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Some factors influencing the level of reducing sugar in the blood of black-tailed deer Whitehead, Philip Edward 1966

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SOME FACTORS INFLUENCING THE LEVEL OF REDUCING SUGAR IN THE BLOOD OF BLACK-TAILED DEER by PHILIP EDWARD WHITEHEAD B.Sc, University of B r i t i s h Columbia, 1962 A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements f o r the Degree of MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the standard required from candidates f o r the Degree of Master of Science. THE UNIVERSITY OF BRITISH COLUMBIA May, 1966 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 o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n Department The U n i v e r s i t y o f B r i t i s h C o l umbia Vancouver 8, Canada i i ABSTRACT Some of the factors that influence the blood reducing sugar l e v e l i n the b l a c k - t a i l e d deer Odeeoileus hemionus columbianus (Richardson) (Vancouver Island genotype), have been investigated. The d i s t r i b u t i o n of reducing sugar i n the blood of these animals was also examined. I t was found that: feed intake during the hour preceeding blood l e t t i n g , short periods of f a s t , nature of the feed, and sex of the animal apparently have no effect on the l e v e l of blood reducing sugar i n deer. Blood samples taken i n the evening generally had a higher reducing sugar l e v e l than those taken e a r l i e r i n the day. The means used to r e s t r a i n the animals during the blood l e t t i n g procedure was also found to have a marked influence on the l e v e l of blood reducing sugar. Deer restrained by physical force exhibited s i g n i f i c a n t l y higher and more variable blood sugar l e v e l s than those immobilized with succinylcholine. The length of time required to draw a blood sample from an animal also influenced the blood sugar l e v e l . The longer the time to l e t a sample, the higher the blood sugar l e v e l i n the sample. The resu l t s indicate that the degree of excitement, fear, and pain experienced by the animals preceeding and during the blood l e t t i n g procedure was the p r i n c i p a l cause of v a r i a b i l i t y found i n the l e v e l of blood reducing sugar. Wo reducing sugar could be detected i n the erythrocytes of these deer. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 EXPERIMENTAL 3 Animals 3 N u t r i t i o n 3 Housing 4 A n a l y t i c a l Techniques 4 Blood c o l l e c t i o n 4 Serum extraction and storage 5 Hematocrits 5 Blood reducing sugar determination 5 EXPERIMENT 1 7 EXPERIMENT 2 32 EXPERIMENT 3 42 EXPERIMENT 4 47 RESUME 49 BIBLIOGRAPHY 53 APPENDICES 1 Glucose metabolism i n ruminants 59 2 Hormonal regulation of blood reducing sugar 61 XV TABLE OF CONTENTS (cont.) APPENDICES (cont.) 3 Succinylcholine chloride 63 4 Ingredients of Peebles V f l e r milk replacer 64 5 Ingredients of the weaning r a t i o n 65 6 Ingredients of the adult r a t i o n 66 7 Absorption curve f o r the Nelson-Somogyi method of 67 determining blood glucose 8 Standard curve f o r the Nelson-Somogyi method of 69 determining blood glucose 9 Recovery of glucose from serum 72 10 Blood sampling record form. 73 LIST OF TABLES TABLE PAGE 1 Treatment for experiment 1 8 2 Results of experiment 1 10 3 Student t test comparing serum reducing sugar levels of fasted and nonfasted deer. 14 4 Student t test comparing serum reducing sugar levels of deer immobilized with succinylcholine and those restrained physically. 18 5 F test for determi ning the reproducibility of serum reducing sugar levels by the two methods of restraint. 20 6 Student t test comparing the serum reducing sugar levels of male deer to those of female deer. 23 7 The influence of time of day on serum reducing sugar levels in deer. 26 8 Student t test comparing serum reducing sugar levels of deer sampled at 0800, 1200, and 1600 hours to those sampled at 2000 hours. 28 8A Feed intake, serum reducing sugar levels, and weight data of U34 throughout experiment 2. 35 9 Feed intake, serum reducing sugar levels, and weight data of U43 throughout experiment 2. 36 10 Feed intake, serum reducing sugar levels, and weight data of U16 throughout experiment 2. 37 11 Feed intake, serum reducing sugar levels, and weight data of U24 throughout experiment 2. 38 12 Reducing sugar levels in sequential blood samples. 43 13 The distribution of reducing sugar in the blood of deer. 48 14 A comparison of blood sugar values reported for deer. 50 v i LIST OF FIGURES FIGURE PAGE 1 The relationship between serum reducing sugar l e v e l s and feed intake the hour p r i o r to blood sampling. 12 2 A comparison between the serum reducing sugar l e v e l s of fasted and nonfasted deer. 15 3. A comparison between the serum reducing sugar l e v e l s of deer restrained p h y s i c a l l y and those immobilized with succinylcholine. 19 4 A comparison between the serum reducing sugar l e v e l s of male and female b l a c k - t a i l e d deer. 24 5 Comparing serum reducing sugar l e v e l s found i n deer at diff e r e n t times during the day. 27 6 I l l u s t r a t i n g the relationship that can occasionally occur between the serum reducing sugar l e v e l s of two animals sampled at the same times on the same day. 31 7 I l l u s t r a t i n g the changes i n serum reducing sugar l e v e l s that occurred i n deer on p e l l e t s and those on hay. 33 8 The effect of immobilization and blood sampling on serum reducing sugar l e v e l s . 44 9 Absorption spectrum: Nelson-Somogyi method of determining blood sugar. 68 10 Standard curve: Nelson-Somogyi method of determining blood sugar. 71 v i i ACKNOWLEDGEMENTS I wish t o thank Dr. I . McT. Cowan, Dean of Graduate Studies, (formerly Head of the Department of Zoology) f o r his permission to undertake t h i s project, f o r supporting i t , and for the use of the f a c i l i t i e s of the Department of Zoology. My thanks also to Dr. A.J. Wood, Dean of Arts and Science at the University of V i c t o r i a , (formerly Professor of Animal Science at the University of B r i t i s h Columbia) for the motivation he applied during the development of t h i s work. To Dr. H.C. Nordan, Assistant Professor i n the Department of Zoology, I wish to express sincere thanks for the i n t e r e s t , encouragement, guidance, and philosophy that he provided throughout our association. F i n a l l y I wish to acknowledge the assistance of Mr. R. Addison and Mr. D. Leckenby, who made the load that much easier to carry. INTRODUCTION 1 INTRODUCTION Subjective descriptions of the relationship of deer and habitat have dominated game management literature for many years. This material has extended our knowledge of these animals and provided the understanding essential for the development of needed management techniques. However, in keeping with the contemporary movement of research to intensive investig-ation, deer, more than ever before, are being studied as unique physiological systems manifesting profound cyclic phenomena. Documentations of changes in antler growth, feed intake, body weight, hormonal levels, and other physiological parameters, coincident with the seasonal sexual cycle are becoming priminant in the literature. French et al (1955), Cowan et al (1955), Kitts et al (1956), Magruder et al (1957) have a l l studied various aspects of the growth and nutrition of deer. Silver et al (1959) reported metabolic rates. Rosen and Bischoff (1952), Kitts et al (1956), Bandy et a l (1956), Terri et al (1958), and Touatt et al (19#5) have carried out studies on the haematology of deer under various circumstances. Investiga-tions into bone and antler development have been reported by Bernhard et al (1953), Meister (1956) and Long (1959). Robinson et al (1965) studied the reproductive cycle of male white-tailed deer. Investigations into the hormonal system of deer have been reported by Browman and Sears (1956), Grieser and Browman (1956), Tyler (1961), and Dawson (1963). French et al (i960) noted the response of white-tailed bucks to added a r t i f i c i a l light. As one reviews literature of this nature i t is apparent that in some cases the investigator has failed to appreciate the f u l l effect of his experimental manipulation on the animal. Techniques of handling, 2 r e s t r a i n i n g , and sampling, used with success on domestic animals are often misapplied to w i l d species. As a consequence results often r e f l e c t the state of agitation of the animal rather than i t s normal, undisturbed, physiological functioning. I n i t i a l l y t h i s study was intended to explore alterations i n the l e v e l of blood reducing sugar that might occur i n deer during the profound physiological changes that are associated with r u t . The findings provide information regarding the factors responsible f o r v a r i a b i l i t y i n the blood reducing sugar l e v e l s i n deer, and also some i n d i c a t i o n of the normal range of these l e v e l s . They also serve as a caution to those engaged i n measuring physiological parameters i n w i l d animals. Goodwin (3956) pointed out that the blood sugar of adult ruminants i s almost exclusively glucose. He suggested, however, that the expression *blood reducing sugar* i s more appropriate as i t acknowledges the other reducing sugars (mostly fructose) thought to be present i n small amounts. This terminology was adopted and appears throughout the thesis . Expression of reducing sugar l e v e l s as mgm.# i n serum rather than as mgm.$ i n whole blood was done f o r two important reasons: 1. i t i s the concentration of sugar i n the serum and ti s s u e f l u i d that the body c e l l s respond t o , and 2. changes i n the d i s t r i b u t i o n of t i s s u e f l u i d can occur rapidly, a l t e r i n g the packed c e l l volume, and subsequently the reducing sugar l e v e l i f expressed i n terms of whole blood. EXPERIMENTAL 3 EXPERIMENTAL 1. ANIMALS Four deer, two male (U16 and U43) and two female (U24 and U34) of the species Odecoileus hemionus columbianus (Vancouver Island genotype) were used for these experiments. The animals were captured as fawns in the spring of 1963 in the vicinity of Courtenay on Vancouver Island, B.C., Canada. Shortly after capture they were shipped, by air, to the Zoology Vivarium on the campus of the University of British Columbia. During the period of experimentation from December 1964 to March 1965 the deer were in the late stages of rut. Normally this is a period when the animals, particularly males, are difficult to handle. However, the close care and attention given them by the experimenter rendered them unusually tractable throughout the experiments. 2. NUTRITION The experimental animals were fed a commercial milk replacer (Appendix 4) until they had reached a weight of approximately 15 pounds. The weaning process was then initiated and the animals were maintained on the weaning ration (Appendix 5) until early September. They were then placed on the adult ration (Appendix 6) which was fed throughout the remainder of the experimental period. Some modifications were introduced as the investiga-tions directed. Animals U16 and U24 were given chopped alfalfa hay exclusively from January 21 1965 to February 12 1965 and from January 21 1965 to March 23 1965, respectively. 4 3. HOUSING The animals used i n t h i s study were housed i n i n d i v i d u a l pens. As fawns they were held i n 4 f by 2 f wooden pens located i n a ground l e v e l steam heated room i n the Vivarium. At weaning the deer were moved to larger pens, 3* by 6* i n the same room. By October 1963 the s i z e of the deer and the onset of rut necessitated a move to the W i l d l i f e Unit (Wood et a l 1 9 6 l ) where they were housed i n adult penes These pens were l a t e r modified to include a s l a t t e d f l o o r . 4 . ANALYTICAL TECHNIQUES ( i ) Blood Collection Blood samples were taken from the jugular vein. Occasionally, when t h i s proved d i f f i c u l t , samples were drawn from the recurrent t a r s a l vein. Blood was l e t i n t o untreated, evacuated, 10 cc. vacutainer tubes*. A record was made of the time required f o r each aspect of every blood drawing operation. f 0 * time was considered to be the i n i t i a l i n t r u s i o n of the technician on the animal i . e . as the pen door was opened. tInjection» time was then noted as the immobilizing agent was administered. The a c t i v i t y i n t h i s i n t e r v a l involved ushering the animal down the h a l l t o the laboratory and the mln1ma,1 r e s t r a i n t necessary f o r i n j e c t i o n . The succeeding notation came as the animal collapsed due to the action of the drug. During the i n t e r v a l between 'inje c t i o n * and ,downT time the deer usually stood q u i e t l y , apparently undisturbed. Most a c t i v i t y i n t h i s period occurred as the animal fought the effects of the drug. The time that blood was f i r s t released i n t o the vacutainer and the time the * B.D. Vacutainer, Beeton, Dickinson and Co., Rutherford, N.J., U.S.A. 5 vacutainer was f i l l e d were also noted. See Appendix 10 f o r an example of the form used. ( i i ) Hematocrits When circumstances allowed, t r i p l i c a t e blood samples were taken i n heparinized c a p i l l a r y tubes f o r the purpose of determining the packed c e l l volume. These samples were a l l taken with a single i n s e r t i o n of the vacutainer needle when a sample f o r glucose analysis was drawn. ( i i i ) Serum Extraction and Storage After c o l l e c t i o n , the blood samples were allowed to stand undisturbed fo r t h i r t y minutes. The tubes were then Ringed* with a wood dowel, freeing the c l o t from the walls of the vessel. They were then placed i n a c e n t r i -fuge* and spun at high speed f o r ten minutes. Following t h i s the serum was c a r e f u l l y drawn o f f the c l o t with a t h i n bore pipette and placed i n t o clean, dry, t est tubes. The serum samples were immediately put i n a freezer and stored u n t i l analyzed. (i v ) Blood Glucose Determination Of the methods f o r determining blood reducing sugar investigated, a Nelson-Somogyi technique modified to s u i t our requirements was chosen. This method was found to provide accurate reproducible results and i s , therefore, described i n d e t a i l . 0.2 cc. of serum was pipetted i n t o 3.0 cc. of d i s t i l l e d water. Deproteinization with 2.0 cc. of 0.06 N ZnSO^ and 2.0 cc. of 0.06_N Ba(0H) 2 followed. I t should be noted that the amount and concentration of these solutions i s not c r i t i c a l but can be varied to accomodate p a r t i c u l a r situations (Somogyi 1945)• As the amount of glucose i n t h i s procedure must not be too high, 2.0 cc. of the serum f i l t r a t e was added to 2.0 cc. * International Model HN, Needham Hts., Mass., U.S.A. 6 of the combined al k a l i n e t a r t r a t e and copper solution (Somogyi 1945). The t a r t r a t e and copper solutions were prepared separately and mixed as required as the mixed reagents show some degree of autoreduction. (Somogyi 1952). After heating i n a b o i l i n g water bath f o r exactly ten minutes followed by immediate cooling to 20 C i n a stream of cold water, the reaction mixture was combined with 2.0 cc. of Nelsons arsenomolybdate reagent (Nelson 1944). The volume was made up to 10 cc. (addition of 4.0 cc. of d i s t i l l e d water) and the color was allowed to develop f o r at least 15 minutes. The transmittance was then measured with a Beckman Model D.U. Spectrophotometer at 650 mu. An absorption curve from 400 mu to 1,000 mu of the coloured end product of the procedure was determined and appears i n Appendix 7« Three standard glucose solutions at concentration of 40, 70, and 100 mgm.# were prepared (Natelson 1961). One of the standards was run with every series of unknowns. Ultimately a number of r e p e t i t i v e determinations of each standard solution was made and used to prepare a standard curve. (Appendix 8). (v) Internal Standard A standard glucose solution (70 mgm.$) was mixed with a serum sample at three different l e v e l s : 2:1, 1:1, and 1:2. The mixtures were frozen and stored f o r a few days p r i o r to analysis. The results appear i n Appendix 9. EXPERIMENT 1 7 EXPERIMENT 1 This experiment was designed to investigate the effects of feed intake, means of r e s t r a i n t , sex of the animal, and time of day, on blood reducing sugar l e v e l s i n deer. Four animals were placed on a feeding regime such that each was offered 1/4 of 80% of i t s d a i l y intake (calculated from the average d a i l y intake of the previous week), at each of O70O, 1100, 1500, and 1900 hours. The d a i l y allowance was cut to 80% of normal so that the deer would clean up each feeding almost immediately after i t was offered. The animals were allowed a week to become accustomed to t h i s program before the experiment was begun. By t h i s means some control was exercised over the amount and time of feed intake p r i o r to blood l e t t i n g . Animals *on feed 1 were main-tained on t h i s regime. Deer f o f f feed 1 received t h e i r usual allotment at 1900 hours the day before blood l e t t i n g . They were given no feed throughout the day that blood was being drawn, the next feeding occurring at 0700 hours the succe<#ding day. In effect then, blood samples were drawn from these animals a f t e r fasts of 12, 16, 20, and 24 hours. The deer were restrained during the blood l e t t i n g procedure by one of two means; immobilization with succinylcholine or physical force. When the l a t t e r method was used, two or three technicians would sieze the animal (recorded as time 'grabbed*), wrestle i t down (time 'held*), and so hold i t u n t i l the operation was completed. Blood was drawn at 0800, 1200, 1600, and 2000 hours from the animals as indicated i n table 1. To assess the influence of each variable, i . e . fon feed* vs. 'off feed* the data was grouped under these headings ir r e s p e c t i v e of the other TABLE 1; TREATMENT FOR EXPERIMENT 1 Date Animals Restraint Feeding Regime 16/ 9/64 16.24 physical on 22/10/64 34.43 physical on 26/10/64 16.24 succinylcholine on 27/10/64 34.43 sue cinylcholine on 2/11/64 16.24 physical o f f 3/11/64 34.43 physical o f f 4/H/64 16.24 sue cinylcholine o f f 25/11/64 34.43 sue cinylcholine o f f 9 variables. In the cases where i t was applicable a Student * t f test was applied to the data to determine the significance of any differences noted. (l) Influence of Feed Intake As can be seen from table 2 and figure 1 there is no correlation between feed intake the hour prior to blood letting and serum reducing sugar level. That monogastric animals exhibit a postprandial hyper-glycemia associated with an increase in glucose absorption through the gut is well documented. Such a rise is not found in cattle (Hodgson et al 1932), or in sheep (Allcroft and Strand 1933). Moreover, i t has been shown that the oral administration of glucose in sheep (Schambye 1951) and cattle (Bell and Jones 1945) does not cause an increase in the blood reducing sugar. The explanation for this relative constancy is based on two facts. Firstly, starch and cellulose, the principal carbohydrates found in the diet of ruminants, are rapidly degraded in the rumen by a variety of microorganisms to simple sugars; mostly glucose. These sugars are fermented to short chain volatile fatty acids which are then absorbed into the energy metabolism of the animal. The major source of ruminant blood reducing sugar was once believed to result from acidic and enzymatic digestion of the carbohydrate of ruminal bacteria and protozoa. This degradation occurs in the abomasum and small intestine, and the liberated glucose is subsequently absorbed. Heald (1952), however, investigated this pathway and found, using sheep fed hay, that no more than 20 gms. of glucose per day could be produced in this manner, Later work by Annison et al (1957) supported this finding. Although glucose can be absorbed directly from the rumen as first shown by Rankin (1940), i t i s unlikely that any great amount TABLE 2; RESULTS OF EXPERIMENT 1: PART 1 THE EFFECTS OF FEED INTAKE ON SERUM REDUCING SUGAR LEVELS IN BEER. Feeding Program Animal Feed Intake (gms.) Serum Sugar (mgm.jS) On U16 426 91.2 426 90.4 426 96.8 426 88.7 182 88.4 182 90.1 182 91.2 182 87.6 U24 283 83.6 198 86.5 142 100.3 57 90.9 3 4 0 89.2 3 4 0 81.6 3 4 0 85.9 266 92.1 U 3 4 66 1 0 2 . 2 73 91-5 87 78.6 76 109.2 250 82.9 250 78.6 250 93.6 U 4 3 228 81.9 226 82.9 328 90.9 2 0 4 78.1 68 81.1 79.5 — 77.0 83.1 Average serum reducing sugar l e v e l *on feed': 2725.6 = 87.9 mgm./K. 31 TABLE 2: (cont.) Feeding Program Animal Hours of Fast Serum Sugar (mgm.5) Off U16 12 85.2 85-4 U24 92.1 75-4 U34 81.6 79.0 U43 81.6 76.3 U16 16 83.0 83.1 U24 90.4 80.7 U34 103.0 83.6 U43 102.5 84-9 U16 20 87-6 78.6 U24 92.1 75.2 U34 82.4 80.9 U43 79-7 88.9 U16 24 86.2 U24 97.1 U34 98.4 87.3 U43 88.7 Average serum reducing sugar level after: (1) 12 hours fast 656.6 =82.1 mgm.# 8 (2) 16 hours fast 711.3 =88.9 mgm.# 8 (3) 20 hours fast 665.4 =83.2 mgm.# 8 (4) 24 hours fast 555.1 =92.5 mgm.# 6 97-4 FIGURE 1: THE RELATIONSHIP BETWEEN SERUM REDUCING SUGAR LEVEL AND FEED INTAKE DURING THE HOUR PRIOR TO BLOOD SAMPLING. 160 150 140 130 < O 3 </> o z o Z> Q 5 Of 120 110 E J 100 90 80 70 60 50 40 30 20 10 0 40 60 100 140 180 220 260 FEED INTAKE (gm.) 300 340 380 420 460 13 follows t h i s route. The glycemic l e v e l of ruminants then i s not nearly so much the r e f l e c t i o n of exogenous glucose absorption that i t i s i n monogastric animals but i s l a r g e l y a product of gluconeogenesis. Secondly, the large s i z e of the rumen and an associated slow rate of passage results i n an even flow of ingesta through the gut. This flow i s r e l a t i v e l y unaffected by temporary changes i n the f i l l of the rumen. This explains i n part why there i s no s i g n i f i c a n t difference (0.200 =»P >0.400) between blood reducing sugar l e v e l s when the animals were on feed as compared to the l e v e l a f t e r 12, 16, 20, and 24 hours of fast (table 3). Figure 2 i l l u s t r a t e s the negligable effect of these r e l a t i v e l y short periods of fast on the blood seducing sugar l e v e l of deer. The effect of fas t i n g on blood reducing sugar l e v e l s i n ruminants i s variabl e . A l l c r o f t and Strand (1933) noted that a seven day fast had l i t t l e e ffect on sheep. Magee (1932), on the other hand, recorded a r i s e i n blood reducing sugar i n goats during a seven day fa s t . Robertson (i960) also found a substantial increase i n the l a c t a t i n g cow. Hodgson et a l (1932), however, noted a considerable decrease i n the l e v e l i n dairy c a t t l e during a seven day f a s t . Reid (1950) recorded that 45 - 46 hour fasts have l i t t l e e ffect on blood reducing sugar l e v e l s i n sheep but that longer periods produced s i g n i f i c a n t decreases. Had the deer used i n t h i s experiment been fasted longer perhaps a si m i l a r effect would have been noted. TABLE 3: RESULTS OF EXPERIMENT 1: PART 1 STUDENT t TEST COMPARING THE SERUM REDUCING SUGAR LEVELS OF FASTED AND  NONFASTED DEER. Animal Treatment Nonfasted Fasted (12-24 hours) X^ x-j-X-x x?£ Xg X2-X-X U16 91.2 3.3 10.89 85.2 1.0 1.00 90.4 2.5 6.25 85.4 .8 .64 96.8 8.9 79.21 83.0 3.2 10.24 88.7 .8 .64 83.1 3.1 9.61 88.4 .5 225 87.6 1.4 1.96 90.1 2.2 4.84 78.6 7.6 57.76 91.2 3.3 10.89 86.2 — 87-6 .4 .16 U24 83.6 4.3 18.49 92.1 5-9 34.81 86.5 1.4 1.96 75.4 10.8 116.64 100.3 12.4 153.76 90.4 4-2 17.64 90.9 3.0 9.00 80.7 5.5 30.25 89.2 1.7 2889 92.1 5.9 34.81 81.6 6.3 39.69 75.2 11.0 121.00 85.9 2.0 4.00 97-1 10.9 118.81 92.1 4-2 17-64 U34 102.2 14.3 204.49 81.6 4.6 21.16 91.5 3.6 12.96 79.0 7.2 51.84 78.6 9.3 86.49 102.9 16.8 282.24 109.2 21.3 453.69 83.6 2.6 6.76 82.9 5.0 25Q00 82.4 3.8 14-44 78.6 9.3 86.19 80.9 5-3 28.09 93-6 5.7 32.49 98.4 12.2 148.84 8723 1.1 1.21 U43 81.9 6.0 36.00 81.6 4.6 21.16 82.9 5.0 25-00 76.3 9.9 98.01 90.9 3.0 9.00 102.6 16.4 268.96 78.1 9.8 96.04 84-9 1.3 1.69 81.1 6.8 46.24 79.7 6.5 42.25 79.5 8.4 70.56 88.9 2.7 7-29 76.9 11.0 121.00 88.7 2.5 6.25 83.1 4.8 23.04 97-4 11.2 125.44 Xx and X2 = serum reducing sugar level (mgm./S) n x = 30, n 2 = 31, xx = 86.2, x 2 = 87-9, d.f. = 59, x 2 - x x = 1.1 = 1,680.80 <fx| = 1,688.75 s 2 = 3,369-55/59 = 57-11 Sx x - x 2 = v/57-H(6l)/930 = 1.94 t = 1.7 = 0.876 ,400<P< .200 1.94 FIGURE 2: A COMPARISON BETWEEN THE SERUM REDUCING SUGAR LEVELS OF FASTED AND NONFASTED DEER SERUM REDUCING SUGAR (mgrn. X) to o o o o oo o o o o o o z I o O c TO CO > 16 (2) Means of Restraint This study was designed to investigate the effect of the means of re s t r a i n t used to f a c i l i t a t e blood sampling on the l e v e l of serum reducing sugar i n deer. Two methods of r e s t r a i n t were investigated: physical force, and immobilization with succinylcholine. Deer p h y s i c a l l y restrained were thrown and held down by the weight of two or three technicians. The process of throwing the animals generally entailed some f i g h t i n g , but there was l i t t l e struggle once they were down and a good hold was secured. I t usually took l e s s time from the f i r s t i n t r u s i o n to drawing of the sample using t h i s technique of r e s t r a i n t compared to succinylcholine. A deer administered a suitable dose of succinylcholine (Cowan 1962), (Nordan, 1962) exhibits a reasonably characteristic response to the onset of paralysis. The 2-3 minutes following i n j e c t i o n i t remains r e l a t i v e l y quiet. Then, just p r i o r to collapse, a b r i e f period of s t i f f - l e g g e d walking, chewing, back-humping and muscle spasms occurs. An inexperienced animal w i l l f i g h t the paralysis and remain standing as long as possible. I t s uncontrolled collapse i s immediately followed by complete relaxation. An experienced animal, on the other hand, does not fi g h t t o the same extent and i t s collapse i s generally more controlled. I t i s worthy of note that the animals used i n t h i s experiment were experienced. Control of the musculature of the jaw, neck, and extremities, usually returned 15-20 minutes a f t e r collapse, and the animal would regain i t s feet apparently unaffected by the paralysis. The differences i n behaviour e l l i c i t e d by the two methods of r e s t r a i n t are c l e a r l y r e f l e c t e d i n the response of the blood sugar. As can be seen 17 from table 4 s i g n i f i c a n t l y lower serum reducing sugar l e v e l s (0.001>P^>0.005) r e s u l t when deer are restrained using succinylcholine as compared to when they are restrained using physical force. The difference i s i l l u s t r a t e d i n figure 3- Moreover, i t i s noteworthy that s i g n i f i c a n t l y l e s s variable l e v e l s are obtained using succinylcholine. This c a l c u l a t i o n i s presented i n table 5-The difference i n blood reducing sugar l e v e l s obtained by the two methods of re s t r a i n t might be accounted f o r i n two ways. F i r s t l y , the degree of muscular energy expended by the animals p h y s i c a l l y restrained i s considerably greater than when succinylcholine immobilization i s used. Solandt and Ferguson (1932) investigated the effects of strenuous exercise of short duration on blood reducing sugar i n man. They found that 30-45 seconds of standing running at top speed caused a noticable increase i n blood reducing sugar f i v e minutes l a t e r . Reichard et a l (1961) using -^C labled glucose noted that there was an increased uptake of blood sugar by working muscle, compensated by increased hepatic glucose output. The struggle of the physically restrained deer, then, could account i n part f o r the high blood sugar l e v e l s recorded. Secondly, though i t i s impossible to assess with certainty the degree of f r i g h t and pain experienced by the animals when restrained by either method, i t i s undoubtedly true that physical r e s t r a i n t i s more traumatic than immobilization with succinylcholine. Therefore, the difference i n blood reducing sugar l e v e l s obtained by the two methods of r e s t r a i n t , might r e f l e c t the degree of stimulation of the sympathicoadrenal complex. The acti v a t i o n of t h i s system has been shown to occur i n almost a l l types of stress situa t i o n s . The fear and pain associated with both means of r e s t r a i n t , then, t r i g g e r the complex r e s u l t i n g 18 TABLE 4: RESULTS OF EXPERIMENT 1; PART 2 STUDENT t TEST COMPARING SERUM REDUCING SUGAR LEVELS OF DEER IMMOBILIZED  WITH SUCCINYLCHOLINE AND THOSE RESTRAINED PHYSICALLY. A n i m a l Treatment U 16 Succinylcholine Physical X l X2=X-x 88.4 4-4 19.36 91.2 1.4 1.96 90.1 6.1 37.21 90.4 .6 •36 91.2 7.2 51.84 96.8 7.0 49.00 87.5 3.5 12.25 88.7 1.1 1.21 85-4 1.4 1.96 85.2 4-6 21.16 83.1 .9 .81 83.0 6.8 46.24 78.6 5.4 29.16 87-6 2.2 4-84 86.2 3-6 12.96 89.2 5.2 27-04 83.6 6.2 38.44 81.6 2.4 5.76 86.5 5-3 10.89 85.9 1.9 3.61 100.3 10.5 110.25 92.1 8.1 65.61 90.9 1.1 1.21 75-4 8.6 73-96 92.1 2.3 5-29 80.7 3.3 10.89 90.4 .6 .36 75-2 8.8 77-44 92.1 2.3 5.29 97.1 7.3 53.29 82.9 1.1 1.21 102.2 12.4 153.76 78.6 5.4 29.16 91.5 1.7 2.89 93.6 9.6 92.16 78.6 11.2 125.44 79-0 5.0 25-00 109.2 19-4 376.89 83.6 .4 .16 81.6 8.2 67.24 80.9 3-1 9.61 103.0 13.2 174.24 87-3 3-3 10.89 82.4 7.4 54-76 98.4 8.6 73.96 81.1 2.9 8.41 81.9 7.9 62.41 79.5 4-5 20.25 90.9 1.1 1. 21 76.9 7.1 50.41 78.1 11.7 136.89 83.1 .9 .81 82.9 6.9 47.61 76.3 7-7 59.29 81.6 8.2 67.24 84-9 .9 -81 102.6 12.8 163.84 88.9 4.9 24.01 79.7 10.1 102.01 97-4 13.4 179-56 88.7 1.1 1.21 and X2 = serum reducing sugar l e v e l (mgm.#). ^=29, n2=32, x1=84-0, x2=89.8, *2~\ =5-8 £x2 = 928.64, = 1,973-82, d.f. = 59 s 2 = 2,902.46/59 = 49.19, Sxj-xf, = \/49.19(6l)/928 = 1.80 t = 5.8 = 3-222 . 005<P<.001 1.8 FIGURE 3: A COMPARISON BETWEEN THE SERUM REDUCING SUGAR LEVELS OF DEER RESTRAINED PHYSICALLY AND THOSE IMMOBILIZED WITH SUCCINYLCHOLINE. TABLE 5: RESULTS OF EXPERIMENT 1; PART 2 F TEST* FOR DETERMINING THE REPRODUCIBILITY OF SERUM REDUCING SUGAR LEVELS BY THE TWO METHODS OF RESTRAINT. Animal ^ X 2 Xg X 2 U16 88-4 7,814.56 91.2 8,317-44 90.1 8,118.01 90.4 8,172.16 91.2 8,317-44 96.8 9,370.24 87.5 7,656.25 88.7 7,867.69 85.4 7,293.16 85.2 7,259.04 83.1 6,905.61 83.0 6,889.00 78.6 6,177-96 87.6 7,673-76 86.2 7,430.44 U24 89.2 7,956.64 83.6 6,988.96 81.6 6,658.56 86.5 7,482.25 85-9 7,378.81 100.3 10,060.09 92.1 8,482.41 90.9 8,262.81 75-4 5,685.16 92.1 8,482.41 80.7 6,512.49 90.4 8,172.16 75-2 5,655-04 92.1 8,482.41 97.1 9,428.41 U34 82.9 6,872.41 102.2 10,444-84 78.6 6,177-96 91-5 8,372.25 93-6 8,760.96 78.6 6,177-96 79.0 6,241.00 109.2 11,924.64 83.6 6,988.96 81.6 6,658.56 80.9 6,544.81 103.0 10,609.00 87.3 7,621.29 82.4 6,789-76 98.4 9,682.56 U43 81.1 6,577-21 81.9 6,707.61 79.5 6,320.25 90.9 8,262.81 76.9 5,916.61 78.1 6,099.61 83.1 6,905-61 82.9 6,872.41 76.3 5,821.69 81.6 6,658.56 84.9 7,208.01 102.6 10,526.76 88.9 7,903.21 79-7 6,352.09 97.4 9,486.76 88.7 7,867.69 X-^  and X2 = serum reducing sugar l e v e l (mgm.#) (x\ = 205,955.84, f x | = 260,346.38 ( ( \ ) 2 = 5,945,794-56, ( ^ X g ) 2 = 8, 267,925.16, d.f.x=28 d.f.2=31 6? - ( ft)2 n F= $ L F = 63.66 = 1.919 n 02 3 3 - 1 6 * Natelson, S. Microtechniques of C l i n i c a l Chemistry (1961) pp.493• C.C. Thomas Spr i n g f i e l d I l l i n o i s , U.S.A. 21 in manifold physiological responses. For example, heart rate, systolic pressure, cardiac output, blood flow through the liver, brain, kidney, and musculature are undoubtedly increased. On the other hand, the activity of the intestine and genital system are slowed. Of principal importance the adrenal medulla is stimulated to cause an increased release of epinepherine. Epinepherine augments the effects of stimulation of the sympathetic system and ell i c i t s an important hyperglycemic response. It also stimulates the adenohypothesis to release ACTH which increases the release of adrenal cortical hormones, potent in promoting gluconeogenesis from protein (Turner I960). Reid (1962) attempted to correlate blood sugar and plasma Cortisol levels in sheep under stress but was unsuccessful. He assumed that the hyperglycemia noted was an effect mediated by epinepherine rather than by Cortisol. E l l i s (1956) stated that the metabolic effects of epinepherine on carbohydrate metabolism are far too complex to allow any precise conclusions as to the metabolic significance of the variations in blood sugar recorded. The data presented in this experiment indicate that the means used to restrain deer during the blood letting procedure has a pronounced effect on the level of serum reducing sugar. Immobilization with succinylcholine results in lower and less variable serum reducing sugar levels. 22 (3) The I n f l u e n c e o f Sex The d a t a o f t h i s experiment ( t a b l e 6 ) , ( f i g u r e 4) i n d i c a t e t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e ( . 2 0 0 > P > .400) between t h e serum r e d u c i n g sugar l e v e l s o f bucks as compared t o t h o s e o f does . T h i s r e s u l t i s suppor ted by t h e f a c t t h a t no d i f f e r e n c e i n b l o o d r e d u c i n g sugar l e v e l s between t h e sexes has been r e p o r t e d i n c a t t l e (Hodgson et a l , 1 9 3 2 ) , i n c a l v e s ( V o e l k e r , 1 9 5 5 ) , o r between wethers and ewes ( R e i d , 1 9 5 0 ) . As i n d i c a t e d i n t a b l e 1 t h e m a j o r i t y o f t h e b l o o d samples f o r t h i s experiment were drawn i n l a t e October and e a r l y November. A l though t h e bucks were e n t e r i n g t h e r u t t i n g p e r i o d a t t h i s t i m e t h e r e was l i t t l e ev idence o f a g g r e s s i v e behav iou r , and no e f f e c t on serum r e d u c i n g sugar l e v e l s o c c u r r e d . 23 TABLE 6: RESULTS OF EXPERIMENT 1: PART 3 STUDENT t TEST COMPARING THE SERUM REDUCING SUGAR LEVELS OF MALE DEER TO  THOSE OF FEMALE DEER. Male Female x 1 =X - x £ Xg=X-x 91.2 5.1 26.01 83.6 4.4 19.36 90.4 4-3 18.49 86.5 1.5 2.25 96.8 10.7 114.49 100.1 12.1 146.41 88.7 2.6 6.76 90.9 2.9 8.41 88.4 2.3 5-39 89-2 1.2 1.44 90.1 4.0 16.00 81.6 6.4 40.96 91.2 5.1 26.01 85.9 2.1 4-41 87.6 1.5 2.25 92.1 4.1 16.81 85.2 .9 .81 92.1 4.1 16.81 83.0 3.1 9.61 90.4 2.4 5.76 87.6 1.5 2.25 92.1 4.1 16.81 86.2 .1 .01 97.1 9.1 82.81 85-4 •7 .49 75.4 12.6 158.76 83.1 3.0 9.00 80.7 7.3 53-29 78.6 7.5 56.25 75.2 12.8 163.84 81.9 4.2 17-64 102.2 14.2 201.64 82.9 3.2 10.24 91.5 3.5 12.25 90.9 4.8 23.04 78.6 9.4 88.36 78.1 8.0 64-00 109.2 21.2 449.44 81.1 5.0 25.00 82.9 5.1 26.01 79-5 6.6 43-56 78.6 9.4 88.36 77.0 9.1 82.81 93.6 5.6 31.36 83-1 3.0 9.00 81.6 6.4 40.96 102.6 16.5 275.25 82.4 5.6 31.36 79.7 6.4 40.96 92.4 4.4 19.36 88.6 2.5 6.25 79.0 9.0 81.00 76.3 9.8 96.04 83.6 4-4 19.36 84-9 1.2 1.44 80.9 7.1 50.41 88.9 2.8 7.84 87.3 .7 .49 97.4 11.3 127-69 X^ and Xg = serum reducing sugar level (mgm.$). *1 = 31, nz - 30 , SQ_ — 86.1, %2 — 88.0, x;,-*! = 1.9 £xf = 1,141.73, = 2,103.49, d.f. = (31 + 30) - 2 = 59 s 2 = 3,245.22/59 = 55-0, S x ^ V/55.0(6l)/928 = 1.9 t = 1^ 2 = 1.0 .400<P<.200 1.9 FIGURE 4: A COMPARISON BETWEEN THE SERUM REDUCING SUGAR LEVELS OF MALE AND FEMALE BLACK-TAILED DEER. SERUM REDUCING SUGAR (mgm.%) to o s 00 o 5 > cn m X > 8 O O 25 (4) The Influence of Time of Day This experiment was designed to determine i f any change in serum reducing sugar level occurs during the day in deer. The animals were on feed as outlined earlier, and blood samples were let at each of 0800, 1200, 1600, and 2000 hours. As shown in table 7 and figure 5 the serum reducing sugar levels in samples drawn at 0800, 1200, and 1600 hours are essentially the same. The calculations presented in table 8 show that the slightly higher levels found in samples drawn at 2000 hours are not significantly different from the mean of the levels found at 0800, 1200, and 1600 hours. This elevation at 2000 hours, which was found in the fasted (figure 2) and nonfasted deer (figure 5) might be due to a mild stimulation of the sympathicoadrenal complex. Although the deer used in this experiment were accustomed to considerable activity in the wildlife unit during the day, the disturbance at night (light, talk, movement) associated with blood sampling was unusual and could conceivably ellicited such a response. Reid (1950) also noted in sheep that the blood sugar level was higher in the late afternoon than in the morning but could offer no explanationfcr this. The findings of this experiment are supported by the investigations of Schuhecker (1925) and by Hitchcock and Phillipson (1946) who found no diurnal variation in the blood sugar of adult ruminants. Teichman (1952) as reported by Reid (1950), and Kennedy et al (1939) reported a marked diurnal variation in the blood sugar levels of calves in response to feeding. Preston and Ndumbe (l96l) studied the diurnal variations in the blood sugar of ruminating calves. They noted a postprandial hyper-glycemia similar to that found in monogastric animals in calves on a milk TABLE 7: RESULTS OF EXPERIMENT 1; PART 4 THE INFLUENCE OF TIME OF DAY ON SERUM REDUCING SUGAR LEVELS IN DEER ON FEED. Serum Reducing Sugar (mgm.#) Time 0800 91.2 88.4 83.6 89.2 102.2 82.9 81.9 81.1 1200 90.4 90.1 86.5 81.6 91.5 82.9 79.5 1600 96.8 91.2 100.1 85'.9 78.6 78.6 90.9 77.0 2000 88.7 87.6 90.9 92.1 109.2 93.6 78.1 83.1 Average serum reducing sugar l e v e l at 0800 =87.6 mgm.$ 1200 = 86.1 mgm.# 1600 = 87-4 mgm.$ 2000 = 90.4 mgm.$ FIGURE 5: COMPARING SERUM REDUCING SUGAR LEVELS FOUND IN DEER ON FEED AT DIFFERENT TIMES DURING THE DAT. SERUM REDUCING SUGAR (mgm.X) o o o o oo o o o o 00 o o o O [ ~1 > o -< o o : o o '-."fii rii^'Vl - r i ••• -'--l-J"-'*^»^^^' '^.^i TABLE 8: RESULTS Of EXPERIMENT It PART 4 STUDENT t TEST COMPARING SERUM REDUCING SUGAR LEVELS OF DEER SAMPLED AT  0800. 1200. and 1600 HOURS TO THOSE SAMPLED AT 2000 HOURS. 0800, 1200, 1600 Hours 2000 Hours x-^X-x d h X2=X-x 91.2 4.2 17-64 88.7 1.7 2.89 88.4 1.4 1.96 87.6 2.8 7.84 83.6 3-4 11.56 90.9 .5 .25 89.2 2.2 4.84 92.1 1.7 2.89 102.2 5.2 27.04 109.2 18.8 353.44 82.9 4.1 16.81 93.6 3.2 10.24 81.9 5-1 26.01 78.1 12.3 151.29 81.1 5.9 34.81 83.1 7.3 53.29 90.4 3-4 11.56 90.1 3.1 9.61 8655 .5 .25 91.5 4-5 20.25 82.9 4.1 16.81 79.5 7-5 56.25 96.8 9.8 96.04 91.2 4-2 17.64 100.1 13.1 171.61 85.9 1.1 1.21 78.6 8.4 70.56 78.6 8.4 70.56 90.9 3-9 15.21 77.0 10.0 100.00 81.6 5-4 29.16 Xj and Xg = serum reducing sugar (mgm.$) n x = 23, x x = 87.0, ^ x j = 827-39, ^2~\ = 3-4, d.f. = 29 n| = 8, X2 = 90.4 = 582.13, s 2 = 1,409.52/29 = 48£6 Sxj-xf = /48.6(31)/184 = \ClI = 2.86 1 = lik = 1.188 .400<P<.200 2.86 29 d i e t . Calves on dried grass, however, showed no response to feeding. Another group on a concentrate r a t i o n exhibited a postprandial hypoglycemia, presumably due to active fermentation i n the rumen immediately following feeding. The re s u l t s of these studies on calves do not detract from the findings of t h i s experiment on deer. I t i s u n l i k e l y that the energy metabolism of calves, even ruminating calves, as ref l e c t e d by blood reducing sugar l e v e l s can be compared to any advantage to that of adult deer. I t i s pertinent to note that blood sugar l e v e l s of animals sampled on the same day at the same time occasionally varied i n the same d i r e c t i o n . Figure 6 shows the blood reducing sugar l e v e l s of U34 and U43 during the fa s t i n g experiment. Both deer were sampled at 0800, 1200, 1600, and 2000 hours, i n every case U34 was sampled f i r s t . In t h i s p a r t i c u l a r example the reducing sugar l e v e l s of both deer are almost i d e n t i c a l and t h e i r coincident v a r i a t i o n i s s t r i k i n g . In other instances, although the reduc-ing sugar l e v e l s of the deer are d i f f e r e n t the same coincidence of v a r i a t i o n occurs. This phenomena i s l i k e l y due to excitement of the animals preceeding blood sampling. I f , f o r example, the deer were undisturbed the hour or so p r i o r to blood sampling the blood sugars of both would be r e l a t i v e l y low. Had some unusual disturbance occurred, on the other hand, then the blood sugars of the deer would be correspondingly higher. Under the conditions of the experiment i t was impossible to control t h i s 'extraneous disturbance* factor. In t h i s regard i t i s noteworthy that the second animal sampled each day did not show consistently higher serum reducing sugar l e v e l s as compared to the f i r s t deer sampled. This can be accounted for i n two ways. F i r s t l y , and most important, the animals were accustomed to some 30 disturbance during the day and blood sampling was carried out quietly in a laboratory somewhat removed from the other animals in the unit. Secondly, it is impossible to assess the starting or normal blood reducing sugar level, therefore, i f the second animal had a lower normal level than the first any increase due to excitement could be masked. FIGURE 6: ILLUSTRATING THE RELATIONSHIP THAT CAN OCCASIONALLY OCCUR BETWEEN THE SERUM REDUCING SUGAR LEVELS OF TWO ANIMALS SAMPLED AT THE SAME TIMES ON THE SAME DAY. 5 1 i 1 i -U34 U43 0800 1200 1 I 1 1600 4? I' "I .5 • 'I I ••a I 2000 TIME (HOURS) EXPERIMENT 2 32 EXPERIMENT 2 This experiment was designed to determine i f a change in diet from a concentrate ration to chopped alfalfa hay would alter the blood reducing sugar levels found in deer. As in the first experiment the animals were placed on a feeding program such that each was offered 1/4 of 80$ of its daily intake (calculated from the daily intake of the previous week) at each of 0700, 1100, 1500, and 2000 hours. As indicated earlier some control was thus exercised over the amount and time of feed intake the hour prior to blood letting. A week was allowed for the deer to become accustomed to this regime before the experiment was begun. A l l blood samples were drawn about 0800 hours, one hour after feeding. The remaining feed of the a n i m a l being sampled was weighed back and a record was kept of the feed intake the hour prior to blood letting. On the basis of the results of experiment 1 a l l animals were immobilized with succinylcholine before blood samples were drawn. 10 cc. samples were let from a l l deer once they were accustomed to the new feeding regime. Following this U16 and U24 were taken off feed for two days and then offered chopped alfalfa hay on the same schedule that the concentrate ration had been given. U34 and U43 were treated as controls, remaining on concentrate throughout the experiment. Blood samples were drawn every second day, on alternate days, from both animals in each group. The deer were weighed regularly throughout the experiment. Figure 7 illustrates the considerable variation found in the serum reducing sugar levels of the deer during the experimental period. As can FIGURE 7: ILLUSTRATING THE CHANGES IN SERUM REDUCING SUGAR LEVELS THAT OCCURRED IN DEER ON PELLETS AND THOSE ON HAY. 34 be seen from tables 8 and 9 no clearly defined trends are apparent in the levels of reducing sugar in the control animals. Both deer exhibit levels ranging quite consistently between 80 mgm.# - 9 0 xogja.%. U 4 3 showed the greatest extremes in levels with a low of 72 mgm.$ and a high of 133 mgjn.%. The serum reducing sugar levels of the experimental deer (U16 and U24) as shown in tables 10 and 11, remained reasonably constant throughout the experiment and were generally within the same range as those of the controls. The data for U24 (table 11) might indicate a slight increase in serum reducing sugar level during the first week on hay. Only three samples, however, contribute to the higher level and considering the general variability encountered i t is unlikely that this is significant. The relatively low levels obtained toward the end of the experiment (see figure 7) are no lower than those exhibited by this animal on the pelleted ration in earlier experiments (see table 3). There was, therefore, no significant change in the levels of serum reducing sugar in this deer after a period of six weeks on hay. The range in the level of reducing sugar (85 mgm.# -95 mgm.$) of Ul6 is generally higher than that of the other animals. As indicated in table 10, however, this deer showed no remarkable change in level of serum reducing sugar despite his serious loss of body weight on hay. The feed intake of the control animals (tables 8 and 9) was relatively constant and apparently sufficient as they maintained their weight with only minor fluctuations throughout the experiment. The hay intake of U16 and U24 was also reasonably constant (tables 10 and 11), both however, suffered considerable weight loss. In the case of U16 this was serious enough to force his return to the pelleted ration after only three weeks on hay. During the last few days on this diet Ul6 was so weak and 35 TABLE 8: FEED INTAKE. SERUM REDUCING SUGAR. IND WEIGHT DATA OF U34  THROUGHOUT EXPERIMENT 2. Date Feed Intake (gms.pellets) Reducing Sugar (mgm./&) Weight (lb.) 19/1/65 834 — — 20 902 82.4 — 21 963 950 22 753 82.9 — 23 753 94-5 24 743 — 25 755 94 26 858 84-4 — 27 865 95 28 825 — 29 946 84-1 94-5 30 427 — - — 31 747 — — 1/2/65 699 93 2 811 77-4 — 3 815 — 93 4 806 80.9 — 5 881 — 92.5 6 806 — — 7 515 — — 8 629 — — 9 847 91.8 — 10 761 — 93 11 805 94.2 — 12 906 — 93 13 714 115-7 — 14 578 — 91 15 772 — 16 946 — 92 17 860 — — 18 914 90.4 93 19 868 ___ — 20 669 — — 21 829 — 92 22 728 — 23 859 — 93 24 1,004 94.9 — 25 1,004 890 — 93.5 26 — — 27 562 — — 28 516 — 93 1/3/65 875 - — — 36 TABLE 9; FEED INTAKE. BLOOD REDUCING SUGAR. AND WEIGHT DATA OF U43  THROUGHOUT EXPERIMENT 2. Date Feed Intake (gms.pellets) Reducing Sugar (mgm.%) Weight (lb.) 19/1/65 1,100 — — 20 1,100 81.1 — 21 1,100 — - 126.5 22 825 83.4 — — 23 825 — 128 24 1,011 — — — — 25 1,100 129 26 1,100 88.9 27 1,100 1,056 128 28 29 1,100 85.2 128 30 550 31 880 1/2/65 1,100 128 2 1,100 71.9 3 1,100 126 4 1,100 133-3 5 1,100 — 124.5 6 1,100 — — 7 905 — — 8 1,100 943 — 9 83.1 10 1,100 126 11 1,100 108.8 — — 12 1,100 825 126.5 13 85.9 _ _ _ 14 970 _ _ _ 124 15 1,032 — 16 1,100 125 17 1,100 — — 18 1,100 88.9 126.5 19 1,100 — 20 1,100 125.5 21 1,100 — 22 1,082 127 23 1,100 — — 24 1,100 85.9 127.5 25 1,100 — - _ — 26 1,100 — - — 27 852 — 28 536 — 126.5 1/3/65 1,235 — 37 TABLE 10; FEED INTAKE. BLOOD REDUCING SUGAR. AM) WEIGHT DATA OF U16 THROUGHOUT EXPERIMENT 2. Date Feed Intake (gms. hay) Reducing Sugar (mgm.%) Weight (lb.) 19/1/65 92.7 20 j „ 21 285 79.2 109 22 5336 23 349 79.7 108.5 24 350 — 25 472 100.8 .I.,,, • -j- — 26 482 27 631 — 105 28 580 100.8 29 810 105 30 403 92.7 31 756 1/2/65 648 94.6 103-5 2 500 . _ 3 333 85.7 102.5 4 246 5 231 99-5 6 420 — — 7 274 8 602 95.8 9 702 10 622 96.4 97 11 590 12 162/549 107.6 97 13 819 14 1070 97-5 15 721 115-7 16 1243 96 17 1168 92.1 18 1268 — — 91 19 1646 96.4 20 1865 94 21 1544 22 1805 96 23 1968 88.9 24 2268 96.5 25 2198 26 1837 100.5 27 2276 88.9 28 1969 _ . — 99.5 1/3/65 2276 82.6 * returned to pellets 38 TABLE 11: FEED INTAKE. BLOOD REDUCING SUGAR. AND WEIGHT DATA OF U2A THROUGHOUT EXPERIMENT""^ P-3fee Feed Intake (gms.hay) Reducing Sugar (mgnff^  Weight (lb.) 19/1/65 _ _ _ 75.2 20 21 234 91.2 107 22 235 23 389 80.9 105-5 24 464 — 25 414 89.2 26 372 27 737 105.5 28 632 92.7 29 699 106.5 30 344 106.8 31 667 1/2/65 499 92.7 104.5 2 416 — 3 570 88.9 103 4 452 _ _ _ 5 630 — 101.5 6 669 7 478 8 676 87.0 9 564 10 559 97.8 101 11 610 12 774 83.1 100.5 13 602 _ _ _ 14 773 — , N , M | | 15 947 80.4 16 507 — _ 98.5 17 901 82.4 18 831 _ _ _ 100.5 19 939 79.7 20 845 _ _ _ 98 21 1,059 — 22 962 100 23 958 77.9 24 891 — 99.5 25 756 — 26 726 — 97-5 27 756 80.7 28 479 — _ 97 1/3/65 1,112 79.5 39 lethargic that he could not walk to the scale but had to be carried. U24 lost weight consistently during the first three weeks of the experiment, thereafter she maintained weight stasis. There are three possible explanations for the inability of these animals to maintain their weight during the early part of the experiment. Firstly, inadequate hay intake, secondly, the poor quality of the hay, and thirdly, slow development of a rumen microflora capable of handling the hay. Although no attempt was made to assess in detail the nutritive quality of the hay i t was apparent from the considerable amount of coarse material left by the deer that i t was poor. This and the physical limitations of the feeding arrangements undoubtedly resulted in insufficient intake. It is well estbalished that a considerable change in rumen microflora is associated with any marked alteration in diet. The time required to effect this change is variable, being dependent on a multitude of factors. Warner (1962) noted that in a sheep switched from a diet of hay alone to one of hay plus concentrate a l l major changes in rumen microflora were completed by ten days. He points out that this is usually the period needed for adaptation to a new diet in ruminants. Gouws (1965) studied the alteration in c e l l -ulytic bacterial species in sheep associated with a change from lucerne to teff hay. He stated that the time lapse between a change of diet and the attainment of a balance characteristic of the new diet varied from animal to animal. In one case he studied, the change was complete in a week, in another there was no change after four weeks. Although i t i s attractive to account for the loss in body weight of the deer during the first three weeks as being due to the development of a rumen microflora capable of fermenting hay, to do so i s complete conjecture. 40 The principal object of this experiment was to determine the effect of a change in ration on the serum reducing sugar level in deer. It i s apparent from the data (tables 8 , 9 , 10, and 11) that there was no change in this parameter associated with the alteration in diet. This finding is supported by that of Hibbs (1956) who noted that calves fed various ratios of hay to grain had essentially the same level of blood reducing sugar. Lambert (1955) reported similar results with calves fed various ratios of calf starter and alfalfa. There were two factors inherent in the experiment that might have worked to alter the serum reducing sugar levels of the deer. One was the nature of the diet. Annison et al (1959) found that when sheep on a diet of hay plus additives were turned out to lush spring grass their blood reducing sugar levels rose from 45 mgm.# to 60 mgm.$. He attributed this increase to either or both the increased availability of proportionate or the abundant supply of lactate. Presumably the hay used in this experiment did not provide a sufficient alteration in nutrients to cause a change in the reducing sugar level. The second factor that might have led to a change in the level of serum reducing sugar was the stress that could have been imposed on the deer by the substitute regime. Reid (1950) and Wright (1962) showed that underfeeding can produce a f a l l in blood sugar levels in pregnant ewes. They also noted that blood reducing sugar levels f a l l during lactation.in these animals. Their explanation for the decrease in levels found was that the demands of, in the first case the feotus, and in the second, milk production, outstripped the ewes exogenous and endogenous sources of glucose. During the experiment reported here, a stress, as reflected by the loss in body weight suffered by the deer, particularly U16, was undoubtedly imposed. 41 However, sufficient exogenous and endogenous material must have been present to maintain the serum reducing sugar level as no decrease was found. EXPERIMENT 3 42 EXPERIMENT 3 This experiment was designed to explore the possibility that the factor of time during the actual drawing of the blood sample had an influence on the blood reducing sugar levels determined. On the basis of the results of experiment 1 no control of feed intake was exercised. The deer were immobilized with succinylcholine before blood samples were drawn. Each animal was immobilized on separate days generally in the morning. As outlined earlier accurate accounts of the time required for each aspect of every blood letting were made. In this experiment sequential rather than single samples were drawn from each animal.- Once the deer was immobilized and the vacutainer needle seated, a series of samples were released at roughly 30 second intervals into separate, un-treated vacutainer tubes. It i s apparent from the data presented in table 12 that the time lapse between i n i t i a l intrusion on the animal and actual drawing of the blood sample has a profound effect on the level of serum reducing sugar. As shown in figure 8 all the deer exhibit the same general pattern although the results are displaced one from the other both in time and in reducing sugar level. The data for U34 represents the trend and presents an almost complete picture. The first sample was drawn from this doe only three minutes and forty seconds after the i n i t i a l intrusion of the technician. This is close to the minimal amount of time necessary to inject the animal, have i t collapse, seat a vacutainer needle, and draw a blood sample. The second sample let slightly over a minute later showed essentially the same 43 TABLE 1 2 ; REDUCING SUGAR LEVELS IN SEQUENTIAL BLOOD SAMPLES. •Time* i s t h e i n t e r v a l ( i n minutes and seconds) f rom i n i t i a l i n t r u s t i o n o f t h e t e c h n i c i a n on t h e anima] t o t h e end o f t h a t p a r t i c u l a r sample. Animal Time Reducing Sugar (mgm.%) U16 U24 U34 U43 4 : 0 2 9 7 . 1 4 : 4 0 1 0 2 . 6 8 : 4 1 1 1 8 . 1 9 : 3 4 1 1 7 . 4 12 :45 1 1 3 . 0 1 3 : 3 0 1 1 3 . 5 1 4 : 2 1 1 1 5 . 3 1 5 : 1 4 1 1 4 . 8 8 : 0 8 9 0 . 9 1 0 : 0 3 9 9 . 4 1 0 : 3 0 1 0 1 . 9 1 1 : 0 0 1 0 2 . 2 1 1 : 4 5 1 0 2 . 6 12 :13 1 0 3 - 3 12:51 1 0 1 . 2 1 3 : 3 0 1 0 2 . 6 3:40 8 3 . 6 4 : 5 2 8 5 . 4 5 :42 9 0 . 1 6 : 3 5 1 0 2 . 2 9 : 0 1 1 0 5 . 6 9:50 1 1 0 . 0 10:07 1 0 5 . 2 1 1 : 1 0 1 0 5 . 2 4 : 5 2 84/-4 5 :17 8 3 . 6 5 : 5 0 8 6 . 2 6 : 2 4 9 1 . 5 6 :52 9 1 . 8 7 : 3 0 9 8 . 1 8 : 1 1 9 7 . 1 9:07 9 9 - 7 45 l e v e l of reducing sugar. I t can be concluded that these l e v e l s are reasonably close to the normal f o r t h i s deer. The following three samples, drawn i n the i n t e r v a l from f i v e minutes to nine minutes a f t e r i n i t i a l i n t r u s i o n are t y p i c a l of the rapid increase that occurred i n a l l the deer with the exception of U24. During t h i s i n t e r v a l U34 exhibited a 25.8$ increase over the i n i t i a l serum reducing sugar l e v e l . The l e v e l s i n blood samples drawn a f t e r ten minutes were generally constant i n a l l the deer. The change i n the serum reducing sugar l e v e l s during the blood sampling procedure found i n t h i s experiment might be due to three factors: f i r s t l y to the excitement and ant i c i p a t i o n experienced by the animals as they were moved into the laboratory, secondly to the fear and pain associated with i n j e c t i o n , immobilization, and withdrawal of the blood samples, and t h i r d l y to the muscular energy expended by the deer throughout the procedure. Unfortunately the r e l a t i v e contributions of these factors cannot be determined from t h i s data. The effects of a l l are mediated through the sympathico-adrenal complex. As pointed out e a r l i e r , the speed and extent of the mobilization of glucose, as reflected i n the serum reducing sugar l e v e l , depends on the degree of stimulation of t h i s complex. That excitement might have an effect on blood reducing sugar l e v e l s has been suggested by others; Hodgson et a l (1932), i n c a t t l e Reid (1962) i n sheep, Wing (1955) i n calves, and Bandy (1957) and Touatt et a l (1965) i n deer. No work has been done, however, to determine the rate and magnitude of the change that excitement during blood sampling can e l l i c i t . The response of the deer i n t h i s experiment was undoubtedly minimal as they had been handled extensively and were r e l a t i v e l y accustomed to immobilization. The pattern f o r l e s s 46 experienced animals could 3how a curve attaining higher levels in a much shorter time. Moreover there might be considerable interspecies variation in the rate and magnitude of change in the blood sugar. Domestic species, for example, might exhibit a slower and smaller increase under a particular blood sampling.procedure than a wild species. The findings here add a new element to the factors that must be controlled i f useful blood sugar values are to be obtained. EXPERIMENT L 47 EXPERIMENT 4 This experiment was carried out to determine the distribution of reducing sugar in the blood of deer. On the basis of the results of earlier experiments no control of feed intake was exercised, a l l the deer were on pellets ad l i b . The deer were immobilized with succinylcholine before blood was drawn. Each animal was immobilized on a different day, generally in the morning. Two blood samples were let from each animal into separate test tubes, the first being followed immediately by the second. The first tube was untreated, and serum was extracted as outlined earlier. The second tube was heparinized. Within minutes of being drawn, 0.2 cc. of whole blood from this tube was pipetted into 3.0 cc. of distilled water, thus laking the cells. The level of blood reducing sugar in this sample was then determined in the usual manner. The results of the experiment appear in table 13. Although only a single determination per deer was done i t appears that no reducing sugar occurs in the erythrocytes of these animals. Goodwin (1956) using the same method of calculation, found that in sheep the corpuscular/plasma glucose concentration was 23-5$. This ratio in cattle was roughly the same, being 24.2$. In other words, he found that there was a considerable amount of reducing material in the erythrocytes of these animals. In vitro permeability studies, however, have shown that the erythrocytes of most nonprimate adults are impermeable to glucose. To rationalize this inconsistancy Somogyi (1933) suggested that the corpuscular glucose of sheep and cattle is due to the plasma retained between the packed cells. Andreen-Svedberg (1933) on the other hand, considered the glucose to be absorbed on the cells. 48 TABLE 13: THE DISTRIBUTION OF REDUCING SUGAR IN THE BLOOD OF DEER Animal U16 U24 U34 U43 Serum Level Hematocrit (mgm.%) 96.1 84.6 96.4 120.1 42.5# 46.5# 48.2^  49.2# Whole Blood (mgm.$) Calculated Actual 55.3 45.3 49.9 61.0 56.5 49.9 47.6 59.6 Sample Calculation: serum reducing sugar l e v e l = 96.0 mgm.$ hematocrit of 45$ (therefore 55$ i s plasma) 96 mgm.ff x 55% = the amount of reducing sugar expected 100 i n whole blood sample assuming that a l l the sugar occurs i n the serum. RESUME 49 RESUME T h i s i n v e s t i g a t i o n has shown t h a t feed i n t a k e d u r i n g t h e hour p r e c e e d i n g b l o o d s a m p l i n g , shor t p e r i o d s o f f a s t , n a t u r e o f t h e d i e t , and sex o f t h e a n i m a l , a p p a r e n t l y have no e f f e c t on b l o o d r e d u c i n g sugar l e v e l s i n d e e r . I t was a l s o found t h a t b l o o d samples t a k e n i n t h e even ing had a s l i g h t l y h i g h e r r e d u c i n g sugar l e v e l t h a n t h o s e drawn e a r l i e r i n t h e day . No r e d u c i n g sugar was found t o occur i n t h e e r y t h r o c y t e s o f t h e s e d e e r . Of t h e f a c t o r s s t u d i e d t h a t c o u l d p o s s i b l y i n f l u e n c e r e d u c i n g sugar l e v e l s , o n l y two, t h e means o f r e s t r a i n t , and t h e t i m e r e q u i r e d t o draw samples were found t o have any e f f e c t . Deer p h y s i c a l l y r e s t r a i n e d e x h i b i t e d h i g h e r and more v a r i a b l e r e d u c i n g sugar l e v e l s t h a n t h o s e i m m o b i l i z e d w i t h s u c c i n y l c h o l i n e . The an imals a l s o showed a p r e c i p i t o u s i n c r e a s e i n r e d u c i n g sugar l e v e l d u r i n g t h e b l o o d sampl ing o p e r a t i o n . I t i s i n t e r e s t i n g t h a t t h e s e two f a c t o r s , means o f r e s t r a i n t , and t i m e r e q u i r e d t o draw a sample , a re r a r e l y noted by i n v e s t i g a t o r s r e p o r t i n g sugar l e v e l s i n a n i m a l s . Tab le 14 p resents a comparison o f t h e b l o o d sugar l e v e l s t h a t have been r e p o r t e d f o r Odeco i leus hemionus s p e c i e s . I n every case t h e deer were r e s t r a i n e d by f o r c e , and b lood sugar v a l u e s were r e p o r t e d as mgm.% i n whole b l o o d . Bandy (1957) found a s i g n i f i c a n t d i f f e r e n c e between t h e b lood sugar l e v e l s o f y e a r l i n g deer on a h i g h p l a n e o f n u t r i t i o n and those on a low p l a n e . He a l s o noted t h a t t h e b l o o d sugar l e v e l s o f fawns (20 - 100 days o l d ) and those o f a d u l t deer were s i g n i f i c a n t l y d i f f e r e n t . Bandy r e s t r a i n e d t h e deer w i t h p h y s i c a l f o r c e , but d i d not note t h e t i m e r e q u i r e d t o draw samples . The b l o o d sugar l e v e l s and v a r i a t i o n t h a t he found i n a d u l t an imals a r e v e r y c l o s e t o t h o s e r e p o r t e d i n t h i s exper iment . 50 TABLE 1 4 : COMPARISON OF BLOOD SUGAR VALUES REPORTED FOR DEER. I n v e s t i g a t o r Bandy, P . J . e t a l (1957) An imal R e s t r a i n t # D e t . Odeco i leus hemionus columbianus P h y s i c a l B lood Sugar (mgm.%) 3 7 - 2 + 5 - 4 (whole b lood) T e r r i . A . et a l Odeco i leus hemionus P h y s i c a l (19585 v i r g i n i a n u s 12 6 6 . 9 + 1 2 . 9 (whole b lood) Y o u a t t , W.G. e t Odeco i leus hemionus a l (1965) v i r g i n i a n u s P h y s i c a l 9 1 . 3 + 8 . 1 20 (whole b lood) T h i s r e p o r t Odeco i leus hemionus  columbianus P h y s i c a l 4 4 - 0 + 4 . 0 32 (whole""blood) T h i s r e p o r t Odeco i leus hemionus S u c c i n y l -columbianus c h o l i n e 29 4 2 . 0 + 2 . 9 (whole b lood ) * C a l c u l a t e d f rom serum v a l u e s on t h e b a s i s o f a h e m a t o c r i t o f 50%. 51 T e r r i et a l (1958) r e p o r t e d b l o o d sugar v a l u e s f o r deer i n , n u t r i t i o n a l l y poor s t a t u s * . The an imals were thrown and b l o o d samples were l e t f rom t h e j u g u l a r v e i n . T e r r i noted t h a t t h e deer were ex t remely nervous , f r o t h i n g a t t h e mouth, and t h a t o c c a s i o n a l l y some were f a t a l l y i n j u r e d by t h i s p rocedure . The l e v e l s and v a r i a t i o n t h a t he found l i k e l y r e f l e c t t h e f e a r and p a i n exper ienced by t h e deer d u r i n g b l o o d s a m p l i n g . The b l o o d sugar l e v e l s r e p o r t e d by Youatt et a l (1965) a re even h i g h e r and more v a r i a b l e t h a n those r e p o r t e d by T e r r i e t a l (1958) . Youatt captured h i s an imals w i t h a net and s h a c k l e d them t o a r e s t r a i n i n g board d u r i n g b l o o d s a m p l i n g . He noted t h a t t h e deer were v e r y e x c i t e d breathed h e a v i l y , and became exhausted f i g h t i n g t h i s p r o c e d u r e . On t h e b a s i s o f t h e r e s u l t s r e p o r t e d i n t h i s t h e s i s t h e v a l u e o f t h e b l o o d sugar l e v e l s o b t a i n e d by t h e s e methods o f r e s t r a i n t a re open t o q u e s t i o n . They most a s s u r e d l y a re not normal l e v e l s , t h e r e f o r e t h e i r comparat ive v a l u e i s d o u b t f u l , moreover , any changes recorded as b e i n g due t o e x p e r i m e n t a l m a n i p u l a t i o n a r e l i k e l y f o r t u i t o u s . I t i s apparent t h e n , t h a t t h e b l o o d r e d u c i n g sugar l e v e l s r e p o r t e d f o r deer t o date r e f l e c t t h e degree o f exc i tement , f e a r , and p a i n exper ienced by t h e an imals d u r i n g t h e b l o o d sampl ing procedure employed. I t i s p e r t i n e n t once a g a i n t o p o i n t out t h e r e l a t i v e l y low and c o n s i s t e n t b l o o d sugar l e v e l s t h a t r e s u l t when s u c c i n y l -c h o l i n e i s used t o i m m o b i l i z e t h e deer d u r i n g b l o o d sampl ing ( t a b l e 1 3 ) . The use o f s u c c i n y l c h o l i n e , however, does not guarantee c o n s i s t e n t , n o r m a l , b lood sugar l e v e l s . A number o f o t h e r f a c t o r s must be c o n s i d e r e d : t h e deer must be c o m p l e t e l y f a m i l i a r w i t h t h o s e i n v o l v e d i n t h e b l o o d sampl ing o p e r a t i o n ; r e c o r d s shou ld be made o f t h e t i m e r e q u i r e d f o r each aspect o f every b l o o d s a m p l i n g , and o f t h e a c t i v i t y o f t h e deer throughout t h e o p e r a t i o n ; and t h e 52 animals shou ld be housed such t h a t extraneous d i s t u r b a n c e i s n d n i m i z e d . Only t h r o u g h t h e a d o p t i o n o f such an i n t e n s i v e program w i l l w o r t h w h i l e d a t a r e l a t i n g b l o o d sugar t o e x p e r i m e n t a l m a n i p u l a t i o n be r e a l i z e d . BIBLIOGRAPHY 53 BIBLIOGRAPHY A l l c r o f t , W.M., and R. Strand. Studies on the l a c t i c acid, sugar and inorganic phosphorous of the blood of ruminants. Biochem. J . 27:512, 1933. Andreen-Svedberg^; A. On the d i s t r i b u t i o n of sugar between plasma and corpuscles i n animal and human blood. Skand. Arch. Physiol. 66:113, 1933. Annison, E.F., D. Lewis, and D.B. Lindsay. Studies on the por t a l blood of sheep. 2. Absorption of v o l a t i l e f a t t y acids from the rumen of the sheep. Biochem. J . 66:592, 1957-Annison, E.F., D. Lewis, and K.J. H i l l . The metabolic changes which occur i n sheep transfered to lush spring grass. 1. Changes i n blood and rumen constituents. J . Agri. S c i . , 53:34, 1959. Armstrong, D.G. Physiology of digestion i n the ruminant. R.W. Dougherty Ed., Butterworth Inc., 1965. Bandy, P.J., W.O. K i t t s , A.J. Wood, and I . McT. Cowan. The effect of age and plane of n u t r i t i o n on the blood chemistry of the Columbian blacktailed deer. Can. J . Zool. 35:283, 1956. Bard, P. Medical physiology. 11 ed. C.V. Mosby Co. 1961. Baxter, C.F., M. Kleiber, and A.L. Black. Glucose metabolism i n the la c t a t i n g dairy cow. Biochem. Biophys. Acta. 17:354, 1955. B e l l , F.R., and E.R. Jones. Glucose tolerance i n the bovine. J . Comp. Path, and Therapeut. 55:117, 1945. Bernhard, K., G. Brubacher, H. Hediger, and H. Bruhin. Untersuchungen uber chemische zusammensetzung und aufbau des hirschgeweihes. Experimentia 9:138, 1953. Browman, L.G., and H.S. Sears. Cyclic v a r i a t i o n i n the mule deer thymus. Proc. Soc. Expertl. B i o l , and Med. 93:161, 1956. C a s t i l l o , J.C., and E.J. deBeer. The neuromuscular blocking action of succinylcholine. J . Pharmacol, and Expertl. Therap. 99:458, 1950. Cowan, I . McT., and A.J. Wood. The growth rate of bla c k t a i l e d deer. J . W i l d l . Mgt. 19:331, 1955. Cowan, I . McT., A.J. Wood, and H.C. Nordan. Studies on the t r a n q u i l i z -ation and immobilization of deer. (Odocoileus) Can. J . Comp. Med. Vet. S c i . 26:57, 1962. 54 15. Dawson, A.B.. The P i t u i t a r y gland of the whitetailed deer (O.v. bo r e a l i s ) . In: 76*" meeting of the Amer. Assoc. of Anatomists, 1962. Anat. Rec. 145:316, 1963. (abst. only). 16. E l l i s , S. The metabolic effects of epinepherine and related amines. Pharmacol. Rev. 8:486, 1956. 17. French, C.E., L.C. McEwen, N.D. Magruder, R.H. Ingram, and R.W. Swift. Nutrient requirements f o r growth and antler development i n the whitetailed deer. J . W i l d l . Mgt. 20:221. 1955. 18. French, C.E., L.C. McEwen, N.D. Magruder, T. Rader, T.A. Long, and R.W. Swift. Response of whitetailed bucks to added a r t i f i c i a l l i g h t . J . Mamm. 41:23, I960. 19. Garner, R.J. Disturbances i n carbohydrate metabolism i n c a t t l e assoc-iated with l i v e r disease. J . Comp. Path, and Therap. 62:292, 1952. 20. Goodwin, R.F.W. The d i s t r i b u t i o n of sugar between red c e l l s and plasma: variations associated with age and species. J . Physiol. 134:88, 1956. 21. Gouws, L., and A. Kistner. Bacteria of the ovine rumen. IV. Effect of change of diet on the predominant type of cell u l o s e digesting bact e r i a . J . Agr. S c i . 64:51, 1965. 22. Grieser, K.C., and L. Browman. Total gonadotrophic potency of mule deer p i t u i t a r i e s . Endocrinology 58:206, 1956. 23. Heald, P.$. The assessment of glucose-containing substances i n rumen microorganisms during a digestion cycle i n sheep. B r i t . J . Nut. 5:84, 1951. 24. Hibbs, J.W., H.R. Conrad, W.D. Pounden, and N. Frank. A high roughage system f o r r a i s i n g calves based on early development of rumen function. 6. Influence of hay to grain r a t i o on c a l f performance, rumen development, and certain blood changes. J. Dairy S c i . 39:171, 1956. 25. Jlxtchcock, M.W.S., and A.T. P h i l l i p s o n . The tolerance of sheep to low concentrations of blood sugar. J . Physiol. 105:42, 1946. 26. Ho, P. and E.F. Reber. Effects of glucagon on hypoglycemia and keton-emia i n pregnant ewes. Amer. J . Vet. Res. 18:342, 1957. 27. Hodgson, R.E., W.H. R i d d e l l , and J.S. Hughes. Factors influencing the blood-sugar l e v e l of dairy c a t t l e . J . Agr. Res. 44:357, 1932. 55 28. Hunt, R. and R. deM. Taveau. On physiological action of certain choline derivatives and new methods for detecting choline. B r i t . Med. J . 2:1788, 1906. 29- Kennedy, W.L., A.K. Anderson, S.I. Bechdel, and J.F. Shigley. Studies on the composition of bovine blood as influenced by gestation, l a c t a t i o n and age. J . Dairy S c i . 22:251, 1939. 30. K i t t s , W.D., I . McT. Cowan, J . Bandy, and A.J. Wood. The immediate post-natal growth i n the Columbian blacktailed deer i n r e l a t i o n to the composition of the milk of the doe. J . W i l d l . Mgt. 20:212, 1956. 31. K i t t s , W.D., P.J. Bandy, A.J. Wood, and I . McT. Cowan. Effect of age and plane of n u t r i t i o n on the blood chemistry of the Columbian blacktailed deer. Can. J . Zool. 34:477, 1956. 32. Krebs, H.A. GLuconeogenesis. Proc. Royal Soc. Series B; 159-545, 1964. -33- Kronfeld, D.S. Growth hormone administration to pregnant sheep. Cornell Vet. 47:255, 1957. 34- Lambert, M.R., N.L. Jacobson, R.S. A l l e n , and M.R. B e l l . The r e l a t i o n of growth, fee consumption and certain blood constituents to changes i n the dietary of young dairy calves. J . Dairy S c i . 38:6, 1955. 35. Lindsay, D.B. The significance of carbohydrate metabolism i n ruminant metabolism. Vet. Rev. 5:103, 1959. 36. Long, T.A., C.E. French, and N.D. Magruder. Effect of seasonal r e s t r i c t i o n s on antler development of whitetailed deer. Penn. Agr. Exp. Stat. Progr. Rept. #209, 1959. 37. Magee, H.E. Observations on digestion i n the ruminant. J . Exp. B i o l . 9:409, 1932. 38. Magruder, D., C.E. French, and T.A. Long. Nutrient requirements of whitetailed deer f o r growth and antler development. Pa. State U. Agr. Exp. Sta. B u l l . 628, 1957. 39. Mayrofer, D.K. Se l f experiments with succinylcholine. A new u l t r a -short-acting muscle relaxant. B r i t . Med. J . 1:1332, 1952. 40. Meister, W.W. Changes i n h i s t o l o g i c a l structure of the long bones of whitetailed deer during the growth of the antlers. Anat. Rec. 124:709, 1956. 41. Natlelson, S. Microtechniques of c l i n i c a l chemistry. CC. Thomas, 1961. 56 42. Nelson, N. A photometric adaptation of the Somogyi method f o r the determination of glucose. J . B i o l . Chem. 153:375, 1944. 43. Nordan, H.C., A.J. Wood, and I . McT. Cowan. Further studies on the immobilization of deer with succinylcholine. Can. J . Comp. Med. 26:246, 1962. 44« Ochs, S., B. Annis, and A.K. Mukherjee. Succinylcholine and muscle e x c i t a b i l i t y . S c i . 131:1679, I960. 45« Pisty,,R.W., and J.F. Wright. The immobilization of captive w i l d animals with succinylcholine;2 . Can. J . Comp. Med. 25:59, 1961. 46. Potter, B.J., and I.G. Jarret. I n s u l i n tolerance and hypoglycemia convulsions i n sheep. Aust. J . E x p t l . B i o l . and Med. S c i . 31:311, 1953. 47. Preston, T.R., and R.D. Ndumbe. Diurnal variations i n blood sugar concentration i n ruminating calves. B r i t . J . Nut. 15:281, 1961. 48. Rankin, A.D. A study of absorption from the rumen of the sheep. Vet. B u l l , (abst.) 11:328, 1940. 49« Reichard, G.A., B. Issekutz, J r . , P. Kimbel, R.C. Putman, N.J. Hochella and S. Weinhouse. Blood glucose metabolism i n man during muscular work. J . Appl. Physiol. 16:1001, 1961. 50. Reid, R.L. Studies on the carbohydrate metabolism of sheep. 1. The range of blood sugar values tinder several conditions. Aust. J . Agr. Res. 1:182, 1950. 51. Reid, R.L. Studies on the carbohydrate metabolism of sheep. 3« The blood glucose during i n s u l i n hypoglycemia. Aust. J . Agr. Res. 2:132, 1950. 52. Reid, R.L. Studies on the carbohydrate metabolism of sheep. 5. The effect of hyperglycemia and of i n s u l i n on the rate of extrahepatic glucose assimilation. Aust. J . Agr. Res. 3:160, 1952. 53. Reid, R.L., and S.C. M i l l s . Studies on the carbohydrate metabolsim of sheep. 14. The adrenal response to psychological stress. Aust. J . Agr. Res. 13:282, 1962. 54. Robertson, A., H. Paver, P. Barden, and T.G. Marr. Fasting metabolism of the l a c t a t i n g cow. Res. Vet. S c i . 1:117, I960. 55. Robinson, R.M., R.G. Marburger, and J.W. Thomas. The reproductive cycle of male whitetailed deer i n central Texas. J . W i l d l . Mgt. 29:53, 1965. 57 56. Rosen, M.N., and A.I. Bischoff. The r e l a t i o n of hematology to condition i n C a l i f o r n i a deer. Trans. N.A. W i l d l . Conf. 17:482, 1952. 57* Schambye, P. V o l a t i l e acids and glucose i n portal blood of sheep. Nord. Vet. Med. 3:1003, 1951. 58. Schuhecker, K. Beobachtungen uber den blutzucker der ziege. Biochem. Z. 156:353, 1925. 59. S e t c h e l l , B.P. and G.L. McClymont. Blood inorganic phosphorous and serum potassium i n i n s u l i n hypoglycemia i n the sheep. Aust. J . Agr. Res. 6:589, 1955. 60. Shaw, J.C., A.C. Chung, and I . Bunding. The effect of p i t u i t a r y growth hormone and adrenocorticotropic hormone on established l a c t a t i o n . Endocrinology 56:327, 1955. 61. S i l v e r , H., N.F. Colovos, and H.H. Hayes. Basal metabolism of whitetailed deer...a p i l o t study. J . W i l d l . Mgt. 23:434, 1959. 62. Soldandt, O.M., and G.C. Ferguson. Ef f e c t of strenuous exercise of short duration on the blood-sugar. Trans. Royal Soc. Can. 26; v:173, 1932. 63- Somogyi, M. The d i s t r i b u t i o n of sugar and rate of gl y c o l y s i s i n the blood of some mammals. J . B i o l . Chem. 103:665, 1933. 64« Somogyi, M., A new reagent f o r the determination of sugars. J . B i o l . Chem. 160-61, 1945. 65. Somogyi, M. Determination of blood sugar. J . B i o l . Chem. 160:69, 1945. 66. Somogyi, M. Notes on sugar determination. J . B i o l . Chem. 195:19, 1952. 67. T e r r i , A., W. Virchow, N.F. Colovos, and F. Greeley. Blood composition of whitetailed deer. J . Mamm. 39:269, 1958. 68. Tyler, C. The effect of prolonged emotional disturbance on the vaso-pressor and oxytcic a c t i v i t i e s contained i n the porterior p i t u i t a r y glands of fallow deer. Arch. Intern. Pharmacodyn. 131:301, 1961. 69 Voelker, H.H., N.L. Jacobson, and R,S. A l l e n . Relationship of a n t i -b i o t i c feeding and the rate of growth to blood reducing sugar l e v e l s and glucose absorption i n dairy calves. A n t i b i o t i c s and Chemotherapy 5:224, 1955. 70. Warner, A.C.I. Some factors influencing the rumen microbial population. J . Gen. Microbiol. 28:129, 1962. 58 71. Whitlock, S.C. Studies on the blood of whitetailed deer. J . W i l d l . Mgt. 3:14, 1939. 72. Wilber, C.G., and P.F. Robinson. Aspects of the blood chemistry of whitetailed deer. J . Mamm. 39:309, 1958. 73. Wing, J.M., N.L. Jacobson, and R.S. A l l e n . The effect of various r e s t r i c t e d diets on the growth and certain blood components of young dairy calves. J . Dairy S c i . 38:1006, 1955. 74. Wright, P.L., A.L. Pope, and P.H. P h i l l i p s . Effect of protein and energy intake on lamb production and certain blood constituents of ewes. J . An. S c i . 21:602, 1962. 75. Youatt, W.G., L.J. Verme, and D.E. U l l r e y . Composition of milk and blood i n nursing does and blood composition of t h e i r fawns. J . W i l d l . Mgt. 29:79, 1965. APPENDICES APPENDIX I G lucose Metabo l i sm Glucose i s t h e major end product o f carbohydrate d i g e s t i o n i n t h e monogastr ic a n i m a l , and p l a y s an impor tant r o l e i n i t s m e t a b o l i c p r o c e s s e s . I t i s e s s e n t i a l f o r t h e maintenance o f c e l l s both as a p r e c u r s o r t o many c e l l u l a r components and as a source o f energy . I n t h e l a c t a t i n g an imal i t i s a p r e c u r s o r o f l a c t o s e . Glucose a l s o p r o v i d e s 2 - c a r b o n fragments f o r f a t s y n t h e s i s and t h e n e c e s s a r y reduced coenzyme (NADPH) f o r t h e i n c o r p o r a -t i o n o f t h e 2 - c a r b o n u n i t s i n t o l o n g c h a i n f a t t y a c i d s (Armstrong 1 9 6 5 ) . I t has l o n g been cons ide red t h a t g lucose metabo l i sm i s q u a n t i t a t i v e l y l e s s impor tant i n ruminants as compared t o n o n - r u m i n a n t s . (Bax ter et a l 1 9 5 5 ) . The f a c t t h a t l i t t l e g lucose i s absorbed from t h e d i g e s t i v e t r a c t o f t h e ruminant , (Schambye 1951) and t h a t i t appears i n low c o n c e n t r a t i o n s i n t h e b lood o f t h e s e an imals suppor ts such a c o n t e n t i o n . U t i l i z a t i o n r a t e s measured by 1 4 c - g l u c o s e i n f u s i o n , and expressed per u n i t o f m e t a b o l i c body s i z e , however, i n d i c a t e t h a t t h e r a t e o f u t i l i z a t i o n o f g lucose i n ruminants i s e s s e n t i a l l y t h e same as i t i s i n s imple -s tomached a n i m a l s . (Armstrong 1965) • G l u c o s e , t h e n , i s aa impor tant i n t h e metabo l i sm o f t h e ruminant as i t i s t h e non -mmi n a n t . ( L i n d s a y 1 9 5 9 ) . I n ruminants d i e t a r y carbohydrates a re fermented r a t h e r t h a n d i g e s t e d i n t h e rumen. The u l t i m a t e p roducts o f t h i s f e r m e n t a t i o n a r e t h e v o l a t i l e f a t t y a c i d s , a c e t i c , p r o p r i o n i c , and b u t y r i c . These a re absorbed i n t o t h e p o r t a l b l o o d system through t h e rumen w a l l and p l a y a major r o l e i n t h e energy metabol ism o f t h e a n i m a l . Some o f t h e a c e t a t e i s o x i d i z e d i n t h e l i v e r , but t h e m a j o r i t y i s passed i n t o t h e p e r i p h e r a l c i r c u l a t i o n where i t i s u t i l i z e d by t h e t i s s u e s as a source o f energy . I t i s i n t e r e s t i n g t o note 60 that many tissues can derive energy from oxid i z i n g f a t t y acids and ketone bodies, but nervous tissue can u t i l i z e only glucose as an energy source. (Krebs 1964). Proprionic acid i s either oxidized or converted to glucose i n the l i v e r . L i t t l e butyric acid i s carried to the l i v e r , the majority i s converted to ketone bodies i n the rumen epithelium. (Armstrong 1965). Glucose i n the ruminant i s derived from non-carbohydrate sources by the process of gluconeogenesis. Proportionate and amino acids are the p r i n c i p a l precursors. The glucose formed i s metabolized i n es s e n t i a l l y the same manner as i t i s i n monogastric animals. L i t t l e , however, i s u t i l i z e d i n the elaboration of long chain f a t t y acids. The abundant supply of r e a d i l y activated acetate i s the primary source of these i n the ruminant. I t has also been shown that glucose i s involved i n the synthesis of lactose, g l y c e r o l , milk c i t r a t e , and some non-essential amino acids i n ruminants. (Armstrong 1965). 61 APPENDIX 2 Hormonal R e g u l a t i o n o f B lood Reducing Sugar The maintenance o f normal l e v e l s o f r e d u c i n g sugar i n t h e b l o o d o f mammals i s a f i n e l y r e g u l a t e d homeostat ic mechanism. The l i v e r p l a y s an e s s e n t i a l r o l e as i t f u n c t i o n s b o t h t o remove and t o add sugar t o t h e b l o o d . The a c t i v i t y o f t h e l i v e r i n m a i n t a i n i n g normal l e v e l s o f sugar i n t h e b l o o d i s i n f l u e n c e d by a number o f hormonal f a c t o r s . 1 . I n s u l i n I n s u l i n i s a s e c r e t i o n f o t h e B - c e l l s o f t h e i s l e t s o f Langerhans. I t f u n c t i o n s t o l o w e r t h e b l o o d r e d u c i n g sugar l e v e l by promoting p e r i p h e r a l u t i l i z a t i o n o f g l u c o s e , g l y c o g e n e s i s i n muscle and l i v e r , and l i p o g e n e s i s . The mechanism o f i n s u l i n a c t i o n remains open t o d i s c u s s i o n , however, e x p e r i m e n t a l ev idence suggests t h a t i t a c t s by a f f e c t i n g 1 . membrane t r a n s p o r t phenomena, and 2 . o x i d a t i v e p h o s p h o r y l a t i o n r e a c t i o n s . (Bard 1 9 6 l ) . That i n j e c t i o n o f i n s u l i n lowers t h e l e v e l o f b lood r e d u c i n g sugar i n sheep was shown by R e i d (1951) and (1952) . The r a t e o f f a l l , however, was c o n s i d e r a b l y s lower than t h a t r e p o r t e d f o r n o n - r u m i n a n t s , and t h e l e v e l d i d not f a l l below 5 mgm.%. Moreover , f u r t h e r i n j e c t i o n o f l a r g e doses o f i n s u l i n (10 U/Kg.) d i d not cause a f u r t h e r r e d u c t i o n i n b l o o d sugar , but mere ly p ro longed t h e e x i s t i n g hypoglycemia . I t i s noteworthy t h a t spontaneous d i a b e t e s i s uncommon i n r u m i n a n t s . 2 . Ep inepher ine Hyperg lycemia has been r e p o r t e d i n sheep ( S a t c h e l l and McClymont 1955) and i n c a t t l e (Garner 1952) when i n j e c t e d w i t h e p i n e p h e r i n e . The importance o f t h i s s e c r e t i o n : ! o f t h e a d r e n a l m e d u l l a becomes apparent when 62 a splanchnic section (Potter 1952) i s carried out on a sheep - the blood sugar l e v e l f a l l s to nothing. 3. Glucagon Glucagon i s a secretion of the occells of the pancreas that operates to r a i s e the l e v e l of blood sugar by promoting glycogenolysis. The s i t e of action of glucagon i s apparently the same as that of epinepherine. The hyperglycemic reaction of glucagon administration has been demonstrated i n sheep (Ho and Reber 1957)• 4. Growth Hormone In monogastric animals, growth hormone acts to decrease peripheral u t i l i z a t i o n of blood sugar and to increase glycogenolysis i n the l i v e r . The net e f f e c t , then, i s to rai s e the l e v e l of blood reducing sugar. This effect was noted i n sheep (Kronfeld 1957), however, the elevated l e v e l did not persist despite repeated i n j e c t i o n of the hormone. 5. ACTH and Cortisone ACTH undoubtedly exerts i t s effect i n d i r e c t l y v i a increased release of 11-oxycorticoids from the adrenal cortex. The 11-oxycorticoids exert t h e i r diabetogenic effect through increased gluconeogenesis from amino acids i n the l i v e r . These hormones are also i n s u l i n antagonists. Satchell and McClymont (1955) i n sheep and Shaw (1955) i n c a t t l e produced hyperglycemia by i n j e c t i n g ACTH and C o r t i s o l . APPENDIX 3 Succinylcholine Chloride Hunt (1906) was the f i r s t to synthesis succinylcholine. The immobil-i z a t i o n action of the drug remained undiscovered f o r f o r t y years. C a s t i l l o and deBeer (1950) f i r s t documented t h i s and since then numerous reports of i t s use i n veterinary and human medicine have appeared. The structure of succinylcholine, which appears below, i s very s i m i l a r to that of ac e t y l -The mechanism of action of succinylcholine i s believed to be enzymatic i n h i b i t i o n at the myoneural junction (Pistey and Wright 1961). The succinylcholine i n h i b i t s the action of acetylcholinesterase r e s u l t i n g i n an accumulation of acetylcholine. This accumulation lowers the resting potential of the muscle, reducing i t s e x c i t a b i l i t y and the muscle i s e f f e c t i v e l y paralyzed (Ochs I960). Pseudocholinesterase, found i n the plasma, hydrolyzes succinylcholine into succinylmonocholine and choline. The succinylmonocholine i s then degraded i n t o succinic acid and choline chloride (Bovet et a l 1949 as reported by Pistey and Wright 1961). Acetylcholinesterase then acts to degrade the accumulated acetylcholine, conduction i s resumed and the paralysis i s terminated. Mayrhofer (1952) induced s e l f paralysis with succinylcholine. Muscle weakness, i n i t i a l l y of the neck, jaw and diaphragm, pain f u l muscle twitching, and double v i s i o n , characterize the sensations that he attributed to the action of the drug. At no time did he become unconscious, and no change i n pulse rate or blood pressure was recorded. Moreover, no af t e r or side effects were experienced. choline. HX O O / C H 3 H 3C-N +CH 2CH 2OCCH 2CH 2COCH 2CH 2N +-CK3 APPENDIX A Peebles V'LER Milk Replacer Ingredients Dried skim milk Dried butter m i l Dried whey-product L e c i t h i n Sodiumbenzoat e Magnesium carbonate Dicalcium phosphate Iron sulphate A n t i b i o t i c supplement (oxytetrcycline and V i t A palmitate V i t D 2 V i t (thiamine) V i t B Q (riboflavine) Guaranteed minimum analysis crude f a t 16. C# crude protein 24-0$ V i t A 1,500 U/lb V i t D 2 3,000 U/lb V i t B^ 11 mgm. V i t Bg 11 mgm. terramycin) APPENDIX 5 Weaning Ration Ingredient lbs/ton Ground barley 200 Ground wheat 585 Oat groats 390 Wheat bran 130 Herring meal 200 Soya meal 100 Lysine Methionine Skim milk powder 200 Brewers yeast 20 Irradiated yeast 2 D i s t i l l e r s solubles Dehydrated grass meal 150 Vit D 2  Dicalcium phosphate 10 Iodized salt 10 Manganous sulphate Zinc sulphate Chromic oxide 1 APPENDIX 6 Adult Ration (36-57) Ingredients lbs/ton Com meal 600 Ground wheat 250 Bran 275 Beet pulp 200 Vitagrass 200 Soyabean meal 175 Herring meal 110 Bone meal 20 Iodized s a l t 20 Molasses 150 67 APPENDIX 7 Absorption Curve for the Nelson-Somogyi Method of Determining Blood Glucose. Beckman Model DU Spectrophotometer Standard Glucose Solution 100 mgms./S Wavelength S l i t w i d t h O.D. 400 1.95 .0752 410 1.15 .0809 420 0.68 .0894 430 0.44 .1007 440 0.30 .n?/, 450 0.22 .1278 460 0.158 .1421 470 0.150 .1586 480 0.150 .1733 490 0.140 .1898 500 0.140 .2083 510 0.140 .2291 520 0.065 .2557 530 0.065 .2865 540 0.065 .3242 550 0.065 .3675 560 0.070 .4179 570 0.080 .4660 580 0.090 .5186 590 0.110 .5686 600 0.14 .6126 605 0.17 .6308 610 0.22 .6478 615 0.28 .6676 620 0.32 .6799 625 0.40 .6946 630 0.075 .7305 635 0.075 •7471 640 0.065 .7696 645 0.065 .7878 650 0.065 .8069 655 0.065 .8327 660 0.065 .8539 665 0.065 .8827 670 0.065 .9136 Wavelength S l i t w i d t h O.D. 675 0.065 .9508 680 0.065 .9914 685 0.065 1.0269 690 0.065 1.0706 695 0.065 1.1079 700 0.065 1.1549 710 0.065 1.2218 720 0.065 1.2924 730 0.065 1.3468 740 0.065 1.3768 750 0.065 1.4089 760 0.065 1.4089 770 0.065 1.3979 780 0.065 1.3768 790 0.065 1.3279 800 0.065 1.3010 810 0.065 1.2596 820 0.065 I.2076 830 0.065 1.1675 840 0.065 1.1249 850 0.065 1.0809 860 0.065 1.0410 870 0.065 1.0000 880 0.065 .9626 890 0.065 .9318 900 0.065 .9031 910 0.065 .8665 920 0.065 .8356 930 0.065 .8097 940 0.065 .7852 950 0.065 .7595 960 0.065 .7375 970 0.065 • 7122 980 0.065 .6946 990 0.065 .6757 1000 0.065 .6576 FIGURE 9: ABSORPTION SPECTRUM: NELSON-SOMOGTI METHOD OF DETERMINING BLOOD GLUCOSE 1 6 0 0 200 I -560 600 640 I I 700 750 .WAVE LENGTH (mu) 800 850 900 <25C 69 APPENDIX 8 Standard Curve f o r the Nelson-Somogyi Method of Determining Blood Glucose. Beckman Model DU Spectrophotometer S l i t w i d t h 0.065 Wavelength 650 mu Cone ent rat ion 0. D. Concentration O.D. Concentration O.D. 100 .8539 100 .8239 100 .8125 • 8539 .8239 .8125 .8477 .8239 .8097 .8477 .8239 .8041 .8477 .8239 .8041 .8477 .8239 .8041 .8477 .8239 .8041 .8477 .8239 .8041 .8477 .8210 .8041 .8416 .8210 .8041 .8416 .8210 .8013 .8416 .8210 .8013 .8416 .8210 .7986 .8416 .8210 .7986 .8386 .8210 •7986 .8386 .8182 .7986 .8386 .8182 .7986 .8386 .8182 • 7986 .8386 .8182 .7959 .8386 .8182 .7959 .8356 .8182 .7959 .8297 .8153 .7905 .8268 .8153 .7878 .8268 .8153 .7878 .8268 .8153 .7878 .8268 .8153 .7852 .8239 .8153 .7852 .8239 .8153 .7852 .8239 • 8153 .7825 .8239 .8125 .7747 .8239 .8125 APPENDIX 8 (cont.) Concentration O.D. Concentration O.D. Concentration O.D. 70 mgm.$ .6055 70 mgm.# .5654 40 mgp\.% .3270 .6055 .5654 .3270 .5918 .5638 .3261 .5918 >5638 .3261 • 5969 .5622 .3261 .5969 .5607 .3251 .5982 .5607 •3251 • 5901 .5591 .3251 .5884 .5591 .3251 .5850 .5560 •3251 .5850 .5560 .3233 • 5834 • 5575 .3233 • 5834 • 5544 .3233 • 5834 .5513 •3224 .5817 .5513 • 3215 .5800 •5513 .3215 .5751 .5513 .3206 • 5735 .5498 .3197 .5735 .5492 .3188 .5735 .5452 .3188 • 5719 .5376 .3179 .5602 40 mgm.% .3468 .3170 .5602 .3382 .3152 .5670 53382 .3152 .5686 •3372 .3152 .5686 •3354 .3143 .5686 .3344 .3143 .5670 .3344 •3143 .5654 .3335 .3125 • 5654 .3316 .3116 .5654 .3316 .3116 .5654 .3316 .3116 • 5654 .3307 .3107 • 5654 .3298 .3054 .5654 .3279 •3045 .3028 FIGURE 10: STANDARD CURVE: NELSON-SOMGYI METHOD OF DETERMINING BLOOD GLUCOSE. OPTICAL DENSITY APPENDIX 9 Recovery of Glucose from Serum. Sample Recovery Calculated % Recovered 70 mgm.% 69-9 70.0 9 9 . 9 2:1 84.1 82.0 102.6 1:1 8753 88.0 99.2 1:2 89-5 94.0 95-2 serum 106.0 APPENDIX 10: BLOOD SAMPLING RECORD FORM. 73 DATE ANIMAL BLOOD ANALYSIS DATA RESTRAINT WEIGHT ANECTINE DOSE SEX TIME 0800 lapse 1200 lapse 1600 lapse 2000 lapse Time approached .  Time grabbed/injected Time held/down Sampling begun Sampling ended Sample ringed Sample centrifuged Sample i n freezer COMMENTS GLUCOSE ANALYSIS  % T. Time Date O.D. mgm. % Glucose 0800 1 2 3 1200 1 2 1600 1 2 2000 1 2 2_ CALCIUM ANALYSIS O.D. mgm. % Calcium Time Date % T. 0800 1 2 3 1200 1 2 JL 1600 1 2 2_ 2000 1 

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