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A starch-gel electrophoretic study of some of the sources of variation in the blood sera of deer of the… Van Tets, Patricia Anne 1964

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A STARCH-GEL ELECTROPHORETIC STUDY OF SOME OF THE SOURCES OF VARIATION IN THE BLOOD SERA OF DEER OF THE GENUS ODOCOILEUS  by  PATRICIA ANNE VAN TETS B.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1961  A t h e s i s submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of MASTER OF SCIENCE i n the Department of Zoology  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  The U n i v e r s i t y of B r i t i s h October, 1964  Columbia  In the  presenting  r e q u i r e m e n t s f o r an a d v a n c e d  British  Columbia, I agree  available mission  f o r reference  f o r extensive  representatives.  cation  Department  of  by the Head  October,  I further  ZOOLOGY  19 6 4 .  Columbia,  of •  make i t f r e e l y ,  agree  that  per-  f o r scholarly  o f my Department  o r by  t h a t , c o p y i n g or p u b l i -  f o r f i n a n c i a l gain  permission*  ?  shall  copying of t h i s thesis  The U n i v e r s i t y o f B r i t i s h Vancouver 8 Canada Date  the L i b r a r y  I t i s understood  of this thesis  w i t h o u t my w r i t t e n  that  fulfilment of  degree a t the U n i v e r s i t y  and s t u d y .  p u r p o s e s may be g r a n t e d his  this thesis i n partial  shall  n o t be a l l o w e d  - i -  Chairman:  Professor  I . McTaggart Cowan ABSTRACT  After standardizing  a starch-gel  electrophoretic  technique, v a r i a t i o n s i n the serum p r o t e i n s  of the genus  Odocoileus due t o the c o n d i t i o n of the sample and the c o n d i t i o n of the animal were  studied.  S i g n i f i c a n t changes i n the serum sample were brought about b y hemolysis, c l o u d i n e s s , Cold  and decomposition.  storage f o r two years of a d u l t deer serum, the  a d d i t i o n of a b a c t e r i o s t a t t o the sample, and the use of a muscle r e l a x a n t  t o procure samples from c a p t i v e  deer  produced no s i g n i f i c a n t changes i n e i t h e r the m o b i l i t y or the p e r c e n t composition of the p r o t e i n f r a c t i o n s . A l a r g e i n d i v i d u a l v a r i a t i o n was found i n both the m o b i l i t y and the p e r c e n t composition o f the p r o t e i n fractions. The  p e r c e n t composition of the p r o t e i n f r a c t i o n s  was a f f e c t e d by sex, age, and season.  The m o b i l i t y of  the p r o t e i n f r a c t i o n s was a f f e c t e d by sex, b u t not by age or season. C a p t i v e deer a t the U n i v e r s i t y of B r i t i s h e x h i b i t e d an a d d i t i o n a l n e g a t i v e l y m i g r a t i n g  Columbia  protein  f r a c t i o n when compared to t h e i r w i l d  counterparts.  Comparisons of the m o b i l i t i e s i n three groups of a d u l t females of the genus Odocoileus i n d i c a t e i n t r a - s u b s p e c i f i c d i f f e r e n c e s than  greater  inter-specific  differences. The therefore,  technique of s t a r c h - g e l may  r e c o g n i t i o n but  electrophoresis,  be u s e f u l i n i n d i v i d u a l and  herd  i t i s not u s e f u l i n the r e c o g n i t i o n  subspecies or species  of  of deer of the genus O d o c o i l e u s .  ACKNOWLEDGMENTS  I wish t o express my a p p r e c i a t i o n t o Dr. I . McTaggart Cowan, Dean of Graduate S t u d i e s , U n i v e r s i t y of B r i t i s h Columbia, who p a t i e n t l y d i r e c t e d the study, t o Dr. W. S. Hoar P r o f e s s o r i n the Department  of Zoology, U n i v e r s i t y of B r i t i s h  Columbia, who t o l e r a t e d my equipment  and presence i n h i s  l a b o r a t o r y , t o Dr. H. T s y u k i , F i s h e r i e s T e c h n o l o g i c a l  Station  Vancouver, B. C , who r e a d i l y imparted h i s knowledge of e l e c t r o p h o r e s i s , and t o Mr., G. M c l n t y r e of Mathematical S t a t i s t i c s , C.S.I.R.O., Canberra, A.C.T., A u s t r a l i a , who checked my method of s t a t i s t i c a l  analysis.  Many thanks are a l s o due t o the f o l l o w i n g who p r o v i d e d the  b l o o d samples:  Mr. G. E . Connolly, Hopland F i e l d S t a t i o n , U n i v e r s i t y of C a l i f o r n i a , Hopland, C a l i f o r n i a , U.S.A. Mr. Wo E . Dodge, U n i t e d States Department  of the I n t e r i o r ,  F i s h and W i l d l i f e S e r v i c e , Bureau of Sport F i s h e r i e s and W i l d l i f e , F o r e s t Research Center, Olympia, Washington, Mr. S. G a l l i z i o l i ,  U.S.A. A r i z o n a Game and F i s h  Department,  Phoenix, A r i z o n a , U.S.A. Mr. W. C. Glazener, Rob and B e s s i e Welder W i l d l i f e Foundation W i l d l i f e Research and E d u c a t i o n , S i n t o n , Texas, U.S.A.  - iv-  Dr.  W. M. Longhurst, Hopland F i e l d S t a t i o n , U n i v e r s i t y of C a l i f o r n i a , Hopland, C a l i f o r n i a ,  Mr. W..A.  U.S.A.  Low, Department of Zoology, U n i v e r s i t y of B r i t i s h  Columbia, Vancouver, B r i t i s h Columbia, Canada. Mr. I . D. Luman, Oregon S t a t e Game Commission, P o r t l a n d , Oregon, U.S.A. Mr. D. McCaughran,  Division  of F i s h  and Game, P r o v i n c e of  B r i t i s h Columbia, Nanaimo, B r i t i s h Columbia, Canada. Mr. E. I . McDougall, The Rowett Research I n s t i t u t e , Bucksburn, Aberdeenshire,  Scotland.  Mr. H. Merriam, S t a t e of A l a s k a Department of F i s h and Game, Petersburg, A l a s k a , U.S.A.  A special friends  thank you must be g i v e n t o the many  and a s s o c i a t e s who f r e e l y gave of t h e i r  i n f o r m a t i o n , and time.  advice,  -  V  -  TABLE OP CONTENTS page Introduction  1  M a t e r i a l s and Methods  6  Source and c o l l e c t i o n date of samples  6  P r e p a r a t i o n of the sample  8  E l e c t r o p h o r e t i c procedure  11  A n a l y s i s of r e s u l t s  20  R e s u l t s and D i s c u s s i o n  25  C o n d i t i o n of sample  25  Hemolysis and c l o u d i n e s s  25  Preservation  32  Succinylcholine chloride  43  C o n d i t i o n of animal  48  Individual variation  48  Sex  54  Age  62  Season  70  Captivity  77  Geographic s e p a r a t i o n , subspecies,  and s p e c i e s  80  Conclusions  88  Summary  91  Literature Cited  93  - vi -  page Appendix A E f f e c t of c a p t i v i t y - m o b i l i t i e s _5 10  2 cm  in  . / v o l t seconds  105  Appendix B E f f e c t of c a p t i v i t y - p e r c e n t  composition  i n a r c s i n p e r c e n t a g e degrees  106  Appendix C Geographic s e p a r a t i o n , subspecies, -5 2 / species - m o b i l i t i e s  i n 10  cm  and  / v o l t seconds  107  specie - p e r c e n t composition n a r c s i n / p e rand centAppendix D s Geographic separation, i subspecies, age degrees  108  - vii -  LIST OF TABLES  Table  Page -5  1.  E f f e c t of hemolysis  - m o b i l i t i e s i n 10  cm  2. /  v o l t seconds 2.  E f f e c t of hemolysis  28 - Student's  t - t e s t comparisons  of the m o b i l i t i e s of the serum p r o t e i n f r a c t i o n s 3.  E f f e c t of hemolysis  - p e r c e n t composition i n  arcsinVpercentage degrees 4.  E f f e c t of hemolysis  30  - Student's  of the p e r c e n t composition  t - t e s t comparisons  ( i n arcsin/percentage  degrees) of the serum p r o t e i n f r a c t i o n s  .31 -5  5.  E f f e c t of c o l d storage - m o b i l i t i e s  i n 10  2 cm  v o l t seconds 6.  29  / 35  E f f e c t of c o l d storage - Student's  t-test  comparisons of the m o b i l i t i e s of the serum protein fractions 7.  E f f e c t of c o l d storage - p e r c e n t  36 composition  i n a r c s i n / p e r c e n t a g e degrees 8.  E f f e c t of c o l d storage - Student's  37 t-test  comparisons of the p e r c e n t composition ( i n arcsinvfeercentage degrees) of the serum protein fractions  38  - viii -  E f f e c t of a b l o o d p r e s e r v a t i v e -5 10  - mobilities in  2  cm / v o l t seconds  E f f e c t of a b l o o d p r e s e r v a t i v e  -  Student's  t - t e s t comparisons of the m o b i l i t i e s of the serum p r o t e i n f r a c t i o n s E f f e c t of a b l o o d p r e s e r v a t i v e  - percent  composition i n a r c s i n / p e r c e n t a g e  degrees  E f f e c t of a b l o o d p r e s e r v a t i v e  Student's  -  t - t e s t comparisons of the p e r c e n t  composition  ( i n a r c s i n v p e r c e n t a g e degrees) of the serum protein fractions E f f e c t of s u c c i n y l c h o l i n e c h l o r i d e - m o b i l i t i e s -5 2 , i n 1 0 cm / v o l t seconds E f f e c t of s u c c i n y l c h o l i n e c h l o r i d e -  Student's  t - t e s t comparisons of the m o b i l i t i e s of the serum p r o t e i n f r a c t i o n s E f f e c t of s u c c i n y l c h o l i n e c h l o r i d e - p e r c e n t composition i n a r c s i n v p e r c e n t a g e  degrees  E f f e c t of s u c c i n y l c h o l i n e c h l o r i d e -  Student's  t - t e s t comparisons of the p e r c e n t composition ( i n a r c s i n ^percentage fractions  degrees) of the serum p r o t e i n  - ix-  Table  Page  2,  -5 17.  I n d x v i d u a l varxatxon - mobxlxtxes  xn 10  cm /  v o l t seconds 18.  Individual comparisons protein  19.  50  v a r i a t i o n - Student's t - t e s t of the m o b i l i t i e s of the serum  fractions  Individual  51  v a r i a t i o n - p e r c e n t composition i n  a r c s i n ^percentage degrees 20.  Individual comparisons  52  v a r i a t i o n - Student's t - t e s t of the p e r c e n t composition ( i n  a r c s i n / p e r c e n t a g e degrees) of the serum p r o t e i n fractions  53 -5  21.  E f f e c t of sex - m o b i l i t i e s i n 10  2 cm / v o l t  seconds 22.  58  E f f e c t of sex - Student's t - t e s t comparisons the m o b i l i t i e s of the serum p r o t e i n  23. 24.  of  fractions  • 59  /percentage degrees E f f e c t of sex - p e r c e n t composition i n a r c s i n  60  E f f e c t of sex - Student's t - t e s t comparisons  of  the p e r c e n t composition ( i n a r c s i n ^percentage degrees) of the serum p r o t e i n  fractions  61  -  X  -  Table  Page -5  25.  E f f e c t of age - m o b i l i t i e s  i n 10  2 cm / v o l t  seconds 2 6.  E f f e c t of age - Student's t - t e s t comparisons the  27.  66  mobilities  of  of the serum p r o t e i n f r a c t i o n s  67  E f f e c t of age - p e r c e n t composition i n a r c s i n ^percentage degrees  28.  68  E f f e c t of age - Student's t - t e s t comparisons the  of  p e r c e n t composition ( i n a r c s i n ^percentage  degrees) of the serum p r o t e i n f r a c t i o n s -5 29.  E f f e c t of season - m o b i l i t i e s  i n 10  69 2  cm / v o l t  seconds 30.  E f f e c t of season - Student's t - t e s t of the m o b i l i t i e s  31.  73 comparisons  of the serum p r o t e i n f r a c t i o n s  E f f e c t of season - p e r c e n t composition i n a r c s i n v/percentage degrees  32.  74  E f f e c t of season - Student's t - t e s t  75 comparisons  of the p e r c e n t composition ( i n a r c s i n p e r c e n t a g e degrees) of the serum p r o t e i n f r a c t i o n s 33.  76  E f f e c t of c a p t i v i t y - t o t a l number of serum protein fractions  79  - xi -  Table 34.  Page Geographic s e p a r a t i o n ,  subspecies,  and  species  t o t a l number of serum p r o t e i n f r a c t i o n s 35.  E f f e c t of geographic s e p a r a t i o n and -5 mobilities  36.  i n 10  2 cm  83  species -  . / v o l t seconds  E f f e c t of geographic s e p a r a t i o n and  84  species  d i s t r i b u t i o n of serum p r o t e i n f r a c t i o n s  E f f e c t of geographic s e p a r a t i o n and Student's t - t e s t comparisons of the  -  into  c a t e g o r i e s a c c o r d i n g to t h e i r m o b i l i t i e s -5 2 . . 10 cm / v o l t seconds) 37.  -  (in  species  85 -  mobilities  of the serum p r o t e i n f r a c t i o n s w i t h i n m o b i l i t y -5 categories 38.  ( i n 10  2 cm / v o l t seconds)  E f f e c t of geographic s e p a r a t i o n and summary of d i f f e r e n c e s and mobilities  86 species  -  s i m i l a r i t i e s i n the  of the serum p r o t e i n f r a c t i o n s  87  INTRODUCTION  M o r p h o l o g i c a l c h a r a c t e r i s t i c s have been used s i n c e the days of A r i s t o t l e to c l a s s i f y organisms and s i n c e Darwin's " O r i g i n of S p e c i e s " to p o s t u l a t e e v o l u t i o n a r y relationships.  More r e c e n t l y , other f a c t o r s such as the  b e h a v i o u r a l , p h y s i o l o g i c a l , and b i o c h e m i c a l a t t r i b u t e s of organisms are a l s o b e i n g c o n s i d e r e d .  The p r e s e n t  study  t r i e s t o e v a l u a t e whether serum p r o t e i n s can h e l p to e l u c i d a t e the phylogeny of the genus Odocoileus. An e x c e l l e n t review of the u n d e r l y i n g p h i l o s o p h y of the a p p l i c a t i o n of p r o t e i n s t u d i e s to phylogeny has been presented by S i b l e y (1960).  I n v e s t i g a t i o n s of the  p r o t e i n s of amphibians and r e p t i l e s  (McGabe & Deutsch,  1952), b i r d s ( S i b l e y , 19 60), and s m a l l mammals e t a l , 1958,  Johnson & Wicks, 1959)  (Johnson  to a l a r g e extent  s u b s t a n t i a t e c l a s s i f i c a t i o n s based on morphology. A v a r i e t y of techniques  f o r the study of the  b l o o d p r o t e i n s of animals have been.elaborated  and  their  a p p l i c a t i o n s t o taxonomy are o u t l i n e d below. Serology Kraus i n 1897  d i s c o v e r e d the p r e c i p i t i n r e a c t i o n  - 2 -  and understood i t t o be a b s o l u t e l y s p e c i f i c This r e a c t i o n was  (Boyden, 1943).  f i r s t e x t e n s i v e l y a p p l i e d to the problems  of animal s y s t e m a t i c s by N u t t a l l (1901) and has s i n c e been w i d e l y employed by s e v e r a l workers.  Decapod Crustacea  have been s t u d i e d i n t h i s r e s p e c t by Leone Stallcup's  (1954) study of the f a m i l y F r i n g i l l i d a e i s the  f i r s t a p p l i c a t i o n of s e r o l o g y taxonomy.  (1954).  to a c t u a l problems i n avian  Mammals i n v o l v e d i n s e r o l o g i c a l i n v e s t i g a t i o n s  i n c l u d e the Orders Rodentia and Lagomorpha (Moody e t a_l, 1949, Moody & Doniger, 1956), C a r n i v o r a  (Leone & Wiens,  1956, Pauly & Wolfe, 1957), and Cetacea and A r t i o d a c t y l a (Boyden & Gemeroy, 1950). S e r o l o g i c a l s t u d i e s d i s t i n g u i s h i n g the c e r v i d genera Odocoileus, A x i s , and Cervus, were c a r r i e d out by Wolfe (1939) and B a i e r & Wolfe  (1942).  Wolfe  (1939)  reports  t h a t N u t t a l l (1904) a l s o noted i n t e r s p e c i f i c d i f f e r e n c e s i n deer. Paper  Chromatography A s i d e from the use of paper chromatography as an  analytical tool,  i t s a p p l i c a t i o n t o taxonomy was not  suggested u n t i l r e l a t i v e l y r e c e n t years.  Micks (1956) and  Micks & Gibson (1957) showed t h a t the b a s i c amino a c i d  - 3 -  patterns and  of v a r i o u s  i n s e c t s and t i c k s were s p e c i e s s p e c i f i c  differed quantitatively.  Paper chromatography was used  by B u z z a t i - T r a v e r s o & R e c h n i t z e r differences fish,  (1953) t o d e t e c t s p e c i f i c  i n several morphologically  and by M a i n a r d i  some G a l l i n a c e o u s  i d e n t i c a l s p e c i e s of  (1958) t o c l a r i f y the phylogeny of  birds.  Electrophoresis Tiselius  (19 37)  designed the b a s i c  apparatus and c a l l e d h i s method " f r e e "  electrophoretic  electrophoresis.  Using horse serum, he compared the p r o t e i n f r a c t i o n s obtained by  s a l t p r e c i p i t a t i o n w i t h those obtained by  electrophoresis  and was the f i r s t " t o apply the terms "albumin, alpha-, b e t a - , and  gamma-globulin" r e s p e c t i v e l y t o f r a c t i o n s of d e c r e a s i n g  electrophoretic mobility. quantitative differences horse, and r a b b i t .  He was a l s o the f i r s t  to n o t i c e  i n the p r o t e i n f r a c t i o n s of human,  The T i s e l i u s apparatus has been used to  i n v e s t i g a t e a wide v a r i e t y of animals:  invertebrates,  fish,  amphibians, and r e p t i l e s (Deutsch & McShan, 1949); b i r d s (Bain & Deutsch, 1947, L a n d s t e i n e r et. a l , 1938); and mammals (Deutsch & Goodloe, 1945, Moore, 1945). Modifications involved  of the T i s e l i u s apparatus have mainly  the a d d i t i o n of a s u p p o r t i n g medium.  In the l a t e  -  4 -  1930*s, f i l t e r paper p r o v i d e d one of the f i r s t media (Wunderly,  supporting  1959) and one of i t s advantages over f r e e  e l e c t r o p h o r e s i s i s t h a t s m a l l samples  can be analysed.  Paper  e l e c t r o p h o r e s i s has been used t o i n v e s t i g a t e the b l o o d p r o t e i n s of the f o l l o w i n g t a x a : various small vertebrates amphibia 1956, al,  i n s e c t a (Stephen, 19 61),  (Gleason & F r i e d b e r g ,  (Dessauer & Fox, 1956), r e p t i l i a  1953),  (Dessauer & Fox,  Zweig & Crenshaw, 1957), and mammalia (Auernheimer e t  19 60, Bangham, 1957, Johnson & Wicks,  van Bekkum, 1958).  1959, W e l l i n g &  The l e n s p r o t e i n s of f i s h  (Smith, 1962)  and the egg white p r o t e i n s of b i r d s (Forsythe & F o s t e r , 1950, McCabe & Deutsch, 1952) have a l s o been s t u d i e d by t h i s method. S t a r c h has l a r g e l y r e p l a c e d f i l t e r paper as a s u p p o r t i n g medium because of i t s g r e a t e r r e s o l u t i o n .  Starch-gel  electro-  p h o r e s i s has been used t o i n v e s t i g a t e the egg white p r o t e i n s of b i r d s ( S i b l e y & Johnsgard, 1959b, S i b l e y , 1960);  whole  serum of i n v e r t e b r a t e s (Woods e t a l , 1958), a l l i g a t o r s et  (Baril  a l , 1961), and domestic mammals (Latner & Z a k i , 1957); and  f r a c t i o n s of whole b l o o d  (haemoglobins, h a p t o g l o b i n s , and  t r a n s f e r r i n s ) of amphibians 19 61), r e p t i l e s  (Dessauer e t a l , 19 62, Fox e t a l ,  (Dessauer e t a l , 19 62), domestic mammals  (Ashton, 1957a, 1957b, 1958, 1958a-e, 1959, 1960, Ashton & F a l l o n , 1962, Ashton & McDougall, 1958, Hickman & Smithies,  - 5 -  1957), mice (Mainardi, 1958, al,  Rosa e t a l , 1958,  1954), and humans ( A l l i s o n e t a l , 1958,  1958a, 1958b, Parker 1957,  1958,  H a r r i s et a l ,  & Bearn, 19 61, Smithies, 1955a, 1955b,  1959b, Smithies & H i l l e r ,  Walker, 1955b, 1956,  Thompson e t  1959,  Sutton e t a l , 1956,  Smithies  1959,  &  1960).  Lowe & McDougall (19 61) used s t a r c h - g e l e l e c t r o p h o r e s i s t o study the serum b e t a - g l o b u l i n types i n Red (Cervus  Deer  elaphus). Other s u p p o r t i n g media i n c l u d e a g a r - g e l  W i l l i a m s , 1955,  R e s s l e r & Jackson,  (Graber  &  1955), c e l l u l o s e a c e t a t e  (Kohn, 1957), and p o l y a c r y l a m i d e g e l (Raymond & Weintraub, 1959).  Besides t h i s development and u t i l i z a t i o n of  media, combinations  new  of t r i e d techniques have a l s o been  shown to y i e l d u s e f u l i n f o r m a t i o n . which i s a combination  Immunoelectrophoresis  of s e r o l o g y and s t a r c h - g e l e l e c t r o -  p h o r e s i s (Williams & Graber, 1956)  has proven to be a  v a l u a b l e t o o l f o r the study and c h a r a c t e r i z a t i o n of v a r i o u s antigen-antibody  systems (Kunkel & Trautman, 1959).  In the p r e s e n t i n v e s t i g a t i o n the s e p a r a t i o n of serum p r o t e i n s was  obtained by u t i l i z i n g the technique of s t a r c h -  gel electrophoresis.  MATERIALS AND METHODS  Source and c o l l e c t i o n date of samples  Odocoileus hemionus - captive  columbianus (Richardson) 1829 i n Vancouver, B r i t i s h Columbia,  O r i g i n a l l y from Wolf Lake, Vancouver B r i t i s h Columbia,  Canada.  Island,  Canada.  C o l l e c t e d June, 19 61 - October, 19 62. - w i l d - Wolf Lake, Vancouver I s l a n d , Columbia,  British  Canada.  C o l l e c t e d December, 19 61. - w i l d - Chemainus, Columbia,  Vancouver I s l a n d ,  British  Canada.  C o l l e c t e d December, 19 61. - w i l d - Mendocino County, C a l i f o r n i a , C o l l e c t e d Octob er, 19 62,  November, 19 62, and  January, 19 63.  Odocoileus hemionus  crooki  U.S.A.  (Mearns) 1897  - w i l d - Southern A r i z o n a , U.S.A. C o l l e c t e d December, 1962.  - 7 -  Odocoileus hemionus s i t k e n s i s Merriam, 1898 - captive  i n Vancouver, B r i t i s h Columbia,  Originally  Canada,  from Petersburg, A l a s k a , U.S.A.  C o l l e c t e d February, 19 61 - March, 19 62. - w i l d - Mitkof I s l a n d and Kupreanof  Island,  A l a s k a , U.S.A. C o l l e c t e d A p r i l , 1963.  Odocoileus v i r g i n i a n u s l e u c u r u s (Douglas) 1829 - wild - Clatskanie  County, Oregon, U.S.A.  C o l l e c t e d December, 19 62.  Odocoileus v i r g i n i a n u s texanus (Mearns) 1898 - w i l d - Sinton,  Texas, U.S.A.  C o l l e c t e d A p r i l - May, 19 63.  - 8 -  P r e p a r a t i o n of the sample C a p t i v e stock a t the U.B.C. deer u n i t was  immobilized  w i t h s u c c i n y l c h o l i n e c h l o r i d e (Anectine - Burroughs Wellcome Canada) as d e s c r i b e d by Cowan e_t al_ (19 62).  Blood was  i n v a c u t a i n e r tubes from the r e c u r r e n t t a r s a l v e i n and  taken was  o allowed to c l o t f o r 24 hours a t 5 C.  After centrifugatxon  f o r 10 minutes a t 3,500 r.p.m., the serum was  removed and  s t o r e d a t - 18°C u n t i l used f o r a n a l y s i s . Workers a t the U n i v e r s i t y of C a l i f o r n i a and A s s o c i a t e d Hopland F i e l d S t a t i o n , Mendocino County, C a l i f o r n i a ,  are  c a r r y i n g out a l a r g e experimental programme i n v o l v i n g tagging, t a k i n g body measurements, and c o l l e c t i n g b l o o d samples from 0. h. columbianus (Leopold e t a l , 1951, communication, 1964).  Leopold,  personal  Sheppard's K e i d e l Vacuum Tubes were  used to draw the b l o o d samples, the s e r a of which were f r o z e n a f t e r removal of the c l o t and were then sent a i r m a i l to U.B.C. The  sample of 0. v. l e u c u r u s was  obtained from the  j u g u l a r v e i n w i t h i n 5 minutes of s h o o t i n g .  The  serum s e p a r a t -  ed from the c l o t a f t e r 12 hours a t room temperature.  I t was  then p i p e t t e d i n t o a c l e a n c o n t a i n e r c o n t a i n i n g p r e s e r v a t i v e and 24 hours l a t e r i t was  s t o r e d a t - 18°C  a t U.B.C.  For those samples o b t a i n e d elsewhere the deer were e i t h e r shot or r e s t r a i n e d p h y s i c a l l y . i n s t r u c t i o n sheet was  For these samples an  prepared which appears on the f o l l o w i n g  -  page.  9  -  The equipment sent w i t h these i n s t r u c t i o n s i n c l u d e d  l a b e l l e d g l a s s t e s t tubes, g l a s s p i p e t t e s , corks wrapped i n aluminum f o i l ,  l a b e l l e d cardboard m a i l i n g c o n t a i n e r s , and  v e r m i c u l i t e 'for p a c k i n g . All U.B.C.  samples were s t o r e d a t - 18°C upon a r r i v a l a t  F o r those samples which c o u l d n o t be f r o z e n  during  shipment t o U.B.C, the b a c t e r i o s t a t , p r o p y l p-hydroxybenzoate, was p u t i n the c o l l e c t i n g v i a l s t o prevent decomposition.  - 10 -  DIRECTIONS FOR OBTAINING BLOOD SAMPLES FROM DEER A.  I f deer k i l l e d : 1.  slit  j u g u l a r v e i n or c a r o t i d a r t e r y or use any  a v a i l a b l e blood vessels  immediately.  2.  allow b l o o d  t o flow i n t o a c l e a n  3.  l e t s i t a t room temperature f o r 12 - 15 hours. a. i f s e p a r a t i o n has occurred,  container.  that i s i f yellowish-  c o l o u r e d f l u i d i s seen d i s t i n c t from a r e d semis o l i d mass (the c l o t ) ,  then pour t h i s f l u i d  i n t o another c l e a n c o n t a i n e r b. i f no s e p a r a t i o n has occurred  only  and d i s c a r d the c l o t . a f t e r 12 - 15 hours,  t h a t i s i f no f l u i d i s seen t o be d i s t i n c t from the c l o t , then c e n t r i f u g e a t 3,000 r.p.m. (or highest minutes.  speed on a hand c e n t r i f u g e ) f o r about 10 Then pour the f l u i d i n t o another c l e a n  c o n t a i n e r and d i s c a r d the c l o t .  B.  4.  complete the l a b e l on the c o n t a i n e r .  5.  f r e e z e or r e f r i g e r a t e u n t i l  mailing.  6.  use cardboard m a i l i n g tube  provided.  7.  tape corks  8.  a i r mail.  into place.  I f deer i s not t o be k i l l e d : 1.  i f p o s s i b l e , immobilize  the deer w i t h s u c c i n y l c h o l i n e  - 11 -  c h l o r i d e before 2.  bleeding.  b l e e d from r e c u r r e n t t a r s a l v e i n  (most prominent  v e i n i n the leg) or from the j u g u l a r v e i n or from an ear v e i n . 3.  use v a c u t a i n e r  More i n f o r m a t i o n obtained 4.  needles and tubes i f a v a i l a b l e .  or m a t e r i a l s r e g a r d i n g  the above may be  i f required.  f o l l o w d i r e c t i o n s of A. no. 3 - no. 8.  Powder i n tubes i s a p r e s e r v a t i v e .  E l e c t r o p h o r e t i c procedure Smithies g i v e s the o r i g i n a l d e s c r i p t i o n of zone e l e c t r o p h o r e s i s i n s t a r c h - g e l i n 1955b, an improved procedure i n 1959a, and a d e t a i l e d diagram of the apparatus i n 1959b. The procedure used i n t h i s study was o n l y s l i g h t l y  modified  from h i s by p l a c i n g the g e l i n a h o r i z o n t a l r a t h e r than a v e r t i c a l p o s i t i o n during  the run and by s u b s t i t u t i n g the  suggested b u f f e r t r a y s and e l e c t r o d e s by those designed by the E-C Apparatus Company (538 Walnut Lane, Swarthmore, Pennsylvania, which constant  U.S.A.). pressure  Wooden p r e s s e s were designed w i t h c o u l d be a p p l i e d t o the s e t t i n g g e l s  inymolds s u p p l i e d by Otto H i l l e r Wisconsin, U.S.A.).  (P.O. Box 1294, Madison 1,  - 12 -  B r i e f l y the procedure was  as f o l l o w s :  A l l mold p i e c e s  except the s l o t s e c t i o n s were v e r y l i g h t l y greased w i t h mineral o i l .  One l i t r e  of s t a r c h s o l u t i o n , the c o n c e n t r a t i o n  depending on l o t s p e c i f i c a t i o n s  (Connaught M e d i c a l Research  L a b o r a t o r i e s , U n i v e r s i t y of Toronto, Toronto, O n t a r i o ) , c o n t i n u o u s l y heated ( t o 95°C, Smithies, 1959b) and for  a c o n s t a n t 20 minutes  was  stirred  (the peak of v i s c o s i t y o c c u r r i n g a t  10 minutes), evacuated f o r about 5 minutes to remove a l l a i r bubbles, and poured i n t o the t r a y s tray).  The cover was  even p r e s s u r e was at  (500 ml. s t a r c h s o l u t i o n /  c a r e f u l l y lowered i n t o p o s i t i o n and  applied.  The s e t t i n g g e l s were then kept  room temperature f o r about 1 hour and a t 10°C u n t i l used -  u s u a l l y about 8 hours At  later.  the b e g i n n i n g of a run the p r e s s and cover were  removed and 50 _ul of serum were i n j e c t e d i n t o each s l o t w i t h a Hamilton m i c r o s y r i n g e .  The s l o t s were s e a l e d w i t h m i n e r a l  011 and "Saran Wrap" was p l a c e d over the g e l s u r f a c e so as t o eliminate a i r bubbles.  Number 17 f i l t e r papers (6" square)  which had j u s t p r e v i o u s l y been wetted w i t h b u f f e r were p l a c e d at  each end of the t r a y which was  then p l a c e d between the two  c o o l i n g p l a t e s of the E-C Apparatus so as t o b r i n g the f i l t e r 1  paper i n t o c o n t a c t w i t h i t s b u f f e r t r a y s . was  a c t i v a t e d and the c u r r e n t  applied.  The c o o l i n g system  - 13  At the end  of the run,  system were disconnectedWrap", and  -  the c u r r e n t and  The  cooling  f i l t e r paper wicks, the  the trough-ends of the g e l were d i s c a r d e d .  remaining .rectangle of s t a r c h - g e l was a s l i c i n g tray. g e l was  the  w i t h a microtome b l a d e two  The  p l a c e d upside down i n t o  A f l a t weight the same s i z e as the  p l a c e d on top and by  "Saran  starch-  slicing in a horizontal direction  equal h a l v e s  of the s t a r c h - g e l were  obtained. Staining followed s u r f a c e uppermost) was (500  immediately.  The  lower h a l f  (inner  s t a i n e d f o r p r o t e i n w i t h Amido Black  mg.. Amido Black / 90 ml. Methanol / 10 ml. Water) f o r 5  minutes and was  then r i n s e d w i t h 8 l i t r e s of 50 ml. Methanol /  50 ml. Water / 10 ml. G l a c i a l A c e t i c A c i d i n a g e l washer (Pert et a l , 1959) upper h a l f was  f o r 13 hours.  stained for l i p i d .  The  inner s u r f a c e of  The  the  r e s u l t s of t h i s w i l l  appear i n a l a t e r p u b l i c a t i o n . The  s t a i n e d s t a r c h - g e l was  then wrapped i n "Saran Wrap"  o and  s t o r e d at 5 C. In order  to s t a n d a r d i z e  the technique, yellow  from c a p t i v e , male, a d u l t , 0. h. columbianus was various  c o n d i t i o n s as e l a b o r a t e d below.  adopted was  The  serum  run under  f i n a l method  based on the number of p r o t e i n bands v i s i b l e  the naked eye  and  the d i s t a n c e the f u r t h e s t band had  to  migrated.  - 14 -  Buffer Since p r o t e i n s are amphoteric,  the pH of the b u f f e r  s o l u t i o n w i l l e x e r t a g r e a t i n f l u e n c e on t h e i r m o b i l i t y . For maximum m i g r a t i o n t o occur a pH should be used which i s g r e a t er than the i s o e l e c t r i c p o i n t s of the m a j o r i t y of the p r o t e i n s i n the s o l u t i o n t o be examined.  As suggested by Smithies  (1959b), a b o r a t e b u f f e r o f pH 8.8 was used. The v e l o c i t y of m i g r a t i o n a l s o depends on the i o n i c s t r e n g t h of the s o l u t i o n , the v e l o c i t y d e c r e a s i n g as the i o n i c s t r e n g t h i n c r e a s e s f o r any given f i e l d decrease  strength.  This  i s due t o the e f f e c t i v e charge of the m i g r a t i n g  molecule b e i n g reduced by ions of o p p o s i t e charge  surrounding  it. Using constant time  (12% hours) and v o l t a g e (800  v o l t s ) v a r i o u s c o n c e n t r a t i o n s of b o r a t e b u f f e r o f pH 8.8 were used t o t e s t the e f f e c t of i o n i c s t r e n g t h r a n g i n g from 0.0610 - 0.0745. noted  Among these t h e r e was l i t t l e d i f f e r e n c e  i n the m i g r a t i o n d i s t a n c e of the f u r t h e s t  fraction.  However, the b u f f e r c o n c e n t r a t i o n , 0.0092 M sodium hydroxide 0.0230 M b o r i c a c i d , of i o n i c s t r e n g t h 0.0667 and pH 8.8 produced the g r e a t e s t s e p a r a t i o n of the p r o t e i n f r a c t i o n s and was t h e r e f o r e s e l e c t e d as the constant b u f f e r c o n c e n t r a t ion.  /  - 15 -  Attempts made u s i n g T r i s  (hydroxymethyl)  standard b u f f e r as suggested by P o u l i k  aminomethane  (1957) were not  successful. The e l e c t r o d e s were r e v e r s e d a f t e r each r u n .  I t was  found t h a t the b u f f e r c o u l d be used 4 times b e f o r e the pH began t o a l t e r .  Consequently, no more than 4 runs were made  b e f o r e the apparatus was cleaned and new b u f f e r  supplied.  A h u m i d i t y of 45 - 50% was found t o prevent condensation  on the apparatus and e v a p o r a t i o n of the b u f f e r d u r i n g the  run. Time Because each p r o t e i n f r a c t i o n i n the s o l u t i o n w i l l differ  i n the n e t number of n e g a t i v e and p o s i t i v e  charges  per u n i t area of molecular s u r f a c e , i t w i l l have a s p e c i f i c m o b i l i t y and w i l l separate from the o t h e r s .  Enough time  must be allowed f o r the f r a c t i o n s t o achieve t h e i r maximum separation.  However, i f allowed t o run i n d e f i n i t e l y , the  f a s t e s t moving f r a c t i o n s a t l e a s t , would migrate i n t o the trough r e g i o n of the g e l t r a y making t h e i r  analysis  impossible. With the standard b o r a t e b u f f e r of i o n i c 0.0667 and pH 8.8, and 900 v o l t s , were t e s t e d .  strength  times from 8 - 18 hours  A t 13 hours and l o n g e r , a y e l l o w l i n e  - 16 -  appeared  on the f i l t e r paper wick a t the n e g a t i v e e l e c t r o d e  suggesting some change o c c u r r i n g i n the b u f f e r s o l u t i o n . This l i n e d i d not appear a t 12% hours or l e s s .  Because a  g r e a t e r number of f r a c t i o n s separated w i t h p r o g r e s s i v e l y i n c r e a s i n g lengths of time, 12% hours running time was made standard. Voltage One advantage i n u s i n g an extremely h i g h v o l t a g e i s t h a t maximum s e p a r a t i o n of the p r o t e i n f r a c t i o n s w i l l be obtained more q u i c k l y than a t a lower v o l t a g e .  In a d d i t i o n  to s a v i n g time, the a p p l i c a t i o n of h i g h e r f i e l d  intensities  (20 - 100 v o l t s / cm.) has the advantage of producing sharper s e p a r a t i o n s of the p r o t e i n f r a c t i o n s s i n c e the i n t e r f e r i n g d i f f u s i o n of the substances  i n the s u p p o r t i n g  medium ( s t a r c h - g e l ) i s of l e s s s i g n i f i c a n c e A disadvantage,  (Wieland,  1959).  however, l i e s i n the f a c t t h a t an i n c r e a s e  i n v o l t a g e means an i n c r e a s e i n the amount of heat  generated  d u r i n g the r u n . Even though t h i s i s p a r t i a l l y negated by the low i o n i c s t r e n g t h of the b u f f e r (Smithies, 1955b), the maintenance of an e f f i c i e n t c o o l i n g system becomes necessary. Using the standard b o r a t e b u f f e r of i o n i c s t r e n g t h 0.0667 and pH 8.8, and a constant time of 12% hours, ages from 130 - 900 v o l t s were t e s t e d .  900 v o l t s  volt-  produced  - 17 -  the g r e a t e s t s e p a r a t i o n and gave a v o l t a g e g r a d i e n t of approximately  28 v o l t s /  cm.  At 900 v o l t s the i n i t i a l amperage was 10 minutes t h i s decreased  20 ma.  After  and remained constant a t 16  ma.  Temperature As p r o t e i n s are t h e r m o l a b i l e they w i l l be at h i g h temperatures  and m i g r a t i o n w i l l cease.  denatured However,  even b e f o r e t h i s c r i t i c a l p o i n t of d e n a t u r a t i o n i s reached, heat may  d i s t o r t the p a t t e r n of e l e c t r o m i g r a t i o n by  d i f f u s i o n w i t h i n the g e l . v o l t s ) i t was  necessary,  At the h i g h v o l t a g e used  t h e r e f o r e , to keep the  i n a constant temperature room a t 10°C.  causing (900  apparatus  The c o o l i n g p l a t e s  o on e i t h e r s i d e of the g e l were constant a t 3 C. Serum a l i q u o t The volume of each s l o t i n the s t a r c h - g e l i s about 55 u l .  Samples of 30,  and i t was  35, 40, 45,  and 50 u l were t e s t e d  found t h a t w i t h i n c r e a s i n g serum volume the  p r o t e i n p a t t e r n became more d i s t i n c t . a l i q u o t was  Therefore, a 50 u l  taken as the standard volume.  The number of a l i q u o t s r e q u i r e d per serum sample Each g e l contained e i g h t s l o t s i n t o which the serum  - 18 -  samples were i n j e c t e d .  When a l l the s l o t s of one g e l were  f i l l e d w i t h a l i q u o t s from one sample, s l o t s 2 - 7 produced p a t t e r n s  inclusive  whose p r o t e i n f r a c t i o n s were extremely  s i m i l a r i n m o b i l i t y and p e r c e n t composition.  Runs i n s l o t s  1 and 8 were o b v i o u s l y  discarded.  i n c o n s i s t e n t and were  This s i m i l a r i t y of runs from s l o t s 2 - 7 the use of the minimum of two a l i q u o t s sample r e q u i r e d  justifies  (100 jil) from each  for s t a t i s t i c a l analysis.  I f only two  a l i q u o t s are a v a i l a b l e , however, i t i s p r e f e r a b l e t o put them i n t o separate g e l s t o allow f o r s l i g h t g e l v a r i a t i o n s (Mclntyre,  personal.communication).  D i a l y s i s of sample D i a l y s i s serves t o remove from the serum v i r t u a l l y a l l d i f f u s i b l e solutes  ( t h a t might i n t e r f e r e w i t h m i g r a t i o n )  other than b u f f e r e l e c t r o l y t e s (Longsworth, 1959). 50 i l l a l i q u o t s were taken from samples d i a l y s e d a t 10°C  from 0 - 8 6  hours.  A few samples were l e f t f o r longer  p e r i o d s w i t h a l i q u o t s taken a t v a r i o u s  intervals.  The  b u f f e r was changed every day on each sample. With no d i a l y s i s few p r o t e i n bands separated and the run was s h o r t e r  than w i t h samples d i a l y s e d 23 - 53 hours.  There were no v i s i b l e d i f f e r e n c e s between samples d i a l y s e d  - 19 -  20 - 69 hours.  A t 86 hours t h e r e was some l o s s i n c l a r i t y  and w i t h s u c c e s s i v e days of d i a l y s i s the runs became p r o g r e s s i v e l y poorer. Therefore, d i a l y s i s c o u l d be no l e s s than 20 hours and no more than 69.  For convenience the samples used f o r  the p r e v i o u s l y mentioned  t e s t s and f o r those d i s c u s s e d  below were d i a l y s e d f o r 27 - 28 hours. Time between completion of run and s t a i n i n g A f t e r the c u r r e n t was d i s c o n n e c t e d , 4 minutes were r e q u i r e d t o remove the two g e l s from the apparatus and the constant temperature room, s l i c e them, and p l a c e them i n staining dishes.  I f the i n t e r v a l between d i s c o n n e c t i n g the  c u r r e n t and s t a i n i n g the g e l s were extended t o 14 minutes, the p r o t e i n bands were s t i l l d i s t i n c t . minute  I f t h e r e were a 20  i n t e r v a l , however, some d i f f u s i o n c o u l d be observed,  i . e . the b o r d e r s of the bands were i n d i s t i n c t .  An i n t e r v a l  of one hour caused d i f f u s i o n g r e a t enough t o d e s t r o y the pattern. Because d i f f u s i o n of the p r o t e i n bands was u n d e s i r a b l e , the g e l s were s t a i n e d immediately a f t e r p l a c i n g them i n the s t a i n i n g d i s h e s - no more than 5 minutes c u r r e n t was d i s c o n n e c t e d .  a f t e r the  - 20 -  Time of s t a i n i n g The time of s t a i n i n g must be s h o r t enough t o prevent the background  g e l from becoming permanently  impregnated  w i t h dye thereby l e s s e n i n g the c o n t r a s t between the p r o t e i n bands and y e t l o n g enough t o s t a i n those f r a c t i o n s p r e s e n t i n the l e a s t amount. When the g e l s were s t a i n e d f o r 10 minutes w i t h Amido Black the background d i d n o t become white w i t h r i n s i n g and t h e r e f o r e d i d not p r o v i d e the g r e a t e s t c o n t r a s t between the p r o t e i n bands.  Because  5 minutes s t a i n i n g produced  d i s t i n c t bands and the background r i n s e d white, t h i s time was  made c o n s t a n t .  Time of r i n s i n g Using a g e l washer ( P e r t e t a l , 1959) the background r i n s e d white a t 12 hours and t h e r e was no v i s i b l e change i n the p r o t e i n p a t t e r n c o l o u r i f r i n s i n g continued f o r 18 hours.  A f t e r t h i s time, however, the p a t t e r n  lighter.  appeared  For convenience the r i n s i n g time was made  constant a t 13 hours. A n a l y s i s of r e s u l t s Photography A view camera c o n t a i n i n g f a s t Pan f i l m and w i t h a  - 21 -  red  f i l t e r was used t o photograph the g e l s .  r a t e was f l 6 a t 1 second by r e f l e c t e d 8" x 10" f i l m was used.  The exposure  and t r a n s m i t t e d l i g h t .  R e p l i c a t e s photographed on the same  day and a t a l a t e r date were found t o be i d e n t i c a l t o the first.  D u p l i c a t e s made on 5" x 7" f i l m l a t e r had to be  d i s c a r d e d as the densitometer c o u l d not r e s o l v e the r e s u l t i n g s h o r t e r d i s t a n c e s between p r o t e i n bands on the s m a l l e r negative.  A c e n t i m e t e r r u l e r was p l a c e d a l o n g s i d e each g e l  to g i v e the s c a l e . Densitometry The n e g a t i v e of the whole g e l was c u t so t h a t the band p a t t e r n emanating from each s l o t c o u l d be run i n d i v i d u a l l y through a Z e i s s densitometer.  B l a c k photography paper  was c u t so as t o l e a v e a r e c t a n g u l a r space 1 cm. wide x 17 cm. l o n g .  The n e g a t i v e was then p l a c e d between a p i e c e of  g l a s s and t h i s paper. top  Another sheet of g l a s s was p l a c e d on  of t h i s and the r e s u l t i n g  densitometer. 1 mm. wide.  sandwich was p l a c e d i n the  The l i g h t s l o t i n the Z e i s s densitometer i s With the photography paper a c o n s t a n t area of  1 x 10 mm. was scanned f o r a d i s t a n c e of 17 cm. a t an approximate speed of 1.5 mm. / second. .Thus the s t a r c h - g e l s , by means of p h o t o g r a p h i c n e g a t i v e s and the densitometer were f i n a l l y r e s o l v e d  into  - 22 -  curves showing the amount of dye taken up by the p r o t e i n i n the g e l (Wunderly, 1959).  Four i d e n t i c a l curves were  obtained f o r each a l i q u o t , t h r e e t o be used i n the p e r c e n t composition d e t e r m i n a t i o n s and the f o u r t h t o be used f o r the measurement of m i g r a t i o n d i s t a n c e s . Calculations In order t o minimize was  any background dye a b a s e l i n e  drawn t o u c h i n g the lowest trough of the curve.  To  find  the r e l a t i v e p r o p o r t i o n s of the area under each peak, i . e . the r e l a t i v e amounts of p r o t e i n p r e s e n t , each of the three curves was  weighed and then the area under each peak  weighed s e p a r a t e l y . was  was  Averages were c a l c u l a t e d and each peak  expressed as a percentage  of the average  total.  Because  of the v e r y s m a l l amounts p r e s e n t i n some cases a l l of the percentages were then . converted t o a r c s i n ^/percentage degrees  (Snedecor,  1956,  pages 318 - 319).  C a l c u l a t i o n s of  the means, standard d e v i a t i o n s , and standard e r r o r s were on the transformed numbers.  Student's  t - t e s t s were  performed  i n the a p p r o p r i a t e circumstances. To c a l c u l a t e the m o b i l i t y of each p r o t e i n f r a c t i o n a l i n e was  drawn p e r p e n d i c u l a r to the b a s e l i n e through  c e n t r e of each peak of the curve. was  the  The d i s t a n c e to each l i n e  then measured along the b a s e l i n e from the o r i g i n .  - 23 -  Because of s l i g h t photographic r e d u c t i o n ment as occurred  i n the s t a r c h - g e l was obtained by a prop-  o r t i o n a l conversion  of the centimeter s c a l e on the n e g a t i v e  t o the measured d i s t a n c e  on the curve.  the c a l c u l a t i o n of the m o b i l i t y E = field V =  the a c t u a l measure-  The f o l l o w i n g i s  (Raymond, 1955).  strength  voltage  D = l e n g t h of medium i n cm. d = d i s t a n c e p r o t e i n has moved i n cm. t = time i n seconds }i = m o b i l i t y  E = V , d = jiEt , t h e r e f o r e , ja = d x D D V t Mean values,  standard d e v i a t i o n s ,  2 , cm / v o l t  and standard  seconds.  errors  were c a l c u l a t e d f o r each m o b i l i t y and Student's t - t e s t s were performed where r e q u i r e d Standard D e v i a t i o n  Standard E r r o r  as f o l l o w s .  (S.D.) =  (S.E.)  .=  S.D.  - 24 -  Student's t - t e s t ( t )  = /  x_ - x A B (S.E.j*  +  (S.E.)2  The method of s t a t i s t i c a l a n a l y s i s was checked by Mr. G. M c l n t y r e (see Acknowledgments) who found t h a t the v a r i a t i o n within gels i s numerically gels.  l e s s than t h a t between  The l a t t e r v a r i a t i o n , i n t u r n , i s n u m e r i c a l l y  less  than t h a t between i n d i v i d u a l s .  He a l s o found no  signific-  ant change i n the method d u r i n g  the course of the study.  - 25 -  RESULTS AND  DISCUSSION  CONDITION OF SAMPLE Hemolysis and c l o u d i n e s s Deer serum i s n o r m a l l y a dark y e l l o w c o l o u r . Haugen (19 60)  As  found no c a r o t e n o i d s i n the b l o o d serum of  n i n e t e e n 0. v i r g i n i a n u s any r e d d i s h c o l o u r a t i o n v i s i b l e to the  naked eye was  assumed t o be an i n d i c a t i o n of h e m o l y s i s .  The appearance of c l o u d i n e s s i n some samples i s p r o b a b l y due t o l i p i d s All  (Cantarow & Schepartz, 1957).  samples were a r b i t r a r i l y c l a s s i f i e d  as yellow,  y e l l o w b u t cloudy, s l i g h t l y hemolysed, hemolysed, and v e r y hemolysed.  Those samples used to t e s t the e f f e c t of  hemolysis and c l o u d i n e s s were run w i t h i n one year of sampling. The term "hemolysis" i m p l i e s t h a t haemoglobin has been r e l e a s e d from the b l o o d c e l l s i n t o the serum.  This  a d d i t i o n a l p r o t e i n i n the serum would be expected t o have some e f f e c t on the m o b i l i t y and/or the p e r c e n t of  composition  the p r o t e i n f r a c t i o n s as separated by e l e c t r o p h o r e s i s . Very hemolysed samples u s u a l l y v a r i e d the e l e c t r o -  p h o r e t i c p a t t e r n by e i t h e r an apparent i n c r e a s e or decrease i n the number of p r o t e i n f r a c t i o n s as compared  t o the  - 26 -  number r e s o l v e d i n yellow, s l i g h t l y hemolysed, and.hemolysed samples.  The t - t e s t comparisons of s l i g h t l y hemolysed and  hemolysed v s . y e l l o w samples (Tables 2 & 4) show no s i g n i f i c ant d i f f e r e n c e s i n e i t h e r the m o b i l i t y or the p e r c e n t composition of the p r o t e i n f r a c t i o n s .  C l o s e r examination of  Tables 1 & 3, however, r e v e a l s t h a t over 50% of the standard e r r o r s of the s l i g h t l y hemolysed and hemolysed samples are g r e a t e r than the corresponding standard e r r o r s of the y e l l o w samples. T h i s i n d i c a t i o n of g r e a t e r v a r i a b i l i t y w i t h  hemolysis  and the change i n f r a c t i o n number i n v e r y hemolysed samples compels the e l i m i n a t i o n of a l l c a t e g o r i e s of hemolysed samples from f u r t h e r  comparisons.  L i p i d s a r e c a r r i e d by the a l p h a - and b e t a - g l o b u l i n s (Moore, 1959). lipid  I f c l o u d i n e s s i s an i n d i c a t i o n of excess  i n the serum then some e f f e c t on the e l e c t r o p h o r e t i c  p r o t e i n p a t t e r n might be expected.  A few cloudy samples  d i d show an apparent decrease i n the number of p r o t e i n f r a c t i o n s as compared t o those r e s o l v e d i n y e l l o w samples. In 0. v. texanus (Table 2) c l o u d i n e s s caused  a significant  i n c r e a s e i n the m o b i l i t y of f r a c t i o n +7 i n the comparison of y e l l o w v s . y e l l o w b u t cloudy samples.  In the same  - 27 -  comparison (Table 4) the p e r c e n t composition of f r a c t i o n increased  +1  significantly.  Examination  of Tables 1 & 3 r e v e a l s t h a t over 50% of  the standard e r r o r s f o r the m o b i l i t y and t h a t 100% of the standard e r r o r s f o r the p e r c e n t composition of the p r o t e i n f r a c t i o n s of cloudy samples are g r e a t e r than those of corresponding y e l l o w samples. The decrease i n f r a c t i o n number i n some cloudy samples, the s i g n i f i c a n t d i f f e r e n c e s i n m o b i l i t y and cent composition,  and the g r e a t e r v a r i a b i l i t y i n the standard  e r r o r s of c l o u d y samples warrant further  per-  comparisons.  e l i m i n a t i n g them from  TABLE 1 EFFECT OF HEMOLYSIS - m o b i l i t i e s  i n 10~  5  cm  2  / v o l t seconds  ".  + FRACTION 10 9 8 . 7 6 5 4 3 2 1 1 2 w i l d j female,, a d u l t , 0. v. texanus, Texas ' yellow X .829 . 4 6 4 . 4 0 6 .371 .327 .254 .190 .158 .079 .015 .011 .143 N = 8 + S.E. .008 .016 .016 .013 .011 .013 .013 .003 .005 .002 .001 .015 yellow + cloudy N = 2  X .772 .515' .444 . 4 l 6 .380 .277 .210 .185 .105 .010 .017 .111 + S.E. .124 .018 .000 .000 .013 .003 .006 .018 .018 .001 .007 .044  s l i g h t l y hemolysed = 2  X .813 .437 .374 .364 .329 .258 .187 .165 .065 .010 .013 .117 + S.E. .013 .011 .006 .002 .013 .003 .000 .004 .016 .000 .002 .008  N"  wild,, male., adult,, 0. h. columbianus, Chemainus, B.C.  ••  N  yellow = 2  X + S.E.  .829 .494 .423 ' .383 .313 .260 .165 .133 .022 .012 - .013 .008 .008 .005 .011 .012 .005 .011 .012 .003  hemolysed N = 3  X" + S.E.  .815 .480 .400 .370 .296 .231 .142 .071 .027 .011 .018 .014 .011 .012 .011 .019 .012 .020 .010 .002  N = number of a n i m a l s  00  TABLE 2 EFFECT OF HEMOLYSIS - S t u d e n t ' s t - t e s t comparisons o f the m o b i l i t i e s FRACTION  .  10  9  8  7  6  5  o f the serum p r o t e i n f r a c t i o n s . + 4 3 2 1 1 2  wild,, f e m a l e , a d u l t s _0. v. t e x a n u s , Texas y e l l o w v s . s l i g h t l y hemolysed y e l l o w v s . y e l l o w + cloudy  =  =  =  =  =  =  = '  =  =  =  =  =  •=  =  =  ++  =  =  =  =  =  =  =  =  wild,, male, a d u l t , _0. h. columbianus„ Chemainusj B.C. y e l l o w v s . hemolysed = ++  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y f a s t e r than the f i r s t i t e m t o the .01 l e v e l .  TABLE 3 EFFECT OF HEMOLYSIS - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n V p e r c e n t a g e FRACTION wild., f e m a l e , a d u l t , _0.• v. texanus, Texas  10  9  8  7  yellow N = 8  X 3 8 . 5 16.2 + S.E. 0.9 1.0  yellow + cloudy N = 2  X 3 3 . l 18.4 l4.7 12.5 + S.E. 3.4 3.4 3.9 1.7  6  13.1 12.9 10.3 0.6 0.9 1.5  5  degrees. 4  3  2  1  1  7-3 0.6  9 . 8 11.6 13.7 0.8 0.5 1.1  13.1 15.1 0.5 1.2  6.9 2.6  9.5 1.5  9.5 1.5  12.6 1.4  s l i g h t l y hemolysed X 35.6 14.0 15.0 13.7 12.5 N = 2 + S.E. 2.2 4.0 0.4 0.3 0.7  9.4 1.0  8.0 0.6  8.4 1.1  6.9 0.8  7.2 1.8  10.4 0.1  8.8 2.1  10.1 13.9 15.9 20.4 0.9 0.4 3.6 4.1  16.6 17.6 2.4 1.0  2  21.1 1.2  16.7 17 A 5.0 5.2  9.4 11.4 13.9 2.0 0.6 1.5  22.7 8.7  w i l d , male, a d u l t , 0. h_.. Columbianus, Chemainus, B.C. yellow N = 2  X + S.E.  4 4 . 6 19.2 14.2 0.1 2.4 0.5  7.4 1.4  hemolysed N = 3  X + S.E.  4 l . 0 18.2 \ 1 3 . 8 2.4 1.6 2.1  12.2 1.2  11.3 1.7  11.3 1.8  18.0 3.2  19.9 0.6  N = number of a n i m a l s o  TABLE 4 EFFECT OF HEMOLYSIS - Student's t - t e s t comparisons of the p e r c e n t a g e c o m p o s i t i o n ( i n a r c s " " i n / p e r c e n t a g e degrees) of the serum p r o t e i n f r a c t i o n s . FRACTION  10  9  8  7  6  5  4  3  wild., f e m a l e , a d u l t , 0. v. texanus, Texas  1  1  2  =  =  =  '  y e l l o w vs.. s l i g h t l y hemolysed  = . =  y e l l o w v s . y e l l o w + cloudy  =  =  =  =  =  =  =  =  =  =  =  =  =  =  w i l d , male, a d u l t , 0. h. columbianus, Chemainus, B.C. y e l l o w v s . hemolysed = ++  +  2  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y g r e a t e r than the f i r s t i t e m t o the .01  level.  =  = • + +  - 32 -  Preservation Serum samples were a r r i v i n g a t U.B.C. t h a t were obviousl y decomposing.  A NIPA e s t e r  (propyl  p-hydroxybenzoate),  because of i t s b a c t e r i o s t a t i c a c t i o n (Merck, I960), was t r i e d and found t o be s u c c e s s f u l i n p r e v e n t i n g decomposition. became important, t h e r e f o r e , t o determine whether t h i s  It ester  had any e f f e c t on the m o b i l i t y or the p e r c e n t composition of the serum p r o t e i n s as separated by e l e c t r o p h o r e s i s . A serum sample from a w i l d , female, columbianus  a d u l t , 0. h-  - C a l i f o r n i a , was d i v i d e d i n t o two samples w i t h  and two samples without the NIPA e s t e r upon a r r i v a l a t U.B.C. They were then a l l kept a t room temperature.  The a d d i t i o n of  the p r e s e r v a t i v e on t h e day of a r r i v a l d i d not s i g n i f i c a n t l y a l t e r e i t h e r the m o b i l i t y or the p e r c e n t composition of the p r o t e i n f r a c t i o n s and a f t e r 16 days a t room  temperature  t h e r e were s t i l l no changes i n the sample c o n t a i n i n g the preservative.  However, a f t e r 16 days a t room  without the p r e s e r v a t i v e ,  temperature  f r a c t i o n +9 was l o s t and f r a c t i o n  +10 e x h i b i t e d a s i g n i f i c a n t l y f a s t e r m o b i l i t y (Table 1 0 ) . There were no s i g n i f i c a n t changes i n the m o b i l i t i e s of the remaining f r a c t i o n s .  The p e r c e n t composition of a l l  comparable f r a c t i o n s a l s o d i d not change (Table 1 2 ) .  - 33 -  B a c t e r i a l decomposition prevented by f r e e z i n g .  of p r o t e i n s can a l s o be  P r e v i o u s work on deer serum u t i l i z -  i n g paper e l e c t r o p h o r e s i s (Johnston, B.Sc.  T h e s i s , 1961)  shown t h a t serum from a c a p t i v e a d u l t , 0. v. ochrourus, be s t o r e d a t - 18°C f o r a t l e a s t 5% months w i t h no  had could  signific-  ant changes i n the p e r c e n t composition of the serum p r o t e i n f r a c t i o n s , w h i l e serum from a c a p t i v e fawn, 0. h. columbianus - Vancouver I s l a n d , changed s i g n i f i c a n t l y a f t e r three weeks storage. In order t o t e s t the e f f e c t of storage a t - 18°C i n the p r e s e n t study, a l i q u o t s were taken a t v a r i o u s time v a l s from a c a p t i v e , female, Lake (Vancouver  I s l a n d ) , B.C.  inter-  a d u l t , 0. h. columbianus - Wolf Storage times b e g i n w i t h the  a r r i v a l of the serum a t U.B.C. and i t s subsequent storage a t - 18°C.  I t was  found t h a t these samples c o u l d be s t o r e d as  l o n g as 7 61 days w i t h no s i g n i f i c a n t changes o c c u r r i n g i n e i t h e r the m o b i l i t y (Tables 5 & 6) or the p e r c e n t (Tables 7 & 8) of the p r o t e i n f r a c t i o n s .  composition  As fawn serum  was  not examined f o r c o l d storage e f f e c t s , o n l y those fawn samples s t o r e d l e s s than three weeks were used i n f u r t h e r analyses. The e f f e c t of f r e e z i n g on the b a c t e r i o s t a t was  tested  - 34 -  by d i v i d i n g a serum sample from a w i l d , female,  fawn, 0.  h.  columbianus - C a l i f o r n i a , i n t o one sample w i t h and one w i t h out the NIPA e s t e r . at - 18°C. (Tables  No  These samples were s t o r e d f o r 58 days  s i g n i f i c a n t differences i n either mobility  9 & 10) or the p e r c e n t composition  (Tables  11 &  12)  of the p r o t e i n f r a c t i o n s were found. In summary, the b a c t e r i o s t a t , p r o p y l p-hydroxybenzoate, during  was  found necessary t o prevent  storage a t room temperature.  decomposition  N e i t h e r the NIPA e s t e r  nor f r e e z i n g had any e f f e c t on the m o b i l i t y or the p e r c e n t composition of the p r o t e i n f r a c t i o n s .  -5 EFFECT OF COLD STORAGE - m o b i l i t i e s i n 10 FRACTION captive, female, adult, 0. h. c o l u m b i a n u s , Wolf Lake, B.C. 2 days N = 2  9  8  TABLE 5 2 cm / v o l t seconds. 7  6  5  + 4  3  2  1  1  2  X + S.E.  .913 .519 .471 .442 .403 .300 .240 .164 .019 .019 .087 .023 .023 .023 .000 .033 .023 .002 .000 .001 .005 .020  221 days N = 2  'X + S.E.  .908 .512 .454 .438 .375 .296 .245 .169 .016 .010 .095 .073 .005 .021 .026 .016 .005 .005 .005 .002 .005 .001  304 days N = 2  X + S.E.  .947 .515 .471 .442 .409 .305 .260 .164 .019 .019 .087 .033 . . 0 0 4 .000 .000 .005 .000 .020 .011 .000 .000 .005  761 days N = 5  X + S.E.  .924 .518 . 4 6 4 .432 .394 .295 .238 .160 .018 .013 .086 .029 .010 .008 .010 . 0 0 9 .018 .018 .022 . 0 0 4 .005 .005  N = number of a l i q u o t s  TABLE 6 EFFECT OF COLD STORAGE - Student's t - t e s t comparisons of the m o b i l i t i e s fractions. 9  FRACTION  8  7  6  5  of the serum p r o t e i n  ^  3  2  + 1  1  c a p t i v e , female, a d u l t , 0. h. columbianus, Wolf Lake, B.C. 2 days  v s . 221 days  =  =  =  =  =  v s . 304 days  =  =  =  =  =  vs. 761 days  =  =  -  =  '=  221 days v s . 304 days  =  =  =  =  vs. 761 days  =  =  =  304 days v s . 761 days  =  = ' =  =  = . =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  = ' '=  =  =.  =  =  =  =  no s i g n i f i c a n t d i f f e r e n c e between the two samples.  = -  = •  =  TABLE 7 . EFFECT OF COLD STORAGE - p e r c e n t c o m p o s i t i o n i n a r c s ~ i n / p e r c e n t a g e  degrees. +  9  FRACTION  8  7  6  5  4  3  2  1  1  2  c a p t i v e , female, a d u l t , 0. h. columbianus, Wolf Lake, B.C. 2 days N = 2  X + S.E.  31.5 1.3  11.5 i4.4 0.6 1.5  9.5 0.7  6.5 0.5  9.8 1.3  10.9 1.1  2 0 . 5 21.7 18.5 2 0 . 4 0.8 l - . o 1.0 0.8  31.9 13.2 15.9 0.2 2.9 0.5  7.9 0.2  8 . 5 10.4 10.8 18.0 19.9 18.6 2 2 . 6 0.2 0.9 0.6 0.3 1.4 0.9 2.0  221 days N = 2  'X + S.E.  304 days N = 2  X + S.E.  3 0 . 1 12.3 12.2 10.6 1.2 0.9 2.6 2.4  8.1 10.5 12.3 2 1 . 8 2.0 0.2 0.2 1.7  761 days N = 5  X + S.E.  32.4 12.7 14.3 1.4 1.0 1.2  7.3 1.0  N = number of a l i q u o t s  9.4 1.0  2 2 . 3 19.6 1.4 1.4  21.3 0.2  9 . 9 11.0 19.7 2 0 . 6 18.8 2 2 . 3 0.6 0.6 0.9 1.3 0.5 0.8  TABLE 8 EFFECT OF COLD STORAGE - Student's t - t e s t comparisons o f the p e r c e n t c o m p o s i t i o n ( i n a r c s ^ i n v/percentage degrees) o f the serum p r o t e i n f r a c t i o n s .  +  9  8  7  6  5  4  3  2  v s . 221 days  =  =  =  =  =  =  =  =  =  =  =  v s . 304 days  =  =  =  =  =  =  =  =  =  =  =  v s . 761 days  =  =  =  =  =  =  .=  =  =  =  =  FRACTION  1  1 . 2  c a p t i v e , female, adult, 0. h. columbianus, Wolf Lake, B.C. 2 days  221 days v s . 304 days  =  =  =  =  =  =  =  =  =  =  =  304 days v s . 761 days  =  =  =  =  =  =  =  =  =  =  =  no s i g n i f i c a n t d i f f e r e n c e between the two samples.  CO  00  TABLE 9 -5 2 EFFECT OF A BLOOD PRESERVATIVE - m o b i l i t i e s i n 10 cm / v o l t 10  FRACTION  9  8  7 . 6  5  seconds. 4  3  '  2  1  1  w i l d , female, a d u l t , _0. h. columbianus, California room temperature,. 1 day s t o r a g e no p r e s e r v a t i v e N = 2  X 1.060 .557 .461 .457 . 4 3 3 .347 .300 .203 .130 .017 .012 +S.E. .018 .023 .023 .013 .018 .005 .005 .023 .000 .005 .003  plus preservative • X 1.062 .570 .480 .457 .423 .345 .294 ' . 2 0 8 .130 .009 .009 N = 2 +S.E. .011 .003 .007 .001 .018 .001 .018 .005 .003 .003 .001 room temperature, 16 days s t o r a g e no p r e s e r v a t i v e N = 4  X +S.E.  plus preservative N = 3  X I . 0 6 3 .569 .489 .459 .433 .345 .305 .208 .120 .010 .011 +S.E. .007 .013 .005 .003 .008 .011 .003 .005 .018 .001 .001  .913 .028  . 4 6 6 .423 .401 .331 .234 .214 .145 .036 .010 .016 .016 .036 .048 .036 .036 .0.32 .023 .000  w i l d , f e m a l e , fawn, 0. h. columbianus, California - 18° C., 58 days s t o r a g e X +S.E.  .866 .491 .419 .390 .347 .258 .184 .170 .097 .014 .010 .018 .004 .015 .018 .013 .001 .004 .001 .028 .009 .000  plus preservative X N = 2 • +S.E.  .871 .496 . 4 l 8 .394 .347 .260 .183 .173 .068 .014 .010 .004 .009 .005 .000 .000 .001 .004 .004 .009 .005 .000  no p r e s e r v a t i v e N = 2  N = number of a l i q u o t s  TABLE 10 EFFECT OF A BLOOD PRESERVATIVE - Student's t - t e s t comparisons o f the m o b i l i t i e s protein fractions. FRACTION  10  o f the serum  9  8  7  6  5  4  3  2  =  =  =  =  =  =  =  =  +  1  1  =  =  w i l d , female, adult," _0. h. columbianus, California room temperature, 1 day s t o r a g e no preservative vs. plus preservative  =  room temperature, 16 days s t o r a g e no preservative vs. plus preservative + + 0  =  =  =  =  =  =  =  room temperature, no p r e s e r v a t i v e 1 day v s . 16 days s t o r a g e  0  =  =  =  =  =  =  =  =  =  =  =  =  =  =  =  = '  =  =  =  =  =  -  -  room temperature, p l u s p r e s e r v a t i v e 1 day v s . 16 days s t o r a g e =  = . =  w i l d , f e m a l e , fawn, _0. h. columbianus, California - l8°C., 58 .days s t o r a g e , no preservative vs. plus preservative = ++ 0  =  =  =  =  =  =  =  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y f a s t e r than the f i r s t i t e m t o the . 0 1 l e v e l . second i t e m s i g n i f i c a n t l y s l o w e r than the f i r s t i t e m t o the . 0 1 l e v e l . p r o t e i n f r a c t i o n p r e s e n t i n one sample but not i n the o t h e r .  =  =  4^  TABLE 11 EFFECT OF A BLOOD PRESERVATIVE - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n ^ p e r c e n t a g e degrees. 10  FRACTION w i l d , female, a d u l t , 0. h. columbianus , California room t e m p e r a t u r e ,  9  8  7  6  5  4  3  + 2  1  13.O l8.6 0.0 1.1  12.6 1.1  1  1 day s t o r a g e  no p r e s e r v a t i v e N = 2  T - + S.E.  plus preservative N = 2  X + S.E.  38.7 1.1  17.2 10.9 11.5 10.0 14.4 13.2 0.9 0.6 0.0 0.9 0.6 1.4  35.9 17.6 0.5 1.9  8.7 12.0 11.0 12.2 12.1 12.0 18.0 10.2 0.3 0.6 0.0 0.6 0.4 0.9 0.9 0.6  19.5 1.4 19.0 0.0  room t e m p e r a t u r e , 16 days s t o r a g e no p r e s e r v a t i v e N = 3  X ± S.E.  36.2 1.5  10.3 11.7 0.8 1.9  10.0 15.0 12.4 10.4 23.6 0.2 3.6 0.6 0.5 1.4  plus preservative N = 2  X + S.E.  3 6 . 9 18.3 0.1 0.0  9.5 0.1  12.3 1.2  10.2 1.1  13.0 0.1  no p r e s e r v a t i v e N = 2  X + S.E.  4l.5 1.4  9.2 15.7 0.6 1.2  6.8 3.4  7.1 1.5  plus preservative N = 2  X + S.E.  4 0 . 9 18.6 10.5 12.5 2.4 0.6 0.0 0.0  7.9 0.2  5.6 0.0  15.1 0.4  23.9 0.9  19.0 11.9 0.0 1.9  20.7 1.5  9.9 0.0  8.1 19.5 12.2 0.6 2.0 1.6  22.6 0.6  8.1 1.1  8 . 9 20.7 10.7 0.9 0.7 2.1  20.8 0.0  13.0 14.5 0.6 2.1  w i l d , f e m a l e , fawn, 0. h. columbianus, California - l8°C., 58 days s t o r a g e  N = number of a l i q u o t s  17.9 0.0  TABLE 12 EFFECT OF A BLOOD PRESERVATIVE - Student's t - t e s t comparisons of t h e p e r c e n t c o m p o s i t i o n ( i n a r c s in/'percentage degrees) o f t h e serum p r o t e i n f r a c t i o n s . +  FRACTION  •  10  9  8  7  6  .  5  4  3  .  =  =  ' =  2  1  1  w i l d , female, a d u l t , _0. h. columbianus, California room temperature, 1 day storage no p r e s e r v a t i v e v s . p l u s p r e s e r v a t i v e =  =  room temperature, 16 'days storage no p r e s e r v a t i v e v s . p l u s p r e s e r v a t i v e =  =  = 0  =  = =  =  =  =  = =  = =  =  =  =  room temperature, no p r e s e r v a t i v e 1 day v s . 16 days storage  =  0  =  =  ='  =  =  =  =  =  =  room t e m p e r a t u r e , p l u s p r e s e r v a t i v e 1 day v s . 16 days storage  =  =  =  =  =  =  =  =  =  =  =  w i l d , f e m a l e , fawn, 0. h. columbianus, California - l 8 ° C., 58 days storage no p r e s e r v a t i v e , v s . p l u s p r e s e r v a t i v e = = 0  no s i g n i f i c a n t d i f f e r e n c e between t h e two samples. p r o t e i n f r a c t i o n present i n one sample b u t n o t i n the o t h e r .  - 43 -  Succinylcholine  chloride  A c c o r d i n g t o Craighead e t a l (I960), c h l o r i d e i s a s k e l e t a l muscle r e l a x a n t  succinylcholine  t h a t a c t s by r e p l a c i n g  a c e t y l c h o l i n e a t the motor end p l a t e s thereby b l o c k i n g transmission  a t the myoneural j u n c t i o n .  nervous  Muscular p a r a l y s i s  persists until  the d i a c e t y l c h o l i n e i s h y d r o l y s e d by  cholinesterase  t o s u c c i n i c a c i d and c h o l i n e .  cardiovascular  system a r e minimal (Goodman & Gelman, 1956).  E f f e c t s on the  In the deer u n i t a t the U n i v e r s i t y of B r i t i s h  Columbia,  i t was p o s s i b l e t o o b t a i n b l o o d samples from the deer by throwing and h o l d i n g  them.  However, the s t r u g g l e was un-  d e s i r a b l e and the technique of i m m o b i l i z i n g succinylcholine chloride  (Anectine  the deer w i t h  - Burroughs Wellcome -  Canada) was used (Cowan e t a_l, 1962, Pearson e_t a l , 1963). Serum samples 7 days apart were taken from a c a p t i v e , a d u l t , male, 0. h. columbianus - Wolf Lake, Vancouver B.C.,  w i t h and without the h e l p  of s u c c i n y l c h o l i n e c h l o r i d e .  These samples when compared showed no s i g n i f i c a n t ences i n e i t h e r the m o b i l i t y composition  (Tables  Therefore,  Island,  (Tables  differ-  13 & 15) or the p e r c e n t  14 & 16) of the p r o t e i n f r a c t i o n s .  serum samples obtained from U.B.C. c a p t i v e  deer w i t h the h e l p of s u c c i n y l c h o l i n e c h l o r i d e can be used i n f u r t h e r comparisons.  TABLE--13 EFFECT OF SUCCINYLCHOLINE CHLORIDE - m o b i l i t i e s i n 10 FRACTION  9  8  7  -5  cm 6  2  / v o l t seconds. 5  4  3  2  +  1  1  2  c a p t i v e , male, a d u l t , 0. h. columbianus, Wolf Lake, B.C. without a i d of succinylcholine chloride N = 2 _ X .854 .654 .582 .458 .425 .372 .315 .134 .043 .015 .083 + S.E. , .005 .004 .000 .000 .004 .000 .000 .000 .005 .004 .003 w i t h a i d of s u c c i n y l c h o l i n e c h l o r i d e N• = 2 _ X .856 .659 .568 .472 .439 .377 .310- .123 .053 .019 .086 + S.E. .005 .009 .016 .005 .009 .005 .005 .004 .004 .000 .000 N = number of a l i q u o t s  TABLE 14 EFFECT OF SUCCINYLCHOLINE CHLORIDE - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum protein fractions. FRACTION  9  8  7  c a p t i v e , male, a d u l t , 0. h. columbianus, Wolf Lake, B.C. w i t h v s . w i t h o u t a i d of succinylcholine chloride  no s i g n i f i c a n t d i f f e r e n c e between the two samples.  6  5  4  3  2  1  1  2  TABLE 15 EFFECT OF SUCCINYLCHOLINE CHLORIDE - p e r c e n t c o m p o s i t i o n i n a r c s i n / p e r c e n t a g e degrees. +  FRACTION  9  8  7  6  5  ^  3  2  1  c a p t i v e , male, a d u l t , 0. h. columbianus, Wolf Lake, B.C. w i t h o u t a i d of s u c c i n y l c h o l i n e c h l o r i d e N = 2 X 39.9 + S.E. 2.0  9.5 12.0 17.3 12.9 0.0 0.2 0.5 0.4  6.5 0.4  8 . 3 15.8 21.6 0.3 0.6 0.6  8.0 2 0 . 0 0.4 0.6  w i t h a i d of s u c c i n y l c h o l i n e c h l o r i d e N - 2 X 37.2 + S.E. 0.1  8 . 8 12.1 19.2 14.9 0.2 0.2 0.0 0.3  7.0 1.1  8.5 15.3 24.3 0.2 0.1 0.1  9.0 0.4  N = number of a l i q u o t s  18.0 0.2  TABLE 16 EFFECT OF SUCCINYLCHOLINE CHLORIDE - Student's, t - t e s t comparisons of the p e r c e n t c o m p o s i t i o n ( i n arcs'^in / p e r c e n t a g e degrees) of the serum p r o t e i n f r a c t i o n s . +  FRACTION .  9  8  7  c a p t i v e , male, a d u l t , 0. h. columbianus, Wolf Lake, B.C.L w i t h v s . w i t h o u t a i d of succinylcholine chloride  no s i g n i f i c a n t d i f f e r e n c e between the two samples.  6  5  4  3  2  1  1  2  - 48 -  CONDITION OF ANIMAL Individual variation Serum samples from e i g h t w i l d , female,  a d u l t , 0.  v.  texanus were o b t a i n e d from S i n t o n , Texas from A p r i l 19 3, 19 63.  May  These y e l l o w samples were s t o r e d f o r s l i g h t l y more  than one year.  The numerical data f o r the m o b i l i t y and f o r  the p e r c e n t composition of the p r o t e i n f r a c t i o n s are g i v e n i n Tables 17 & 19  respectively.  . I n d i v i d u a l v a r i a t i o n i s found i n a l l parameters used i n taxonomy.  That there i s a l s o a v e r y g r e a t i n d i v i d u a l  v a r i a t i o n i n the serum p r o t e i n s i s r e a d i l y seen i n Tables  18  & 20 where the t - t e s t comparisons f o r m o b i l i t y and p e r c e n t composition r e s p e c t i v e l y are g i v e n .  M o b i l i t y appears  a more v a r i a b l e parameter than p e r c e n t Each i n d i v i d u a l animal has  t o be  composition.  i t s own  p a r t i c u l a r immune  h i s t o r y which w i l l have an e f f e c t on the gamma-globulins  and  p o s s i b l y a l s o on the a l p h a - and b e t a - g l o b u l i n s (see s e c t i o n on Age).  T h i s immune response must account f o r some of the  v a r i a b i l i t y between i n d i v i d u a l s which i s shown here i n the m a j o r i t y of the p r o t e i n f r a c t i o n s . albumin  That f r a c t i o n +10  may  be  (as i t i s the f r a c t i o n of g r e a t e s t m o b i l i t y ) i s  supported by i t s s t a b i l i t y i n m o b i l i t y . There i s no obvious e x p l a n a t i o n as to why  the m o b i l i t -  i e s of f r a c t i o n +1 and +2 and the p e r c e n t compositions  of  - 49 -  fractions  +1 and +3 do not v a r y s i g n i f i c a n t l y between  individuals.  The e f f e c t of i n d i v i d u a l v a r i a t i o n has been  accounted f o r i n f u r t h e r l e a s t two  individuals.  comparisons by u s i n g groups of a t  INDIVIDUAL VARIATION - m o b i l i t i e s i n 10 10  FRACTION  9  -5  cm 8  2  TABLE 17 / volt 7  seconds. 6  5  4  3  2  + 1  1  2  w i l d , female, a d u l t , _0. v. t e x a n u s , Texas D 210 N = 4  + s.E.  D 237 N = 2  -x + S.E.  .863 .481 .435 .359 .330 .254 .197 .155 .092 .026 .010 .190 .057 .004 .001 .010 .000- .011 .006 .000 .023 .016 .000 .000  D 242 N = 2  X + S.E.  .804 .412 .349 .33^ .291 .222 .164 .154 .064 .016 .010 .154 .000 .021 .010 .006 .006 .000 .006 .006 .011 .006 .000 .006  D 243 N = 2  X + S.E.  .809 .391 .339 .317 .285 .217 .159 .148 .084 .010 .016 .164 .005 .000 .000 .000 .000 .006 .000 .000 .000 .000 .006 .006  D 247 N = 4  x + S.E.  .821 .439 .381 .352 .321 .259 .177 .15^ .060 .014 .010 .111 .012 .013 .009 .009 .005 .016 .011 .000 .006 .005 .000 .020  D 249 N = 5  X + S.E.  .803 .509 .469 .436 .392 .339 .275 .169 .097 .011 .009 .064 .007 .004 .002 .005 .003 .004 .009 .009 .022 .002 .000 .003  D 250 N = 4  X + S.E.  .841 .469 .407 .379 .332 .254 .161 .149 .072 .010 .010 .128 .032 .021 .012 .012 .002 .012 .001 .002 .007 .000 .000 .030  X  .850 .510 .439 .401 .338 .256 .211 .175 .077 .011 .011 .036 .011 .007 .000 .012 .017 .004 .009 .009 .001 .001  .143  .010  N = number of a l i q u o t s  o  TABLE 18 INDIVIDUAL VARIATION - Student's t - t e s t comparisons FRACTION  10  .9  = =  =  = — --= =  =  =  = =  -=  ---  =  ++  =  =  of the m o b i l i t i e s  of the serum p r o t e i n  8  7  6  — ---  — — ---  = --  5  4  3 - 2  =  = =  = — -=  1  1  fractions. 2  w i l d , female, a d u l t , 0. v. texanus, Texas D 210 v s . vs. vs. vs. vs . vs.  D D D D D D  237 242 243 247 249 250  D 237 v s . D 242 v s . D 243 v s . D 247 v s . D 249 v s . D 250 D 242 v s . vs. vs. vs.  D D D D  =  =  243 247 249 250  =  D 243 v s . D 247 v s . D 249 v s . D 250  = —  ++  D 247 vs . D 249 v s . D 250  =  D 250  =  D 249 v s . =  ++ --  =  ++  ++ =  ++ =  =  =  =  =  =  -=  — =  =  =  =  =  =  ++  ++  ++ ++  ++  =  =  =  ++ ++ ++  ++ ++  ++  ++  ++  =  =  --  --  = -  =  =  =  ++  =  = = =  ++  -=  ++  =  --  = = ++  =  =  ++  =  =  =  =  =  ++ =  =  ++  --  —  = ++ =  =  --= = — = ---  -  —  = = :  -  =  —  — = = = =  -  = -  -  :  = = =  =:  =  -  =  ++ • ++ ++  ++ =  =  =  =:  ++ =  ++ =  ++  —  =  --  --  ++  ++  =  =  =  no slg;nif i c a n t d i f f e r e n c e between the two 3 a m o l e s . second i t e m Si£ ; n i f I c a n t l y f a s t e r than the :f i r s t i t e m t o the . 01 l e v e l . second i t e m si£? n i f i c a n t l y slower than the :f i r s t i t e m t o the . 01 l e v e l .  =  -  ++ -  =  = =  -  -  — -= -  = = --  = =  --  =  :  -  — --  = =  =  =  — =  =  -  =  ++  =  TABLE 19 INDIVIDUAL VARIATION - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n / p e r c e n t a g e 10  FRACTION  9  8  7  6  degrees. 5  4  3  2  +  1  1  2  w i l d , female, a d u l t , 0. v. texanus, Texas X + S.E.  36.3 0.2  17.8 0.9  14.9 0.7  10.6 0.7  8.0 1.3  5.3 1.1  X + S.E.  42.7 1.1  17.6 1.7  15.4 0.6  15.1 1.1  6.9 0.6  8.1 1.6  X + S.E.  37.6 0.1  12.5 2.1  H.3 0.3  11.0 0.0  4.1 2.0  X + S.E.  39.6 ^199  12.2 lA  13.5 oo-.-4  11.6 1.1  X~ + S.E.  37.3 0.6  17.9 2.1  13.1 1.0  D 249 N=5  X + S.E.  36.6 0.5  19.9 0.3  D 250 N = 4  X + S.E.  42.0 0.3  17.5 1.2  D 237 N = 3  D 243 N = 2  N = number o f a l i q u o t s  9.9 1.1  13.3 1.4  13.5 1.0  18.7 2 3 . 0 1.6 l.i  11.6 1.0  11.8 0.7  16.7 0.4  12.3 1.5  14.7 20.0 0.8 O.t  7.1 1.7  10.8 0.1  10.6 0.6  16.9 0.9  13.3 0.1  21.1 22.6 0.3 OA  10.7 0.2  10.3 0.0  10.3 0.2  11.5 o.o  10.8 0.9  12.7 1.8  15.8. 23.O 3.2 h.t  15.5 0.3  10.3 0.4  5.6 0.7  11.4 0.8  10.7 0.2  11.8 0.5  10.4 2.1  12.5 24.0 1.8 2.C  14.0 1.0  12.0 0.6  18.1 1.4  8.7 0.5  5.7 0.9  13.1 2.6  11.5 1.2  15.3 1.7  14.0 0.5  10.6 1.4  10.5 0.6  10.9 1.4  5.0 0.3  7 A  10.5 1.7  10.4 1.6  12.9 0.8  12.9 23.5 0.9 0.8  j  9.2 0.8  0.9  14.6 0.5  TABLE 20 INDIVIDUAL VARIATION - Student's t - t e s t comparisons of the p e r c e n t c o m p o s i t i o n ( i n a r c s ^ i n / p e r c e n t a g e degrees) of the serum p r o t e i n f r a c t i o n s . 10  FRACTION  9  8  7  6  5  4  3  +  2  1  1  2  w i l d , fe'male, a d u l t , 0_. v. texanus, Texas D 210 vs .. D vs. D vs. D vs. D vs. D vs. D  237 242 243 247 249 250  D 237 v s . D vs. D vs. D vs. D vs. D  242 243 247 249 250  D 242 vs. D 243 vs. D 247 vs. D 249 v s . D 250 D 243 vs. D 247 vs . D 249 vs. D 250  ++ =  ++  = =  — —  =  = =  =  ++ •  =  = = =  _  =  = =  -  —  ++  D 249 vs. D 250  ++  ++ ++ ++  ++  —  = = =  =  _  _  =  =  =  —  _  _  —  =  = = :  =  —  =  -  n ++ = =  =  =  --  =  =  —  -  _  _  _  — — —  —  =  —  =  =  =  =  -  = =  :  = =  = =  =  = = =  = = =  ++ =  -  =  — —  =  — -  =  --  =  =  —  = =  =  —  —  -:  =  = =  _ =  = —  — -  _  —  =  —  -  -  = =  =  = --  --  _  = =  =  = =  =  —  =  -  —  ;  =  =  =  =  = ==  = =  ++  -  =  —  =  -  =  ++ _  = =  ++ =  — =  —  = ---  D 249 D 250  D 247 vs. vs.  = -  — (JO  = +-+-  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y g r e a t e r than the f i r s t i t e m t o the .01 second Item s i g n i f i c a n t l y s m a l l e r than the f i r s t i t e m t o the .01  level, level.  - 54 -  Sex E l e c t r o p h o r e t i c s t u d i e s c o n s i d e r i n g the e f f e c t of the sex of the animal on a serum sample are few.  Using micro-  e l e c t r o p h o r e s i s i n a g a r - g e l , van Sande and Karcher  (1960)  found no s i g n i f i c a n t sexual d i f f e r e n c e s i n the p r o t e i n patterns  of many s p e c i e s of i n s e c t s .  Free e l e c t r o p h o r e s i s  was u t i l i z e d by Brandt e t a l (1950), Deutsch & Goodloe (1945), and Moore (1945) t o d e t e c t sexual d i f f e r e n c e s i n the p r o t e i n patterns  of c h i c k e n s .  Brandt e t a l (op. c i t . ) found t h a t  l a y i n g hens e x h i b i t e d i n c r e a s e d t o t a l p r o t e i n , an e x t r a pre-albumin f r a c t i o n ,  i n c r e a s e d a l p h a - g l o b u l i n , and decreas-  ed albumin when compared t o n o n - l a y i n g S i b l e y and Johnsgard  hens and c o c k e r e l s .  (1959a) found t h a t i n one year o l d  pheasants the l a y i n g females had v e r y low albumin compared t o those of male pheasants and i n a d u l t fowl the l a y i n g and n o n - l a y i n g albumin l e v e l s than the males.  levels jungle  females e x h i b i t e d lower J u v e n i l e pheasants,  however,  evinced no sex d i f f e r e n c e i n serum p r o t e i n p a t t e r n s . Moore (1945) examined the f r e e e l e c t r o p h o r e t i c p r o f i l e s of a wide v a r i e t y of mammals and c o u l d d e t e c t no sexual d i f f e r e n c e s .  U t i l i z i n g f i l t e r paper e l e c t r o p h o r e s i s ,  Cowan & Johnston (19 62) found sexual d i f f e r e n c e s i n the  - 55 -  p r o t e i n p a t t e r n s of the c a p t i v e deer 0. h. s i t k e n s i s and v. ochrourus.  0.  The a d u l t female deer had a s i g n i f i c a n t l y -  lower p e r c e n t composition of f r a c t i o n I (albumin?) and a s i g n i f i c a n t l y h i g h e r p e r c e n t composition of f r a c t i o n s I I and V ( g l o b u l i n s ? ) than the males. Thus i t appears  t h a t where sexual d i f f e r e n c e s i n  p r o t e i n c o n c e n t r a t i o n are d e t e c t e d they w i l l u s u a l l y be e x h i b i t e d by lowered albumin and i n c r e a s e d g l o b u l i n i n the  levels  female. Yellow samples of w i l d a d u l t , 0. h. columbianus  Chemainus, Vancouver I s l a n d , B.C.  -  ( c o l l e c t e d i n December,  19 61 and s t o r e d l e s s than two years) and of w i l d fawns of 0. h. columbianus October,  - Mendocino County, C a l i f o r n i a  (collected i n  19 62 and s t o r e d l e s s than t h r e e weeks) were examin-  ed e l e c t r o p h o r e t i c a l l y f o r the e f f e c t of sex on the serum proteins.  The w i l d , female, fawns of 0. h. columbianus  Mendocino County, C a l i f o r n i a  (Tables 23 & 24) have a  s i g n i f i c a n t l y lower p e r c e n t composition of f r a c t i o n ( g l o b u l i n ? ) than the c o r r e s p o n d i n g male fawns. p r e c i s e age of the fawns i s not known t h i s one  As  +3 the  significant  d i f f e r e n c e may be l i n k e d not o n l y w i t h sex but a l s o w i t h maturation  (see s e c t i o n on  -  Age).  - 56 -  The w i l d , female, a d u l t s of 0. h. columbianus  -  Chemainus, Vancouver I s l a n d , B.C. show a s i g n i f i c a n t l y lower p e r c e n t composition of f r a c t i o n +10 than the c o r r e s p onding male a d u l t s p o s s i b l y albumin  (Tables 23 & 24).  As t h i s f r a c t i o n i s  (see s e c t i o n on I n d i v i d u a l v a r i a t i o n )  then t h i s r e s u l t i s i n agreement w i t h p r e v i o u s work. Cowan & Johnston  (19 62) found no s i g n i f i c a n t  d i f f e r e n c e s i n the m o b i l i t i e s of the p r o t e i n f r a c t i o n s of a d u l t male and female c a p t i v e 0. h . s i t k e n s i s and 0. v. ochrourus u s i n g f i l t e r paper e l e c t r o p h o r e s i s . starch-gel electrophoresis  The use of  i n the p r e s e n t study i n d i c a t e s  t h a t the w i l d , a d u l t 0. h. columbianus  - Chemainus,  Vancouver I s l a n d , B.C. d i f f e r s i g n i f i c a n t l y i n the m o b i l i t i e s of t h r e e f r a c t i o n s , the female h a v i n g s i g n i f i c a n t l y slower f r a c t i o n s +3, +6, and +8 (Tables 21 & 22).  Table  22 a l s o shows no s i g n i f i c a n t d i f f e r e n c e s between the m o b i l i t y of any of the p r o t e i n f r a c t i o n s of male and female fawns of 0. h. columbianus  - Mendocino County, C a l i f o r n i a .  In summary, s e x u a l d i f f e r e n c e s i n the m o b i l i t y of the p r o t e i n f r a c t i o n s were not observed between male and female fawns b u t were observed between male and female  adults.  Sexual d i f f e r e n c e s i n the p e r c e n t composition of the p r o t e i n  - 57 -  f r a c t i o n s were observed i n b o t h male and female fawns and adults.  Therefore, the sex of the deer must be taken i n t o  consideration i n further  comparisons.  TABLE 21 -5 EFFECT OF SEX - m o b i l i t i e s  i n 10  2 cm  10  FRACTION  volt 9  seconds. 8  7  6  4  + 1  wild, 0. h. columbianus, California male, fawn N = 3  X + S.E.  .977 .542 .460 .431 .394 .330 .224 .182 .101 .015 .011 .037 .021 .002 .005 .014 .031 .031 .005. .015 .005 .001  f e m a l e , fawn N = 3  X + S.E.  .893 .527 .443 .407 .371 .272 .183 .179 .095 .034 . O i l .024 .043 .020 .023 .017 .007 .006 .006 .014 .013 .001  male, a d u l t N = 2  X + S.E.  .829 .494 .423 .383 .313 .260 .165 .133 .022 .012 .013 .008 .008 .005 - O i l .0H2 .005 .011 .012 .003  female, a d u l t • N = 2  X + S.E.  .775 .421 .383 .340 .286 .221 .115 .080 .017 .013 .006 .006 .006 .001 .013 .007 .006 .006 .001 .001  wild, 0. h. columbianus, Chemainus, B.C.  N = number of animals  Ul CO  TABLE 22 EFFECT OF SEX - Student's t - t e s t comparisons of the m o b i l i t i e s  of the serum p r o t e i n f r a c t i o n s . 4-  FRACTION  10  9  8  7  6 . 5  4  3  2  1  1  wild, O.h. columbianus, California male fawn v s . female fawn wild, _0.h. columbianus, Chemainus, B.C. male a d u l t v s . female a d u l t  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y slower than the f i r s t i t e m t o the .01  level.  U3  TABLE 23 EFFECT OF SEX - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n / p e r c e n t a g e d e g r e e s . FRACTION  10  7  6  5  + 1  4  wild, 0. h. columbianus, California male, fawn N = 3 ' f e m a l e , fawn N = 3 t  X ± S.E.  35.0 17.3 13.6 11.2 2.2 0.8 0.3 2.1  9.6 11.2 13.1 0.9 3.4 1.5  12.2 20.8 0.6 0.5  9.8 1.6  23.7 0.9  X + S.E.  41.0 19.1 10.9 13.7 0.8 0.3 0.6 1.5  8.7 0.8  6.2 1.0  8.8 1.4  9.1 22.8 o.l 1.2  11.7 0.8  17.1 2.0  7.4 1.4  6.9 0.8  7.2 1.8  10.4 11.3 o.l 1.8  18.0 3.2  19.9 0.6  8.1 12.8 14.7 12.7 15.5 0.3 3.7 3.2 2.6 3.1  22.9 2.4  wild, Q. h. columbianus, Chemainus, B.C. male, a d u l t N = 2 female, a d u l t N = 2  X . + S.E. X + S.E.  44.6 0.1  19.2 14.. 2 2.4 6.5  39.2 16.3 13.7 l l . l 0.5 0.5 0.9 1.4  N = number of a n i m a l s  O  TABLE 24 EFFECT OF SEX - Student's t - t e s t comparisons of the p e r c e n t c o m p o s i t i o n ( i n a r c s ^ i n ^/percentage degrees) of the serum p r o t e i n f r a c t i o n s . +  FRACTION  10  9  wild, _0.h. columbianus, California male fawn v s . female fawn  =  =  8  =  7  =  6  =  5  =  4  3  2  =  -  1  -  =  1  =  =  wild, O.h. columbianus, Chemainus, B.C. male a d u l t v s . female a d u l t  - - =  =  =  =  =  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y s m a l l e r than the f i r s t i t e m t o the .01  =  level.  =  =  =  - 62 -  Age Examinations of the b l o o d serum of chickens by means of f r e e e l e c t r o p h o r e s i s  (Brandt e_t aJL, 1950, M a r s h a l l &  Deutsch, 1950) and of pheasants b y means of f i l t e r electrophoresis  paper  ( S i b l e y & Johnsgard, 1959a) a l l show an  i n c r e a s e i n gamma-globulin  w i t h age.  The r e s u l t s  involving  the e f f e c t of the animal's age on albumin c o n c e n t r a t i o n are varied:  Brandt e t a l found no change i n albumin c o n c e n t r a t -  i o n w h i l e M a r s h a l l & Deutsch and Moore found albumin  increased  w i t h age i n chickens and S i b l e y & Johnsgard found i t decreased w i t h age i n pheasants. ' Brandt et. a l a l s o found an i n c r e a s e i n t o t a l p r o t e i n and a l p h a - g l o b u l i n w i t h no change i n the b e t a - g l o b u l i n c o n c e n t r a t i o n of c h i c k e n s . •I  Konig e t a l (1949) used f r e e e l e c t r o p h o r e s i s t o observe an i n c r e a s e i n gamma-globulin Munkacsi  as lambs mature.  (19 61) used paper e l e c t r o p h o r e s i s t o study horses  from the newborn stage t o twenty year o l d s .  He found t h a t  the t o t a l p r o t e i n r o s e s t e a d i l y w i t h i n c r e a s i n g age, t h a t t h i s i n c r e a s e was due t o the a d d i t i o n of b e t a - g l o b u l i n up t o 1% years, and t h a t the i n c r e a s e t h e r e a f t e r was due t o the a d d i t i o n of gammaglobulin. or a l p h a - g l o b u l i n .  He found no change i n albumin  Cowan & Johnston  (19 62) used paper  e l e c t r o p h o r e s i s t o study the e f f e c t of age on the serum  - 63 -  proteins B.C.  of c a p t i v e 0. h. columbianus - Vancouver I s l a n d ,  The  p e r c e n t composition of the p r o t e i n f r a c t i o n s  changed up  to the age  of f i f t e e n months.  (albumin?) decreased. F r a c t i o n s and  F r a c t i o n IV and The  increase  Fraction V  Fraction I  I I & I I I remained constant, (gamma-globulin?)  increased.  o n l y c o n s i s t e n t f i n d i n g i n the above work i s the  i n gamma-globulin as the animal ages.  i g a t o r s agree t h a t t h i s i n c r e a s e  The  invest-  i s p r o b a b l y a response t o  the i n c r e a s i n g number of a n t i g e n i c substances the  animal  comes i n t o c o n t a c t w i t h as i t grows o l d e r . Most samples r e c e i v e d f o r the p r e s e n t study were labelled either  "fawn" or  "adult".  However, y e l l o w samples  taken from w i l d , female, 0. h. columbianus - Mendocino County, C a l i f o r n i a i n October, 1962,  were c l a s s i f i e d  fawns, 1 year o l d s , 2 year o l d s , 4 year o l d s , and olds.  Due  to the  the l a t t e r three  as  6 year  small number of samples i n each age ages were grouped and  " a d u l t s " i n the f o l l o w i n g t a b l e s .  are considered  These " a d u l t s "  the p o s s i b l e range of ages t h a t would be  included  i n the  fawn sera were s t o r e d l e s s than three weeks w h i l e the years.  as  represent  " a d u l t " l a b e l s on samples r e c e i v e d from other sources.  i n g sera were s t o r e d l e s s than two  group,  The remain-  - 64  -  Numerical data f o r the m o b i l i t y and  the p e r c e n t  composition of the p r o t e i n f r a c t i o n s are given 25 & 27 r e s p e c t i v e l y .  i n Tables  A l l ages e x h i b i t the same t o t a l  number of p r o t e i n f r a c t i o n s . Table 2 6 shows t h a t there, i s no  s i g n i f i c a n t change  i n the m o b i l i t y of the p r o t e i n f r a c t i o n s from fawns to adults. (19 62)  T h i s i s i n agreement w i t h Cowan & Johnston's f i n d i n g s on  mobility.  That the p e r c e n t composition of the p r o t e i n f r a c t i o n s does change w i t h age  i s seen.in  which i s assumed t o be  Table 28.  albumin, has  Fraction  decreased w i t h  +10, age  s u f f i c i e n t l y to show a s i g n i f i c a n t d i f f e r e n c e between fawns and  adults.  T h i s decrease i n albumin agrees w i t h  t h a t found i n pheasants ( S i b l e y & Johnsgard, 1959a) and c a p t i v e O. h. columbianus - Vancouver I s l a n d , B.C.  in  (Cowan &  Johnston, 19 62). The  fawns and  the one  year o l d s are s i m i l a r i n the  p e r c e n t composition of a l l t h e i r p r o t e i n f r a c t i o n s . fawns and but  The  the a d u l t s d i f f e r not o n l y i n the albumin f r a c t i o n  the a d u l t s a l s o show an i n c r e a s e  ( g l o b u l i n ? ) over the fawns. i n f r a c t i o n s +3  and  +4  The  i n f r a c t i o n +3  a d u l t s a l s o show an  ( g l o b u l i n s ? ) over the one  increase  year o l d s .  - 65 -  As the i d e n t i t y of the g l o b u l i n s w i t h a p a r t i c u l a r f r a c t i o n number has not been determined  f r a c t i o n s +3  and  +4 must remain o n l y " g l o b u l i n s " . In summary, no changes i n the m o b i l i t y of the p r o t e i n f r a c t i o n s are shown t o occur w i t h age.  Changes i n  p e r c e n t composition are found between fawns, one year o l d s , and a d u l t s .  As the m a j o r i t y of the serum samples  i n t h i s study were from a d u l t s , the fawns and one o l d s were e l i m i n a t e d  from f u r t h e r  comparisons.  received year  TABLE 25 EFFECT OF AGE - m o b i l i t i e s i n 10  -5  cm  10  FRACTION  2  / volt  seconds. 9  8  7  6  5  4  3  2  41  1  w i l d , female, 0. h. co.lumbianus, California fawn N = 4  X + s.E.  .901 .517 . 4 4 2 .411 .375 .290 .212 . l 8 l .099 .029 .011 .019 .032 .014 .017 .013 .019 .023 .005 .011 ...Oil .001  1 year o l d N=4  .+ s.E.  X  .952 .515 .^60 .431 .403 .295 .228 .174 .077 .012 .011 .046 .027 .025 .026 .025 .019 .017. .024 .017 .002 .001  adult. N = 3  X + S.E.  .979 .540 .475 .434 .406 .288 .225 . 1 4 9 .087 .012 .012 .044 .016 .012 .012 .013 .028 .040 .042 .024 .002 .001  N = number of animals  TABLE 26 EFFECT OF AGE - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum p r o t e i n  fractions.  + 10  9  8  7  6  5  4  3  2  fawn v s . 1 y e a r o l d  =  =  =  =  =  =  =  = .  =  =  -  fawn v s . a d u l t  = .  =  =  =  -  =  =  =  =  =  =  1 year o l d vs. adult  =  '=  =  =  =  =  =  =  =  =  FRACTION  1  1  w i l d , female 0 . h. columbianus, California  •  no s i g n i f i c a n t d i f f e r e n c e between the two samples.  CTi  TABLE 27 EFFECT OF AGE - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n / p e r c e n t a g e 10  FRACTION  9  7  degrees. 6 - 5  ^  3  +  1  1  w i l d , female, 0 . h. columbianus, California fawn N = 4  X + S.E.  41.2 18.9 11.7 12.8 0.6 0.3 0.9 1.5  8.3 0.7  7.0 1.0  9.0 9.2 2 2 . 2 11.6 l . l 0.2 l . l 0.6  1 year o l d N = 4  X + S.E.  40.4 20.0 0.3 1.3  7.0 0.8  6.4 1.7  9.5 0.2  adult N = 3  X + S.E.  37.2 16.7 13.I 0.8 0.9 1.9  N = number  12.2 1.9  8.8 0.8  10.6 7.9 12.2 l . l 2.5 2.7  17.6 1..  9 . 8 18.5 13.3 22.2 0.3 3.5 0.3 1.6  12.9 13.3 19.6 10.9 0.2 0.6 0.6 1.1  21.2 4.1  o f animals  en 00  TABLE 28 EFFECT OF AGE - Student's t - t e s t comparisons of the p e r c e n t c o m p o s i t i o n ( i n a r c s ^ i n degrees) of the serum p r o t e i n f r a c t i o n s .  /percentage  +  10  9  8 ' 7  fawn v s . 1 y e a r o l d  =  =  =  fawn v s . a d u l t  - - =  1 year o l d v s . adult  =  FRACTION  6  5  .4  3  2  1  1  =  =  =  =  =  =  =  =  =  =  =  =  =  + + =  =  =  =  =  =  =  + + + + =  =  =  w i l d , female, _0. h. columbianus, California  = ++  =  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y g r e a t e r than the f i r s t i t e m t o the .01 second i t e m s i g n i f i c a n t l y s m a l l e r than the f i r s t i t e m t o the .01  level, level.  - 70 -  Season Deer, l i k e many other animals, are a f f e c t e d by seasonal changes i n the q u a n t i t y  and the q u a l i t y of t h e i r food and  m a i n t a i n an annual c y c l e of r e p r o d u c t i o n . patterns diet there  of mammalian  serum p r o t e i n s  As e l e c t r o p h o r e t i c  are a f f e c t e d both by  (see s e c t i o n on C a p t i v i t y ) and pregnancy (Moore, 1959) i s the p o s s i b i l i t y of the deer's p r o t e i n  pattern  showing seasonal e f f e c t s . Reports of seasonal e f f e c t s on b l o o d p r o t e i n s are scarce.  S i b l e y & Johnsgard (1959a) g i v e the f o l l o w i n g  i l l u s t r a t i o n of the e f f e c t of season on the e l e c t r o p h o r e t i c pattern  of the serum p r o t e i n s  of m a l l a r d s .  They found t h a t  female m a l l a r d s had lower albumin l e v e l s i n November than i n July.  However, the f i r s t  samples were from w i l d b i r d s i n  J u l y which were subsequently kept i n c a p t i v i t y u n t i l the November sampling (see s e c t i o n on C a p t i v i t y ) . Johnston (19 62)  Cowan &  i n a paper e l e c t r o p h o r e t i c study of c a p t i v e ,  a d u l t , male, 0. h. columbianus r e p o r t no s i g n i f i c a n t d i f f e r e n c e s i n e i t h e r the m o b i l i t y or the p e r c e n t compositi o n of the serum p r o t e i n s  a s s o c i a t e d w i t h the onset of r u t .  In the p r e s e n t study, serum samples were compared from w i l d , female, a d u l t , 0. h. columbianus - Mendocino County, C a l i f o r n i a ;  three  i n October, 1962 and two i n  - 71 -  January, 19 63. In January the n u t r i t i o n a l s t a t u s of the deer i n Mendocino County, October  C a l i f o r n i a i s markedly poorer than i n  ( B i s s e l & Strong, 1955,  Taber & Dasmann, 1958).  That t h e r e were no s i g n i f i c a n t d i f f e r e n c e s observed i n the m o b i l i t i e s of any of the p r o t e i n f r a c t i o n s  (Tables 29 &  i s i n agreement w i t h the f i n d i n g s of Cowan & Johnston  30)  (1962).  S i g n i f i c a n t d i f f e r e n c e s were found, however, i n the p e r c e n t composition of the p r o t e i n f r a c t i o n s as separated by gel electrophoresis  (Tables 31 & 32).  shows an i n c r e a s e d albumin globulin  The January  ( f r a c t i o n +10)  and  ( f r a c t i o n +4) over the October group.  r e s u l t s agree w i t h those of G o l d s t e i n & S c o t t  starch-  group  lowered These (1956) who  s t u d i e d the e f f e c t s of a V i t a m i n E d e f i c i e n c y i n c h i c k e n s . Taber  (1953) found the peak of c o n c e p t i o n i n 1949  O. h. columbianus  - Mendocino County,  C a l i f o r n i a occurred  d u r i n g the l a s t week i n October.and the f i r s t November.  of  two weeks i n  I t i s not known i f the groups of females used i n  the p r e s e n t study were pregnant a t the time of sampling. I t i s d o u b t f u l , however, t h a t e a r l y pregnancy would have a g r e a t e r e f f e c t on the e l e c t r o p h o r e t i c p a t t e r n than the d r a s t i c change i n n u t r i t i o n . In summary, a change of season does not a f f e c t the  - 72 -  m o b i l i t y but does a f f e c t the percent protein fractions.  composition of the  These changes can be l i n k e d t o the  n u t r i t i o n a l s t a t u s of the season.  I t i s apparent, t h e r e -  f o r e , t h a t s t u d i e s i n t e n d i n g t o explore  serum p r o t e i n  v a r i a t i o n i n deer can o n l y get comparable samples by u s i n g phenologically equivalent i n d i v i d u a l s .  TABLE 29 EFFECT OF SEASON - m o b i l i t i e s FRACTION  i n -10 10  -5  cm  2  / volt 9  seconds. 7  5  4  3  +  l  w i l d , female, a d u l t , 0_. h. columbianus, California October, N = 3  1962  X + S.E.  979 .540 .475 .434 .406 .288 .225 .149 .087 .012 .012 .044 .016 .012 .012 .013 .028 .040 .042 .024 .002 .001  January, N = 2  1963  X + S.E.  961 .533 .463 .435 .402 .297 .209 .156 .099 .010 . o i l .083 .049 .032 .037 .o4o .001 .001 .025 .006" .001 .001  N = number of animals  to  TABLE 30 EFFECT OF SEASON - Student's t - t e s t comparisons of the m o b i l i t i e s  of the serum p r o t e i n  fractions. +  10  FRACTION  9  8 . 7  6  5  4  3  2  1  =  =  =  =  =  1  w i l d , female., a d u l t , P.. h. columbianus, California October, 1962 v s . January, 1963 =  no s i g n i f i c a n t d i f f e r e n c e  ' = .  =  =  =  =  between the two samples.  ^1  TABLE 31 EFFECT OF SEASON - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n / p e r c e n t a g e  degrees. +  10  FRACTION  9  8  7  6  5  4  3  2  1  1  w i l d , female, a d u l t , 0. h. columbianus, California October, 1962 N = 3  X ± S.E.  37.2 16.7 13.1 0.8 0.9 1.9  10.6 7.9 12.2 l . l 2.5 2.7  January, 1963 N = 2  X + S.E.  4 6 . 4 2 0 . 5 11.5 0.5 0.4 1.4  11.2  1.3  6.3 1.1  9.2 0.9  12.9 13.3 19.6 10.9 0.2 0.6 0.6 1.1 8.5 0.4  9.5 13.7 10.9 0.5 3.5 0.6  21.2 4.1 16.9  1.9  N = number of a n i m a l s  Ul  TABLE 32 EFFECT OF SEASON - Student's t - t e s t comparisons of the p e r c e n t c o m p o s i t i o n ( i n a r c s ^ i n / p e r c e n t a g e degrees) of the serum p r o t e i n f r a c t i o n s . +  FRACTION  10  9  8  7  6  5  4  w i l d , female, a d u l t , _0. h. columbianus, California October, 1962 v s . January, 1963  = ++  ++  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y g r e a t e r than the f i r s t i t e m t o the .01 l e v e l , second i t e m s i g n i f i c a n t l y s m a l l e r than the f i r s t i t e m t o the .01 l e v e l .  3  2  1  1  - 77 -  Captivity The b l o o d p r o t e i n f r a c t i o n s of a wide v a r i e t y of animals have been found t o be a f f e c t e d b o t h by d i e t et a l , 1957,  G o l d s t e i n & S c o t t , 1956,  Martinsons, 1958,  K i t t s e t a l , 1956,  Helgebostad  &  Weech et a l , 1935)  by d i s e a s e (Foreman, 19 60, Gleason & F r i e d b e r g , 1953, 195 6, Jencks et. a l , 1955, 1959,  Johnson  Sanders e t a l , 1944,  (Bandy  e t a l , 1958,  S c h i n a z i , 1957).  and  Ingram,  Pert et a l ,  C a p t i v i t y has  been shown to have an e f f e c t on b o t h s t r u c t u r e and growth (Darwin, 1859,  Spurway,  1952).  At the U n i v e r s i t y of B r i t i s h Columbia  (U.B.C.) deer  were brought from the w i l d and put under c a p t i v e c o n d i t i o n s (Wood e t a l , 19 61) at the age of two t o t h r e e weeks.  These  c a p t i v e c o n d i t i o n s are such t h a t the c a p t i v e deer used i n the p r e s e n t study are a l l i n good h e a l t h and are a l l on an i d e n t i c a l h i g h plane d i e t  (Wood e t a l , 19 61).  The food and  the s t a t e of h e a l t h are v e r y d i f f e r e n t from those found i n the w i l d s t a t e ( L i n s d a l e & Tomich, 1953).  Bandy e t a l (1956)  found i n c r e a s e d body s i z e i n U.B.C. c a p t i v e 0. h.  columbianus  on a h i g h plane d i e t as compared t o those on a low plane d i e t . Dasmann & Dasmann (1963) found a s i m i l a r r e l a t i o n s h i p between food q u a l i t y and deer s i z e i n w i l d p o p u l a t i o n s of mule deer (0. hemionus) i n C a l i f o r n i a .  - 78 -  Table 33 shows the number of e l e c t r o p h o r e t i c a l l y separated p r o t e i n f r a c t i o n s f o r c a p t i v e and w i l d 0. h . columbianus Wolf Lake, Vancouver I s l a n d , B.C. (samples c o l l e c t e d December, 19 61)  and from c a p t i v e  (samples c o l l e c t e d March, 19 62)  (samples c o l l e c t e d A p r i l ,  19 63)  0. h. s i t k e n s i s - A l a s k a .  c a p t i v e deer of both races d i f f e r o b v i o u s l y counterparts  The  from t h e i r w i l d  i n h a v i n g an a d d i t i o n a l n e g a t i v e l y  serum p r o t e i n f r a c t i o n .  and w i l d  migrating  As i t i s a l s o obvious t h a t the l i m i t e d  data w i l l not allow f u r t h e r numerical comparisons they are l i s t e d o n l y i n Appendices A & B. Disease a f f e c t s the e l e c t r o p h o r e t i c p a t t e r n  significantly  o n l y i n the acute stages and then o n l y i n the p e r c e n t compositi o n of the p r o t e i n f r a c t i o n s .  I t i s suspected t h a t the great  d i f f e r e n c e seen between c a p t i v e and w i l d animals (Table  33) i s  not due to d i s e a s e b u t i s due mainly t o the extreme d i f f e r e n c e s in diet. In summary, the U.B.C. c a p t i v e c o n d i t i o n s  are such t h a t  serum samples from c a p t i v e deer cannot be u t i l i z e d i n f u r t h e r comparisons which a l s o i n v o l v e serum samples from deer i n the wild state.  N u t r i t i o n a l d i f f e r e n c e s are suspected of causing  the great d i f f e r e n c e s between c a p t i v e and w i l d deer.  79  TABLE 33  EFFECT OF CAPTIVITY - t o t a l number of serum p r o t e i n f r a c t i o n s . * number of p o s i t i v e l y migrating fractions  wild  number of n e g a t i v e l y migrating fractions  female,adult O.h.columbianus, Wolf Lake, B.C.  N = 1  .9  1  male, a d u l t , 0_.h. columbianus, Wolf Lake, B.C.  N = 1  9  1  male, a d u l t , O.h. s i t k e n s i s , N = 1 Kupreanof I s . A l a s k a  9  1  captive female, adult O.h. columbianus, Wolf Lake, B.C.  N = 1  9  2  male, a d u l t , .O.h. columbianus, Wolf Lake, B.C.  N = 1  9  2  female, a d u l t , O.h. s i t k e n s i s , Petersburg,Alaska  N = 1  9  2  male, a d u l t , O.h. s i t k e n s i s , . Petersburg,Alaska  N = 1  9  2  N = number of a n i m a l s see Appendix A f o r the m o b i l i t i e s and Appendix B f o r the p e r c e n t c o m p o s i t i o n of the serum p r o t e i n f r a c t i o n s .  - 80 -  Geographic s e p a r a t i o n , s u b s p e c i e s , and s p e c i e s A f t e r removal of a l l the hemolysed  and c l o u d y samples,  samples from c a p t i v e deer, and samples from w i l d fawns and one year o l d deer, v e r y l i t t l e  comparable m a t e r i a l  remained.  Furthermore, as the m a j o r i t y of t h i s remaining m a t e r i a l c o n s i s t e d of one sample from one i n d i v i d u a l of each sex the numerical comparisons i n v o l v i n g the m o b i l i t y and the p e r c e n t composition c o u l d not be made.  These data are given o n l y i n  Appendices C & D. One p o s s i b l e e l e c t r o p h o r e t i c c r i t e r i o n f o r comparing these subspecies and s p e c i e s i s the number of p r o t e i n fractions  (Table 34).  There appear t o be 9 t o 10 f r a c t i o n s  t h a t w i l l migrate i n a p o s i t i v e d i r e c t i o n i n the genus Odocoileus.  A l l the O. hemionus group have but 1 n e g a t i v e l y  m i g r a t i n g f r a c t i o n w h i l e the 0. v i r g i n i a n u s group have up to 4 negatively migrating f r a c t i o n s .  0. h. columbianus from Wolf  Lake and from Chemainus, Vancouver  I s l a n d , B.C. have a t o t a l  number of 10 f r a c t i o n s .  T h i s number i s the same as t h a t  found f o r two other subspecies, 0. h. c r o o k i and 0. h. sitkensis.  However, 0. h. columbianus from C a l i f o r n i a  from the other subspecies and from Vancouver  Island  differs  specimens  p r e s e n t l y regarded as of the same subspecies i n h a v i n g a t o t a l number of 11 f r a c t i o n s .  Thus, the p o s s i b l e c r i t e r i o n of the  - 81 -  number of f r a c t i o n s f o r comparing subspecies or s p e c i e s of Odocoileus i s not v a l i d .  I t has a l r e a d y been shown t h a t  c a p t i v i t y can a l t e r the number of f r a c t i o n s . Three groups remain f o r q u a n t i t a t i v e comparison. These are 0. h. columbianus - Chemainus, Vancouver B.C.  Island,  (2 females - samples c o l l e c t e d December, 1961), 0. h.  columbianus - C a l i f o r n i a  (5 females - samples  collected  October, 19 62 - January, 19 63), and 0. v. texanus - Texas (8 females - samples c o l l e c t e d A p r i l - May,  1963).  Because  seasonal d i f f e r e n c e s were e x h i b i t e d i n the p e r c e n t compositi o n b u t not i n the m o b i l i t y of the p r o t e i n f r a c t i o n s , these t h r e e groups were compared o n l y on the b a s i s of t h e i r mobilities.  These n u m e r i c a l data are g i v e n i n Table 35.  These groups are c l e a r l y separated i n t o t h e i r  respective  taxonomic c a t e g o r i e s on the b a s i s of morphology Kellogg,  (Cowan, 1956,  1956).  As each group of animals has i t s p a r t i c u l a r number of p r o t e i n f r a c t i o n s , the f r a c t i o n s of each group were separated i n t o 14 c a t e g o r i e s on the b a s i s of t h e i r m o b i l i t i e s 36).  (Table  Those f r a c t i o n s of each group t h a t belonged to the  same c a t e g o r y of m o b i l i t y were then compared by means of Student's t - t e s t  (Table 37).  ed i n t o 4 c a t e g o r i e s : ences, l b .  The r e s u l t s were f u r t h e r  l a . the number of s i g n i f i c a n t  condensdiffer-  the number of times no comparison c o u l d be made  - 82 -  due  t o a f r a c t i o n o c c u r r i n g i n one group of animals b u t not  i n the other, 2a.  the number of s i m i l a r i t i e s ,  number of common absences.  and 2b.  the  These c a t e g o r i e s gave r i s e t o  the t o t a l number of d i f f e r e n c e s and s i m i l a r i t i e s between g e o g r a p h i c a l l y separated groups of the subspecies, columbianus, and between these and another  0. h.  species represent-  ed by 0. v. texanus (Table 38). I t was found  t h a t d i f f e r e n c e s i n m o b i l i t y were g r e a t -  er between females from C a l i f o r n i a and Vancouver I s l a n d genotypes of Odocoileus  hemionus than between e i t h e r of  these and the females of the s p e c i e s 0. v i r g i n i a n u s r e p r e s e n t ed by 0. v. texanus. In summary, there are 9 - 1 0  p o s i t i v e l y migrating  and 1 - 4 n e g a t i v e l y m i g r a t i n g f r a c t i o n s i n the genus Odocoileus.  Comparisons of m o b i l i t i e s i n three groups of  a d u l t females of the genus Odocoileus  i n d i c a t e greater  i n t r a - s p e c i f i c d i f f e r e n c e s than i n t e r - s p e c i f i c d i f f e r e n c e s .  83  TABLE 34  GEOGRAPHIC SEPARATION, SUBSPECIES, AND SPECIES - t o t a l number of serum protein fractions.* number of p o s i t i v e l y number o f n e g a t i v e l y migrating fractions migrating fractions  w i l d , female, a d u l t , O.hemionus columbianus, N = 1 Wolf Lake, B.C. w i l d , male, a d u l t , O.hemionus columbianus, N = 1 Wolf Lake, B.C. w i l d , female, a d u l t , O.hemionus columbianus, N = 2 Chemainus, B.C.  9  1  w i l d , male, a d u l t , O.hemionus columbianus, N = 2 Chemainus, B.C.  9  1  w i l d , female, a d u l t , O.hemionus columbianus, N = 5 California  10  1  w i l d , female, a d u l t , O.hemionus c r o o k i , Arizona  N = 1  9  1  w i l d , male, a d u l t , O.hemionus c r o o k i , Arizona  N = 1  9  1  w i l d , male, a d u l t , O.hemionus s i t k e n s i s , Kupreanof I s . , A l a s k a  N = 1  9  w i l d , male, a d u l t , 0.virginianus leucurus, N = 1 Oregon  10  w i l d , female, a d u l t , 0 . v i r g i n i a n u s texanus, Texas  10  N = 8  N = number of a n i m a l s * For a l l samples, f e m a l e , N = 1, male, N = 1, N = 2, see Appendix C f o r the m o b i l i t i e s and Appendix D f o r the p e r c e n t c o m p o s i t i o n of the serum p r o t e i n f r a c t i o n s . For a l l samples, female, N = 2, N = 5, N = 8, see Table 35 f o r the m o b i l i t i e s of the serum p r o t e i n f r a c t i o n s and T a b l e s 36 - 38 f o r the a n a l y s i s of these m o b i l i t i e s .  TABLE 35 EFFECT OF GEOGRAPHIC SEPARATION AND SPECIES - m o b i l i t i e s FRACTION  10  6  9  i n 10 5  -5  cm 4  2  / volt  seconds. + 1  w i l d , female, a d u l t , 0. v i r g i n i a n u s texanus, Texas N =  X  + S.E.  .829 . 4 6 4 .406 .371 .327 .254 .190 .158 .079 .015 .011 .143 .008 .016 .016 .013 .011 .013 .013 .003 .005 .002 .001 .015  w i l d , female, a d u l t , 0. hemionus columbianus, 'California N = 5  x .972 .537 .470 .434 .405 . 2 9 2 , .218 .151 .092 .011 .011 + S.E. .036 .017 .013 .013 .015 .016 .022 .024 .014 .001 .001  w i l d , female, a d u l t , 0_. hemionus columbianus, Chemainus, B.C. N = 2  X  + S.E.  N = number of a n i m a l s  .775 .006  .421  .006  .383 .340 .286 .221 .115 .080 .017 .013 .006 .001 .013 .007 .006 .006 .001 .001  TABLE 36 EFFECT OF GEOGRAPHIC SEPARATION AND SPECIES - d i s t r i b u t i o n of serum p r o t e i n f r a c t i o n s i n t o c a t e g o r i e s "5 2 a c c o r d i n g t o t h e i r m o b i l i t i e s ( i n 10 cm / v o l t s e c o n d s ) . MOBILITY  o o o  o o  O O  vo  L  O n  o  LT\ J  O O  O L  n  o  O u  n  O h  -  LT\ o  O '  O L  +  O  0  n  O 0  O 0  H  wild,female,adult, 0 . v i r g i n i a n u s texanus, Texas  1  0  1  0  1  1  1  1  1  1  1  1  1  1  wild,female,adult, 0.hemionus columbianus, California  1  1  1  1  1  0  0  1  1  1  1  1  1  0  wild,female,adult, 0.hemionus columbianus, Chemainus, B.C.  1  0  0  0  1  1  1  1  1  1  1  1  1  0  1 0  serum p r o t e i n f r a c t i o n p r e s e n t . serum p r o t e i n f r a c t i o n absent.  00  TABLE 37 EFFECT OF GEOGRAPHIC SEPARATION AND SPECIES - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum -5 2 p r o t e i n f r a c t i o n s w i t h i n m o b i l i t y c a t e g o r i e s ( i n 10  MOBILITY  o o o  O  o  •  ^  O L  o  O  seconds).  O i  L  L  O  n  ^  t  o -  ^  j t  ^  O  o J  O  L  -  O  n  o  0 O 0 O  O  LP,  O  O  LO,  i>-  o  LP,  CV)  rH  H  O  cm / v o l  +o-  o  o  o  o  o  O  rH  0J  wild,female,adult 0 . v i r g i n i a n u s texanus, Texas vs.  vs.  wild,female,adult, O.hemionus columbianus, California  0  wild,female,adult, O.hemionus columbianus, Chemainus, B.C.  0  0  0  0  0  0  wild,female,adult, O.hemionus columbianus, California vs.  = ++ 0 /  wild,female,adult, O.hemionus columbianus, Chemainus, B.C.  0  0  0  0  0  no s i g n i f i c a n t d i f f e r e n c e between the two samples. second i t e m s i g n i f i c a n t l y f a s t e r than the f i r s t i t e m t o the .01 l e v e l . second i t e m s i g n i f i c a n t l y slower than the f i r s t i t e m t o the .01 l e v e l . p r o t e i n f r a c t i o n p r e s e n t i n one sample but not i n the o t h e r . i n d i c a t e s the common absence of a p r o t e i n f r a c t i o n .  ++  TABLE 38 EFFECT OF GEOGRAPHIC SEPARATION AND SPECIES - summary of d i f f e r e n c e s and s i m i l a r i t i e s of the serum p r o t e i n f r a c t i o n s . * significant differences -)*  no comparison ( 0 •)*  s u b t o t a l of differences  similarities ( = )*  common absence ( / )*  i n the m o b i l i t i e s s u b t o t a l of similarities  tota  w i l d , female, a d u l t , 0 . . v i r g i n i a n u s texanus, Texas vs. w i l d , female, a d u l t , O.hemionus columbianus, California  0  +  5  =  5  9  +  0  vs. w i l d , female, a d u l t , O.hemionus columbianus, Chemainus, B. C.  2  +  2  =  4  8  +  2  2  +  5  =  7  6  +  1  =  =  9  14  10  14  7  14  w i l d , female, a d u l t , O.hemionus columbianus, California vs. w i l d , female, a d u l t , O.hemionus columbianus, Chemainus, B.C. *  see T a b l e 37  =  - 88 -  CONCLUSIONS  The  o r i g i n a l aim of t h i s study was to compare, by  means of s t a r c h - g e l e l e c t r o p h o r e s i s ,  the serum p r o t e i n s  of as many subspecies of the genus Odocoileus as p o s s i b l e i n an attempt t o c l a r i f y t h e i r phylogeny. The  study showed t h a t many independent v a r i a b l e s  a f f e c t the e l e c t r o p h o r e t i c p a t t e r n  of the serum.  I t also  showed t h a t f o r taxonomic purposes c l e a r y e l l o w samples from a t l e a s t two w i l d i n d i v i d u a l s of the same sex and age of each subspecies should be obtained a t i d e n t i c a l seasons. The  e f f e c t s of c a p t i v i t y and season i n d i c a t e t h a t the  n u t r i t i o n a l h i s t o r y of the deer a l s o has a major on the e l e c t r o p h o r e t i c  influence  pattern.  These s t r i c t u r e s render i t most d i f f i c u l t t o o b t a i n t r u l y comparable samples t h a t w i l l permit an examination of d i f f e r e n c e s t h a t may be considered  of g e n e t i c  r e l a t e d t o the e v o l u t i o n of d i f f e r e n t s p e c i e s There i s no p u b l i s h e d  o r i g i n and or subspecies.  evidence to suggest t h a t the same  causes of v a r i a t i o n of non g e n e t i c nature do not occur i n other mammals.  Under these circumstances one must approach  w i t h some s k e p t i c i s m apparent s p e c i e s  the s e v e r a l p u b l i s h e d  r e p o r t s of  r e l a t e d or subspecies r e l a t e d d i f f e r e n c e s  - 89  -  (Cowan & Johnston, 19 62) between mammals. In t h i s study i t was between 2 p o p u l a t i o n s currently bearing  p o s s i b l e to make v a l i d  comparisons  of b l a c k t a i l deer, 0. hemionus, b o t h  the same s u b s p e c i f i c d e s i g n a t i o n ,  and  a  w h i t e t a i l 0. v i r g i n i a n u s . The  two  s p e c i e s d i f f e r e d from each other  of i d e n t i f i a b l e f r a c t i o n s . .0. v i r g i n i a n u s had negatively migrating  fractions.-  i n the number 2 i n s t e a d of 1  Comparing the two  species  p o i n t of m o b i l i t y the d i f f e r e n c e s are of the same order magnitude as they are between the 2 p o p u l a t i o n s I t i s of g r e a t i n t e r e s t t h a t two  on  of  of 0. hemionus.  populations  of  0.  hemionus p r e s e n t l y known by the same s u b s p e c i f i c name are shown to be markedly d i f f e r e n t from each other  i n terms of  serum p r o t e i n m o b i l i t y c h a r a c t e r i s t i c s . T h i s supports observations  other  made at t h i s l a b o r a t o r y r e v e a l i n g t h a t these  two  genotypes d i f f e r i n many f e a t u r e s of body form, behaviour  and  physiology,  as  any  other  and  two  are p r o b a b l y as d i f f e r e n t from each other  populations  w i t h i n t h i s s p e c i e s c u r r e n t l y given  taxonomic r e c o g n i t i o n . T h i s study s t r o n g l y suggests t h a t e l e c t r o p h o r e t i c s t u d i e s of serum p r o t e i n s i n d i c a t e important d i f f e r e n c e s between even the minor taxa.  They are, t h e r e f o r e ,  p o t e n t i a l value  However, the l a r g e assortment  i n taxonomy.  of  - 90 -  of f a c t o r s t h a t may be r e s p o n s i b l e f o r a l t e r a t i o n s i n serum p r o t e i n values of the same order as those t h a t can be of taxonomic value make i t most d i f f i c u l t t o o b t a i n comparable samples.  Other body p r o t e i n s may be  v a r i a b l e and of more value to the s y s t e m a t i s t .  truly less  SUMMARY  1.  After standardization  of the s t a r c h - g e l  electrophoretic  technique, v a r i a t i o n i n the serum p r o t e i n s of the genus Odocoileus due  to the c o n d i t i o n of the sample and  c o n d i t i o n of the animal could be 2.  the  studied.  S i g n i f i c a n t changes i n the serum sample were brought  about by hemolysis, c l o u d i n e s s ,  and  decomposition.  storage of a d u l t deer serum f o r two b a c t e r i o s t a t to the relaxant  sample, and  years,  the use  Cold  the a d d i t i o n of a  of a muscle  to procure samples from c a p t i v e deer produced  no  s i g n i f i c a n t changes i n e i t h e r the m o b i l i t y or the p e r c e n t composition of the p r o t e i n f r a c t i o n s . 3.  A l a r g e i n d i v i d u a l v a r i a t i o n was  m o b i l i t y and 4.  The  found i n both  the  the p e r c e n t composition of the p r o t e i n f r a c t i o n s .  p e r c e n t composition of the p r o t e i n f r a c t i o n s  a f f e c t e d by  sex,  age,  p r o t e i n f r a c t i o n s was  and  season.  a f f e c t e d by  The  m o b i l i t y of  was  the  sex, but not by age  or  season. 5.  Deer under the c a p t i v e regimen at the U n i v e r s i t y of  B r i t i s h Columbia e x h i b i t e d an a d d i t i o n a l n e g a t i v e l y p r o t e i n f r a c t i o n when compared to t h e i r w i l d 6.  counterparts.  Comparisons of the m o b i l i t i e s i n three groups of  females of the genus Odocoileus i n d i c a t e  migrating  adult  intra-specific  - 92 -  d i f f e r e n c e s of the same order  as those between s p e c i e s .  The  s p e c i e s however, d i f f e r a d d i t i o n a l l y i n the number of p r o t e i n fractions. 7.  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Wolf  -5  female, a d u l t , columbianus, N = 2 Lake, B.C. male, a d u l t , columbianus, N = 2 Lake, B.C.  seconds.  8  7  6  + 5  4  3  2  1  1  2  _ X  .778  .501  .448  .382  .301  .258  .l8l  .144  .015  .010  _ X  .839  .541  .447  .400  .345  .282  .216  .114  .019  .009  captive,female,adult, _ O.h. columbianus, N = 11 X Wolf Lake, B.C.  .924  .517  .465  .439  .395  .297  .241  .164  .018  .015  .087  captive,male,adult, O.h. columbianus, Wolf Lake, B.C.  _ X  .855  .656  .575  .465  .432  .375  .313  .128  .048  .017  .085  w i l d , male, a d u l t , O.h. s i t k e n s i s , N = 3 Kupreanof I s . , A l a s k a  _ X  .884  .540  .487  .421  .381  . 3 3 5 - .279  .176  .032  .010  captive,female,adult, O.h. s i t k e n s i s , N = 2 Petersburg,Alaska  _ X  .892  .582  .466  .436  .359  .262  .165  .068  .039  .010  .142  captive,male,adult, O.h. s i t k e n s i s ,  _ X  .961  .538  .452  .423  .336  .288  .163  .059  .012  .009  .163  N = 4  N = 2  N = number of a l i q u o t s from one sample from one a n i m a l  H  O Ul  APPENDIX B EFFECT OF CAPTIVITY - p e r c e n t c o m p o s i t i o n i n a r c s ^ i n / p e r c e n t a g e d e g r e e s .  + 9  FRACTION wild, O.h. Wolf wild, O.h. Wolf  female, a d u l t , columbianus, N = 2 Lake, B.C. male, a d u l t columbianus, N = 2 Lake, B.C.  8  7  6  5  4  3  2  1  _ X  3 9 . l 26.O  10.3  6.2  8.4  3.7  17.0  22.4  11.6  _ X  32.6  20.4  12.3  10.4  12.6  10.9  ' 18.4  20.6  15.8  captive,female,adult, O.h. columbianus, N = 11 Wolf Lake, B.C.  _ X  32.0  12.6  14.3  9.4  ' 7.4  10.6  11.1  19.8  captive,male,adult, O.h. columbianus, Wolf Lake, B.C.  _ X  38.6  9.1  12.0  18.2  13.7 '  6.7  8.4  w i l d , male, a d u l t , N = 3 .O.h. s i t k e n s i s , Kupreanof I s . , A l a s k a  X  38.6  10.4  19.8  12.9  8.1  5.1  captive,female,adult, O.h. s i t k e n s i s , N = 2 Petersburg, Alaska  _ X  30.0  15.4  14.5  16.7  10.3  _ _X.  36.7  10.1  10.1  10.7  7.7  captive,male,adult, O.h. s i t k e n s i s , Petersburg, Alaska  N = 4  N = 2  •  1  2  15.5  0.0  20.3  0.0  20.8  18.8  22.0  15.5  22.9  8.5  19.0  12.4  13.3  20.9  22.8  0.0  5.7  15.9  14.4  22.4  20.2  13.4  11.2  12.4  13.5  26.2  21.4  14.6  •  N = number of a l i q u o t s f r o m one sample from one a n i m a l H O  APPENDIX C GEOGRAPHIC SEPARATION, SUBSPECIES, AND SPECIES - m o b i l i t FRACTION  10  wild,female,adult, 0.h.c olumb i a n u s , Wolf Lake, B.C._ N = 2 X w i l d , male, a d u l t , O.h.columbianus, Wolf Lake, B.C._ N = 2 X wild,male 1,adult, 0.h.c olumb i a n u s , Chemainus, B.C._ N = 3 X wild,male 2 , a d u l t , O.h.columbianus, Chemainus, B.C._ N = 2 X wild,female,adult, _0.h. c r o o k i , Arizona _ N = 3 x wild,male,adult, O.h. c r o o k i , Arizona _ N = 2 X wild,male,adult, _0.h. s i t k e n s i s , Kupreanof I s . , A l a s k a N = 3 X wild,male,adult, O.v. l e u c u r u s , Oregon N = 4 X .829  9  8  7  6  -5 s i n 10  5  4  2 cm  / v o l t seconds. +  —  1  3  2  1  .778  .501  .448  .382  .301  258  .181  .144  .015  .010  .839  .541  .447  .400  .345  282  .216  .114  .019  .009  .843  .503  .414  .378  .324  272  .170  .144  .009  .009  .815  .485  .432  .388  .301  247  .160  .121  .034  .015  .870  .504  .436  .4o4  .381  252  .171  .145  .035  .010  .878  .499  .427  .383  .359  282  .165  .073  .019  .010  .884  .540  .487  .421  .381  335  .279  .176  .032  .010  .606  .466  .403  .379  .339  262  .204  .146  .010  N = number of a l i q u o t s f r o m one sample f r o m one a n i m a l  .010  2  .063  3  .131  APPENDIX D GEOGRAPHIC SEPARATION, SUBSPECIES, AND  SPECIES - p e r c e n t c o m p o s i t i o n I n a r c s ^ i n / p e r c e n t a g e d e g r e e s . +  FRACTION  10  wild,female,adult, O.h.c olumb i a n u s , Wolf Lake, B.C._ N = 2 X wild,male,adult, O.h.columbianus, Wolf Lake, B.C._ N = 2 X wild,male 1 , a d u l t , O.h.columbianus, Chemainus, B.C._ N = 3 X w i l d , m a l e 2, a d u l t , O.h. columbianus, Chemainus, B.C._ N = 2 X wild,female,adult, O.h. c r o o k i , Arizona _ N = 3 X wild,male,adult, O.h. crooki, Arizona _ N = 2 X wild,male,adult, O.h.sitkensis, Kupreanof I s . , A l a s k a N = 3 X wild,male,adult, 0.y_. l e u c u r u s , Oregon N = 4 X 31.8  9  8  7  6  39.1  26.0  10.3  6.2  8.4  32.6  20.4  12.3  10.4  12.6  5  4  3  2  1  1  3.7  17.0  22.4  11.6  15.5  10.9  18.4  20.6  15.8  20.3  2  3  4  > 44.7  21.5  14.7  8.8  7.7  5.4  10.5  9.5  14.8  20.6  44.4  16.8  13.6  6.0  6.1  9.0  10.3  13.1  21.2  19.2  37.1  17.2  9.2  9.1  9.6  8.2  8.7  15.0  23.6  26.2  34.3  19.3  H.8  10.7  10.0  6.7  12.9  12.6  21.2  38.6  10.4  19.8  12.9  8.1  5.1  12.4  13.3  20.9  17.3  18.4  18.4  13.2  8.4  13.2  22.4  13.3  4.9  N = number of a l i q u o t s f r o m one sample f r o m one a n i m a l  i  28.3  | !  22.8  6.6  5.4  12.6  9.1 H  O CO.  

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