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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., University of B r i t i s h Columbia, 1961 A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements for the degree of MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia October, 1964 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of • B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y , a v a i l a b l e f o r reference and study. I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t , c o p y i n g or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission* Department of ZOOLOGY The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 ? Canada Date O c t o b e r , 19 64. - i -Chairman: Professor I. McTaggart Cowan ABSTRACT After standardizing a starch-gel electrophoretic technique, variations i n the serum proteins of the genus Odocoileus due to the condition of the sample and the condition 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 by hemolysis, cloudiness, and decomposition. Cold storage for two years of adult deer serum, the addition of a bacteriostat to the sample, and the use of a muscle relaxant to procure samples from captive deer produced no s i g n i f i c a n t changes i n either the mobility or the percent composition of the protein f r a c t i o n s . A large 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 mobility and the percent composition of the protein f r a c t i o n s . The percent composition of the protein f r a c t i o n s was affected by sex, age, and season. The mobility of the protein fractions was affected by sex, but not by age or season. Captive deer at the University of B r i t i s h Columbia exhibited an additional negatively migrating protein f r a c t i o n when compared to t h e i r wild counterparts. Comparisons of the m o b i l i t i e s i n three groups of adult females of the genus Odocoileus indicate greater i n t r a - s u b s p e c i f i c differences than i n t e r - s p e c i f i c differences. The technique of starch-gel electrophoresis, therefore, may be useful i n i n d i v i d u a l and herd recognition but i t i s not useful i n the recognition of subspecies or species of deer of the genus Odocoileus. ACKNOWLEDGMENTS I wish to express my appreciation to Dr. I. McTaggart Cowan, Dean of Graduate Studies, University of B r i t i s h Columbia, who p a t i e n t l y directed the study, to Dr. W. S. Hoar Professor i n the Department of Zoology, University of B r i t i s h Columbia, who tolerated my equipment and presence i n his laboratory, to Dr. H. Tsyuki, Fisheries Technological Station Vancouver, B. C , who r e a d i l y imparted his knowledge of electrophoresis, and to Mr., G. Mclntyre of Mathematical S t a t i s t i c s , C.S.I.R.O., Canberra, A.C.T., Au 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 also due to the following who provided the blood samples: Mr. G. E. Connolly, Hopland F i e l d Station, University 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, United States Department of the Interior, F i s h and W i l d l i f e Service, Bureau of Sport Fisheries and W i l d l i f e , Forest Research Center, Olympia, Washington, U.S.A. Mr. S. G a l l i z i o l i , Arizona Game and F i s h Department, Phoenix, Arizona, U.S.A. Mr. W. C. Glazener, Rob and Bessie Welder W i l d l i f e Foundation W i l d l i f e Research and Education, Sinton, Texas, U.S.A. - i v -Dr. W. M. Longhurst, Hopland F i e l d Station, University 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. W..A. Low, Department of Zoology, University of B r i t i s h Columbia, Vancouver, B r i t i s h Columbia, Canada. Mr. I. D. Luman, Oregon State Game Commission, Portland, Oregon, U.S.A. Mr. D. McCaughran, D i v i s i o n of Fi s h and Game, Province 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 Institute, Bucksburn, Aberdeenshire, Scotland. Mr. H. Merriam, State of Alaska Department of Fi s h and Game, Petersburg, Alaska, U.S.A. A special thank you must be given to the many friends and associates who f r e e l y gave of th e i r advice, information, and time. - V -TABLE OP CONTENTS page Introduction 1 Materials and Methods 6 Source and c o l l e c t i o n date of samples 6 Preparation of the sample 8 Electrophoretic procedure 11 Analysis of re s u l t s 20 Results and Discussion 25 Condition of sample 25 Hemolysis and cloudiness 25 Preservation 32 Succinylcholine chloride 43 Condition of animal 48 Individual v a r i a t i o n 48 Sex 54 Age 62 Season 70 Capti v i t y 77 Geographic separation, subspecies, and species 80 Conclusions 88 Summary 91 Literature Cited 93 - v i -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 i n _5 2 . 10 cm / v o l t seconds 105 Appendix B E f f e c t of c a p t i v i t y - percent composition i n arcsinpercentage degrees 106 Appendix C Geographic separation, subspecies, and -5 2 / species - m o b i l i t i e s i n 10 cm / v o l t seconds 107 Appendix D Geographic separation, subspecies, and species - percent composition i n arcsin/percent-age degrees 108 - v i i -LIST OF TABLES Table Page -5 2 . 1. E f f e c t of hemolysis - m o b i l i t i e s i n 10 cm / v o l t seconds 28 2. E f f e c t of hemolysis - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum protein frac t i o n s 29 3. E f f e c t of hemolysis - percent composition i n arcsinVpercentage degrees 30 4. E f f e c t of hemolysis - Student's t - t e s t comparisons of the percent composition (in arcsin/percentage degrees) of the serum protein fractions .31 -5 2 5. E f f e c t of cold storage - m o b i l i t i e s i n 10 cm / v o l t seconds 35 6. E f f e c t of cold storage - 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 36 7. E f f e c t of cold storage - percent composition i n arcsin/percentage degrees 37 8. E f f e c t of cold storage - Student's t - t e s t comparisons of the percent composition (in arcsinvfeercentage degrees) of the serum protein frac t i o n s 38 - v i i i -E f f e c t of a blood preservative - m o b i l i t i e s i n -5 2 1 0 cm / v o l t seconds E f f e c t of a blood preservative - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum protein f r a c t i o n s E f f e c t of a blood preservative - percent composition i n arcsin/percentage degrees E f f e c t of a blood preservative - Student's t - t e s t comparisons of the percent composition (in arcsinvpercentage degrees) of the serum protein f r a c t i o n s E f f e c t of succinylcholine chloride - 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 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 f r a c t i o n s E f f e c t of succinylcholine chloride - percent composition i n arcsinvpercentage degrees E f f e c t of succinylcholine chloride - Student's t - t e s t comparisons of the percent composition (in arcsin ^ percentage degrees) of the serum protein frac t i o n s - i x -Table Page -5 2, 17. Indxvidual varxatxon - mobxlxtxes xn 10 cm / v o l t seconds 50 18. Individual v a r i a t i o n - Student's t - t e s t comparisons of the m o b i l i t i e s of the serum protein f r a c t i o n s 51 19. Individual v a r i a t i o n - percent composition i n arcsin ^ percentage degrees 52 20. Individual v a r i a t i o n - Student's t - t e s t comparisons of the percent composition (in arcsin/percentage degrees) of the serum protein f r a c t i o n s 53 -5 2 21. E f f e c t of sex - m o b i l i t i e s i n 10 cm / v o l t seconds 58 22. E f f e c t 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 protein f r a c t i o n s • 59 23. E f f e c t of sex - percent composition i n arcsin /percentage degrees 60 24. E f f e c t of sex - Student's t - t e s t comparisons of the percent composition (in arcsin ^percentage degrees) of the serum protein f r a c t i o n s 61 - X -Table Page -5 2 25. E f f e c t of age - m o b i l i t i e s i n 10 cm / v o l t seconds 66 2 6. E f f e c t 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 protein fractions 67 27. E f f e c t of age - percent composition i n arcsin ^percentage degrees 68 28. E f f e c t of age - Student's t - t e s t comparisons of the percent composition (in arcsin ^percentage degrees) of the serum protein fractions 69 -5 2 29. E f f e c t of season - mo b i l i t i e s i n 10 cm / v o l t seconds 73 30. E f f e c t 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 protein fractions 74 31. E f f e c t of season - percent composition i n arcsin v/percentage degrees 75 32. E f f e c t of season - Student's t - t e s t comparisons of the percent composition (in arcsin percentage degrees) of the serum protein fractions 7 6 33. 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 fract i o n s 79 - x i -Table Page 34. Geographic separation, subspecies, and species -t o t a l number of serum protein fractions 83 35. E f f e c t of geographic separation and species --5 2 . mo b i l i t i e s i n 10 cm / v o l t seconds 84 36. E f f e c t of geographic separation and species -d i s t r i b u t i o n of serum protein fractions into categories according to t h e i r m o b i l i t i e s (in -5 2 . . 10 cm / v o l t seconds) 85 37. E f f e c t 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 protein fract i o n s within mobility -5 2 categories (in 10 cm / v o l t seconds) 86 38. E f f e c t of geographic separation and species -summary of differences and s i m i l a r i t i e s i n the mo b i l i t i e s of the serum protein fractions 87 INTRODUCTION Morphological c h a r a c t e r i s t i c s have been used since the days of A r i s t o t l e to c l a s s i f y organisms and since Darwin's "Origin of Species" to postulate evolutionary r e l a t i o n s h i p s . More recently, other factors such as the behavioural, physiological, and biochemical attributes of organisms are also being considered. The present study t r i e s to evaluate whether serum proteins can help to elucidate the phylogeny of the genus Odocoileus. An excellent review of the underlying philosophy of the application of protein studies to phylogeny has been presented by Sibley (1960). Investigations of the proteins of amphibians and r e p t i l e s (McGabe & Deutsch, 1952), birds (Sibley, 19 60), and small mammals (Johnson et a l , 1958, Johnson & Wicks, 1959) to a large extent substantiate c l a s s i f i c a t i o n s based on morphology. A var i e t y of techniques for the study of the blood proteins of animals have been.elaborated and t h e i r applications to taxonomy are outlined below. Serology Kraus i n 1897 discovered the p r e c i p i t i n reaction - 2 -and understood i t to be absolutely s p e c i f i c (Boyden, 1943). This reaction was f i r s t extensively applied to the problems of animal systematics by N u t t a l l (1901) and has since been widely employed by several workers. Decapod Crustacea have been studied i n th i s respect by Leone (1954). Stallcup's (1954) study of the family F r i n g i l l i d a e i s the f i r s t application of serology to actual problems i n avian taxonomy. Mammals involved i n s e r o l o g i c a l investigations include the Orders Rodentia and Lagomorpha (Moody et a_l, 1949, Moody & Doniger, 1956), Carnivora (Leone & Wiens, 1956, Pauly & Wolfe, 1957), and Cetacea and Artiodactyla (Boyden & Gemeroy, 1950). Serological studies distinguishing the cervid genera Odocoileus, Axis, and Cervus, were carried out by Wolfe (1939) and Baier & Wolfe (1942). Wolfe (1939) reports that N u t t a l l (1904) also noted i n t e r s p e c i f i c differences i n deer. Paper Chromatography Aside from the use of paper chromatography as an a n a l y t i c a l t o o l , i t s application to taxonomy was not suggested u n t i l r e l a t i v e l y recent years. Micks (1956) and Micks & Gibson (1957) showed that the basic amino acid - 3 -patterns of various insects and t i c k s were species s p e c i f i c and d i f f e r e d q u a n t i t a t i v e l y . Paper chromatography was used by Buzzati-Traverso & Rechnitzer (1953) to detect s p e c i f i c differences i n several morphologically i d e n t i c a l species of f i s h , and by Mainardi (1958) to c l a r i f y the phylogeny of some Gallinaceous b i r d s . Electrophoresis T i s e l i u s (19 37) designed the basic electrophoretic apparatus and c a l l e d his method "free" electrophoresis. Using horse serum, he compared the protein fractions obtained by s a l t p r e c i p i t a t i o n with those obtained by electrophoresis and was the f i r s t " t o apply the terms "albumin, alpha-, beta-, and gamma-globulin" respectively to fractio n s of decreasing electrophoretic mobility. He was also the f i r s t to notice quantitative differences i n the protein f r a c t i o n s of human, horse, and rabbit. The T i s e l i u s apparatus has been used to investigate a wide var i e t y of animals: invertebrates, f i s h , amphibians, and r e p t i l e s (Deutsch & McShan, 1949); birds (Bain & Deutsch, 1947, Landsteiner et. a l , 1938); and mammals (Deutsch & Goodloe, 1945, Moore, 1945). Modifications of the T i s e l i u s apparatus have mainly involved the addition of a supporting medium. In the late - 4 -1930*s, f i l t e r paper provided one of the f i r s t supporting media (Wunderly, 1959) and one of i t s advantages over free electrophoresis i s that small samples can be analysed. Paper electrophoresis has been used to investigate the blood proteins of the following taxa: insecta (Stephen, 19 61), various small vertebrates (Gleason & Friedberg, 1953), amphibia (Dessauer & Fox, 1956), r e p t i l i a (Dessauer & Fox, 1956, Zweig & Crenshaw, 1957), and mammalia (Auernheimer et  a l , 19 60, Bangham, 1957, Johnson & Wicks, 1959, Welling & van Bekkum, 1958). The lens proteins of f i s h (Smith, 1962) and the egg white proteins of birds (Forsythe & Foster, 1950, McCabe & Deutsch, 1952) have also been studied by t h i s method. Starch has la r g e l y replaced f i l t e r paper as a supporting medium because of i t s greater resolution. Starch-gel e l e c t r o -phoresis has been used to investigate the egg white proteins of birds (Sibley & Johnsgard, 1959b, Sibley, 1960); whole serum of invertebrates (Woods et a l , 1958), a l l i g a t o r s ( B a r i l et a l , 1961), and domestic mammals (Latner & Zaki, 1957); and frac t i o n s of whole blood (haemoglobins, haptoglobins, and transf e r r i n s ) of amphibians (Dessauer et a l , 19 62, Fox et a l , 19 61), r e p t i l e s (Dessauer et a l , 19 62), domestic mammals (Ashton, 1957a, 1957b, 1958, 1958a-e, 1959, 1960, Ashton & Fallon, 1962, Ashton & McDougall, 1958, Hickman & Smithies, - 5 -1957), mice (Mainardi, 1958, Rosa et a l , 1958, Thompson et a l , 1954), and humans ( A l l i s o n et a l , 1958, Harris et a l , 1958a, 1958b, Parker & Bearn, 19 61, Smithies, 1955a, 1955b, 1957, 1958, 1959b, Smithies & H i l l e r , 1959, Smithies & Walker, 1955b, 1956, Sutton et a l , 1956, 1959, 1960). Lowe & McDougall (19 61) used starch-gel electrophor-esis to study the serum beta-globulin types i n Red Deer (Cervus elaphus). Other supporting media include agar-gel (Graber & Williams, 1955, Ressler & Jackson, 1955), c e l l u l o s e acetate (Kohn, 1957), and polyacrylamide gel (Raymond & Weintraub, 1959). Besides t h i s development and u t i l i z a t i o n of new media, combinations of t r i e d techniques have also been shown to y i e l d useful information. Immunoelectrophoresis which i s a combination of serology and starch-gel e l e c t r o -phoresis (Williams & Graber, 1956) has proven to be a valuable t o o l for the study and characterization of various antigen-antibody systems (Kunkel & Trautman, 1959). In the present investigation the separation of serum proteins was obtained by u t i l i z i n g the technique of starch-gel electrophoresis. MATERIALS AND METHODS Source and c o l l e c t i o n date of samples Odocoileus hemionus columbianus (Richardson) 1829 - captive i n Vancouver, B r i t i s h Columbia, Canada. O r i g i n a l l y from Wolf Lake, Vancouver Island, B r i t i s h Columbia, Canada. Collected June, 19 61 - October, 19 62. - wi l d - Wolf Lake, Vancouver Island, B r i t i s h Columbia, Canada. Collected December, 19 61. - wild - Chemainus, Vancouver Island, B r i t i s h Columbia, Canada. Collected December, 19 61. - wild - Mendocino County, C a l i f o r n i a , U.S.A. Collected Octob er, 19 62, November, 19 62, and January, 19 63. Odocoileus hemionus crooki (Mearns) 1897 - wild - Southern Arizona, U.S.A. Collected December, 1962. - 7 -Odocoileus hemionus sit k e n s i s Merriam, 1898 - captive i n Vancouver, B r i t i s h Columbia, Canada, O r i g i n a l l y from Petersburg, Alaska, U.S.A. Collected February, 19 61 - March, 19 62. - wild - Mitkof Island and Kupreanof Island, Alaska, U.S.A. Collected A p r i l , 1963. Odocoileus virginianus leucurus (Douglas) 1829 - wild - Clatskanie County, Oregon, U.S.A. Collected December, 19 62. Odocoileus virginianus texanus (Mearns) 1898 - wild - Sinton, Texas, U.S.A. Collected A p r i l - May, 19 63. - 8 -Preparation of the sample Captive stock at the U.B.C. deer unit was immobilized with succinylcholine chloride (Anectine - Burroughs Wellcome -Canada) as described by Cowan e_t al_ (19 62). Blood was taken i n vacutainer tubes from the recurrent t a r s a l vein and was o allowed to c l o t for 24 hours at 5 C. After centrifugatxon for 10 minutes at 3,500 r.p.m., the serum was removed and stored at - 18°C u n t i l used for analysis. Workers at the University of C a l i f o r n i a and Associated Hopland F i e l d Station, Mendocino County, C a l i f o r n i a , are carrying out a large experimental programme involving tagging, taking body measurements, and c o l l e c t i n g blood samples from 0. h. columbianus (Leopold et a l , 1951, Leopold, personal communication, 1964). Sheppard's Keidel Vacuum Tubes were used to draw the blood samples, the sera of which were frozen after removal of the c l o t and were then sent airmail to U.B.C. The sample of 0. v. leucurus was obtained from the jugular vein within 5 minutes of shooting. The serum separat-ed from the c l o t after 12 hours at room temperature. I t was then pipetted into a clean container containing preservative and 24 hours l a t e r i t was stored at - 18°C at U.B.C. For those samples obtained elsewhere the deer were either shot or restrained p h y s i c a l l y . For these samples an i n s t r u c t i o n sheet was prepared which appears on the following - 9 -page. The equipment sent with these instructions included l a b e l l e d glass t e s t tubes, glass pipettes, corks wrapped i n aluminum f o i l , l a b e l l e d cardboard mailing containers, and vermiculite 'for packing. A l l samples were stored at - 18°C upon a r r i v a l at U.B.C. For those samples which could not be frozen during shipment to U.B.C, the bacteriostat, propyl p-hydroxy-benzoate, was put i n the c o l l e c t i n g v i a l s to prevent decomposition. - 10 -DIRECTIONS FOR OBTAINING BLOOD SAMPLES FROM DEER A. If deer k i l l e d : 1. s l i t jugular vein or carotid artery or use any available blood vessels immediately. 2. allow blood to flow into a clean container. 3. l e t s i t at room temperature for 12 - 15 hours. a. i f separation has occurred, that i s i f yellowish-coloured f l u i d i s seen d i s t i n c t from a red semi-s o l i d mass (the c l o t ) , then pour t h i s f l u i d only into another clean container and discard the c l o t . b. i f no separation has occurred after 12 - 15 hours, that i s i f no f l u i d i s seen to be d i s t i n c t from the c l o t , then centrifuge at 3,000 r.p.m. (or highest speed on a hand centrifuge) for about 10 minutes. Then pour the f l u i d into another clean container and discard the c l o t . 4. complete the l a b e l on the container. 5. freeze or r e f r i g e r a t e u n t i l mailing. 6. use cardboard mailing tube provided. 7. tape corks into place. 8. a i r mail. B. If deer i s not to be k i l l e d : 1. i f possible, immobilize the deer with succinylcholine - 11 -chloride before bleeding. 2. bleed from recurrent t a r s a l vein (most prominent vein i n the leg) or from the jugular vein or from an ear vein. 3. use vacutainer needles and tubes i f available. More information or materials regarding the above may be obtained i f required. 4. follow directions of A. no. 3 - no. 8. Powder i n tubes i s a preservative. Electrophoretic procedure Smithies gives the o r i g i n a l description of zone electrophoresis i n starch-gel i n 1955b, an improved procedure i n 1959a, and a detailed diagram of the apparatus i n 1959b. The procedure used i n thi s study was only s l i g h t l y modified from h i s by placing the gel i n a horizontal rather than a v e r t i c a l p o s i t i o n during the run and by substituting the suggested buffer trays and electrodes by those designed by the E-C Apparatus Company (538 Walnut Lane, Swarthmore, Pennsylvania, U.S.A.). Wooden presses were designed with which constant pressure could be applied to the setting gels inymolds supplied by Otto H i l l e r (P.O. Box 1294, Madison 1, Wisconsin, U.S.A.). - 12 -B r i e f l y the procedure was as follows: A l l mold pieces except the s l o t sections were very l i g h t l y greased with mineral o i l . One l i t r e of starch solution, the concentration depending on l o t s p e c i f i c a t i o n s (Connaught Medical Research Laboratories, University of Toronto, Toronto, Ontario), was continuously heated (to 95°C, Smithies, 1959b) and s t i r r e d for a constant 20 minutes (the peak of v i s c o s i t y occurring at 10 minutes), evacuated for about 5 minutes to remove a l l a i r bubbles, and poured into the trays (500 ml. starch solution / t r a y ) . The cover was c a r e f u l l y lowered into p o s i t i o n and even pressure was applied. The se t t i n g gels were then kept at room temperature for about 1 hour and at 10°C u n t i l used -usually about 8 hours l a t e r . At the beginning of a run the press and cover were removed and 50 _ul of serum were injected into each s l o t with a Hamilton microsyringe. The s l o t s were sealed with mineral 011 and "Saran Wrap" was placed over the gel surface so as to eliminate a i r bubbles. Number 17 f i l t e r papers (6" square) which had just previously been wetted with buffer were placed at each end of the tray which was then placed between the two cooling plates of the E-C Apparatus 1 so as to bring the f i l t e r paper into contact with i t s buffer trays. The cooling system was activated and the current applied. - 13 -At the end of the run, the current and the cooling system were disconnected- The f i l t e r paper wicks, the "Saran Wrap", and the trough-ends of the gel were discarded. The remaining .rectangle of starch-gel was placed upside down into a s l i c i n g tray. A f l a t weight the same size as the starch-gel was placed on top and by s l i c i n g i n a horizontal d i r e c t i o n with a microtome blade two equal halves of the starch-gel were obtained. Staining followed immediately. The lower h a l f (inner surface uppermost) was stained for protein with Amido Black (500 mg.. Amido Black / 90 ml. Methanol / 10 ml. Water) for 5 minutes and was then rinsed with 8 l i t r e s of 50 ml. Methanol / 50 ml. Water / 10 ml. G l a c i a l Acetic Acid i n a gel washer (Pert et a l , 1959) for 13 hours. The inner surface of the upper half was stained for l i p i d . 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 stained starch-gel was then wrapped i n "Saran Wrap" o and stored at 5 C. In order to standardize the technique, yellow serum from captive, male, adult, 0. h. columbianus was run under various conditions as elaborated below. The f i n a l method adopted was based on the number of protein bands v i s i b l e to the naked eye and the distance the furthest band had migrated. - 14 -Buffer Since proteins are amphoteric, the pH of the buffer solution w i l l exert a great influence on the i r mobility. For maximum migration to occur a pH should be used which i s great-er than the i s o e l e c t r i c points of the majority of the proteins i n the solution to be examined. As suggested by Smithies (1959b), a borate buffer of pH 8.8 was used. The v e l o c i t y of migration also depends on the i o n i c strength of the solution, the v e l o c i t y decreasing as the i o n i c strength increases for any given f i e l d strength. This decrease i s due to the e f f e c t i v e charge of the migrating molecule being reduced by ions of opposite charge surrounding i t . Using constant time (12% hours) and voltage (800 volts) various concentrations of borate buffer of pH 8.8 were used to tes t the e f f e c t of i o n i c strength ranging from 0.0610 - 0.0745. Among these there was l i t t l e difference noted i n the migration distance of the furthest f r a c t i o n . However, the buffer concentration, 0.0092 M sodium hydroxide / 0.0230 M bo r i c acid, of i o n i c strength 0.0667 and pH 8.8 produced the greatest separation of the protein fract i o n s and was therefore selected as the constant buffer concentrat-ion. - 15 -Attempts made using T r i s (hydroxymethyl) aminomethane standard buffer as suggested by Poulik (1957) were not successful. The electrodes were reversed after each run. I t was found that the buffer could be used 4 times before the pH began to a l t e r . Consequently, no more than 4 runs were made before the apparatus was cleaned and new buffer supplied. A humidity of 45 - 50% was found to prevent condensat-ion on the apparatus and evaporation of the buffer during the run. Time Because each protein f r a c t i o n i n the solution w i l l d i f f e r i n the net number of negative and p o s i t i v e charges per unit area of molecular surface, i t w i l l have a s p e c i f i c mobility and w i l l separate from the others. Enough time must be allowed for the fractio n s to achieve th e i r maximum separation. However, i f allowed to run i n d e f i n i t e l y , the fas t e s t moving fractions at least, would migrate into the trough region of the gel tray making the i r analysis impossible. With the standard borate buffer of i o n i c strength 0.0667 and pH 8.8, and 900 vo l t s , times from 8 - 18 hours were tested. At 13 hours and longer, a yellow l i n e - 16 -appeared on the f i l t e r paper wick at the negative electrode suggesting some change occurring i n the buffer solution. This l i n e did not appear at 12% hours or l e s s . Because a greater number of fractio n s separated with progressively increasing lengths of time, 12% hours running time was made standard. Voltage One advantage i n using an extremely high voltage i s that maximum separation of the protein fra c t i o n s w i l l be obtained more quickly than at a lower voltage. In addition to saving time, the application of higher f i e l d i n t e n s i t i e s (20 - 100 vol t s / cm.) has the advantage of producing sharper separations of the protein fractions since 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 supporting medium (starch-gel) i s of less significance (Wieland, 1959). A disadvantage, however, l i e s i n the fac t that an increase i n voltage means an increase i n the amount of heat generated during the run. Even though t h i s i s p a r t i a l l y negated by the low io n i c strength of the buffer (Smithies, 1955b), the maintenance of an e f f i c i e n t cooling system becomes necessary. Using the standard borate buffer of i o n i c strength 0.0667 and pH 8.8, and a constant time of 12% hours, v o l t -ages from 130 - 900 vol t s were tested. 900 v o l t s produced - 17 -the greatest separation and gave a voltage gradient of approximately 28 volts / cm. At 900 v o l t s the i n i t i a l amperage was 20 ma. After 10 minutes t h i s decreased and remained constant at 16 ma. Temperature As proteins are thermolabile they w i l l be denatured at high temperatures and migration w i l l cease. However, even before t h i s c r i t i c a l point of denaturation i s reached, heat may d i s t o r t the pattern of electromigration by causing d i f f u s i o n within the g e l . At the high voltage used (900 volts) i t was necessary, therefore, to keep the apparatus in a constant temperature room at 10°C. The cooling plates o on either side of the gel were constant at 3 C. Serum aliquot The volume of each s l o t i n the starch-gel i s about 55 u l . Samples of 30, 35, 40, 45, and 50 u l were tested and i t was found that with increasing serum volume the protein pattern became more d i s t i n c t . Therefore, a 50 u l aliquot was taken as the standard volume. The number of aliquots required per serum sample Each gel contained eight slo t s into which the serum - 18 -samples were injected. When a l l the s l o t s of one gel were f i l l e d with aliquots from one sample, s l o t s 2 - 7 i n c l u s i v e produced patterns whose protein fractions were extremely similar i n mobility and percent composition. Runs i n slot s 1 and 8 were obviously inconsistent and were discarded. This s i m i l a r i t y of runs from s l o t s 2 - 7 j u s t i f i e s the use of the minimum of two aliquots (100 jil) from each sample required for s t a t i s t i c a l analysis. If only two aliquots are available, however, i t i s preferable to put them into separate gels to allow for s l i g h t gel variations (Mclntyre, personal.communication). D i a l y s i s of sample D i a l y s i s serves to 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 (that might i n t e r f e r e with migration) other than buffer e l e c t r o l y t e s (Longsworth, 1959). 50 i l l aliquots were taken from samples dialysed at 10°C from 0 - 8 6 hours. A few samples were l e f t for longer periods with aliquots taken at various i n t e r v a l s . The buffer was changed every day on each sample. With no d i a l y s i s few protein bands separated and the run was shorter than with samples dialysed 23 - 53 hours. There were no v i s i b l e differences between samples dialysed - 19 -20 - 69 hours. At 86 hours there was some loss i n c l a r i t y and with successive days of d i a l y s i s the runs became progressively poorer. Therefore, d i a l y s i s could be no less than 20 hours and no more than 69. For convenience the samples used for the previously mentioned tests and for those discussed below were dialysed for 27 - 28 hours. Time between completion of run and staining After the current was disconnected, 4 minutes were required to remove the two gels from the apparatus and the constant temperature room, s l i c e them, and place them i n staining dishes. If the i n t e r v a l between disconnecting the current and staining the gels were extended to 14 minutes, the protein bands were s t i l l d i s t i n c t . If there were a 20 minute i n t e r v a l , however, some d i f f u s i o n could be observed, i . e . the borders 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 great enough to destroy the pattern. Because d i f f u s i o n of the protein bands was undesir-able, the gels were stained immediately after placing them i n the staining dishes - no more than 5 minutes after the current was disconnected. - 20 -Time of staining The time of staining must be short enough to prevent the background gel from becoming permanently impregnated with dye thereby lessening the contrast between the protein bands and yet long enough to stain those fractions present i n the lea s t amount. When the gels were stained for 10 minutes with Amido Black the background did not become white with r i n s i n g and therefore did not provide the greatest contrast between the protein bands. Because 5 minutes staining produced d i s t i n c t bands and the background rinsed white, t h i s time was made constant. Time of r i n s i n g Using a gel washer (Pert et a l , 1959) the background rinsed white at 12 hours and there was no v i s i b l e change i n the protein pattern colour i f r i n s i n g continued for 18 hours. After t h i s time, however, the pattern appeared l i g h t e r . For convenience the r i n s i n g time was made constant at 13 hours. Analysis of r e s u l t s  Photography A view camera containing f a s t Pan f i l m and with a - 21 -red f i l t e r was used to photograph the gels. The exposure rate was f l 6 at 1 second by r e f l e c t e d and transmitted l i g h t . 8" x 10" f i l m was used. Replicates photographed on the same day and at a l a t e r date were found to be i d e n t i c a l to the f i r s t . Duplicates made on 5" x 7" f i l m l a t e r had to be discarded as the densitometer could not resolve the r e s u l t -ing shorter distances between protein bands on the smaller negative. A centimeter r u l e r was placed alongside each gel to give the scale. Densitometry The negative of the whole gel was cut so that the band pattern emanating from each s l o t could be run i n d i v i d -u a l l y through a Zeiss densitometer. Black photography paper was cut so as to leave a rectangular space 1 cm. wide x 17 cm. long. The negative was then placed between a piece of glass and thi s paper. Another sheet of glass was placed on top of t h i s and the r e s u l t i n g sandwich was placed i n the densitometer. The l i g h t s l o t i n the Zeiss densitometer i s 1 mm. wide. With the photography paper a constant area of 1 x 10 mm. was scanned for a distance of 17 cm. at an approximate speed of 1.5 mm. / second. .Thus the starch-gels, by means of photographic negatives and the densitometer were f i n a l l y resolved into - 22 -curves showing the amount of dye taken up by the protein i n the gel (Wunderly, 1959). Four i d e n t i c a l curves were obtained for each aliquot, three to be used i n the percent composition determinations and the fourth to be used for the measurement of migration distances. Calculations In order to minimize any background dye a baseline was drawn touching the lowest trough of the curve. To f i n d the r e l a t i v e proportions of the area under each peak, i . e . the r e l a t i v e amounts of protein present, each of the three curves was weighed and then the area under each peak was weighed separately. Averages were calculated and each peak was expressed as a percentage of the average t o t a l . Because of the very small amounts present i n some cases a l l of the percentages were then . converted to arcsin ^/percentage degrees (Snedecor, 1956, pages 318 - 319). Calculations of the means, standard deviations, and standard errors were on the transformed numbers. Student's t-tests were performed i n the appropriate circumstances. To calculate the mobility of each protein f r a c t i o n a l i n e was drawn perpendicular to the baseline through the centre of each peak of the curve. The distance to each l i n e was then measured along the baseline from the o r i g i n . - 23 -Because of s l i g h t photographic reduction the actual measure-ment as occurred i n the starch-gel was obtained by a prop-o r t i o n a l conversion of the centimeter scale on the negative to the measured distance on the curve. The following i s the c a l c u l a t i o n of the mobility (Raymond, 1955). E = f i e l d strength V = voltage D = length of medium i n cm. d = distance protein has moved i n cm. t = time i n seconds }i = mobility 2 , E = V , d = jiEt , therefore, ja = d x D cm / v o l t seconds. D V t Mean values, standard deviations, and standard errors were calculated for each mobility and Student's t-tests were performed where required as follows. Standard Deviation (S.D.) = Standard Error (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 analysis was checked by Mr. G. Mclntyre (see Acknowledgments) who found that the v a r i a t i o n within gels i s numerically less than that between gels. The l a t t e r v a r i a t i o n , i n turn, i s numerically less than that between i n d i v i d u a l s . He also found no s i g n i f i c -ant change i n the method during the course of the study. - 25 -RESULTS AND DISCUSSION CONDITION OF SAMPLE Hemolysis and cloudiness Deer serum i s normally a dark yellow colour. As Haugen (19 60) found no carotenoids i n the blood serum of nineteen 0. virginianus any reddish colouration v i s i b l e to the naked eye was assumed to be an i n d i c a t i o n of hemolysis. The appearance of cloudiness i n some samples i s probably due to l i p i d s (Cantarow & Schepartz, 1957). A l l 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, yellow but cloudy, s l i g h t l y hemolysed, hemolysed, and very hemolysed. Those samples used to test the e f f e c t of hemolysis and cloudiness were run within one year of sampling. The term "hemolysis" implies that haemoglobin has been released from the blood c e l l s into the serum. This additional protein i n the serum would be expected to have some e f f e c t on the mobility and/or the percent composition of the protein f r a c t i o n s as separated by electrophoresis. Very hemolysed samples usually varied the e l e c t r o -phoretic pattern by either an apparent increase or decrease i n the number of protein fractions as compared to the - 26 -number resolved 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 vs. yellow samples (Tables 2 & 4) show no s i g n i f i c -ant differences i n either the mobility or the percent composition of the protein f r a c t i o n s . Closer examination of Tables 1 & 3, however, reveals that over 50% of the standard errors of the s l i g h t l y hemolysed and hemolysed samples are greater than the corresponding standard errors of the yellow samples. This i n d i c a t i o n of greater v a r i a b i l i t y with hemolysis and the change i n f r a c t i o n number i n very hemolysed samples compels the elimination of a l l categories of hemolysed samples from further comparisons. Lipids are carried by the alpha- and beta-globulins (Moore, 1959). If cloudiness i s an in d i c a t i o n of excess l i p i d i n the serum then some e f f e c t on the electrophoretic protein pattern might be expected. A few cloudy samples did show an apparent decrease i n the number of protein fractions as compared to those resolved i n yellow samples. In 0. v. texanus (Table 2) cloudiness caused a s i g n i f i c a n t increase i n the mobility of f r a c t i o n +7 i n the comparison of yellow vs. yellow but cloudy samples. In the same - 27 -comparison (Table 4) the percent composition of f r a c t i o n +1 increased s i g n i f i c a n t l y . Examination of Tables 1 & 3 reveals that over 50% of the standard errors for the mobility and that 100% of the standard errors for the percent composition of the protein fractions of cloudy samples are greater than those of corresponding yellow 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 differences i n mobility and per-cent composition, and the greater v a r i a b i l i t y i n the standard errors of cloudy samples warrant eliminating them from further comparisons. TABLE 1 EFFECT OF HEMOLYSIS - m o b i l i t i e s i n 10~ 5 cm2 / 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 ' ye l l o w X .829 .464 .406 .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 y e l l o w + cloudy X .772 .515' .444 . 4 l 6 .380 .277 .210 .185 .105 .010 .017 .111 N = 2 + S.E. .124 .018 .000 .000 .013 .003 .006 .018 .018 .001 .007 .044 s l i g h t l y hemolysed X .813 .437 .374 .364 .329 .258 .187 .165 .065 .010 .013 .117 N " = 2 + S.E. .013 .011 .006 .002 .013 .003 .000 .004 .016 .000 .002 .008 wild,, male., adult,, 0. h. columbianus, Chemainus, B.C. •• yel l o w X .829 .494 .423 ' .383 .313 .260 .165 .133 .022 .012 N = 2 + S.E. - .013 .008 .008 .005 .011 .012 .005 .011 .012 .003 hemolysed X" .815 .480 .400 .370 .296 .231 .142 .071 .027 .011 N = 3 + S.E. .018 .014 .011 .012 .011 .019 .012 .020 .010 .002 N = number of animals 00 TABLE 2 EFFECT OF HEMOLYSIS - 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 . + FRACTION . 10 9 8 7 6 5 4 3 2 1 1 2 wild,, female, adults _0. v. texanus, Texas ye l l o w vs. s l i g h t l y hemolysed = = = = = = = ' = = = = = yellow vs. yellow + cloudy •= = = ++ = = = = = = = = wild,, male, a d u l t , _0. h. columbianus„ Chemainusj B.C. yell o w vs. 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 item 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 item to the .01 l e v e l . TABLE 3 EFFECT OF HEMOLYSIS - percent composition i n arcs^inVpercentage degrees. FRACTION 10 9 8 7 6 5 4 3 2 1 1 2 wild., female, a d u l t , _0.• v. texanus, Texas yellow X 38.5 16.2 13.1 12.9 10.3 7-3 9.8 11.6 13.7 13.1 15.1 21.1 N = 8 + S.E. 0 . 9 1.0 0 .6 0 . 9 1.5 0 .6 0 . 8 0 . 5 1.1 0 . 5 1.2 1.2 yellow + cloudy X 3 3 . l 18.4 l 4 . 7 12.5 6.9 9.5 9.5 12.6 16.6 17.6 16.7 17 A N = 2 + S.E. 3.4 3.4 3.9 1.7 2.6 1.5 1.5 1.4 2.4 1.0 5.0 5.2 s l i g h t l y hemolysed X 35.6 14.0 15.0 13.7 12.5 9.4 8.0 8.4 9.4 11.4 13.9 22.7 N = 2 + S.E. 2.2 4.0 0 .4 0 . 3 0.7 1.0 0 . 6 1.1 2.0 0 . 6 1.5 8.7 w i l d , male, a d u l t , 0. h_.. Columbianus, Chemainus, B.C. yellow X 44.6 19.2 14.2 7.4 6.9 7.2 10.4 11.3 18.0 19.9 N = 2 + S.E. 0 .1 2.4 0 . 5 1.4 0 . 8 1.8 0 . 1 1.8 3.2 0 .6 hemolysed X 4 l . 0 18.2 \13 .8 12.2 11.3 8 . 8 10.1 13.9 15.9 20.4 N = 3 + S.E. 2.4 1.6 2.1 1.2 1.7 2.1 0 . 9 0 . 4 3 .6 4.1 N = number of animals o TABLE 4 EFFECT OF HEMOLYSIS - Student's t - t e s t comparisons of the percentage composition ( i n arcs""in /percentage 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 2 1 1 2 wild., female, a d u l t , ' 0. v. texanus, Texas yellow vs.. s l i g h t l y hemolysed = . = = = = = = = = = = = yellow vs. yellow + cloudy = = = = = = = = = • + + w i l d , male, a d u l t , 0. h. columbianus, Chemainus, B.C. yellow vs. 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 item s i g n i f i c a n t l y greater than the f i r s t item to the .01 l e v e l . - 32 -Preservation Serum samples were a r r i v i n g at U.B.C. that were obvious-l y decomposing. A NIPA ester (propyl p-hydroxybenzoate), because of i t s b a c t e r i o s t a t i c action (Merck, I960), was t r i e d and found to be successful i n preventing decomposition. I t became important, therefore, to determine whether t h i s ester had any e f f e c t on the mobility or the percent composition of the serum proteins as separated by electrophoresis. A serum sample from a wild, female, adult, 0. h-columbianus - C a l i f o r n i a , was divided into two samples with and two samples without the NIPA ester upon a r r i v a l at U.B.C. They were then a l l kept at room temperature. The addition of the preservative on the day of a r r i v a l did not s i g n i f i c a n t l y a l t e r either the mobility or the percent composition of the protein fractions and after 16 days at room temperature there were s t i l l no changes i n the sample containing the preservative. However, after 16 days at room temperature without the preservative, f r a c t i o n +9 was l o s t and f r a c t i o n +10 exhibited a s i g n i f i c a n t l y faster mobility (Table 10). There were no s i g n i f i c a n t changes i n the mo b i l i t i e s of the remaining f r a c t i o n s . The percent composition of a l l comparable fractions also did not change (Table 12). - 33 -B a c t e r i a l decomposition of proteins can also be prevented by freezing. Previous work on deer serum u t i l i z -ing paper electrophoresis (Johnston, B.Sc. Thesis, 1961) had shown that serum from a captive adult, 0. v. ochrourus, could be stored at - 18°C for at l e a s t 5% months with no s i g n i f i c -ant changes i n the percent composition of the serum protein f r a c t i o n s , while serum from a captive fawn, 0. h. columbianus - Vancouver Island, changed s i g n i f i c a n t l y after three weeks storage. In order to test the e f f e c t of storage at - 18°C i n the present study, aliquots were taken at various time i n t e r -vals from a captive, female, adult, 0. h. columbianus - Wolf Lake (Vancouver Island), B.C. Storage times begin with the a r r i v a l of the serum at U.B.C. and i t s subsequent storage at - 18°C. I t was found that these samples could be stored as long as 7 61 days with no s i g n i f i c a n t changes occurring i n either the mobility (Tables 5 & 6) or the percent composition (Tables 7 & 8) of the protein f r a c t i o n s . As fawn serum was not examined for cold storage e f f e c t s , only those fawn samples stored less than three weeks were used i n further analyses. The e f f e c t of freezing on the ba 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 wild, female, fawn, 0. h. columbianus - C a l i f o r n i a , into one sample with and one with-out the NIPA ester. These samples were stored for 58 days at - 18°C. No s i g n i f i c a n t differences i n either mobility (Tables 9 & 10) or the percent composition (Tables 11 & 12) of the protein fractions were found. In summary, the bacteriostat, propyl p-hydroxy-benzoate, was found necessary to prevent decomposition during storage at room temperature. Neither the NIPA ester nor freezing had any e f f e c t on the mobility or the percent composition of the protein f r a c t i o n s . TABLE 5 -5 2 EFFECT OF COLD STORAGE - m o b i l i t i e s i n 10 cm / v o l t seconds. + -FRACTION 9 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 X .913 .519 .471 .442 .403 .300 .240 .164 .019 .019 .087 N = 2 + S.E. .023 .023 .023 .000 .033 .023 .002 .000 .001 .005 .020 221 days ' X .908 .512 .454 .438 .375 .296 .245 .169 .016 .010 .095 N = 2 + S.E. .073 .005 .021 .026 .016 .005 .005 .005 .002 .005 .001 304 days X .947 .515 .471 .442 .409 .305 .260 .164 .019 .019 .087 N = 2 + S.E. .033 . .004 .000 .000 .005 .000 .020 .011 .000 .000 .005 761 days X .924 .518 .464 .432 .394 .295 .238 .160 .018 .013 .086 N = 5 + S.E. .029 .010 .008 .010 .009 .018 .018 .022 .004 .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 of the serum p r o t e i n f r a c t i o n s . + FRACTION 9 8 7 6 5 ^ 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 vs. 221 days = = = = = = = = . = = vs. 304 days = = = = = - = = = • = vs. 761 days = = - = '= = = = = = 221 days vs. 304 days = = = = = = = = = = vs. 761 days = = = = = = = = = = 304 days vs. 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 - percent composition i n a r c s ~ i n /percentage degrees. + FRACTION 9 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 X 31.5 11.5 i 4 . 4 9.5 6.5 9.8 10.9 20.5 21.7 18.5 20.4 N = 2 + S.E. 1.3 0 .6 1.5 0.7 0 . 5 1.3 1.1 0 . 8 l - . o 1.0 0 . 8 221 days ' X 31.9 13.2 15.9 7.9 8.5 10.4 10.8 18.0 19.9 18.6 22.6 N = 2 + S.E. 0 .2 2 .9 0 . 5 0 .2 0 . 2 0 . 9 0 . 6 0 . 3 1.4 0 . 9 2.0 304 days X 30.1 12.3 12.2 10.6 8.1 10.5 12.3 21.8 22.3 19.6 21.3 N = 2 + S.E. 1.2 0 . 9 2.6 2.4 2.0 0 . 2 0.2 1.7 1.4 1.4 0 .2 761 days X 32.4 12.7 14.3 9.4 7 .3 9.9 11.0 19.7 2 0 . 6 18.8 22.3 N = 5 + S.E. 1.4 1.0 1.2 1.0 1.0 0 . 6 0 .6 0 . 9 1.3 0 .5 0 . 8 N = number of a l i q u o t s TABLE 8 EFFECT OF COLD STORAGE - Student's t - t e s t comparisons of the percent composition ( i n a r c s ^ i n v/percentage degrees) of the serum p r o t e i n f r a c t i o n s . + FRACTION 9 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 vs. 221 days = = = = = = = = = = = vs. 304 days = = = = = = = = = = = vs. 761 days = = = = = = .= = = = = 221 days vs. 304 days = = = = = = = = = = = 304 days vs. 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 seconds. 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, C a l i f o r n i a room temperature,. 1 day storage no p r e s e r v a t i v e X 1.060 .557 .461 .457 . 4 3 3 .347 .300 .203 .130 .017 .012 N = 2 +S.E. .018 .023 .023 .013 .018 .005 .005 .023 .000 .005 .003 plus p r e s e r v a t i v e • 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 storage no p r e s e r v a t i v e X .913 . 4 6 6 .423 .401 .331 .234 .214 .145 .036 .010 N = 4 +S.E. .028 .016 .016 .036 .048 .036 .036 .0.32 .023 .000 plus p r e s e r v a t i v e X I . 0 6 3 .569 .489 .459 .433 .345 .305 .208 .120 .010 .011 N = 3 +S.E. .007 .013 .005 .003 .008 .011 .003 .005 .018 .001 .001 w i l d , female, fawn, 0. h. columbianus, C a l i f o r n i a - 18° C., 58 days storage no p r e s e r v a t i v e X .866 .491 .419 .390 .347 .258 .184 .170 .097 .014 .010 N = 2 +S.E. .018 .004 .015 .018 .013 .001 .004 .001 .028 .009 .000 plus p r e s e r v a t i v e X .871 .496 . 4 l 8 .394 .347 .260 .183 .173 .068 .014 .010 N = 2 • +S.E. .004 .009 .005 .000 .000 .001 .004 .004 .009 .005 .000 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 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 . + FRACTION 10 9 8 7 6 5 4 3 2 1 1 w i l d , female, adult," _0. h. columbianus, C a l i f o r n i a room temperature, 1 day storage no pr e s e r v a t i v e vs. plus p r e s e r v a t i v e = = = = = = = = = = = room temperature, 16 days storage no pr e s e r v a t i v e vs. plus p r e s e r v a t i v e + + 0 = = = = = = = = . = room temperature, no pres e r v a t i v e 1 day vs. 16 days storage - - 0 = = = = = = = = = room temperature, plus p r e s e r v a t i v e 1 day vs. 16 days storage = = = = = = = = ' = = = w i l d , female, fawn, _0. h. columbianus, C a l i f o r n i a - l8°C., 58 .days storage, no pr e s e r v a t i v e vs. plus p r e s e r v a t i v e = = = = = = = = = = = = 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 item 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 item to the .01 l e v e l . second item s i g n i f i c a n t l y slower than the f i r s t item to the .01 l e v e l . 0 p r o t e i n f r a c t i o n present i n one sample but not i n the other. 4^ TABLE 11 EFFECT OF A BLOOD PRESERVATIVE - percent composition i n a r c s ^ i n ^percentage degrees. + 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 , C a l i f o r n i a room temperature, 1 day storage no p r e s e r v a t i v e T 38.7 17.2 10.9 11.5 10.0 14.4 13.2 13.O l 8 . 6 12.6 19.5 N = 2 - + S.E. 1.1 0 . 9 0 . 6 0 . 0 0 . 9 0 .6 1.4 0 . 0 1.1 1.1 1.4 plus p r e s e r v a t i v e X 35.9 17.6 8.7 12.0 11.0 12.2 12.1 12.0 18.0 10.2 19.0 N = 2 + S.E. 0.5 1.9 0 . 3 0 . 6 0 . 0 0 . 6 0 . 4 0 . 9 0 . 9 0 . 6 0 . 0 room temperature, 16 days storage no p r e s e r v a t i v e X 36.2 10.3 11.7 10.0 15.0 12.4 10.4 23.6 15.1 23.9 N = 3 ± S.E. 1.5 0 . 8 1.9 0 . 2 3.6 0 . 6 0 . 5 1.4 0 . 4 0 . 9 plus p r e s e r v a t i v e X 36.9 18.3 9.5 12.3 10.2 13.0 13.0 14.5 19.0 11.9 20.7 N = 2 + S.E. 0 .1 0 .0 0 .1 1.2 1.1 0 .1 0 .6 2.1 0 . 0 1.9 1.5 w i l d , female, fawn, 0. h. columbianus, C a l i f o r n i a - l8°C., 58 days storage no p r e s e r v a t i v e X 4 l . 5 17.9 9.2 15.7 6.8 7.1 9.9 8 .1 19.5 12.2 22.6 N = 2 + S.E. 1.4 0 .0 0 . 6 1.2 3.4 1.5 0 . 0 0 . 6 2.0 1.6 0 . 6 plus p r e s e r v a t i v e X 4 0 . 9 18.6 10.5 12.5 7 .9 5 .6 8.1 8 . 9 20.7 10.7 20.8 N = 2 + S.E. 2.4 0 .6 0 .0 0 . 0 0 . 2 0 . 0 1.1 0 . 9 0.7 2.1 0 . 0 N = number of a l i q u o t s TABLE 12 EFFECT OF A BLOOD PRESERVATIVE - Student's t - t e s t comparisons of the percent composition ( i n arcs-in/'percentage 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 . 2 1 1 w i l d , female, a d u l t , _0. h. columbianus, C a l i f o r n i a room temperature, 1 day storage no p r e s e r v a t i v e vs. plus preservative = = = = = = = ' = = = = room temperature, 16 'days storage no p r e s e r v a t i v e vs. plus pr e s e r v a t i v e = 0 = = = = = = = = = room temperature, no preserv a t i v e 1 day vs. 16 days storage = 0 = = =' = = = = = = room temperature, plus p r e s e r v a t i v e 1 day vs. 16 days storage = = = = = = = = = = = w i l d , female, fawn, 0. h. columbianus, C a l i f o r n i a - l 8 ° C., 58 days storage no p r e s e r v a t i v e , vs. plus preservative= = 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. 0 p r o t e i n f r a c t i o n present i n one sample but not i n the other. - 43 -Succinylcholine chloride According to Craighead et a l (I960), succinylcholine chloride i s a sk e l e t a l muscle relaxant that acts by replacing acetylcholine at the motor end plates thereby blocking nervous transmission at the myoneural junction. Muscular p a r a l y s i s p e r s i s t s u n t i l the diacetylcholine i s hydrolysed by cholinesterase to succinic acid and choline. E f f e c t s on the cardiovascular system are minimal (Goodman & Gelman, 1956). In the deer unit at the University of B r i t i s h Columbia, i t was possible to obtain blood samples from the deer by throwing and holding them. However, the struggle was un-desirable and the technique of immobilizing the deer with succinylcholine chloride (Anectine - Burroughs Wellcome -Canada) was used (Cowan et a_l, 1962, Pearson e_t a l , 1963). Serum samples 7 days apart were taken from a captive, adult, male, 0. h. columbianus - Wolf Lake, Vancouver Island, B.C., with and without the help of succinylcholine chloride. These samples when compared showed no s i g n i f i c a n t d i f f e r -ences i n either the mobility (Tables 13 & 15) or the percent composition (Tables 14 & 16) of the protein f r a c t i o n s . Therefore, serum samples obtained from U.B.C. captive deer with the help of succinylcholine chloride can be used i n further comparisons. TABLE--13 -5 2 EFFECT OF SUCCINYLCHOLINE CHLORIDE - m o b i l i t i e s i n 10 cm / v o l t seconds. + FRACTION 9 8 7 6 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 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 .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 p r o t e i n f r a c t i o n s . FRACTION 9 8 7 6 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. with vs. without 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 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 15 EFFECT OF SUCCINYLCHOLINE CHLORIDE - percent composition i n arcs i n /percentage 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. without 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 9.5 12.0 17.3 12.9 6.5 8 . 3 15.8 21.6 8.0 2 0 . 0 + S.E. 2.0 0 .0 0 .2 0 .5 0.4 0 . 4 0 . 3 0 . 6 0 . 6 0 .4 0 .6 with 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 8 .8 12.1 19.2 14.9 7 .0 8 .5 15.3 24 .3 9.0 18.0 + S.E. 0 . 1 0 .2 0 .2 0 .0 0 . 3 1.1 0 . 2 0 . 1 0 . 1 0 .4 0 .2 N = number of a l i q u o t s TABLE 16 EFFECT OF SUCCINYLCHOLINE CHLORIDE - Student's, t - t e s t comparisons of the percent composition ( i n arcs'^in /percentage degrees) of the serum p r o t e i n f r a c t i o n s . + FRACTION . 9 8 7 6 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.L with vs. without 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 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. - 48 -CONDITION OF ANIMAL Individual v a r i a t i o n Serum samples from eight wild, female, adult, 0. v. texanus were obtained from Sinton, Texas from A p r i l 19 - May 3, 19 63. These yellow samples were stored for s l i g h t l y more than one year. The numerical data for the mobility and for the percent composition of the protein fractions are given i n Tables 17 & 19 respectively. .Individual 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 also a very great i n d i v i d u a l v a r i a t i o n i n the serum proteins i s r e a d i l y seen i n Tables 18 & 20 where the t - t e s t comparisons for mobility and percent composition respectively are given. Mobility appears to be a more variable parameter than percent composition. Each i n d i v i d u a l animal has i t s own p a r t i c u l a r immune hi s t o r y which w i l l have an e f f e c t on the gamma-globulins and possibly also on the alpha- and beta-globulins (see section on Age). This immune response must account for some of the v a r i a b i l i t y between individuals which i s shown here i n the majority of the protein f r a c t i o n s . That f r a c t i o n +10 may be albumin (as i t i s the f r a c t i o n of greatest mobility) i s supported by i t s s t a b i l i t y i n mobility. There i s no obvious explanation as to why the mobilit-ies of f r a c t i o n +1 and +2 and the percent compositions of - 49 -fra c t i o n s +1 and +3 do not vary s i g n i f i c a n t l y between in d i v i d u a l s . 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 for i n further comparisons by using groups of at le a s t two i n d i v i d u a l s . TABLE 17 -5 2 INDIVIDUAL VARIATION - m o b i l i t i e s i n 10 cm / v o l t seconds. + FRACTION 10 9 8 7 6 5 4 3 2 1 1 2 w i l d , female, a d u l t , _0. v. texanus, Texas D 210 X .850 .510 .439 .401 .338 .256 .211 .175 .077 .011 .011 . 1 4 3 N = 4 + s.E. .036 .011 .007 .000 .012 .017 .004 .009 .009 .001 .001 .010 D 237 - x .863 .481 .435 .359 .330 .254 .197 .155 .092 .026 .010 .190 N = 2 + S.E. .057 .004 .001 .010 .000- .011 .006 .000 .023 .016 .000 .000 D 242 X .804 .412 .349 .33^ .291 .222 .164 .154 .064 .016 .010 .154 N = 2 + S.E. .000 .021 .010 .006 .006 .000 .006 .006 .011 .006 .000 .006 D 243 X .809 .391 .339 .317 .285 .217 .159 . 1 4 8 .084 .010 .016 .164 N = 2 + S.E. .005 .000 .000 .000 .000 .006 .000 .000 .000 .000 .006 .006 D 247 x .821 .439 .381 .352 .321 .259 .177 .15^ .060 .014 .010 .111 N = 4 + S.E. .012 .013 .009 .009 .005 .016 .011 .000 .006 .005 .000 .020 D 249 X .803 .509 .469 .436 .392 .339 .275 .169 .097 .011 .009 .064 N = 5 + S.E. .007 .004 .002 .005 .003 .004 .009 .009 .022 .002 .000 .003 D 250 X .841 .469 .407 .379 .332 .254 .161 .149 .072 .010 .010 .128 N = 4 + S.E. .032 .021 .012 .012 .002 .012 .001 .002 .007 .000 .000 .030 N = number of a l i q u o t s o TABLE 18 INDIVIDUAL VARIATION - 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 . FRACTION 10 .9 8 7 6 5 4 3 - 2 1 1 2 w i l d , female, a d u l t , 0. v. texanus, Texas D 210 vs. D 237 = — = = = ++ vs. D 242 — — — = — = — -vs. D 243 = -- -- -- -- = -- = = = = = vs. D 247 = -- -- -- = = = = = = vs . D 249 = ++ ++ ++ ++ = — : -vs. D 250 = = = = = = -- = - = = D 237 vs. D 242 = = = = = = — — -vs. D 243 = -- -- = -- = -- -- - = - = vs. D 247 = = -- = = = = -- = = — = vs. D 249 = ++ ++ ++ ++ ++ ++ = - = -- --vs. D 250 = = = = = = -- = = = = D 242 vs. D 243 = = — = = — — - : - = vs. D 247 = = = = = = = = =: vs. D 249 ++ ++ ++ ++ ++ ++ --- = = --vs. D 250 = = = = ++ = = - = = : D 243 vs. D 247 = ++ - ++ • ++ — vs. D 249 = ++ ++ ++ ++ ++ ++ = - - --vs. D 250 — = ++ ++ ++ = = = =: = = D 247 vs . D 249 = ++ ++ ++ ++ ++ ++ — = = vs. D 250 = = = = = = = - = D 249 vs. D 250 = = -- -- -- -- = = = ++ = = no slg ;nif i c a n t d i f f e r e n c e between the two 3 a m oles. ++ second item Si£ ; n i f I c a n t l y f a s t e r than the : f i r s t item to the . 01 l e v e l . -- second item si£ ? n i f i c a n t l y slower than the : f i r s t item to the . 01 l e v e l . TABLE 19 INDIVIDUAL VARIATION - percent composition i n a r c s ^ i n /percentage degrees. + FRACTION 10 9 8 7 6 5 4 3 2 1 1 2 w i l d , female, a d u l t , 0. v. texanus, Texas X 3 6 . 3 17.8 14.9 10.6 8 . 0 5 .3 9.2 9.9 13.3 13.5 18.7 23 .0 + S.E. 0 . 2 0 . 9 0 .7 0 .7 1.3 1.1 0 . 8 1.1 1.4 1.0 1.6 l.i D 237 X 42.7 17.6 15.4 15.1 6 .9 8.1 11.6 11.8 16.7 12.3 14 .7 2 0 . 0 N = 3 + S.E. 1.1 1.7 0 . 6 1.1 0 . 6 1.6 1.0 0.7 0 . 4 1.5 0 . 8 O.t X 37.6 12.5 H . 3 11.0 4 . 1 7.1 10.8 10.6 16.9 13.3 21 .1 22 .6 + S.E. 0 . 1 2.1 0 . 3 0 . 0 2.0 1.7 0.1 0 . 6 0 . 9 0 . 1 0 . 3 OA D 243 X 39 .6 12.2 13.5 11.6 10.7 10.3 10.3 11.5 10.8 12.7 15.8. 23.O N = 2 + S.E. ^199 lA oo-.j-4 1.1 0 . 2 0 . 0 0.2 o.o 0 . 9 1.8 3 .2 h.t X~ 37 .3 17.9 13.1 15.5 10.3 5.6 11.4 10.7 11.8 10.4 12.5 24 .0 + S.E. 0 . 6 2.1 1.0 0 . 3 0 . 4 0 .7 0 . 8 0 . 2 0 . 5 2.1 1.8 2.C D 249 X 36 .6 19.9 14.0 12.0 18.1 8.7 5.7 13.1 11.5 15.3 14.0 14.6 N = 5 + S.E. 0 . 5 0 . 3 1.0 0 . 6 1.4 0 . 5 0 . 9 2.6 1.2 1.7 0 . 5 0.5 D 250 X 42.0 17.5 10.6 10.5 10.9 5.0 7 A 10.5 10.4 12.9 12.9 23 .5 N = 4 + S.E. 0 . 3 1.2 1.4 0 . 6 1.4 0 . 3 0 .9 1.7 1.6 0 . 8 0 . 9 0.8 N = number of a l i q u o t s TABLE 20 INDIVIDUAL VARIATION - Student's t - t e s t comparisons of the percent 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 . + FRACTION 10 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 237 — = — = — - = : vs. D 242 ++ = — = — = = vs. D 243 = - = = ++ — ; vs. D 247 = ++ = = — vs. D 249 = • - ++ = = = - = = -vs. D 250 ++ = = — = = — = = = D 237 vs. D 242 — _ _ _ _ _ _ ++ vs. D 243 = = = - ++ — - = vs. D 247 -- = = ++ = — — — vs. D 249 -- = == ++ — = - -vs. D 250 = = = = = = = -- = = = D 242 vs. D 243 _ — _ _ — = _ - _ vs. D 247 = n — — — vs. D 249 = = = ++ = = — -vs. D 250 ++ - = = = = = = -- = D 243 vs. D 247 = = = : = = — = = — vs . D 249 ++ = - = = = = - : = vs. D 250 = = = = -- = = = = = D 247 vs. D 249 — = = = — — _ vs. D 250 ++ = - -- = = = = = = D 249 vs. D 250 ++ — — = — (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 item s i g n i f i c a n t l y greater than the f i r s t item to the .01 l e v e l , second Item s i g n i f i c a n t l y smaller than the f i r s t item to the .01 l e v e l . - 54 -Sex Electrophoretic studies considering the e f f e c t of the sex of the animal on a serum sample are few. Using micro-electrophoresis i n agar-gel, van Sande and Karcher (1960) found no s i g n i f i c a n t sexual differences i n the protein patterns of many species of insects. Free electrophoresis was u t i l i z e d by Brandt et a l (1950), Deutsch & Goodloe (1945), and Moore (1945) to detect sexual differences i n the protein patterns of chickens. Brandt et a l (op. c i t . ) found that laying hens exhibited increased t o t a l protein, an extra pre-albumin f r a c t i o n , increased alpha-globulin, and decreas-ed albumin when compared to non-laying hens and cockerels. Sibley and Johnsgard (1959a) found that i n one year old pheasants the laying females had very low albumin l e v e l s compared to those of male pheasants and i n adult jungle fowl the laying and non-laying females exhibited lower albumin l e v e l s than the males. Juvenile pheasants, however, evinced no sex difference i n serum protein patterns. Moore (1945) examined the free electrophoretic p r o f i l e s of a wide v a r i e t y of mammals and could detect no sexual differences. U t i l i z i n g f i l t e r paper electrophoresis, Cowan & Johnston (19 62) found sexual differences i n the - 55 -protein patterns of the captive deer 0. h. sitkensis and 0. v. ochrourus. The adult female deer had a significantly-lower percent 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 higher percent composition of fract i o n s II and V (globulins?) than the males. Thus i t appears that where sexual differences i n protein concentration are detected they w i l l usually be exhibited by lowered albumin and increased globulin l e v e l s i n the female. Yellow samples of wild adult, 0. h. columbianus -Chemainus, Vancouver Island, B.C. (collected i n December, 19 61 and stored less than two years) and of wild fawns of 0. h. columbianus - Mendocino County, C a l i f o r n i a (collected i n October, 19 62 and stored less than three weeks) were examin-ed e l e c t r o p h o r e t i c a l l y for the e f f e c t of sex on the serum proteins. The wild, 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 percent composition of f r a c t i o n +3 (globulin?) than the corresponding male fawns. As the precise age of the fawns i s not known t h i s one s i g n i f i c a n t difference may be linked not only with sex but also with maturation (see section on Age). - 56 -The wild, female, adults of 0. h. columbianus -Chemainus, Vancouver Island, B.C. show a s i g n i f i c a n t l y lower percent composition of f r a c t i o n +10 than the corresp-onding male adults (Tables 23 & 24). As th i s f r a c t i o n i s possibly albumin (see section on Individual variation) then t h i s r e s u l t i s i n agreement with previous work. Cowan & Johnston (19 62) found no s i g n i f i c a n t differences i n the mo b i l i t i e s of the protein fractions of adult male and female captive 0. h. sitkensis and 0. v. ochrourus using f i l t e r paper electrophoresis. The use of starch-gel electrophoresis i n the present study indicates that the wild, adult 0. h. columbianus - Chemainus, Vancouver Island, 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 three f r a c t i o n s , the female having 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 also shows no s i g n i f i c a n t differences between the mobility of any of the protein 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, sexual differences i n the mobility of the protein f r a c t i o n s were not observed between male and female fawns but were observed between male and female adults. Sexual differences i n the percent composition of the protein - 57 -fractio n s were observed i n both male and female fawns and adults. Therefore, the sex of the deer must be taken into consideration i n further comparisons. TABLE 21 -5 2 EFFECT OF SEX - m o b i l i t i e s i n 10 cm FRACTION w i l d , 0. h. columbianus, C a l i f o r n i a male, fawn N = 3 female, fawn N = 3 X + S.E. X + S.E. 10 v o l t seconds. 9 8 7 6 4 + 1 .977 .542 .460 .431 .394 .330 .224 .182 .101 .015 .011 .037 .021 .002 .005 .014 .031 .031 .005. .015 .005 .001 .893 .527 .443 .407 .371 .272 .183 .179 .095 .034 . O i l .024 .043 .020 .023 .017 .007 .006 .006 .014 .013 .001 w i l d , 0. h. columbianus, Chemainus, B.C. male, a d u l t X N = 2 + S.E. female, a d u l t • X N = 2 + S.E. .829 .494 .423 .383 .313 .260 .165 .133 .022 .012 .013 .008 .008 .005 - O i l .0H2 .005 .011 .012 .003 .775 .421 .383 .340 .286 .221 .115 .080 .017 .013 .006 .006 .006 .001 .013 .007 .006 .006 .001 .001 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 w i l d , O.h. columbianus, C a l i f o r n i a male fawn vs. female fawn w i l d , _0.h. columbianus, Chemainus, B.C. male a d u l t vs. 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 item s i g n i f i c a n t l y slower than the f i r s t item to the .01 l e v e l . U3 TABLE 23 EFFECT OF SEX - percent composition i n a r c s ^ i n /percentage degrees. FRACTION 10 7 6 5 4 + 1 w i l d , 0. h. columbianus, C a l i f o r n i a male, fawn X N = 3 ' ± S.E. female, t fawn X N = 3 + S.E. 35.0 17.3 13.6 11.2 9.6 11.2 13.1 12.2 2 0 . 8 9 .8 23.7 2.2 0 .8 0 . 3 2.1 0 . 9 3.4 1.5 0 . 6 0 . 5 1.6 0 . 9 41.0 19.1 10.9 13.7 8.7 6.2 8.8 9.1 22 .8 11.7 17.1 0 . 8 0 .3 0 .6 1.5 0 . 8 1.0 1.4 o . l 1.2 0 . 8 2.0 w i l d , Q. h. columbianus, Chemainus, B.C. male, a d u l t X N = 2 . + S.E. female, a d u l t X N = 2 + S.E. 44.6 19.2 14.. 2 7 .4 6.9 7.2 10.4 11.3 18.0 19.9 0.1 2.4 6.5 1.4 0 . 8 1.8 o . l 1.8 3.2 0 .6 39.2 16.3 13.7 l l . l 8.1 12.8 14.7 12.7 15.5 22 .9 0 .5 0 .5 0 .9 1.4 0 . 3 3.7 3.2 2.6 3.1 2.4 N = number of animals O TABLE 24 EFFECT OF SEX - Student's t - t e s t comparisons of the percent 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 . + FRACTION 10 9 8 7 6 5 4 3 2 1 1 w i l d , _0.h. columbianus, C a l i f o r n i a male fawn vs. female fawn = = = = = = = - - = = = w i l d , O.h. columbianus, Chemainus, B.C. male a d u l t vs. female ad 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 item s i g n i f i c a n t l y smaller than the f i r s t item to the .01 l e v e l . - 62 -Age Examinations of the blood serum of chickens by means of free electrophoresis (Brandt e_t aJL, 1950, Marshall & Deutsch, 1950) and of pheasants by means of f i l t e r paper electrophoresis (Sibley & Johnsgard, 1959a) a l l show an increase i n gamma-globulin with age. The res u l t s involving the e f f e c t of the animal's age on albumin concentration are varied: Brandt et a l found no change i n albumin concentrat-ion while Marshall & Deutsch and Moore found albumin increased with age i n chickens and Sibley & Johnsgard found i t decreased with age i n pheasants. ' Brandt et. a l also found an increase i n t o t a l protein and alpha-globulin with no change i n the beta-globulin concentration of chickens. •I Konig et a l (1949) used free electrophoresis to observe an increase i n gamma-globulin as lambs mature. Munkacsi (19 61) used paper electrophoresis to study horses from the newborn stage to twenty year olds. He found that the t o t a l protein rose s t e a d i l y with increasing age, that t h i s increase was due to the addition of beta-globulin up to 1% years, and that the increase thereafter was due to the addition of gammaglobulin. He found no change i n albumin or alpha-globulin. Cowan & Johnston (19 62) used paper electrophoresis to study the e f f e c t of age on the serum - 63 -proteins of captive 0. h. columbianus - Vancouver Island, B.C. The percent composition of the protein f r a c t i o n s changed up to the age of f i f t e e n months. Fraction I (albumin?) decreased. Fractions II & III remained constant, and Fraction IV and Fraction V (gamma-globulin?) increased. The only consistent f i n d i n g i n the above work i s the increase i n gamma-globulin as the animal ages. The invest-igators agree that this increase i s probably a response to the increasing number of antigenic substances the animal comes into contact with as i t grows older. Most samples received for the present study were l a b e l l e d either "fawn" or "adult". However, yellow samples taken from wild, 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 as fawns, 1 year olds, 2 year olds, 4 year olds, and 6 year olds. Due to the small number of samples i n each age group, the l a t t e r three ages were grouped and are considered as "adults" i n the following tables. These "adults" represent the possible range of ages that would be included i n the "adult" labels on samples received from other sources. The fawn sera were stored less than three weeks while the remain-ing sera were stored less than two years. - 64 -Numerical data for the mobility and the percent composition of the protein f r a c t i o n s are given i n Tables 25 & 27 respectively. A l l ages exhibit the same t o t a l number of protein f r a c t i o n s . Table 2 6 shows that there, i s no s i g n i f i c a n t change i n the mobility of the protein f r a c t i o n s from fawns to adults. This i s i n agreement with Cowan & Johnston's (19 62) findings on mobility. That the percent composition of the protein f r a c t i o n s does change with age i s seen.in Table 28. Fraction +10, which i s assumed to be albumin, has decreased with 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 difference between fawns and adults. This decrease i n albumin agrees with that found i n pheasants (Sibley & Johnsgard, 1959a) and i n captive O. h. columbianus - Vancouver Island, B.C. (Cowan & Johnston, 19 62). The fawns and the one year olds are similar i n the percent composition of a l l t h e i r protein f r a c t i o n s . The fawns and the adults d i f f e r not only i n the albumin f r a c t i o n but the adults also show an increase i n f r a c t i o n +3 (globulin?) over the fawns. The adults also show an increase i n fractions +3 and +4 (globulins?) over the one year olds. - 65 -As the i d e n t i t y of the globulins with a p a r t i c u l a r f r a c t i o n number has not been determined fractions +3 and +4 must remain only "globulins". In summary, no changes i n the mobility of the protein fractions are shown to occur with age. Changes i n percent composition are found between fawns, one year olds, and adults. As the majority of the serum samples received i n t h i s study were from adults, the fawns and one year olds were eliminated from further comparisons. TABLE 25 -5 2 EFFECT OF AGE - m o b i l i t i e s i n 10 cm / v o l t seconds. 4-FRACTION 10 9 8 7 6 5 4 3 2 1 1 w i l d , female, 0. h. co.lumbianus, C a l i f o r n i a fawn X .901 .517 . 4 4 2 .411 .375 .290 .212 . l 8 l .099 .029 .011 N = 4 + s.E. .019 .032 .014 .017 .013 .019 .023 .005 .011 ...Oil .001 1 year o l d X .952 .515 .^60 .431 .403 .295 .228 .174 .077 .012 .011 N = 4 .+ s.E. .046 .027 .025 .026 .025 .019 .017. .024 .017 .002 .001 a d u l t . X .979 .540 .475 .434 .406 .288 .225 . 1 4 9 .087 .012 .012 N = 3 + S.E. .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 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 0 . h. columbianus, C a l i f o r n i a fawn vs. 1 year o l d = = = = = = = = . = = -fawn vs. a d u l t = . = = = - = = = = = = 1 year o l d vs. 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. CTi TABLE 27 EFFECT OF AGE - percent composition i n a r c s ^ i n /percentage degrees. FRACTION 10 9 7 6 - 5 ^ 3 + 1 1 w i l d , female, 0 . h. columbianus, C a l i f o r n i a fawn N = 4 X + S.E. 41.2 18.9 11.7 12.8 8.3 7 .0 9.0 9.2 22.2 11.6 17.6 0.6 0 . 3 0 . 9 1.5 0.7 1.0 l . l 0 .2 l . l 0 . 6 1.. 1 year o l d N = 4 X + S.E. 40.4 20.0 12.2 8.8 7.0 6.4 9.5 9 .8 18.5 13.3 22.2 0 .3 1.3 1.9 0 . 8 0 . 8 1.7 0 . 2 0 . 3 3.5 0 . 3 1.6 a d u l t N = 3 X + S.E. 37.2 16.7 13.I 10.6 7.9 12.2 12.9 13.3 19.6 10.9 21.2 0 .8 0 . 9 1.9 l . l 2.5 2.7 0.2 0 . 6 0 . 6 1.1 4 .1 N = number of animals en 00 TABLE 28 EFFECT OF AGE - Student's t - t e s t comparisons of the percent 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 . + FRACTION 10 9 8 ' 7 6 5 .4 3 2 1 1 w i l d , female, _0. h. columbianus, C a l i f o r n i a fawn vs. 1 year o l d = = = = = = = = = = = fawn vs. a d u l t - - = = = = = = + + = = = 1 year o l d vs. adu 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 item s i g n i f i c a n t l y greater than the f i r s t item to the .01 l e v e l , second item s i g n i f i c a n t l y smaller than the f i r s t item to the .01 l e v e l . - 70 -Season Deer, l i k e many other animals, are affected by seasonal changes i n the quantity and the q u a l i t y of t h e i r food and maintain an annual cycle of reproduction. As electrophoretic patterns of mammalian serum proteins are affected both by die t (see section on Captivity) and pregnancy (Moore, 1959) there i s the p o s s i b i l i t y of the deer's protein pattern showing seasonal e f f e c t s . Reports of seasonal e f f e c t s on blood proteins are scarce. Sibley & Johnsgard (1959a) give the following i l l u s t r a t i o n of the e f f e c t of season on the electrophoretic pattern of the serum proteins of mallards. They found that female mallards 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 wild birds i n July which were subsequently kept i n c a p t i v i t y u n t i l the November sampling (see section on C a p t i v i t y ) . Cowan & Johnston (19 62) i n a paper electrophoretic study of captive, adult, male, 0. h. columbianus report no s i g n i f i c a n t differences i n either the mobility or the percent composit-ion of the serum proteins associated with the onset of rut. In the present study, serum samples were compared from wild, female, adult, 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 status of the deer i n Mendocino County, C a l i f o r n i a i s markedly poorer than i n October (B i s s e l & Strong, 1955, Taber & Dasmann, 1958). That there were no s i g n i f i c a n t differences observed i n the mo b i l i t i e s of any of the protein f r a c t i o n s (Tables 29 & 30) i s i n agreement with the findings of Cowan & Johnston (1962). S i g n i f i c a n t differences were found, however, i n the percent composition of the protein f r a c t i o n s as separated by starch-gel electrophoresis (Tables 31 & 32). The January group shows an increased albumin ( f r a c t i o n +10) and lowered globulin ( f r a c t i o n +4) over the October group. These r e s u l t s agree with those of Goldstein & Scott (1956) who studied the effects of a Vitamin E deficiency i n chickens. Taber (1953) found the peak of conception i n 1949 of O. h. columbianus - Mendocino County, C a l i f o r n i a occurred during the l a s t week i n October.and the f i r s t two weeks i n November. I t i s not known i f the groups of females used i n the present study were pregnant at the time of sampling. It i s doubtful, however, that early pregnancy would have a greater e f f e c t on the electrophoretic pattern 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 -mobility but does a f f e c t the percent composition of the protein f r a c t i o n s . These changes can be linked to the n u t r i t i o n a l status of the season. It i s apparent, there-fore, that studies intending to explore serum protein v a r i a t i o n i n deer can only get comparable samples by using phenologically equivalent i n d i v i d u a l s . TABLE 29 -5 2 EFFECT OF SEASON - m o b i l i t i e s i n -10 cm / v o l t seconds. FRACTION 10 9 7 5 4 3 + l w i l d , female, a d u l t , 0_. h. columbianus, C a l i f o r n i a October, 1962 N = 3 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, 1963 N = 2 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 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 , P.. h. columbianus, ' C a l i f o r n i a October, 1962 vs. 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 - percent composition i n a r c s ^ i n /percentage degrees. + 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, C a l i f o r n i a October, 1962 X 37.2 16.7 13.1 10.6 7 . 9 12.2 12.9 13.3 19.6 10.9 21 .2 N = 3 ± S.E. 0 .8 0 . 9 1.9 l . l 2.5 2.7 0 . 2 0 . 6 0 . 6 1.1 4 .1 January, 1963 X 46 .4 20.5 11.5 11.2 6.3 9.2 8.5 9.5 13.7 10.9 16.9 N = 2 + S.E. 0 .5 0.4 1.4 1 . 3 1.1 0 . 9 0 . 4 0 . 5 3.5 0 .6 1 . 9 N = number of animals Ul TABLE 32 EFFECT OF SEASON - Student's t - t e s t comparisons of the percent composition ( i n arcs^in/percentage 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 2 1 1 w i l d , female, a d u l t , _0. h. columbianus, C a l i f o r n i a October, 1962 vs. 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 item s i g n i f i c a n t l y greater than the f i r s t item to the .01 l e v e l , second item s i g n i f i c a n t l y smaller than the f i r s t item to the .01 l e v e l . - 77 -Cap t i v i t y The blood protein f r a c t i o n s of a wide var i e t y of animals have been found to be affected both by di e t (Bandy et a l , 1957, Goldstein & Scott, 1956, Helgebostad & Martinsons, 1958, K i t t s et a l , 1956, Weech et a l , 1935) and by disease (Foreman, 19 60, Gleason & Friedberg, 1953, Ingram, 195 6, Jencks et. a l , 1955, Johnson et a l , 1958, Pert et a l , 1959, Sanders et a l , 1944, Schinazi, 1957). C a p t i v i t y has been shown to have an e f f e c t on both structure and growth (Darwin, 1859, Spurway, 1952). At the University of B r i t i s h Columbia (U.B.C.) deer were brought from the wild and put under captive conditions (Wood et a l , 19 61) at the age of two to three weeks. These captive conditions are such that the captive deer used i n the present study are a l l i n good health and are a l l on an i d e n t i c a l high plane di e t (Wood et a l , 19 61). The food and the state of health are very d i f f e r e n t from those found i n the wild state (Linsdale & Tomich, 1953). Bandy et a l (1956) found increased body size i n U.B.C. captive 0. h. columbianus on a high plane di e t as compared to those on a low plane d i e t . Dasmann & Dasmann (1963) found a similar r e l a t i o n s h i p between food q u a l i t y and deer size i n wild populations 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 separat-ed protein fractions for captive and wild 0. h. columbianus -Wolf Lake, Vancouver Island, B.C. (samples col l e c t e d December, 19 61) and from captive (samples col l e c t e d March, 19 62) and wild (samples c o l l e c t e d A p r i l , 19 63) 0. h. sitkensis - Alaska. The captive deer of both races d i f f e r obviously from t h e i r wild counterparts i n having an additional negatively migrating serum protein f r a c t i o n . As i t i s also obvious that the l i m i t e d data w i l l not allow further numerical comparisons they are l i s t e d only i n Appendices A & B. Disease affects the electrophoretic pattern s i g n i f i c a n t l y only i n the acute stages and then only i n the percent composit-ion of the protein f r a c t i o n s . I t i s suspected that the great difference seen between captive and wild animals (Table 33) i s not due to disease but i s due mainly to the extreme differences i n d i e t . In summary, the U.B.C. captive conditions are such that serum samples from captive deer cannot be u t i l i z e d i n further comparisons which also involve serum samples from deer i n the wild state. N u t r i t i o n a l differences are suspected of causing the great differences between captive and wild 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 . * w i l d number of p o s i t i v e l y number of n e g a t i v e l y migrating f r a c t i o n s migrating f r a c t i o n s female,adult O.h.columbianus, N = 1 .9 1 Wolf Lake, B.C. male, a d u l t , 0_.h. columbianus, N = 1 9 1 Wolf Lake, B.C. male, a d u l t , O.h. s i t k e n s i s , N = 1 9 1 Kupreanof Is.Alaska captive female, a d u l t O.h. columbianus, N = 1 9 2 Wolf Lake, B.C. male, a d u l t , .O.h. columbianus, N = 1 9 2 Wolf Lake, B.C. female, a d u l t , O.h. s i t k e n s i s , N = 1 9 2 Petersburg,Alaska male, a d u l t , O.h. s i t k e n s i s , . N = 1 9 2 Petersburg,Alaska N = number of animals see Appendix A f o r the m o b i l i t i e s and Appendix B f o r the percent composition of the serum p r o t e i n f r a c t i o n s . - 80 -Geographic separation, subspecies, and species After removal of a l l the hemolysed and cloudy samples, samples from captive deer, and samples from wild fawns and one year old deer, very l i t t l e comparable material remained. Furthermore, as the majority of th i s remaining material consisted of one sample from one i n d i v i d u a l of each sex the numerical comparisons involving the mobility and the percent composition could not be made. These data are given only i n Appendices C & D. One possible electrophoretic c r i t e r i o n for comparing these subspecies and species i s the number of protein fractions (Table 34). There appear to be 9 to 10 fractions that 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 negatively migrating f r a c t i o n while the 0. virginianus 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 Island, B.C. have a t o t a l number of 10 f r a c t i o n s . This number i s the same as that found for two other subspecies, 0. h. crooki and 0. h. s i t k e n s i s . However, 0. h. columbianus from C a l i f o r n i a d i f f e r s from the other subspecies and from Vancouver Island specimens presently regarded as of the same subspecies i n having a t o t a l number of 11 f r a c t i o n s . Thus, the possible c r i t e r i o n of the - 81 -number of fractions for comparing subspecies or species of Odocoileus i s not v a l i d . I t has already been shown that 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 for quantitative comparison. These are 0. h. columbianus - Chemainus, Vancouver Island, B.C. (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 c o l l e c t e d 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 differences were exhibited i n the percent composit-ion but not i n the mobility of the protein f r a c t i o n s , these three groups were compared only on the basis of t h e i r m o b i l i t i e s . These numerical data are given i n Table 35. These groups are c l e a r l y separated into t h e i r respective taxonomic categories on the basis of morphology (Cowan, 1956, Kellogg, 1956). As each group of animals has i t s p a r t i c u l a r number of protein f r a c t i o n s , the fr a c t i o n s of each group were separated into 14 categories on the basis of th e i r m o b i l i t i e s (Table 36). Those fracti o n s of each group that belonged to the same category of mobility were then compared by means of Student's t - t e s t (Table 37). The r e s u l t s were further condens-ed into 4 categories: l a . the number of s i g n i f i c a n t d i f f e r -ences, l b . the number of times no comparison could be made - 82 -due to a f r a c t i o n occurring i n one group of animals but not i n the other, 2a. the number of s i m i l a r i t i e s , and 2b. the number of common absences. These categories gave r i s e to the t o t a l number of differences and s i m i l a r i t i e s between geographically separated groups of the subspecies, 0. h. columbianus, and between these and another species represent-ed by 0. v. texanus (Table 38). It was found that differences i n mobility were great-er between females from C a l i f o r n i a and Vancouver Island genotypes of Odocoileus hemionus than between either of these and the females of the species 0. virginianus represent-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 negatively migrating fractions i n the genus Odocoileus. Comparisons of mob i l i t i e s i n three groups of adult females of the genus Odocoileus indicate greater i n t r a - s p e c i f i c differences than i n t e r - s p e c i f i c differences. TABLE 34 83 GEOGRAPHIC SEPARATION, 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 . * 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. number of p o s i t i v e l y number of n e g a t i v e l y migrating f r a c t i o n s migrating f r a c t i o n s w i l d , female, a d u l t , O.hemionus columbianus, N = 2 9 1 Chemainus, B.C. w i l d , male, a d u l t , O.hemionus columbianus, N = 2 9 1 Chemainus, B.C. w i l d , female, a d u l t , O.hemionus columbianus, N = 5 10 1 C a l i f o r n i a w i l d , female, a d u l t , O.hemionus c r o o k i , N = 1 9 1 Ariz o n a w i l d , male, a d u l t , O.hemionus c r o o k i , N = 1 9 1 Ar i z o n a w i l d , male, a d u l t , O.hemionus s i t k e n s i s , N = 1 9 Kupreanof I s . , Alaska w i l d , male, a d u l t , 0 . v i r g i n i a n u s leucurus, N = 1 10 Oregon w i l d , female, a d u l t , 0 . v i r g i n i a n u s texanus, N = 8 10 Texas N = number of animals * For a l l samples, female, 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 percent composition 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 Tables 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 -5 2 EFFECT OF GEOGRAPHIC SEPARATION AND SPECIES - m o b i l i t i e s i n 10 cm / v o l t seconds. FRACTION 10 9 6 5 4 + 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 .829 . 4 6 4 .406 .371 .327 .254 .190 .158 .079 .015 .011 .143 + S.E. .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, ' C a l i f o r n i a 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. .775 . 4 2 1 .383 .340 .286 .221 .115 .080 .017 .013 .006 .006 .006 .001 .013 .007 .006 .006 .001 .001 N = number of animals 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 ategories "5 2 according to t h e i r m o b i l i t i e s ( i n 10 cm / v o l t seconds). MOBILITY o o o o o vo H O O LT\ O O O O LT\ O O + O - O O O L n o J O L n o u n h - o ' L n 0 0 0 w i l d , f e m a l e , a d u l t , 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 wil d , f e m a l e , a d u l t , 0.hemionus columbianus, C a l i f o r n i a 1 1 1 1 1 0 0 1 1 1 1 1 1 0 wil d , f e m a l e , a d u l t , 0.hemionus columbianus, Chemainus, B.C. 1 0 0 0 1 1 1 1 1 1 1 1 1 0 1 serum p r o t e i n f r a c t i o n present. 0 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 categories ( i n 10 cm / v o l seconds). MOBILITY o o o • O O O L O O O O O LP, O O + o - o o o o L n o j o L n o LO, i>- o LP, o o o ^ L O i ^ t - ^ t ^ J - 0 O 0 O CV) rH H O O rH 0J wild,female,adult 0 . v i r g i n i a n u s texanus, Texas vs. wild,female,adult, O.hemionus columbianus, C a l i f o r n i a 0 0 0 0 0 vs. wild,female,adult, O.hemionus columbianus, Chemainus, B.C. 0 0 wild , f e m a l e , a d u l t , O.hemionus columbianus, C a l i f o r n i a vs. w i l d , f e m a l e , a d u l t , 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 item 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 item to the .01 l e v e l . second item s i g n i f i c a n t l y slower than the f i r s t item to the .01 l e v e l . 0 p r o t e i n f r a c t i o n present i n one sample but not i n the other. / 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 i n 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 . * s i g n i f i c a n t no s u b t o t a l of common s u b t o t a l of d i f f e r e n c e s comparison d i f f e r e n c e s s i m i l a r i t i e s absence s i m i l a r i t i e s t o t a - ) * ( 0 • ) * ( = ) * ( / ) * 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, 0 + 5 = 5 9 + 0 = 9 14 C a l i f o r n i a vs. w i l d , female, a d u l t , O.hemionus columbianus, 2 + 2 = 4 8 + 2 = 10 14 Chemainus, B. C. w i l d , female, a d u l t , O.hemionus columbianus, C a l i f o r n i a vs. w i l d , female, a d u l t , O.hemionus columbianus, 2 + 5 = 7 6 + 1 = 7 14 Chemainus, B.C. * see Table 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 starch-gel electrophoresis, the serum proteins of as many subspecies of the genus Odocoileus as possible i n an attempt to c l a r i f y t heir phylogeny. The study showed that many independent variables a f f e c t the electrophoretic pattern of the serum. I t also showed that for taxonomic purposes clear yellow samples from at lea s t two wild individuals of the same sex and age of each subspecies should be obtained at i d e n t i c a l seasons. The effects of c a p t i v i t y and season indicate that the n u t r i t i o n a l h i s t o r y of the deer also has a major influence on the electrophoretic 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 to obtain t r u l y comparable samples that w i l l permit an examination of differences that may be considered of genetic o r i g i n and related to the evolution of d i f f e r e n t species or subspecies. There i s no published evidence to suggest that the same causes of v a r i a t i o n of non genetic nature do not occur i n other mammals. Under these circumstances one must approach with some skepticism the several published reports of apparent species related or subspecies related differences - 89 -(Cowan & Johnston, 19 62) between mammals. In t h i s study i t was possible to make v a l i d comparisons between 2 populations of b l a c k t a i l deer, 0. hemionus, both currently bearing the same subspecific designation, and a w h i t e t a i l 0. virginianus. The two species d i f f e r e d from each other i n the number of i d e n t i f i a b l e f r a c t i o n s . .0. virginianus had 2 instead of 1 negatively migrating fractions.- Comparing the two species on point of mobility the differences are of the same order of magnitude as they are between the 2 populations of 0. hemionus. I t i s of great i n t e r e s t that two populations of 0. hemionus presently known by the same subspecific name are shown to be markedly d i f f e r e n t from each other i n terms of serum protein mobility c h a r a c t e r i s t i c s . This supports other observations made at t h i s laboratory revealing that these two genotypes d i f f e r i n many features of body form, behaviour and physiology, and are probably as d i f f e r e n t from each other as any other two populations within t h i s species currently given taxonomic recognition. This study strongly suggests that electrophoretic studies of serum proteins indicate important differences between even the minor taxa. They are, therefore, of p o t e n t i a l value i n taxonomy. However, the large assortment - 90 -of factors that may be responsible for alt e r a t i o n s i n serum protein values of the same order as those that can be of taxonomic value make i t most d i f f i c u l t to obtain t r u l y comparable samples. Other body proteins may be less variable and of more value to the systematist. SUMMARY 1. After standardization of the starch-gel electrophoretic technique, v a r i a t i o n i n the serum proteins of the genus Odocoileus due to the condition of the sample and the condition of the animal could be studied. 2. S i g n i f i c a n t changes i n the serum sample were brought about by hemolysis, cloudiness, and decomposition. Cold storage of adult deer serum for two years, the addition of a bacteriostat to the sample, and the use of a muscle relaxant to procure samples from captive deer produced no s i g n i f i c a n t changes i n either the mobility or the percent composition of the protein f r a c t i o n s . 3. A large 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 mobility and the percent composition of the protein f r a c t i o n s . 4. The percent composition of the protein fractions was affected by sex, age, and season. The mobility of the protein f r a c t i o n s was affected by sex, but not by age or season. 5. Deer under the captive regimen at the University of B r i t i s h Columbia exhibited an additional negatively migrating protein f r a c t i o n when compared to t h e i r wild counterparts. 6. 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APPENDIX A -5 2 EFFECT OF CAPTIVITY - m o b i l i t i e s i n 10 cm / v o l t seconds. + FRACTION 9 8 7 6 5 4 3 2 1 1 2 w i l d , female, a d u l t , _ O.h. columbianus, N = 2 X .778 .501 .448 .382 .301 .258 . l 8 l .144 .015 .010 Wolf Lake, B.C. w i l d , male, a d u l t , _ O.h. columbianus, N = 2 X .839 .541 .447 .400 .345 .282 .216 .114 .019 .009 Wolf Lake, B.C. captive,female,adult, _ O.h. columbianus, N = 11 X .924 .517 .465 .439 .395 .297 .241 .164 .018 .015 .087 Wolf Lake, B.C. captive,male,adult, _ O.h. columbianus, N = 4 X .855 .656 .575 .465 .432 .375 .313 .128 .048 .017 .085 Wolf Lake, B.C. w i l d , male, a d u l t , _ O.h. s i t k e n s i s , N = 3 X .884 .540 .487 .421 .381 .335- .279 .176 .032 .010 Kupreanof Is.,Alaska captive,female,adult, _ O.h. s i t k e n s i s , N = 2 X .892 .582 .466 .436 .359 .262 .165 .068 .039 .010 .142 Petersburg,Alaska captive,male,adult, _ O.h. s i t k e n s i s , N = 2 X .961 .538 .452 .423 .336 .288 .163 .059 .012 .009 .163 N = number of a l i q u o t s from one sample from one animal H O Ul APPENDIX B EFFECT OF CAPTIVITY - percent composition i n a r c s ^ i n /percentage degrees. + FRACTION 9 8 7 6 5 4 3 2 1 1 2 w i l d , female, a d u l t , _ O.h. columbianus, N = 2 X 3 9 . l 26.O 10.3 6.2 8 .4 3.7 17.0 22.4 11.6 15.5 0 . 0 Wolf Lake, B.C. w i l d , male, a d u l t _ ' • O.h. columbianus, N = 2 X 32.6 20.4 12.3 10.4 12.6 10.9 18.4 20.6 15.8 2 0 . 3 0 . 0 Wolf Lake, B.C. captive,female,adult, _ O.h. columbianus, N = 11 X 32.0 12.6 14.3 9.4 ' 7 .4 10.6 11.1 19.8 2 0 . 8 18.8 22.0 Wolf Lake, B.C. captive,male,adult, _ O.h. columbianus, N = 4 X 38.6 9.1 12.0 18.2 13.7 6.7 8.4 15.5 2 2 . 9 8 .5 19.0 Wolf Lake, B.C. • ' w i l d , male, a d u l t , N = 3 X 38.6 10.4 19.8 12.9 8 .1 5.1 12.4 13.3 2 0 . 9 22.8 0 . 0 .O.h. s i t k e n s i s , Kupreanof Is.,Alaska captive,female,adult, _ O.h. s i t k e n s i s , N = 2 X 30.0 15.4 14.5 16.7 10.3 5.7 15.9 14.4 22.4 20.2 13.4 Petersburg, Alaska captive,male,adult, _ O.h. s i t k e n s i s , N = 2 _X. 36.7 10.1 10.1 10.7 7.7 11.2 12.4 13.5 26.2 21.4 14.6 Petersburg, Alaska N = number of a l i q u o t s from one sample from one animal H O APPENDIX C GEOGRAPHIC SEPARATION, SUBSPECIES, AND SPECIES - m o b i l i t FRACTION 10 9 8 7 6 5 w i l d , f e m a l e , a d u l t , 0.h.c olumb ianus, Wolf Lake, B.C._ N = 2 X .778 .501 .448 .382 .301 w i l d , male, a d u l t , O.h.columbianus, Wolf Lake, B.C._ N = 2 X .839 .541 .447 .400 .345 wild,male 1,adult, 0.h.c olumb ianus, Chemainus, B.C._ N = 3 X .843 .503 .414 .378 .324 wild,male 2 , a d u l t , O.h.columbianus, Chemainus, B.C._ N = 2 X .815 .485 .432 .388 .301 w i l d , f e m a l e , a d u l t , _0.h. c r o o k i , A r i z o n a _ N = 3 x .870 .504 .436 .4o4 .381 wild,male,adult, O.h. c r o o k i , A r i z o n a _ N = 2 X .878 .499 .427 .383 .359 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 .884 .540 .487 .421 .381 wild,male,adult, O.v. leucurus, Oregon N = 4 X .829 .606 .466 .403 .379 .339 N = number of a l i q u o t s from one sample from one animal -5 2 s i n 10 cm / v o l t seconds. + — 4 3 2 1 1 2 3 258 .181 .144 .015 .010 282 .216 .114 .019 .009 272 .170 .144 .009 .009 247 .160 .121 .034 .015 252 .171 .145 .035 .010 282 .165 .073 .019 .010 335 .279 .176 .032 .010 262 .204 .146 .010 .010 .063 .131 APPENDIX D GEOGRAPHIC SEPARATION, SUBSPECIES, AND SPECIES - percent composition In a r c s ^ i n /percentage degrees. + FRACTION 10 9 8 7 6 5 4 3 2 1 1 2 3 4 w i l d , f e m a l e , a d u l t , O.h.c olumb ianus, Wolf Lake, B.C._ N = 2 X 39.1 26.0 10.3 6.2 8.4 3.7 17.0 22.4 11.6 15.5 wild,male,adult, O.h.columbianus, Wolf Lake, B.C._ N = 2 X 32.6 20.4 12.3 10.4 12.6 10.9 18.4 20.6 15.8 2 0 . 3 wild,male 1 , a d u l t , > O.h.columbianus, Chemainus, B.C._ N = 3 X 44.7 21.5 14.7 8 . 8 7.7 5 .4 10.5 9.5 14.8 2 0 . 6 wild,male 2, a d u l t , i O.h. columbianus, Chemainus, B.C._ N = 2 X 44 .4 16.8 13.6 6.0 6.1 9.0 10.3 13.1 21.2 19.2 w i l d , f e m a l e , a d u l t , O.h. c r o o k i , A r i z o n a _ N = 3 X 37.1 17.2 9.2 9.1 9 .6 8 .2 8.7 15.0 23.6 26.2 wild,male,adult, O.h. c r o o k i , A r i z o n a _ N = 2 X 34.3 19.3 H . 8 10.7 10.0 6.7 12.9 12.6 21.2 2 8 . 3 | wild,male,adult, ! O. h . s i t k e n s i s , Kupreanof I s . , A l a s k a N = 3 X 38.6 10.4 19.8 12.9 8.1 5.1 12.4 13.3 20.9 22 .8 wild,male,adult, 0.y_. leucurus, Oregon N = 4 X 31.8 17.3 18.4 18.4 13.2 8.4 13.2 22 .4 13.3 4.9 6.6 5 .4 12.6 9.1 N = number of a l i q u o t s from one sample from one animal H O C O . 

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