produce more marked changes i n the standard curves.  3»  Evaluation of the Precision of the In-Vivo A n a l y t i c a l Methods Table 7 presents an estimate of the s e n s i t i v i t y of the present  densitometric method f o r determining deuterium oxide i n water. pooled within-aliquots variance f o r values of l / T x 10^ ± within-aliquots variance of l / T x 1(P Tables k and  s  The  based on the  shown f o r the standard curves i n  The calculations i n Table 7 indicate that differences i n  deuterium oxide concentration of*0.0087$ could be detected between two aliquot runs, each consisting of s i x r e p l i c a t e drops.  Calculations shown i n Table 8 indicate that the above a n a l y t i c a l s e n s i t i v i t y could be expected to lead to a v a r i a t i o n i n per cent t o t a l body water of 2.6$,  and that additional variations i n per cent body  water of approximately i 0.5 and 0.7 per cent respectively could be due to errors i n weighing the animals and i n i n j e c t i n g the heavy water during the in-vivo analysis.  Such errors would lead to a t o t a l predicted  - 21 v a r i a t i o n of i  3,8 per cent body water, and examination of the agreement  between values under the X and Y  2  columns of Table 1 1 suggests that t h i s  estimate i s a f a i r l y good expression of f a c t . k*  E q u i l i b r a t i o n of Deuterium Oxide i n the Pig  Figure 6 describes the e q u i l i b r a t i o n of i n t r a c a r d i a l l y i n j e c t e d deuterium oxide i n two small pigs.  Tables 9 and 1 0 summarized tests  c a r r i e d out to determine the extent of e q u i l i b r a t i o n a t the time of taking the second blood sample i n pigs i n j e c t e d i n t r a p e r i t o n e a l l y with oxide.  deuterium  In a l l cases but one i t was found that s t a t i s t i c a l e q u i l i b r a t i o n  had been achieved by the time the second blood sample was taken.  Table 9  also indicates that by the time the second blood sample was removed, deuterium oxide was d i s t r i b u t e d equally i n the water of blood, muscle and f a t , but not i n the urine.  5.  Comparison of In-Vitro and In-Vivo Body Water Values  Table 1 1 l i s t s per cent body water values found f o r the same animals by in-vivo and i n - v i t r o methods, with the in-vivo values calculated on the basis of both i n i t i a l and f i n a l weights being shown.  Table 1 2  shows the l i n e a r regression of body water values of each method of in-vivo body water c a l c u l a t i o n against the i n - v i t r o data, using the l a t t e r as a standard.  Since a perfect c o r r e l a t i o n between the two methods would y i e l d  a regression l i n e having a positive slope of one, the f i n a l weight was chosen as being the most s a t i s f a c t o r y basis f o r c a l c u l a t i o n of in-vivo body water values on the grounds that the regression of these values against the i n - v i t r o r e s u l t s gave a higher c o r r e l a t i o n c o e f f i c i e n t  FIGURE  6  THE CHANGES IN CONCENTRATION OFTJ^O IN BLOOD WATER FOLLOWING THE 1NTR/\~ CARCMAJL JlMTtTCTIOrv OFT^O,  T IG  Mo.  A  — 0-  %  D  7  0  P/G /Vo. 73  trw BJLOOTJ  <s>-  WATER.  C2.  WciGHfX; PIG A PIG -B  O  B  |  INITIAL APPARENT  O  *To TJW  60  0-882 K G . M25  TLo  HQ.  CoNceNTK/moNS  i~fcO  l3o  IN / V / / V 0 T E 5 A T 7 E R  iNieCTIOfM  O F 97-74% 2*0  260 OF  2-0  ML.  - 22 -  (0.8185  as  (0.8191; a s  coefficient on  in-vivo  6.  Extent  values  of  against  calculated  the  were  noticed of  the  the  occasions  a  the  times  sample  the  of  injection  represented  d i d the  of  cage  Injection  so  that  of  regression  weight.  and Sampling  number o f  urine  Period  tranquillized  excreted  during  and sampling of  heavy water  was  was  occurred  loss  regression  similar  i n i t i a l  passed.  It  during or  second blood sample.  marked weight  greater  representative  injection  u r i n a t i o n sometimes  concluded that  blood  than  basis  v i r t u a l l y no u r i n e  withdrawal of  the  on the  respective  collected, that  between  0.7517)  placed i n a metabolism  period of  c o u l d be  significantly  Urination by Animals During  When on t h r e e piglets  0.8013) a n d  compared w i t h  after  On t h i s  usually noticed  heavy water  and of  p r i m a r i l y an i n s e n s i b l e  the  the  taking  loss  of  blood  however, excitement  basis, i n  and  i t  was  animals the  second  water  from  the  lungs.  7.  The  Effects  In-Vivo  on  of the  the  Subsequent  No o b v i o u s c o u l d be The  to  the  t r a n q u i l i z a t i o n d i d not  visual  their  examination  quilizer damage  to  injection  caused  the  heart  of  dams the  punctures,  on  prevent after  site  the  the  Determination  of  of  the  growth and development  in-vivo  showed no  d i r e c t l y by  Used f o r  Growth and Development  i l l effects  attributable  when r e t u r n e d  that  Techniques  body water the  animals  analysis. the  increase needle.  In  Water  were  noted  which  technique.  from suckling vigorously the  animals  damage  Autopsy of  when p r o p e r l y c a r r i e d  Body  Pig  determination  previous week's i n muscle  of  gross  intramuscular over  sacrificed  out  killed  left  the  tran-  mechanical  animals  almost  no  showed  visible  - 23 -  damage this  of  the  musculature  respect,  puncture  due  however, to  a  tear  made  an unexpected  pigs  which were  in  injections consume of  not  pigs.  the  the  of  creep  in-vivo  several i n  the  yet  the  pigs wall  eating  levels  However,  iron  either  of  the  ration.  technique  heart  of  the  effect  the  further  on growth i s  largely  presented  of  those  off-set  the  (In heart  animal  b l o o d samples  creep r a t i o n  time when the  discussion  during  caused when the  s l i g h t l y below was  body w a l l .  instantly  iron-containing  up u n t i l A  or  The w i t h d r a w a l o f  w h i c h were  this  dextran  the  heart  died almost  sudden movement.)  blood haemoglobin  untreated  of  i n  frequent  animals  began  possible  i n Section  resulted  found  by  of  II.  to  effects  - 2h TABLE NO. 1 SUMMARY OF ANALYSIS OF VARIANCE TO DETECT DIFFERENCES IN DROPPING TIMES BETWEEN SAMPLES OF DISTILLED WATER ( I ) , UNPURIFIED WATER RECOVERED FROM PIGS NOT INJECTED WITH D 0 (II) AND PURIFIED WATER FROM THE SAME BIOLOGICAL SOURCES (III) 2  Computing o r i g i n - 720 seconds  No. of drops run per aliquot = 8  6 Treatment  Aliquot  Aliquot Mean  Treatment Mean  I Distilled H0  1 2 3 h 5  23.3 25.3 20.1 15.1 21.2  21.0  II Unpurified Biological Samples  l 2 3 h 5  -ill. 6 -21.3 -12.1 -21.1  III Purified Biological Samples  l 2 3 h 5  16.5 22.6 13.1  2  19.1  18.9  2U.3  d.f.  Within aliquots sj -  92.5  105  s|r173•1  Test F-x _ s| S?  12  Between Treatments s| =• 170l|2.  F  2  173.1 . 1 . 8 7  II\  III  * -  )  I  ) P = 0.01 )  i  p-0.05  Significance Just s i g n i f i c a n t at P r 0.05  92.5  S< _ 98.5  Duncan's Multiple Range Test on Treatment Means Shows:  III  8.2  - 9.1  Variance  Between Aliquots  -15.6  Total Mean  Highly s i g n i f i cant a t P- 0.01  - 25 TABLE NO. 2  SUMMARY OF ANALYSIS OF VARIANCE TO DETECT DIFFERENCES IN DROPPING TIMES OF PURIFIED AND UNPURIFIED ALIQUOTS OF STANDARD 0.1% D 0 RESULTING FROM CHANGE IN CONCENTRATION CAUSED BY FRACTIONAL DISTILLATION IN THE PURIFICATION TRAIN 2  No. of drops run per aliquot - 6  Computing o r i g i n - 670 seconds  6 Treatment  Aliquot  Aliquot Mean  1 2 3  7.00 6.17 1.67 6.33 6.87  5.61  12.33 5.50 7.66 U.50 5.50  7.10  Purified  h  5 l 2 3  II Unpurified  h  5  S^z 66.5  Significance >2 - 57.2  66.5 Between Aliquots  S^r57.1  Between Treatments  S^r33.8  33.8  S2 2  Conclusion: A  Total Mean  6.35  d.f.  Variance Within Aliquots  Treatment Mean  57.2  Not s i g n i f i c a n t a t P= 0.05  Not s i g n i f i c a n t at P - 0.05  I - II  In t h i s test, 3 drops of each aliquot were run, and then several hours l a t e r the remaining 3 .drops were run. Since there was a highly s i g n i f i c a n t , cons i s t e n t difference between the dropping times of the f i r s t and second sets of 3 drops i n each aliquot, S^ i s calculated on the basis of sets of 3 drops instead of 6. This further d i v i s i o n of the data absorbs 10 degrees of freedom.  -  26  -  TABLE NO.  3  SUMMARY OF A N A L Y S I S OF V A R I A N C E TO D E T E C T D I F F E R E N C E S I N D R O P P I N G T I M E S OF P U R I F I E D A N D U N P U R I F I E D A L I Q U O T S OF S T A N D A R D 0 . 5 $ D - 0 R E S U L T I N G F R O M C H A N G E I N C O N C E N T R A T I O N CAUSED BY F R A C T I O N A L D I S T I L L A T I O N I N THE P U R I F Y I N G T R A I N  No.  of  drops  run  per  aliquot  -  6  Computing  origin  U82  -  seconds  6  Treatment  Aliquot  Aliquot  1 Purified  2  -1.66 -5.0  3  -U.33  5  1.83 0.0  k  Mean  Treatment  Mean  Total  Mean  -1.83  -2.11 II  1  -3.17 -0.33 -1.33 -1.83 -5.33  2 Unpurified  3 h 5  Variance  Within  Between  aliquots  aliquots  Between Treatments  -2.U0  d.f.  s| -  18  50  s|  -  36  8  S^  =•  Significance  36 _ 2  Not  18  at  ii.8  Not  36~  at  significant P =  0.05  significant  U.8  Conclusion:  1=  II  P r  0.05  - 27 TABLE NO. k  Standard Curve No. 1 Least Squares Linear Regression of l / T x 10-3 (Y) against % D2O i n Standard Solutions (X). T i s dropping time measured i n sixths of a second. "Y at each point i s the mean of the r e c i p r o c a l dropping times of 6 r e p l i c a t e drops. M  X %  Y l/T x 103  D0 o  0.0817 0.21+27 0.3216 0.0950 0.2188 0.3675 Equation:  1.1|132 1.6U80 1.750U 1.U15U 1.6100 1.83U9 Y ^ 1.U813 X -v 1.281+3  -  0.999  Variance about Regression Line (S|) d.f. r It  = 0.000125  Standard Error of Estimate  r 0.0112  Variance of Regression C o e f f i c i e n t S g (d.f.- U)  - 0.00031  Pooled Variance of Within Aliquot Reciprocal Drop Times (s|) - O.OOOlUO Test f o r Significance of Regression:t  1 . 9 9 8 . 36.5  0.05U6 Highly s i g n i f i c a n t at P -  0.01  - 28 TABLE NO. 5  Standard Curve No. 2 Least Squares Linear Regression of l / T x 10^ (Y) against % D2O i n Standard Solutions (X). T i s dropping time measured i n sixths of a second. Y" at each point i s the mean of the r e c i p r o c a l dropping times of 6 r e p l i c a t e drops. M  Y D  l/T x  2°  1.2566 1.5152 1.6239 1.2901 1.1+833 1.6959  0.0817 0.21+27 0.3216 0.0950 0.2188 0.3675 Equation:  103  I r  1.5121+ X  -+  1.11+30  - 0.999 Variance about Regression Line (s|) ( d . f . r 1+)  r 0.0000825  Standard Error of Estimate  - 0.00907  Variance of Regression C o e f f i c i e n t S (d.f. x. k) 2  - 0.00020  Pooled Variance of Within Aliquot Reciprocal Drop Times (S )  0.000057  Test f o r Significance of Regression:t  Vn - 2, d.f. ~ 1+  2  =  r  1.998 _36.5 0.051+6 Highly s i g n i f i c a n t at P =: 0.01  - 29 TABLE NO. 6 Comparison o f t h e S l o p e s of T y p i c a l S t a n d a r d Curves  t . (b  where b]_ and b  2  x  -  b ) 2  and S^ and S  2  are t h e  r e s p e c t i v e s l o p e s and v a r i a n c e s o f r e gression coefficients  o f t h e two l i n e s .  Comparing S t a n d a r d Curves 1 and 2,  ( S | +- s|)s  ~ (0.00031 + 0.00020)^ = (0.00051)^ c 0.0225  .\  t  =  1.512U - 1.U813 0.0225 0.0311 - 1.382, 8 d.f. 0.0225 Not s i g n i f i c a n t a t P =: 0.05  - 30 TABLE NO. 7 Calculation of the S e n s i t i v i t y of the Determination of Deuterium Oxide i n Water  A representative within-aliquots variance f o r values of l / T x 10-^ was calculated, based on the within-aliquots variances shown i n Tables h and 5 f o r S£.  This pooled variance was 0.0001.  The standard deviation was  therefore i 0 . 0 1 . The smallest s i g n i f i c a n t difference between the l / T x leP mean values of two 6-drop aliquots a t P — 0 . 0 5 i s therefore as follows: t  Q < C  £ , 10 d.f.  2.23  *1 - x  2  (2.23) (0.01) ^ 0.0129  X-j-X-  From equation 1, Table h, a change of 0.1$ D 0 gives a difference i n l / T x 2  IO  3  of 0.11*81.  Therefore the Method w i l l detect differences i n % D 0 i n 2  water o f : 0-0129  0.lli8l  x  0.1-^0.0087$  - 31 TABLE NO. 8 The E f f e c t s of A n a l y t i c a l S e n s i t i v i t y and of Weighing and Injecting Errors on the Precision of In-vivo Body Water Measurement Consider a 15 kg. p i g , containing 65$ body water, and receiving 19.5 cc, of D 0 to give a f i n a l blood water concentration of 0.2000 $ D 0 . 2  2  $ B.W.  =  19.5 x 10  _ 65$  0.2000 x 15 1.  The E f f i c e of A n a l y t i c a l Sensitivity} Assume difference i n blood water D 0 of 0.0087$ 2  $ B.W.  =  19.5  x 10  - 62.1*$  0.2087 x 15  Thus, one can expect v a r i a t i o n of+2.6$ body water due to a n a l y t i c a l insensitivity. 2.  E f f e c t of Making a \ l b . (O.llU kg.) Error i n Weighing the Animal:  $ B.W. _  19.5 x 10 - 6Lu5$ 0.2000 x 15.11U  This error would lead to a t 0.5$ v a r i a t i o n i n per cent body water. 3.  E f f e c t of Losing or Overestimating 0.2 cc. of D 0 during Injection: 2  $ B.W._  19.3 x 10 ~ 6U.3$ 0.2000 x 15  This error would lead to a t 0.7$ v a r i a t i o n i n per cent body water. 1+.  Total Variation: On the basis of the above calculations, a maximum v a r i a t i o n of l l 3 . 8 $ of body water can be expected, when the present method i s used.  - 32 TABLE NO. 9  Summary o f  Pig No.  Final Weight in kg.  122  T2  Equilibration Analyses  - T0 (min.)  T - T (min.)  22.25  315  515  2, 3 , 3M, 3 F  F  N.S.  135  20.70  305  hk5  2, 3 , 3M, 3 F  F  N.S.  131  19.07  255  Uio  2, 3, 3M, 3F  F  N.S.  13U  1U.35  198  2h3  3M, 3F  F  N.S.  12U  13.39  176  230  2, 3 , 3M, 3F  F  N.S.  130  25.20  315  U55  2, 3, 3M,  3F  F  N.S.  133  10.71  226  270  2, 2 - U , 3 2, 2 - U 2, 3  F  132  29.96  310  510  2, 3, 3M, 3F  F  i  Blood sample  2: 2-U:  3,  /  3M,  N.S.:  Urine  3F:  Not  taken  sample  Water  3  at  of  . 2, 3,  Tg m i n . a f t e r  collected  from blood,  significant  Comparison* Dropping Times  2  at  D2O  Significance Test  injection  at  P=0.05  Significant Significant N.S. N.S.  at  T^  Tg  muscle  and f a t  tissue  collected  at  T.  /  -  33  -  TABLE NO.  Summary o f  Pig  Final Weight in kg.  No.  (mm.  7  9  Equilibration Analyses  T - T (min.)^  of  Comparison* D r o p p i n g Times  Significance Test  at  P=0.05  /  13U  5.28  56  135  2,  t  N.S.  110  8.22  90  165  2, 3  t  N.S.  110  10.67  65  9k  2, 3  t  120  k.99  70  102  2, 3  t  N.S.  127  6.81  79  128  2, 3  t  N.S.  121  13.62  228  296  2, 3  t  N.S.  130  20.70  275  kk6  2, 3  t  N.S.  116  19.30  230  295  2, 3  t  N.S.  12U  9.99  228  2, 2 - U  t  Very significan a t P- 0.01  122  19.98  325  k60  2, 3  t  N.S.  132  25.88  310  U50  2, 3  t  N.S.  122  15.21  300  U60  2, 3  t  N.S.  39c  /  2,  3 s  Blood samples  2-U:  Urine at  N.S.:  Not  T  taken  2  significant  at  T  2  and  T^  Significant ( N . S . a t P = 0.(  -  - 3h TABLE NO.  Equilibration  Body Weight  in  kgs.  Times  for  10  Intraperitoneal  D2O  Injections  Time i n M i n u t e s w i t h i n w h i c h S t a t i s t i c a l E q u i l i b r a t i o n of D2O with Fluids other than Urine Occurred  102  k.99  5.28  56  6.81  79  8.22  90  10.67  Not  equilibrated  10.71  226  13.39  176  13.62  228  lii.35  198  15.21  300  19.07  225  19.30  230  19.98  325  20.7  275  20.7  305  22.25  315  25.20  315  25.88  310  29.96  310  i n  9k  min.  - 35 TABLE N O .  11  C O M P A R I S O N O F BODY W A T E R V A L U E S D E T E R M I N E D I N - V I T R O B Y D E S S I C A T I O N ( X ) A N D I N - V I V O ON T H E S A M E A N I M A L S J U S T P R I O R TO S A C R I F I C E B Y T H E D E U T E R I U M O X I D E B I L U T I O N T E C H NIQUE. T H E I N - V I V O V A L U E S A R E C A L C U L A T E D ON B O T H A N I N I T I A L WEIGHT ( Y ) AND F I N A L W I G H T ( Y ) B A S I S . x  Pig No.  % H 0 by 2  Drying  2  %  H 0by D 0 2  2  X  Y  W  1-A 1-B  x  %  H 0by D 0 2  l Basis  7U.0  2  Y  W  2  Basis  52 5U 56 59 57 118 llU 110 113 116 125 136 120 127 119 123 121 133 12U 13U 129 131 135 122  79.9 6U.5 63. h 65.1 61.U 63.3 68.1 63.U 67.U 65.6 62.7 71.1 72.0 68.3 66.9 67. U 66.8 66.8 65.9 68.5 68.1 63.5 63.7 63.5 65.3  77.9 6ii.O  62.5 67.5 6U.6 62.9 62.9 63.8 61.U 67.2 59.5 6U.7 68.1 63.5 6U.8 65.5 62.2 61.6 65.U 67.8 62.2 58.0 60.5 63.5 62. U  77.6 78.7 6U.3 61.2 67.5 . 65.9 6U.0 63.3 6Ii.li 63.9 67.8 60.3 6U.7 70.0 6U.6 67.5 68. h 6U.0 62.7 66.5 69.1 63.1 59.5 62.0 65.8 63.8  Mean:-  66.99  6U.55  65.79  79.0  - 36 T A B L E N O . 12  Linear  Regressions  o f Y-j_ a n d Yg  Y  Equations s-  1  =  0.7517  (Significant  Standard Error of Estimate:-  of  Coefficient  X +  X from Table  1U.197  Regression ( S g ) : -  Test  at  (b! -  =  just  =  0.819U  X + 10.90+  0.8185  (Significant  0.01)  at  P s  2.761  0.00050  0.00059  i n Difference  of  b&  0.0677 ~ 2.05; d . f . ? ii8 0.033  Test  2  2.557  of Significance  t  P =  N o . 11  Y  0.8013  Correlation Coefficient:  Variance  against  significant  at  P —  0.05  Slopes:  0.QL )  D.  1.  DISCUSSION  Densitometric Method f o r Deuterium Oxide a)  Determination  L i n e a r i t y of c a l i b r a t i o n curves Stokes law states that when a sphere f a l l s i n a viscous medium, V_  2 ga  2  (dx  -  d ) 2  9N where  V  i s the rate of a l l , measured i n cms/second  a  i s the radius of the sphere  g  i s the acceleration due to gravity i n cms/second  d  and d  N  i s the c o e f f i c i e n t of v i s c o s i t y i n dyne-sec/cm or poises.  1  2  2  are the densities of the sphere and the medium i n Gm/cm3 2  On the basis of the above r e l a t i o n s h i p , i t would be expected that the r e c i p r o c a l of the time of f a l l of water drops containing d i f f e r e n t amounts of deuterium oxide would be d i r e c t l y proportional to the concentration of heavy water i n the water samples, providing the size of the drops and the density and v i s c o s i t y of the viscous medium are kept constant.  Keston  et a l . (1+9) have shown that the. r e l a t i o n s h i p of the r e c i p r o c a l dropping time through orthofluorotoluene to deterium oxide concentration i s not l i n e a r when considered over a concentration range of 0 to 8,0$  deuterium  oxide, with the rate of change of dropping time with respect to concentrat i o n becoming less as the l a t t e r increases.  These workers, however, found  that the r e l a t i o n s h i p was e s s e n t i a l l y l i n e a r i n the concentration range of 0 to 0.7$  deuterium oxide, and t h i s l i n e a r i t y has been substantiated i n  the present study.  I t i s possible that n o n - l i n e a r i t i e s i n the above r e l a -  tionship may be due to the exchange of heavy water and water between the small dissolved aqueous phase of the orthofluorotoluene and the f a l l i n g  - 38 -  drop, r e s u l t i n g i n a small change i n the density of the drop as i t f a l l s . Such a p o s s i b i l i t y i s suggested by the f a c t that good r e p r o d u c i b i l i t y of dropping time was not obtained with pure dry orthofluorotoluene u n t i l a number of drops containing d i f f e r e n t concentrations of deuterium oxide has been allowed to f a l l through the fresh medium. b)  Micro-dropping pipette The micropipette used (Figure U) was of very simple  construction  as compared to the elaborate design described by Keston et a l . (1+9). I t was f e l t , however, that good r e p r o d u c t i b i l i t y of drop s i z e was obtained, and that provided the pipette was kept scrupulously clean, that i t d i d not contribute appreciably to the error of deuterium oxide measurement compared with the thermostat errors discussed below. c)  Water bath Probably the major contribution to the variance of the dropping  procedure arose from the thermostatic control of the water bath.  On the  tsesis of Stokes Law expressed i n 1. above, i t w i l l be seen that the radius of the drop, the densities of both sample and medium and the c o e f f i c i e n t of v i s c o s i t y of the medium are a l l subject to v a r i a t i o n due to temperature fluctuation.  Due to the low c o e f f i c i e n t of cubic expansion of water, the  most thermally susceptible of the factors mentioned above are the density and c o e f f i c i e n t of v i s c o s i t y of the orthofluorotoluene.  Since the v i s c o s i t y  of orthofluorotoluene i s decreased by only about one tenth as much per u n i t increase i n temperature as i n the density of the dropping medium, the net e f f e c t of a r i s e i n water bath temperature i s an increase i n the value of 1. T  - 39 The thermostat used was s p e c i f i e d by the manufacturer as being able to hold a constant temperature of — 0.01°C.  Such a f l u c t u a t i o n would give a  f l u c t u a t i o n of - 0.001 per cent of apparent deuterium oxide concentration f o r a single dropping run, or a difference o f i 0.002 per cent of apparent deuterium oxide when the difference between two dropping run means i s considered.  On the other hand, a 0.1°C. v a r i a t i o n i n temperature could  lead to a 0.02 per cent change i n apparent deuterium oxide concentration or a f l u c t u a t i o n i n per cent body water of — 6 per cent.  Figure £ shows  the difference i n c a l i b r a t i o n curves r e s u l t i n g from a non-measurable change i n thermostat s e t t i n g .  A c y c l i c f l u c t u a t i o n of the bath temperature occurred and was accentuated by overheating of the bath due to heat which dissipated from the heating c o i l a f t e r the thermostat had cut out.  This f l u c t u a t i o n ,  which was r e f l e c t e d i n rythmic changes i n the dropping times within a single sample run was minimized, but not eliminated by allowing two hours f o r the bath to e q u i l i b r a t e a f t e r i t had reached the desired temperature of 27.9°C. before any analyses were carried out.  The e f f e c t of t h i s f l u c -  tuation on the f i n a l body water calculations was further minimized because a complete thermostat cycle usually occurred within the time required to drop and record s i x r e p l i c a t e drops of a sample.  The f a c t remains however  that these fluctuations were probably the major source of variance i n the present densitometric procedure.  The following are some methods by which the thermal contributions to the dropping time variance could be reduced.  - ko i) ii)  The use of a more sensitive thermostat, The use of a smaller heating element •which would cause less overheating i n the water bath,  iii)  A more refined adjustment than the screw clamp used i n the present work f o r c o n t r o l l i n g the rate of flow of cold water through the cooling c o i l of the water bath to o f f s e t the heating e f f e c t of the s t i r r i n g blade motor. These adjustments could probably be made without any further  improvement i n the thermal i n s u l a t i o n of the water bath being necessary. S t i l l another means of c o n t r o l l i n g temperature more c l o s e l y would be to f i n d a dropping medium which had the r i g h t densitometric properties at some e a s i l y maintained constant temperature such as that of an ice-water mixture or of b o i l i n g alcohol.  d)  S e n s i t i v i t y of the densitometric method  I t has already been mentioned that the densitometric method used i n the present work was able to distinguish differences i n deuterium oxide concentration of - 0.0087$, and that t h i s s e n s i t i v i t y , with considerations being made f o r weighing and i n j e c t i n g errors lead to an expected p r e c i s i o n i n body water measurement of?3.8 per cent of body water, as compared to a p r e c i s i o n of ± 2.0 per cent reported by Schloerb (9k) other hand, Keston et a l . (k9)  from work done on humans.  On the  using a water bath thermostated to - 0.001°C.  reported that the densitometric method f o r determining heavy water i n water was able to distinguish differences of i 0.001  per cent of deuterium oxide which  -la  -  would lead to a v a r i a t i o n of - O.k per cent of body water i f this were the only source of v a r i a t i o n .  One obvious way  to increase the p r e c i s i o n of the present method  without making any other changes would be to increase the number of r e p l i cate drops per sample dropping run.  On consideration of Table 7, i t  i s evident that twelve drops per run instead of 6 would reduce the error of a single dropping mean f r o m - O.OOI4I to —  standard  0.0029. This would have  led to an a n a l y t i c a l d e f i n i t i o n of - 0.0061 per cent of deuterium oxide, but would have doubled the time spent on the f a l l i n g drop analysis, a factor which merits some consideration when a large number of samples are to be  analysed.  A further consideration i n this respect i s that, while i t i s possible and very desirable to increase the p r e c i s i o n of the densitometric method, the further increase i n t o t a l p r e c i s i o n i s eventually l i m i t e d by one's a b i l i t y to accurately weigh the l i v e p i g at the time of blood sampling.  2.  The E q u i l i b r a t i o n of Deuterium Oxide i n the Pig  A)  E q u i l i b r a t i o n time  In Figure 6 the e q u i l i b r a t i o n curves indicate that e q u i l i b r a t i o n of deuterium oxide i n the blood and tissues i n the new born pig following i n t r a c a r d i a l i n j e c t i o n occurs within about 80 minutes.  I t i s reasonable  to expect that e q u i l i b r a t i o n time increases with body s i z e , and that deuterium oxide i n j e c t e d i n t r a p e r i t o n e a l l y would e q u i l i b r a t e more slowly than would that i n j e c t e d intravenously or i n t r a c a r d i a l l y , since a d d i t i o n a l time would be required f o r the heavy water to d i f f u s e into the blood from  - Ii2 from the i n j e c t i o n s i t e .  Since the a n a l y t i c a l methods f o r deuterium oxide  had not been adequately tested by the author at the time when the pigs were a v a i l a b l e to enable optimum e q u i l i b r a t i o n times to be established before the in-vivo analysis, i t was necessary to estimate the e q u i l i b r a t i o n times on the basis of the l i t e r a t u r e (2k, that the times allowed were adequate.  36, 79, 9U).  Table 10 indicates  However, absolute e q u i l i b r a t i o n  i s not necessarily indicated, nor i s the p o s s i b i l i t y that s t a t i s t i c a l e q u i l i b r a t i o n occurred i n a shorter time than shown.  Some i n d i c a t i o n  of the l i m i t s are given by the f a c t that one ten kilogram p i g d i d not equilibrate i n ninety-four minutes.  b)  D i s t r i b u t i o n of heavy water i n the body a f t e r i n j e c t i o n Although heavy water was found to be d i s t r i b u t e d evenly i n f a t  and muscle tissue, slower e q u i l i b r a t i o n was found to occur with the urine, (2k).  I t i s probably that the rate of e q u i l i b r a t i o n of heavy water with ihe  urine depends i n v e r s e l y with the amount of urine i n the bladder at the time of i n j e c t i o n , since dueterium oxide would tend to enter the urine v i a the kidneys and not to any great extent by d i f f u s i o n through the fibrous walls of the bladder.  The lack of e q u i l i b r a t i o n of the heavy  water with urinary water a t the time of the second heart puncture may well be a major contributing factor to the f a c t that the in-vivo body water values tend to be s l i g h t l y lower than expected.  In such a case,  the deuterium oxide concentration i n the blood i s measuring a water volume which i s less than t o t a l body water approximately by that which i s i n the bladder as urine.  - Ii3 c)  E q u i l i b r a t i o n and loss of weight during the sampling  schedule  Table 12 indicates on an empirical basis that in-vivo body water values calculated using the f i n a l weight of the animal a f t e r the i n j e c t i o n and sampling schedule than those using the i n i t i a l weight using the i n i t i a l weight.  Theoretical considerations also support t h i s  finding, f o r u n t i l complete tissue e q u i l i b r a t i o n of heavy water has occurred, water l o s t insensibly contains l i t t l e or no deuterium oxide, so that the i n i t i a l weight contained some water with which the injected deuterium oxide never equilibrated and consequently a lower apparent r e l a t i v e body water content w i l l r e s u l t .  Examination of Table 11 shows that on the average the in-vivo body water values were one per cent lower than the corresponding i n - v i t r o values, whereas on a theoretical basis (50, 97, 107) s l i g h t l y higher values were anticipated.  This apparent underestimation can p a r t l y be explained  on the basis of the slow e q u i l i b r a t i o n of deuterium oxide with u r i n a r y water.  However, i t i s possible that the i n - v i t r o determinations are not  an absolute standard, since any loss of dry matter i n handling or drying of the carcass would be expressed as being part of the body water.  -hkE.  SUMMARY AMD CONCLUSIONS  A method f o r the in-vivo determination of t o t a l body water by the deuterium oxide d i l u t i o n technique has been tested on growing p i g l e t s , and the results obtained were i n ,g>od agreement with those obtained f o r the same animals by carcass dessication.  The f a l l i n g - d r o p densitometric  method f o r the determination of deuterium oxide i n the water recovered from blood or tissue has been c a r e f u l l y evaluated, and some suggestions for the further modification of the apparatus and techniques involved are presented.  I t i s f e l t that the r e l a t i v e s i m p l i c i t y of the techniques and  equipment used i n this investigation may render the method u s e f u l f o r more extensive sequential studies of body composition i n growing animals.  - U5 II.  CHANGES IN BODY COMPOSITION IN THE SUCKLING PIG  A.  INTRODUCTION  The phenotype of a l i v i n g animal represents the absolute value of an i n t r i c a t e complex of b i o l o g i c a l processes and sequences. Por many years now, studies i n animal body composition have been c a r r i e d out i n order to gain a more quantitative knowledge of animal growth, and of the extent of the genetic and environmental contributions to the l i v i n g state of animals. composition.  There are two main avenues of approach to studies of body The f i r s t has involved the d i r e c t physical and chemical  analysis of carcasses, and the data c o l l e c t e d by these laborious methods serve as the basis f o r the present knowledge of body composition (15, 32, 37, 57b, 59, 72, 77, 102).  27,  More recently, i n d i r e c t methods of measure-  ment have been devised by which the body composition of l i v i n g animals may by studied (19, 50).  As a r e s u l t of some early studies i n which the carcasses of r e l a t i v e l y mature animals were analysed, (57b), i t was concluded that the f a t free body mass had a constant composition, and that variations i n t o t a l body composition were e s s e n t i a l l y due to v a r i a t i ons i n the f a t compartment (76).  Several of the present day i n d i r e c t methods of  measuring body composition are based on this assumption (85, 50).  On the  other hand, Moulton et a l . (75) demonstrated that the f a t free body mass has a gross chemical composition which changes i n a d e f i n i t e way as animals develop from conception to maturity.  These findings have since been  substantiated by other workers, using a wide v a r i e t y of d i f f e r e n t species (87) and (102).  -he The most s t r i k i n g changes i n the composition of the whole animal body during growth are the decrease i n the r e l a t i v e water content and the increase i n the r e l a t i v e f a t content.  In the foetus, there i s a  very high water content and v i r t u a l l y no f a t . In some species, such as the human and the guinea p i g , fattening begins i n the foetus towards the end of  the gestation, so that at b i r t h the young have a r e l a t i v e l y high f a t  content (113).  In other species, such as the r a t and the p i g , v i r t u a l l y  no fattening occurs i n utero.  However, i n the baby p i g , i t has been  observed that considerable fattening occurs during the f i r s t few days of post natal l i f e (102).  A f t e r b i r t h , there i s a continued increase i n the  t o t a l f a t percentage of the body with a corresponding r e l a t i v e decrease i n t o t a l body water, with the rate at which these changes occur being l a r g e l y a function of the plane of n u t r i t i o n (63, 8 l ) . In the f a t free body, there i s once again a progression from high r e l a t i v e amounts of water i n the foetus to lower l e v e l s of water i n the adult.  Moulton (73>) and Reid (87) working with the bovine have shown  that the per cent water of the f a t free body f a l l s  very r a p i d l y from  l e v e l s i n excess of ninety-five per cent shortly a f t e r conception to about seventy-four per cent at an age of about four months a f t e r b i r t h , and that thereafter there i s a more gradual decrease i n the per cent body water. Nitrogen and ash showed a r e c i p r o c a l change r e l a t i v e to water, both showing a very rapid r e l a t i v e increase i n the f a t free body mass, from conception up to the post natal age of about four months, a f t e r which there was a continued, but gradual increase.  These changes have been demonstrated i n  many other species, with the exception that the point where rapid change gives way to slower change varies i n t e r s p e c i f i c a l l y i n terms of the time  - U7 elapsed between b i r t h and maturity (102). These changes i n body composition are n e c e s s a r i l y the r e s u l t of progressive changes a t the tissue and c e l l u l a r l e v e l s (38).  The  developing embryo a t one e a r l y stage consists of three primary germ layers, ectoderm, mesoderm, and endoderm.  A portion of the developing  mesoderm (mesenchyme) i n f i l t r a t e s between structures which are developing from non-mesenchymal sources.  This mesenchymal tissue eventually d i f f e r e n -  t i a t e s into various forms of connective t i s s u e .  I n i t i a l l y , mesenchymal  tissue consists of undifferentiated c e l l s rather widely dispersed i n an amorphous i n t e r s t i t i a l c o l l o i d a l substance which contains a high proportion of water.  As the embryo develops, the mesenchymal tissue d i f f e r e n t i a t e s  i n t o more h i g h l y s p e c i a l i z e d types of connective tissue having an i n t e r c e l l u l a r substance which contains less water and an increasing proportion of ash and s t r u c t u r a l proteinaceous constituents.  This general trend,  most rapid during the d i f f e r e n t i a t i o n and development of tissues, continues throughout the l i f e of the i n d i v i d u a l , causing a decrease i n i n t e r c e l l u l a r f l u i d (67). material.  These changes are not l i m i t e d to i n t e r c e l l u l a r  Thus deRobertis (90) i n a discussion of c e l l senescence points  out that i n aging i n c e l l s , there tends to be an increase i n i n e r t proteinaceous materials such as keratin and p l a s t i n , a decrease i n water content, and an o v e r a l l increase i n protoplasmic consistency as w e l l as an i n f i l t r a t i o n of the c e l l s by various l i p i d s .  In 1923, Moulton (75) noticed although the composition of mammals varies i n t e r s p e c i f i c a l l y a t b i r t h , that the body composition of a l l mammals changed i n a s i m i l a r way a t s i m i l a r times r e l a t i v e to t h e i r respective l i f e  -Ingrowth curves.  More p a r t i c u l a r l y , he noticed that while the i n i t i a l rate  of change o f i a t free body composition was very rapid, there came a point i n the animal's post n a t a l development a f t e r which body composition changes occurred at a very slow rate. of chemical maturity of the animal.  Moulton c a l l e d t h i s point the point More recently, Spray andWiddowson  (102) have contended that although the concept of chemical maturity i s a useful one i t i s not a true generalization, since d i f f e r e n t components of the body reach a"mature" rate of change at d i f f e r e n t times. not s u r p r i s i n g i n the l i g h t of the findings of Hammond (39), (63)  This i s  and McMeekan  as c i t e d by Palsson (81) which show that the d i f f e r e n t parts of the  animal body grow and mature at d i f f e r e n t rates and that the sequence of growth and development, though s i m i l a r , i s not exactly the same i n d i f f e r e n t species.  Carrel (17) has discussed the differences between s i d e r i a l time measured i n constant, a r b i t r a r y units and p h y s i o l o g i c a l time which i s measured on a scale depending on the rate of " i r r e v e r s i b l e change" i n the animal body.  Carrel (17) pointed out that the rate of p h y s i o l o g i c a l  aging was greatest a t conception, and that thereafter the rate decreased u n t i l i n the mature animals physiological aging was very slow.  Thus, i t  would seem that p h y s i o l o g i c a l aging and Moulton's chemical maturation are e s s e n t i a l l y expressions of the same thing.  Physiological aging i s also  dealt with extensively by Brody ( l l ) , who defines physiological time as "a measure of the rate of change i f the organism", and demonstrates by p l o t t i n g growth and other data f o r many d i f f e r e n t species on axes of equivalent scale, that the sequence of physiological events has great interspecific similarity.  - 49 -  The concept of physiological aging has profound implications i n the understanding of growth and development of animals.  Although i t  has been shown ( 7 5 , 7 7 , 8 7 , 1 0 2 ) that mammals tend to age i n a s i m i l a r p h y s i o l o g i c a l manner, r e l a t i v e to t h e i r t o t a l l i f e spans from conception through maturity to o l d age and death, there i s great v a r i a b i l i t y i n the physiological ages of various species at b i r t h , depending, as Palsson (81) has observed, on the proportion of the t o t a l growth period spent by i n d i v i d u a l s of d i f f e r e n t species i n utero.  Thus, calves, f o a l s , lambs  and baby guinea pigs are considerably older i n the p h y s i o l o g i c a l sense than are the young of marsupials, mice, rabbits and r a t s , while baby pigs and human infants are of an intermediate p h y s i o l o g i c a l age as compared to the two extreme groups above.  A r e s u l t of the difference of p h y s i o l o g i c a l  age at b i r t h between d i f f e r e n t species i s a difference i n the rate of change of post-natal s i z e and conformation.  One of the most s i g n i f i c a n t features of physiological aging i s that the rate of i r r e v e r s i b l e change i n an animal body i s very dependent on the environment.  Thus, although the p o t e n t i a l mature phenotype  of a species must be genetically determined, the extent to which the genotype i s f u l f i l l e d i s i n a large measure environmentally controlled. Many workers ( l l , 1 2 , 3 9 ,  6 1 ,63,  70,  103, 108,  1 1 5 , 1 1 7 ) have studied  &he effects of plane of n u t r i t i o n on animal growth and development. These workers have demonstrated that n u t r i t i o n a l stress w i l l retard growth and rate of change of body composition and that even a f t e r extended periods of deprivation, realimentation results i n a resumption of growth and development ( 6 1 , 7 8 ) .  These studies have also shown that the effects  - 5© of n u t r i t i o n a l stress are not completely r e v e r s i b l e , and that permanent deviations from normal development may be produced, which are more pronounced as the physiological age of the affected i n d i v i d u a l i s decreased. This phenomenon i s explainable on the basis of the concept of heterogonic growth and the d i f f e r e n t i a l aging and nutrient p r i o r i t y of d i f f e r e n t tissues and organ systems (39). i n b r i e f , heterogonic growth involves a progressive anterior-to-posterior development and maturation with a secondary wave of a x i a l development beginning a t the matacarpals and metatarsals and extending i n both directions along the limbs.  The body  composition changes concomitant with the above sequence of growth involve broadly the development and maturation of neural, s k e l e t a l , muscular and adipose tissues i n that order ( 8 l ) .  I t has been demonstrated that severe  n u t r i t i o n a l stress a f f e c t s the organ systems and body compartments i n the order of t h e i r r e l a t i v e maturity, ( 8 l ) .  Thus, i f a low plane of  n u t r i t i o n i s imposed, fattening and then muscle growth are retarded before there i s any appreciable change i n the rate of s k e l e t a l development (81, 6 7 ) .  I f a submaintenance regimen i s imposed, f i r s t f a t and then  muscle tissues w i l l be degraded to provide energy f o r maintenance and even growth of the e a r l i e r maturing systems.  Another e f f e c t of n u t r i t i o n a l  stress i s that i r r e v e r s i b l e changes i n systems occur i n d i r e c t proportion . to the i n t e n s i t y of development of the systems at the time the stress i s imposed.  In this respect, i f an animal i s subjected to a low plane of  n u t r i t i o n at a stage of intensive muscular or s k e l e t a l development, permanent muscular or s k e l e t a l stunting may occur, while the ultimate a b i l i t y to fatten may not be affected.  Thus, i t i s frequently observed  that animals which have suffered unfavourable n u t r i t i o n a l conditions during  - 51 early l i f e and are subsequently realimented do not quite a t t a i n t h e i r expected mature stature or weight ( 6 l ) , but at the same time have a tendency to be r e l a t i v e l y f a t t e r at maturity than i f they had not been retarded (U, 63, 103)•  I t has also been observed that early i n h i b i t i o n  of growth leads to s t r i k i n g changes i n adult conformation, s p e c i f i c a l l y involving the under development of the pelvic region and hind quarters  (12, 63, 70). The time at which environmental i n h i b i t i o n of growth may occur i s not l i m i t e d to the post natal period.  In the developing foetus, the  p r i o r i t y f o r nutrients from the dam's blood stream i s very great (39), whereas the actual amount of energy required i s small r e l a t i v e to the t o t a l energy intake of the dam  (ll).  Thus, as Wallace has  demonstrated  with sheep (108), the foetus i n i t s early stages i s not affected by the plane of n u t r i t i o n of the dam.  Wallace (108) however has  demonstrated  that a low maternal plane of n u t r i t i o n over the f i n a l weeks of pregnancy results i n a decrease i n size and vigour of the lambs at b i r t h and i n a decrease i n t h e i r subsequent growth rates due to reduced mammary development i n the dam.  The gestation period was not affected by the low plane  of maternal n u t r i t i o n ; however, the reduced nutrient supply available to the foetus slowed i t s rate of physiological change, so that at b i r t h , the lambs were smaller, p h y s i o l o g i c a l l y younger, and more poorly equipped to deal with t h e i r post-natal environment.  The effects on the foetus of the  changes i n intrauterine environment due to the maternal d i e t are more pronounced i n the case of multiple b i r t h s (108), and p a r t i c u l a r l y i n the case of species having large l i t t e r s .  Widdowson (113) has analysed the  largest and smallest p i g l e t s i n several newborn l i t t e r s of pigs and the  r e s u l t s obtained indicate that the large p i g l e t s are considerably the p h y s i o l o g i c a l sense than are the younger ones.  older i n  In smaller multiparous  species, such as the mouse, the maternal plane of n u t r i t i o n has more e f f e c t on the developing foetuses as a r e s u l t of the r e l a t i v e l y higher proportion of the d a i l y energy intake of the dam which must be used to meet her own maintenance needs.  Barry (6), working with rats and more  recently McClure (62) with mice, have studied some of the adverse in-utero effects on foetuses due to maternal i n a n i t i o n .  Also, i n t h i s respect  Warkany ( l l O ) has reviewed an expanding l i t e r a t u r e on the  teratogenic  effects of various s p e c i f i c maternal n u t r i t i o n a l stresses such as deficiencies and antimetabolites  on f o e t a l development.  vitamin  I t should be  pointed out that the treatments applied i n these studies a f f e c t the embryonic and f o e t a l animals at a stage when physiological aging i s occurring at a near maximal rate, so that the effects of such stress are much more d r a s t i c i n terms of l a t e r growth and development as compared to post n a t a l stresses, p a r t i c u l a r l y with respect to the e a r l i e r maturing body systems.  In order that the concept of physiological aging may  find  useful a p p l i c a t i o n i n quantitative studies of animal growth and development, some measurable index of the passage of physiological time must be found. The above discussion shows the independence of physiological and chronol o g i c a l times, and i t follows that the former cannot be measured by latter.  S i m i l a r l y , since fattening may  throughout growth (75,  occur to such a variable degree  1$) i t i s evident that body weight i s a poor i n d i -  cator of physiological change. index (75)  the  Fat free body mass i s a more r e l i a b l e  but would have l i m i t e d value i n i n t e r s p e c i f i c comparisons of  p h y s i o l o g i c a l time.  Other more u s e f u l expressions of r e l a t i v e p h y s i o l o g i c a l  - 53 state are those i n which present development are expressed as percentages of expected mature development ( l l ) .  Many other attempts to quantitate  rate of change i n animals have been made ( l , 11), including measurements of metabolic rate, s k e l e t a l development and chronaxy. On the basis of evidence presented by Moulton (75)  i t would  seem that an index which measured chemical maturity i n animals would have the broadest value i n describing p h y s i o l o g i c a l age. (8,  25, 66)  I t has been recognized  that there i s a d e f i n i t e r e l a t i o n s h i p between the amounts of  nitrogen and water i n the animal body, and that the rate of the two narrows as the animal ages chronologically.  Reasoning that protein and  water are the major constituents of the metabolic mass of the animal, Bailey (U) has recently proposed the nitrogen-to-water r a t i o as a v a l i d index of physiological age i n mammals. composition data (75)  Recalculation of e x i s t i n g body  shows that d i f f e r e n t species which undergo equivalent  p h y s i o l o g i c a l changes tend to have equal nitrogen to water r a t i o s .  This  index i s made a l l the more useful by the i n d i r e c t methods, described i n d e t a i l elsewhere, f o r the measurement of the body nitrogen and water compartments i n - v i v o .  To complete the foregoing discussion, some mention should be made of the energetic implications of body composition changes.  Initially,  i n pre-natal development, the gross energetic e f f i c i e n c y i s very high ( l l ) . I t , however, f a l l s sharply at b i r t h from s i x t y to t h i r t y per cent and then decreases progressively ( l l , 64, 65).  Blaxter (9)  the gross c a l o r i c values of dry beef f a t and muscle are calories per kilogram.  has reported that  9250 and 5878  Since muscle tissue contains about eighty per cent  -  $1*  -  water and f a t only s i x per cent, the stored energy represented by equal masses of body protein and f a t i s i n the r a t i o of about 1:7.1*.  It is  evident then, that the economic e f f i c i e n c y of l i v e weight gained i s dependent on the composition of the gain, which i n turn i s influenced both by the plane of n u t r i t i o n and by the p h y s i o l o g i c a l age of the animal.  In t h i s l a s t respect Brody ( l l ) has observed that animals of  equal p h y s i o l o g i c a l ages tend to have equal compositions of gain, regardless of differences i n body s i z e provided the r e l a t i v e plane of n u t r i t i o n of the animals i s the same.  Thus, a p h y s i o l o g i c a l l y young  animal tends to l a y on protein gain having a low energy content, while a p h y s i o l o g i c a l l y older animal stores more energy per unit gain because of  the higher f a t content of the gain.  This generalization i s compli-  cated by the factthat, as has been stated e a r l i e r , some animals are born f a t (113) l i f e (102).  or fatten rapidly f o r a short time i n early post natal This phenomenon may be accounted f o r i n one of two ways,  namely, that there i s some s p e c i f i c mechanism which causes the animal to consume more energy than i t needs and therefore to fatten at t h i s stage, or  else that t h i s early fattening r e s u l t s from the occurrence of some sort  of  environmental growth l i m i t a t i o n which does not decrease the appetite.  I t i s possible that the extent of fattening which occurs a t this early stage i s r e l a t e d to the development of the homeostatic mechanism (lCU). During the prenatal period, there i s a s l i g h t increase i n the c a l o r i c content of gain.  However, since there i s not homeothermic maintenance  cost, the gross e f f i c i e n c y of gain i s high ( l l ) .  At b i r t h , the complica-  t i o n of maintenance energy loss a r i s e s , causing the gross e f f i c i e n c y of growth to f a l l sharply from about s i x t y per cent to about t h i r t y per cent.  - 55 This l e v e l of e f f i c i e n c y i s maintained f o r the e a r l y part of the growth period and then declines to a low value as maturity i s approached. has been demonstrated  It  (I4., 11) that metabolic rate i n the young animal  tends to increase a t a rate almost proportional to body weight, and that at about the time of puberty the homeothermic mechanism becomes s t a b i l i z e d a f t e r which metabolic rate increases a t a slower rate proportional to a smaller f r a c t i o n a l power of body weight.  I t i s approximately at t h i s  break, which also coincides with the maximum of absolute growth rate, that the gross e f f i c i e n c y of growth begins to decrease.  I t would seem  then, that up u n t i l the point of i n f l e x i o n of the absolute growth curve, an animal on an adequate plane of n u t r i t i o n stores about the same amount of energy r e l a t i v e to i t s energy intake regardless of the composition of the gain, since although there has been a decrease i n the relative growth rate there has been an increase i n the f a t , and hence energy content, of the gain made.  A f t e r t h i s point of maximum metabolic rate, the rate of  decline i n r e l a t i v e growth rate i s greater than the rate of increase of the energy content of gain r e s u l t i n g i n a decreased gross e f f i c i e n c y of growth.  I t i s probable that high or low planes of n u t r i t i o n at any point  i n the growth period would raise or lower the energetic e f f i c i e n c y of gain by increasing or decreasing the proportion of the available energy to the maintenance requirement.  In the foregoing, the changes i n body composition which occur i n animals as they grow have been discussed i n r e l a t i o n to physiological aging, to the heterogenic nature of growth, and to the gross energetic e f f i c i e n c y of weight gain and energy storage.  I t i s hardly necessary to  - 56 point out that these p r i n c i p l e s have profound economic significance i n a l l phases of the modern animal industry, p a r t i c u l a r l y i n the production and marketing of meat animals, and i n the s e l e c t i o n of superior animals for breeding.  However, i n view of the f a t that many of the above p r i n -  c i p l e s have been w e l l established s c i e n t i f i c a l l y f o r at l e a s t two decades or longer, i t i s amazing that more p r a c t i c a l use has not been made of them.  For this reason i t i s perhaps important that research i n  body composition and animal growth must continue to demonstrate e x i s t i n g facts as w e l l as to investigate new  the  theories.  In the present studies i t has been recognized that there i s a paucity of data i n which body composition has been sequentially measured i n the same growing i n d i v i d u a l s .  I t was f e l t that t h i s type  of investigation, applied to young animals i n the early post-natal stages of growth would contribute to a more comprehensive knowledge of the f i n e structure of animal growth, and might at the same time, reveal some of the factors which may influence the subsequent conformation and compos i t i o n of meat animals at the time of marketing.  The baby p i g has been  chosen f o r study since i t i s born at a younger physiological age than most other domestic animals (81) and hence undergoes a more rapid and more extensive change i n body composition during the suckling period thai do other  domestic species.  The p i g also has the advantage that body  composition studies i n this species are not complicated by v a r i a b l e gastroi n t e s t i n a l f i l l to the extent met with i n ruminants. pig,  In addition, the  when young i s of a convenient size f o r laboratory study, may  be  obtained i n genetically s i m i l a r l i t t e r groups, and has many aspects of  i t s growth and development already well documented i n the l i t e r a t u r e  (63).  The objectives of t h i s study are as follows: 1.  To evaluate a method suitable f o r the sequential in-vivo measurement of body composition i n the growing p i g l e t .  2.  To establish by i n - v i t r o chemical analysis the relationships between the t o t a l water, protein, ash and f a t compartments of growing p i g l e t s of d i f f e r e n t ages.  3.  To use the above methods and relationships to estimate at frequent i n t e r v a l s the in-vivo body composition of suckling p i g l e t s growing under e s s e n t i a l l y normal management and n u t r i t i o n a l conditions.  I t should be pointed out that the experiment was  designed to  investigate methodology and to establish a base l i n e i n tenns of body composition changes i n the normally developing p i g l e t , and was  thus a  preliminary experiment to future investigations of the e f f e c t s of various environmental stresses such as plane of n u t r i t i o n on subsequent growth. Therefore, although a considerable  amount of sequential in-vivo body com-  p o s i t i o n data has been obtained, the experiment has been p r i m a r i l y designed to f u l f i l the f i r s t two objectives mentioned above.  B.  1.  METHODS AND MATERIALS  Animals Four l i t t e r s of p i g l e t s , a l l s i r e d by the same registered Lan-  drace boar and out of h a l f - s i b Yorkshire sows were available f o r study. The l i t t e r size was adjusted to eight p i g l e t s a t one week post partum and when possible such adjustment provided equal numbers of males and females.  The t h i r d and fourth l i t t e r s of eight and twelve p i g l e t s res-  p e c t i v e l y were born simultaneously and the number of p i g l e t s i n e ach l i t t e r was evened to ten at one day post partum.  Pertinent farrowing  data i s presented i n Table 1 of the appendix. 2.  Rations Sow No. 36 was inadvertantly f e d U.B.C. 10-59, a r a t i o n designed  for small animal production.  The remaining three sows were f e d the  standard U.B.C. swine rations (U.B.C. 26-59 and 28-59). (U.B.C. 18-59) was provided f o r the p i g l e t s .  A creep r a t i o n  The proximate  composition  and net energy contents of the rations are presented i n Table 2 of the appendix. 3.  Management Throughout the experiment,  the sows and l i t t e r s were housed  i n 8' x 8' pens, each of which opened i n t o a farrowing crate.  The sows  were placed i n the pens one week p r i o r to the anticipated farrowing date and were treated with lindane to control l i c e .  A further lindane  treatment was administered two weeks a f t e r farrowing.  The p i g l e t s were weighed, tattooed, and treated with lindane at fourty-eight hours from b i r t h .  In addition, each p i g l e t received 2.0  cc. of Imferon* (injectable iron-dextran) by intramuscular i n j e c t i o n at this time.  Haemoglobin l e v e l s were determined a t i n t e r v a l s u n t i l the  p i g l e t s were eating the creep r a t i o n s a t i s f a c t o r i l y .  A d d i t i o n a l Imferon  was i n j e c t e d when the haemoglobin l e v e l s were found to be low.  I t was  assumed that haemoglobin tests or Imferon i n j e c t i o n s would be unnecessary a f t e r the p i g l e t s began to eat the creep r a t i o n , since the l a t t e r contained adequate amounts of available i r o n .  The young boars were not castrated, and the p i g l e t s were allowed to remain with the dams throughout the experiment.  The sows and p i g l e t s  were weighed weekly, and these weights have been recorded i n Tables 3 and 5 of the appendix.  The feed consumption of the sows was measured d a i l y  (Table 6 of the appendix). The feed consumption of the p i g l e t s was not measured, because i t was impossible to t e l l how much of the sow's feed they had eaten i n addition to t h e i r own creep r a t i o n .  Fresh water was  provided f o r both the sows and t h e i r l i t t e r s a t a l l times. cleaned out d a i l y .  The pens were  S u f f i c i e n t bedding was provided i n the form of dry  f i r shavings.  4.  Analytical  a)  In-vitro At weekly intervals one animal from each l i t t e r was selected at  random and k i l l e d f o r t o t a l chemical and physical analysis of body water, &  Benger Laboratories Ltd., Toronto, Canada.  - 60 -  f a t , nitrogen and ash.  The r e s u l t s obtained are tabulated i n Tables  13  and l i u i)  Total body water The carcass was  freeze.  c a r e f u l l y weighed, and was  When frozen, i t was  placed i n a deep  cut up into t h i n s l i c e s with a band  A f t e r each p i g had been s l i c e d , the bandsaw was  saw.  c a r e f u l l y cleaned,  the t i s s u e debris were added to the s l i c e d material which was  and  then spread  i n metal c r i s p e r dishes and d r i e d to constant weight i n a tunnel dryer set at 160°F.  This usually required f i v e days.  terms of the weight l o s t during drying.  Body water was  expressed i n  The dried carcasses were then  stored i n a i r - t i g h t p l a s t i c bags i n a r e f r i g e r a t o r .  ii)  Total body f a t  The d r i e d carcasses were crushed i n a large cast i r o n mortar, and representative 1000  Gm.  samples from each animal were placed i n tared  canvas bags, weighed, and then extracted f o r f o r t y - e i g h t hours with petroleum ether i n a large Soxhlet apparatus.  The samples were then placed f o r  t h i r t y minutes i n a low-humidity a i r stream to remove the petroleum ether. The canvas bags and t h e i r contents were then c a r e f u l l y weighed. f a t free material was  then ground, f i r s t i n an Enterprise Model  The  coarse  2522 meat  grinder having a face plate with three-sixteenth diameter holes, then i n a Hobart coffee grinder set at the f i n e s t grind adjustment, and f i n a l l y i n a Webber Bros. No. 22 Laboratory Pulverizing M i l l to pass a O.Ol; inch screen.  A f t e r these processes, the f a t free material had a f l o u r - l i k e  consistency; duplicate two gram samples of this material were then tested f o r moisture content by a standard method (1. a ) ) .  The percentage of f a t  - 61 i n the dry matter was calculated a f t e r having f i r s t corrected the f a t free material f o r moisture content, assumed to represent the moisture taken up by the f a t free sample during the evaporation of the petroleum ether. iii)  Total nitrogen  T r i p l i c a t e one gram samples of the a i r - d r i e d f a t free dry matter were analysed f o r t o t a l nitrogen by the Kjeldahl method ( l - a ) .  The per-  centages of t o t a l body nitrogen were corrected f o r the moisture content of the f a t - f r e e dry matter before f i n a l tabulation. iv)  Body ash  The ash content of duplicate two gram aliquot samples of the f a t free dry matter was determined by a standard i g n i t i o n method ( l - a ) , and the r e s u l t i n g ash values were corrected f o r r e s i d u a l moisture content as above.  b)  In-vivo Total body water was determined at weekly i n t e r v a l s i n most of  the p i g l e t s using the deuterium oxide d i l u t i o n technique discussed f u l l y i n Section I.  Prediction equations, based on the body composition  relationships determined by the i n - v i t r o analysis were calculated which related t o t a l body nitrogen, f a t , and ash to t o t a l body water, and were used to estimate the other body compartments a f t e r body water has been determined.  - 62 C. 1.  EXPERIMENTAL RESULTS  Changes i n Body Composition, Determined i n - V i t r o The r e s u l t s of the i n - v i t r o analyses are presented i n Tables  13,  l u and 15,  with the A part of each table showing the percentage body  composition of the pigs and the B part showing the actual composition by weight.  Figures 7 to 10 i n c l u s i v e show graphically the changes of water  nitrogen, ash and f a t as the animals aged chronologically.  a)  Body water The percentage of body water f e l l r a p i d l y from about eighty-  three to about sixty-seven i n the f i r s t f i f t e e n days of post-natal l i f e , and thereafter decreased at a much slower rate to about sixty-three per cent at s i x t y days.  A f t e r the fifteen-day point, there was a considerable  scatter i n the data. per  However, there were indications of r e l a t i v e minima i n  cent body water at about f i f t e e n , t h i r t y - t h r e e and f i f t y - f i v e days, and  r e l a t i v e maxima at twenty-two and forty-two days.  The minimum and maximum  at f i f t e e n and twenty two days were not as marked i n l i t t e r s one and two s s i n l i t t e r s three and four.  b)  Body nitrogen The percentage of body nitrogen increased r a p i d l y from about  1.6  at b i r t h to a r e l a t i v e maximum of 2.1; at f i f t e e n days, then declined to a r e l a t i v e minimum of about 2.0 at thirty-two days and f i n a l l y increased to about 3.0 at s i x t y days.  The changes i n per cent bodyp r o t e i n (N x  necessarily follow the same pattern.  6.25)  FIGURE THE CHANCES AGE  f  7  IN PtfiCC/MT  ^ O T 7 V  W/nxft  ' / V - V / T ^ C DATA 1  A \  8  o  o A  A  ®  E  Bo  "  O  ^  |3  r  "  2'O  IO  AGE-  INHA^S  5  30  ffiom  0 BIRTH  =  I. m e t ?  A  8  I  LITTER'S  6 O  WITH  - 63 -  c)  Body ash The percentage of body ash declined r a p i d l y frommore than U.O s  at b i r t h to a minimum of 2.5 at f i f t e e n days.  A f t e r this point, there i s  too much scatter i n the data to describe changes d e f i n i t e l y , although there i s a h i n t i n Figure 9 of a maximum and minimum at twenty-five and t h i r t y f i v e days respectively. d)  Body f a t The percentage of body f a t rose r a p i d l y from .almost zero at b i r t h  to a r e l a t i v e maximum of about sixteen at twenty days, then decreased to about f i f t e e n at f o r t y days and increased again to eighteen at f i f t y days, a f t e r which there was some evidence of a further s l i g h t decrease.  e)  Fat-free dry mass I t i s evident from the above, that up u n t i l twenty days of age,  during which time the most marked changes occurred, that the r e l a t i v e changes i n body water and body protein were almost exactly opposite to the r e l a t i v e changes i n body f a t and ash, respectively.  Further changes con-  tinued to show t h i s mirror image e f f e c t , but to a l e s s marked degree.  In  t h i s respect, i t i s i n t e r e s t i n g to note that the percentage f a t - f r e e dry matter, composed primarily of protein and ash, remained almost constant from b i r t h to s i x t y days.  In spite of the fluctuations i n the r e l a t i v e proportions of the gross body compartments, a l l compartments increased i n the absolute sense as age and body weight increased. 13B  An examination of the data i n Tables  to l£B i n c l u s i v e shows that there was great v a r i a t i o n i n body weight at  o a  d  Q  o  Q  <  to  0 •  (/>  or .« u y r  £ f  O  •  o c  w  s  K  K  -J  -i  t  "  <  o <  u  o  to  t f  a  o  h  o  o  Q  r—. B  0  00 QC 3  o QC  CO  13 Q  > f-1  <  tm 00 Q%0  —r  Ln  Q O  5  O QC  o  *o  0  QQ.  o  >-  •  Q  Q  PQ  Q  «  O in  0  h  P o ac  Q 0 QQ  0-  CL  -5*  0  2  H  a  GJ  CC  O  Q lO  a:  <  P  2  lu  13  (5  0  <  0 .O Q  0  4 O —r o  0  0 —r  Q  o  to ro  r  o  —r  ^ 0  4  •  0  < ca  (3 •  o  2  O  \0 10  or a:  f-i  UJ  a -J  O  I' Q  -J  -4  n  i,  o  in  < a  3 t-  O  ro  O  0  2 UJ  B  H G  Ul Ul  Q  O  4  Q Q  UJ  004  I Ui  O CN  7-  Ffl  -6kany given age, i n d i c a t i n g generally that there was  a v a r i a t i o n between  pigs i n the rate of t h e i r development, and suggesting a reason f o r the considerable scatter seen i n the data points i n Figures 7 to 10 i n c l u s i v e . 2.  Relationship of Body Nitrogen, to Body Water a)  The nitrogen-to-water r a t i o The data i n Tables 17  and 18 i n d i c a t e i n general that the nitrogen  to water r a t i o tends to increase with both chronological age and body weight. However, due to the v a r i a t i o n i n nitrogen-to-water r a t i o between i n d i v i d u a l s of s i m i l a r ages and weights, neither of these are s a t i s f a c t o r y bases f o r the comparison of these r a t i o s .  Fat-free dry matter was  therefore chosen  as the most v a l i d parameter against which to express the nitrogen-to-water ratios. An examination of the data i n Figures 11, the f a t - f r e e dry body mass increased, there was  and 12 shows that, as  a rapid increase i n nitrogen  to water r a t i o u n t i l the abcissa had a value of about 0.955 kg. and that thereafter there was  a much more gradual increase.  Figure 12 and Table  16  show the l e a s t squares logarithmic r e l a t i o n s h i p between nitrogen-to-water r a t i o (Y) and kilograms of f a t free dry mass (X) i n the i n t e r v a l s 0 . 1 ^ . 0.955 kgj 0.955 4  °\0  kg.  nitrogen to water r a t i o was  x^»  The difference i n the rate of change of the  highly s i g n i f i c a n t at P — 0.01.  Figures 11  and  12 also indicate a possible break i n the rate of change of the nitrogen to water r a t i o at X s  2.5  kgj however, the s c a t t e r i n the data did not  this further d i v i s i o n i n the regression l i n e s .  justify  THE  / IGUHI  Rt  uxi  Ii  IO/VSH/P  of THI NITROGEN  P)f\Tio(Y) EG FATFREE'DHI  M A T T E R  / W A ( X )  V - O. 0 7><l  0.03  J  C-.2375"  O 0 2  0-03/25 X  V  00  TV  2-0  AG.  EE EM.  5-0  4 -O  - 65 b)  The regression of body water against body nitrogen Although a preliminary p l o t of the r e l a t i o n s h i p of grams of body  nitrogen (Y) to kilograms of body water (X) on an arithmetic g r i d showed near l i n e a r i t y , i t was  obvious that the variance of the r e l a t i o n s h i p was  d i r e c t l y proportional of the amount of body water present.  On a r e l a t i v e  basis, the variance was much more constant, and hence the logarithmic r e l a t i o n s h i p shown i n Figure 13 was fact.  On the basis of Figures 11,  used as a more v a l i d description of 12 and the data i n Table 16,  f e l t that a d i v i s i o n of the data at an amount of body water to 0.955 kilograms of f a t - f r e e dry mass was  When this was  i t was  corresponding  justified.  done and l e a s t squares logarithmic regression l i n e s  were calculated, using data points from a l l of the l i t t e r s , i t was  found  that the exponent of the r e l a t i o n s h i p i n the i n t e r v a l 0.5 4. x ^. 3.9 was 3.94  kg.  1.2598, while the exponent of the r e l a t i o n s h i p i n the i n t e r v a l x < 20 kg. was  I.O867.  The difference between these two exponents  was h i g h l y s i g n i f i c a n t at P =1 0.01.  Similar regressions calculated f o r  the i n d i v i d u a l l i t t e r s gave s i m i l a r exponents i n the two i n t e r v a l s , but non s i g n i f i c a n t or only s l i g h t l y s i g n i f i c a n t differences between these exponents due to the decreased number of the points on the regression l i n e s . No s i g n i f i c a n t differences were found between the exponents of the regressions of nitrogen against water f o r a l l males and a l l females i n the two  intervals.  In addition, there were not enough data points to determine -whether or not rate of increase of body nitrogen r e l a t i v e to body water changed e a r l i e r i n females than i n males.  These results are summarized i n Table  19.  - 66 3.  The Relationship of Body Ash to Body Yfetter a)  The ash-to-water  ratio  The data i n Figures lU and 15 show on arithmetic and logarithmic grids, respectively, the r e l a t i o n s h i p of the ash-to-water r a t i o (Y) to k i l o grams of f a t - f r e e dry mass (X).  Although there i s much more scatter i n  this data as compared with that f o r the nitrogen-to-water r a t i o , probably due to a n a l y t i c a l v a r i a t i o n , i t i s once again evident that a d i v i s i o n of the data can j u s t i f i a b l y be made, and that the point at which this d i v i s i o n occurs comes e a r l i e r with respect to f a t - f r e e dry mass than i n the case of the nitrogen-to-water r a t i o .  The data i n Table 20 shows the regression  curves and the highly s i g n i f i c a n t difference between the regression exponents calculated f o r the i n t e r v a l s 0.1^.  x C 0.85 kg. and 0.85^. x < 6 kg.  As  i n the case of the nitrogen-to-water r a t i o , there i s some i n d i c a t i o n that a further d i v i s i o n of the data could be made at X=. 0.85,  but t h i s i s not  j u s t i f i e d due to the amount of scatter i n the data.  b)  The regression of body ash against body water In the l i g h t of the significance of the difference between the  exponents of the two above ash-to-water r a t i o vs. f a t - f r e e mass regressions, the data used to calculate the r e l a t i o n s h i p of grams of body ash (Y) to kilograms of body water (X) was divided i n t o two parts, and separate l e a s t squares logarithmic regression l i n e s were calculated f o r the two i n t e r v a l s 0.5 ^ x <^2.9  and 2.5 4  x  ^ > 20  where 2.5 kilograms i n the weight of water  i n a p i g having 0.85 kg. of f a t - f r e e dry mass.  The equations f o r these  regressions are shown i n Table 21 and plotted i n Figure 16.  The exponents  of these two regressions are s i g n i f i c a n t l y d i f f e r e n t at P — 0.01.  Since no  FIGURE 15  THE RELATIONSHIP OF THE ASM /WATER RATIO ( *%G) TO F/\T FFIEEDM / ^ A S S (KG} (X)  (Y)  FIGURE / 6  500  %  lHE r\EU\Tl0N5HIP OF B O D Y ASH  TOTOTMBODY  WATER /.037T +-12.5%  2ooi 1  ioo  1  Y*  47 O S 3 X  Vo  50  30  L,  Ol  1 — 1 — 1 — 1 — ' — ' —  Ol  IO  50  20 /CG5.  /OT/\L  Bony  WATER  1  —  fO-0  2 0 0  - 67 s i g n i f i c a n t differences i n the exponents of the nitrogen vs. water regressions were found f o r i n d i v i d u a l l i t t e r s due to the lack of data points, and since the s c a t t e r of the ash vs. water data points i s r e l a t i v e l y great, no attempt to c a l c u l a t e regressions of i n d i v i d u a l l i t t e r s or f o r the sexes was made. U.  Relationships Involving the Fat-Free Dry Body Mass  a  )  The r e l a t i o n s h i p of f a t - f r e e dry mass to body water The relationship of kilograms of f a t - f r e e body dry mass (Y) to  kilograms of body water (X) i s shown i n Figure 17 and i n Table 29 Part A. There i s r e l a t i v e l y l i t t l e scatter i n the data points, and since the regression of a l l of the points together has a high c o r r e l a t i o n c o e f f i c i e n t and r e l a t i v e l y low standard error, no j u s t i f i c a t i o n was  seen f o r d i v i d i n g  the data as i n the cases of the nitrogen and ash vs. water regressions. There i s , however, some i n d i c a t i o n of f i n e - s t r u c t u r e i n the r e l a t i o n s h i p s , probably r e f l e c t i n g the changes i n the nitrogen and ash components described earlier.  b)  The r e l a t i o n s h i p of per cent f a t - f r e e dry mass to body weight I t has been noted e a r l i e r that the percentage of f a t - f r e e dry mass  remained almost constant i r r e s p e c t i v e of the age of the pigs.  The data i n  Table 22 Part B shows the l e a s t squares l i n e a r regression of the percentage of f a t - f r e e dry mass (Y) against kilograms of body weight (X) on an arithmetic g r i d .  Due  to the almost n e g l i g i b l e regression e f f e c t and non-  s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t , i t was  f e l t that f o r the purpose of  c a l c u l a t i n g body composition, very l i t t l e error would be introduced i f the average percentage f a t - f r e e body mass, 17.92 the regression curve.  per cent, was used instead of  The v a l i d i t y of this choice i s i n d i c a t e d by the  18  FIGURE  7c /ATFfiff DRY MATTER  VS.  WEIGHT  B o n y  2SO. JoF.F.DM  (Y) 20-O  Y-Y-17-12%  /SO  Y= Y- 2 s  I  1  1  O  1  1  1  1  B o n y  Y  =  1  1  1  1  8O  4 0  T?£TGRE55IOfV  1  17*12%  1  1  I 2-0  WEIGHT  COEFFICIENT  1  IN  1  1  1  1—1  1 6 0  1—1  2 O 0  1—1  ^  1——i  {  1  2 4 0  KG. ( X )  B y ^ = 0 . 0 3 4 $ ; t = 0- 2 3 3 ) , N . S . A T P - 0 . / 0  ; STAivupifsD  DCV/AT/O/V  « +  M 8 %  1  1  2.&-0  r  0  - 68 f a c t that the standard deviation of the average i s only 1.7 per cent greater than i s the standard error of estimate of the regression l i n e .  An  examination of the two expressions shown i n Table 22 shows that each estimates the f a t - f r e e dry mass with the same accuracy, since — 1.18  per  cent of body weight i s equal to — 6.7 per cent of the actual f a t - f r e e dry mass. 5.  The Calculation of Body Composition a)  In-Vivo  The prediction equations These equations have been summarized i n Table 23.  Total body  water, determined as described e a r l i e r by the deuterium oxide d i l u t i o n method has been used as a parameter f o r the estimation of t o t a l body ash and t o t a l body protein.  I t w i l l be noticed that the equations f o r estimating  body protein are the same as these f o r estimating t o t a l nitrogen, except that the constants of the former are 6.25  times those of the l a t t e r .  Total  body f a t has been estimated by taking the difference between the t o t a l body weight and the sum of the weights of body water and f a t - f r e e dry mass. The value of f a t - f r e e dry mass used i n t h i s c a l c u l a t i o n has been estimated on the basis of 17.92  per cent of body weight rather than as a function of  t o t a l body water, since the former r e l a t i o n s h i p i s , at l e a s t i n the weight range encountered i n the present experiment, as v a l i d as the l a t t e r , and i s moreover independent  of any errors incident with the in-vivo measurement of  body water. b)  The in-vivo body composition by weight The data i n Table 2h shows the body composition of t h i r t y - t h r e e  - 69  -  growing p i g l e t s estimated in-vivo at weekly i n t e r v a l s .  The column headed  "Per cent Water" shows the percentage of t o t a l body water as determined the deuterium oxide d i l u t i o n method.  by  The difference between the t o t a l body  weight and the sum of the weights of the estimated body water, ash, protein and f a t compartments i s shown both as an absolute weight and as a percentage, with t h i s l a s t being only very s l i g h t l y greater than that f o r the i n - v i t r o body composition data shown i n Tables 13 to 15 i n c l u s i v e .  Probably about  one h a l f of t h i s difference represents glycogen and other  non-nitrogenous  constituents not measured i n either the i n - v i t r o or in-vivo determinations. I t should be pointed out that i f the f a t - f r e e dry mass had been estimated from body water, the apparent "error" would have been l e s s .  Consider the  expression:  weight of)  Body Fat — Body Weight - (Weight of body water (  -t- f a t - f r e e dry mass  )  I t i s evident that error i n the c a l c u l a t i o n of body water would lead to ai error i n the same d i r e c t i o n i n c a l c u l a t i n g the amount of f a t - f r e e dry mass, and that the error i n the calculated body f a t would be equal to the sum of these errors and i n the opposite d i r e c t i o n .  Thus, while the apparent t o t a l  error i n body composition estimation i s decreased by c a l c u l a t i n g f a t - f r e e dry mass from body water, the true error may a c t u a l l y be quite great, since i n the present analyses, an error i n the measurement of body water of about two per cent can be expected.  Since body f a t contains about one and a h a l f  times as much energy as protein, such true errors may be very serious from an energetic point of view.  I t i s contended then, that within the weight  range of the pigs used i n the present experiment,  the c a l c u l a t i o n f a t - f r e e  dry mass from body weight and not body water produces an "error" i n t o t a l body composition estimation which r e f l e c t s the true error i n the measurement of body water and provides a minimum error i n the energetic sense.  - 70 The data i n Figures 19 to 22 i n c l u s i v e show the pattern of body composition changes i n the absolute sense i n ten representative pigs as they increase i n age.  The Data i n Figures 23 to 31 i n c l u s i v e show the r e l a t i v e rates of  change i n the body compartments and i n t o t a l body weight of some of the same pigs as they age chronologically. 6.  The Estimation of the Chemical and Energetic Composition of Gain i n Growing Figs The data i n Table 25 calculated from Table 2k,  presents the e s t i -  mated composition of d a i l y gain f o r the thirty-three pigs which were subjected to in-vivo body water determinations.  I t should be pointed out that  the values i n the column marked t o t a l d a i l y gain do not represent the sums of the values of the d a i l y protein, f a t and water gains, but are calculated d i r e c t l y on the basis of changes i n body weight.  In some cases, the sum of  the apparent differences i n protein, f a t and water are a c t u a l l y s l i g h t l y greater than the t o t a l change i n weight, due to the errors mentioned i n 5 b above.  The energy content of gain has been calculated from the chemical composition, using the energy contents of beef f a t and muscle protein given by Blaxter and Rook (9).  These were 9U00 and 5950 Calories per Kilogram for  f a t and muscle respectively.  In these calculations, i t was assumed that  stored energy was derived s o l e l y from f a t and protein gain. t i o n was  given to the "error" value given i n Table 2k,  No considera-  since t h i s was  an  unknown combination of f a t , protein, ash, glycogen and water, and was small energetically speaking since at l e a s t h a l f of t h i s "error" vrould represent water and ash.  FIGURE  IS  CunuLMm C O H  PIG  KG.  CHANGE IN T H F BODY T S  P A HTtlEN  W/FH  A G F  No. 5 ^  8 4  WATER  J  FA-  PROTON  o 1  PIG /VO  ASH  5 4  12 J  WATER  KG.  FAT  PROTEAN A S H  0  '0  20  30  AGE IN BAYS FROM BIRTH  4 0  50  FIGURE H - A CUMULATIVE  CHANGES  COMPARTMENTS  O  WITH  IN THE  Act"  BODY  57  PIG  WO.  P/G  No. 110  10  2 0  A G E IN D A Y S  3 0  ffiow  4 0  B / R T H  5 0  FIGURE  2  0  C U M U L A T I V E C/IA/VGL5  0  10  AGEINUAIS  2 0  IN THE BOTJY  Ffton  60  B I R T H  40  50  FIGURE  21  CUMULATIVE  CHANGES IN THE Con PART VENTS W I T H A G E PIG  Bony  No. 17 2  TROTEIN  * 20  O  IO  3  O  4o  5 O  20  3 0  AQ  A G E IN UATS FKOM B / R T H  ASH  6 O  5o  FIGURC  22  A G E IN  U A T S  ffior? B / K T H  - 71 The most s t r i k i n g revelation of the data i n Table 25 i s that while the protein, ash, and water compartments continue to increase as body weight increases, there are many instances i n which increase i n body weight a c t u a l l y i s accompanied by a negative gain i n the f a t compartment, which connot a l l be dismissed as simply representing a n a l y t i c a l errors.  These  effects are demonstrated graphically i n Figures 23 to 31 i n c l u s i v e l y . These figures show the s e r i a l body weight and estimated weights of body f a t and protein of several representative pigs, plotted on semilogarithmic paper against age i n days from b i r t h .  The slopes of the curves are equal  to the K values f o r the instantaneous r e l a t i v e growth rate expression ( l l )  dw =. KDT dt where  ¥  i s the weight of the animal or body compartment;  dw dt  i s the rate of change of the weight with respect to time; and  K  i s the instantaneous r e l a t i v e growth rate constant having the units of gain per u n i t of mass per u n i t of time.  TABULATED RESULTS  TABLE NO. 13^A. L i t t e r No. 1  Pig No.  Age i n Days  —  Percentage Body Composition  Sex  % Total Body Water  % Total Dry Matter  % Total Fat-Free D.M.  —  Based on In-Vitro Analysis  % Total Body Fat  % Total Body Nitrogen  % Total Body Protein N x 6.25  % Not Accounted For % Total Water & Fat & Ash Protein & Ash  1~A  1  M  79.0  21.0  18.4  2.6  1.96  12.3  3.95  2.2  1-B  1  M  79.9  20.1  17.8  2.3  1.83  11.4  3.85  2.5  53  5  F  7U.3  25.7  17.1  8.6  2.06  12.9  3.32  0.9  55  5  F  73.8  26.2  16.8  9.4  2.01  12.6  3.12  1.1  51  12  F  68.1  31.9  18.2  13.7  2.31  lil.4  2.85  0.9  50  12  F  68.2  31.8  19.1  12.7  2.U3  15.2  3.12  0.8  58  19  F  65.6  34.4  17.3  17.1  2.27  14.2  2.35  0.8  52  26  M  64.5  35.5  17.7  17.8  2.21;  lU.O  2.95  0.7  5U  3k  M  63.U  36.6  15.2  21.U  1.89  11.8  2.18  1.2  56  Uo  F  65.1  3U.9  17.1  17.8  2.01  12.6  2.36  2.1  59  U8  F  6l.lt  38.6  18.0  20.6  2.25  lU.l  2.50  I.U  57  53  M  63.3  36.7  18.2  18.5  2.29  llw3  2.92  0.9  - 73 TABLE NO. L i t t e r No.  Pig No.  Age i n Days  1-A  l  1-B  l  1  —  13-B  Body Composition by Weight  —  Kg. Kg. Body F.F.D.M. Fat  Based on In-Vitro Analysis  Sex  Kg. Weight a t Sacrifice  Kg. Body Water  Kg. Body P.M.  Kg. Body Ash  Kg. Not Accounted For  M  0.925  0.731  0.19U  0.170  0.02U0 0.0182  o.llU  0.0365  0.011  M  1.173  0.937  O.236  0.209  0.027  0.0215  0.13U  0.0U52  0.030  Kg. Body Nitrogen  Kg. Body Protein N x 6.25  53  5  F  1.816  1.350  0.U66  0.311  0.156  0.037U  0.23U  0.0603  0.016  55  5  F  2.063  1.522  0.5U1  0.3U7  0.19U  0.0U15  0.259  0.0812  0.007  51  12  F  3.3UO  2.275  1.065  0.608  0.U58  0.0772  0.U83  0.0952  0.029  50  12  F  3.2U6  2.21U  1.032  0.620  0.U12  0.789  O.U93  0.1013  0.026  58  19  F  U.99U  3.276  1.718  0.86U  0.85U  0.113U  0.709  0.117U  0.038  52  26  M  7.U90  U.831  2.659  1.326  1.333  0.1678  1.0U9  0.2217  0.055  5U  3U  M  10.030  6.359  3.671  1.525  2.1U6  O.I896  1.185  0.2186  0.111  56  Uo  F  13.393  8.719  U.67U  2.290  2.38U  0.269  1.681  0.3161  0.293  59  U8  F  16.U80  10.119  6.36I  2.966  3.395  0.3708  2.318  0.U120  0.236  57  53  M  17.920  11.3U3  6.577  3.261  3.315  0.U10U  2.565  0.5233  0.17U  - 74 T A B L E NO.  L i t t e r No. 2  Pig No.  Age i n Days Sex  —  14-A  Percentage Body Composition  % Total Body Water  % Total Dry Matter  % Total Fat-Free -P.M.  —  Based on In-Vitro Analysis  I Total Body Fat  % Total Body Nitrogen  % Total Body Protein N x 6.25  % Total Ash  % Not Accounted For Water & Fat & Protein & Ash  -1-2A  1  M  80.0  20.0  18.2  1.8  1.85  11.5  4.64  2.1  111  6  F  71.8  28.2  16.9  11.3  2.12  13.3  2.81  0.8  118  13  F  68.1  31.9  16.6  15.3  2.12  13.3  2.40  0.9  114  20  F  63.4  36.6  17.3  19.3  2.05  12.8  3.54  0.9  117  20  F  68.0  32.0  19.1  12.9  2. 42  15.1  3.07  0.9  110  41  M  67.4  32.6  17.3  15.3  2.10  13.1  2.67  1.5  113  48  M  65.6  34.4  17.U  17.0  2.2U  1U.0  2.65  0.7  116  56  M  62.7  37.3  18.8  18.5  2.27  14.2  2.82  1.8  P-l  2  F  81.3  18.7  16.6  2a  1.91  11.9  3.45  1.2  P-2  3  M  80.6  19.4  17.8  1.6  2.13  13.3  3.83  0.7  - 75 TABLE NO. L i t t e r No. 2  Pig No.  Age i n Days Sex  —  lii-B  Body Composition by Weight  Kg. Weight a t Sacrifice  Kg. Body Water  Kg. Body P.M.  Kg. F.F.D.M.  —  Based on In-Vitro Analysis  Kg. Body Fat  Kg. Body Nitrogen  Kg. Body Protein N x 6.25  Kg. Body Ash  Kg. Not Accounted For  L-1-2A  1  M  1.055  O.&kh  0.211  0.192  0.0190  0.0195  0.122  0.01*90  0.021  111  6  F  2.736  1.96k  0.772  0.U62  0.309  o.o58o  0.363  0.0769  0.023  118  13  F  U.025  2.7ltl  1.28U  0.668  0.616  o.o853  0.533  0.0966  0.038  111;  20  F  7.070  U.U82  2.588  1.223  1.365  o.iU5  0.906  0.250  0.067  117  20  F  U.990  3.393  1.597  0.953  0.6UU  0.121  0.756  0.153  0.01*1*  110  Ul  M  10.670  7.192  3.U78  1.8U6  1.633  0.22U  1.1*00  0.285  0.160  113  1*8  M  15.920  5.1*76  2.770  2.706  0.357  2.231  0.1*22  0.117  116  56  M'  19.270  12.082  7.188  3.623  3.565  0.1*37  2.731  0.5U3  0.21*9  P-l  2  F  1.299  1.056  0.2U3  0.216  0.027  0.021*8  0.155  O.0UI18  0.016  P-2  3  M  0.950  0.766  0.181*  0.169  0.015  0.0203  0.127  0.036U  0.006  -  76  -  TABLE NO. 15-A L i t t e r s 3 and 4  Pig No.  Age i n Days Sex  —  Percentage Body Composition  % Total Body Water  % Total Dry Matter  % Total Fat-Free D.M.  % Total Body Fat  —  Based on In-Vitro Analysis  % Total Body Nitrogen  % Total Body Protein % Total N x 6.25 Ash  % Not Accounted For Water & Fat& Protein & Adi  125  8  M  71.1  28.9  18.6  10.3  2.30  I4.4  2.70  1.5  136  8  F  72.0  28.0  17.8  10.2  2.27  1U.2  3.02  0.6  126  15  M  67.2  32.8  17.1  15.7  2.16  13.5  2.56  1.0  120  22  F  68.3  31.7  18.5  13.2  2.36  I4.8  2.78  0.9  127  22  F  66.9  33.1  18.0  15.1  2.27  lU.2  2.87  0.9  119  29  M  67.5  32.5  18.0  Lu.5  2.2k  1U.0  2.93  1.1  123  29  M  66.8  33.2  15.U  17.8  1.95  12.2  2.h$  0.7  133  37  F  65.9  3U.1  17.6  16.5  2.25  -lU.1  2.99  0.5  121  37  M  66.8  33.2  19.2  I4.0  2.1*0  15.0  2.92  1.3  \2k  43  M  68.4  31.5  18.8  12.7  2.30  I4.4  2.97  1.5  - 77 TABLE NO. L i t t e r s 3 and 1* (cont'd.)  Pig No.  Age i n Days Sex  M  —  15-A contd.  Percentage Body Composition  % Total Body Fat  —  Based on In-Vitro Analysis  % Total Body Nitrogen  % Total Body Protein N x 6.25  % Not Accounted H>r Water & Fat & Protein & Ash  % Total Body Water  % Total Dry Matter  % Total Fat-Free D.M.  68.1  31.9  18.8  12.1  2.1*8  15.5  2.39  1.9  % Total Ash  13U  1*3  129  51  63.5  36.5  15.8  20.7  1.95  12.2  2.67  0.9  131  51  63.7  36.3  18.1*  17.9  2.29  lU.3  2.83  1.3  130  58  6U.1  35.9  17.6  18.3  2.22  13.9  2.38  1.3  135  58  63.5  36.5  18.1*  18.1  2.33  lU.6  2.90  0.9  122  65  65.3  3U.7  18.6  16.1  2.21*  ll*.0  3.13  1.5  132  65  63.9  36.1  18.8  17.3  2.36  ll*.8  3.18  0.8  M  M  - 78 TABLE NO. 15-B Litters 3 and k  Pig No.  Age i n Days Sex  —  Body Composition by Weight  Kg. Weight at Sacrifice  Kg. Body Water  Kg. Body D.M.  Kg. F.F.D.M.  —  Based on In-Vitro Analysis  Kg. Body Fat  Kg. Body Nitrogen  Kg. Body Protein N x 6.25  Kg. Not Kg. Body Accounted Ash For  125  8  M  3.630  2.581  1.01*9  0.675  0.374  0.0834  0.098  0.098  0.056  136  8  F  3.170  2.282  0.888  0.561*  O.323  0.0720  0.1*50  0.0957  0.019  126  15  M  5.81*0  3.92U  1.916  0.999  0.917  0.126  0.789  0.150  0.060  120  22  F  4.990  3.408  1.582  0.923  0.659  0.118  0.738  0.139  O.O46  127  22  F  6.800  4.549  2.251  1.224  1.027  0.154  O.963  0.195  0.066  119  29-  M  7.U80  5.049  2.431  1.346  1.085  0.168  1.050  0.219  0.077  123  29  M  7.710  5.150  2.560  1.187  1.372  0.150  0.938  0.189  0.061  133  37  F  10.700  7.051  3.6U9  1.883  1.766  0.2U1  1.506  0.320  0.057  121  37  M  13.600  9.085  4.515  2.611  1.90U  0.326  2.038  0.397  0.176  U  43  M  13.370  9.1u5  4.212  2.511*  1.613  0.308  1.925  0.397  0.290  12  - 79 TABLE NO. i£-B contd. L i t t e r s 3 and 1* (cont'd.)  Pig No.  Age i n Days Sex  —  Body Composition by Weight  Kg. Weight a t Sacrifice  Kg. Body Water  Kg. Body P.M.  Kg. F.F.D.M.  Kg. Body Fat  —  Based on In-Vitro Analysis  Kg. Body Nitrogen  Kg. Body Protein N x 6.25  Kg, Body Ash  Kg. Not Accounted For  13U  1*3  M  1U.330  9.759  U.572  2.691*  1.73U  0.355  2.219  0.31*2  0.276  129  51  F  15.650  9.938  5.712  2.1x73  3.21*0  0.305  1.906  O.lp.8  0.ll*8  131  51  F  19.050  12.135  6.915  3.505  3.1*10  0.1*36  2.725  0.539  0.21*1  130  58  M  25.150  16.121  9.029  U.l*26  1*.602  0.558  3.U88  0.599  0.31*0  135  58  F  20.700  13.11*5  7.555  3.809  3.71*7  0.1*82  3.013  0.600  0.195  122  65  F  22.210  ll*.503  7.707  1*.131  3.576  0.1*98  3.113  0.695  0.323  132  65  M  29.900  19.106  10.791* 5.621  5.173  0.706  1*.1*13  0.951  0.257  -  80  -  TABLE NO. 1 6 Relationship Between N/H2O (Y) Ratio (kg/kg) and Kg. Fat Free Dry Mass (X)  No. of Pigs  Line No.  Variance of Exponent  Equation  Significance of Difference Between Exponents  1  A l l Pigs X  <  0.955  kg.  16  Y = O.036O4 x ' 0  2 3 7 5  k-  7.3  - 6 . 8  0.910  5.17  x  ,-5 10  Highly S i g n i f i c a n t at  A l l Pigs X  >  0.955  A l l Pigs Total Line  +-6.7 Kg.  23  T  39  Y =• 0.0311a X  =•  0.03125  X°*°938  0.1086  - 5 . 9  +  0.582  x  3.U  IO"*  9.1  -8.U  O.8I4  r i n a l l lines i s significantly)  0  at  P-=0.01  P = 0 . 0 1  - 81 TABLE NO. 17 DIFFERENCES IN BODY COMPOSITION AND PHYSIOLOGICAL AGE EETWEEN NEW BORN PIGLETS IN THE SAME LITTER * Litter A  Weight kg. % Water % Fat % Protein  Litter B  Litter C  L  S  L  S  L  S  1.236  CU72  1.331  0.890  1.1*48  1.035  83.9 1.17 12.0  8U.3  85.9  0.89  0.85  11.7  10.2  86.0  0.89  0.69 10.0  % Nitrogen  1.92  1.63  1.87  1.60  N/H 0  0.0229  0.0189  0.0222  0.0186  2  81*.9  L  Largest P i g l e t i n L i t t e r  5  Smallest P i g l e t i n L i t t e r  6  Body Composition extracted from  11.0  Widdowson, E.M., Chemical Composition of Newly Born Mammals, Nature, 1950, 166; 626  86.8 0.53 9.0  1.76  1.1*1*  0.0207  0.0166  - 82 TABLE NO. 18 Differences Between the Nitrogen to Water Ratios of Pigs of the Same Chronological Ages  N/R" 0  Pig No.  Age i n Days  Weight i n Kg.  1-A  1  0.925  0.021*8  1-B  1  1.17  0.0229  1-1-2A  1  1.06  0.0231  53  5  1.82  0.0277  55  5  2.06  9.0272  111*  20  7.07  0.0323  117  20  h.99  0.0356  120  22  U.99  0.031*6  127  22  6.80  0.0339  122  65  22.2  0.031*3  132  65  29.9  0.0369  2  - 83 TABLE NO. 19 The Relationship of Gms. Total Body N to Kg. Total Body H 0 2  No. of Description  Line No.  Variance of Exponent  s %  Equation  e  Significance of Difference Between Exponents  Pigs Y = 20.32U X *  L i t t e r No. 1  1  3 0 2 8  7 Y = 20.389 X * 1  0 2 3 1  5 Total  Litters 3 & k  12  6  Y  27.UOU X ' 1  Y = 25.281 X 1  0 8 1 1  2301  *  •+• 7.k - 6.8 + 30  -2h  0.8U*  +20 -16.5  0.985  +16.U -LU.l  Total  h  Yr  26.573  X -  1 1 0 2  10  T =  25.8U6  X '  1 3 6 1  3  L i t t e r No. 2  Hi  Total  17  3  1  Y = 25.9U8 X * 1  Y = 28.151 X * 1  2 3  ^  0 8 2 3  Y - 30.151 Xl.0583 Line i , X < 3.9 kg.;  0.996  ^5.2  0.985  - k.9  0.997  + 13.0 -11.5  0.99U  + 0.9 - 0.7  0.999  + 6.0 - 5.5  0.995  -+ 5 . 9 - 5.U  0.99U  0.00014; )  ) )  N.S. P =  0.10  0.02808 )  0.0019U ) ) )  Significant P=0.10j N.S. P = 0 . 0 5  0.00109 )  0.00021 ) ]  0.0007U )  Line 2 X > 3-9 kg.  Significant  P-0.01  - Bh TaBLE NO. 19 (cont'd.) The Relationship of Gms. Total Body N to Kg. Total Body H 0 2  Variance of Exponent  No. o f Description  Line No.  A l l Pigs  Pigs 1 6  23  Total  A l l Males A l l Females A l l Males A l l Females  39  5  11  1 4  9  Equation  I  Y  Y  Y  Y  Y  Y  -  =  =  =  -  -  =  25.509 27.631  27.077  25.592  2U.871 27.033 28.615  S  X  X  X  X  X  X  X  -  1  *  1  1  1  1  1  1  2  ,  0  1  *  -  1  0  6  i  2  2  9  8  0  *  -  5  2  9  0  *  8  7  i  2  1  ^  8  l  9  6  0  1  e*  + 10.0 - 9.0  0 . 9 9 1  + 12.2 -11.0  0 . 9 7 4  + 13.0 -11. k  0 . 9 5 2  + 9.3 - 8.5  0 . 9 9 3  0.00013 ) ) ) 0.00013 )  0 . 0 0 1 4 0  Highly Significant at P= 0.01  )  ) )  + 11.0 -10.2  0 . 9 8 0  0.00069 )  + 6.0 - 5.9  0 . 9 9 4  0 . 0 0 0 0 9  + 19.5 -16.3  Significance of Difference Between Exponents  N.S. P = 0 . 1  )  N.S. P= 0 . 1 0 . 9 4 0  0.002U1 )  TABLE NO. 20 Relationship Between the Ash/Water Ratio (kg/kg) 00 and Kg. of Fat Free Dry Mass (X)  Line No.  No. of Figs  1 A l l Pigs F.F.D.M. < 0.85 kg.  13  A l l Pigs F.F.D.M.> 0.85 kg.  A l l Pigs Total Line  Equation  Y =: 0.03729 X  - 0  ' ' 1  3 0  _  1 0 >  5  -0.6711  Y = 0.03938 X°*°7°3  + 10.8 - 9.5  0.373  38  Y = 0.01+263 X *  +~ 11.5 -10*3  0.085  0 0 3 2  0.000255)  Highly S i g n i f i c a n t  ) ) )  25  0  Variance of Exponent  Significance of Difference Between Exponents  0.000058  Significance of Correlation C o e f f i c i e n t (r) Line 1  r i s s i g n i f i c a n t at P - 0.02; N.S. at P - 0.01  Line 2  r i s s i g n i f i c a n t a t P - O.Olj N.S. at P = 0.05  Total Line  r i s not s i g n i f i c a n t at P = 0 . 5 0  at P = 0.01  - 86 TABLE NO.  21  Relationship Between Gms. T o t a l Body Ash (Y) and Kgs. Total Body Water (X)  Line No.  No. of Pigs  Equation  13  Y •= 1*7.083 X ° « ° 9  Se$  r  Variance of Exponent  Significance of Difference Between Exponents  1 A l l Pigs X < 2.9 kg.  8l  -+ 32.0 -21*. 2  0.831  0.0020^8 Highly S i g n i f i c a n t at P = 0.01  M2.5  A l l Pigs X ) 2.9 kg.  26  Y ^ 38.981*  A l l Pigs T o t a l Line  39  Y = i*5.3U2 X°'?638  X*™ 1  0  -11.0  0.978  _^2.1*  0.993  0.000080  A l l Correlation Coefficients S i g n i f i c a n t a t P =. 0.01  - 87 TABLE NO. 22 A.  Relationship of Kg. Fat Free Dry Mass (Y) to Kg. Body Water (X)  Y = 0.2312 X - '? 1  0  %  r * 0.998  r i s s i g n i f i c a n t at P - 0.01  B.  Relationship of Per Gent Fat Free Dry Mass (Y) to Kg. Live Weight (X)  Y = 0.035 X + 17.59 - 1.16  r -0.233  r i s not s i g n i f i c a n t a t P = 0.10  For calculations i n Live Weight Range X ^ 29 kg. Y = Y =-17.92 t.1,18 % F.F.D.M.  - 88 TABLE NO. 23 Summary of the Prediction, Equations Used i n Calculating Body Composition In-Vivo  1.  2.  3.  Kg. of Protein (T) from Kg. of Y/ater (X)  0.1 < x < 3.9 kg.  Y ^ 0.159U X - ^  3.9 4 x <20  I : 0.1727 X '  1  kg.  1  2  8  0 8 6 7  Grams of Ash (Y) from Kg. of Water (X)  0.1 < x < 2.9 kg.  Y = U7.083 X°- °9  2.9 4 x< 20  Y = 38.984 X '  8l  kg.  1  0 3 7 7  Fat-Free Dry Mass Kg. F.F.D.M.  -  17.92 x Body Weight (kg.)  100 k. Body Fat Kg. Body Fat - Kg. Body Weight - (Kg. Body Water-vKg. F.F.D.M.)  -  89  -  TABLE NO. 2k Body Composition of Growing Piglets Estimated In Vivo L i t t e r No. 1  Pig No.  Sex  1-A  M  1  0.882  77.6  0.158  0.681;  0.099  0.035  O.OUO  0.024  2.72  1-B  M  1  1.125  78.7  0.202  0.885  0.137  0.01+3  0.038  0.022  1.95  58  F  5  1.617  69.3  0.290  1.120  0.182;  0.052  0.207  0.051;  3.3U  52  M  19  5.362  62.8  0.961  3.367  0.736  0.137  1.03U  0.088  1.61;  26  7.605  6U.3  1.363  U.890  0.969  0.202  1.352  0.192  2.52  5  1.871  75.1  0.335  1.U05  0.2U5  0.062  0.131  0.028  1.U9  19  5.533  58.9  0.992  3.259  0.707  0.133  1.282  0.152  2.75  26  7.9U5  60.2  1.U2U  lw783  0.9U6  0.198  1.738  0.280  3.52  3k  10.U22  61.2  1.868  6.378  1.293  O.l^iO  2.176  0.135  1.29  5U  M  Weight Kg.  % HgO  F.F.D.M. Kg.  H0 Kg.  Protein (N x 6.25) Kg.  Ash Kg.  Fat Kg.  Not Accounted For and/or Error Kg.  Age i n Days From Birth  2  % of Total  - 90 TABLE NO. 2U (cont'd.) Litter  Pig No.  56  59  57  Sex F  F  M  Age i n Days From Birth  Weight Kg.  H0  %  13  3. U05  19  F.F.DJ £.  H0 2  No.  1 -  Protein (N x 6.25) Ash Kg. Kgj.  Fat Kg.  Not Accounted For and/or Error Kg.  % of Total  **•  Kg^  57.5  0.610  1.958  0.367  0.081 0.0837  0.162  U.75  U.796  63.8  0.859  3.060  0.652  0.12U 0.877  0.083  1.71*  26  7.26U  67.2  1.302  U.881  0.967  0.25U 1.081  0.081  1.12  Ui  13.620  67.5  2.UU1  9.191*  1.755  0.390 1.985  0.295  2.17  5  1.801  82.2  0.323  1.1*80  0.261  0.065 0.000  0.005  o.U5  13  3.788  6U.5  0.679  2.UU3  0.U91  0.099 0.666  O.O69  1.82  34  11.350  63.7  2.03U  7.230  1.1*82  0.30U 2.086  0.2U8  2.18  Ul  1U.07U  57.1  2.522  8.036  1.662  0.339 3.516  0.521  3.71  U8  16.U80  65.9  2.953 10.860  2.25U  0.U63 2.667  0.236  1.1*3  19  5.136  63.3  0.920  3.251  0.70U  0.133 0.965  0.083  1.62  26  7.U63  6U.5  1.337  U.81U  0.818  0.172 1.312  0.3U7  U.65  3U  9.761  61.9  1.7U9  6.0U2  1.220  0.252 1.970  0.277  2.8U  Ul  12.712  66.7  2.278  8.1*79  1.762  0.358 1.955  0.158  1.23  U8  15.209  6U.3  2.725  9.779  2.058  0.1*16 2.705  0.251  1.61*  53  17.933  6U.0  3.211*  11.U77  2.UU9  0.1*91 3.2U2  0.27U  1.53  2  -  91  -  TABLE NO. 2k (cont'd.) L i t t e r No. 2 Not Accounted For and/or Error Kg.  Pig No.  Sex  Age i n Days From Birth  Ill  F  2  1.715  72.7  0.307  1.2U7  0.211  0.056 0.161  o.oUo  2.33  118  F  6  2.783  66.9  O.U99  1.862  0.3U9  0.078 0.U22  0.072  2.58  13  U.058  63.3  0.727  2.569  0.523  0.101  0.762  0.103  2.5U  2.831  6U.2  0.507  1.818  0.339  0.077 0.506  0.091  3.22  U.5U0  6U.U  0.81U  2.92U  0.616  0.119 0.802  0.070  1.5U  2.180  72.9  0.391  1.589  0.278  0.067 0.200  0.0U6  2.11  13  U.209  65.9  0.722  2.655  0.5U6  0.10U 0.652  0.072  1.79  6  2.276  71.1  0.U08  1.618  0.292  0.070 0.250  0.0U6  2.03  13  U.U83  68.9  0.803  3.089  0.660  0.126 0.591  0.017  0.38  20  6.21U  6U.3  l.llU  3.996  0.778  0.16U l . i o U  0.172  2.76  3k  8.217  67.6  1.U72  1.113  0.231 1.190  0.128  1.56  Ul  10.669  63.9  1.912  1.390  0.286  0.236  2.22  11U  F  6 13  117  110  F  M  6  Weight Kg.  %  M.  F.F.D.M. Kg.  H0 2  6.817  Protein (N x 6.25) Ash Kg.  Fat  lEli  1.9U0  % of Tota]  TABLE NO. 2k (cont'd.) L i t t e r No. 2  Pig No.  Sex  Age i n Days From Birth  113  M  6  2.710  13  116  115  M  F  Not Accounted For and/or Error Kg.  F.F.D.M. Kg.  H0 K|.  Protein (N x 6.25) Kg.  69.2  0.U86  1.875  0.352  0.078  0.349  0.056  2.06  5.335  61.7  0.956  3.292  0.715  0.13U  1.087  0.107  2.01  20  6.697  62.3  1.200  4.172  0.815  0.172  1.325  0.213  3.18  27  8.172  65.0  1.464  5.312  1.060  0.221  1.396  0.183  2.21*  3k  9.307  67.8  1.668  6.310  1.278  0.263  1.329  0.127  1.36  la  11.804  65.5  2.115  7.732  1.594  0.326  1.957  0.195  1.65  U8  15.935  67*8  2.856  IO.8O4  2.293  O.46I  2.275  0.102  O.64  2  2.050  71.9  0.367  l.klk  0.260  O.O64  0.209  0.043  2.10  56  19.295  60.3  3.U58  11.635  2.486  0.498  4.202  0.47U  2.1*5  6  2. U71  67.0  0.443  1.656  0.301  0.071  0.372  0.071  2.87  13  4.682  6U/5  0.839  3.020  0.642  0.123  0.823  0.074  1.58  20  6.697  60.1  1.200  4.025  0.78U  0.165  1.472  0.251  3.75  27  7.718  65.3  1.383  5.0U0  1.001  0.209  1.295  0.173  2.1*1  Weight Kg.  % H0 2  9  Ash  Fat Kg.  % of Total  TABLE NO. 2k (cont'd.) L i t t e r s 3 and k  Sex  125  M  8  3.602  65.3  0.6U5  2.352  0.2*68  0.09k  0.605  O.O83  2.30  136  F  3  1.911  78.3  0.3I+2  1.2*96  0.265  0.065  0.073  0.073  2.03  8  3.178  70.1  0.569  2.228  0.1*37  0.090  0.381  0.012  0.38  3  1.880  69.3  0.337  1.303  0.223  0.058  0.2U0  0.056  2.98  8  3.292  69.7  0.590  2.295  0.2*52*  0.092  0.U07  o.oiOi  1.3U  3  1.766  70.2  O.316  1.22*0  0.209  0.056  0.210  0.051  2.89  8  2.U68  72.0  0.1*1*2  1.777  0.329  0.075  0.2U9  0.0U0  1.62  15  3.9kh  67.3  0.707  2.651*  0.5U5  0.10U  0.583  0.058  1.U7  22  U.99U  6k.6  0.895  3.226  0.697  0.131  0.873  0.067  1.3U  8  2.980  69.O  0.53U  2.056  0.395  0.082*  0.390  0.055  1.85  15  5.079  72.0  0.910  3.657  O.707  0.150  0.512  0.035  0.69  22  6.810  67.5  1.220  1+.597  0.906  0.190  0.993  0.12U  1.82  *ig No.  126  120  127  M  F  F  Weight kg.  H2O  %  -  F.F.D.M. kg.  H0 Mil  Protein N x 6.25 kg.  Ash kg.  Fat kg.  Not Accounted and/or Error  Age i n Days From Birth  2  HSi  - 9k TABLE NO. 2k (cont'd.) L i t t e r s 3 and k  Pig No.  135  122  132  Sex F  F  M  Age i n Days From Birth  Protein N x 6.25 kg.  Ash Kg.  Fat Kg.  10.232  2.162  O.U35  3.742  o.U5ii  2.67  3.710  13.622  2.950  0.586  3.370  0.174  0.84  71.6  0.534  2.134  O.iilU  0.088  0.312  0.032  1.07  4.495  65.0  O.806  2.922  0.616  0.118  0.767  0.072  1.60  29  6.129  74.4  1.098  U.560  0.898  0.188  0.471  0.012  0.19  37  9.352  66.5  1.676  6.220  1.259  0.260  1.U56  0.157  1.68  k3  11.577  57.9  2.075  6.703  1.365  0.281  2.799  0.429  3.71  51  15.209  68.5  2.725  IO.4I8  2.20U  O.I444  2.066  0.077  0.51  58  19.976  6U.3  3.580  12.8U5  2.768  0.551  3.551  0.261  1.31  65  22.2U6  63.8  3.986  14.193  3.085  0.612  4.067  0.289  1.30  3  2.121  73.9  O.380  1.567  0.281  0.068  0.174  0.031  I.46  51  21.202  65.8  3.799  13.951  2.959  0.601  3.452  0.239  1.13  Weight kg.  % H0  F.F.D.M.  51  17.025  60.1  3.051  58  20.702  65.8  15  2.980  22  2  KG.  H0 2  kg.  Not Acoaunted For and/or Error kg., JL  - 95 TABLE NO. 2i* (cont'd.) L i t t e r s 3 and 1+  Pig No.  Sex  Age i n Days From Birth  129  F  8  130  Protein N x 6.25 kg.  % H0  1.731  69.1  0.310  1.196  0.200  0.052+ 0.225  15  3.150  68.0  0.562*  2.11+2  0.2+16  0.087  22  5.221  0.936  3.519  0.678  29  6.810  67. 1 +  1.220  37  2  F.F.D.M. kg.  H0  Weight kg.  2  kg.  Ash  Fat  Not Accounted For and/or Error k£i  JL  0.056  3.22+  0.061  1.92*  01 .2+2+  02 .+2+2+ O.766  0.212+  0.905  0.190  1.000  0.125  2*. 10 1.82*  1.188  0.22+6  2.116  0.315  3.22  9.761  1.72*9  2*. 590 5.896  2*3  67.2* 60. U  11.986  67.7  2.12+8  8.115  1.680  0.32+2  1.723  0.126  1.05  51  15.663  59.5  2.801  9.319  1.953  0.395  3.52*3  0.2+53  2.89  3.772*  73.9  0.676  2.789  0.580  0.108  O.309  0.012  3.17  15  6.158  62. U  l.lOii  0.72+6  0.158  1.211  0.200  3.22*  22  8.263  67.5  5.576  1.108  0.232  1.206  0.12+1  1.71  29  9.91+3  1.1+81  3.81+3  63.3  1.782  6.29U  1.275  0.263  1.867  02 .2+1+  2.2*5  8  37  13.257  68.5  2.376  9.081  1.899  0.306  1.800 0.171  51  20.702  61.6  3.710  12.752  2.72*6  0.52*7  2*. 22+0  0.2+17  1.31 2.01  -96  -  TABLE NO. 2k (cont'd.) L i t t e r s 3 and k Age i n Days From Pig No. Sex Birth  119  123  121  133  M  M  M  F  Weight kg.  % H 0 2  .F.D.M. kg.  H0 2  Protein N x 6.25 kg.  Ash kg.  Fat kg.  Not Accounted For and/or Error  HT  JET  8  2.866  71.8  0.51U  2.058  0.396  0.085  0.29k  0.033  1.15  22  6.220  67.9  1.115  4.223  0.826  0.174  0.882  0.115  1.85  29  7.355  68.1;  1.318  5.031  0.999  0.209  1.006  0.110  1.50  8  2.781  72.6  0.498  2.019  0.386  O.O83  0.26k  0.028  1.01  15  U.058  67.5  0.727  2.739  0.567  0.107  0.592  0.053  1.31  22  6.038  67.9  1.082  4.100  0.800  O.I69  0.856  0.113  1.80,  29  7.718  64.O  1.383  4.940  0.980  0.205  1.395  0.198  2.57  3  1.968  68.4  0.353  1.3U6  0.237  0.060  0.269  0.056  2.84  37  13.620  62.7  2.441  8.5UO  1.776  0.361  2.639  0.30k  2.23  3  1.796  71.1  0.322  1.277  0.217  0.057  0.197  0.048  2.67  37  10.71U  66.5  1.920  7.125  1.U59  0.299  1.669  0.172  1.61  - 97 T A B L E N O . 2i* ( c o n t ' d . ) Litters  3 and k  Pig No.  Sex  Age i n Days From Birth  12k  M  3  1.511  72.0  0.271  1.088  0.177  0.050  0.152  0.02*2*  2.91  8  2.299  71.3  0.1*12  1.639  0.297  0.070  0.22*8  0.02*5  1.96  15  3.81*5  73.5  0.689  2.826  0.590  0.109  0.330  0.010  0.26  22  5.675  70.8  1.017  U.018  0.783  o.i65  0.62*0  0.069  1.22  29  7.128  72.1*  1.277  5.161  1.192*  0.22*7  O.690  0.162*  2.30  37  9.988  65.7  1.790  6.562  1.33U  0.275  I.636  0.181  1.81  k3  13 .'393  69.1  2.1*00  9.255  1.938  0.392  1.738  0.070  0.52  8  3.3U8  76.1*  0.600  2.558  0.521  0.100  0.190  0.021  0.63  15  5.278  61*.3  0.9U6  3.392*  0.72*3  0.139  0.938  0.062*  1.21  22  6.855  68.9  1.228  2*.  723  0.933  0.195  0.902*  0.100  1.2*6  29  8.217  65.7  1.1*72  5.399  1.079  0.222*  1.31*6  0.159  1.92*  37  11.1*1*1  62.1*  2.050  7.139  1.1*62  0.300  2.252  0.288  2.52  k3  li*.3l*6  63.1  2.571  9.052  1.892  0.383  2.723  0.296  2.06  37  11.801*  66.0  0.328  1.898  11+.800 19.068  62.1*  7.791 9.235 11.822  1.608  1*3  2.115 2.652 3.2*17  1.932* 2.529  0.392 0.506  2.913 3.829  0.179 0.326 0.382  1.52 2.20 2.00  13U  131  M  F  Weight kg.  % H 0 2  62.0  F.F.D.M.  H 0 2  kg.  Protein N x  6.25  Not Accounted F o r Ash  Fat  kg.  and/6 r kg.  Error %  - 98 TABLE NO. 25 Composition of Gain U t t e r No. 1  Pig No.  Age i n Days  52  19  5.362  26  7.605  5  1.871  19  54  56  59  A  Weight kg.  Protein  D a i l y Gain kg. Fat Water  Total*  Energy Stored/Day Cals. Protein Fat, Total  Cals./ Kg. Gain  0.033  0.01*5  0.217  0.320  198  1*27  625  1951  5.533  0.033  0.082  0.132  0.262  196  772  968  3705  26  7.945  0.03U  0.065  0.217  0.3U5  203  612  815  2367  34  10.1*22  0.043  0.055  0.199  0.310  258  515  773  21*96  19  4.796  0.0l*8  0.007  0.181*  0.232  283  63  3U6  11*90  26  7.26k  0.01*5  0.029  0.260  0.353  268  274  51*2  1536  0.035  0.060  0.288  0.1*21*  313  567  880  2075  0.028  O.083  0.120  0.21*8  171  783  95U  3839  13  41  13.620  5  1.801  13  3.788  This i s not the sum of the preceeding d a i l y gains of protein, f a t , and water, but i s calculated from the increment i n live-weight.  -99  -  TABLE NO. 25 (cont'd.) Composition of Gain L i t t e r No. 1 Pig No.  59  (cont'd)  57  Age i n Days  Weight kg.  Protein  3h  11.350  0.01+7  0.068  0.228  0.360  281  635  916  251+U  i+i  Hi. 071+  0.026  0.201+  0.115  0.389-  153  1920  2073  5329  1+8  16.1+80  0.085  -0.121  0.1+03  0.31+3  503  -lll+O  -637  -186  19  5.136  26  7.1+63  0.022  0.050  0.223  0.332  100  1+66  566  1701+  3k  9.761  0.050  0.082  0.151+  0.287  299  773  1072  3732  kl  12.712  0.077  -0.002  0.31+8  0.1+22  1+60  -20  1+8  15.209  O.Oi+2  0.107  0.185  0.357  252  1007  1259  3529  53  17.933  0.078  0.107  0.339  0.51+5  1+65  1010  11+75  2707  0.261+  228  607  835  3160  Daily Gain kg. Fat Water  Total  Energy Stored/Day Cals. Protein Fat Total  l+l+o  Cals. Kg. Gain  101+5  L i t t e r No. 2  117  6 13  2.180 1+.029  0.038  0.065  0.152  .  - 100 TABLE NO. 25 (cont'd.) Composition of Gain L i t t e r No. 2 P i g No.  Age i n Days  110  6  2.276  13  4.U83  0.053  20  6.21U  3k la  11U  118  113  Weight kg.  D a i l y Gain kg. fat Water  Total  Energy Protein  0.049  0.210  0.315  313  457  770  2444  0.016  0.073  0.130  0.247  100  689  789  3191  8.217  0.021;  0.006  0.111  0.143  lk2  58  200  1398  10.669  0.039  0.107  0.350  235  1007  12U2  35U7  0.039  0.042  0.180  0.244  235  397  64I  2592  0.025  0.015  0.101  0.182  1U8  U57  605  332U  6  2.831  13  4.51*0  6  2.783  13  u.058  6  2.710  Protein  Stored/Days Cals Fat Total  Cals./ Kg. Gain  13  5.335  0.052  0.105  0.202  0.375  308  991  1299  3k6k  20  6.697  O.OLU  0.034  0.126  0.195  85  319  kok  2072  27  8.172  0.035  0.001  O.llU  0.211  208  9  217  1028  3k  9.307  0.031  -01001  0.143  0.162  186  -9  177  1093  - 101 TABLE NO. 25 (cont'd.) Composition of Gain L i t t e r No. 2  Pig No. 113 (cont'd.)  115  Age i n Days  Weight kg.  Protein  i+i  11.802+  o.oi+5  2+8  15.935  D a i l y Gain kg. Fat Water  Total  Energy Stored/Day Cals. Protein Fat. Total  0.090  0.203  0.357  268  81+3  1111  3112  0.099  0.01+5  0.1+39  0.590  59U  1+27  1021  1731  Cals./ Kg. Gain  6  2.1+71  13  IN 6 8 2  0.01+9  0.061+  0.195  0.316  290  606  896  2835  20  6.697  0.020  0.093  0.11+1+  0.288  121  872  993  324+8  27  7.718  0.031  0.025  o.iU5  O.li+6  181+  -238  -5H  -370  L i t t e r s 3 and 1+ 136  126  3  1.911  8  3.178  3  1.880  8  3.292  0.031+  0.062  o.li+6  0.253  205  579  781+  3099  0.01+6  0.033  0.198  0.282  271+  311+  588  2085  - 102 TABLE NO. 25 (cont'd.) Composition of Gain L i t t e r s 3 and 1+ Pig No. 120  127  135  119  Age i n Days  Weight kg.  Protein  Daily Gain kg. Fat Water  Total  Energy Stored/Day Cals. Fat Total Protein  Cals./ Kg. Gain  3  1.766  8  2.U68  0.021*  0.008.  0.107  0.11*0  11+3  73  216  151+3  15  3.91+1+  0.031  0.01+8  0.125  0.211  181;  10+8  573  2716  22  U.99U  0.022  o.oia  0.082  0.150  129  389  518  3U53  8  2.980  15  5.079  0.01+1+  0.017  0.228  0.300  265  161;  1*29  11+30  22  6.810  0.028  0.069  0.131+  0.21*7  169  61*6  815  3299  51  17.025  58  20.702  -0.053  0.1+81;  0.525  669  -1*99  170  32l+  8  2.866  22  6.220  0.031  0.01*2  0.155  0.2l|0  183  395  578  21+08  29  7.355  0.025  0.018  0.072  0.162  H*7  166  313  1932  0.113  - 103 TABLE NO. 25 (cont'd.) Composition o f Gain L i t t e r s 3 and k Age i n Days  122  15  2.980  22  4.495  0.030  0.065  0.113  0.216  180  611  791  3662  29  6.129  O.O4O  -0.01+2  0.234  0.233  2k0  -397  -157  -673  37  9.352  0.045  0.123  0.208  0.U03  269  1157  1426  3538  43  11.577  0.017  0.244  0.080  0.371  105  210U  2209  5954  51  15.209  0.104  -0.091  O.464  0.U5U  620  -861  -241  -531  58  19.975  0.085  0.212  0.347  0.681  479  1994  2473  3631  65  22.21+6  0.045  0.074  0.192  0.324  269  693  962  2969  129  Weight kg.  D a i l y Gain kg. Fat Water  Pig No.  Protein  Total  Energy Stored/Day Cals. Total Protein Fat  Cals., Kg. Ga:  8  1.731  15  3.150  0.031  0.031  0.135  0.203  I84  294  U78  2355  22  5.221  0.037  O.O46  0.197  0.296  222  432  65U  2209  29  6.810  0,032  0.033  0.153  0.227  193  314  507  2233  37  9.761  0.035  0.139  0.163  0.369  211  1311  1522  U125  U3  11.986  0.082  -0.065  0.370  0.371  488  -616  -128  -3U5  51  15.663  0.034  0.228  0.151  0.460  203  2139  2342  5091  - 101* TABLE NO. 25 (cont'd.) Composition of Gain B i t t e r s 3 and 1* Pig No.  123  130  124  Age i n Days  Weight kg.  Protein  Daily Gain kg. Fat Water  Total  Energy Stored/Day Cals Fat Total Protein  Cals./ Kg. Gain  8  2.781  15  4.058  0.026  0.047  0.103  0.182  154  kko  591*  3261*  22  6.038  0.033  0.038  0.19k  0.283  198  355  553  2851  29  7.718  0.026  0.077  0.120  0.240  153  724  877  7308  8  3.774  15  6.158  0.02U  0.129  0.151  0.340  1U3  1212  1335  3927  22  8.263  0.052  -0.001  0.21*7  0.301  309  -9  300  996  29  9.943  0.021*  0.09k  0.103  O.24O  143  884  1027  1*279  37  13.257  0.078  -0.008  0.348  O.4I4  464  -75  389  9k0  51  20.702  0.060  0.174  0.262  0.532  357  161*  521  979  3  1.5H  8  2.299  0.024  0.019  0.078  0.158  11*2  179  321  2031  15  3.8U5  0.042  0.015  0.169  0.221  250  11*1  391  1769 •  22  5.675  0.028  0.044  0.170  0.261  166  1*13  579  2218  - 105 TABLE NO. 25 (cont'd.) Composition of Gain L i t t e r s 3 and 1+ Pig No.  Age i n Days  12k  (cont'd.)  131  Protein  Daily Gain kg. Fat Water  Total  Energy Stored/Day Cals. Protein Fat Total  Cals./ Kg. Gain  29  7.128  0.059  0.007  0.163  0.208  351  66  37  9.988  0.018  0.118  0.175  0.358  107  1109  1216  3397  13.393  0.100  0.017  0.1+1+9  0.568  595  159  751+  1327  1186  1+298  k3  13U  Weight Kg.  1+17  2005  8  3.31+8  15  5.278  0.032  0.106  0.119  0.276  190  996  22  6.855  0.027  -0.005  0.189  0.225  161  -kl  111+  507  29  8.217  0.021  O.63  0.097  0.195  125  592  717  3677  37  11.1+1+1  0.01+7  0.113  0.218  0.1+03  280  1062  131+2  3330  1+3  11+.3U6  0.072  0.078  0.319  0.1+81+  1+28  733  1161  2399  37  11.801+  k3  lit. 800  0.051t  0.169  0.21+1  O.i+99  321  1589  1910  3828  51  19.068  0.071+  0.115  0.323  0.53U  14+0  1081  1521  281+8  - 106 D. DISCUSSION  1.  The Changes i n the Relationships of Nitrogen, Ash, and Water i n Relation to Physiological Aging In Table 19 and Figure 13, i t i s indicated that immediately  a f t e r b i r t h , the baby pig gains nitrogen (protein) a t a rate which i s about twenty-six per cent f a s t e r than the rate of increase i n i t s body water compartment.  Then, when the p i g l e t has 3.9 kilograms of body water,  the rate of nitrogen increase r e l a t i v e to water drops rather abruptly to a rate only nine per cent greater than the rate of body water increase. This event occurs when the p i g weighs about f i v e and a h a l f kilograms and i s about f i f t e e n days o l d .  In Figures 12 and 13, there i s some i n d i c a -  t i o n that there i s a further such drop i n the rate of change of nitrogen r e l a t i v e to water when the p i g has ten kilograms of water and weighs about f i f t e e n kilograms.  Similar nitrogen to water relationships have been  reported by Robertson (91) and Bailey(U),  using rats and mice respectively.  I t i s i n t e r e s t i n g to note that Robertson (91) shows a break i n the nitrogen to water r e l a t i o n s h i p i n rats weighing t h i r t y grams, and having the same nitrogen to water r a t i o (0.03U) as do the pigs i n the present study a t the point where they contain 3.9 kilograms of water.  Figures 23 to 31  i n c l u s i v e show that there i s an abrupt decrease i n the r e l a t i v e rate of liveweight gain i n most of the pigs a t a body weight of between f i v e and s i x kilograms, and that there i s u s u a l l y a further, less marked decrease i n r e l a t i v e growth rate a t a body weight of between nine and f i f t e e n kilograms. These changes correspond quite c l o s e l y to the changes i n the nitrogen to water r e l a t i o n s h i p .  - 107 The data i n Table 21 and i n Figure 16 i n d i c a t e that the newborn p i g l e t gains ash constituents at a rate which i s nineteen per cent slower than the rate of increase of i t s body water compartment.  Yfflien the p i g l e t  contains 2.9 kilograms of body water, the rate of ash accumulation  increases  to a l e v e l which i s three per cent greater than the rate of increase i n water.  In Figure 16, there i s some i n d i c a t i o n of a second increase i n the  rate of ash gain r e l a t i v e to water gain when the p i g l e t s contain ten k i l o grams of water, and weigh between thirteen and f i f t e e n kilograms.  These  changes also agree c l o s e l y with the changes i n the o v e r a l l growth rate of the animal, with the exception that the f i r s t "break" i n the rate of ash gain r e l a t i v e to water increase occurs at a s l i g h t l y e a r l i e r time than does the corresponding  "break" i n the nitrogen to water r e l a t i o n s h i p .  To r e l a t e these observations to the processes involved i n growth and p h y s i o l o g i c a l aging, i t i s reasonable to assume that the rapid rate of increase of nitrogen r e l a t i v e to water during the i n i t i a l phase of rapid post-natal growth, i s associated with rapid hypertrophy of muscle tissue (63), growth of immature bone, the formation of collagenous and e l a s t i c f i b r e s i n the amorphous i n t e r c e l l u l a r matrix i n connective tissue (38), and an increase of i n e r t proteinaceous material i n the c e l l s themselves (90).  A t the same time, the r e l a t i v e l y low i n i t i a l rate of change of body  ash r e l a t i v e to frody water indicates that during t h i s f i r s t post-natal growth phase mineralization of the i n t e r c e l l u l a r matrix i n bone and other connective tissue does not keep pace with the rate of growth of these tissues.  A f t e r the f i r s t break i n the r e l a t i v e growth rate of the p i g l e t  which occurs at about f i f t e e n days, the increase i n body nitrogen r e l a t i v e  - 108  -  to body water continues a t a slower rate, and at the same time the s i m i l a r increase of body ash occurs at a more rapid rate than before, i n d i c a t i n g an acceleration i n the rate of mineralization of the skeleton and probably of other connective tissue r e l a t i v e to t o t a l body growth.  The f a c t that  the breaks i n the ash and nitrogen to water relationships do not coincide tends to support Hammond's (39)  concept of heterogonic growth according  to which the s k e l e t a l system matures at an e a r l i e r time than the musculature, since ash and nitrogen may be considered to represent primarily the s k e l e t a l and muscle systems respectively. The data presented i n Figures 11 and 12, and i n Tables 16, 17, and 18 also seem to support Bailey's contention (k) that the nitrogen-towater r a t i o i s a v a l i d index of physiological age i n mammals.  Spray and  Widdowson (102) have c r i t i c i z e d the concept of chemical maturation  (75)  on the grounds that the d i f f e r e n t components of the growing body do not mature at the same time.  Obviously, the nitrogen-to-water r a t i o , measuring  the changes i n the protein compartment r e l a t i v e to body water does not necessarily indicate the exact physiological changes i n other compartments such as that of body ash.  The nitrogen-to-water r a t i o , does, however, tend  to increase as the animal increases i n s i z e and development, and increases at a rate which r e f l e c t s the rate of change i n the animal as indicated by the r e l a t i v e t o t a l growth rate.  Figure 11 shows, however, that just a f t e r  the f i r s t "break" i n the rate of increase of the nitrogen to water r a t i o , that there may a c t u a l l y be a decrease i n the actual value of the r a t i o . In the present argument, this would mean that the animal suddenly decreased i n physiological age, an i n t e r e s t i n g p o s s i b i l i t y , which, though d i f f i c u l t to  - 109  -  v i s u a l i z e credibly, cannot nevertheless be discarded as being completely unlikely.  I t should however be noted that, as Tables 17  and 18 indicate,  the nitrogen-to-water r a t i o i s independent of both body weight and chronological time.  Although f a t - f r e e dry mass i s the most consistent  parameter against which to express nitrogen-to-water r a t i o s , i t i s probable that physiological aging i s also independent of changes i n f a t - f r e e body mass.  Thus, at l e a s t some of the scatter of data points shown i n Figures  11 and 12 i s caused by the f a c t that d i f f e r e n t animals having d i f f e r e n t p o t e n t i a l mature weights due to sex or other genetic differences may  be  expected to be of d i f f e r e n t p h y s i o l o g i c a l age when they contain the same amount of f a t - f r e e dry mass.  In spite of the above arguments, however,  i t would appear that the nitrogen-to-water r a t i o can be used to indicate the physiological state of the animal. 2.  The Constancy of Proportion of Fat-Free Dry Mass Relative to Body Weight The constancy of the percentage of the body weight represented  by the f a t - f r e e dry mass of the growing pigs used i n t h i s experiment  can  only be considered as being a b i o l o g i c a l coincidence due to the opposite r e l a t i v e changes i n the body compartments as described e a r l i e r .  The prac-  t i c a l use of this constant percentage has already been demonstrated, since apparently, i n r a p i d l y growing animals i n the range of one to t h i r t y k i l o grams of body weight the percentage of f a t - f r e e body mass i s r e l a t i v e l y i n s e n s i t i v e to changes i n conformation or body composition.  I t would seem,  however, that i n mature animals which the only body compartment subject to any appreciable change i s body f a t , that a high or low plane of n u t r i t i o n would tend to lower or raise the r e l a t i v e amount of the f a t - f r e e dry mass.  -110 Thus, i n the more mature animal i t would probably be desirable to estimate the f a t - f r e e dry matter on the basis of t o t a l body water despite the arguments r a i s e d i n an e a r l i e r section to the 3.  contrary.  The Composition of Gain i n Relation to and Growth i n P i g l e t s The data i n Figures 23 to 31 i n c l u s i v e show the r e l a t i v e rates  of gain of t o t a l body weight and of the estimated f a t and protein compartments f o r several representative pigs.  No p a r t i c u l a r attempt has been made  to impose l i n e a r i t y on any of the data, however, i t i s evident that the growth of the pigs can be divided i n t o several periods i n which the r e l a t i v e rate of growth i s almost constant, and that as has been mentioned e a r l i e r between each growth phase there i s an abrupt change i n the r e l a t i v e growth rate. Brody ( l l ) has demonstrated s i m i l a r breaks i n the r e l a t i v e growth rates of many species including man.  Actually, i t i s perhaps more exact to consider  the pattern of growth i n these plots as a series of shallow concave upwards curves, and these have been drawn wherever the data permitted, rather than regressing a l i n e a r approximation.  I t i s assumed that these abrupt breaks i n r e l a t i v e growth rate have some profound b i o l o g i c a l s i g n i f i c a n c e ; however, a complete explanation f o r t h e i r occurrence i s not r e a d i l y given, although Brody ( l l ) has demonstrated that one such break coincides with the onset of puberty. respect, i t i s i n t e r e s t i n g that the f i r s t "break" evident i n the  In t h i s present  data agrees quite c l o s e l y i n time with the rather major change i n the digestive enzymes of the pig observed by Bailey et a l (Ua) and Walker (108a) i n animals of about twenty-one days of age.  One plausible explanation f o r  FIGURE  23  CH ANGES IN PROTEIN. WITH AGE  FAT  AA/A B O D Y  WEIGHT  PIG NO. 51  £  fo  io  30  4o  so  feo  FIGURE 24 CHANGES IN TROTEIN. FAT AND'Bony WEIGHT WJTH PIG/VO.  5 7  AGE"  F I G U R E  CHANGCS  2  5  . : _ _ . . ( . . .  IN TRQTCIN.FATAND WITH  AGE  2oJ  BOBY  '  WEIGHT  L 2.0 KG. PROTEIN AND FAT J  10.  5 J  . 10  Lo-5  4 - FAT  O =• PROTEIN X  2  - BODV  Wf/GHT  J  A G F /A/ZMY5 I—  •o  —I—  20  30  WEIGHT ...  P/G No. / / O KG  BODY  :  — I —  AO  —1— 50  FIGURE  26  CHANGES IN P R O T O N , WEIGHT W / T H AGE PIG  NO.  113  FAT A N D B O T J Y  FIGURE 27 CHANGES IN TRQTEIN, FAT, AND WITH PIG  NO.  122  AGE  B O Z ? Y  WEIGHT  FIGURE  No.  28  CHANGES IN PROTEIN , FAT, AND WITH  TIG  NO.  12 H  AGE  BODY  WE/GHT  FIGURE  2.1  CHANGES IN VROJEIN , EAT, AND BODY WF/ WITH  TIG  AGE  130  NO.  AGE  - i —  vo  SO  30  IN  77AN5  AO  50  FIGURE 3 0 C H A N G E S  I N  WEIGHT  WITH  PIG NO. 3 0 _| 1.3.0  20  PROTEIN . FAT, AIVD "BODY A G E  121  •20 KG.  KG. BODY  PROTEIN  AND  FAT  I  10.  5_  10  O- 5 F*T P R O T C / / V BOI>Y  W E I G H T  2}  / AGE  —  O  10  20  IN  t  —  30  TMYS  FIGURE 3 / CHANGES  IN  PROTEIN,  M O .  J 3 / I  A.  - FAT  X  = Bo'QY  AGE —I—  B O D Y  AGE  W E I G H T WITH PIG  F A T , AND  IN  WEIGHT  DAY 5  50  - Ill these grovrth breaks i s that the animal reaches a point when i t can no longer consume enough energy to maintain i t s present growth rate, causing i t to begin to gain at a slower rate.  That t h i s i s an o v e r s i m p l i f i c a t i o n  i s indicated however by the f a c t that i t has been noticed ( l l 8 a ) that a n i mals may  v o l u n t a r i l y reduce t h e i r feed intake at the time when the growth  break occurs.  The f a c t that the two breaks evident i n most of the  present  data are coincident with changes i n the nitrogen-to-water and ash-to-water relationships indicates that each such break i n r e l a t i v e growth rate must be an i n t e g r a l part of the o v e r a l l process of p h y s i o l o g i c a l aging.  That  these breaks occur at d i f f e r e n t body weights i n d i f f e r e n t animals indicates that they are probably g e n e t i c a l l y determined, i n that a pig having a greater p o t e n t i a l mature body weight would be expected to have growth rate changes at higher body weights than a p i g having a smaller mature weight. I t i s also almost c e r t a i n that these changes i n r e l a t i v e grovrth rate are affected by the environment, but inasmuch as the optimum environmental conditions f o r growing p i g l e t s are not yet known, further experimentation i s necessary before the genetic and environmental e f f e c t s on these changes can be d e f i n i t e l y separated.  The data i n Figures 23 to 31 i n c l u s i v e shows that the r e l a t i v e growth rate of the protein compartment r e f l e c t s c l o s e l y the growth rate of the t o t a l body mass.  The r e l a t i v e rate of increase of the t o t a l body  water compartment has not been plotted, but must show the same r e l a t i o n s h i p as protein, since t h i s was  calculated on the basis of body water.  The  rate  of gain of the body f a t compartment showed quite a d i f f e r e n t r e l a t i o n s h i p . In Figure 10 i t was noticed that there seemed to be peaks i n the i n - v i t r o  - 112 per cent body f a t data at approximately twenty and f i f t y days post partum. An examination of the data i n Figures 23 to 31 shows that t h i s i s supported by the in-vivo data.  observation  Although each of the pigs shown i n these  graphs gave a somewhat d i f f e r e n t picture of f a t and protein gain, the following general pattern was  demonstrated.  While the rate of protein gain  c l o s e l y followed that of the gain of the body whole, the body f a t compartment exhibited a series of alternate low or even negative rates of gain followed by high rates of gain.  Usually, the high rate of fattening con-  tinued u n t i l just p r i o r to the time of the f i r s t growth rate break. this point, there was  After  usually f i r s t a s h o r t - l i v e d increase and then a steady  decrease i n the rate of f a t gain which p e r s i s t e d u n t i l the rate of protein gain decreased abruptly, a f t e r which the rate of fattening increased. second peak of fattening was  also s h o r t - l i v e d and was  This  followed by a second  decline i n rate which persisted u n t i l a f t e r the next break i n the rate of protein gain.  Although the in-vivo data i n ths experiment does not deal with pigs older than s i x t y days, i t i s suspected that t h i s pattern of leaning and fattening i s continued throughout the whole growth period.  Before further discussion of these effects can be entered, i t i s f i r s t necessary to e s t a b l i s h that these fluctuations i n fattening are not artifacs.  F i r s t l y , i t must be considered that as already shown error i n  the measurement of water, and hence of protein w i l l lead to an  opposite  error i n the stimation of body f a t , and indeed the r e l a t i v e rates of protein and f a t gain do occur i n an opposite manner.  Secondly, i t i s possible that  an underestimation followed by an overestimation i n the water compartment  - 113 could r e s u l t i n an apparent decrease i n the rate of body f a t gain.  Since  the possible error of body water measurement i n t h i s experiment was  about  two per cent, successive under and over estimations  of t h i s magnitude i n  the body water compartment of a f i v e kilogram p i g would lead to a d a i l y decrease i n apparent f a t gain of twenty-nine grams, giving a corresponding energy decrement of 272 would be doubled.  Calories.  In a ten kilogram pig, these quantities  F i n a l l y , i f such error i n the estimation existed, an  opposite change i n the rate of protein gain would occur, and i n almost a l l instances where negative fattening occurred, t h i s i s the case. In spite of the above, the following strong arguments e x i s t to support the v a l i d i t y of the r e s u l t s shown i n the above figures and i n Table 25.  F i r s t l y , an examination of the data i n Table 25 indicates that  while many of the f a t d e f i c i t s are of the same order or l e s s than those possible due to the a n a l y t i c a l errors described above, many such d e f i c i t s are much greater than those shown above.  In addition, the r e g u l a r i t y of the  fluctuations i n the rate of f a t gain indicates that these events are probably not caused by the random a n a l y t i c a l errors which could occur on the basis of the data i n Table 8.  At the same time the f a c t that a l l of the f a t decre-  ments i n a given l i t t e r do not occur between the same time i n t e r v a l s indicates that these changes r e s u l t from actual changes i n the pigs and are not due systematic  errors i n the measurement of body composition.  to  F i n a l l y , although  i t i s evident that changes i n the rate of fattening do seem to be  opposite  to changes i n the rate of protein gain, i t must also be r e a l i z e d that as has already been noted, the rate of protein gain corresponds c l o s e l y with the o v e r a l l growth rate,, which i s independent of any of the errors involved  - Ilk  -  i n the estimation of body composition in-vivo except those concomitant with weighing the pigs.  The following calculations i l l u s t r a t e that the observed  fluctuations i n body composition are reasonable i n terms of p r a c t i c a l p o s s i bilities. F i r s t , l e t us consider l i t t e r No. 1, 53 days.  between the ages of 3U and  During t h i s time there were no evidences of creep feeding, and  since the piglets were never observed to eat the sow's feed, i t must be assumed that a l l of t h e i r energy intake was derived from the sow's milk. In the following calculations, the approximate energy requirements f o r r e s t i n g metabolism have been extrapolated from the values given f o r swine by Brody ( l l ) .  The amounts of energy required f o r the grovrth of the p i g l e t s  have been extracted from Table 25. When the l i t t e r was between 3h and U l days o l d , the sow was suckling three p i g l e t s , numbers 56, 57 and 59, having average weights of 11.0,  11.2  and 12.6  kilograms respectively.  These p i g l e t s had d a i l y resting  metabolism energy requirements of approximately 1300,  1300,  and 1350  Calories  and d a i l y gains of 880, I4I4.O and 2073 Calories, giving t o t a l d a i l y energy requirements of 2180,  17U0 and 3U-23 Calories respectively.  If i t i s  assumed that the net energy content of sows' milk i s 500 Calories per pound as consumed, (Table 2, Appendix) the above energy requirements would be met by the consumption of U.36, the  three p i g l e t s .  3.U8,  and 6.85 pounds of milk d a i l y by  The sow must therefore be producing l U . 6 9 pounds of  milk per day, a high but s t i l l reasonable milk y i e l d .  Table 6 of the  appendix shows that during t h i s period, Sow No. 36 consumed 12 pounds of Ration 10-59  d a i l y , or about IO.76U net Calories (Table 2, Appendix).  - 115  -  I f the 177 Kilogram sow's r e s t i n g metabolism requirement i s 3962 Calories, 6802 Calories of the net energy consumed may be used f o r producing milk. If the net energetic e f f i c i e n c y of milk production by the sow i s 60 per cent, as i t i s i n the cow, each pound of sows milk requires 833 net Calories f o r synthesis, and the sow can produce 8.12 energy i t consumed i n the feed.  pounds of milk on the basis of the  The a d d i t i o n a l 6.57  above could be produced i f the sow used 1.28  pounds of milk indicated  pounds of her body f a t to  provide the additional 5U72 net Calories required by the p i g l e t s .  In the period when the l i t t e r was between U l and U8 days of age, sow number 36 was only suckling two p i g l e t s , numbers 57 and 59. l e t s had average weights of 13.9  and 15.2  metabolism requirements of lUOO and 1550  These p i g -  Kilograms, and had d a i l y r e s t i n g Calories.  They gained 1259  and  186 Calories each day, and therefore had t o t a l d a i l y energy requirements of 2659 and 136U Calories respectively, which could be met by the d a i l y consumption of 5.32  and 2.73  pounds of milk.  The sow's d a i l y milk output i n  this period was thus 8.03 pounds, which would require no depletion of her body f a t reserves.  When the l i t t e r was between the ages of U8 to 53 days, only one p i g l e t , No. 57 remained.  This p i g had an average weight of 16.5  Kilograms  and required 1700 and 1U75 Calories f o r r e s t i n g metabolism and growth respectively. the  The t o t a l energy requirement of 3175  consumption of 6.35 pounds of milk.  Calories could be met by  During t h i s l a s t period the sow  reduced her feed intake to 9.U pounds per day, which would supply UU08 Calories above her own resting metabolism requirement, enabling her to produce 5.29 pounds of milk i/ithout depleting her f a t reserves.  In t h i s  - 116 case, i t i s very possible that the p i g l e t , now weighing 36 pounds, made up the a d d i t i o n a l 530 Calories per day be eating a small amount of e i t h e r the creep r a t i o n or the sow r a t i o n , although t h i s was not observed. On the basis of the above discussion, i t would appear that a l l of the values shown above are of a reasonable magnitude, there i s a remarkably good agreement between the calculated energy requirements of the growing p i g l e t s and the amount of energy available to them i n the sow's milk.  The  f a c t that the p i g l e t s i n l i t t e r 1 refused to eat any creep ration appears to stem from two causes.  F i r s t l y , the sow was  consuming a r a t i o n which was  of a s l i g h t l y higher energy content than the regular sow r a t i o n and therefore would allow her to have a greater maximum milk production than i f she had been consuming the regular ration.  In addition, t h i s sow was,  during  most of her l a c t a t i o n , feeding a small number of pigs and since these were developing at d i f f e r e n t rates t h e i r energy requirements were complementary. Thus, when Pig No. 59 was f a t t e n i n g r a p i d l y and had a high energy requirement, pigs number 56 and 57 were f a t t e n i n g slowly or negatively and had a low net energy requirement.  When, i n the next time i n t e r v a l , pig number  57 began to f a t t e n r a p i d l y and to have a high energy requirement, pig number 59 was  entering a negative fattening stage and required only a small amount  of energy from the sow. Figures 23 and  These l a s t effects are demonstrated by the data i n  2k.  Similar calculations may be made f o r the other l i t t e r s . now  the p i g l e t s raised by sow number 16,  was between 37 and U3, k3 and 51,  Consider  during the i n t e r v a l s when the l i t t e r  51 and 58 days of age, and numbered four,  three, and two p i g l e t s respectively. A complete energy balance cannot be  - 117 calculated since body composition data i s not available f o r one of the pigs, number 132.  However, calculations f o r the other piglets i n the l i t t e r 1.  give a s i m i l a r pattern to that shown above l i t t e r In the f i r s t period, pigs number 122, 11.1,  and 10.9  12l* and 129 weighed  Kilograms respectively and had t o t a l energy requirements of  345>9, 205U, and 1152  Calories.  These requirements could be met by the  energy i n 6.90,  4.12,  pigs number 122  and 129 weighed 13.3  requirements of 1159  and 2.31 pounds of sow milk. and 13.9  In the second period,  Kilograms and had t o t a l energy  and 3792 Calories respectively, which would be  ingested i n 2.32  and 7.59  122 weighed 17.1  Kilograms and required 1*073 Calories and 8.15  milk.  10.5,  pounds of milk.  In the f i n a l period, p i g number pounds of  During a l l three periods, the sow, weighing approximately  206  Kilo-  grams consumed an average of 15 pounds of r a t i o n 28-59, which provided 13,200 Calories.  This would lead to an excess of 9150  Calories over the  4050 Calories required f o r r e s t i n g metabolism, which would lead to an average milk production by the sow of 11.0  pounds of milk per day without  causing any depletion of net body f a t reserves. there was an average of approximately produced per p i g l e t .  2.75,  3.67,  Thus, i n the three periods, and 5.50  pounds of milk  Although i t i s evident that i n d i v i d u a l p i g l e t s  consumed more or less than these average values, i t i s obvious that the t o t a l milk requirements shown above were greater than the milk production of the sow.  This may have been o f f s e t by the depletion of the sow's f a t reserves  to produce more milk, although i t i s more l i k e l y that the a d d i t i o n a l energy was  derived from the creep ration, since i t was observed that the piglets  i n t h i s l i t t e r began to eat considerable quantities of r a t i o n 18-59 the age of twenty days.  In t h i s respect, i f pig number 122  after  d i d receive only  - 118 5.50  -  pounds of milk d a i l y instead of 8.15  pounds between the ages of 51  58 days, i t would be able to make up the a d d i t i o n a l 1325 by consuming 1.U5  and  Calories required  pounds of the creep r a t i o n , an intake which would not be  impossible f o r a p i g weighing 38 pounds. The foregoing discussion indicates that the fluctuations i n the rates of fattening shown by the data i n Table 25 and i n Figures 23 to i n c l u s i v e are energetically possible.  31  Apart from the differences between  i n d i v i d u a l pigs i n the positions of the end points of the d i f f e r e n t growth phases, r e l a t i v e to age and body weight and i n the absolute proportions  of  the body f a t and protein compartments, a remarkably consistent pattern of r e l a t i v e body composition changes during each grovrth phase i s demonstrated. This pattern has already been described, and i s summarized as follows.  At the beginning of each phase of growth, the r e l a t i v e rate of protein gain i s at a minimum, and this rate increases u n t i l i t reaches a maximum f o r the growth phase j u s t before the next growth phase begins. The r e l a t i v e rate of fattening, however, reaches a maximum soon a f t e r the beginning of the growth phase, then begins to decrease at about the midpoint of the growth phase and i s minimal at the end of the period.  In  this respect, there i s an anomaly i n the case of the l i t t e r 2 pigs, which showed a minimum i n the rate of f a t gain at t h i r t y - s i x days, but did not exhibit the expected corresponding break i n either the rate of protein or of t o t a l body gain as shown i n Figures 25 and 26.  In these pigs i t i s  possible that s l i g h t breaks i n the above rates did occur, or else that the observed depression of fattening rate i n this case was  due to some environ-  mental e f f e c t , at this time these pigs were found to be very anemic and were  - 119 given an i n j e c t i o n of iron-dextran.  -  In addition, both pigs began to eat  creep r a t i o n s h o r t l y thereafter, possibly accounting f o r the observed In p i g number 113,  increase i n fattening rate.  a further decrease i n  fattening occurred just p r i o r to f o r t y - e i g h t days, -where the upsweep of the body weight and protein gain curves suggests the approach of a genuine growth rate break.  An explanation f o r the t y p i c a l body composition may be as follows.  changes observed  In the beginning of the growth phase, when the r e l a t i v e  rate of protein gain i s minimal, the maintenance energy requirement i s also low.  At the same time the feed intake i s maximal at the beginning of each  growth phase as evidenced by the t o t a l energy gain of the pigs at t h i s time. Thus, a maximum amount of energy i s a v a i l a b l e f o r growth, and since protein gain i s proceeding slowly, r a p i d fattening occurs.  Towards the end of the  growth phase, the rate of protein gain increases, causing an increase i n the energy cost of growth and maintenance.  At the same time the data i n  Table 25 indicates that f o r some reason, there i s a decrease i n the t o t a l amount of energy consumed.  Thus, the energy required f o r the increased  rate of protein synthesis must come from body f a t .  The consequent decrease  i n the rate of f a t gain does not e f f e c t the r e l a t i v e rate of body weight gain because the increased rate of protein gain i s accompanied by an increase i n t o t a l body water, at l e a s t some of which arises metabolically from the breakdown of body f a t . The pattern of fattening and the reduction of feed energy intake shown by the p i g l e t s during the f i n a l part of each growth phase are s i m i l a r to the changes observed i n deer at the time of t h e i r winter growth rate break ( l l 8  a).  - 120 -  I t i s i n t e r e s t i n g that while the rate of protein synthesis i s maximal during the f i n a l part of each growth phase, the apparent r e s t r i c t i o n of energy intake by the p i g must r e s u l t i n a r e s t r i c t i o n i n i t s protein intake.  Thus, while the pig has enough energy i n the form of f a t  to support the accelerated rate of protein synthesis, there must come a point where the protein intake of the p i g becomes inadequate to allow t h i s rate of protein gain to be continued.  The rate of gain of protein and water  would then p r a c t i c a l l y stop u n t i l the mechanism which controlled the pig's appetite readjusted, causing an increased feed intake i n order to support further growth.  In such a case, when the protein intake of the p i g l e t f e l l  below that required f o r the rate of protein gain, the animal might, i n the following period of adjustment continue to gain some tissue protein at the expense of reserve protein i n the l i v e r and i n the blood plasma. events would lead to a s i t u a t i o n i n which there was proteinaceous  These  f o r a short time,  growth with no net increase of body protein, with body f a t  being broken down to provide the necessary energy and a concomitant release of metabolic water i n the c e l l s .  Such a s i t u a t i o n could lead to the observed  apparent increase of body water r e l a t i v e to body nitrogen at the time of the f i r s t growth break as shown i n Figures 11 and 12.  This l a s t i s pure conjec-  ture, and further explanation of the observed breaks i n the r e l a t i v e growth curves i s beyond the present scope of t h i s i n v e s t i g a t o r . The following calculations, however, lend some truth to the argument. tions i t was  In previous c a l c u l a -  shown that pig number 59 at the age of between 3k  fattened r a p i d l y and consumed an estimated 6.85  and Ul days  pounds of sows milk.  In the  following week, t h i s pig showed a negative f a t gain, and only consumed an estimated 2.73  pounds of milk.  I f sow's milk i s 5.9  per cent protein  (69),  - 121 these amounts o f m i l k would p r o v i d e the p i g l e t w i t h kOk and 73 grams o f p r o t e i n d a i l y i n t h e two p e r i o d s .  A t t h e same time, the d a i l y p r o t e i n  g a i n d u r i n g t h e s e p e r i o d s as shown i n T a b l e respectively.  25 was  26.0  and 73.0  Thus, i t w j u l d seem t h a t w h i l e the p r o t e i n i n t a k e  grams during  the f i r s t p e r i o d above was more than adequate t o meet the requirements f o r maintenance and growth, t h a t o f the second p e r i o d was n o t .  I n t h e above d i s u c s s i o n , a t t e n t i o n has been g i v e n t o the f l u c t u a t i o n s i n the r e l a t i v e r a t e s o f f a t t e n i n g and p r o t e i n g a i n d u r i n g the growth o f t h e young p i g .  I t s h o u l d be remembered t h a t w h i l e  the p i g gains f a t  more s l o w l y , o r even may l o s e some f a t a t the end o f each growth phase, t h e a b s o l u t e v a l u e o f the body f a t compartment  o f an a d e q u a t e l y  nourished p i g  tends t o i n c r e a s e w i t h age as shown b y t h e data i n T a b l e s 13-B, 2k,  and i n F i g u r e s 19 t o 22 i n c l u s i v e .  p r e s e n t d a t a , and by the l i t e r a t u r e  11+-B, l £ - B ,  I t i s a l s o demonstrated b y the  (39, 63, 72, 87) t h a t as an animal  matures, a g r e a t e r , and g r e a t e r p r o p o r t i o n o f t h e g a i n made i s f a t . Thus, as t h e a n i m a l matures and the r e l a t i v e r a t e o f p r o t e i n g a i n i n each  succes-  s i v e growth phase becomes l e s s , a g r e a t e r and g r e a t e r amount o f the i n g e s t e d energy which exceeds the animal's maintenance requirement i s s t o r e d as f a t , and t h e g r e a t e r the r e l a t i v e p r o p o r t i o n o f the body f a t compartment  becomes.  On the b a s i s o f t h e p r e s e n t work, i t would seem t h a t the r a t e o f f a t t e n i n g depends e s p e c i a l l y on t h e amount o f f e e d energy a v a i l a b l e t o t h e p i g l e t i n the i n i t i a l to  p a r t o f each growth phase.  I n a d d i t i o n , i t would seem l o g i c a l  b e l i e v e t h a t any f a c t o r which i n t e r f e r e d w i t h t h e r a t e o f p r o t e i n s y n -  t h e s i s would a l s o l e a d t o an i n c r e a s e d r a t e o f f a t t e n i n g p r o v i d e d the appet i t e was n o t i n h i b i t e d .  These c o n s i d e r a t i o n s c o u l d e x p l a i n t h e f a c t t h a t  - 122 while almost a l l of the p i g l e t s exhibited the same basic r e l a t i v e pattern of grovrth, the absolute quantities of the various body compartments during corresponding grovrth phases varied between individuals and between l i t t e r s . Thus, the animals i n L i t t e r 1, which received more milk than d i d the other p i g l e t s , tended to fatten more rapidly and to stay f a t t e r than the other pigs.  At the same time, a l l of the animals were more or less anemic at some  time during the experiment  and the effects of t h i s , though d i f f i c u l t  to assess  may have also contributed to some of the i n d i v i d u a l differences i n body composition change. Palseon (8l) indicates that the state of maturity of new animals i s a function of t h e i r physiological age at b i r t h .  born  Thus, i t can  be expected that body f a t , shown by Hammond (37) and McMeekan (63) to be the l a t e s t maturing of the body compartments would be present i n the greatest quantities i n animals which were p h y s i o l o g i c a l l y o l d at b i r t h .  Widdowson  (113) shows i n studies on a v a r i e t y of species including the p i g , the guinea pig  and the human that t h i s i s so.  In addition, her data shows that p i g l e t s  which were large at b i r t h had greater amounts of f a t than smaller, physiol o g i c a l l y younger p i g l e t s (Table 17).  In the present data, i t i s indicated  (Figures 23 to 31 i n c l u s i v e ) that within a given l i t t e r , the l a r g e r pigs at b i r t h tend to reach the f i r s t growth break at an e a r l i e r age than those smaller at b i r t h .  I t would seem therefore that the large p h y s i o l o g i c a l l y  older p i g l e t s , begin t h e i r f i r s t post-natal growth phase with i t s high rate of fattening, i n utero.  The extent to which the p i g l e t s mature i n utero i s  probably very dependent on the plane of n u t r i t i o n of the sow (108).  In this  respect, i t i s i n t e r e s t i n g that the guinea p i g which at b i r t h has a nitrogen-  - 123 tc—water r a t i o equal to that of a twenty-one day o l d p i g , has apparently almost completed the f i r s t u s u a l l y post-natal phase of growth i n utero. Thus, the data of Widdowson and McCance ( l l U )  shows that the guinea p i g  undergoes the same r e l a t i v e body composition changes at between one and seven days as the twenty-one day o l d p i g , including the r e l a t i v e decrease i n f a t and s l i g h t decrease i n the nitrogen-to-water r a t i o . 4.  The E f f e c t s of Restricted Feed Intake Many workers ( 4 , 57c, 63, 103) have observed that animals which  are  subjected to a low plane of n u t r i t i o n and are subsequently realimented,  produce carcasses which have a higher r e l a t i v e f a t content than those of animals grown on a high plane of n u t r i t i o n .  Bailey (4) working with mice  has noted that during such periods of realimentation the gross energetic e f f i c i e n c y of gain i s greater than that of normal animals of the same body weight despite the high f a t content of the gain.  In addition, he shows that  at equal weights, the realimented animals had lower nitrogen-to-water r a t i o s and hence were p h y s i o l o g i c a l l y younger than were the normal mice even though the former were chronologically older.  The low plane of n u t r i t i o n therefore  had the e f f e c t of d r a s t i c a l l y slowing the rate of physiological aging.  A  possible explanation of these effects i n the l i g h t of the present findings i s as follows.  The low plane of n u t r i t i o n i n h i b i t s the rate of fattening and  allows protein and s k e l e t a l growth to proceed at sub-normal rates, with the protein gain being more s e r i o u s l y i n h i b i t e d than that of the skeleton (39). If the plane of n u t r i t i o n i s very low, the animal may remain i n the same protein growth phase as i t was i n when f i r s t retarded.  I f s l i g h t l y more  feed energy i s provided the animal may pass slowly from one protein growth  - 12U phase to another with l i t t l e or no net fattening, since any f a t gained would be almost completely used to support protein gain p r i o r to the growth rate break.  In any case when such an animal i s realimented, and growth  resumes with approximately the vigour of a normal animal of s i m i l a r physiol o g i c a l age, the f a c t that the rate of protein gain i s i n i t i a l l y lower than normal would decrease the i n i t i a l energy cost of maintenance and growth. The animal then i s able to store more of i t s t o t a l energy intake than could a normal animal of comparable p h y s i o l o g i c a l age.  Although both animals  w i l l probably show a s i m i l a r decrease i n r e l a t i v e fattening rates as they approach t h e i r next growth curve breaks, the realimented animal w i l l nevertheless have made a greater absolute f a t gain than the normal animal, and w i l l thus remain f a t t e r .  5.  The Effects of the A n a l y t i c a l Methods on the Growth Rates of the Piglets  As had been mentioned i n Section I, no obvious i l l effects on the pigs were noticed which could s p e c i f i c a l l y be attributed to any of the techniques involved i n the determination of body composition i n vivo.  There  wasatendency f o r the pigs which had had blood removed to be s l i g h t l y more anemic than those which had not been sampled, and t h i s could be expected to have some e f f e c t on the rate of growth.  Pigs number 16 and 132 were  sampled on only a few occasions and both of these animals grew f a s t e r than most of the others.  This e f f e c t could be accounted f o r at l e a s t i n part by  the f a c t that both of these pigs were l a r g e r than most of t h e i r l i t t e r mates at b i r t h and so could be expected to show greater gains.  In addition,  pig number 130, which was sampled regularly grew nearly as r a p i d l y as p i g number 132 which was not.  In the present experiment, there were therefore  - 125 too many differences between i n d i v i d u a l pigs and too few pigs involved to be able to say whether or not the procedure produced any l a s t i n g harm to the animals. in litters  S u f f i c e i t to say that at eight weeks, a l l of the surviving pigs  3 and k weighed between f o r t y - f i v e and f i f t y - e i g h t pounds - weights  which were at l e a s t average from a commercial point of view.  6.  The P r a c t i c a l Significance of the Present Findings At present, the marketing standards f o r bacon pigs require a long  lean carcass at a l i v e weight of two hundred pounds, with shorter, f a t t e r animals being subject to price discrimination.  The present data indicate that  i f the alternate fattening and leaning noticed f o r suckling pigs  continues  throughout the growth period, i t would be possible f o r the same p i g on the same plane of n u t r i t i o n to be either f a t or lean at market weight, depending on the stage of growth i t was  i n , and that i f i t were too f a t f o r the top bacon grade  at a weight of two hundred pounds, i t might make an excellent bacon carcass when i t weighed either a few pounds less or more than this weight.  The e f f e c t s  of n u t r i t i o n a l stress on the subsequent conformation and carcass composition of animals have been well established (U, 12, 39, 57, 63, 67, 81, 108),  and  i t i s demonstrated that the e a r l i e r the i n i m i c a l conditions are imposed on the animal, the more marked the effects of such treatment are l i k e l y to be. In the present study, i t was  seen that the animals i n l i t t e r 1 fattened more  r a p i d l y i n the f i r s t twenty days and remained f a t t e r than did the animals i n the other l i t t e r s .  While the p o s s i b i l i t y of genetic differences  between these animals cannot be excluded, differences i n environmental conditions between l i t t e r s ,  and even between i n d i v i d u a l pigs i n a  - 126 l i t t e r probably accounted f o r almost a l l of the observed differences i n conformation and composition.  In t h i s experiment  an attempt was made to  study the body composition changes of normally growing p i g l e t s under standard environmental conditions. The results of the experiment show that while each p i g l e t showed the same r e l a t i v e pattern of growth, the actual changes i n the p i g l e t s were d i f f e r e n t , subject to various environmental differences such as the physiological age of the p i g l e t at b i r t h , the number of piglets i n the l i t t e r , the rate of l a c t a t i o n by the sow, possible differences i n the composition of the sow's milk (98, the present and past n u t r i t i o n of the sow, other factors not even considered.  99)  r e s u l t i n g from  the ambient a i r temperature  and  I t i s evident then, that while n u t r i -  t i o n a l and other environmental factors a f f e c t i n g the p i g l e t i n i t s e a r l i e s t and most rapid growth phases can be expected to have serious effects on the animal's subsequent growth and development, neither the exact nature of early growth i n the p i g , or the effects of the environment on i t are p r e c i s e l y known.  The d i f f i c u l t y i n assessing the true genetic merit of  swine on the basis of either conformation or growth rate i n the post-weaning period can therefore be appreciated.  Thus, studies such as this which attempt  to c l a r i f y the patterns of animal growth have practical significance i n that they may contribute both to an easier assessment of genetic worth f o r the purpose of s e l e c t i n g superior breeding stock and to the more e f f e c t i v e use of present strains of swine f o r food production.  - 127 E.  SUMMARY AND CONCLUSIONS  In this study, the t o t a l body composition of four l i t t e r s of rapidly growing p i g l e t s between the ages of one and s i x t y - f i v e days has been determined.  One p i g from each l i t t e r was k i l l e d each week, and the body comp o s i t i o n of the carcasses was determined by c l a s s i c a l methods.  The data  obtained r e - i l l u s t r a t e s the gross changes i n body composition already observed i n the l i t e r a t u r e (75, 87, 77, 39, 63).  Thus, the r e l a t i v e amounts  of body water and body ash tend to decrease, while the r e l a t i v e amounts of body protein and f a t tend to increase during the f i r s t eight weeks of post-natal l i f e . continuous rates.  I t i s evident however that these changes do not occur at Close examination of the data reveals that the rate of  increase of body nitrogen r e l a t i v e to body water i s greater during the f i r s t f i f t e e n days of growth than subsequently, while the corresponding increase of body ash i s less before than i t i s a f t e r this age.  I t i s also shown that  the changes i n r e l a t i v e rates of increase of nitrogen and ash are r e l a t i v e l y abrupt, and that the change i n the ash r e l a t i o n s h i p occurs at a s l i g h t l y e a r l i e r age and weight than that f o r the nitrogen-to-water r e l a t i o n s h i p . This tends to support the concept of heterogonic growth (39) by which the d i f f e r e n t systems of the body grow and mature at d i f f e r e n t i a l rates.  It  was also observed i n this respect that the nitrogen-to-water r a t i o increased as the animals increased i n size and maturity, and that the rate of such increase i n the nitrogen-to-water r a t i o p a r a l l e l e d that of the o v e r a l l rate of change of body composition i n the animal i r r e s p e c t i v e of the chronological  - 128 -  age of the animal.  Therefore, i t seems that the contention that the  nitrogen-to-water r a t i o may be used as an index of physiological age (U) is valid. On the basis of the weights of the compartments of body nitrogen, water, and ash found i n the carcasses of p i g l e t s of d i f f e r e n t s i z e s , pred i c t i o n equations were calculated which allowed f o r the estimation of body nitrogen and ash on the basis of body water.  A further equation was  calculated f o r the estimation of the f a t free body dry mass, so that body, f a t could be estimated as being the difference between the weight of the t o t a l body and the sum of the t o t a l body water and f a t - f r e e dry mass.  I t was  noticed that c o i n c i d e n t a l l y the f a t free dry body mass was a remarkably constant percentage of t o t a l body weight, regardless of the absolute value of body weight or of the t o t a l body composition.  This f a c t was used  i n subsequent calculations to provide an estimate of f a t free dry mass which was independent of errors i n body water measurement, f o r the purpose of estimating body f a t as shown above*  Total body water was determined at weekly i n t e r v a l s by the deuterium oxide d i l u t i o n technique described i n Section I.  The amounts  of the other body compartments were then estimated using the prediction equations mentioned above.  I t was found that there was good agreement  between the in-vivo and i n - v i t r o methods of body composition determination.  The average d a i l y composition of gain f o r each weekly period was calculated on the basis of the body composition determined i n - v i v o .  When  the weekly weights of the t o t a l body and of the protein and f a t constituents  - 129 -  were p l o t t e d on a semi-logarithmic grid i t was seen that t o t a l body growth occurred i n d i s t i n c t phases.  In each such phase, the growth rate was  minimal at the beginning and slowly increased to a maximum at the end. Then the next phase began, and there was an abrupt decrease i n the . relative growth rate.  The average rate of gain i n each successive growth phase  decreased from b i r t h onwards.  I t was noticed that the f i r s t growth curve  break coincided with the observed break i n the ash-to-water and nitrogento-water relationships, i n d i c a t i n g that these breaks i n the r e l a t i v e growth rate are i n t e g r a l parts of the o v e r a l l process of p h y s i o l o g i c a l aging. The pattern of the rate of protein gain followed c l o s e l y that shewn by the t o t a l body weight.  However, the body f a t compartment c o n s i s t e n t l y  showed an inverse r e l a t i o n s h i p to body weight gain.  Thus, at the beginning  of each growth phase there was a low rate of protein gain and a high rate of f a t gain, while a t the end of each phase there was a high rate of protein gain and a lower or even negative f a t gain r e f l e c t i n g an apparent breakdown of body f a t to supply the energy necessary to support the accelerated protein gain.  Although feed consumption was not measured f o r the p i g l e t s ,  the estimated composition of gain indicated that although there was a high feed intake at the beginning of each growth phase, there was a greatly reduced feed intake at the end of each phase, and i t i s speculated that the termination of the growth phase occurred when the protein intake f e l l below the amount required by the p i g l e t f o r maintenance and f o r the accelerated rate of protein gain. As f a r as could be determined, the technique involved i n the determination of body composition i n - v i v o , had no adverse effects on the piglets.  - 130 TABLE NO. 1 Farrowing Data  L i t t e r No. Sow No.  4  1  2  3  k  36  3  12  16  Sire  The same Registered Landrace Boar  Number of Pigs Born  11  10  9  12  Number Born A l i v e  11  10  8  12  ft A l l sows were h a l f - s i b purebred Yorkshires  - 131  -  TABLE NO. 2 Percentage Proximate Compositions of the Rations*  Ration No.  18-59  10-59  28-59  Crude Protein  21. U  26.0  21.U  5.9  Nitrogen Free Extract  65.3  56.7  60.U  5.4  Fibre  2.9  U.3  5.9  -  Ash  7.0  8.9  8.5  1.0  -  -  -  91k  897  880  Water Approximate Net Energy Cals/lb /  Sow's Milk  81 5oo +  &  Calculated from values given by Morrison (69).  /  Assuming that net energy i s 50$ of gross energy i n rations 18-59, 10-59, and 28-59.  +  This i s the gross energy calculated from the percentages above. However, i t i s also 75$ of the gross energy content of sow's milk reported by Smith (98) and i s considered to be a reasonable estimate of the net energy of sow's milk.  - 132  -  TABLE NO. 3 Growth Data - L i t t e r No. 1 Pig No.  53  55  51  50  58  Sex  F  F  F  F  F  Age i n Days from B i r t h  Weights i n kg.  0  1.135  1.362  1.135  1.180  1.0UU  2  1.317  1.63U  1.2U9  1.362  1.180  5  1.816  2.088  1.771  1.861  1.771  3.269  3.269  12  *  13  3.178  19  U.99U  - 133 TABLE NO. 3 Growth Data - L i t t e r No. 2  P i g No.  I l l  118  lilt  Sex  F  F  F  Age i n Days from Birth  117 F  Weights i n kg.  l  1.589  1.U53  1.51*4  1.562  6  2.738  2.819  2.878  2.211  4.1U5  4.585  4.032  7.037  4.994  13  20  -  13U  -  TABLE N0. 3  Grovrth Data - L i t t e r Wo.  1  Pig No.  52  5U  56  59  57  Sex  M  F  F  F  M  Age i n Days from B i r t h  Weights i n kg.  0  1.275-  1.226  0.953  1.135  1.135  2  1.362  1.362  0.953  1.362  1.271  5  1.930  1.930  I.63U  1.861  1.771  13  3.723  3.799  3.U6U  3.768  3.723  19  5.362  5.589  U.79U  5.761  5.167  26  7.565  8.01+5  7.291  8.286  7.1+91  10.215  9.988  11.305  9.91+3  13.620  13.8U7  12.712  16.798  15.209  3k Ul U8 53  18.27U  - 135 TABLE NO. 3 Growth Data - L i t t e r No. 2  Pig No. Sex  115  HO  113  116  F  M  M  M  Age i n Days from B i r t h  Weights i n kg.  1  1.335  1.510;  1.562  1.875  6  2.515  2.315  2.7U7  3.196  13  lu713  U.5U0  5.362  U.99U  20  6.610  6.270  6.720  7.151  27  7.718  6.583  8.172  9.080  8.217  9.761  3h Ul U8 56  11.12  11.58  12.03  1U.98  16.12  18.38 19.52  -  -  136  TABLE NO. 3 Growth Data - L i t t e r s 3 and 1+  Pig No. Sex From Sow No. Raised by Sow No.  125  136  126  120  M  F  M  F  16 16  12 12  16 16  16 12  Age i n Days from B i r t h  127 F  119  16 16  16 12  Weights i n kg.  2  1.81+1+  1.930  1.703  1.787  1.67k  1.81+1+  8  3.60U  3.263  3.292  2.1+68  2.98O  2.866  5.81+5  3.972  5.136  4.625  5.085  7.082  6.311  15 22 29  7.673  - 137 TABLE NO. 3 Growth Data - L i t t e r s 3 and 1*  Pig No.  123  121  133  121*  13U  M  F  Sex From Sow No. Raised by Sow No.  16 12  16 16  Age i n Days from Birth  12 12  129  16 16  12 12  16 16  1.U76  1.930  0.738  2.298  3.3U8  1.731  Weights i n kg.  2  I.67I+  8  2.838  15  l*.lli*  5.533  U.199  3.859  5.335  3.151*  22  6.129  8.172  5.993  5.766  6.91*6  5.266  29  7.9U5  7.581  7.1*00  8.308  7.082  37  1*3  51  1.816  10.62 13.85  1.731  10.90  9.988 13.62  11.58 1U.53  9.988 12.17 16.30  - 138 TABLE NO. 3 Growth Data - L i t t e r s 3 and 1+  131  130  135  122  132  Sex  F  M  F  F  M  From Sow No. Raised by Sow No.  12 12  12 12  12 12  16 16  12 16  Pife No.  Age i n Days from B i r t h  2  Weights i n kg.  1.986  8  2.129  1.21+9  3.830  1.21+9  2.01+3  1.792  15  5.U20  6.157  2.721+  3.008  5.902  22  7.037  8.308  1+.222  U.5U0  8.898  29  8.671  9.988  6.038  6.536  11.08  9.080  9.716  11+.30  37  12.03  13.39  1+3  14.98  16.1+8  12.1+9  11.80  16.80  51  19.75  21.11  17.62  16.12  21.70  58  65  26.21+  21.1+3  20.61  22.93  26.79  31.10  - 139 TABLE NO. k Blood Haemoglobin and Iron Injection Data - L i t t e r No. 1  Age i n Days from B i r t h  8  Pig No.  15  29  37  k3  U8  51  Haemoglobin Measured i n Gm./lOO ml. Blood  51  15.3  50  11.5  58  10.0  13.2  52  13.5  15.5  5U  lii.o  10.2  7.5  56  iU.5  iU.5  12.0  59  12.5  1U.5  8.5  16.5  li.o  13.6  57  12.5  13.5  8.5  16.5  10.5  8.8  1U.5  15.8  Imferon Injection Schedule  globin determination  Note:  There was no evidence of creep feeding by t h i s l i t t e r ; the older p i g l e t s may, however, have eaten some of the sow's r a t i o n .  - lUo TABLE NO. k Blood Haemoglobin and Iron Injection Data - L i t t e r No. 2  Age i n Days 3h  13  from B i r t h Pig No.  h3  Haemoglobin Measured i n Gm./lOO ml. Blood  118  lS.o  llU  li.U  117  10.8  115  11.2  110  10.2  5.0  113  11.2  5.5  12.5  116  14.6  6.5  12.5  Imferon Injection Schedule  Note;  Age of Pigs i n Days  Ml. Imferon Injected  1  2.0  3k  4.0  Injected a f t e r Haemoglobin determination  F i r s t evidences of creep feeding observed when p i g l e t s were 37 days o l d  - lill TABLE NO. k Blood Haemoglobin and Iron Injection Data - L i t t e r s 3 and i*  Pig No.  Haemoglobin Level a t 23 Days  P i g No.  119  7.6  129  9.h  123  8.0  131  6.6  121  8.5  130  7.9  133  12.0  135  16.0  12k  8.5  122  13.U  131;  8.2  132  5.5  Imferon I n j e c t i o n Schedule Age of Pigs i n Days  Note:  Ml. Imferon Injected  2  2.0  23  3.0  Piglets began eating creep r a t i o n at 20 days.  Haemoglobin Level at 23 Days  - Ik2 TABLE NO. 5 Sow Weights  1  L i t t e r No.  3 and k  2  12  Sow No.  16  T  W  T  W  T  W  T  W  1  185  8  182  12  206  12  210  11  177  19  170  22  20l|  22  199  18  170  31  177  36  205  36  211  25  177  55  175  6k  208  6k  202  37  189  51  179  T  Time i n Days after Farrowing  W  Weight i n kg.  - 1U3 TABLE NO. 6 D a i l y Feed Intake of Sows  i  No.  36  3  12  16  10-59  28-59  28-59  28-59  i n Days Farrowing  Feed Intake i n Pounds  0  lu5  6.0  U.o  U.o  1  6.0  8.0  6.0  7.0  2  11.0  9.0  8.0  8.0  3  11.0  10.0  10.0  10.0  U  11.0  10.0  10.0  10.0  5  11.0  10.0  10.0  10.0  6  6.0  10.0  10.0  10.0  7  11.0  10.0  12.0  10.0  8  11.0  10.0  12.0  10.0  9  11.0  10.0  12.0  12.0  10  11.0  10.0  13.0  12.0  11  11.0  10.0  13.0  12.0  12  6.0  10.0  13.0  13.0  13  9.0  10.0  15.0  15.0  lU  9.0  10.0  15.0  10.0  15  11.0  10.0  i5.o  15.0  16  9.0  11.0  15.0  15.0  17  9.0  10.0  15.0  i5.o  18  9.0  10.0  15.0  15.0  19  9.0  10.0  15.0  i5.o  - 144 TABLE NO. 6 (cont'd.) D a i l y Feed Intake of Sows Sow No. Ration No.  36  3  10-59  28-59  Time i n Days A f t e r Farrowing  12 28-59  16 28-59  Feed Intake i n Pounds  20  9.0  10.0  15.0  15.0  21  11.0  10.0  15.0  15.0  22  11.0  10.0  15.0  15.0  23  11.0  10.0  15.0  15.0  2k  11.0  12.0  15.0  15.0  25  11.0  12.0  15.0  15.0  26  11.0  15.0  15.0  15.0  27  12.0  13.0  15.0  15.0  28  12.0  12.0  15.0  15.0  29  11.0  12.0  15.0  15.0  30  11.0  12.0  15.0  15.0  31  11.0  12.0  15.0  15.0  32  11.0  12.0  15.0  15.0  33  11.0  12.0  15.0  15.0  3k  12.0  12.0  15.0  15.0  35  12.0  12.0  15.0  15.0  36  12.0  12.0  15.0  15.0  37  12.0  12.0  15.0  15.0  38  12.0  12.0  15.0  15.0  39  12.0  12.0  15.0  15.0  - lU5 TABLE NO. 6 (cont'd.) D a i l y Feed Intake of Sows Sow No. Ration No.  36  3  10-59  28-59  Time i n Days A f t e r Farrowing  12  28-59  16 28-59  Feed Intake i n Pounds  Uo  13.0  12.0  15.0  15.0  Ul  12.0  12.0  15.0  15.0  U2  12.0  12.0  15.0  15.0  U3  12.0  12.0  15.0  15.0  UU  11.0  12.0  15.0  15.0  U5  11.0  12.0  15.0  15.0  U6  11.0  12.0  15.0  15.0  U7  10.0  5.0  15.0  15.0  U8  10.0  8.0  15.0  15.0  U9  9.0  10.0  15.0  15.0  50  9.0  10.0  15.0  15.0  51  9.0  10.0  15.0  15.0  52  9.0  10.0  15.0  15.0  53  9.0  10.0  15.0  15.0  5U  10.0  15.0  15.0  56  10.0  15.0  15.0  57 58  15.0 15.0  15.0 15.0  59 60 62  15.0 15.0 15.0 15.0  15.0 15.0 15.0 15.0  63  15.0  15.0  6U 65  15.0  15.0  61  It'  A  146  TRANQUI LLI ZATTON OF THE PIGLETS Tranquillization of the piglets was carried out i n order to f a c i l i t a t e the heart puncturing procedure, and to reduce the danger to the piglet of heart damage during the heart puncture. No systematic effort was made to establish dosage rates; however the following observations have been recorded because of their possible practical application. Without tranquillization, a successful heart puncture could only be carried out i f the animal was very firmly held on i t s back i n a V-shaped  trough by two persons, while a third person took a blood sample.  Under these conditions, the piglet was s t i l l capable of some movement, which i n some cases led to fatal damage to the heart during the heart puncture. In addition, the pectoral musculature was always tense, so that i t was d i f f i c u l t to feel the heart beat and to find the correct place to insert the blood taking needle. After a suitable level of tranquillization had been achieved the piglet, although conscious, was easily heart punctured by one person. The pectoral musculature was relaxed, so that the heart beat was easily palpated and the correct intercostal space for the insertion of the needle was readily found.  Furthermore, the piglets seemed f a i r l y insensible to  pain from the needle and during the blood sampling operation, were not prone to violent movement which might result i n injury* The following dosages were used and the following degrees of tranquillization i n piglets weighing from three to seventy pounds were obtained: 1. Aceprcmazine maleate: (Atravet* , an injectible product manufactured by 1  Ayerst McKenna and Harrison Ltd., Montreal, containing 10 mg^of aceprcmazine maleate per cc.)  ,147 a.  - -  0*25 mg* acepromazine maleate per pound of body weight: The t r a n q u i l l i z e r took e f f e c t within f i v e minutes, with the  the p i g l e t s assuming a head-down, sway-backed attitude with eyes p a r t i a l l y closed.  The p i g l e t s were p a r t i a l to l y i n g down, but were also quite able  to walk about.  Ten minutes a f t e r the intramuscular i n j e c t i o n ,  tranquillization  was deep enough f o r the p i g l e t to be quite e a s i l y heart-punctured by one person, although the best results were obtained i f one person l i g h t l y steadied the p i g while another drew the blood. In two hours, however, the effect of the t r a n q u i l l i z e r had p a r t i a l l y worn o f f , and the heart puncture was  effected  with much more d i f f i c u l t y . b.  0*5 mg* acepromazine maleate per pound of body weight* The t r a n q u i l l i z e r took e f f e c t within f i v e minutes a f t e r i n t r a -  muscular i n j e c t i o n . in  The head-down attitude described above was more pronounced  standing animals, and there was a much greater tendency f o r the p i g l e t s  to sleep, although they were s t i l l able to get up on t h e i r feet*  Ten minutes  a f t e r the i n j e c t i o n , a heart puncture could e a s i l y be made by one person* Further heart punctures could e a s i l y be achieved by one person at i n t e r v a l s up t o f i v e hours a f t e r the i n j e c t i o n of the t r a n q u i l l i z e r , although the p i g l e t s were seen to have become more a l e r t than they were at the time of the i n i t i a l heart puncture*  A f t e r f i v e hours, the p i g l e t s which had been  separated from t h e i r dams during the blood sampling schedule, were placed back with the sows, and were seen to suckle aggressively, even though they were s t i l l obviously t r a n q u i l l i z e d , as evidenced by t h e i r postural  attitude*  Twelve hours a f t e r i n j e c t i o n , no obvious signs of t r a n q u i l l i z a t i o n persisted* 2* Promethazine hydrochloride ("Phenergan",  an i n j e c t i b l e  solution  manufactured by Poulenic, Montreal, containing 25 mg. o f promethazine per cc.)  148  When injected intramuscularly at the rate of 0>5  mg.  per pound of  body weight, the p i g l e t s were t r a n q u i l l i z e d to a l e s s e r degree than when i n j e c t e d with acepromazine maleate at a s i m i l a r rate* t r a n q u i l l i z a t i o n were e s s e n t i a l l y the same as above*  The  signs of  However,  although  one person could quite e a s i l y effect a heart puncture ten minutes a f t e r the t r a n q u i l l i z e r was  injected, the e f f e c t had l a r g e l y worn o f f a f t e r three  hours* A macroscopic examination of the intramuscular i n j e c t i o n s i t e s i n p i g l e t s k i l l e d one week a f t e r t r a n q u i l l i z a t i o n did not show any obvious t i s s u e damage other than that caused mechanically by the hypodermic needle* No difference  could be seen between a one-week old t r a n q u i l l i z e r i n j e c t i o n  s i t e and a one week old s i t e where a s i m i l a r volume of p e n i c i l l i n had been i n j e c t e d with a needle of the same s i z e * As indicated e a r l i e r t r a n q u i l l i z a t i o n and manipulation of the pigs had no deleterious  e f f e c t s on feed consumption or growth rate*  BIBLIOGRAPHY  l-a  Association of O f f i c i a l A g r i c u l t u r a l Chemists, O f f i c i a l Methods of Analysis, Washington, 1950  1.  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