UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A comparison of the effects of carbohydrate and fat as energy sources in trout and chick diets on tissue… Hickling, David Robert 1981

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1981_A6_7 H53.pdf [ 7.97MB ]
Metadata
JSON: 831-1.0095237.json
JSON-LD: 831-1.0095237-ld.json
RDF/XML (Pretty): 831-1.0095237-rdf.xml
RDF/JSON: 831-1.0095237-rdf.json
Turtle: 831-1.0095237-turtle.txt
N-Triples: 831-1.0095237-rdf-ntriples.txt
Original Record: 831-1.0095237-source.json
Full Text
831-1.0095237-fulltext.txt
Citation
831-1.0095237.ris

Full Text

A COMPARISON OF THE EFFECTS OF CARBOHYDRATE AND FAT AS ENERGY SOURCES IN TROUT AND CHICK DIETS ON TISSUE GLYCOGEN CONCENTRATION AND ON THE RATE OF GLYCOGEN DEPLETION FROM THE TISSUES DURING A SUBSEQUENT PERIOD OF FAST by DAVJID ROBERT HICKLING B . S c , The Univers i ty of B r i t i sh Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF \ THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Poultry Science) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1981 (c) David Robert H ick! ing, 1981 In presenting t h i s thesis 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 University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or pu b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^CSAAJKA^ ^ The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i -ABSTRACT Rainbow trout, about one-year-old, were fed d iets containing e i ther glucose (C) or herring o i l (F) as the non-protein energy source for a period of two weeks. As we l l , they were fed each d ie t at sa t ia t ion (C-2, F-2) and at leve ls hal f that (C - l , F - l ) . The trout were subsequently fasted and sampled for t issue glycogen, prote in, dry matter and glucose-6-phosphatase a c t i v i t y at f u l l feeding and at 2, 4, 8, 10, 13 and 16 days of f as t ing . The l i v e r s of the C-fed f i s h had 12% wet weight glycogen and the l i v e r s of the F-fed f i s h had 3% wet weight glycogen at f u l l feeding. Upon fas t i ng , glycogen concentrations in the F-fed f i s h l i v e r s f e l l to basal leve ls of 1% by 2 days while glycogen concentrations in the C-fed f i s h l i v e r s f e l l to basal leve ls only after 10 days. The protein concentration in the trout l i v e r s was inversely related to the glycogen concentration. The amount of l i v e r protein in the t rout carcass, however, was d i r e c t l y related to d ietary carbohydrate l eve l s . Trout l i v e r glucose-6-phosphatase a c t i v i t y increased as fast ing progressed but there were no treatment differences in enzyme a c t i v i t y . The muscle of the C-2 fed trout contained .4% glycogen at f u l l feeding. The muscle of the other d ietary treatments contained .1-.15% glycogen. It appears that trout muscle stored dietary carbohydrate that was not taken up by the l i v e r or that was not ox id ized. Upon fast ing there was a depletion in muscle glycogen to basal leve ls of .05% after 4 days. Thereafter there occurred a rebound in muscle glycogen to leve ls at or greater than f u l l - f e d leve ls followed by a decl ine back to basal values. The rebound was greater and peaked ea r l i e r (8 days) in the C-fed trout than in the F-fed trout (10 days). B ro i l e r (BR) and White Leghorn (WL) pu l le t chicks were fed d i e t s , where 25% of the energy was supplied as corn starch (C) or corn o i l (F), for a three-week period. The chicks were then fasted and sampled for t issue glycogen, protein and dry matter at f u l l feeding and at 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours of f as t ing . The BR l i v e r s contained more glycogen (3%) than the WL l i v e r s (2%) and the C-fed chicks had greater l i v e r glycogen concentrations (3%) than the F-fed chicks (2%) at f u l l feeding. Upon fas t i ng , l i v e r glycogen f e l l to basal leve ls of .03% af ter 8 hours. Thereafter there was a rebound increase in l i v e r glycogen leve ls to a peak of 1% and a subsequent tapering o f f . The rebound occurred ea r l i e r in the C-fed chicks than in the F-fed chicks. The peak was attained ea r l i e r in the WL (32-44 hours) than in the BR (50 hours). The amount of l i v e r protein in the C-fed chicks was higher than that in the F-fed chicks over the ent i re fast ing period. Chick muscle glycogen concentrations were i n i t i a l l y higher in the C-fed than in the F-fed chicks and higher in BR (1.2%) than in WL (.8%). Upon fas t i ng , BR muscle glycogen concentrations were maintained while those of WL f e l l to .3%. There were e r ra t i c f luctuat ions in muscle glycogen l eve l s . - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES ix LIST OF APPENDIX TABLES x i ACKNOWLEDGEMENTS xiv 1. INTRODUCTION 1 2. LITERATURE REVIEW 3 2.1 Early History of Glycogen 3 2.2 Forms and D is t r ibut ion of Glycogen 5 2.3 Control and Regulation of Glycogen Metabolism. • • -7 2.3.1 Enzymes of Glycogen Metabolism 7 2.3.2 Hormones of Glycogen Metabolism 10 2.3.2.1 Insul in and Glucagon 10 2.3.2.2 Other Control 12 2.4 Dietary Influence on Glycogen Metabolism 14 2.5 Fasting Influence on Glycogen Metabolism 18 3. EXPERIMENT 1. A COMPARISON OF GLUCOSE AND HERRING OIL AS THE PRINCIPAL SOURCES OF NON-PROTEIN ENERGY IN A TROUT DIET ON TISSUE GLYCOGEN CONCENTRATION AND ON THE RATE OF GLYCOGEN DEPLETION FROM THE TISSUES DURING A SUBSEQUENT PERIOD OF FAST 24 3.1 Objectives 24 3.2 Materials and Methods . .24 - v -3.2.1 Animals and Maintenance 24 3.2.2 Diets 2 5 3.2.3 Experimental Treatment 26 3.2.4 Chemical Analysis 2 7 3.2.5 Calculat ions 2 8 3.2.6 S t a t i s t i c a l Analysis • • • 2 8 3.3 Results and Discussion 2 9 3.3.1 Trout Body Weight 2 9 3.3.2 Trout L iver 3 0 3.3.3 Trout Muscle 3 6 3.3.4 Trout V i a b i l i t y 41 4. EXPERIMENT 2. A COMPARISON OF CORNSTARCH AND CORN OIL AS SOURCES OF 25 PERCENT OF THE ENERGY IN A CHICK DIET ON TISSUE GLYCOGEN CONCENTRATION AND ON THE RATE OF GLYCOGEN DEPLETION FROM THE TISSUES DURING A SUBSEQUENT PERIOD OF FAST 57 4.1 Objectives 57 4.2 Materials and Methods 57 4.2.1 Animals and Maintenance 57 4.2.2 Diets - 5 8 4.2.3 Experimental Treatment- 58 4.2.4 Chemical Analysis 59 4.2.5 Calculat ions 59 4.2.6 S t a t i s t i c a l Analysis 59 4.3 Results and Discussion 60 - vi -4.3.1 Chick Growth 60 4.3.2 Chick Fasting Body Weights 61 4.3.3 Chick L iver . . .61 4.3.4 Chick Muscle 68 5. SUMMARY 86 REFERENCES 90 APPENDIX TABLES 99 - v i i -LIST OF TABLES Table Page I Composition of trout d iets . . .43 II Calculated nutr ient composition of trout d iets 44 III Body weight and condit ion factor of trout in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 45 IV L iver weight, hepato-somatic index and l i v e r dry matter of trout in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 46 V L iver glycogen of trout in response to d i f fe rent d ietary treatments after d i f ferent periods of fast ing 47 VI L iver protein of trout in response to d i f fe rent d ietary treatments af ter d i f ferent periods of fast ing 48 VII L iver glucose-6-phosphatase a c t i v i t y of trout in response to d i f fe rent dietary treatments after d i f fe rent periods of fast ing 49 VIII Muscle glycogen, muscle protein and muscle dry matter of trout in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 50 IX Composition of chick d iets 70 X Calculated nutr ient composition of chick d iets • • • • • -71 XI Body weights of chicks in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fas t ing 72 XII Estimated i n i t i a l body weights and estimated retention of i n i t i a l body weights of chicks in response to d i f fe rent d ietary treatments after d i f ferent periods of fast ing 73 XIII L iver weights of chicks in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fas t ing 74 XIV Hepato-somatic indices of chicks in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing . 75 - v i i i -XV L iver dry matters of chicks in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fast ing 76 XVI L iver glycogen of chicks in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fas t ing 77 XVII L iver protein of chicks in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 78 XVIII Muscle dry matters of chicks in response to d i f ferent d ietary treatments af ter d i f fe rent periods of fast ing . 79 XIX Muscle glycogen of chicks in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 80 - ix -LIST OF FIGURES Figure Page la Glycogen synthesis and catabolism 8 lb Cascade control of glycogen synthesis and catabolism 8 2a Trout l i v e r weight (g) over the fast ing period 51 2b Trout hepato-somatic index (g l iver/100 g body weight) over the fast ing period 51 3a Trout l i v e r glycogen (g/100 g l i v e r ) over the fast ing period 52 3b Trout l i v e r glycogen (g/100 g body weight) over the fast ing period 52 4a Trout l i v e r protein (g/100 g l i v e r ) over the fast ing period 53 4b Trout l i v e r protein (g/100 g body weight) over the fast ing period 53 5a Trout l i v e r glycogen + protein (g/100 g l i v e r ) over the fast ing period 54 5b Trout l i v e r glycogen + protein (g/100 g dry l i v e r ) over the fast ing period 54 6 Trout l i v e r glycogen + protein (g/100 g body weight) over the fast ing period 55 7 Trout muscle glycogen (mg/g muscle) over the fast ing period 56 8a Bro i le r hepato-somatic index (g l iver/100 g body weight) over the fast ing period 81 , 8b White Leghorn hepato-somatic index (g l i v e r / 100 g body weight) over the fast ing period 81 9a Bro i le r l i v e r glycogen (g/100 g l i v e r ) over the fast ing period 82 9b White Leghorn l i v e r glycogen (g/100 g l i v e r ) over the fast ing period 82 - X -10a Bro i le r l i v e r protein (g/100 g l i v e r ) over the fas t ing period 83 10b White Leghorn l i v e r protein (g/100 g l i v e r ) over the fast ing period . . . 83 11a Bro i l e r l i v e r protein (g/100 g body weight) over the fast ing period 84 l i b White Leghorn l i v e r protein (g/100 g body weight) over the fas t ing period . .84 12a Bro i le r muscle glycogen (g/100 g muscle) over the fast ing period 85 12b White Leghorn muscle glycogen (g/100 g muscle) over the fast ing period 85 - x i -LIST OF APPENDIX TABLES Table Page IA S t a t i s t i c a l analysis of trout body weight in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 99 IIA S t a t i s t i c a l analys is of trout condit ion factor in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 100 IIIA S t a t i s t i c a l analys is of trout l i v e r weight in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fas t ing 101 IVA S t a t i s t i c a l analysis of trout hepato-somatic index in response to d i f ferent dietary treatments af ter d i f fe rent periods of fast ing 102 VA S t a t i s t i c a l analys is of trout l i v e r dry matter in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fast ing 103 VIA S t a t i s t i c a l analysis of trout l i v e r glycogen (g/100 g l i v e r ) in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 104 VIIA S t a t i s t i c a l analysis of trout l i v e r glycogen (mg/100 g body weight) in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 105 VIIIA S t a t i s t i c a l analysis of trout l i v e r protein (g/100 g l i v e r ) in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 106 IXA S t a t i s t i c a l analysis of trout l i v e r protein (mg/100 g body weight) in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 107 - x i i -XA S t a t i s t i c a l analysis of trout l i v e r glucose-6-phosphatase a c t i v i t y (ug phosphate released/ 10 minutes/g l i v e r ) in response to d i f fe rent dietary treatments af ter d i f ferent periods of fas t ing 108 XIA S t a t i s t i c a l analysis of trout l i v e r glucose-6-phosphatase a c t i v i t y (ug phosphate released/10 minutes/g l i v e r protein) in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 109 XIIA S t a t i s t i c a l analysis of trout muscle glycogen in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing "110 XIIIA S t a t i s t i c a l analysis of trout muscle protein in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fas t ing I l l XIVA S t a t i s t i c a l analysis of trout muscle dry matter in response to d i f fe rent dietary treatments a f ter d i f fe rent periods of fast ing 112 IB S t a t i s t i c a l analysis of chick body weights in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 113 11B S t a t i s t i c a l analysis of chick l i v e r weights in response to d i f fe rent dietary treatments after d i f fe rent periods of fast ing . 114 11 IB S t a t i s t i c a l analysis of chick hepato-somatic indices in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 115 IVB S t a t i s t i c a l analys is of chick l i v e r dry matters in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 116 VB S t a t i s t i c a l analys is of chick l i v e r glycogen (g/100 g l i v e r ) in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 117 - xi i i -VIB S t a t i s t i c a l analys is of chick l i v e r glycogen (mg/100 g body weight) in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fast ing 118 VIIB S t a t i s t i c a l analysis of chick l i v e r protein (g/100 g l i v e r ) in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing 119 VII IB S t a t i s t i c a l analysis of chick l i v e r protein (mg/100 g body weight) in response to d i f fe rent d ietary treatments after d i f fe rent periods of fast ing • • 120 IXB S t a t i s t i c a l analysis of chick muscle dry matters in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fas t ing 121 XB S t a t i s t i c a l analys is of chick muscle glycogen in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fast ing .122 - x iv -ACKNOWLEDGEMENTS I would l i k e to thank Professor B.E. March for her o r ig ina l idea which 'spawned/laid' th i s thesis and her support and advice which made i t s completion poss ib le. Her insp i ra t ion and sharing have added great ly to my knowledge and are personally appreciated. Secondly, I would l i k e to thank the members of my committee: Dr. D. Bragg, Dr. B. Owen and Dr. D. Higgs for the i r time and e f fo r t spent on th i s thes is . I would espec ia l ly l i k e to thank Dr. Higgs, Ian McCallum and the s ta f f of the West Vancouver Laboratory (Canada Fisher ies and Oceans) for assistance with the trout experiment and for the use of the f a c i l i t i e s of that i n s t i t u t i o n . I would also thank the s ta f f and students of the Poultry Science Department for assistance read i ly given. Special thanks were due to the V |Tate'Professor J . B ie ly for his constant encouragement. F i na l l y , I would l i k e to thank Sylv ia Chan for her expert typing of th i s thes i s . - 1 -1. INTRODUCTION Glycogen i s a branched chain glucose polymer of the formula ( C g H 1 0 0 5 ) n , having a molecular weight range of 2.7 x 10 5 to 1.0 x 10 8 . There are l inear sections formed from a l -4 g lycos id ic l inkages, and after every 8 to 12 units there are a l -6 g lycos id ic l inkage branch points. Glycogen i s found only in animal t i ssues; i t i s the counterpart of plant starch. I t i s found in varying leve ls and in various forms depending, in part, on the animal species and the type of t i ssue. The l i v e r can contain a large amount of glycogen, as can the muscle when i t s tota l body mass i s considered. Glycogen serves as a storage form of energy. It can be quick ly degraded to glucose because i t s highly branched structure provides many act ive s i tes for the catabol ic enzyme, glycogen phosphorylase. This i s espec ia l ly important in skeleta l muscle t issues because "burst" muscle contract ion requires large amounts of ATP in a short time period, and th i s fuel i s derived from the rapid breakdown of glycogen to lac ta te . In the l i v e r , glycogen serves as a source of blood glucose. I t can be catabol ized and released into the c i r cu la t i on at a var iable rate; slowly, in response to normal metabolic needs ( i . e . growth and maintenance), or qu ick ly , in response to the extraordinary needs of s t ress . The control of glycogen metabolism i s complex, as expected from i t s central ro le in metabolic processes. Its synthesis and degradation are under humoral cont ro l , and i t s degradation i s under nervous control as we l l . - 2 -One of the striking aspects of glycogen metabolism is that diet can markedly influence the amount of glycogen that is stored in the tissues, especially in the l i ver . In balanced diets with only the fat and carbohydrate levels varying, increasing the amount of carbo-hydrate in the diet may increase l iver glycogen tenfold. There are ways, one could imagine, in which different levels of glycogen in the tissues could influence an animal's metabolism in an energy demanding situation. The more glycogen stored could mean that an animal is able to rely upon i t longer in a stress situation before turning to fat and protein for energy. Conceivably this could affect survival. As well , different levels of glycogen in the tissues usually have related levels of the appropriate synthetic and catabolic enzymes associated with them. Thus the rapidity with which an animal adapts to a stress situation could be indirect ly related to glycogen levels. Information is very scarce as to the effects of different glycogen levels in the tissues, as determined by d iet , on the subsequent glycogen metabolism in a state of stress. The study reported in this thesis provides such information with respect to the chick and the trout. - 3 -2. LITERATURE REVIEW 2.1 Early History of Glycogen The existence and function of glycogen were discovered by Claude Bernard in a ser ies of experiments, in the 1840's and 1850's, on dogs. In the f i r s t experiments Bernard demonstrated, by analyzing the sugar content of blood entering and leaving the l i v e r , that the l i v e r can produce sugar. This was an important discovery because the preva i l ing theory of the time said that only plants could synthesize complex molecules - animal metabolism was confined to breaking down substances in the d ie t and using the resu l t ing products d i r e c t l y as components of the body. Bernard thus hypothesized (1853) an internal secretion in l i v e r which gives r i se to glucose. In subsequent experiments he determined, p a r t i a l l y by accident (he l e f t a dupl icate l i v e r sample overnight before analyzing i t for glucose), that glucose in the l i v e r comes from a substance there which i s broken down. Bernard iso lated th i s substance and characterized i t as s ta rch- l i ke . He termed i t 'matiere glycogene' (1857). Later, he extracted from the l i v e r a ' d i a s ta t i c ferment' which was capable of breaking down glycogen to glucose. He reported (1877) the sum of his f ind ings: there i s a substance 'glycogen' present in animal l i v e r s . I t i s broken down to glucose which then enters the blood. Joseph von Mering (1877) found that many sugars; glucose, sucrose, lactose, fructose as Well as g l yce ro l , led to glycogen deposit ion in the - 4 -l i v e r . This was acceptable as i t was general ly postulated that glycogen arose from a condensation of glucose molecules. He also found that egg albumin and casein ingestion led to l i v e r glycogen deposit ion. At f i r s t i t was hard to accept that glycogen could be made from proteins. This ear ly work contributed to the concept of gluconeogenesis. Much of our knowledge of glycogen metabolism was attained between 1900 and 1940. Insul in and i t s ro le in the control of blood sugar leve ls was discovered by Banting and Best. The pathways of g lyco lys i s and gluconeogenesis were mapped. Concerning glycogen i t s e l f , i t s branched structure, the nature of i t s l inkages as well as i t s molecular weight were determined by Haworth and Freudenberg in the 1930's (see Meyer 1943). Perhaps the most s i gn i f i can t contr ibut ions to the understanding of glycogen since Claude Bernard, were made by Carl and Gerty Cor i . In a ser ies of experiments s tar t ing in 1925, concerning the re lat ions among l a c t i c ac id , glucose and glycogen in ra t s , they derived the 'Cor i c y c l e ' . This cycle charts the glucose recyc l ing between the t issues and the l i v e r . L iver glycogen i s broken down to glucose which i s released into the c i r cu l a t i on . The blood glucose i s taken up by the muscle where i t may be converted to glycogen. The muscle glycogen i s broken down to l a c t i c acid which moves out of the t issue and back to the l i v e r v ia the blood stream. There i t i s converted back to glycogen. The Cor i ' s determined that epinephrine functions to accelerate the cycle from muscle glycogen to l i v e r glycogen and to i nh i b i t i t from blood glucose to muscle glycogen. - 5 -The resu l t i s an accumulation of sugar in the blood. Insu l in , on the other hand, was found to accelerate the cycle from blood glucose to muscle glycogen, which leads to hypoglycemia (Cori and Cori 1929). The other major contr ibut ion of the Cor i ' s in th i s area was the i r study of the enzymes involved in the interconversion of glucose and glycogen. They found that glucose-1-phosphate and glucose-6-phosphate are intermediates between glycogen and glucose. They ident i f i ed glycogen phosphorylase and determined i t s regulatory importance (Cori ejt al_. 1939). The biochemical energy transfer mechanisms demonstrated in these studies marked a turning point in the understanding of polymeric molecule synthesis. 2.2 Forms and D is t r ibut ion of Glycogen Glycogen can be stored in d i f fe rent forms. One form i s the glycogen granule or 6 -pa r t i c l e . These par t i c les are 150 to 400 Angstroms in diameter and have a molecular weight range of 1 to 5 m i l l i o n . Another glycogen form i s ca l led the rosette or a - pa r t i c l e and i t i s anywhere from 2 to 5 times larger than the 3-particle. These are the two major types of glycogen in most t i ssues. There are two other glycogen forms that appear to be quite spec ia l i zed: i n t r a ce l l u l a r glycogen bodies in which granules are associated with muscle endoplasmic reticulum or sarcoplasmic ret iculum, and glycogen seas which are made up of small granules sequestered in peripheral regions of the muscle c e l l s . Both 'bodies' and 'seas' are - 6 -associated with highly anaerobic muscle; 'bodies are in high concentration in tuna white muscle, and 'seas' are most prominant in the red muscle of the South American lungf i sh . See Hochachka (1980) for a descr ipt ion of glycogen forms. The basic unit of a l l these forms of glycogen i s the 3 -pa r t i c l e . I t appears that a l l the other forms are aggregates or d i s t r ibu t ions of th i s basic glycogen granule. The a - pa r t i c l e , for example, i s an aggregate of several B-par t i c les (Drochmans and Danton 1968). The means by which granules aggregate i s not known but may be re lated to the glycoprotein nature of glycogen. Current evidence indicates that glycogen i s bu i l t on a protein primer which can be the backbone for many glycogen molecules. The structure i s unknown but i t i s certa in that glycogen i s always associated with a protein component. There i s evidence that the associat ion of 6 -par t i c les into rosettes occurs by disulphide bonds between the d i f fe rent protein f ract ions (Matchem ejt aj_. 1978). The d i s t r i bu t i on of glycogen i s dependent on the type of t i ssue. In muscle, glycogen granules are normally found in the i n t e r f i b r i l i a r spaces and along the Z bands. In skeleta l muscle there are two types of f i b re s : 'wh i te ' , characterized by high glycogen content, low myoglobin and s ingle innervat ion, and ' r e d ' , which is characterized by low glycogen content, high myoglobin and mult ip le innervat ion. The 'white' muscle functions anaerobical ly and i s useful for short term, power response, while the ' red ' muscle operates ox idat ive ly and i s more suited for durat iona l , low power work. Actua l l y , no one skeleta l muscle i s made up of a l l white or a l l red f i b r e s , but i s made proport ional ly from both depending on the muscle's funct ion. - 7 -Liver can be divided into functional areas ca l led a c i n i . The acinus is a mass of c e l l s arranged around an axis cons ist ing of a terminal hepatic a r t e r i o l e , portal venule, b i l e ductule and lymph vessels. The acinus can be divided into three zones defined by the i r distance from the axis vessels and characterized by spec i f i c metabolic a c t i v i t y (Rappaport 1963). Glycogen may or may not be homogenously dispersed throughout the zones. The ind icat ion i s that the greatest glycogen turnover occurs in the peripheral zone (the zone c losest to the axis vesse ls ) , while the intermediary and central acinar zones are more concerned with 'excess' glycogen storage (den Otter and de Minger 1972, Sasse 1975). This subject w i l l be discussed further subsequently. 2.3 Control and Regulation of Glycogen Metabolism See Figure 1 for a diagrammatic representation of glycogen metabolic pathways and cont ro l . 2.3.1 Enzymes of Glycogen Metabolism Synthesis of glycogen from glucose i s accomplished by the j o i n t action of several enzymes, the most important of which i s glycogen synthetase. The glucose must f i r s t be converted to glucose-6-phosphate (G-6-P), then to glucose-l-phosphate (G-l-P) and then to ur idine diphosphate glucose (UDPG) before the rate l im i t i ng enzyme glycogen synthetase may exert i t s cont ro l . - 8 -GLYCOGEN G l y c o g e n s y n t h e t a s e G l y c o g e n p h o s p h o r y l a s e UDP-GLUCOSE <: UDP g l u c o s e p h o s p h o r y l a s e 1 i v e r b l o o d GLUCOSE-1-PH0SPHATE P h o s p h o g l u c o m u t a s e GLUC0SE-6-PH0SPHATE ADP G l u c o s e - 6 - p h o s p h a t a s e F i g u r e l a . G l y c o g e n s y n t h e s i s a n d c a t a b o l i s m . A d e n y l c y c l a s e G l u c a g o n , E p i n e p h r i n e G l y c o g e n < -s y n t h e t a s e k i n a s e ( a c t i v e ) ATP 8 G l y c o g e n — s y n t h e t a s e ( a c t i v e ) ( I ) G l y c o g e n s y n t h e t a s e k i n a s e ( i n a c t i v e ) ADP 2. G l y c o g e n s y n t h e t a s e i n a c t i v e ) 0) G l y c o g e n p h o s p h o r y l a s e k i n a s e k i n a s e ( i n a c t i v e ) ADP G l y c o g e n p h o s p h o r y l a s e k i n a s e ( a c t i v e ) > G l y c o g e n p h o s p h o r y l a s e k i n a s e k i n a s e a c t i v e ) 6 ATP G l y c o g e n p h o s p h o r y l a s e k i n a s e ( i n a c t i v e ) ATP ADP G l y c o g e n ^ — p h o s p h o r y l a s e b ( i n a c t i v e ) -f> G l y c o g e n p h o s p h o r y l a s e a ( a c t i v e ) 9 s i g n i f i e s s t i m u l a t i o n F i g u r e l b . C a s c a d e c o n t r o l o f g l y c o g e n s y n t h e s i s and c a t a b o l i s m . - 9 -Glycogen synthetase adds glucose units onto a glycogen base in a l - 4 l inkage. To accomplish the a l -6 l inkage branch points, there i s another enzyme ca l led amylo (1-4—> 1-6) transglycosidase or 'branching enzyme'. Glycogen synthetase ex is ts in an inact ive (D) form and an act ive (I) form. The I form i s nonphosphorylated and the D form i s phosphorylated. Conversion to the act ive form i s stimulated by increasing G-6-P concentrations, and conversion to the inact ive form i s stimulated by increasing cyclic-AMP concentrations v ia a cascade react ion. Glycogen catabolism to glucose i s accomplished by several enzymes, some of which are also in the synthetic pathway. Glycogen phosphorylase i s the key enzyme and i t catalyzes the removal of glucose units from the glycogen molecule with the formation of G- l-P. As glycogen phosphorylase can only break a l - 4 l inkages, there i s also a 'debranching enzyme' to break the a l -6 l inkages. Glycogen phosphorylase ex i s t s , l i k e glycogen synthetase, in an act ive and inact ive form. Only for the phosphorylase, the act ive form i s phosphorylated which i s the reverse of the synthetase s i tua t ion . Thus a method of control i s achieved whereby glycogen synthetase i s act ivated and glycogen phosphorylase i s inact ivated at the same time, and vice versa. It i s therefore not surpr is ing to learn that the same cascade react ion that inact ivates glycogen synthetase, act ivates glycogen phosphorylase. Glucose-6-phosphatase i s another key enzyme in that i t regulates the release of glucose into the blood stream. Its a c t i v i t y increases in states of gluconeogenesis though, ind icat ing that i t exerts i t s regulatory role in th i s process rather than in glycogenolysis. Further mention w i l l be made l a t e r , but i t i s in teres t ing to note here that the enzyme that - 10 -catalyzes the reverse react ion of glucose to G-6-P; hexokinase, i s missing in the chicken and at low leve ls in the t rout . 2.3.2 Hormones of Glycogen Metabolism 2.3.2.1 Insul in and Glucagon The primary hormones concerned with glycogen metabolism are insu l in and glucagon. These two hormones are antagonist ic to each other and i t i s the i r ra t io in the body at any one time that decides the d i rect ion of glycogen metabolism. Bas i ca l l y , i nsu l i n stimulates glycogen deposit ion and glucagon stimulates glycogen breakdown. The release of i nsu l i n from the pancreas i s stimulated by increasing glucose leve ls in the c i r cu l a t i on , which would ar ise from dietary carbohydrate. The function of i n su l i n i s to 'dispose' of th i s extra glucose, i .e . enable the body to to lerate a glucose load, by enhancing i t s uptake by the t i ssues. Insul in acts pr imar i ly at peripheral t i ssues , most notably muscle. In these areas i nsu l i n improves the permeabil ity of the membranes to glucose so that more glucose enters the ce l l s and, v ia st imulat ion by increasing G-6-P concentrat ions, i s made into glycogen. It appears that i n su l i n also d i r e c t l y stimulates l i v e r glycogen synthesis by an as yet unclear mechanism. M i l l e r ejt al_. (1973) and Bergman and Bucolo (1974) have shown that i nsu l i n reduces hepatic i n t r a ce l l u l a r cAMP leve ls in the rat and dog respect ive ly . This would have the ef fect of i nh ib i t i ng glycogen phosphorylase and st imulat ing - 11 -glycogen synthetase . The mechanism f o r t h i s decrease i n cAMP concen t ra t i ons may be a t the de novo RNA syn thes i s l e v e l . Glucagon r e l ease from the pancreas i s s t imu la ted by dec reas ing blood g lucose l e v e l s . The a c t i o n of glucagon i s to i nc rease blood g lucose l e v e l s by break ing down hepat i c g lycogen. I t s t imu l a te s the a c t i v a t i o n of glycogen phosphorylase by i n i t i a t i o n o f the ' cascade ' r e a c t i on s (see F igure l b ) . Glucagon operates a t the l e v e l o f adenyl c y c l a se so as to i nc rease the l e v e l s o f cAMP. Th is has the u l t ima te e f f e c t o f i n c r e a s i ng glycogen phosphorylase a c t i v i t y and decreas ing glycogen synthetase a c t i v i t y . Glucagon has no e f f e c t on muscle g lycogen. There appears to be spec ies d i f f e r en ce s i n t i s s u e s e n s i t i v i t y to i n s u l i n and i n s u l i n response to g lucose l oads . Raheja ejt al_. (1972) found tha t feed ing a high f a t d i e t to r a t s and c h i c k s , or f a s t i n g them, decreases panc rea t i c i n s u l i n content and impa i rs g l u cose - s t imu l a t ed i n s u l i n s e c r e t i o n i n the r a t but not the ch i c k . Simon and Rosse l i n (1978) found tha t ch ickens g iven a g lucose load and then f a s t ed f o r 65 hours showed an inc rease i n plasma i n s u l i n l e v e l s . Hazelwood (1976) s t a t ed tha t ch ickens are very r e s i s t a n t to exogenous i n s u l i n - even to pharmacologic ( convu l s i ve producing) doses. And Naber and Hazelwood (1977) demonstrated t ha t ch icken panc rea t i c 3-cells, i n con t r a s t to mammalian, are i n s e n s i t i v e to a l l but very high g lucose concen t ra t i ons and are incapab le of a sus ta ined i n s u l i n response. These repo r t s i n d i c a t e tha t the ch i c ken , compared w i th the mammal, r equ i r e s h igher l e v e l s o f i n s u l i n to e x h i b i t the same g lycogen i c response to a g lucose l o ad . - 12 -As an aside, the study of Colca and Hazelwood (1976) ra ises the po s s i b i l i t y of an extrapancreatic source of i n su l i n in chickens. These invest igators demonstrated that 99% pancreatectomy diminishes but does not stop insu l i n response to a glucose load in chickens. Some f i s h have an apparent glucose-intolerance that may be insu l i n dependent. Furuichi and Yone (1971) and Palmer and.Ryman (1972), in studies with red sea bream and trout respect ive ly , have shown that these f i s h poorly to lerate glucose loads. And Shimeno et al_. (1978) showed that the carnivorous ye l l owta i l to lerated a glucose load more poorly than the omnivorous carp. A high fa t d iet or f a s t i ng , w i l l increase glucagon l eve l s . This i s because, as well as st imulat ing glycogenolysis, glucagon stimulates l i v e r gluconeogenesis v ia increasing a c t i v i t y of the regulatory enzyme phosphoenolpyruvate carboxy kinase (PEPCK). PEPCK i s stimulated by increasing cAMP l eve l s . 2.3.2.2 Other Control Thyroid hormones cause l i v e r glycogen catabolism (Cal las and Cannon 1975, Reheja et al_. 1971a).. Thyroid hormone def ic iency, as achieved by radiothyroidectomy or feeding propy l th iourac i l (PTU), allows an increase in l i v e r glycogen leve ls (Snedecor 1968, Raheja e_t al_. 1971b, and Raheja and Linscheer 1978, 1980). The mechanism of th i s act ion i s probably v ia an inf luence of thyroid hormones on glucagon l eve l s . The general action of thyroid hormones i s to increase oxidat ive metabolic rate. This means - 13 -increased glucose u t i l i z a t i o n in the t issues which would cause a decrease in blood glucose. The f a l l i n g blood glucose leve ls would stimulate glucagon mediated glycogenolysis. This i s consistent with the resu l ts of Raheja and Linscheer (1980) who found that PTU treatment of chickens caused a decrease a plasma glucagon to one quarter of the normal concentration, and an increase in plasma insu l i n to 6 times the euthyroid values. Lenzen (1978) found that tr i iodothyronine and thyroxine i nh i b i t glucose-induced insu l i n secretion in ra t s . A number of invest igators have found that feeding a high fat d ie t w i l l increase thyroid hormone production; Suzuki and Fuwa (1971) and Fe l t (1973) in ra t s , March and B ie ly (1957) in chickens, and Higgs et al_. (1981) in f i s h . This is perhaps due to the necessity for thyroid hormones in hepatic gluconeogenesis, a process which would be induced by feeding a high fat d i e t . Llobera et a]_. (1978) found that hypothyroid rats exh ib i t a greater decrease in l i v e r glycogen leve ls upon fast ing than do euthyroid ra ts . It was subsequently determined that hypothyroid rats cannot achieve adequate gluconeogenesis when fasted (Llobera and Herrera 1980). Epinephrine causes a mobi l i zat ion of glycogen. As for glucagon the mechanism is at the level of adenyl cyclase. Epinephrine release from the adrenal medulla is stimulated by the nervous system, and e f fec t i ve l y mobil izes energy reserves in a short term, emergency stress s i tua t ion . Epinephrine stimulates the catabolism of both l i v e r and muscle glycogen, whereas glucagon affects only the l i v e r . This is funct iona l l y useful because as muscle has no glucose-6-phosphatase i t cannot release glucose into the c i r cu l a t i on . Therefore, a glucagon mediated catabolism of muscle glycogen could not serve whole animal - 14 -energy needs. The adrenal cortex releases glucocort icoids which have the opposite e f fect of epinephrine: glycogen deposit ion i s i nd i r ec t l y enhanced v ia a decreased u t i l i z a t i o n of glucose by the t i ssues. 2.4, Dietary Influence on Glycogen Metabolism Dietary composition af fects glycogen deposit ion great ly. Numerous references for many species are ava i lab le which show that increasing the amount of carbohydrate in the d iet increases the amount of glycogen deposited in the t i ssues . Concerning glycogen in rats ; Akrabawi et al_. (1974) showed that animals fed a fa t ty acid based d ie t supplemented with graded leve ls of starch increased the i r l i v e r glycogen content as d ietary starch increased. H i l l e_t al_. (1974) and den Otter and deMinjer (1972) found that increasing the amount of carbohydrate in the d iet increased l i v e r glycogen l eve l s . Vrana et al_. (1978a, b) showed that the type of d ietary carbohydrate influenced the amount of glycogen deposited in the rat l i v e r ; starch and glucose had a greater l i v e r glycogenic e f fect than sucrose or fructose. Kang et_ al_. (1979) had s l i g h t l y d i f fe rent resu l t s . They found that starch enhanced glycogen stores the most, followed by sucrose, glucose and fructose respect ive ly . The glycogen content of chickens i s great ly affected by dietary composition. Brambila and H i l l (1966), in a study with male b ro i l e r ch icks, found that a high carbohydrate d ie t gave a l i v e r glycogen content of 3.8% wet weight, while a high fa t d ie t gave a glycogen content of 2.0%. - 15 -Renner and Elcombe (1967), in feeding experiments with male b ro i l e r chicks, observed l i v e r glycogen to range from 2.3% to 3.5% on high carbohydrate d iets and from .5% to .9% on low carbohydrate, fa t based d ie t s . When the glycerol portion of the fat based d iets was removed, i . e . when the non-protein energy source of the diets was fa t ty acids rather than g lycer ides, the l i v e r glycogen content was further reduced to .1%. No d ietary difference in breast muscle glycogen was detected. Rosebrough and colleagues have conducted several studies on the effects of d ietary composition on poultry glycogen metabolism. Rosebrough and Begin (1975) reported that meat-type chicks had a higher concentration of glycogen in the gastrocnemius muscle on a high carbohydrate than on a high fa t d i e t . Rosebrough et a]_. (1978) found that turkey hens have lower l i v e r glycogen leve ls and higher glycogen phosphorylase, glycogen synthetase and glucose-6-phosphatase a c t i v i t i e s on a high fat d ie t . They also found that feeding a high carbohydrate d ie t caused a greater act ivat ion of glycogen synthetase than did feeding a high fat d i e t . In the developing turkey poult , Rosebrough et al_. (1979a), found an increase in l i v e r glycogen upon feeding a high carbohydrate d i e t , as well as an increase in glucose-6-pnosphate concentrations. They also observed an increase in glycogen synthetase a c t i v i t y but no increase in a c t i v i t y of glycogen phosphorylase. Rosebrough e_t al_. (1979b), in a study with White Leghorn chicks, found that glucose or sucrose supplementation of the d ie t resulted in higher l i v e r and carcass glycogen contents. They also confirmed that increasing dietary carbohydrate increased the synthetase/phosphorylase. It has been - 16 -demonstrated by Schwartz and Rail (1973) that the interact ion of the act ive forms of glycogen synthetase and glycogen phosphorylase control glycogen degradation in the ra t . Watts and Gain (1976) suggested that the ra t io of act ive synthetase to act ive phosphorylase regulates glycogenolysis. Seaton ejt al_. (1978) s im i l a r l y found that chicks fed diets high in carbohydrate had higher l i v e r glycogen leve ls than when dietary carbohydrate was low. Raheja et a]_. (1978, 1980) showed that the PTU-induced glycogen accumulation in chicken l i v e r s i s greater on a high carbohydrate d iet than on a high fa t d i e t . The potent ia l for glycogen deposit ion in f i s h i s very high: greater than 16% wet weight in rainbow trout l i v e r (Hochachka and S i n c l a i r , 1962), 12% in Chinook l i v e r (Buhler and Halver, 1961) and greater than 12% in the hepatopancreas of carp (Nagai and Ikeda, 1971). These three studies examined the ef fects of increasing dietary carbohydrate on glycogen deposit ion. The high l i v e r concentrations suggest that f i s h cannot read i ly metabolize dietary carbohydrate and accordingly store i t in the t i ssues , espec ia l ly in the l i v e r . Early studies concerning the ef fects of d ietary carbohydrate on glycogen deposit ion were conducted by Tunison et aj_. (1939) and Ph i l l i p s ejt al_. (1948). They found that glycogen deposit ion in trout l i v e r s was enhanced by increasing dietary carbohydrate. S imi lar resu l ts have subsequently been reported for carp, sea bream and ye l l owta i l (Furuichi and Yone, 1980), ca t f i sh (Andrews and Davis, 1979), rainbow trout (M i l l e r et al_. 1959, and Bergot 1979) and p la ice (Cowey e ta l _ . 1975). The study by Cowey et al_. (1975) i s in terest ing because i t approached the ef fect - 17 -more d i r e c t l y . After feeding the p la ice on high carbohydrate and low carbohydrate d iets for several weeks, the f i s h were injected with 14 C-glucose. The uptake of the labe l led glucose into l i v e r glycogen was greater in the high carbohydrate than in the low carbohydrate fed f i s h . This indicates that the a c t i v i t y of the glycogen synthesizing 'machinery' was higher in the l i v e r s of the former group. There are other studies which i l l u s t r a t e the effect- of dietary carbohydrate on increasing glycogen deposit ion: Shimeno et al_. (1978) with ye l lowta i l and carp, Nagai and Ikeda (1971) with carp and Pieper and Pfeffer (1978) with t rout . However, these invest igat ions cannot r e l i ab l y estimate the extent of th i s ef fect because carbohydrate was added to the d iets at the expense of prote in, Lee and Wales (1973) have shown that d ietary protein can inf luence glycogen deposit ion. In trout experiments, they found that increasing d ietary protein content from 36% to 53%, with a concomitant decrease in ' d i ges t i b l e ' carbohydrate from 27% to 9%, caused an increase in l i v e r glycogen from 3.1% to 6.2% of wet weight. Therefore the three 'confounded' reports have probably underestimated the ef fect of d ietary carbohydrate on glycogen deposit ion. Indeed, Nagai and Ikeda expected a greater difference in carp hepatopancreatic glycogen between d iets than actua l ly occurred. Fish muscle glycogen content may also be affected by dietary composition. M i l l e r et al_. (1959) and Hochachka and S i n c l a i r (1962) found that increasing the carbohydrate content of rainbow trout d iets w i l l increase the glycogen concentrations in white muscle. Furuichi and Yone (1980) observed the same ef fect in ye l l owta i l but not in sea bream and carp. - 18 -The impl icat ion of th i s l a t t e r study i s that the carnivorous ye l lowta i l i s less able to u t i l i z e d ietary carbohydrate than the omnivorous sea bream and carp. This i s supported by the ea r l i e r glucose tolerance studies of Furuichi and Yone (1971) and Shimeno et aj_. (1978). The study by Shimeno ejt al_. l ikewise showed that the ye l lowta i l has a higher gluconeogenic capacity, as evidenced by higher glucose-6-phosphatase a c t i v i t y , than the carp. Black ejt al_. (1961) reported that coho salmon and steel head trout have very low leve ls of hexokinase in the t i ssues , espec ia l l y muscle. Nagayama ejt al_. (1972) found s im i l a r l y low hexokinase levels for carp and ee l , as well as for t rout . This would indicate that f i s h cannot read i ly metabolize, i . e . take up, glucose at the t i ssue l e ve l . This may be the reason for the apparent glucose intolerance of f i s h . Interest ing ly , chickens also have no hexokinase (Hazelwood 1976) and th i s i s consistent with the i r apparent i n sens i t i v i t y to i n su l i n . 2.5 Fasting Influence on Glycogen Metabolism Liver glycogen serves as a source of blood glucose for animal energy requirements during fas t ing . It i s thought that the depletion of l i v e r glycogen during a fast ing stress i s a measure of an animal's a b i l i t y to adapt to and withstand th i s s t ress . In the ra t , Freedland (1967) observed a f a l l in l i v e r glycogen from 4.35% of wet weight at f u l l feeding to .07% after one day of fas t ing; - 19 -thereafter there was an increase to .77% after 4 days fas t ing . Goldstein and Curnow (1977) found that rat l i v e r glycogen f e l l from 2.1% to .7% after 24 hours f a s t i ng , and then increased to 25% of fed leve ls from 48 to 120 hours. In hamsters, Sasse (1975) noted a decrease in l i v e r glycogen at 16, 36 and 48 hours, and a rebound at 72 and 96 hours fas t ing . Hazelwood and Lorenz (1959) followed l i v e r glycogen depletion in chickens. In the 8-week o ld , male White Leghorn they measured l i v e r glycogen to be 1.85%, 0.57%, 0.23%, 0.25%, 0.43% and 0.47% of wet weight af ter 0, 6, 12, 24, 48 and 72 hours fast ing respect ive ly . In the 10-week o ld , female White Leghorn, l i v e r glycogen was 1.84%, 0.31%, 0.42%, 0.63%, 0.80%, 0.42% and 0.19% af ter 0, 1, 3, 4, 6, 7 and 8 days fas t ing . Davison and Langslow (1975) found that l i v e r glycogen in the fasted, 6-week old Rhode Island Red was 2.81%, 0.14%, 0.27%, 0.14% and 0.09% after 0, 12, 24, 48 and 72 hours. Most examinations of l i v e r glycogen depletion in f i s h have shown a steady f a l l in leve ls upon fas t ing - with no rebound. Stimpson (1965) found a loss in goldf ish l i v e r glycogen of 50% over an 8-day fas t . Swallow and Fleming (1969) found that l i v e r glycogen in Tilapia-mossambica was p a r t i a l l y depleted over a 19-day fas t . Black et'al_. (1966) starved trout for 84 hours and found that l i v e r glycogen decreased from 3.66% to 1.38%. Nagai and Ikeda (1971) fasted carp for 101 days and observed that hepatopancreatic glycogen did not vary appreciably from or ig ina l concentrations of 8.51% during the f i r s t 22 days, but declined to 1.55% by day 101. - 20 -Milne et al_. (1979) found that rainbow trout l i v e r glycogen f e l l to basal leve ls at 20 days fast ing and stayed there unt i l the end of the experiment at 65 days. Vernier and Sire (1978) found that trout l i v e r glycogen f e l l over a 7-day fas t . Shaff i (1.979) noted a decl ine in ca t f i sh l i v e r glycogen over a 40-day fast along with an increase in glucose-6-phosphatase a c t i v i t y - ind icat ive of increased gluconeogenesis. The data of Shimma et_ al_. (1976) on fasted trout indicate that l i v e r glycogen f a l l s to 20 days and is s l i g h t l y higher at 31 days fas t ing . After longer fast ing the concentrations vary. Sakaguchi (1976) reported l i v e r glycogen leve ls of 2.6%, 0.2%, 0.1%, 0.1%, 0.1%, 0.1%, 0.2% and 1.3% at 0, 2, 3, 5, 10, 22, 32 and 50 days fast ing in the yellow ee l . Dave et al_. (1975) measured l i v e r glycogen in the European eel to be 4.00%, 5.24%, 4.30% and 0.71% at 11, 47, 96 and 164 days fast ing respect ive ly . In eels there appears to be a rebound in l i v e r glycogen leve ls af ter about 50 days. In rats and chickens, the 'rebound' in l i v e r glycogen under fast ing stress appears to be a common phenomenon. It i s not so in f i s h , however, or i t occurs only a f ter a longer fast ing time. The reasons for the rebound are unknown. One may speculate that the rebound i s coincident with act ive gluconeogenesis, both processes reaching a maximum at the same time. Then as body reserves become depleted and body weight decreases, gluconeogenesis f a l l s of f and l i v e r glycogen i s depleted for the f i na l time before death. The peak in glycogen rebound i s l i k e l y influenced by factors such as age, sex, body weight and temperature. - 21 -Glycogen may also be depleted from the muscle of fasted animals even though muscle glycogen i s not a source of blood glucose. In the monkey, Rivera and Martinez-de Jesus (1974) observed a f a l l in muscle glycogen af ter 3 days fas t ing . M ig l i o r i n i et al_. (1973) observed a fal-1 in the pectoral muscle glycogen of- the vulture from i n i t i a l levels of 0.691.to 0.28% at 3 days fas t ing . The data regarding the ef fects of fast ing on f i s h muscle glycogen are not consistent. No depletion of muscle glycogen was noted i n : goldf ish (Stimpson 1965), t i l a p i a (Swallow and Fleming 1969) or trout (Black et al_. 1966). On the other hand, Shaff i (1979) and Hochachka and S i n c l a i r (1962) found that muscle glycogen content in ca t f i sh and trout respect ive ly , f e l l upon fas t ing . Dave et al_. (1975) noted that European eel muscle glycogen leve ls were 0.15%, 0.14%, 0.23% and 0.15% at 11, 47, 96 and 164 days of fas t ing . It i s uncertain whether the glycogen increase observed at 96 days represents a true rebound. Blood glucose leve ls were also elevated at that time. In summary, there i s no pattern re la t ing muscle glycogen to fast ing in the data that has so far been reported. One i s tempted to speculate that muscle has a preferred glycogen concentration at which to operate in any spec i f i c metabolic s i tua t ion ; e.g. f as t i ng , and th i s level i s attained independently of the s tar t ing concentration. Therefore, i n i t i a l glycogen concentration would determine whether there i s a decrease in muscle glycogen content upon fast ing and i n i t i a l muscle glycogen concentration i s influenced by d ietary carbohydrate. - 22 -There have been very few studies invest igat ing the ef fect of d ietary h is tory on the subsequent glycogen metabolism of a fas t ing animal. Akrabawi et al_. (1974) fed rats d iets high in carbohydrate or fa t t y acids and achieved glycogen leve ls of 1.39% and 0.97% respect ive ly in the l i v e r . After fast ing for 18 hours, l i v e r glycogen leve ls were 0.71% and 0.97% respect ive ly; i . e . there was a decl ine for the high carbohydrate-fed and no change for the fat- fed ra ts . The study of den Otter and de Minjer (1972) i s more i l l u s t r a t i v e . They fed rats and mice d iets high in e i ther carbohydrate or f a t . In the rats th i s establ ished i n i t i a l l i v e r glycogen levels of 16% and 1%. After 1 day of fast ing the glycogen of the carbohydrate-fed rats was v i r t u a l l y zero while that of the fa t - fed rats remained at about 1%. Upon further f as t i ng , both groups increased l i v e r glycogen content to the same peak leve ls at the same time. Subsequently, glycogen was depleted and the animals d ied. S imi lar resu l ts were observed for the mice. The authors were able to corre late the peak rebound time with s ta r t ing body weight by the formula: ^ i........ .i.-.i I... . J i i V Body Weight Number of Days to Peak = Constant In th i s study as in that of Akrabawi et_ al_. (1974), there was no decrease in the l i v e r glycogen of the fat- fed animals. This i s probably because these animals were already in a state of extreme gluconeogenesis at the s ta r t of f a s t i ng . The animals of den Otter and de Minjer were fed a 100% fa t d iet - hardly well balanced! The Akrabawi - 23 -d iets were l i t t l e better. H i l l et a]_. (1974) demonstrated that rats fed a fa t ty acid based d ie t showed growth retardat ion, hypoglycemia, severe ketonemia and depletion of l i v e r glycogen. When the animals were fasted for 24 hours the ketonemia was reduced and both l i v e r glycogen and blood glucose increased. Thus the energy demands of metabolizing a highly gluconeogenic d iet can be greater than those of fas t ing . This i s the normal s i tuat ion for some animals. Veiga et a l . (1978) found that in the carnivorous vu l ture, gluconeogenesis decreases upon fas t ing . The study of Hochachka and S inc l a i r (1962) on rainbow trout also touches upon the focus of th i s thes is . They found that feeding high and s l i g h t l y lower leve ls of d ietary carbohydrate resulted in l i v e r glycogen contents of 4.13% and 3.31% respect ive ly . At 7 and 14 days fas t i ng , trout on the f i r s t d ie t had glycogen leve ls of 1.31% and 0.93% in the l i v e r . On the other d iet the leve ls were 1.60% and 1.19%. There was no s ign i f i can t dif ference in the depletion patterns between d ie t s . - 24 -3. EXPERIMENT 1. A COMPARISON OF GLUCOSE AND HERRING OIL AS THF PRINCTPAI SOURCES OF NON-PROTEIN ENERGY IN A TROUT DIET ON TISSUE  GLYCOGEN CONCENTRATION AND ON THE RATE OF GLYCOGEN DEPLETION  FROM THE TISSUES DURING A SUBSEQUENT PERIOD OF FAST 3.1 Objectives This experiment was conducted to examine the ef fects of feeding nu t r i t i ona l l y adequate d i e t s , where the only var iable was carbohydrate versus fa t as the non-protein energy source, to trout on the subsequent depletion of glycogen from the l i v e r and skeleta l muscle under fast ing st ress . It was postulated that a high concentration of glycogen in the l i v e r and muscle at the s ta r t of a fast ing period would give the trout an extra energy supply which would be of advantage in withstanding the fast ing s t ress . I t was also speculated that d i f fe rent concentrations of glycogen in the t issues might have d i f fe rent a c t i v i t i e s of glycogen metabolizing enzymes associated with them. The interact ion of feeding level with d ietary carbohydrate in terms of ef fect on glycogen deposit ion was also invest igated. 3.2 Mater ia ls and Methods 3.2.1 Animals and Maintenance On Apr i l 9, 1979, 160 rainbow trout (Salmo ga i r dne r i i ) , about one-year-old, were received from Sun Val ley Trout Farms (Mission, B.C.) - 25 -into the West Vancouver Laboratory (Canada Fisher ies and Oceans). They were evenly d is t r ibuted into three f iberg lass tanks. The tanks each contained 1,100 l i t e r s of aerated well water at 10° + .5°C. Water exchange was a 100% flow through system at a rate of about 10 l i t e r s / minute. The tanks were i l luminated by overhead 40W fluorescent l i gh t s timed to a natural photoperiod. 3.2.2 Diets Three diets were formulated; an accl imation d ie t in which non-protein ca lor ies came from both fa t and carbohydrate, a high carbohydrate d ie t (C) having glucose as the primary non-protein energy source and a high fa t d ie t (F) having herring o i l as the main non-protein energy source. The composition of these d i e t s , in terms of feed ingredients, i s given in Table I and, in terms of nutr ient concentrations, in Table I I . A l l ingredients used were commercial products except the pol lock meal which was prepared at the West Vancouver Laboratory. It was made from whole pollock (Theragra chalcogramma) by cooking at 100°C, pressing and drying at 75-83°C in a steam jacketed d r i e r . A l l feedstuffs were ground, i f necessary, to pass through a U.S. 20 sieve (840 um). The d iets were blended in a Hobart mixer for 20 minutes and then cold pel leted in a Ca l i fo rn ia model CL-type 2 laboratory pe l l e t m i l l having a 3.18 mm d ie . In the high fa t d i e t , only half of the herring o i l was added pr io r to pe l l e t i ng ; the remainder was sprayed on the pe l le ts af ter pe l l e t i ng . The diets were stored at -40°C unt i l a few days pr ior to feeding. - 26 -3.2.3 Experimental Treatment The trout were fed the accl imation d ie t for two weeks at a level of 2% of body weight (BW)/day. The feeding level was estimated on the basis of 53 f ish/tank with an average BW of 250 g / f i sh . On Apr i l 22, the trout were randomly d is t r ibuted into 4 tanks (1,100 l i t e r s ) so that there were 39 or 40 f i sh/tank. The f i s h were anaesthetized, in 0.5 ml 2-phenoxyethanol per l i t e r of water, and ind i v idua l l y weighed. The feeding of the C and F d iets was i n i t i a t ed : one tank of f i sh (n = 40) was fed C at 1% BW/day (C - l ) , one tank of f i s h (n = 39) was fed C at 2% BW/day (C-2), one tank of f i s h (n = 40) was fed F at 0.81% BW/day (F- l ) and the f i na l tank of f i s h (n = 40) was fed F at 1.62% BW/day (F-2). • I t was decided that the C and F d iets should be fed iso-nitrogenously; therefore the F d iets were fed at leve ls 81% of the C d iets ( i . e . 845/1040 = 0.81). At the end of two weeks, feeding was stopped and a period of starvat ion was i n i t i a t e d . Six f i s h from each of the four treatments were sampled on the l as t day of feeding and at 2, 4, 8, 10 and 13 days of fas t ing . At 16 days of fast ing the f i na l three of four trout in each tank were sampled. A l l f i s h were sampled at the same time of day. The decision as to when to sample the f i s h was based on the resu l ts of l i v e r glycogen analys is of the previous sample. The trout were k i l l e d by a sharp blow to the s k u l l . The f i s h were ind iv idua l l y weighed and measured for fork length. The l i v e r was excised, frozen in l i qu i d nitrogen, weighed, bagged and kept frozen in i ce . A 0.2-0.3 g section of white muscle was cut from the area jus t anter ior to the dorsal f i n on the r ight side of the trout - 27 -and placed into l i qu i d nitrogen. It was then bagged and kept frozen in i ce . Freezing of the l i v e r was accomplished within 45 seconds from the time of the blow to the head while the muscle sample was frozen within 90 seconds of th i s time. 3.2.4 Chemical Analysis L iver and muscle glycogen were determined by a modif icat ion of the anthrone method of Hassid and Abraham (1957). A 0.5 g section of frozen l i v e r was digested in 3.0 mis of 30% (w/v) potassium hydroxide for 20 minutes in a bo i l i ng water bath. The glycogen was prec ip i tated by addit ion of 5.0 mis of 95% ethanol and iso lated by centr i fugat ion at 10,000 rpm for 10 minutes and disposal of the supernatant. After reconst i tut ion and d i l u t i on of the pure glycogen with water, 2.0 mis of th i s sample so lut ion was added to a test tube containing 4.0 mis of f resh ly prepared 0.2% anthrone in concentrated sulphuric ac id . The solut ions were vortex mixed and placed in a bo i l i ng water bath for 10 minutes. The solut ions were cooled and the absorbance was read immediately in a Unicam SP 1800 U l t r av i o l e t Spectrophotometer at a wavelength of 620 mu. By the use of a glucose standard, the amount of glucose that had reacted with anthrone was ca lcu lated. A factor of 1.11 was used to convert th i s f igure to the amount of glycogen in the sample. L iver glucose-6-phosphatase (EC 3.1.3.9) was measured by the method of Baginski et al_. (1974) in which a l i v e r homogenate i s incubated with glucose-6-phosphate and the enzyme a c t i v i t y i s measured as the amount of phosphate released per unit weight of t issue during the incubation time. - 28 -The only modif icat ion of the procedure was that uncentrifuged homogenates were used in th i s assay so that pa r t i t i on of enzyme a c t i v i t y into subce l lu lar f ract ions was not poss ib le. L iver and muscle dry matter was determined, and preparation for protein analysis was accomplished, by l yoph i l i za t i on of the frozen t issues in a V i r t i s 10-145 MR-BA type Freeze-Mobile. L iver and muscle protein was determined by the Kjeldahl macro method for nitrogen determination (AOAC, 1970) on a Buchi 430 Digestor and a Buchi 325 Nitrogen D i s t i l l a t i o n Unit . 3.2.5 Calculat ions Three f i s h from the f u l l fed sample (one each from treatments C - l , C-2 and F-l) were found to have l i v e r glycogen concentrations of less than 0.5 g/100 g l i v e r . These f i s h were excluded from a l l ca l cu la t i ons : A l l chemical analyses, physical measurements and ca lcu lat ions were made on ind iv idual f i s h - there was no pool ing. 3.2.6 S t a t i s t i c a l Analysis The design of the overal l experiment was 2x2x7 f a c t o r i a l . Factor 'A' was dietary energy source (C vs F) , factor 'B' was feeding level ha l f -sa t ia t ion versus sat ia t ion) and factor ' C was fast ing time (0-16 days). The data was subjected to analysis of variance by UBC AN0VAR s t a t i s t i c a l computer package for the 2x2x7 fac to r i a l design, for the 2x2 - 29 -f ac to r i a l design (differences between diets and feeding leve ls within each fas t ing period) and, where appl icab le, for the s ingle factor design (differences between d iet or feeding level within each fast ing per iod). The Newman-Keuls mult ip le range test was used to determine which treatment means were s t a t i s t i c a l l y d i f f e ren t . Pr ior to the analysis of variance, data expressed as proportions was subjected to angular (arcsine) transformation, and data judged to be heteroscedastic was subjected to logarithmic transformation (Zar 1974). 3.3 Results and Discussion 3.3.1 Trout Body Weight At the s ta r t of the two week experimental feeding, the average body weights of the f i sh were 239 + 5 g (SEM), 258 + 6 g, 246 + 5 g and 237 + 4 g for C - l , C-2, F-l and F-2 d ietary treatments respect ive ly . At the end of the feeding period the body weights were 235 +15 g, 328 + 8 g, 263 + 9 g and 264 + 8 g. Thus the body weight gains for C - l , C-2, F-l and F-2 were -4 g, 70 g, 17 g and 27 g respect ive ly . The differences in body weight gain were not s t a t i s t i c a l l y s i gn i f i can t . Likewise, the decrease in trout body weights over the fast ing period (Table I I I ) , while being evident in the general sense, i s not resolvable into consistent depletion differences between treatments because of the differences in indiv idual body weights. Calculat ion of the 'condit ion f a c t o r ' ; - 30 -the resu l ts of which are given in Table I I I , shows that ' cond i t ion 1 decreased for trout over the f i r s t 8-10 days af ter feed withdrawal and thereafter leveled of f . The C-fed trout had a higher condit ion factor overal l than the F-fed trout . There was no difference in condit ion factor between feeding l e ve l . 3.3.2 Trout Liver The l i v e r weights of the C d ie t fed f i sh were larger , at f u l l feeding, than those of the F d iet fed f i s h (Table IV and Figure 2a). As we l l , the C-2 l i v e r s were heavier than the C-l l i v e r s . Over the fast ing period these differences were maintained unt i l day 8 when the l i v e r weights of the C f i s h decl ined to the leve ls of the F f i s h . The l i v e r weights of the F. trout decreased only s l i g h t l y over the fast ing period. L iver weight i s commonly related to body weight by the anthropo-metric measure ca l led 1hepato-somatic index' (HSI) in order to make comparisons of l i v e r weight independent of body weight var ia t ions . These HSI values are given in Table IV and Figure 2b. The resu l ts are s imi lar to those of absolute l i v e r weights. Differences between f i s h fed C and F at f u l l feeding were enhanced in the HSI measurements and differences between C-l and C-2 were diminished. HSI decreased f a i r l y uniformly over the fast ing period. Differences between treatments were no longer apparent by day 10 of fas t ing . These resu l ts indicate that in fast ing t rout , l i v e r t issue i s depleted before the rest of the body. - 31 -The moisture content of the F trout l i v e r s was constant at about 75% over the' fas t ing period (Table IV). The water content of the C l i v e r s was i n i t i a l l y lower; about 72% for C-l and about 70% for C-2, but by day 8 of the fast ing period the l i v e r s increased in water content to the 75% level of the F groups. The reason for the i n i t i a l l y lower water content in the C l i v e r s was probably due to the i r higher glycogen content - discussed below. As expected, the l i v e r s of f u l l - f e d trout on the C d ie t contained more glycogen than those on the F d ie t (Table V and Figure 3). On a percentage bas is , the C l i v e r s had roughly 4 X more glycogen than the F l i v e r s (Figure 3a). When the trout were starved, the glycogen in the F l i v e r s f e l l to a basal level of approximately 1% after 2-4 days. In the C l i v e r s , the glycogen content f e l l to th i s basal level only after 10 days fas t ing . There was no di f ference in l i v e r glycogen content between feeding l eve l s . When glycogen content i s expressed in terms of body weight (Table V and Figure 3b), the differences in glycogen content between C-l and C-2 become evident. This i s of course because the C-2 trout had a larger HSI than the C-l t rout . Expressing l i v e r glycogen in terms of body weight also accentuates the dif ference between C and F. At f u l l feeding the C trout had roughly 8 X more l i v e r glycogen in the i r bodies than the F t rout . The depletion pattern for l i v e r glycogen over the fast ing period was the same whether expressed on a per body or on a percent l i v e r basis. The C-l trout ingested amounts of dietary carbohydrate midway between the C-2 and the F d ietary treatments. The amount of glycogen stored in C-l l i v e r s was nevertheless, c loser to C-2 than F l eve l s . - 32 -There are two possible explanations. It may be that the re lat ionsh ip between l i v e r glycogen deposit ion and amount of carbohydrate ingested increases l i nea r l y and then levels of f as the l i v e r i s unable to store any more glycogen. We don't know where on such a glycogen deposit ion curve th i s experiment l i e s but the C diets were probably pushing glycogen deposit ion in the l i v e r to a maximum. The resu l ts of Hochachka and S i n c l a i r (1962) indicate that trout l i v e r s can store up to 16% glycogen. The other explanation i s that C-l trout were growing more slowly than C-2 t rout , because they received only half the dietary nutr ients , and would hence have a lower energy requirement and would thus u t i l i z e r e l a t i v e l y less carbohydrate for energy than the C-2 t rout . This would tend to diminish differences in l i v e r glycogen deposit ion between C-l and C-2. An a l te rnat ive way of looking at th i s nu t r i t i ona l def ic iency scenario of the C-l trout i s to say that they were u t i l i z i n g r e l a t i v e l y more of the i r d ietary carbohydrate for energy than the C-2 trout because these C-l trout had a re l a t i ve def ic iency in the other energy sources; protein and fa t . In such a s i tuat ion there would be less glucose avai lable for l i v e r glycogen deposit ion in the C-l fed f i s h and there would be an enhancement of the difference in l i v e r glycogen leve ls between C-l and C-2. It i s most l i k e l y that the curve model of glycogen deposit ion i s r e a l . The resu l t i s , however, that there i s extra glucose in the bodies of the C-2 trout that cannot be put into l i v e r glycogen stores. What happens to th i s glucose, i s i t oxidized or stored elsewhere? It i s general ly thought that trout cannot read i ly catabol ize dietary carbohydrate. - 33 -Cowey et al_. (1975) found that p la ice oxidized only 12-23% of a radioact ive dose of glucose (10% was converted to l i v e r glycogen and 57% stayed as glucose) over an 18-hour period at 15°. In contrast, mice oxidized 80% of a s imi la r trace glucose infusion in 8 hours. The authors claimed that the difference in the rate of glucose catabolism between mice and pla ice was greater than could be accounted for by the Q-|Q e f fec t . Experiments s imi la r to that of Cowey et al_. (1975) with trout would be useful to estab l i sh the a b i l i t y of trout to catabol ize glucose and to measure the re la t ionsh ip between carbohydrate loading and glycogen deposit ion. It seems l i k e l y that the t rout , l i k e p la i ce , cannot read i ly metabolize glucose. I f that i s the case, then where did the extra carbohydrate in the C-2 fed trout go? The next section w i l l show that muscle glycogen i s a l i k e l y storage area. The overal l p icture of trout l i v e r glycogen (g/100 g body weight) depletion i s very s imi lar to that of HSI (Figure 2b). This would seem to indicate that there i s no need to ac tua l l y determine glycogen concentrations to gauge i t s changes over a fast ing period - that a measure of HSI w i l l do. There are, however, other constituents in l i v e r besides glycogen. Liver protein measurements are given in Table VI and Figure 4. L iver protein concentration (Figure 4a), i s an apparent mirror image of l i v e r glycogen concentration (Figure 3a). On a dry weight bas is , l i v e r percent glycogen + l i v e r percent protein (Figure 5b) remained f a i r l y constant over the 16-day period of feed withdrawal. When th i s ca lcu la t ion i s expressed on a wet weight basis (Figure 5a), there i s an i n i t i a l d i f ference between C and F because of the previously mentioned difference in l i v e r dry matter between these treatments. - 34 -Again, a more informative way of looking at l i v e r protein i s to express i t in terms of body weight (Table VI and Figure 4b). Here we see that there was i n i t i a l l y more l i v e r protein in the C trout than in the F t rout , and there was more l i v e r protein in the high feeding level trout than in the low feeding level t rout . Over the fast ing period, l i v e r protein was depleted at a steady rate from the f i s h on a l l treatments except F - l , in which the f i s h showed no deplet ion. I t i s probable that the increased amounts of l i v e r protein in the C trout represents l i v e r st ructura l prote in. On the other hand i t could be protein that i s s t ruc tu ra l l y associated with glycogen and/or extra amounts of enzymes associated with the extra glycogen. It i s reasonable that animals on a high plane of nu t r i t i on would have larger l i ve r s with which to metabolize the ingested food and th i s could be the reason for the observed dif ference in l i v e r s izes between feeding l eve l s . Perhaps the extra protein in the l i v e r s of the C trout ar ises from a st imulat ion by increased l i v e r glycogen or increased blood glucose thereby enhancing the a b i l i t y of the l i v e r to store glycogen; The resu l t of adding the trout l i v e r glycogen and protein contents i s given in Figure 6. The s im i l a r i t y of th i s set of curves to those of HSI (Figure 2b) i s remarkable. The conclusion i s therefore that changes in l i v e r weight, in terms of body weight, over the fast ing period could be accounted for by changes in glycogen + prote in. Fat and ash, the other major proximate constituents of the l i v e r together with water, remained approximately constant. - 35 -Glucose-6-phosphatase (G-6-Pase) a c t i v i t y in the trout l i v e r s was assayed and the resu l ts are given in Table VII . When enzyme a c t i v i t y was expressed as ug inorganic phosphate released in 10 minutes per gram of l i v e r , then there was i n i t i a l l y a higher G-6-Pase a c t i v i t y in the F f i s h than in the C f i s h . As the fast ing period proceeded, leve ls rose unt i l 4 days of fast ing and thereafter remained constant - there being no longer a di f ference in enzyme a c t i v i t y between C and F. When a c t i v i t y was expressed in terms of l i v e r protein (enzyme a c t i v i t y i s conventionally expressed in terms of t issue protein) then there was no longer an i n i t i a l d i f ference between C and F. A l l other aspects of the change in a c t i v i t y with fast ing were s imi la r to the above. The difference in the extent of enzyme a c t i v i t y differences between C and F as a resu l t of method of expression i s due to the var iable nature of l i v e r glycogen contents. This i l l u s t r a t e s the value of expressing l i v e r enzyme a c t i v i t y in terms of prote in . Perhaps better s t i l l would be to express l i v e r G-6-Pase a c t i v i t y in terms of body weight because the normal function of the enzyme i s to supply l i v e r glucose to the c i r cu la t i on upon demand by the body. G-6-Pase a c t i v i t y i s related to the rate of gluconeogenesis. Gluconeogenesis i s most operative in the fast ing s tate, when the d ie t i s high in fat,and in carnivorous animals. Under such conditions there i s l i t t l e or no d ietary carbohydrate to replenish blood glucose. The animal must accordingly re ly on glucose produced by the l i v e r and released into the c i r cu la t i on under the regulat ion of G-6-Pase. A l l r ed and Roehrig (1970) have shown with chicks that l i v e r G-6-Pase a c t i v i t y increased under - 36 -condit ions of fast ing or a high fat d i e t . The resu l ts of the present experiment indicate that fast ing increases G-6-Pase a c t i v i t y in trout s im i l a r l y to chicks, but d ietary carbohydrate has l i t t l e e f fec t . Trout l i v e r has very low leve ls of hexokinase (Negayama et a l . 1972). I t i s possible then, that G-6-Pase catalyzes the movement of glucose both into and out of the l i v e r . Therefore, G-6-Pase a c t i v i t y would be increased by a high carbohydrate d i e t . Any increase in G-6-Pase a c t i v i t y resu l t ing from increased gluconeogenesis on a high fa t d ie t would thus be obscured. Since chickens also have l i t t l e hexokinase (O'Nei l l and Langslow 1978) one would expect s im i la r d i f f i c u l t i e s in detecting dietary differences in G-6-Pase a c t i v i t y . This i s not the case. I t i s l i k e l y that there i s enough hexokinase present in trout l i v e r s to enable trout to deposit large amounts of glycogen. A more l i k e l y explanation of the s im i l a r i t y of G-6-Pase a c t i v i t i e s in the C and F trout l i v e r s i s that both C and F l i v e r s were at about the same low leve l of gluconeogenesis during f u l l feeding. There was some d iges t ib le carbohydrate in the F d ie t in the wheat middlings. The amount may have been su f f i c i en t to maintain blood glucose at adequate concentrations without gluconeogenesis. 3.3.3 Trout Muscle Black et al_. (1961) showed that trout muscle glycogen varies from s i t e to s i t e and that in the dorsal white muscle increases poster io r ly . - 37 -In the present experiment, trout muscle was sampled from an area jus t anter ior to the dorsal f i n . Muscle in th is area, according to the above authors, contains intermediate leve ls of glycogen. The resu l ts of the glycogen analyses are given in Table VIII and Figure 7. There was no dif ference in glycogen deposit ion between F-l and F-2. C-l fed trout deposited s l i g h t l y more glycogen in the i r muscle than did the F-fed trout although th i s difference jus t missed being s t a t i s t i c a l l y s i gn i f i c an t . The most remarkable difference in glycogen deposit ion was between C-l and C-2. The C-2 deposited 3 X more glycogen in the i r muscles than did C - l . Herein may l i e the answer of where the extra carbohydrate in d ie t C-2 went, i f not into l i v e r glycogen or i f not ox id ized. There i s ce r ta in ly enough muscle mass on the trout in which to store th i s extra d ietary carbohydrate but to be certa in that th i s i s what i s happening, proper k inet i c and tracer studies should be conducted. Upon fas t i ng , trout muscle glycogen f e l l to basal leve ls after 4 days for a l l treatments with the possible exception of C-2 which may have taken s l i g h t l y longer to reach basal glycogen l eve l s . Thereafter, for each treatment, there was a rebound in muscle glycogen leve ls to a peak and a subsequent decl ine to basal values. The time of the rebound peak was ea r l i e r , 8 versus 10 days, and the magnitude of the rebound was greater in the C treatments than in the F treatments. There was no dif ference in the magnitude of the rebound between C-l and C-2. The F-2 rebound peak appeared to be greater than the F-l peak but the difference was not s t a t i s t i c a l l y s i gn i f i c an t . - 38 -The rebound probably occurred faster for the C muscle than the F muscle because the C muscle most l i k e l y had more or higher a c t i v i t y glycogen synthesizing enzymes present in the t issue i n i t i a l l y , and thus these t issues would have a 'head s t a r t ' on the rebound. Presumably the signal to stop rebounding i s some sort of negative feedback control on the synthetase enzymes. It could take longer to stop the r e l a t i v e l y more act ive enzymes in the C muscles. This could explain the higher peak values of glycogen for f i s h fed the C than the F d ie t . This i s a l l very speculative as l i t t l e i s documented on the subject in general. Muscle glycogen rebound during fast ing in trout does not appear to have been previously invest igated. There i s evidence, however, that a rebound of muscle glycogen occurs a f ter exhaustive exercise upon real imentation with a high carbohydrate d i e t . Bergstrom and Hultman (1966) found that in strenuously exercised man there was a rebound in muscle glycogen leve ls a f ter depletion and that there was actua l ly a 'supercompensation' where rebound muscle glycogen leve ls exceeded or ig ina l rest ing l eve l s . There are several requirements in order that super-compensation w i l l occur: depletion of-glycogen stores (-Bergstrom et al_. 1972, Gorski et a l . 1976), normal i n su l i n response (Roch-Norlund 1972, Machlum et al_. 1977) and recovery on a high carbohydrate d ie t (Hultman et al_. 1971). Kochan ejt al_. (1979) proposed a mechanism for muscle glycogen supercompensation fol lowing exerc ise. It i s based on the f ind ing that mult ip le phosphorylated forms of skeleta l muscle glycogen synthetase can be generated in v i t r o during i t s conversion from a t o t a l l y dephosphorylated (active) to a highly phosphorylated ( inact ive) form. The resu l t i s several - 39 -d i f fe rent k inet i c forms of the enzyme which vary in the i r s ens i t i v i t y to act ivat ion by glucose-6-phosphate (Brown et al_. 1977). Kochan et a l . proposed that supercompensation i s i n i t i a t ed by low G-6-P concentrations (as would be found in glycogen depleted, exercised muscle). Low concentrations of G-6-P favour the formation of intermediate forms of glycogen synthetase which have r e l a t i v e l y low a c t i v i t i e s but are more sens i t ive to act ivat ion by G-6-P. These intermediate forms of glycogen synthetase cause supercompensation. The requirements for the feeding of a high carbohydrate d ie t and for a normal insu l in response would be to ensure that adequate substrate i s ava i lab le to the muscle for glycogen synthesis. I t i s possible that in the trout muscle, rebound of glycogen during fast ing i s accomplished by some such change in the forms of glycogen synthetase. The general theory needs experimental va l i da t ion . The above i s compatible with the theory that the faster rebound in the C muscle was due to the presence o r i g i na l l y of higher a c t i v i t i e s of glycogen synthesizing enzymes in these t issues than in the F muscle. What i s the source of rebound muscle glycogen in the fast ing trout? It i s possible that l i v e r glycogen could be catabol ized and the resu l t ing glucose transported to the muscles. Likewise l i v e r glucose could be formed v ia gluconeogenesis and th i s glucose could be transported to the muscles. But surely l i v e r glucose could be put to better use in the fast ing trout than to serve as a substrate for muscle glycogen. The most l i k e l y sources of muscle rebound glycogen are the metabolites of muscle i t s e l f . Recent evidence has shown that the Cori cycle i s not as important as once thought. The muscle lactate derived - 40 -from exercise i s converted d i r e c t l y back to glycogen in s i tu rather than leaving the muscle f i r s t . Hermansen and Vaage (1977) studied lactate disappearance and glycogen synthesis in human muscle af ter strenuous exerc ise. They found that only 5% of the muscle glycogen accumulation after exercise could be accounted for by blood glucose uptake. The remainder had to be from lac ta te . McLane and Holloszy (1979) further c l a r i f i e d the issue. They found that 44% of the lactate produced in white f i b re muscle (rat) af ter exercise was reconverted to glycogen, 7% was ox id ized, 20% went to blood pyruvate and 35% went to amino acid intermediates. Interest ingly enough, red f i b re muscle showed no glycogen accumulation from lac ta te . The enzyme fructose diphosphatase is missing in red muscle and therefore red muscle r e l i e s on the Cori cycle to rebui ld glycogen stores. Batty and Wardle (1979) found that the anaerobic swimming muscle of p la ice r e l i e s on gluconeogenesis in the muscle to reconvert lactate to glycogen fo l lowing exerc ise. These reports indicate that the rebound phenomenon in muscle glycogen observed after exercise i s the resu l t of an extra surge of lactate conversion to glycogen. The ro le of high carbohydrate real imentation in supercompensation may be that th i s addit ional substrate allows glycogen leve ls to exceed the o r i g i n a l . I t i s extremely un l ike ly that the rebound muscle glycogen observed in the fast ing trout ar ises from muscle lac ta te . I t i s energet ica l ly more e f f i c i e n t for the fast ing trout to completely oxid ize muscle glycogen in the deplet ion phase. Rebound muscle glycogen in the fast ing trout probably ar ises from metabolites in the muscle derived from amino acid catabolism. - 41 -Protein and dry matter concentrations were also measured in the t rout . The resul ts are given in Table VI I I . Muscle protein was s l i gh t l y lower in the C-fed trout i n i t i a l l y . By two days fast ing however, the protein content had increased to the values of the F-fed fas ts . There was a s l i gh t increase in protein concentration over the fast ing period. Muscle dry matter stayed r e l a t i v e l y constant over the fas t . 3.3.4 Trout V i a b i l i t y The l i ve r s of the C-fed trout were uniformly paler in colour than the l i v e r s of the F-fed t rout . Discolouration of trout l i ve r s upon feeding a high carbohydrate d iet was also reported by Austreng et a l . (1977). This l i v e r d isco lorat ion i s no doubt associated with high glycogen concentrations. I t has been suggested that excessive glycogen deposit ion in f i sh l i ve r s has a negative ef fect on the animal's health. Ph i l l i p s et al_. (1948) reported that trout fed high leve ls of carbohydrate showed growth i nh i b i t i o n , pathological glycogen deposit ion and morta l i ty . These resul ts are confounded, however, by possible nutr ient de f i c ienc ies . Hi l ton and Dixon (1981) reported that l i v e r function i s impaired in trout l i v e r s containing large amounts of glycogen. They found that recovery time from l i v e r metabolized anaesthetics (2-phenoxyethanol and tert-amyl alcohol) and plasma clearance time of 35 S-sulphobromophthalein was d i r e c t l y related to l i v e r glycogen concentration. On the other hand, Buhler and Halver (1961) found that d ietary carbohydrate levels up to 48% had no deleter ious ef fects on the growth, morta l i ty or l i v e r pathology of chinook salmon. - 42 -There was considerable research conducted with trout in the years around 1960 concerning glycogen deposit ion in the t i s sues . I t was postulated that glycogen could serve as an extra energy source for hatchery raised f i sh that were released into the w i l d , so as to enhance the i r su rv i va l . The resu l ts obtained then were cond i t iona l ly pos i t i ve . The resu l ts of the present experiment indicate that over a two-week fast ing per iod, the l i v e r glycogen of adult trout previously fed a high carbohydrate d iet i s avai lab le to the f i sh for a longer time than for trout fed a low carbohydrate d ie t . I t i s unclear whether th i s confers any advantage to the f i sh in terms of v i a b i l i t y or ' cond i t i on ' . - 43 -Table I . Composition of trout d i e t s . Ingredients Diets (grams) A C F (Acclimation) (High carbohydrate) (High fat) Pollock meal 500 500 500 Wheat middlings 100 100 100 Herring o i l 1 60 10 120 Glucose 110 250 Cel1ulose 2 80 60 80 Carboxymethyl-eel 1ulose 20 20 20 Lec i th in 10 10 10 Vitamins and minerals 3 15 15 15 Water 60 75 Total 955 1040 845 1Herr ing o i l was s tab i l i zed with 0.333% butylated hydroxyanisole-butylated hydroxytoluene (BHA-BHT) (1:1). 2 A lphace l , ICN Pharmaceuticals, Inc. Cleveland, OH. 3Each 15 g of vitamin-trace mineral premix supplement provided the fo l lowing: D-calcium pantothenate 164 mg, pyridoxine HC1 36 mg, r i bo f l av i n 60 mg, n iac in 300 mg, f o l i c acid 10 mg, thiamine HC1 34 mg, b io t in 3 mg, cyanocobalamin 60 pg, menadione 80 mg, chol ine chlor ide 1,728 mg, dl-alpha tocopheryl acetate 600 IU, re t iny l palmitate 10,000 IU, cho leca lc i fe ro l 1,000 IU, ascorbic acid 1,200 mg, inos i to l 400 mg, manganese sulphate 69 mg, zinc sulphate 78 mg, potassium iodate 5.6 mg, sodium chlor ide 5,000 mg. - 44 -Table I I . Calculated nutr ient composition of trout diets Component Diets A C F (Acclimation) (High carbohydrate) (High fat) (per 955 g) (per 1040 g) (per 845 g) Crude protein (g) Crude fat (g) Digesti ble carbohydrate (g) Crude f ib re (g) Ash (g) Dry matter (g) Metabolizable energy (kcal) 417 99 127 107 51 859 2926 417 49 267 87 51 929 3086 417 159 17 107 51 809 2966 Calculat ions were based on the fol lowing component concentrations in the ingredients: Pollock meal protein 80%, fa t 5%, ash 10%, water 5%; Wheat middlings protein 17%, fat 4%, ash 6%, water 11%, f ib re 7%, nitrogen free extract 55%. A l l values were calculated on the basis of 100% d i g e s t i b i l i t y except for the nitrogen free extract f ract ion of wheat middlings which was assigned a 30% d i g e s t i b i l i t y . The coef f i c ien ts for estimating metabolizable energy values were: 3.9 kcal/g prote in, 4.0 kcal/g carbohydrate and 8.0 kcal/g fat in the feedstuff . - 45 -Table I I I . Body weight and condition f a c t o r of tro u t i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 0 2 4 8 10 13 16 Body weight C-l 235 a 243 a 248 a 245 a 229 a 230 a 257 a (grams) - ± 15 ± 6 ± 21 ± 8 ± 14 ± 11 ± 13 (±SEM) 268 a 256 a C-2 328° 279 a 234 a 226 a 256 a ± 8 ± 18 ± 9 ± 16 ± 7 ± 20 ± 7 F-l 263 a 242 a 233 a 229 a 251 a 244 a 226 a ± 9 ± 16 ± 11 ± 17 ± 17 ± 9 ± 5 F-2 264 a 250 a 239 a 242 a 257 a 226 a 263 a ± 8 ± 16 ± 11 ± 14 ± 4 ± 2 ± 13 Condition f a c t o r 3 C-l 5.05a 5.15a 5.01 a 5.06 b 4.74a 4.88 a 4.91 a (±SEM) ±.18 ±.06 ±.13 ±.10 ±.17 ±.05 ±.30 C-2 5.73 b 5.20a 5.25a 4.74 a' b 4.78 a 4.75 a 4.80 a ±.06 ±.07 ±.14 + .12 ±.18 ±.13 ±.15 F-l 5.18a 4.94a 4.98a 4.49a 4.69a 4.80a 4.71 a ±.17 ±.12 ±.11 ±.09 ±.16 ±.15 ±.10 F-2 5.26a 5.27a 4.94a 4.77 a> b 4.69 a 4.94a 4.87a ±.13 ±.09 ±.17 ±.14 ±.13 ±.17 ±.11 1C-1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 2% of body weight per day r e s p e c t i v e l y , while F - l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Means i n each column followed by d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . 3Condition f a c t o r c a l c u l a t e d as: (1000 x body weight (g) . fork length (cm) - 46 -Table IV. Liver weight, hepato-somatic index and l i v e r dry matter of trout i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 0 2 4 8 10 13 16 Liver weight C--1 6 .19 b 2 4.37 b 3.99 b 3.59 a 2.77 a 2.30 a' b 2.51 a (grams) ± .87 ±.20 ±.40 ±.33 ±.25 ±.12 ±.11 (±SEM) C--2 9 .62 c 6.37C 5.93 c 3.68a 2.97 a 2.80 b 2.82 a + .29 ±.72 ±.58 ±.39 ±.13 ±.31 ±.11 F--1 3 .20 a 2.65 a 2.40a 2.36 a 2.57 a 2.32 a , b 2.29 a + .08 ±.22 ±.30 ±.15 ±.36 ±.09 ±.21 F-•2 3 .98 a 3.04a 2.99 a' b 2.57 a 2.56 a 1.92a 2.30 a + .15 ±.30 ±.24 ±.21 ±.11 ±.09 ±.10 Hepato-somatic C-•1 2 .58 c 1.80 b 1.64b 1.48 b 1.23a 1.00a .99 a index + .25 ±.06 ±.18 ±.14 ±.14 ±.03 ±.07 »grams l i v e r > 400 g body weight' C-•2 2, .94c 2.38 C 2.11 c 1.44 b 1.28a 1.25 b 1.10a (±SEM) +, .11 ±.18 ±.17 ±.08 ±.07 ±.11 ±.06 F-•1 • 1. .22a 1.09a 1.02a 1.04a 1.01 a .96 a 1.01 a +, .03 ±.06 ±.08 ±.02 ±.08 ±.04 ±.07 F-2 1. ,51 b 1.21 a 1.26 a' b 1.06a .99 a .85 a .88 a +, .06 ±.08 ±.11 ±.04 ±.04 ±.04 ±.04 Live r dry matter C-l 28.1 (g/100 g l i v e r ) ± .7 (±SEM) C-2 29.2° ± .5 F-l 25.5 a ± .4 F-2 25.8 a ± .7 28.0 b 26.5 b 26.6 b 26.2 a 25.7 a 25.6 a ± .9 ± .6 ± .7 ± .7 ± .3 ± .2 30.2 b 27.6 b 24.9 a 24.2 a 26.7 a 23.0 a ±1.0 ± .8 ± .4 ± .4 ± .7 ±1.6 25.3 a 23.8 a 23.3 a 24.7 a 26.4 a 25.0 a ± .5 ± .2 ± .3 ± .5 ±. .4 ± .9 25.8 a 24.8 a 24.7 a 2 6 . l a 26.3 a 24.3 a ± .4 ± .3 ± .4 ± .3 ± .3 ± .8 iQ-1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 256 of body weight per day r e s p e c t i v e l y , while F - l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Means i n each column followed by d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . - 47 -Table V. Liv e r glycogen of trou t i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 0 2 4 8 10 13 16 Liv e r glycogen C-l 12.5 b 2 7.8 b 5.8 b 3.2 a 1.7a .5 a .6 a (g/100 g l i v e r ) ±1.1 ±1.2 ±1.0 ±1.1 ± .9 ± 0 + .1 (±SEM) L. 1.7a C-2 12.3 b 10.4 b 7.5 b 2.9 a 1.4a .5 a ± .5 ±1.0 ±1.0 ± .8 ± .4 ± .8 + 0 F-l 3.3 a 1.2a .7 a .7 a .7 a 1.0a .5 a ± .4 ± .2 ± .2 ± .2 ± .1 ± .2 + 0 F-2 2.8 a 1.4a .7 a .9 a .8 a .4 a .9 b ± .3 + .3 ± .2 ± .2 ± 0 ± 0 + .1 Liver glycogen C-l 327 b 143 b 103 b 53 b 27 a 5 a 6 a (mg/100 g body ± 48 ± 24 ± 27 ± 19 ± 17 ± 1 + 1 weight) (±SEM) C-2 363 b 252 c 165 b 43 b 23 a 21 a 5 a ± 28 ± 34 ± 33 ± 13 ± 6 ± 12 + 1 F- l 40 a 13 a 7 a 7 a 7 a 9 a 5 a + 5 ± 3 ± 3 + 2 ± 1 ± 2 + 0 F-2 42 a 17 a 9 a 9 a 8 a 3 a 8 a ± 5 ± 4 ± 1 ± 2 ± 0 ± 0 + 1 XC-1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 2% of body weight per day r e s p e c t i v e l y , while F - l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Means i n each column followed by d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . - 48 -Table VI. Li v e r protein o f trout i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 10 13 16 Live r protein (g/100 g l i v e r ) (±SEM) . C - l 10 .4 3 14.0 b 14.5 a 15.5 a 17.0 a 18.5 a 18.9 a + .5 ± .8 ± .6 ±1.0 ±1.1 ± .4 ±1.1 C-2 10 .6 a 12.0 a 13.0 a 16.5 a 16.6 a 17.4 a 16.5 a + .6 ± .7 ± .9 ± .4 ± .3 ± .8 ±1.1 F - l 15, .9 b 17.6 C 16.8 b 16.8 a 17.8 a 18.3 a 18.0 a + .3 ± .5 ± .2 ± .3 ± .4 ± .3 ± -7 F-2 16. , l b 17.2 C 17.3 b 17.4 a 18.2 a 18.9 a 17.7 a + .2 ± .3 ± .2 + .3 ± .3 ± .2 ± .5 Live r protein (mg/100 g body weight) (±SEM) C - l 2 6 7 b , c 251 b' c 233 b 223 b 202 a 1 8 5 a , b 186 a ± 26 ± 15 ± 17 ± 14 ± 13 ± 4 + 14 C-2 310 C 282 C 268 b 236 b 212 a 211 b 181 a ± 11 ± 24 ± 11 ± 10 ± 13 ± 12 ± 5 F - l 193 a 193 a 170 a 173 a 179 a 175 a 180 a ± 6 ± 11 ± 11 ± 4 ± 11 ± 10 ± 7 F-2 243 b 2 1 4 a , b 218 b 185 a 180 a 161 a 150 a ± 9 ± 13 ± 18 ± 6 ± 6 ± 6 ± 5 XC-1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 1% of body weight per day respectTvely, while F - l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Tif1l IV?0" EllT !2 1 12W F I D b y d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . ' - 49 -Table V I I . Li v e r glucose-6-phosphatase a c t i v i t y of tr o u t i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 0 2 4 8 10 13 16 pg inorganic phosphate released/10 minutes/g l i v e r Glucose-6- C-l 1 6 a , b 2 20 a 38 a 38 a 50 a 64 b 41 a phosphatase + 4 ± 3 + 4 + 5 + 4 + 3 + 3 a c t i v i t y a a a -.a (±SEM) C-2 13 a 16 a 35 a 41 a 42 a 51 a 41 d + 2 ± 1 + 2 + 4 + 3 + 9 + 9 F-l 25 b 35 b 50 a 52 a 59 a 44 a 48 a + 3 ± 3 + 4 + 2 + 5 + 4 + 5 F-2 25 b 38 b 48 a 47 a 50 a 45 a 58 a + 1 ± 4 + 4 + 3 + 4 + 2 + 6 yg inorganic phosphate released/10 minutes/g l i v e r protein Gl ucose-6- C-l 157 a 136 a 260 a 244 a 304 a 346 b 217 a phosphatase ± 35 ± 16 ± 22 + 26 ± 40 ± 17 ± 16 a c t i v i t y (±SEM) C-2 125 a 142 a 270 a 250 a 256 a 289 a 244 a ± 22 ± 21 ± 15 ± 23 ± 23 ± 42 ± 41 F-l 158 a 1 9 9 a , b 294 a 311 a 332 a 241 a 267 a ± 17 ± 22 + 21 ± 11 ± 26 ± 23 ± 30 F-2 154 a 222 b 276 a 271 a 273 a 239 a 328 a ± 8 ± 25 ± 24 ± 13 ± 22 ± 13 + 40 1 C - 1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 2% of body weight per day r e s p e c t i v e l y , while F - l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Means i n each column followed by d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . - 50 -Table V I I I . Muscle glycogen, muscle protein and muscle dry matter of trout i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Dietary treatment 1 Fasting time (days) 0 2 4 8 10 13 16 Muscle glycogen C-l 1 . 5 a 2 1.5a .4 a 3.0 b 1.4 a' b .9 a > 5a,b (mg/g muscle) + .1 ± .5 ± .1 ± .5 ± .3 ± .3 ± 0 (±SEM) C-2 4 .2 b 3.4b 1.6b 2.8 b 2.1 b l . l a .7 b + .7 ± .5 ± .4 ± .6 + .3 ± .2 ± .1 F-l 1 .0 a .6 a .4 a .7 a 1.0a .9 a .4 a + .2 ± .1 ± .2 ± .1 ± .2 ± .2 ± 0 F-2 1 .0 a .6a .4 a 1.2a 1.6 a' b .7 a .4 a + .1 ± .1 ± 0 ± .3 ± .4 + .1 ± 0 Muscle protein C-l 17 .9 a 19.0 a 19.5 a 19.0 a 19.0 a' b 18.6 a 19.0 a (g/100 g muscle) + .3 ± .5 ± .5 ± .3 ± .3 + .2 ± .2 (+SEM) C-2 17.8 a 18.2 a 19.0 a 18.6 a 18.6 a 18.8 a 19.7 a + .2 ± .5 + .1 + .3 ± .2 ± .3 ± .3 F-l 18.8 a' b 18.8 a 18.9 a 18.7 a 19 2***^  19.4 a 19.5 a + .4 ± .2 + .2 ± .2 ± !3 ± .2 ± .3 F-2 18 .9 b 18.3 a 19.0 a 18.9 a 19.7 b 18.5 a 19.5 a + .1 ± .5 ± .3 ± .3 + .2 ± .4 ± .3 Muscle dry matter C-l 22 . l a 23.4 a 24.3 a 24.0 a 22.8 a 23.0 a 23.2 a (g/100 g muscle) + .5 ± .4 ± .3 ± .5 ± .4 ± .5 ± .4 (±SEM) L. C-2 23 .7 b 23.8 a 24.6a 23.4 a 22.9 a 23.0 a 24.0 a + .4 ± .8 ± .4 ± .6 ± .3 ± .8 ± .4 F-l 24 .2 b 24.0 a 22.8 a 22.8 a 23.6a 24.0 a 23.4 a + .6 ± .6 ± .5 ± .5 ± .6 ± .3 + .4 F-2 24 .4 b 23.6a 24.0 a 23.4 a 24.6 a 22.2 a 23.3 a + .2 ± .8 ± .7 ± .3 ± .5 ± .5 ± .2 XC-1 and C-2 are feeding of the high carbohydrate d i e t at 1% and 21 of body weight per day r e s p e c t i v e l y , while F-l and F-2 are s i m i l a r feeding treatments of the high f a t d i e t . 2Means i n each column followed by d i f f e r e n t superscripts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . - 51 -10 9 « 7H i i i K 44 Ul * 3 5 H 2 H --B a f t e r C - 1 d i e t — • a f t e r C - 2 d i e t ...A a f t e r F - 1 d i e t 4 a f t e r F - 2 d i e t —i 16 8 10 S T A R V A T I O N T I M E C d a y s ) 13 Figure 2a . Trout l i ver weight (g) over the fasting period. a f t e r C - 1 d i e t • a f t e r C - 2 d i e t A a f t e r F - 1 d i e t A a f t e r F - 2 d i e t S T A R V A T I O N T I M E ( d a y s ) Figure 2b. Trout hepato-somatic index (g l iver /100 g body weight) over the fasting period. - 52 -Figure 3a. Trout l i v e r glycogen (g/100 g l i v e r ) over the fast ing per iod. o » o •• o s K ' U l >o j o •2H "oT.H 3' O t-\ --B a f t e r C - 1 d i e t — • a f t e r C - 2 d i e t . ~ A a f t e r F - 1 d i e t a f t e r F - 2 d i e t 8 10 S T A R V A T I O N T I M E C d a y s ) •t "t Figure 3b. Trout l i v e r glycogen (g/100 g body weight) over the fast ing per iod. 53 -• i 6 * -/ / --a a f te r C - 1 d ie t — • after C - 2 diet — A af ter F -1 diet —A. a f ter F - 2 diet -•a ' , 1 -4 8 10 S T A R V A T I O N T IME ( d a y s ) 13 16 Figure 4a. Trout l i v e r protein (g/100 g l i v e r ) over the fast ing per iod. e a f te r C - 1 d ie t • a af ter C - 2 diet A- -A af ter F - 1 diet A A a f ter F - 2 diet 1 r— 8 10 S T A R V A T I O N T I M E ( d a y s ) —r— 13 16 Figure 4b. Trout l i v e r protein (g/100 g body weight) over the fast ing per iod. - 54 -Figure 5a. Trout l i v e r glycogen + protein (g/100 g l i v e r ) over the fast ing per iod. B a f te r C - 1 d ie t STARVATION TIME Cdays) Figure 5b. Trout l i v e r glycogen + protein (g/100 g dry l i v e r ) over the fast ing per iod. - 55 -a f t e r C - 1 d i e t • a f t e r C - 2 d i e t A a f t e r F - 1 d i e t * a f t e r F - 2 d i e t "1 r 8 10 STARVATION TIME Cdays) 13 16 Figure 6. Trout l i v e r glycogen + protein (g/100 g body weight) over the fast ing per iod. - 56 -Figure 7. Trout muscle glycogen (mg/g muscle) over the fasting period. - 57 -4. EXPERIMENT 2. A COMPARISON OF CORN STARCH AND CORN OIL AS SOURCES OF  25 PERCENT OF THE ENERGY IN A CHICK DIET,ON TISSUE "GLYCOGEN CONCENTRATION AND ON THE' RATE- OF GLYCOGEN . DEPLETION FROM THE TISSUES DURING A SUBSEQUENT PERIOD  OF FAST 4.1 Objectives The general objectives and design of th i s experiment on chicks were the same as for the trout experiment. Fasting glycogen metabolism in the Bro i le r and White Leghorn stra ins of domestic chicken was to be compared to , and contrasted with that in the trout . A more deta i led look at the 'rebound' of l i v e r glycogen, af ter i n i t i a l deplet ion, was sought. 4.2 Mater ia ls and Methods 4.2.1 Animals and Maintenance On July 30, 1979, 150 bro i le r - type (Hubbard), pu l le t day-old chicks were obtained from Western Hatchery (Abbotsford, B.C.) and placed in e l e c t r i c a l l y heated Petersime Brood-Unit Model 2SD battery brooders, in the Poultry Science nut r i t i on laboratory of the Univers i ty of B r i t i sh Columbia. They were provided with commercial chick s tar ter feed and fresh water on an ad l ib i tum basis. The da i l y photoperiod was 24 hours. On September 24, 1979, 154 White Leghorn-type, pu l le t day-old chicks were obtained from Sanders Hatchery (Abbotsford, B.C.) and placed in the same battery brooders, under the same management condit ions, as the bro i l e rs were previously. - 58 -The subsequent experimental treatments, described below, were the same for both s t ra ins of chicks. 4.2.2 Diets Two diets were formulated; a high carbohydrate d iet (C), and a high fat d iet (F). The composition of the diets i s given in Tables IX and X. The diets were fed in mash form. 4.2.3 Experimental Treatment The chicks were randomly d i s t r i bu ted , at 4 days of age, into 8 brooder compartments, each with 19 or 20 b i rds . The chicks were ind i v idua l l y weighed and wing banded. Chicks in 4 compartments were provided with the C d iet and 4 with the F d i e t , a l l on an ad l ib i tum basis. Fresh water was provided ad 1ibitum. Lighting was continuous on a 24-hour basis for the duration of the experiment. The chicks were weighed at 7, 14 and 21 days af ter imposition of the experimental d ietary treatments. On the 22nd day, the brooder heaters were turned o f f . Feed was withdrawn at 1500 hours on the same day. The chicks were sampled for t issue analysis at 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours after feed withdrawal. Eight birds from each of the two dietary treatments were k i l l e d at each sampling time - with the exception of 128 hours fast ing when the f i na l 5 or 6 chicks on the C d ie t and 4 chicks on the F d ie t were sampled. - 59 -Sampling was conducted by the fol lowing procedure. Each bird was weighed with a minimum of stress and then k i l l e d by a respiratory administered overdose of d iethyl ether. The l i v e r was excised, frozen in l i qu i d nitrogen and weighed. A 3 gram sample of pectoral muscle was excised, frozen in l i qu i d nitrogen and weighed. Freezing of the l i v e r was accomplished within 90 seconds af ter i n i t i a t i o n of anaesthesia while the muscle was frozen within 120 seconds. 4.2.4 Chemical Analysis The t issues were analyzed for dry matter, glycogen and protein as previously described in Section 3.2.4. 4.2.5 Calculat ions A l l measurements were conducted and analyses.made on an indiv idual b i rd basis - as in Experiment 1. The deta i l s of spec i f i c ca lcu lat ions are given in Section 4.3 where appl icable. 4.2.6 S t a t i s t i c a l Analysis The resu l ts were s t a t i s t i c a l l y analyzed by methods described in Section 3.2.6. The overal l design of the experiment was 2x2x10 fac to r i a l where factor A was chick breed (bro i le r versus White Leghorn),factor B was d ie t (Cversus F j and factor C was.fasting time (0 through 128 hours). - 60 -4.3 Results and Discussion 4.3.1 Chick Growth The chicks were ind iv idua l l y weighed after 1, 2 and 3 weeks on the experimental d ie t s . Within each of these time in te rva l s , the b ro i l e r (BR) chicks gained an average of lOOg, 177g and 227g respect ively on the C d i e t , and 103g, 186g and 246g on the F d ie t . Total body weight gain over the 3 weeks was 504g on the C d iet and 535g on the F d ie t . During the respective weekly i n te rva l s , the e f f i c i ency of feed conversion (body weight gain/feed consumption) of the BR chicks was .74, .63 and .54 on the C d i e t , and .77, .66 and .57 on the F d ie t . The overal l e f f i c i ency of feed conversion for the 3 weeks was .60 on the C d ie t and .63 on the F d i e t . These resu l ts indicate that the BR chicks on the F d iets s l i g h t l y outperformed those on the C d ie t in terms of body weight gain and e f f i c iency of feed conversion. Although the diets were formulated to be i soca lo r i c and isonitrogenous, there may have been an ex t ra -ca lo r i c ef fect from the corn o i l in the F d iet which conferred an advantage on the chicks fed th i s d i e t . The weekly weight gains of the White Leghorn (WL) chicks were 46g, 69g and 91g on the C d ie t (at 0-1, 1-2 and 2-3 weeks respect ive ly) and 49g, 72g and 91g on the F d ie t . The tota l body weight gain was 206g on the C d iet and 212g on the F d i e t . The weekly values for e f f i c iency of feed conversion were .61, .52 and .44 on C and .63, .55 and .44 on F. The overa l l values for feed conversion were .49 on C and .52 on F. Again the F chicks outperformed the C chicks. - 61 -4.3.2 Chick Fasting Body Weights Body weights during the fast ing period are given in Table XI. In both stra ins of chicks the measured losses in body weight during fast ing were e r r a t i c because of the v a r i a b i l i t y of body weights in the treatment populations. Fortunately th i s v a r i a b i l i t y can be corrected. A l l chicks were weighed at day 21 - the day pr io r to the i n i t i a t i o n of f as t ing . The average weight gain, over th i s one-day per iod, was calculated for those birds sac r i f i ced at Starvation Time 0. This was determined to be an 8.5% increase in body weight for the BR, and a 7.5% increase, for the WL. The body weights of a l l the chicks were then adjusted by these growth factors to give an estimate of the chick weights at fast ing time 0. These resu l ts are given in Table XII . F i na l l y , the difference in sample body weight, at the various fast ing times, from estimated i n i t i a l weight was calculated and expressed as a percent of the i n i t i a l weight (Table X I I ) . These values show that percent weight loss was l inear over the fast ing period and that there was no difference in the weight loss patterns of BR and WL. Neither was there any difference in weight loss between the birds fed C- and F-diets. 4.3.3 Chick L iver Chick l i v e r weights are given in Table XI I I . There was no difference in l i v e r weights between C and F for e i ther s t ra in of chick. - 62 -L iver weights decreased stead i ly over the fast ing period except for a stat ionary phase at 32 hours for BR-F and a s l i gh t increase at 44 hours for both WL-C and -F. The reasons for these 'hiccups' i s that the estimated i n i t i a l body weight of these samples increased considerably at these times. This is due to the error of random sampling and has no physiological meaning. The s i tuat ion does point out the value of expressing l i v e r weight in terms of body weight however. HSI values are given in Table XIV and in Figures 8a and 8b. In both BR and WL there was a faster decrease in l i v e r weight than in body weight during the f i r s t 16 hours af ter withdrawal of feed. After th i s time there was a l e ve l l i ng off of HSI in BR and an increase of HSI in WL. Other body t issues may have been depleted faster than the l i v e r , or the l i v e r was actua l ly enlarging. The HSI of C-fed chicks was cons istent ly higher than that of F-fed chicks at a l l fast ing times and in both stra ins of chicks. The reason C-fed chicks had a higher HSI than the F-fed chicks was that C chicks contained more l i v e r protein than F chicks. This w i l l be discussed l a te r . There was no dif ference in HSI between BR and WL. Liver dry matter content i s given in Table XV. BR l i v e r moisture was i n i t i a l l y about 72% for F-fed chicks and 74% for C-fed chicks. Over the fast ing period BR l i v e r moisture content f e l l cont inual ly to a f i na l level of 70-71% for both C- and F-fed chicks. WL l i v e r water content was i n i t i a l l y 72-73% for both C- and F-fed chicks (the water content of the C l i v e r s was marginal ly higher than that of the F l i v e r s ) . The water content of the WL l i v e r s f e l l to about 70% at 56 hours fast ing for both C and F. - 63 -L iver moisture content i s s l i gh t l y lower in the chick (72%) compared with the trout (75%). Whereas the trout had a lower moisture content in the C l i v e r than the F l i v e r , the chick had a higher moisture content in the C l i v e r . As we l l , chick l i v e r moisture content f e l l over the fast ing period while that of trout remained constant or increased. The reasons for these dif ferences in l i v e r moisture trends between species are not known. The resu l ts of l i v e r glycogen analysis are given in Table XVI and in Figures 9a and 9b. For both BR and WL, the l i v e r s of the C-fed chicks contained more glycogen than the l i v e r s of the F-fed chicks at f u l l feeding. This was expected. In add i t ion, Bro i le rs had more glycogen in the i r l i v e r s than White Leghorns: 1.5 x more on the C d ie t and 1.9 x more on the F d ie t . Within 8 hours f a s t i ng , l i v e r glycogen was depleted to zero levels in a l l treatments. There followed a gradual rebound in glycogen l eve l s , peaking at 56 hours for BR and 32-44 hours for WL. The magnitude of th is rebound was the same for both stra ins of chicks. Following the rebound peak there was a gradual depletion of l i v e r glycogen, the rate of depletion being slower in BR than WL. At and a f ter the rebound peak there was no d ietary e f fect on the concentration of l i v e r glycogen in chicks of e i ther s t r a i n . An attempt was made to re late the timing of the rebound peak to the body weight of the chicks, as was done by Den Otter and De Minjer (1972) with the rat and the mouse. Using the formula: i n i t i a l body weight ^ / days to peak - 64 -the value obtained for BR was 5.6 and for WL was 5.1 (based on body weights of 640g and 280g, and times of 2.33 days and 1.67 days for BR and WL respect ive ly) . The constant that Den Otter and De Minjer obtained for the rat and the mouse was 2.2-2.3. The rat ionale behind th i s measurement i s that metabolic rate i s related to body surface area in the homeotherm because one of the functions of metabolism i s to maintain body temperature. As glycogen i s used as an oxidat ive energy source for th i s purpose the amount of glycogen oxidized depends on the surface area across which body heat i s l o s t . I f surface area could be reduced or better insulated the peak of glycogen rebound would be prolonged as less glycogen would be oxid ized. Because surface increases by the square and body weight increases by the cube, Den Otter and De Minjer postulate that the above ra t i o must be constant. Since th i s i s not true in the resu l ts of th i s experiment when the formula i s tested across stra ins there must be some difference in fast ing l i v e r glycogen rebound between BR and WL which over-r ides th i s re lat ionsh ip of rebound to surface area. For both BR and WL, the rate of l i v e r glycogen rebound was faster in the birds fed C-diet than F-diet in spite of the probab i l i ty that l i v e r glycogen was depleted to zero leve ls f i r s t in the F-fed chicks and thus these chicks had an ea r l i e r stimulus for rebound than did the C-fed chicks. The faster rebound in the C-fed chicks was probably due to the fact that these l i ve r s had higher a c t i v i t i e s of glycogen synthesizing enzymes associated with the i r i n i t i a l l y higher glycogen content. Evidence for th i s theory i s obtained from studies of the metabolism of the l i v e r acinus. - 65 -As previously described in section 2.2; the l i v e r acinus can be divided into 3 zones defined by the i r distance from a central core of serv ic ing vessels. The zone farthest from that core i s ca l led the central zone and i s known as zone 3. The intermediary zone i s known as zone 2 and the peripheral zone i s ca l led zone 1. Den Otter and De Minjer (1972) found that rats and mice fed a high fa t d iet l o ca l i ze l i v e r glycogen mainly in zone 1 whereas animals fed a high carbohydrate d ie t deposit glycogen evenly in a l l three zones of the acinus. Zones 2 and 3 thus serve as a storage depot for 'excess' glycogen. Upon fas t i ng , the high carbohydrate fed rats showed depletion of l i v e r glycogen evenly over a l l three zones of the acinus. The high fa t fed animals did not show any depletion of the i r already low glycogen stores and there was no change in i t s l o ca l i z a t i on . Sasse (1975) found that hamsters which had an i n i t i a l l y even d i s t r i bu t i on of l i v e r glycogen over a l l three acinar zones had an uneven depletion pattern when fasted. After 16 hours fas t i ng , glycogen was found only in zone 3. Over the next 80 hours of f as t i ng , new glycogen was found in zones 2 and 3. Staining of the t issues showed an increase in glycogen synthetase a c t i v i t y in zones 2 and 3. No glycogen was found in zone 1 at any fast ing time. Sasse ejt al_. (1975) reported s imi la r observations in fasted ra t s : glycogen in zones 2 and 3 of the hepatic acinus increased over a 36-hour fas t . It i s l i k e l y that a s imi la r s i tuat ion occurs in chick l i v e r s . High carbohydrate feeding might thus induce greater glycogen deposit ion in zones 2 and 3 of the l i v e r acinus accompanied by increased a c t i v i t i e s of the glycogen synthesizing enzymes. Again, th i s increased enzyme a c t i v i t y - 66 -in the l i v e r s of C-fed chicks could account for the faster rebound in glycogen l eve l s . This question could be studied by comparing the hepatic acinar glycogen loca l i za t i on in chicks during fast ing glycogen rebound a f ter accl imation to C and F d ie t s . L iver glycogen rebound may be affected by the nature of the glycogen molecule i t s e l f . Geddes and Stratton (1977) reported that two forms of glycogen are present in rabbit l i v e r : one of high molecular weight with a protein backbone capable of forming dissulphide bonds and the other of low molecular weight with no protein backbone or at l eas t , with a backbone incapable of forming dissulphide bonds. The authors did not speci fy, but i t i s l i k e l y that these two species of glycogen are a -par t i c les and 6 -part ic les respect ive ly . Geddes and Stratton found that in rabbits that were fasted and refed, glycogen was synthesized to greater than normal concentrations and that th i s glycogen was predominantly the low molecular weight species. I t i s l i k e l y that there are d i f ferent species of glycogen in the chick l i v e r and that the ra t io of d i f fe rent species i s d i f fe rent in the fast ing rebound s i tuat ion than in the f u l l fed metabolic state. It i s further possible that d ietary energy source would af fect the ra t io of glycogen species and that th i s ra t io could help explain differences in chick l i v e r glycogen metabolism between feeding the C and F d ie t s . I t i s interest ing to note in Figures 9a and 9b that the graph of the rebound of l i v e r glycogen was smoother in F-fed than C-fed chicks in both BR and WL. Livers of C-fed chicks seemed to rebound in glycogen smoothly up to a certa in point and then abruptly stopped rebounding before - 67 -they reach the i r natural peak. Perhaps th is disrupt ion can be explained by changes in zonal metabolism of the l i v e r acinus or synthesis of d i f fe rent forms of glycogen. It should also be mentioned that the model of d i f fe rent forms of glycogen synthetase that has been postulated for muscle glycogen rebound could also be operational in the l i v e r glycogen rebound of fast ing chicks. The resu l ts of the l i v e r protein analysis are given in Table XVII and in Figures 10a and 10b and 11a and l i b . The amount of protein in the BR and WL l i v e r s increased quick ly over the f i r s t 8-16 hours fas t ing and then slowly leveled of f (Figures 10a and 10b). Livers of the C-fed chicks had less protein than l i v e r s of the F-fed chicks at f u l l feeding, and th i s re f l e c t s the dif ference in glycogen contents. When l i v e r protein i s expressed in terms of body weight (Figure 11a and l i b ) we see that there were s ign i f i can t differences between the d ietary treatments in both st ra ins of chicks. The C-fed chicks had more l i v e r protein than the F-fed chicks at a l l times during the fas t . The reverse s i tuat ion was true at f u l l feeding. This i s the cause of the differences in HSI noted previously. The reasons for th i s difference in body l i v e r protein between C and F i s unknown. The WL chicks, on average, had more l i v e r protein per body weight than the BR chicks. - 68 -4.3.4 Chick Muscle The resu l ts of the chick muscle analysis are given in Table XVIII. There was no dif ference in the i n i t i a l moisture concentration of the muscles between BR and WL or between C-fed and F-fed b i rds . The WL muscle moisture concentration increased s l i g h t l y , 75-77% over the fas t ing period. The resu l ts of the glycogen analysis are given in Table XVIX and in Figures 12a and 12b. The i n i t i a l muscle glycogen concentration of the C-fed chicks was higher than that of the F-fed chicks in BR and was almost higher in WL. The muscle glycogen concentration in BR was higher than that in WL. Aberle et a]_. (1979) showed that BR have r e l a t i v e l y more white f ib res in the i r thigh muscles than WL have. I f the same i s true for breast muscle then i t i s obvious why BR stores more muscle glycogen than WL. In general, there appeared to be no differences in muscle glycogen over the fast ing period between the C-fed and the F-fed chicks. I t does appear that glycogen concentration was maintained for a longer time af ter feed withdrawal in BR chicks than in WL chicks. In fac t , i t seems that BR muscle glycogen actua l ly rebounded after an ear ly decrease. I f WL breast muscle has r e l a t i v e l y more red f ib res than BR muscle, as postulated, then according to McLane and Holloszy (1979) i t i s less able to support gluconeogenesis in s i t u . This could account for the lack of maintenance of muscle glycogen stores in WL. A l te rna t i ve ly , the WL have a greater re la t i ve surface area and hence expend proport ional ly more of the i r energy - 69 -output in maintaining body temperature than the BR. It may be that WL cannot afford to maintain muscle glycogen leve ls while BR can. Muscle glycogen concentration f luctuated e r r a t i c a l l y over the fast ing period. The sudden depletions may have resulted from shiver ing associated with heat production. I t i s in terest ing to note that these f luctuat ions were more numerous in WL than BR, and that the onset of these f luctuat ions occurred ea r l i e r in WL than BR. This i s consistent with the large energy expenditure for homeothermy in WL. It appears that the f luctuat ions in muscle glycogen were more evident in F-fed than C-fed chicks. The reasons for th i s are unknown. - 70 -Table IX. Composition of chick d i e t s . Ingredients Diets (grams) C F (High carbohydrate) (High fat) Ground wheat 466.5 466.5 Soybean meal 170 170 Herring meal 120 120 A l f a l f a meal 20 20 Corn starch 200 Corn o i l 83 Ce l lu lose 1 117 Iodized sa l t 3.5 3.5 Calcium phosphate 10 10 Limestone 10 10 Vitamins and minerals * * Total 1000 1000 1 Alphace l , ICN Pharmaceuticals, Inc. Cleveland, OH. *Per kg of d i e t : D-calcium pantothenate 10 mg, r i bo f l av in 3.6 mg, niac in 20 mg, f o l i c acid 0.55 mg, b io t in 0.09 mg, chol ine chlor ide 650 mg, dl-alpha tocopheryl acetate 10 III, cho leca lc i fe ro l 400 ICU, re t iny l palmitate 4,000 IU, manganese sulphate 170 mg, amprolium (25%) 500 mg. - 71 -Table X. Calculated nutr ient composition of chick d i e t s . Component Diets (per 1000 g) C F (High carbohydrate) (High fat) Crude protein (g) 224 224 Ether extract (g) 19 102 Digest ib le carbohydrate (g) 559 379 Crude f ib re (g) 28 133 Ash (g) 33 33 Dry matter (g) 886 894 Metaboli zable energy (kcal) 2866 2869 Calcium (g) 10 10 Avai lable phosphorus (g) 5 5 Calculat ions were based on the fol lowing component concentrations in the ingredients: Ingredient Component CP EE DC CF A DM ME C AP (%) (%) (%) (%) {%) («) (kcal)(%) (*) Ground wheat 12. 5 1.5 67. 9 2.4 1 .7 86 2800 .05 .12 Soybean meal 45 .5 32 6.5 6 .0 90 2440 .25 .2 Herring meal 70 8.5 1 1 10 .5 91 3190 2 .0 1.5 A l f a l f a meal 25 3.5 33 21 10 .5 93 1630 1 .5 .22 Corn starch — 90 — 90 3650 - — Corn o i l — 10C — — 100 8820 - — Cel 1 ulose — — — 90 — 90 — — Calcium phosphate — — — — 100 100 - 30 18 Limestone — — — • — 100 100 - 38 — Table XI. Body weights of chicks in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fas t ing . Diets 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 BR-C 597" 2 596 b 563 b 559 b 537 b 521 c 500 b 461 b 469 b 429 b ± 11 ± 18 ± 10 ± 16 ± 15 ± 10 ± 21 ± 10 ± 11 ± 15 BR-F 680° 604 b 609C 568 b 58 3 b 556° 551 b 521 C 466 b 452 b ± 24 ± 17 ± 7 ± 16 ± 11 ± 14 ± 24 ± 18 ± 20 ± 11 WL-C 282 a 268 a 260 a 246 a 228 a 231 a 212 a 201 a 184 a 180 a ± 7 ± 8 ± 5 ± 7 + 10 ± 8 ± 6 ± 5 ± 7 ± 9 WL-F 274 a 254a 262 a 248 a 235 a 252 a 230 a 214 a 192 a 183 a ± 5 ± 7 ± 4 ± 9 ± 7 ± 9 ± 8 ± 6 ± 4 ± 4 Body weight (grams) (±SEM) ^R-C and BR-F are feeding of the high carbohydrate and high fat d iets to b ro i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f fe rent (p<.05) by the Newman-Keuls tes t . Table XII . Estimated i n i t i a l body weights and estimated retent ion o f i n i t i a l body weights of chicks in response to d i f f e r e n t d ietary treatments a f t e r d i f f e r e n t periods o f f a s t i n g . D i e t s 1 Fasting time (hours) 8 16 24 32 44 56 80 104 128 Estimated i n i t i a l body weight 2 (grams) BR-C 597 BR-F 680 WL-C 282 WL-F 274 630 613 626 637 658 625 281 281 274 266 281 273 616 608 607 653 647 665 261 274 260 266 293 278 596 635 625 651 " 625 646 266 261 280 280 269 280 of I n i t i a l body weight? (grams) BR-C 100 95 92 89 87 86 82 77 74 69 BR-F 100 95 93 91 89 86 83 80 75 70 WL-C 100 96 93 90 87 84 82 76 70 64 WL-F 100 95 93 91 88 86 83 76 71 65 XBR-C and BR-F are feeding o f the high carbohydrate and high f a t d ie ts to b r o i l e r s , while WL-C and WL-F are feeding of these d iets to White Leghorns. Es t imated i n i t i a l body weights were ca lcu lated as 8.5% greater than the previous weighing for bro i l e rs and 7.5% greater than the previous weighing for White Leghorns. Est imated retent ion of i n i t i a l body weight was ca lcu lated as : (100 x , i?°<?y J?f1?h|; J r - r r ) . estimated i n i t i a l body weight' Table XII I . Liver weights of chicks in response to d i f fe rent d ietary treatments a f ter d i f fe rent periods of fas t ing . D iets 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 BR- C 18 .14 b 2 16.13 b 14.31 b 13.87 b 13.75 b 12.49 b 11 .79 b 10 .30 b 10 .55 b 9.94 b + .65 + .73 ±.41 ±.50 ±.60 ±.39 + .47 + .30 ±.24 ±.46 BR- F 17 .75 b 14.66 b 13.93 b 12.99 b 13.35 b 12.47 b 11 .90 b 10 .73 b 10.24 b 9.64 b + .65 + .54 ±.40 ±.58 ±.32 ±.35 + .49 + .63 ±.51 ±.63 WL-C 8 .08 a 6.82 a 6.19 a 5.86 a 5.64 a 5.81 a 5 . l l a 4 .70 a 4.54 a 4.37 a + .35 ±.28 ±.16 ±.11 ±.15 ±.20 + .17 + .13 ±.18 ±.33 WL-F 7 .43 a 6.09 a 5.81 a 5.43 a 5.46 a 5.83 a 5 .05 a 4 .67 a 4.56 a 4.21 a + .21 ±.23 ±.13 ±.18 ±'.14 ±.20 + .22 + .12 ±.16 ±.17 Liver weight (grams) (±SEM) 1BR-C and BR-F are feeding of the high carbohydrate and high fat d iets to b r o i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f fe rent (p<.05) by the Newman-Keuls tes t . Table XIV. Hepato-somatic indices of chicks in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fas t ing . D iets 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 Hepato-•somatic BR- C b2 3.04° 2 .70b 2.54 b 2 .48° 2.56 b 2 .40 a , b 2 .36 b 2 .23 b 2.25 a 2.32 a index ±.10 ± .06 ±.06 + .06 ±.07 + .04 + .04 + .02 ±.04 ±.05 /grams 400 g l i v e r % body weight' BR- F 2.61 a 2 .44 a 2.28 a 2 . 2 8 a ' b 2 . 29 a 2 .25 a 2 .17 a 2 .06 a 2.20 a 2.13 a (±SEM) ±.04 ± .10 ±.06 + .05 ±.05 + .08 + .05 + .07 ±.07 ±.09 WL-C 2 .86 a ' b 2 .54 a ' b 2 . 3 8 a 2 . 3 9 b ' C 2 . 49 a .52 b 2 .41 b 2 .33 b 2.48 b 2.42 a ±.09 ± .04 ±.04 + .05 ±.06 ± .06 + .04 + .01 ±.05 ±.09 WL- F 2.71 a 2 .40 a 2.21 a 2 .20 a 2.33 a 2 .32 a 2 .20 a 2 .18 b 2.38 a ' b 2 . 3 0 a ±.06 ± .05 ±.05 + .05 ±.05 + .04 + .05 + .05 ±.06 ±.04 ^R-C and BR-F are feeding of the high carbohydrate and high fat d iets to b ro i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f fe rent (p<.05) by the Newman-Keuls t es t . Table XV. Liver dry matters of chicks in response to d i f fe rent d ietary treatments after d i f ferent periods of fas t ing . D iets 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 BR- C 26 25 . 2 a 26 . o a 2 5 . 7 a 26 . 7 a 2 7 . 2 a 2 8 . 2 a 28. l a 29 . 0 a 2 9 . l a + .8 + .5 + .2 ± .6 ± .2 ± .2 ± .1 ± .2 ± .2 ± .5 BR-•F 28 . l b 26 . 7 b 26 . 4 a 2 7 . 6 b 2 7 . 2 a 2 7 . 9 a 2 8 . 6 a 2 8 . 5 a 28 . 7 a 29 . 6 a + .3 + .2 + .3 ± .3 ± .3 ± .4 ± .4 ± .5 ± .5 ± .4 WL-C 27 . 5 b 27 C 27 . 3 b 2 8 . 5 b 2 8 . 0 b 2 9 . 4 b 2 9 . 5 b 30 . 4 b 3 0 . 5 b 2 9 . 5 a + .5 + '.2 + .2 ± .4 ± .3 ± .3 ± .3 ±;'.l ± .3 ± .4 WL-F 27 . 9 b 28 . 0 C 27 . 9 b 2 7 . 9 b 2 8 . 4 b 2 8 . 9 b 3 0 . 2 b 3 0 . l b 3 0 . 2 b 3 0 . 3 a + .3 + .3 + .2 ± .3 ± .2 ± .3 ± .4 ± -3, ± .3 ± .3 Liver dry matter (g/100 g l i ve r ) (±SEM) ^R-C and BR-F are feeding of the high carbohydrate and high fat d iets to b ro i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f fe rent (p<.05) by the Newman-Keuls tes t . Table XVI. L iver glycogen of chicks in response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods of f a s t i n g . D i e t s 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 Liver glycogen BR-C c 2 3.75 c .03 a .13 C .39 a . 6 9 b . 67 a . 89 a . 5 5 a . 6 8 b .50 b (g/100 g l i v e r ) ±.30 ±.01 ±.02 ±.10 + .10 ±.05 ±.11 ±.12 ±.11 ±.14 (±SEM) BR-F 2 .41 b .04 a .05 b . 17 a .34 a . 59 a .82 a . 63 a . 6 8 b .52 b ±.36 ± 0 ±.01 ±.05 + .10 + .14 ±.12 ±.10 ±.11 ±.22 WL-C 2.52 b . 05 b . 0 9 b ' c . 2 5 a . 6 1 b . 4 7 a .53 a .50 a .28 a . l l a ±.42 ± 0 ±.02 ±.06 ±.08 ±.06 ±.03 ±.10 ±.06 ±.04 WL-F 1.50 a .03 a .02 a .14 a .48 a ' b .86 a .78 a . 47 a . 25 a . 2 1 a ±.18 ± 0 ± 0 ±.03 ±.08 ±.12 ±.11 ±.06 ±.06 + .04 Liver glycogen BR-C 113.4° . 8 a 3 . 3 C 9 . 7 b 1 7 . 5 b 1 6 . 0 a 2 1 . 0 a 12 .2 a 1 5 . 2 b 1 1 . 3 a (mg/100 g body ±8.8 ± .2 ± .5 ±2.6 ±2.2 ±1.2 ±2.5 ±2.6 ±2.3 ±3.0 weight) L (±SEM) BR-F 6 3 . 7 b 1.0 a l . l b 4 . 0 a 7 . 8 a 1 2 . 8 a 1 7 . 5 a 12 .6 a 1 4 . 6 b 10.7 a ±10.0 ± .1 ± .2 ±1.1 ±2.4 ±2.9 ±2.3 ±1.8 ±2.2 ±4.3 WL-C 7 3 . 4 b 1.4 b 2 . 1 b 6 . 0 a ' b 1 5 . 2 a ' b 1 1 . 8 a 12 .6 a 11 .6 a 6 . 9 a 2 .6 a ±13.1 ± .1 ± .4 ±1.4 ±2.2 ±1.4 ± .7 ±2.3 ±1.4 ±1.0 WL-F 4 0 . 6 a . 6 a . 3 d 3 . 1 a 11.4 a 2 0 . 0 a 1 7 . 0 a 10 .2 a 6 . 0 a 4 . 8 a ±5.0 ± .1 ± .1 ± .7 ±201 ±2.8 +2.2 ±1.3 +1.4 ±1.0 ^R-C and BR-F are feeding of the high carbohydrate and high f a t d ie ts to b r o i l e r s , while WL-C and WL-F are feeding of these d iets to White Leghorns. 2Means in each column followed by d i f f e r e n t superscr ipts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . Table XVII. L iver protein o f chicks in response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods o f f a s t i n g . D ie ts 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 Liver protein (g/100 g l i v e r ) (+SEM) BR- C 15 .9 a 18.6 a 19 . 7 a 19.4 a 2 0 . l a 20 . 6 a 2 0 . 8 a 21. l a 21 .8 a ' b 2 1 . 7 a ± .5 ± .3 + .2 ± .6 ± .2 + .2 ± .3 ± .3 ± .2 + .4 BR- F 19 .2 C 2 0 . 3 b 19 . 7 a 2 1 . l b 2 0 . 5 a 20 . 8 a 2 1 . 2 a • b 2 1 . 4 a 2 0 . 9 a 21.8 a ± .3 ± .3 + .4 ± .2 ± .2 + .4 ± .5 ± .5 ± .6 ± .4 WL- C 1 7 . 8 b 2 0 . 5 b 20 . 2 a , b 2 0 . 9 b 20. l a 21 . 5 a 2 1 . 6 a > b 22.6 b 2 2 . 9 b 2 2 . 5 a ± .3 + .3 + .2 ± .3 ± .3 + .2 ± .2 + .2 ± .3 ± .2 WL- F 19 .3 C 2 0 . 8 b 20 . 9 b 2 0 . 7 b 2 0 . 5 a 21 . l a 2 2 . l b 2 2 . 2 a • b 2 2 . 1 b 22 .6 a ± .2 + .2 + .2 ± .3 ± .2 + .6 + .2 ± .3 ± .3 ± .2 Liver protein (mg/100 g body weight) (±SEM) BR- C 481 a 500 a 495 a 482 a ' b 514 b 492 a 492 a » b 470 b 489 a 501 a ± 18 ± 11 ± 16 ± 17 ± 15 ± 6 ± 12 ± 8 ± 7 ± 6 BR- F 499 a 494 a 450 a 482 a • b 470 a 469 a 459 a 439 a 459 a 462 a ± 7 ± 16 ± 1 5 ± 11 ± 8 ± 16 ± 16 ± 14 ± 15 ± 14 WL- C 509 a 522 a 480 a 498 b 500 a ' b 543 b 518 b 527 c 570 c 544 b ± 12 ± 10 ± 7 ± 6 ± 9 ± 12 ± 9 ± 5 ± 12 ± 23 WL- F 522 a 498 a 461 a 454 a 478 a »b 4 9 0 a 486 a • b 486 b 525 b 520 b ± 11 ± 12 ± 7 ± 6 ± 11 ± 14 ± 9 + 14 ± 10 ± 10 ^R-C and BR-F are feeding of the high carbohydrate and high f a t d iets to b r o i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means 1n each column followed by d i f f e r e n t superscr ipts were found to be s i g n i f i c a n t l y d i f f e r e n t (p<.05) by the Newman-Keuls t e s t . Table XVIII. Muscle dry matters of chicks in response to d i f fe rent d ietary treatments after d i f ferent periods of fas t ing . (±SEM) Diets 1 Fasting time (hours) 8 16 24 32 44 56 80 104 128 dry matter BR-•C 24 .8 a 24 .4 a 24 .6 a 24 . l a 24 . 3 a 24 .4 a ' b 24 .4 a 24 . 3 a 24 . 5 b 24 . 2 b g muscl e) + .2 + .1 + .1 + .1 + .2 + .2 + .2 + .2 + .2 + .1 BR-•F 24 .6 a 24 .2 a 24 . 3 a 24 .4 a 24, . 5 a 24 . 7 b 24 .4 a 24 .2 a 24 .4 b 24 . 3 b + .1 + .2 + .1 + .1 + .2 + .3 + .1 + .2 + .2 + .1 WL-•C 25 .0 a 24 . 5 a 24, .5 a 24 . l a 24, . l a 23 .9 a 24 . o a 23 .8 a 23, . l a 23 . o a + .3 + .1 ± . .1 + .2 + .1 + .2 + .2 + .2 + .3 + .2 WL-•F 25 . o a 24 .5 a 24, .5 a 24 .4 a 24, . 3 a 24, . l a 24 . l a 23 . 7 a 23, .0 a 22 . 3 a + .1 + .1 + .1 + .1 + .1 + .1 + .1 + .2 + .2 + .4 1BR-C and BR-F are feeding of the high carbohydrate and high fat d iets to b ro i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f ferent (p<.05) by the Newman-Keuls tes t . Table XVIX. Muscle glycogen of chicks in response to d i f fe rent d ietary treatments af ter d i f fe rent periods of fas t ing . Diets 1 Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 BR- C 1 c2 .28 c 1 .18° 1 .12 b .97 b 1.05 b .94 b 1.03 C 1.16 C 1.27° 1.14 b + .03 + .05 + .04 + .03 ±.03 + .05 ±.06 ±.06 ±.04 ±.06 BR-•F 1 .04 b 1 .04° 1 .02 b 1 .10° .47 a 1 .14 b 1.07C 1.05C 1.26° 1.26 b + .04 + .04 • + .03 + .03 ±.08 + .05 ±.05 ±.05 ±.04 ±.09 WL-C .84 a .77 b .54 a .57 a .52 a .63 a .25 b .21 a .41 b .31 a + .04 + .04 + .03 + .04 ±.05 + .05 ±.05 ±.03 ±.08 ±.07 WL- F .76 a .25 a .60 a .62 a .51 a .42 a .08 a .72 b .23 a .26 a + .03 + .06 + .05 + .02 ±.04 + .05 ±.01 ±.09 ±.04 ±.03 Muscle glycogen (g/100 g muscle) (±SEM) ^R-C and BR-F are feeding of the high carbohydrate and high fat diets to b ro i l e r s , while WL-C and WL-F are feeding of these diets to White Leghorns. 2Means in each column followed by d i f ferent superscripts were found to be s i gn i f i c an t l y d i f fe rent (p<.05) by the Newman-Keuls tes t . - 81 -S T A R V A T I O N T I M E ( h o u r s ) F igure 8 a . B r o i l e r hepato-somat ic index (g l i v e r / 1 0 0 g body weight) over the f a s t i n g p e r i o d . F igure 8 b . White Leghorn hepato-somat ic index (g l i v e r / 1 0 0 g body weight) over the f a s t i n g p e r i o d . - 82 -'1 S T A R V A T I O N T I M E C h o u r s ) Figure 9a . B ro i l e r l i v e r glycogen (g/100 g l i v e r ) over the fast ing per iod. Figure 9b. White Leghorn l i v e r glycogen (g/100 g l i v e r ) over the fast ing per iod. - 83 -23-Z 22-ui r- 2 H O l L 2 0 i • £ - 19-> W •J g 18-| o HI Oi 17-/ / o K m - • a f t e r C d i e t -A a f t e r F d i e t —i 1 1 1 24 32 44 56 STARVATION TIME Chours) — l — 80 — | — 104 I 128 16 Figure 10a. Bro i le r l i v e r protein (g/100 g l i v e r ) over the fast ing period. STARVATION TIME (hours) Figure l o b . White Leghorn l i v e r protein (g/100 g l i v e r ) over the fast ing per iod. - 84 -5 2 -. 5 1 -Z ^ Ul r-o a K Ul > ae ui O K a S.50-01 C M 7 - | e S..46 OB .44 . 4 3 H 0 A . - a a f t e r C d i e t \ M > \ K / \ A A a f t e r F d i e t V 128 16 24 I 32 — r -4 4 —r— 56 80 104 S T A R V A T I O N T I M E (hours ) Figure 11a. Broiler l iver protein (g/100 g body weight) over the fasting period. Ul H O K a ae ui > z ae O z (9 ui Ul I-z 5 . 5 7 -. 5 6 -. 5 5 -. 5 4 -.53 . 5 2 4 « .51 X 0) • .50H I 49-. 4 8 ' 0> e 0 . 4 7 -f •45H A / / / / V -- • a f t e r C d i e t A A a f t e r F d i e t 16 - 1 — 24 32 I 1 1 44 5 6 8 0 S T A R V A T I O N T I M E C hours) — i — 104 - 1 128 Figure l i b . White Leghorn l i ver protein (g/100 g body weight) over the fasting period. - 85 -4 „ J X I i.H z ui (9 O 7-§.*-s J -u « .4 . s ui d . 2 -O after C diet after F diet — I 128 16 -r— 24 - T -32 —T-44 56 8 0 — I — 104 S T A R V A T I O N T I M E ( h o u r s ) Figure 12a. B ro i l e r muscle glycogen (g/100 g muscle) over the fast ing per iod. x ^ o-| 1 1 — - — i i 1 1 r 8 16 24 32 44 56 8 0 S T A R V A T I O N T I M E ( h o u r s ) Figure 12b. White Leghorn muscle glycogen (g/100 g muscle) over the fast ing per iod. - 86 -5. SUMMARY This thesis was undertaken with the object ives of comparing the ef fects of carbohydrate and fat as non-protein energy sources in the diets of trout and chicks on t issue glycogen concentrations and on glycogen depletion from the t issues during a subsequent fas t . A tota l of 160 rainbow trout,-about one-year-old,were fed balanced d iets containing glucose or herring o i l as the non-protein energy source for a two-week period. Both the high carbohydrate (C) and the high fat d iet (F) were fed at sa t ia t ion level (C-2, F-2) and at levels hal f that (C - l , F - l ) . The trout were then fasted and sample f i s h were taken from each treatment group at the time of feed withdrawal and at 2, 4, 8, 10, 13 and 16 days thereafter . The sample f i s h were analyzed for l i v e r and muscle glycogen, protein and moisture content. The l i v e r s were also assayed for glucose-6-phosphatase a c t i v i t y . The l i v e r s of the C-fed trout were larger than those of the F-fed t rout . This was mostly due to the high glycogen concentrations in the C trout l i v e r s which stored 4 x more glycogen than the F trout l i v e r s did (12% versus 3% wet weight). There was l i t t l e or no difference in l i v e r glycogen concentrations between dietary feeding l eve l s . Upon fas t i ng , l i v e r glycogen f e l l to basal leve ls af ter two days in the F-fed trout while i t only reached the same basal leve ls af ter 10 days in the C-fed t rout . - 87 -On a percent liver basis, as glycogen concentrations increased, there was an equal decrease in liver protein concentrations. If, however, the total liver protein in the animal is calculated, more liver protein was found in the C-fed trout than in the F-fed trout. Over the fasting period, the body liver protein was depleted steadily but more slowly than liver glycogen. The combination of liver glycogen plus l i v e r protein accounted for the changes observed in hepato-somatic index over the fasting period. This indicates that liver mass depletion over the fasting period parallels depletion of liver glycogen plus protein, and HSI is an adequate measure of changes in liver glycogen plus protein. Trout liver glucose-6-phosphatase activity increased over the fasting period. When enzyme activity was expressed in terms of liver weight, the F trout livers had a higher G-6-Pase activity in the i n i t i a l stages of fasting than the C trout livers. When enzyme activity was expressed in terms of liver protein, however, these differences dis-appeared. This indicates the importance of expressing enzyme activity in terms of a standard variable. Liver protein is preferable to liver weight because i t is less variable. Trout muscle glycogen concentrations were directly related to dietary carbohydrate levels. It appears that muscle stored dietary carbohydrate that the liver cannot store as glycogen and that the body cannot metabolize. Upon fasting, muscle glycogen was depleted to basal levels over the f i r s t 4 days. Thereafter there was a rebound increase in glycogen concentrations to a peak and a subsequent depletion back to basal - 88 -l eve l s . The rebound of the C trout was larger and peaked ea r l i e r (8 days) than the rebound of the F trout (10 days). There was no s t a t i s t i c a l l y s ign i f i can t difference in the muscle glycogen rebound between dietary feeding leve ls although by appearances the F-2 fed trout had a greater rebound than the F-l fed t rout . A tota l of 150bro i ler- type, pu l l e t , day-old chicks and 154 White Leghorn, pu l l e t , day-old chicks were fed d i e t s , where 25% of the rat ion energy was in the form of cornstarch (C) or corn o i l (F), for three weeks. The chicks were then fasted and sample chicks were taken at the time of feed withdrawal and at 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours of fas t ing . The sample chicks were analyzed for l i v e r and muscle glycogen and dry matter. The l i v e r s were also analyzed for prote in. As with the t rout , the l i v e r s of the C-fed chicks had i n i t i a l l y more glycogen than the l i v e r s of the F-fed chicks although these differences were r e l a t i v e l y small (1% wet weight). Add i t iona l l y , the bro i l e rs (BR) had more l i v e r glycogen than the White Leghorns (WL) at f u l l feeding. Upon fas t i ng , l i v e r glycogen f e l l to basal leve ls af ter 8 hours. Subsequently there was a rebound in l i v e r glycogen leve ls to a peak and a subsequent tapering o f f . The rebound occurred more quick ly for the C-fed than the F-fed chicks although there was no dif ference in the extent of the rebound or the time of the peak between dietary treatments. The rebound peak occurred ea r l i e r for the WL than the BR and the WL showed a greater subsequent depletion but there was no di f ference in the magnitude of the rebound peak in glycogen leve ls between BR and WL. - 89 -There was no d ietary di f ference in l i v e r protein concentrations but the amount of l i v e r protein was higher in the C-fed than the F-fed chicks at a l l times during the fast ing period. The reasons for th is remarkable s i tuat ion are unknown. Chick muscle glycogen concentrations were i n i t i a l l y higher in the C-fed than the F-fed chicks and higher in BR than WL. Upon fas t ing , muscle glycogen f luctuated e r r a t i c a l l y , espec ia l ly in the F-fed chicks. Generally there was a decrease in WL muscle glycogen concentrations and a maintenance or actual rebound increase in BR muscle glycogen concentrations. There was no dietary dif ference in muscle glycogen leve ls during the fas t . - 90 -REFERENCES Aberle, E.B., P.B. Addis and R.N. Shaffner. 1979. Fibre types in skeletal muscle of b ro i l e r and layer type chickens. Poult. S c i . 58: 1210-1212. Akrabawi, S.S., M.M. Saegert and J .P . S a l j i . 1974. Studies on the growth and changes in metabolism of rats fed on carbohydrate de f i c i en t , fa t ty -ac id based diets supplemented with graded levels of maize starch. Br. J . Nutr. 32: 209-217. A l l r ed , J .B . and K.L. Roehrig. 1970. Hepatic gluconeogenesis and g lyco lys i s in chickens fed "carbohydrate-free" d ie t s . J . Nutr. 100: 615-622. Andrews, J.W. J r . and J.M. Davis. 1979. Relat ive u t i l i z a t i o n of d ietary energy sources by ca t f i sh . Fed. Proc. 38: 448. Associat ion of O f f i c i a l Agr icu l tura l Chemists. 1960. O f f i c i a l Methods of Analysis of the Associat ion of O f f i c i a l Agr icu l tura l Chemists, 9th ed. Washington, D.C. p.94. Austreng, E., S. R isa, D.J. Edwards and H. Hvidsten. 1977. Carbohydrate in rainbow trout d i e t s . I I . Influence of carbohydrate leve ls on chemical composition and feed u t i l i z a t i o n of f i s h from d i f fe rent fami l i es . Aquaculture 11: 39-50. Baginski, E.S., P.P. Foa and B. Zak. 1974. Glucose-6-phosphatase i n : Methods of Enzymatic Analysis V. 2. ed. by Hans U l r i ch Bergmeyer. Academic Press: New York. pp. 876-880. Batty, R.S. and C.S. Wardle. 1979. Restoration of glycogen from l a c t i c acid in the anaerobic swimming muscle of p la i ce , Pleuronectes platessa L. J . Fish B i o l . 15: 509-519. Bergman, R.N. and R.J. Bucolo. 1974. Interaction of i nsu l i n and glucose in the control of hepatic glucose balance. Am. J . Phys io l . 227: 1314-1322. Bergot, F. 1979. Carbohydrate in rainbow trout d ie t s : Effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture 18: 157-167. Bergstrom, J . and E. Hultman. 1966. Muscle glycogen synthesis af ter exercise: an enhancing factor loca l i zed to the muscle ce l l s in man. Nature 210: 309-310. - 91 -Bergstrom, J . , E. Hultman and A.E. Roch-Norlund. 1972. Muscle glycogen synthetase in normal subjects: basal values, ef fect of glycogen depletion by exercise and of a carbohydrate r i ch d ie t fol lowing exerc ise. Scand. J . C l i n . Lab. Invest. 29: 231-236. Bernard, C. 1853. Nouvelle Fonction du Foie Considere comme Organe Producteur de Matiere Sucree chez 1'Homme et les Animaux. Par is : B a i l l i e r e . Bernard, C. 1857. Sur le mecanisme physiologique de la formation du sucre dans le f o i e . Comp. Rend. 44: 578-586. Bernard, C. 1877. Lecons sur Ie Diabete et l a Glycogenese Animale. Par is : B a i l l i e r e . Black, E.G., A.C. Robertson and R.R. Parker. 1961. Some aspects of carbohydrate metabolism in f i s h . In: Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals, ed. by Arthur W. Mart in. Univers i ty of Washington Press; Seatt le , pp.89-124. Black, E.G., N.J. Bosomworth and G.E. Docherty. 1966. Combined ef fect of starvat ion and severe exercise on glycogen metabolism of rainbow trout , Salmo ga i rdner i . J . F i sh . Res. Bd. Can. 23: 1461-1463. Brambila, S. and F.W. H i l l . 1966. Comparison of neutral fat and free fa t t y acids in high l i p i d - low carbohydrate d iets for the growing chicken. J . Nutr. 88: 84-92. Brown, J .H . , B. Thompson and S.E. Mayer. 1977. Conversion of skeleta l muscle glycogen synthase to mult ip le glucose-6-phosphate dependent forms by c y c l i c adenosine monophosphate dependent and independent protein kinases. Biochemistry 16: 5501-5508. Buhler, D.R. and J .E . Halver. 1961. Nutr i t ion of salmonid f ishes IX. Carbohydrate requirements of chinook salmon. J . Nutr. 74: 307-318. Ca l las , G. and M.S. Cannon. 1975. A l terat ions in the f ine structure of hepatocytes in hyperthyroid rats . Anat. Rec. 181: 71-82. Colca, J.R. and R.L. Hazelwood. 1976. Pancreatectomy in the chicken: does an extra-pancreatic source of i n su l i n ex is t? Gen. Comp. Endocrinol. 28: 151-162. Cor i , C F . and G.T. Cor i . 1929. Glycogen formation in the l i v e r from d- and 1- lac t i c ac id . J . B i o l . Chem. 81: 389-403. - 92 -Cor i , G.T., C F . Cori and G. Schmidt. 1939. The ro le of glucose-6-phosphate in the formation of blood sugar and synthesis of glycogen in the l i v e r . J . B i o l . Chem. 129: 629-639. Cowey, C.B., J.W. Adron, D.A. Brown and A.M. Shanks. 1975. Studies on the nut r i t i on of marine f l a t f i s h . The metabolism of glucose by p la ice (Pleuronectes-platessa) and the ef fect of d ietary energy source on protein u t i l i z a t i o n in p la i ce . Br. J . Nutr. 33: 219-231. Dave, G., M.L. Johansson-Sjobeck, A. Larsson, K. Lewander and U. Lidman. 1975. Metabolic and hematological e f fects of starvat ion in the European ee l , Angui l la-Angui l la L.-I. Carbohydrate, l i p i d , protein and inorganic ion metabolism. Comp. Biochem. Phys io l . A. Comp. Phys io l . 52: 423-430. Davison, T.F. and D.R. Langslow. 1975. Changes in plasma glucose and l i v e r glycogen fol lowing the administrat ion of gluconeogenic precursors to the starving fowl. Comp. Biochem. Phys io l . A. Comp. Phys io l . 52: 645-649. Den Otter, W. and A. De Minjer. 1972. Glycogen in the l i v e r during starvat ion: A histochemical and biochemical invest igat ion. Anat. Rec. 173: 141-150. Drochmans, P. and E. Danton. 1968. Size d i s t r ibu t ions of l i v e r glycogen pa r t i c l e s . In: Control of Glycogen Metabolism, ed. by W.J. Whelan.Academic Press, New York. pp.187-201. Fe l t , V. 1973. Effect of thyroid hormones and dietary fat on the in te r re la t i on of l i p i d and carbohydrate metabolism in ra ts . Endocrinologia Experimental i s 7: 283-291. Freedland, R.A. 1967. Effect of progressive starvat ion on rat l i v e r enzyme a c t i v i t i e s . J . Nutr. 91: 489-495. Furu ich i , M. and Y. Yone. 1971. Studies on nut r i t i on of red sea bream - IV. A study of carbohydrate u t i l i z a t i o n by glucose-and insu l in glucose-tolerance tes t . Rep. F ish. Lab., Kyushu Univ. 1: 101-106. Furu ich i , M. and Y. Yone. 1980. Effect of d ietary dextr in leve ls on the growth and feed e f f i c i ency , the chemical composition of l i v e r and dorsal muscle and the absorption of d ietary protein and dextr in i n f i shes . Bu l l . Jap. Soc. Sc i . F i sh . 46: 225-230. Geddes, R. and G.C Stratton. 1977. Molecular and metabolic heterogeneity of 1 iver glycogen. Carbohydr. Res. 57:291-299. - 93 -Goldste in, D.E. and R.T. Curnow. 1977. Effects of starvat ion on glucose tolerance and hepatic glycogen metabolism. C l i n . Res. 25: 391 A. Gorski , J . , U. Puch and K. Kiczka. 1976. Post-adrenaline glycogen recovery in the rat skeleta l muscle. Bu l l . Acad. Po l . S c i . Ser. S c i . B i o l . 24: 185-187. Hassid, W.Z. and S. Abraham. 1957. Chemical procedures for analysis of polysaccharides. I. Determination of glycogen and starch. In: Methods of Enzymology V.3. eds. S.P. Colowick and N.A. Kaplan .Academic Press, New York. pp. 34-37. Hazelwood, R.L. 1976. Carbohydrate metabolism. In: Avian Physiology. ed. by P.D. Sturk ie . Springer-Verlag, New York. pp. 210-232. Hazelwood, R.L. and F.W. Lorenz. 1959. Effects of fast ing and insu l i n on carbohydrate metabolism of the domestic fowl. Am. J . Phys io l . 197: 47-51. Hermansen, L. and 0. Vaage. 1977. Lactate disappearance and glycogen synthesis in human muscle after maximal exerc ise. Am. J . Phys io l . 233: E422-E429. Higgs, D.A. ,.U.H.M. Fagerkmd, J .G. Eales and J.R. McBride. 1981. Appl icat ion of thyroid ..and stero id hormones as anabolic agents in f i s h cu l ture, ( in press) H i l l , F.W., R.B. Rucker and A.M. Faqih. 1974. Fasting responses of rats fed a carbohydrate-free d ie t . Fed. Proc. 33(3 Part 1): 718. H i l ton , J.W. and D.G. Dixon. 1981. Effect of increased l i v e r glycogen and l i v e r weight on l i v e r function in rainbow trout (Salmo gairdneri R.): Recovery from anaesthesia and S-sulphobromophthalein plasma clearance. In: Canadian Federation of B io log ica l Soc iet ies . Programme and Proceedings. Vo l . 24. Montreal, Que. Abstract 455. Hochachka, P.W. 1980. L iv ing without Oxygen. Closed and Open Systems in Hypoxia Tolerance. Harvard Univers i ty Press: Cambridge Mass. pp. 2-5. Hochachka, P.W. and A.C. S i n c l a i r . 1962. Glycogen stores in trout t issues before and after stream plant ing. J . F i sh . Res. Bd. Can. 19: 127-137. Hultman, E., J . Bergstrom and A.E. Roch-Norlund. 1971. Glycogen storage in human skeletal muscle. Adv. Exp. Med. B i o l . 11: 273-288. - 94 -Kang, S.S. , K.R. Bruckdorfer and J . Yudkin. 1979. Influence of d i f f e ren t , d ietary carbohydrates on l i v e r and plasma constituents in rats adapted to meal feeding. Nutr. Metab. 23: 301-315. Kochan, R.G., D.R. Lamb, S.A. Lutz, C.V. P e r r i l l , E.M. Reimann and K.K. Schlender. 1979. Glycogen synthase act ivat ion in human skeleta l muscle: ef fects of d iet and exerc ise. Am. J . Phys io l . 236: E660-E666. Lee, D.J. and J .H . Wales. 1973. Observed l i v e r changes in rainbow trout (Salmo gairdner i) fed varying leve ls of a casein-ge lat in mixture and herring o i l in experimental d i e t s . J . F ish. Res. Bd. Can. 30: 1017-1020. Lenzen, S. 1978. Dose-response studies on the inh ib i to ry ef fect of thyroid hormones on insu l i n secretion in the rat . Metab. C l i n . Exp. 27: 81-88. Llobera, M., M.J. Seibel and E. Herrera. 1978. Metabolic response to short periods of starvat ion in hypothyroid and hyperthyroid male ra ts . Horm. Metab. Res. 10: 319-324. Llobera, M. and E. Herrera. 1980. Effects of starvat ion on in-v ivo gluconeogenesis in hypothyroid and hyperthyroid ra ts . Endocrinology 106: 1628-1633. McLane, J.A. and J . 0 . Hol loszy. 1979. Glycogen synthesis from lactate in three types of skeleta l muscle. J . B i o l . Chem. 254: 6548-6553. Machlum, S., A.T. Hostmark and L. Hermansen. 1977. Synthesis of muscle glycogen during recovery af ter prolonged severe exercise in d iabet ic and non-diabetic subjects. Scand. J . C l i n . Lab. Invest. 37: 309-316. March, B.E. and J . B ie ly . 1957. The ef fect of d ietary fa t level oh thyroid a c t i v i t y in the growing chick. Poult. S c i . 26: 1270-1277. Matcham, G.W.J., N.B. P a t i l , E.E. Smith and W.J. Whelan. 1978. The glycoprotein nature of l i v e r glycogen. In: Regulatory Mechanisms of Carbohydrate Metabolism, ed. by Viggio Esmann. Pergammon Press, Toronto, pp. 305-315. Mering, J . von. 1877. Zur Glycogenbildung in der Leber. Pflugers Arch. 14: 274-284. Meyer, K.H. 1943. The chemistry of glycogen. Adv. Enzymol. 3: 109-135. - 95 -M i g l i o r i n i , R.H., C. Linder, J . L . Moura and J .A.S. Veiga. 1973. Gluconeogenesis in a carnivorous bird (black vu l tu re) . Am. J . Phys io l . 225: 1389-1392. M i l l e r , R.B., A.C. S i n c l a i r and P.W. Hochachka. 1959. Diet, glycogen reserves and resistance to fat igue in hatchery rainbow trout . J . F i sh . Res. Bd. Can. 16: 321-328. M i l l e r , T.B. and J . Larner. 1973. Mechanism of control of hepatic glycogenesis by i n su l i n . J . B i o l . Chem. 248: 3483-3488. Mi lne, R.S., J .F . Leatherland and B.J. Holub. 1979. Changes in plasma thyroxine, t r i - iodothyronine and Cort iso l associated with starvat ion in rainbow trout (Salmo ga i rdner i ) . Environ. B i o l . Fishes 4: 185-190. Naber, S.P. and R.L. Hazelwood. 1977. In v i t r o i n su l i n release from chicken pancreas. Gen. Comp. Endocrinol. 32: 495-504. Nagai, M. and S. Ikeda. 1971. Carbohydrate metabolism in f i s h - 1 . Effects of starvat ion and d ietary composition on the blood glucose level and the hepatopancreatic glycogen and l i p i d contents in carp. Bu l l . Jap. Soc. S c i . F i sh . 37: 404-409. Nagayama, F., H. Ohshima and K. Umezawa. 1972. D is t r ibut ion of glucose-6-phosphate metabolizing enzymes in f i s h . Nippon Suisan Gakkaishi 38: 589-593. O 'Ne i l l , I .E. and D.R. Langslow. 1978. Glucose phosphorylation and dephosphorylation in chicken l i v e r . Comp. Biochem. Phys io l . 59B: 317-325. Palmer, T.N. and B.E. Ryman. 1972. Studies on oral glucose intolerance in f i s h . J . Fish B i o l . 4: 311-319. P h i l l i p s , A.M., A.V. Tunison and D.R. Brockway. 1948. The u t i l i z a t i o n of carbohydrates by t rout . F i sh . Res. Bu l l . N.Y. 11:44pp. Pieper, A. and E. Pfef fer . 1978. Carbohydrates as possible sources of d ietary energy for rainbow trout (Salmo gairdneri Richardson). In: Proceedings of the World Symposium on F i r i f i sh Nutr i t ion and Fishfeed Technology. Hamburg 20-23 June 1978 Ber l in Vo l . 1. pp. 209-219. Raheja, K.L., J .G. Snedecor and R.A. Freedland. 1971a. A c t i v i t i e s of some enzymes involved in l ipogenesis, gluconeogenesis, g lyco lys i s and glycogen metabolism in chicks (Gal 1 us  domesticus) from day of hatch to adulthood. Comp. Biochem. Phys io l . 39B: 237-246. - 96 -Raheja, K.L., J .G. Snedecor and R.A. Freedland. 1971b. Effect of propy l th iourac i l feeding on glycogen metabolism and malic enzyme in the l i v e r of the chick. Comp. Biochem. Phys io l . 39B: 833-839. Raheja, K.L., H.M. Tepperman and J . Tepperman. 1972. Effect of fast ing and high-fat d ie t feeding on plasma and pancreatic i n su l i n contents in young chick given an oral glucose load. Horm. Metab. Res. 4: 337-341. Raheja, K.L. and W.G. Linscheer. 1978. Effect of d ietary composition on l i v e r glycogen accumulation and l i p i d metabolism in the hypothyroid chick (Gallus domesticus). Comp. Biochem. Phys io l . A Comp. Phys io l . 61: 31-34. Raheja, K.L. and W.G. Linscheer. 1980. Modulation of propy l th iourac i l induced hypothyroid syndrome in chick by dietary composition: ro le of i n su l i n and glucagon. Fed. Proc. 39: Abstract 2447. Rappaport, A.M. 1963. Acinar units in the pathophysiology of the l i v e r . In: The L iver . Morphology, Biochemistry, Physiology, Vol . 1. ed. by C. Rou i l l e r . Academic Press, London, pp. 265-328. Renner, R. and A.M. Elcombe. 1967. Metabolic ef fects of feeding "carbohydrate-free" d iets to chicks. J . Nutr. 93: 31-36. Rivera, A. J r . and J . Martinez-de Jesus. 1974. Starvation and the glycogen of the brain and v i t a l organs of the Rhesus monkey. J . Nutr. 104: 1189-1194. Roch-Norlund, A.E. 1972. Muscle glycogen and glycogen synthetase in d iabet ic man. Scand. J . Lab. C l i n . Invest. 29 Suppl. 125: 7-27. Rosebrough, R.W. and J . J . Begin. 1975. The ef fect of nonprotein energy source and age on the blood glucose and the muscle glycogen content of young chicks. Poult. Sc i . 54:1327-1329. Rosebrough, R.W., E.G. Geis, K. Henderson and L.T. Frobish. 1978. Effect of d ietary energy on hepatic glycogen metabolism in the turkey hen. Poult. Sc i . 57: 1652-1657. Rosebrough, R.W., E. Geis, K. Henderson and L.T. Frobish. 1979a. Control of glycogen metabolism in the developing turkey poult. Growth 43: 188-201. Rosebrough, R.W., R.H. Rosebrough, E. Geis and K. Henderson. 1979b. Effect of supplemental glucose or sucrose on l i v e r and carcass glycogen metabolism of young chicks. Poult. S c i . 58: 1524-1528. - 97 -Sakaguchi, H. 1976. Changes of biochemical components in serum, hepatopancreas and muscle of ye l lowta i l during starvat ion. B u l l . Jap. Soc. S c i . F i sh . 42: 1267-1272. Sasse, D. 1975. Dynamics of l i v e r glycogen. The topochemistry of glycogen synthesis, glycogen content and glycogenolysis under the experimental condit ions of glycogen accumulation and deplet ion. Histochemistry 45: 237-254. Sasse, D., N. Katz and K. Jungermann. 1975. Functional heterogeneity of rat l i v e r parenchyma and of i so lated hepatocytes. FEBS-Letters 57: 83-88. Seaton, K.W. , O.P. Thomas, R.M. Gous and E.H. Bossard. 1978. The of d ie t on l i v e r glycogen and body composition in the chick. Poult. Sc i . 57: 692-698. ef fect Schwartz, A.L. and T.W. R a i l . 1973. Hormonal regulat ion of glycogen metabolism in neonatal rat l i v e r . Biochem. J . 134: 985-993. Sha f f i , S.A. 1979. Effect of starvat ion on t issue and serum gluconeogenic enzymes, a lka l ine phosphatase and t issue glycogen in the fresh water ca t f i sh (Heteropneustes  f o s s i l i s ) . Acta. Phys io l . Acad. S c i . Hung. 53: 501-506. Shimeno, S. , H. Hosokawa and M. Takeda. 1978. The importance of carbohydrate in the d ie t of a carnivorous f i s h . In: Proceedings of the World Symposium on F in ish Nutr i t ion and Fishfeed Technology. Hamburg 20-23 June 1978. Ber l in Vo l . 1 pp. 127-143. Shimma, Y. , H. Ichimura and N. Shibata. 1976. Effects of starvat ion on body weight, l i p i d contents and plasma constituents of maturing rainbow t rout . Bu l l . Jap. Soc. S c i . F ish. 42: 83-89. Simon, J . and G. Rossel in. 1978. and food intake on in Horm. Metab. Res. 10: Effect of f as t i ng , glucose, amino acids vivo i nsu l i n release in the chicken. 93-98. Snedecor, J .G. 1968. Liver hypertrophy, l i v e r glycogen accumulation and organ weight changes in radiothyroidectomized and goitrogen treated chicks. Gen. Comp. Endocrinol. 10: 277-291. Stimpson, J .H . 1965. Comparative aspects of the control of glycogen u t i l i z a t i o n in vertebrate l i v e r . Comp. Biochem. Phys io l . 15: 187-197. - 98 -Suzuki, H. and H. Fuwa. 1971. Interact ion of d ietary fat and thyroid function with hepatic and renal gluconeogenesis of ra t s . J . Nutr. 101: 919-926. Swallow, R.L. and W.R. Fleming. 1969. The ef fect of s tarvat ion, feeding, glucose and ACTH on the l i v e r glycogen leve ls of T i l ap ia mossambica. Comp. Biochem. Phys io l . 28: 95-106. Tunison, A.V., A.M. P h i l l i p s , CM . McCay, C R . Mitchel l and E.O. Rodgers. 1939. The nut r i t i on of t rout . Cortland Hatchery Report No. 8. J .B . Lyon: Albany, N.Y. 33pp. Veiga, J .A .S . , E.S. Roselino and R.H. M i g l i o r i n i . 1978. Fast ing, adrenalectomy and gluconeogenesis in the chicken and a carnivorous b i rd . Am. J . Phys io l . 234: R115-R121. Vernier, J.M. and M.F. S i re . 1978. Etude in vivo des ef fects de 1'adrenaline, des gluco^ticoids et du jeune sur les a c t i v i t i e s de la glycogene phosphorylase, de la glucose-6-phosphatase et sur l a teneur en glycogene du fo ie de la t r u i t e a r c -en - c i e l . Gen. Comp. Endocrinol. 34: 370-376. Vrana, A. , P. Fabry.and L. Kazdova. 1978. Liver glycogen synthesis and glucose tolerance in rats adapted to d iets with a high proportion of fructose of glucose. Nutr. Metab. 22: 262-268. Vrana, A. , P. Fabry, L. Kazdova and K. Zvolankova. 1978. Effect of the type and proportion of d ietary carbohydrate on serum glucose leve ls and l i v e r and muscle glycogen synthesis in the ra t . Nutr. Metab. 22: 313-320. Watts, C. and K.R. Gain. 1976. Glycogen metabolism in the l i v e r of the developing ra t . Biochem. J . 160: 263-270. Zar, J .H . 1974. B i o s t a t i s t i c a l Ana lys i s . , P rent i ce-Ha l l , Inc.: Englewood C l i f f s , N.J. pp. 182-186. Table IA. S t a t i s t i c a l analys is of t rout body weight in response to d i f f e r e n t d i e t a r y treatments a f ter d i f f e r e n t periods of f a s t i n g . Analysis of Variance Transformation: log 10(body weight) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8, 10, 13 and 16 days D x L x T: Source df Mean square ( x l O ) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) D l 3.758 1.29 .257 L l 23.298 7.99 .006 2>1 D x L l 5.297 1.82 .177 T 6 9.743 3.34 .004 0,16,2,4>16,2, 4,10,8,13 D x T 6 5.231 1.79 .104 L x T 6 4.093 1.40 .217 D x L x T 6 5.386 1.85 .094 Error 128 2.916 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D l 1 1 1 1 1 1 L l 1 1 1 1 1 1 D x L l 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 2.564 1.872 12.775 4.682 9.735 1.702 1.818 square L 25.504 3.962 7.373 2.696 1.330 2.942 4.047 / 1 /-\—3 \ D x L 28.836 .942 2.932 .162 .015 .437 4.290 ( x l O ) Error 1.573 3.929 3.066 3.977 2.623 3.036 1.257 F value D 1.63 .48 4.17 1.18 3.71 .56 1.45 L 16.22 1.01 2.41 .68 .51 .97 3.22 D x L 18.34 .24 .96 .04 .01 .14 3.41 F prob. D .217 .505 .052 .291 .066 .469 .254 L .000 .329 .133 .425 .491 .338 .097 D x L .000 .634 .342 .823 .898 .708 .089 Conclusion 2>1 Table I IA. S t a t i s t i c a l analys is of t rout condit ion factor in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analysis of Variance Transformation: log 10((arcs in ^condit ion fac tor )+l ) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus hal f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: ~ — Source df Mean square (xl0~ •) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) D 1 1.283 4.26 .039 C>F L 1 4 .147 3.81 .050 D x L 1 .216 .72 .404 T 6 3.007 9.98 .000 0,2>2,4>4,13,16>13,16,8,10 D x T 6 .195 .65 .695 L x T 6 .248 .82 .555 D x L x T 6 .670 2.23 .044 Error 128 .301 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 4.017 .854 4.740 13.070 .884 .481 .469 square L 18.343 6.099 1.705 .061 .054 .009 .105 ( x i o ' S D x L 14.227 3.243 3.557 16.139 .049 3.380 1.779 Error 3.052 1.295 3.325 2.262 4.651 3.048 3.826 F value D 1.32 .66 1.43 5.78 .19 .16 .12 L 6.01 4.71 .51 .03 .01 .00 .03 D x L 4.66 2.50 1.07 7.14 .01 1.11 .47 F prob. D .267 .431 .245 .025 .670 .696 .729 L .024 .040 .489 .846 .880 .912 .846 D x L .043 .126 .315 .014 .883 .306 .465 Conclusion 2>1 2>1 C>F Table 111A. S t a t i s t i c a l analys is of trout l i v e r weight In response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Analysis of Variance Transformation: log 10(Hver weight) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4 , 8 , 10, 13 and 16 days D x L x T: Source df Mean square (xlO' " 2 ) F value F prob. Conclusion (Newman-Keuls mu l t ip le range tes t ) D 1 118.231 155.47 .000 C>F L 1 18 .155 23.87 .000 2>1 D x L 1 5 .863 7.71 .006 C-2>C-1>F-2,F-1 T 6 32.443 42.66 .000 0>2,4>8,10>10,16>16,13 D x T 6 7 .123 9.37 .000 L x T 6 1 .702 2.24 .043 D x L x T 6 .731 .96 .455 Error 128 .760 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 54.036 44.288 40.180 16.053 1.621 3.304 1.487 square L 9.559 6.683 11.275 .235 .409 .010 .197 / i n " 2 \ D x L 3.975 1.420 .723 .112 .078 3.672 .269 (xlO ) Error .641 .879 1.036 .944 .792 5.430 .233 F value D 84.24 50.40 38.80 17.01 2.05 6.08 6.39 L 14.90 7.61 10.89 .25 .52 .02 .85 D x L 6.20 1.62 .70 .12 .10 6.76 1.16 F prob. D .000 .000 .000 .001 .165 .022 .027 L .001 .012 .004 .628 .487 .864 .381 D x L .022 .216 .418 .731 .752 .016 .306 Conclusion C>F C>F C>F C>F C>F C>F 2>1 2>1 2>1 Table IVA. S t a t i s t i c a l analysis o f trout hepato-somatic index (HSI) in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analys is of Variance Transformation: log 10((arcs in VHSI)+l) Diet (D) = high carbohydrate (C) versus high fa t (F) Feeding level (L) = s a t i a t i o n (2) versus h a l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x_J± Source df Mean square (xlO" ) F value F prob. Conclusion (Newman-Keuls mul t ip le range test) D 1 16.657 223.67 .000 C>F L 1 1.268 17.02 .000 2>1 D x L 1 .483 6.48 .012 C-2>C-1>F-2,F- 1 T 6 3.719 49.93 .000 0>2,4>8>10,13,16 D x T 6 .942 12.64 .000 L x T 6 .139 1.87 .090 D x L x T 6 .074 1.00 .431 Error 128 .074 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 87.507 62.899 42.437 16.191 7.045 6.203 .784 square L 3.821 6.964 9.771 .001 .073 .366 .042 (x lO" 4 ) D x L 1.918 1.896 .314 .075 .141 3.633 1.302 Error .656 .667 1.351 .668 .845 .492 .339 F value D 133.41 94.34 31.42 24.24 8.34 12.60 2.31 L 5.82 10.45 7.23 .00 .09 .74 .12 D x L 2.92 2.84 .23 .11 .17 7.38 3.84 F prob. D .000 .000 .000 .000 .009 .002 .154 L .026 .004 .014 .923 .764 .404 .729 D x L .102 .104 .639 .737 .689 .013 .073 Conclusion C>F C>F C>F C>F C>F C>F 2>1 2>1 2>1 Table VA. S t a t i s t i c a l analys is of trout l i v e r dry matter in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analysis of Variance Transformation: log 10((arcs1n V l l v e r dry matter)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: Source df Mean i square (xlO' " 4 ) F value F prob. Conclusion (Newman-Keuls mul t ip le range tes t ) D 1 5.706 51.67 .000 O F L 1 .197 1.79 .180 D x L 1 .254 2.30 .128 T 6 1.444 13.08 .000 2,0>13,4>4,10,8,16 D x T 6 .876 7.93 .000 L x T 6 .254 2.30 .038 D x L x T 6 .396 3.59 .003 Error 128 .110 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D M 27.124 41.833 28.212 12.116 .138 .071 .112 square L .760 5.813 3.674 .062 .294 .579 6.061 D x L .953 2.333 .000 8.898 10.518 1.014 2.576 (.xiu ; Error .656 1.760 .992 .864 .973 .776 2.081 F value D 41.37 23.77 28.45 14.02 .14 .09 .05 L 1.16 3.30 3.71 .07 .30 .75 2.91 D x L 1.45 1.33 .00 10.29 10.81 1.31 1.24 F prob. D .000 .000 .000 .001 .709 .759 .803 L .297 .081 .066 .781 .595 .402 .113 D x L .243 .263 .940 .004 .004 .266 .290 Conclusion C>F C>F C>F C>F Table VIA. S t a t i s t i c a l analys is o f trout l i v e r glycogen (g/100 g l i v e r ) In response to d i f f e r e n t d ietary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Analysis o f Variance Transformation: log 10((arcs in Ol iver glycogen)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus h a l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: Source df Mean square (xlO" ) F value Fprob. Conclusion (Newman-Keuls mul t ip le range tes t ) D 1 43.932 230.87 .000 C>F L 1 .471 2.48 .114 D x L 1 .553 2.91 .087 T 6 11.952 62.82 .000 0>2>4>8.10>10.13.16 D x T 6 4.798 25.21 .000 L x T 6 .098 .52 .797 D x L x T 6 .126 .66 .682 Error 128 .190 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 18.021 25.671 23.071 4.758 1.099 .021 .079 square L .151 .429 .330 .015 .067 .009 .059 ( x l O " 3 ) D x L -.083 .211 .121 .027 .006 .903 .126 Error 1.167 .189 .180 .373 .213 .196 .014 F value D 262.50 136.01 127.95 12.75 5.15 .11 5.52 L 2.20 2.28 1.83 .04 .32 .05 4.11 D x L -1.21 1.12 .67 .07 .03 4.61 8.78 F prob. D .000 .000 .000 .002 .033 .743 .037 L .154 .144 .189 .826 .588 .814 .065 D x L 1.000 .303 .428 .781 .844 .042 .012 Conclusion C>F C>F C>F C>F C>F F>C Table VIIA. S t a t i s t i c a l analys is of trout l i v e r glycogen (mg/100 g body weight) in response to d i f f e r e n t d ietary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analysis of Variance Transformation: log 10((arcs in O l i v e r glycogen)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4 , 8 , 10, 13 and 16 days D x L x T: Source df _2 Mean square (xlO ) F value F prob. Conclusion (Newman-Keuls mu l t ip le range tes t ) D 1 11.147 285.57 .000 C>F L 1 .248 6.35 .013 2>1 D x L 1 .194 4.97 .026 C-2>C-1>F-2,F-•1 T 6 2.799 71.71 .000 0>2>4>8,10>10,13,16 D x T 6 1.185 30.36 .000 L x T 6 .025 .64 .698 D x L x T 6 .030 .76 .603 Error 128 .039 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 62.549 60.424 47.129 9.820 2.385 .242 .025 square L .001 2.249 1.610 .005 .070 .006 .040 D x L .226 1.282 .629 .105 .001 1.434 .049 (xl0~ 3 ) Error .250 .399 .526 .639 .396 .318 .013 F value D 250.67 151.35 89.62 15.36 6.02 .76 2.01 L .01 5.63 3.06 .01 .18 .02 3.14 D x L .91 3.21 1.20 .16 .00 4.51 3.87 F prob. D .000 .000 .000 .001 .022 .397 .182 L .899 .027 .092 .888 .680 .860 .101 D x L .357 .085 .288 .691 .911 .044 .073 Conclusion C>F C>F C>F C>F C>F Table VIIIA. S t a t i s t i c a l analys is of trout l i v e r protein (g/100 g l i v e r ) in response to d i f f e r e n t d ietary treatments a f t e r d i f f e r e n t periods o f f a s t i n g . Analys is of Variance Transformation: log 10((arcs in O l i v e r protein)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding leve l (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4 , 8 , 10, 13 and 16 days D x L x T: Source df -4 Mean square (xlO ) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) D 1 27.019 103.72 .000 F>C L 1 .593 2.28 .130 D x L 1 .699 2.68 .100 T 6 8.302 31.87 .000 13,10,16>10,16,8>4,2>0 D x T 6 4.612 17.71 .000 L x T 6 .566 2.17 .049 D x L x T 6 .328 1.26 .280 Error 128 .261 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 25.635 16.403 8.966 .958 1.009 .316 1.405 square L .183 1.376 .254 .583 .003 .050 1.541 1 ln"4\ D x L -.144 .717 .858 .048 .062 .548 .578 (xlO ) Error .173 .353 .282 .240 .307 .150 .341 F value D 148.26 46.43 31.84 3.99 3.28 2.11 4.12 L 1.06 3.89 .90 2.43 .01 .33 4.52 D x L - .83 2.03 3.05 .20 .20 3.66 1.70 F prob. D .000 .000 .000 .057 .082 .159 .065 L .319 .060 .357 .131 .883 .577 .055 D x L 1.000 .166 .093 .661 .663 .067 .218 Conclusion F>C F>C F>C Table IXA. Analysis of Variance S t a t i s t i c a l analysis of trout l i v e r protein (mg/100 g body weight) i n response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods of f a s t i n g . Transformation: log 10((arcsin J l i v e r protein)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding le v e l (L) = s a t i a t i o n (2) versus h a l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8, 10, 13 and 16 days D x L x T: Source x L x T Error df 1 1 1 6 6 6 6 128 Mean square (xlO" 4) F value F prob. Conclusion (Newman-Keuls m u l t i p l e range t e s t ) 64.192 10.475 1.134 12.952 1.074 1.729 .501 .671 95.73 15.62 1.69 19.31 1.60 2.58 .75 .000 .000 .193 .000 .151 .022 .614 C>F 2>1 0,2>2,4>4,8>8,10>10,13,16 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D L D x L Error 1 1 1 17 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 11 Mean square ( x l O - 4 ) D L D x L Error 15.212 7.034 1.143 .675 16.685 2.703 .021 1.007 15.405 •8.370 .318 1.019 12.663 .844 .000 .411 4.057 .200 .059 .656 5.211 .159 2.021 .437 1.405 1.541 .578 .341 F value D L D x L 22.52 10.41 1.69 16.57 2.68 .02 15.12 8.22 .31 30.78 2.05 .00 6.19 .30 .09 11.92 .36 4.62 4.12 4.52 1.70 F prob. D L D x L .000 .005 .209 .001 .113 .858 .001 .009 .589 .000 .164 .943 .021 .593 .759 .003 .560 .042 .065 .055 .218 Conclusion C>F 2>1 C>F C>F 2>1 C>F C>F C>F Table XA. S t a t i s t i c a l analys is of t rout l i v e r glucose-6-phosphatase a c t i v i t y (ug phosphate released/10 m1nutes/g l i v e r ) in response to d i f f e r e n t d ietary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Transformation: log I0((arcs1n \lG-6-Pase a c t i v i t y ) + l ) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0 , 2, 4, 8, 10, 13 and 16 days Analysis of Variance D x L x T: Source x L x T Error df 1 1 1 6 6 6 6 128 ,-4 Mean square (xl0~ ) F value F prob. 27.713 1.927 1.043 31.106 5.147 .471 .573 .710 39.05 2.72 1.47 43.76 7.25 .66 .81 .000 .098 .226 .000 .000 .681 .568 Conclusion (Newman-Keuls mul t ip le range tes t ) F>C 13,10,16,8,4>2>0 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D L D x L Error 1 1 1 17 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 20 1 1 1 11 Mean square ( x l O - 4 ) D L D x L Error 11.159 .108 .385 .739 26.017 .006 .699 .653 6.421 .239 .015 .590 4.686 .003 .763 .634 2.326 2.405 .014 .666 4.703 1.316 2.239 .940 3.280 .676 .363 .785 F value D L D x L 15.11 .15 .52 39.82 .01 1.07 10.89 .41 .03 7.39 .01 1.20 3.50 3.61 .02 5.00 1.40 2.38 4.18 .86 .46 F prob. D L D x L .001 .706 .487 .000 .889 .314 .004 .538 .848 .013 .901 .286 .073 .069 .856 .035 .250 .135 .063 .377 .517 Conclusion F>C F>C F>C F>C F>C Table XIA. S t a t i s t i c a l analys is o f trout l i v e r glucose-6-phosphatase a c t i v i t y (ug phosphate released/10 minutes/ g l i v e r protein) in response to d i f f e r e n t d ietary treatments a f t e r d i f f e r e n t periods o f f a s t i n g . Analys is of Variance Transformation: log 10((arcs in ^G-6-Pase a c t i v i t y ) + l ) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8, 10, 13 and 16 days. D x L x T: Source df Mean square (xlO" ) F value F prob. Conclusion (Newman-Keuls mul t ip le range tes t ) D 1 16.734 7.02 .009 F>C L 1 5.234 2.19 .137 D x L 1 .244 .10 .745 T 6 59.957 24.98 .000 10,13,4,8,16>2,0 D x T 6 9.589 4.02 .001 L x T 6 2.070 .87 .522 D x L x T 6 1.144 .48 .824 Error 128 2.385 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 5.456 33.559 1.304 8.043 1.882 18.420 5.600 square L 2.574 1.164 .042 .856 8.332 3.062 1.624 D x L .951 .394 .848 1.897 .129 3.274 -.383 ^xiu ; Error 3.453 2.774 1.512 1.461 2.727 2.351 2.735 F value D 1.58 12.10 .86 5.51 .69 7.84 2.05 L .75 .42 .03 .59 3.06 1.30 .59 D x L .28 .14 .56 1.30 .05 1.39 - .14 F prob. D .224 .002 .367 .028 .421 .010 .178 L .404 .531 .845 .459 .092 .267 .463 D x L .612 .710 .469 .267 .813 .251 1.000 Conclusion F>C F>C Table XIIA. S t a t i s t i c a l analys is of trout muscle glycogen 1n response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Analys is of Variance Transformation: log 10((arcs in^muscle glycogen)+l) Diet (D) = high carbohydrate (C) versus high fa t (F) Feeding level (L) = s a t i a t i o n (2) versus h a l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: Source df Mean square (xlO~ 3 ) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) D 1 53.229 89.20 .000 C>F L 1 14.688 24.61 .000 2>1 D x L 1 8.858 14.84 .000 C-2>C-1>F-2,F- 1 T 6 11.447 19.18 .000 8,0,10,2>13>4, 16 D x T 6 3.047 5.11 .000 L x T 6 .795 1.33 .247 D x L x T 6 1.961 3.29 .005 Error 128 .597 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 16.118 25.823 4.305 21.562 2.497 .728 .479 square L 4.600 5.179 5.908 .404 3.324 .014 .030 D x L 6.575 5.026 7.062 1.282 5.467 .592 .084 ( x l O - 3 ) Error .356 .639 .594 .850 7.169 .690 .048 F value D 45.28 40.39 7.25 25.36 3.48 1.06 10.03 L 12.92 8.10 9.95 .47 4.64 .02 .64 D x L 18.47 7.86 11.89 1.51 .01 .86 1.76 F prob. D .000 .000 .014 .000 .074 .318 .009 L .002 .010 .005 .505 .042 .860 .447 D x L .001 .011 .003 .232 .892 .368 .209 Conclusion C>F C>F C>F C>F 2>1 C>F 2>1 2>1 2>1 Table XIIIA. S t a t i s t i c a l analys is of trout muscle protein in response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Analys is of Variance Transformation: log 10((arcsin*Jmuscle prote in)+l) Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus ha l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: Source df Mean square (xl0~ ) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) D 1 1.920 3.67 .055 L 1 .907 1.73 .187 D x L 1 .287 .55 .466 T 6 2.346 4.49 .000 16,10,4,13,8>10,4,13,8,2>13,8,2,0 D x T 6 1.120 2.14 .053 L x T 6 .515 .98 .439 D x L x T 6 .666 1.27 .273 Error 128 .523 D x L: Source Fasting time (days) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 5.183 .122 .404 .004 2.588 .285 .050 square L .078 2.474 .307 .034 .009 .634 .459 D x L .014 .089 .555 .427 1.025 1.812 .360 (xlO ) Error .391 1.117 .479 .507 .288 .487 .244 F value D 13.25 .11 .84 .01 9.00 .58 .20 L .20 2.21 .64 .07 .03 1.30 1.88 D x L .04 .08 1.16 .84 3.56 3.72 1.48 F prob. D .002 .740 .372 .892 .007 .459 .663 L .663 .149 .438 .787 .840 .267 .195 D x L .829 .772 .295 .373 .071 .065 .249 Conclusion F>C F>C Table XIVA. S t a t i s t i c a l analys is of trout muscle dry matter in response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods o f f a s t i n g . Analys is of Variance Transformation: log 1 0 ( ( a r c s i n J m u s c l e dry matter)+l) ' Diet (D) = high carbohydrate (C) versus high f a t (F) Feeding level (L) = s a t i a t i o n (2) versus h a l f s a t i a t i o n (1) Fasting time (T) = 0, 2, 4, 8 , 10, 13 and 16 days D x L x T: Source df Mean square (xlO ) F value F prob. Conclusion (Newman-Keuls mul t ip le range tes t ) D 1 .450 .42 .525 L 1 1.141 1.07 .304 D x L 1 .205 .19 .606 T 6 1.264 1.18 .318 D x T 6 3.068 2.87 .012 L x T 6 1.610 1.50 .179 D x L x T 6 1.476 1.38 .225 Error 128 1.067 D x L: Source Fasting time (days, ) 0 2 4 8 10 13 16 df D 1 1 1 1 1 1 1 L 1 1 1 1 1 1 1 D x L 1 1 1 1 1 1 1 Error 17 20 20 20 20 20 11 Mean D 7.617 .105 4.476 1.377 5.167 .005 .109 square L 3.377 .000 2.123 .001 1.289 3.791 .221 D x L 1.346 .696 .803 1.714 .662 3.388 .453 (xlO 3 ) Error .749 1.802 1.006 .962 .819 1.336 .490 F value D 10.17 .06 4.45 1.43 6.31 .00 .22 L 4.51 .00 2.11 .00 1.58 2.84 .45 D x L 1.80 .39 .80 1.78 .81 2.54 .93 F prob. D .005 .798 .046 .244 .020 .907 .650 L .047 .940 .158 .924 .220 .104 .522 D x L .195 .548 .386 .194 .383 .123 .359 Conclusion F>C O F F>C Table IB. S t a t i s t i c a l analys is of chick body weights 1n response to d i f f e r e n t d ie tary treatments a f t e r d i f f e r e n t periods of f a s t i n g . Analys is of Variance Transformation: log 10(body weight) Stra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8 , 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: ~~ ~ Source df Mean square (xlO" ) F value F prob. Conclusion (Newman-Keuls mul t ip le range tes t ) s 1 10397 7910 .000 B>W D 1 31.449 23 .93 .000 F>C S x D 1 9.774 7 .44 007 B-F>B-C>W-F, ,W-C T 9 84.356 64, .18 000 0>8,16>24,44,32>56>80>104,128 S x T 9 5.745 4 .37 .000 D x T 9 1.475 1, .12 347 S x D x T 9 .897 .68 .726 Error 267 1.314 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1. 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S 1033 1048 985 1030 1184 979 1126 1112 1257 696 square D 3.688 .595 2.833 .175 5.038 8.599 11.223 12.134 .429 .005 ( x l 0 " 3 l S x D 8.869 1.621 1.907 .040 .976 .203 .159 1.244 1.287 1.540 \ A I U ; Error 1.005 1.215 .387 1.323 1.595 1.349 2.225 1.074 1.656 1.313 F value S 1028 862 2544 779 742 725 506 1035 759 530 D 3.67 .49 7.32 .13 3.16 6.37 5.04 11.30 .26 .00 S x D 8.83 1.33 4.93 .03 .61 .15 .07 1.16 .78 1.17 F prob. S .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 D .063 .497 .011 .717 .083 .017 .031 .002 .620 .905 S x D .006 .257 .033 .839 .446 .701 .780 .291 .389 .296 Conclusion B>W B>W B>W B>W B>W B>W B>W B>W B>W B>W F>C F>C F>C Table IIB. Analys is of Variance Source S 0 S x D T S x T D x T S x D x T Error S t a t i s t i c a l ana lys is of chick l i v e r weights in response to d i f f e r e n t d i e t a r y treatments a f t e r d i f f e r e n t periods o f f a s t i n g . Transformation: log 10( l i ve r weight) S t ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fast ing time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours df 1 1 1 9 9 9 9 267 1014 1.392 -.108 17.883 .646 .254 .013 .199 F value F prob 5108 .000 7.01 .008 - .54 1.000 90.02 .000 3.25 .001 1.28 .249 .07 .999 Conclusion (Newman-Keuls mul t ip le range test) B>W C>F 0>8>16,24,32>24,32,44>56>80,104,128 df Mean square (xl0~ 3 ) F value F prob. Conclusion Source S D S x D Error Fasting time (hours) 1 1 1 28 1 1 1 28 16 1 1 1 28 24 1 1 1 28 32 1 1 1 28 44 1 1 1 28 56 1 1 1 28 80 1 1 1 28 104 1 1 1 28 128 1 1 1 15 S D S x D Error 1063 3.915 1.222 2.106 1139 15.797 .120 2.412 1105 3.109 .503 1.302 1126 8.002 .037 1.785 1197 1.222 .015 1.478 881 .004 .009 1.567 1079 U012 .205 2.523 973 .261 .553 2.030 1029 .'405 .636 2.271 612 4.011 -3.214 3.246 S D S x D 505 1.86 .58 472 6.55 .05 1070 3.01 .49 631 4.48 .02 810 .83 .01 562 .00 .01 428 .00 .08 479 .13 .27 453 .18 .28 188 1.24 - .99 S D S x D .000 .181 .459 .000 .016 .809 .000 .090 .498 .000 .041 .858 .000 .374 .884 .000 .914 .899 .000 .902 .769 .000 .721 .612 .000 .678 .607 .000 .284 1.000 B>W B>W C>F B>W B>W C>F B>W B>W B>W B>W B>W B>W I Table IIIB . S t a t i s t i c a l analys is of chick hepato-somatic Indices (HSI) in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analys is o f Variance Transformation: log 10((arcsin^HsT)+l) Stra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high fa t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df Mean square ( x l O - 4 ) F value F prob. Conclusion (Newman-Keuls mul t ip le range t e s t ) S 1 .263 1.51 .218 D 1 18.301 105.22 .000 C>F S x D 1 .381 2.19 .136 T 9 5.680 32.66 .000 0>8>32,44,16,24,104,128>44,16,24,104,128,56>128,56,80 S x T 9 .642 3.69 .000 D x T 9 .161 .93 .502 S x D x T 9 .119 .68 .726 Error 267 .174 S x D: — Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S .054 .435 .779 .391 .010 .521 .091 .872 2.328 .552 square D 3.568 2.187 2.577 2.144 2.366 1.725 2.385 1.696 .333 .746 (x lO" 4 ) S x D .828 .181 -.014 .067 .153 .033 .001 .018 .024 .117 Error .258 .235 .142 .149 .184 .176 .120 .119 .184 .177 F value S .21 1.85 5.50 2.63 .05 2.97 .76 7.31 12.66 3.11 D 13.83 9.30 18.19 14.43 12.87 9.82 19.91 14.21 1.81 4.20 S x D 3.21 .77 -.10 .45 .83 .19 .00 .15 .13 .66 F prob. S .653 .182 .025 .113 .805 .092 .394 .011 .001 .095 D .001 .005 .000 .001 .001 .004 .000 .001 .186 .056 S x D .081 .392 1.000 .514 .372 .669 .902 .701 .719 .434 Conclusion B>W W>B W>B C>F C>F C>F C>F C>F C>F C>F C>F 1 3 1 , 1 6 duSfe^SlrJ? fistic "™ ^ ^ P M p o n " t o d ^ t r e . t n . n t s a f t e r Analysis of Variance Transformation: log I0((arcs1n O l i v e r dry matter)+l) S t r a i n S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source x D T T D Error x T df 1 1 1 9 9 9 9 267 Mean square (xlO ) F value F 83.657 9.616 4.179 20.184 .773 1.243 1.090 .511 163.57 18.80 8.17 39.46 1.51 2.42 2.13 prob. Conclusion (Newman-Keuls m u l t i p l e range t e s t ) .000 .000 .005 .000 .143 .012 .027 W>B F>C W-F,W-C>B-F>B-C 104,128,80,56>44>32,24,0>24,0,16>16,8 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S D S x D Error 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 28 1 1 1 15 Mean square (xlO' 5) S D S x D Error 2.216 7.909 3.737 1.164 12.969 6.055 .814 .452 9.437 1.077 .046 .219 11.432 2.343 7.407 .747 7.502 .783 .036 .338 11.379' .020 1.362 .386 8.882 1.090 .111 .348 15.708 .003 .484 .440 10.207 .388 .002 .533 .879 1.129 -.014 .468 F value S D S x D 1.90 6.79 3.21 28.68 13.39 1.80 43.11 4.92 .21 15.31 3.14 9.92 22.18 2.32 .11 29.52 .05 3.53 25.51 3.13 .32 35.70 .01 1.10 19.16 .73 .00 1.88 2.41 -.03 F prob. S n .175 .014 .081 .000 .000 .001 .000 .000 .000 .000 .000 .188 .138 1.000 U S x D .001 .188 .033 .653 .084 .004 .136 .741 .802 .068 .084 .583 .891 .304 .405 .910 Conclusion F>C W>B F>C W>B F>C W>B W>B W>B W>B W>B W>B i Table VB. S t a t i s t i c a l analys is o f chick l i v e r glycogen (g/100 g l i v e r ) in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods o f f a s t i n g . Analys is o f Variance Transformation: log 10((arcs in O l i v e r glycogen)+l) S t ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8 , 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T f " ~ ~ 3 Source df Mean square (xlO" ) F value F prob. Conclusion (Newman-Keuls mu l t ip le range t e s t ) s 1 9.947 28.46 .000 B>W D 1 4.574 13.09 .001 C>F S x D 1 ,1.029 2.94 .083 T 9 48.301 138.20 .000 0>56,44>44,80,32>80,32,104>128,24>16,8 S x T 9 1.578 4.51 .000 D x T 9 1.487 4.25 .000 S x D x T 9 .476 1.36 .205 Error 267 .349 S x D: Source Fasting time (hours) 16 24 32 44 56 80 104 128 df S 1 1 1 1 D 1 1 1 1 S x D 1 1 1 1 Error 28 28 28 28 Mean S 72.664 .752 64.270 42.840 square D 87.866 4.194 168.831 195.371 , 1 n - 3 > S x D .811 17.317 4.201 19.063 U 1 U ; Error 7.706 .989 4.767 28.805 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 28 28 28 28 15 1.514 .471 9.134 2.833 70.974 38.085 26.526 3.339 1.752 .402 .103 1.101 6.645 19.214 6.046 1.056 .138 3.356 3.204 2.859 2.061 2.984 3.231 4.428 F value S 9.43 .76 13.48 D 11.40 4.24 35.42 S x D .11 17.52 .88 F prob. S .005 .395 .001 D .002 .047 .000 S x D .743 .000 .359 Conclusion B>W B>W C>F C>F C>F 1.49 .46 .16 4.43 .95 21.97 8.60 6.78 8.13 1.17 .85 .13 .03 .25 .66 2.04 6.72 2.93 .35 .04 .76 .231 .508 .689 .042 .340 .000 .010 .014 .008 .289 .367 .715 .837 .630 .428 .161 .014 .094 .564 .819 .402 B>W B>W B>W C>F C>F Table VIB. S t a t i s t i c a l analys is of chick l i v e r glycogen (mg/100 g body weight) in response to d i f f e r e n t d ietary treatments a f ter d i f f e r e n t periods of f a s t i n g . . Analys is o f Variance Transformation: log 10((arcs in O l i v e r glycogen)+l) S t ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8 , 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df Mean square (xlO' ) F value F prob. Conclusion (Newman-Keuls mul t ip le range tes t ) S 1 1.743 16, .18 .000 B>W D 1 2.499 23, .21 .000 C>F S x D 1 .392 3, .64 .054 T 9 18.001 167, .15 .000 0>56,44>44,32,80>32,80,104>128,24>16,8 S x T 9 .506 4.70 .000 D x T 9 .672 6.24 .000 S x D x T 9 .146 1.35 .209 Error 267 .108 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S 3.017 .005 .295 .173 .066 .052 .298 .043 2.252 .097 square D 5.027 .033 .911 .960 1.402 .040 .004 .001 .009 .158 / i r> *~ 3 \ S x D .118 .079 .007 .057 .321 .741 .234 .028 .007 .111 (xlO ) Error .339 .005 .021 .106 .132 .100 .072 .104 .122 .048 F value S 8.90 1.01 13.78 1.63 .50 .52 4.12 .42 18.44 .2.01 D 14.83 6.77 42.47 9.04 10.66 .40 .06 .01 .07 3.28 S x D .35 16.03 .34 .54 2.44 7.41 3.24 .27 .06 2.31 F prob. S .006 .325 .001 .211 .491 .484 .050 .530 .000 .174 D .001 .014 .000 .006 .003 .540 .801 .892 .782 .087 S x D .566 .001 .572 .475 .126 .011 .079 .612 .801 .147 Conclusion B>W B>W B>W C>F C>F C>F C>F C>F Table VIIB. S t a t i s t i c a l analys is of chick l i v e r protein (g/100 g l i v e r ) in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analysis o f Variance Transformation: log 10((arcs in O l iver prote in)+l) St ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high fa t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df Mean square (xlO" ) F value F prob. Conclusion (Newman-Keuls mul t ip le range test) S 1 4.509 64.92 .000 W>B D 1 1.437 20.68 .000 F>C S x D 1 .603 8.68 .004 W-F,W-C>B-F>B-C T 9 4.048 58.29 .000 128,104,80,56>56,44>24,32,16,8>0 S x T 9 .115 1.66 .098 D x T 9 .578 8.32 .000 S x D x T 9 .167 2.41 .012 Error 267 .695 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S 1.007 1.092 .498 .190 .000 .281 .443 .870 .899 .265 square D 4.729 .743 .076 .415 .099 .004 .129 .001 .432 .007 ( x K f 4 ) S x D .816 .421 .075 .639 .001 .091 .003 .062 .004 -.003 Error .100 .058 .043 .096 .044 .085 .062 .073 .080 .041 F value S 10.05 18.75 11.67 1.97 .00 3.33 7.14 11.90 11.31 6.49 D 47.20 12.76 1.77 4.30 2.27 .05 2.07 .02 5.44 .16 S x D 8.15 7.23 1.75 6.62 .02 1.08 .05 .85 .05 - .07 F prob. S .004 .000 .002 .168 .907 .075 .012 .002 .002 .021 D .000 .001 .192 .045 .139 .812 .158 .862 .026 .693 S X D .008 .012 .193 .015 .867 .308 .808 .369 .804 1.000 Conclusion W>B W>B W>B W>B W>B W>B W>B F>C F>C F>C C>F Table VIIIB. S t a t i s t i c a l analys is of chick l i v e r protein (mg/100 g body weight) in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analys is o f Variance Transformation: log 10((arcs1n O l i v e r prote in)+l) S t ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high fa t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df Mean square (xlO" '*) F value F prob. Conclusion (Newman-Keuls mu l t ip le range test) S 1 14.241 43.80 .000 W>B D 1 15.443 47.49 .000 O F S x D 1 .320 .98 .324 T 9 1.585 4.87 .000 104,128,8,0,44,32,56>128,8,0,44,32,56,80,24>32,56,80,24,16 S x T 9 1.565 4.81 .000 D x T 9 .596 1.83 .062 S x D x T 9 .278 .86 .566 Error 267 .325 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S 14.614 3.851 .040 .072 .024 29.077 16.731 64.770 122.510 30.691 square D 5.879 5.211 24.661 11.194 25.443 34.415 25.356 31.958 32.420 11.518 S x D .212 1.536 3.623 11.886 2.621 4.621 .004 .325 .785 2.646 (XlO ) Error 3.801 3.428 3.478 .287 2.785 3.659 3.399 2.895 3.221 2.757 F value S 3.85 1.12 .01 .25 .09 7.95 4.92 22.37 38.04 11.13 D 1.55 1.52 7.09 3.91 9.14 9.41 7.46 11.04 10.07 4.18 S x S .06 .45 1.04 4.15 .94 1.26 .00 .11 .24 .96 F prob. S .057 .299 .880 .626 .764 .009 .033 .000 .000 .005 D .222 .226 .012 .055 .005 .005 .011 .003 .004 .057 S x D .801 .516 .317 .049 .343 .270 .925 .737 .630 .960 Conclusion W>B W>B W>B W>B W>B C>F C>F C>F C>F C>F C>F Table IXB. S t a t i s t i c a l analys is of chick muscle dry matters in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analys is o f Variance Transformation: log 10((arcs in Jmuscle dry matter)+l) Stra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df Mean square (x lO" 5 ) F value F prob S 1 6.205 44.60 .000 D 1 .025 .18 .673 S x D 1 .003 .02 .861 T 9 2.624 18.86 .000 S x T 9 1.930 13.87 .000 D x T 9 .204 1.46 .161 S x D x T 9 .089 .64 .763 Error 267 .139 Conclusion (Newman-Keuls mul t ip le range test) B>W 0>16,8,32,44,24,56>32,44,56,80>104>128 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S .411 .375 .003 .010 .200 1.805 .549 1.211 11.034 7.975 square D .043 .042 .238 .434 .153 .503 .011 .017 .048 .367 ( x l O " 5 ) S x D .026 .054 .163 .002 .017 .014 .006 .005 .005 .513 Error .176 .078 .092 .095 .100 .162 .077 .203 .241 .194 F value S 2.34 4.79 .03 .11 2.00 11.11 7.17 5.97 45.79 41.16 D .25 .53 2.60 4.58 1.54 3.10 .15 .08 .20 1.90 S x D .15 .69 1.78 .02 .17 .09 .07 .02 .02 2.65 F prob. S .134 .035 .837 .743 .165 .003 .012 .020 .000 .000 D .628 .477 .114 .039 .223 .086 .705 .767 .663 .186 S x D .703 .417 .190 .868 .684 .763 .780 .853 .859 .121 Conclusion W>B B>W B>W B>W B>W B>W F>C Table XB. S t a t i s t i c a l analys is of chick muscle glycogen in response to d i f f e r e n t d ie tary treatments a f ter d i f f e r e n t periods of f a s t i n g . Analys is of Variance Transformation: log 10((arcs in Jmuscle glycogen)+l) St ra in (S) = b r o i l e r (B) versus White Leghorn (W) Diet (D) = high carbohydrate (C) versus high f a t (F) Fasting time (T) = 0, 8, 16, 24, 32, 44, 56, 80, 104 and 128 hours S x D x T: Source df -3 Mean square (xlO ) F value F prob. Conclusion (Newman-Keuls mul t ip le range) S 1 109.244 1078 .000 B>W D 1 2.391 23.59 .000 C>F S x D 1 -.057 -.56 1.000 T 9 2.077 20.50 .000 0>24,16,44,8,80,104,128>80,104,128,32>56 S x T 9 2.748 27.12 .000 D x T 9 1.198 11.82 .000 S x D x T 9 1.504 14.84 .000 Error 267 .101 S x D: Source Fasting time (hours) 0 8 16 24 32 44 56 80 104 128 df S 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 S x D 1 1 1 1 1 1 1 1 1 1 Error 28 28 28 28 28 28 28 28 28 15 Mean S 26.001 129.699 64.409 49.850 13.767 76.245 378.898 142.610 296.015 162.284 square D 4.537 53.294 .027 2.272 27.051 1.026 10.188 26.080 6.481 .181 S x D .796 31.141 1.213 .094 27.088 12.661 13.576 41.795 5.472 .669 ( X W ) Error .268 1.568 .477 .293 1.359 .905 .967 1.491 1.742 1.110 F value S 97.05 82.73 135.12 169.98 10.13 84.27 391.85 95.68 169.88 146.25 D 16.94 34.00 .06 7.75 20.35 1.13 10.54 17.50 3.72 .16 S x D 2.97 20.04 2.54 .32 19.94 13.99 14.04 28.04 3.14 .60 F prob. S .000 .000 .000 .000 .004 .000 .000 .000 .000 .000 D .000 .000 .799 .009 .000 .296 .003 .000 .061 .693 S x D .092 .000 .118 .583 .000 .001 .001 .000 .084 .454 Conclusion B>W B>W B>W B>W B>W B>W B>W B>W B>W B>W C>F C>F F>C C>F C>F F>C 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095237/manifest

Comment

Related Items