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Carbohydrate digestion in the chinchilla Smith, Diana Claire 1970

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CARBOHYDRATE DIGESTION IN THE CHINCHILLA by ... DIANA CLAIRE SMITH B.S.A.- U n i v e r s i t y o f B r i t i s h C olumbia, 1 9 6 7 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of A n i m a l S c i e n c e We a c c e p t t h i s t h e s i s as conforming t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1 9 7 0 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada ABSTRACT Carbohydrase activity in the chinchilla (Chinchilla  lanigera) was investigated from birth to post-weaning. Crude homogenates of the small intestinal wall and pancreas were prepared in order to study intestinal lactase, maltase and sucrase, and pancreatic amylase. The study was based on the assumption that, at different stages of growth, the carbohy-drase levels reflect the a b i l i t y of the animal to u t i l i z e specific carbohydrates. Small intestinal lactase activity was highest from birth to three weeks of age, at which time i t decreased sharply reaching the f a i r l y constant low levels found in the post-weaned animal by four weeks. Maltase activity at birth was appreciable, increas-ing significantly at four weeks of age and attaining maximum levels by twelve weeks of age. The adult animal retained this high activity. In contrast to maltase, sucrase activity was negli-gible at birth and did not increase significantly u n t i l five weeks of age, at which time a steady increase was noted to the adult levels attained by the'twelve week old animal. Pancreatic amylase was similarly negligible at birth. The highest increase in activity occurred between three and eight weeks at a time when intestinal lactase activity was decreasing. This also corresponded to the time of most rapid increase in maltase activity. The digestion of more complex carbohydrates was also investigated in the adult chinchilla. Cellulose, com-prising 18.7$ of a pelleted ration, was 54$ digestible, the main sites of cellulose breakdown being the cecum and large intestine. Total volatile fatty acids (VFA) throughout the alimentary tract of animals on a normal ranch ration of pellets and hay. were quantitated, as were the individual acids. The cecum and large intestine were the only sites of VFA production, while the low levels found in the stomach were attributed to coprophagy. ACKNOWLEDGEMENTS I wish to thank Dr. W. D. Ki t t s , Professor of Animal Science and Chair-man of the Department of Animal Science, for his direction and support of this study. I am also very grateful to my fellow students for their valuable assistance and criticism. TABLE OF CONTENTS Page I INTRODUCTION . . . . . . . . 1 I I LITERATURE REVIEW . 3 A. C h i n c h i l l a 3 1 . Growth 3 2 . D i g e s t i o n 4 3 . N u t r i e n t Requirements 5 B. D i g e s t i o n o f D i s a c c h a r i d e s 7 1 . Recent Advances 7 2 . Development w i t h Age . . . . . . . 14 -3. A d a p t a t i o n o f D i s a c c h a r i d a s e s . . . 19 C. D i g e s t i o n o f P o l y s a c c h a r i d e s 23 1 . D i g e s t i o n o f S t a r c h . 2 3 2 . D i g e s t i o n o f C e l l u l o s e 26 I I I GENERAL METHODS 32 A. M a t e r i a l s 32 B. Methods . 3 3 I V .. EXPERIMENTAL . . . " 34 P a r t A. Enzyme S t u d i e s 34 1 . D i s a c c h a r i d a s e s 34 (a) Methods and M a t e r i a l s (b) R e s u l t s and D i s c u s s i o n Page 2. Amylase .• • . . 47 (a) Methods and Materials (b) Results and Discussion Part B. D i g e s t i b i l i t y Studies . . . . . . . . . 53 1. Time of Passage and Site of Cellulose 53 Digestion (a) Methods and Materials (b) Results and Discussion 2. V o l a t i l e Fatty Acid Production . . . 6 l (a) Methods and Materials (b) Results and Discussion i V SUMMARY 67 VI BIBLIOGRAPHY 69 VII APPENDICES 73 1. Composition of C h i n c h i l l a P e l l e t s . 7f 2. Growth Curve of C h i n c h i l l a lanigera 3. Organ Weights as Per Cent of Live Body Weight at Various Ages i n the Ch i n c h i l l a •. . . . .' . . . • & X LIST OF TABLES Table ' Page I Weights of C h i n c h i l l a lanigera 4 II Concentrations of VFA i n the ceca of several species of animals 31 I I I A c t i v i t y of maltase, sucrase and lactase i n the alimentary t r a c t of the c h i n c h i l l a 39 IV Amylase a c t i v i t y i n the c h i n c h i l l a . . . . . . . . 51 V Time of passage of a pelleted feed through the digestive t r a c t of the c h i n c h i l l a 57 VI Cellulose d i g e s t i b i l i t y and VFA production i n the alimentary t r a c t of c h i n c h i l l a fed a commercial c h i n c h i l l a p e l l e t 60 VII VFA's i n the digestive t r a c t s and feces of four adult c h i n c h i l l a fed a l f a l f a hay and a commer-c i a l c h i n c h i l l a p e l l e t 64 VIII Dry matter and c e l l u l o s e d i g e s t i b i l i t y of a commercial c h i n c h i l l a p e l l e t with and without a l f a l f a hay • 65 LIST OF FIGURES Figure Page 1 Outline of carbohydrate digestion and absorption in man 9 2 Lactase activity in intestinal mucous membranes from normal and scouring calves . . . 13 3 Optimum pH of intestinal lactase, sucrase and maltase in the chinchilla 43 4 Intestinal lactase, sucrase and maltase activity as a function of age in the chinchilla. 4 6 5 Pancreatic amylase activity as a function of age in the chinchilla 52 6 Time of passage of a pelleted feed through the digestive tract of the chinchilla 5$ I INTRODUCTION C h i n c h i l l a have been domesticated f o r nearly f i f t y years and t h e i r economic importance as a luxury f u r animal i s now well established. D i s t i n c t i v e features of the f u r enabling the c h i n c h i l l a to compete successfully with other f u r bearers such as mink, sable and fox, are i t s extreme softness of texture and colour. However, f o r the c h i n c h i l l a to remain i n the competitive market, research i s es s e n t i a l to constantly improve f u r quality and e f f i c i e n c y of produc-t i o n . I t has been said that l e s s i s known about the n u t r i t i o n of c h i n c h i l l a than any other aspect of t h e i r pro-duction, and i t i s very apparent that research i s lacking i n t h i s area. Nutrient requirements f o r maintenance, growth, and production have not been determined, so that the growing animal, the pregnant or l a c t a t i n g female, and the pelter are a l l fed the same d i e t , while t h e i r respective requirements are bound to d i f f e r . This means not only that the animal's po t e n t i a l f o r growth and production i s not expressed, due to n u t r i t i o n a l d e f i c i e n c i e s , but also that at r e l a t i v e l y unpro-ductive stages some animals are being fed more than t h e i r requirements, which i s obviously not economical. Before i n -vestigating the energy requirements, i t v/ould help to know more about the digestive processes i n the c h i n c h i l l a , and the type of feedstuffs this animal can u t i l i z e most efficiently for energy production. With this in mind, i t was decided to concentrate on certain features of carbohydrate digestion. Growth of the animal i s associated with corres-ponding changes in the anatomy and physiology of the diges-tive system. These changes create limits on the amount and type of feed that the animal can consume and u t i l i z e . On the assumption that the carbohydrase acti v i t i e s in the i n -testine reflect the a b i l i t y of the animal to u t i l i z e various carbohydrates, the f i r s t experiment was carried out to determine the activities of specific carbohydrases from birth to post-weaning. These were intestinal sucrase, maltase, and lactase, and pancreatic amylase. Since fibre has been shown to be very important in the diet of the chinchilla ( 6 9 ) , both as a nutritive and as a bulk source*, the second experiment was conducted to deter-mine the extent of fibre digestion of a commercial chinchilla ration, with and without a l f a l f a hay, and to quantitate the utilizable end-products, namely volatile fatty acids. Time of passage of feedstuffs through the chinchilla digestive tract was determined before doing the d i g e s t i b i l i t y study, i t being an important factor affecting the efficiency of diges-tion and uti l i z a t i o n of carbohydrates. The purpose of this study was therefore twofold: f i r s t , to measure changes in carbohydrase levels with growth of the chinchilla; and, second, to determine the site and end-products of cellulose digestion. II LITERATURE REVIEW A. CHINCHILLA Chinchilla were practically unknown in North America before 1923 when eleven animals were introduced by an American mining engineer returning from the Andes (59)-Forty years later, in 1967, British Columbia alone had 140 ranches with approximately 16,000 animals (91) • Despite the rapid increase in numbers, there i s l i t t l e s c i e n t i f i c information available on the chinchilla. 1. Growth Young chinchilla are born f a i r l y mature, possess-ing hair and teeth and open eyes, and are usually weaned at sixty days (2). Weights of Chinchilla lanigera from birth to one year of age are given by Bickel (15)• Chinchilla  brevicaudata, a short-tailed, blockier type of lesser im-portance in North America, are ten to fifteen grams heavier than those described by Bickel (59). TABLE I (Bickel ( 1 5 ) ) Weights of Chinchilla Lanigera Age Weight (g.) (months) Maximum Minimum Mean at birth 57 3 5 4 4 1 2 0 0 8 5 1 4 5 2 3 2 6 1 8 5 2 5 5 3 4 2 5 2 9 0 3 4 0 .4 4 8 9 3 1 2 3 9 9 5 5 2 5 3 4 0 4 3 8 6 * 6 5 0 3 6 9 5 5 6 1 2 7 2 0 3 9 7 5 5 6 The average weight of normal adult males on a ranch diet in the United States i s about 5 0 0 grams, with females weighing slightly more. Somewhat lower weights were reported for 6 7 chinchilla by Larrivee and Elvehjem ( 6 9 ) . Most of the animals began to level off in weight at approximately nine to eleven months of age, and the range at maturity was from 3 2 0 to 4 6 0 grams for males, and 3 5 0 to 5 0 0 grams for females. 2 . Digestion The chinchilla i s an herbivorous animal with a large cecum. In the wild state, chinchilla live mainly on grasses, seeds, and the bark of trees, while most captive animals are fed a pelleted ration and hay, sometimes supplemented with green feed. Farmer ( 4 4 ) in 1 9 5 7 found that commercial rabbit pellets as the sole diet of chinchilla during pregnancy and lactation were satisfactory, but Bickel ( 1 5 ) - i n I 9 6 2 advo-cated the use of hay, cereals and fresh green feed, claim-ing they were less l i k e l y to cause digestive disturbances than pellets. According to K i r i s and Barantseva ( 6 6 ) in a report on acclimatisation of chinchilla in the U.S.S.R., hay constitutes two-thirds of the diet and, to a great extent, determines the quality of the fur. 3. Nutrient Requirements No conclusive studies could be found on the pro-tein and energy requirements of chinchilla, but some work has been reported on vitamin and roughage requirements. Using guinea pig diets as a guide, Larrivee and Elvehjem ( 6 9 ) devised four synthetic rations varying in roughage and salt content which were fed to sixty-seven chinchilla. The control ration was a commercial chinchilla pellet. Roughage, either cellulose (solka floe) or gum arabic, at the 1 5 $ level was insufficient and caused constipation, but this was par-t i a l l y corrected at 20% levels of gum arabic and completely eliminated at 20% levels of cellulose. The ration contain-ing 20% cellulose supported apparently normal growth of both young and mature chinchillas for periods of fifteen weeks or longer. Previously, King and Orcutt ( 6 5 ) demonstrated that vitamin C was not necessary for the growing chinchilla, an observation confirmed by Larrivee and Elvehjem ( 6 9 ) in 1 9 5 4 * Withdrav/al of v i t a m i n caused a l o p e c i a and l a c k of co-o r d i n a t i o n . The minimum d a i l y requirement f o r weanling c h i n c h i l l a was found t o be between 0.1 and 0.4 mg. There i s evidence, too, that the c h i n c h i l l a does not need to be given r i b o f l a v i n (70). Animals on a p u r i f i e d d i e t , con-t a i n i n g 0.39 mg r i b o f l a v i n per g, showed no signs of de-f i c i e n c y , and grew as w e l l as those given a supplement to r a i s e the r i b o f l a v i n content of the d i e t t o 14.4 mg per g B. DIGESTION OF DlSACCHARIDES 1. Recent Advances This topic and the ones following .will be dis-cussed with reference to work done with other animals, since no such work using chinchilla has been reported. An understanding of the physiology of digestion and absorp-tion of disaccharides has increased rapidly in the last ten years, and some of the most significant advances have been outlined in several review articles (51, 86, 3 2 ) . (a) Site of oligosaccharide hydrolysis. The theory of intraluminal digestion implying that intestinal disaccharides, such as sucrase, maltase and lactase, are secreted into the intestinal lumen and hydrolyse ingested sugars in the succus entericus has been disproved. Several workers have demonstrated that the hydrolytic activity measured in intestinal contents was far too low to account for the observed rate of digestion of disaccharides (18, 38, 3 1 ) . Also, results obtained in animal studies, using sacs or rings of everted small intestine, have shown that the disacchari-dases are not released into the incubation medium, but exert their action in the small intestinal wall ( 1 6 ) . According to Semenza ( 8 6 ) , evidence for this was actually reported as early as 1880 by Brown and Heron, who showed that the sucrase and maltase activities were higher in the intact gut than in ex-tracts from the intestinal juice. The observation that these enzymes are apparently bound to some structures of the tissue was confirmed later by a number of workers at the turn of the century. It has now been established with l i t t l e doubt that disaccharidases are located in the brush borders of cylindrical cells ( 8 6 ) . Miller and Crane (75) in 1961 isolated brush borders having 90;S of sucrase and lactase associated with them. For a time thereafter, i t was believed that the dietary disaccharides entered the intestinal c e l l prior to hydrolysis, but there i s recent evidence that hydrolysis occurs on the outside of the intestinal membrane at a site exterior to the permeability barrier of the c e l l ( 2 2 ) . No specific mechanism of entry into the intestinal wall has been found for disaccharides, a requirement i f they are to be hydrolysed intracellularly ( 5 1 ) . It has been postulated that the glycocalyx, the outer fuzzy coat of the brush bor-der covering the mi c r o v i l l i , serves as a partial diffusion barrier for the entry and exit of sugars, so that the bulk of monosaccharides released upon hydrolysis at the brush border, rather than diffusing back through the glycocalyx to the lumen, i s preferentially taken up by the intestinal c e l l Gray (51) concluded that the hydrolytic enzymes are located both in the glycocalyx and in the micro-villar membrane i t -self* so that they may bind and hydrolyse disaccharides coming in contact with the c e l l surface. Probably the most plausible answer to the sometimes discrepant theories reported, i s that provided by Crane (22) He suggests an overall digestive-absorptive function that i s not separated physiologically into the two different functions of digestion and absorption. Instead, the ce l l i s probably organized so that sequential reactions of hydrolysis and transport are physically in close proximity, making i t possible for the products of hydrolysis to become the substrates for transport before there i s a chance for them to diffuse away. In support of this theory, i t has been shown that the membrane transport systems allow much better absorption of the monosaccharide products of brush border hydrolases than that of free monosaccharide added to the lumen. An outline of the intraluminal and brush border digestion of carbohydrates in man i s given in Figure 1 . FIGURE 1 (Gray ( 5 1 ) ) Outline of Carbohydrate Digestion and Absorption in Man Intraluminal Starch- Maltose » Maltotriose-amylase D e x t r i n s . . Lactose Sucrose Intestinal Brush Border maltase maltase _ ^ G l u c o s e dextrinase^, lactase < sucrase Galactose, Glucose Glucose Fructose Cell  Transport (b) Characterization of Intestinal Disaccharidases. A second significant advance in the last few years has been the characterization of several specific disaccharide s p l i t -ting enzymes. Evidence has been obtained for the existence of four or five maltases ort the basis of heat stability and kinetics of mutual inactivation by various substrates ( 2 8 ) , but the techniques used have been questioned. Dahlqvist and Telenius (33) have suggested that one of the two human suc-rases isolated may be an artifact produced by biochemical purification. However, several workers have separated and characterized at least two lactases in human intestine, only one of which has the characteristics of a digestive enzyme. This lactase, with a pH optimum of 6 . 0 and a specificity for lactose, i s markedly depressed or absent in man with lactose intolerance. The second lactase has a lower pH optimum and has a specificity for both lactose and synthetic substrates. Although i t i s present at normal levels in man with lactose intolerance, i t does not appear to be of any importance in the digestion of dietary lactose ( 5 0 ) . There i s also an enzyme with activity against 1,6-CX-glucosidase links which is usually called isomaltase ( 5 1 ) . Gray suggests a better name would be ©<(-dextranase, since the only substances i t acts on are the c<-dextrins released by the action of amylase on amylopectin. Isomaltose, the disaccharide with a 1,6-tx. link i s readily hydrolyzed by <X-dextranase,- but i s not a physiological substrate present in the intestine. (c) Deficiency of S p e c i f i c Disaccharidases. C l i n i c a l conditions have recently been recognized i n which the l e v e l s of a c t i v i t y of a s p e c i f i c disaccharidase are i n -adequate to digest the usual dietary intake of that disac-charide. These conditions can either be congenital defects, or acquired conditions r e s u l t i n g from an underlying disorder with loss or damage of i n t e s t i n a l tissue (32). (i) Acquired forms. I n t e s t i n a l lactase i s absent i n adults of most mammals except man, who usually retains measurable l e v e l s of a c t i v i t y . But some do lose t h e i r lactase and therefore cannot t o l e r a t e lactose or milk. According to Dahlqvist (32), these people have a history of normal milk tolerance during infancy, and develop the condition as a sequel to some kind of damage to the mucosa. Lactase a c t i -v i t y has a tendency to be more severely affected by i n t e s t -i n a l disease conditions than the other disaccharidases, and also to return more slowly to normal values. Many reports describing t h i s condition i n man have appeared, i t being present i n 5-10% of the adult white population i n the United States and 70-100$ of Negroes and Orientals (13). Cuatre-casas (24) fed 150 grams of lactose per day to fourteen sub-jects f o r three months and was unable to change lactase l e v e l s i n e i t h e r normal or lactase d e f i c i e n t persons. Gen-e r a l disaccharidase deficiency i s found in diseases such as g i a r d i a s i s , c y s t i c f i b r o s i s , coeliac disease, e n t e r i t i s , and other malabsorption states associated with, blunting of the m i c r o v i l l i (24)• In animal studies, calves dying after scouring (seven days of age) had lower lactase activity in the upper small intestine than did healthy calves (19)' This d i f f e r -ence in activity can be seen in Figure 2 . Radotstits (7$) in 1965 noted a flattened appearance of intestinal epithe-l i a l c ells from scouring calves and suggested that these may be impaired in their a b i l i t y to digest and absorb nutrients. When the lactase in the intestine i s inadequate to cope with the lactose in the diet, undigested lactose i s fermented by micro-organisms in the lower part of .the intestine, result-ing in fermentative diarrhoea which aggravates any existing diarrhoea (19). This i s in keeping with the findings of Blaxter and Wood (17) who showed that scouring animals had a lowered fecal pH and a raised level of fecal fatty acids typical of an abnormal carbohydrate fermentation, ( i i ) Congenital forms. In this condition the small intes-t i n a l mucosa is normal and the symptoms rapidly disappear on a lactose free diet. The disease is believed to be heredi-tary, the deficiency remaining throughout l i f e but the symptoms growing milder with time. Durand (42) found the condition to start in infancy with severe diarrhoea, malnu-t r i t i o n , f l a t lactose tolerance curve but normal galactose plus glucose tolerance, and acid feces containing large amounts of lactose and l a c t i c acid after ingestion of lactose. Newborn human babies sometimes have d i f f i c u l t y digesting the large amounts of lactose ingested, but this should not be mistaken for a deficient condition (32). Aurrichio and co-workers (8) report that lactase activity in man.and other animals develops late in foetal l i f e and seems to increase somewhat after birth. FIGURE 2 (Bywater (19)) Lactase Activity in Intestinal Mucous Membrane from Normal and Scouring Calves Vertical bars represent S.E.M. -p •H > •H •P O <: <D CO as •P o CO i-3 10 8 0 normal scouring 20 duodenum 40 60 80 100 i.c.v. Distance from duodenum to ileo-cecal valve {%) Congenital sucrpse-isomaltose intolerance i s another inheritable condition, the two enzymes always being absent or inactive simultaneously. Maltase activity i s also reduced but lactase remains the same. Symptoms appear only when sucrose, isomaltose and related oligosaccharides containing the same °<-l,6 bonds as form the branch links of starch are in the diet. Although the condition has been found in only a very few people, i t has incited considerable interest since i t is transmitted by a single recessive auto-somic genetic factor. According to. Semenza ( S 6 ) this i s one of the f i r s t examples, i f not the f i r s t , in human genetics of a single genetic factor controlling more than one enzyme. 2. Development With Age Disaccharidase activity during the pre-weaning stage of growth has been studied in recent years to a con-siderable extent. Although factors controlling the develop-ment of enzyme activity are not clearly understood, i t appears that the disaccharidases develop in a way that allows u t i l i -zation of the carbohydrates that the animal i s lik e l y to encounter under normal feeding conditions Thus, in most young suckling mammals, ^ -galactosidase activities in the small intestine are high, allowing efficient u t i l i z a t i o n of the lactose in the dam's milk. One major exception to this i s the Californian seal, the small intestine of which does not contain lactase. Correspondingly, seal milk con-tains no lactose ( 6 8 ) . After weaning, the lactase activity increases. This i s consistent with the change in carbohydrate in the diet, although i t i s not indicative of dietary con-t r o l of disaccharidase activity. Attempts to induce activity by feeding specific disaccharides have generally been incon-clusive (88). Studies with rats, rabbits, calves, and humans have shown that the small intestinal disaccharidases are formed long before they are normally required for the diges-tion of disaccharides (88, 8, 37). Lactase and sucrase have been demonstrated in two to three month old human foetuses by Auricchio and co-workers (8), but in the chick, marked increases in activity occurred only during the last few days of embryonic development (88). This i s the time of elonga-tion and increase in number of the small-intestinal v i l l i . Similarly in the rat,^-galactosidase activity i s present at day eighteen of gestation, the period of rapid differentia-tion of intestinal cells (37). Siddons (88) therefore suggests that the development of enzyme activity reflects morphological changes in the developing intestine. The i n -crease in lactase activity before birth in rats and rabbits xvas also correlated with a rise in the level of lactose in the blood. Doell and Kretchmer (37) surgically removed the mammary glands of pregnant rabbits, but were unable to prevent the rise in lactase activity. Koldovsky et a l . (67) studied extensively the post-natal changes of @-galactosidase acti v i t i e s in the mouse, rat, guinea pig and rabbit. They had previously identified two ^-galactosidases which differed in their a f f i n i t y for lactose and a r t i f i c i a l substrates, and also in their distribution along the digestive tract. The ratio of these two enzymes during development changed in the ileum and jejunum in the rat, mouse, and rabbit, but activity developed similarly in both parts of the small intestine in the guinea pig. Huber et a l . (6l) also found a changing relationship in the calf; lactase activity in the ileum decreasing twenty times post-natally while the decrease in the jejunum was only by 10$. Higher lactase levels in the anterior than in the posterior areas of the small intestine of calves have also been reported by Heilskov (55). Kidder et a l . (64) measuring the absorption of various sugars by the piglet as a means of determining nutritive value, found that most of the sugar disappeared in •the f i r s t half of the small intestine. Since i t is established that disaccharides are mainly s p l i t by enzymes located within or on the surface of the mucosa (75), the absorption of sugars might be expected to correspond to mucosal enzyme concentra-tions (64). In the four species studied by Koldovsky (67), galactosidase activity decreased during postnatal development as expected. In rats, the decrease was f a i r l y sudden between the fourteenth and tv/enty-first days. However, the decrease was almost negligible in guinea pigs, which i s in agreement with the findings of De Groot and Hoogedoorn (34). Koldovsky (67), relates the level of postnatal activity to the degree of maturity of the animal at birth. M i c e , r a t s , and r a b b i t s a r e born immature and weaning o c c u r s between t h e second and t h i r d p o s t n a t a l week a t a time when - g a l a c t o s i d a s e a c t i v i t y d e c r e a s e s . The i n s i g n i f i c a n t changes i n l a c t a s e a c t i v i t y i n the g u i n e a p i g might t h e r e -f o r e be due t o t h e c o m p a r a t i v e l y l o w e r consumption o f m i l k , and t h u s o f l a c t o s e . S i m i l a r l y i n t h e c a l f , an a n i m a l b o r n r e l a t i v e l y mature, Huber ( 6 l ) found i n t e s t i n a l l a c t a s e t o be h i g h e s t a t one day o f age and t o d e c r e a s e t h e r e a f t e r . L a c -t a s e a c t i v i t i e s i n young p i g s have a l s o been o b s e r v e d t o de-c r e a s e w i t h age, but a t b i r t h , t h e a c t i v i t i e s were a p p r o x i -m a t e l y f i v e t i m e s as h i g h p e r u n i t body weight as t h o s e o f one day o l d c a l v e s ( 5 5 , 5 4 ) . Siddons ( 8 8 ) , i n 1968, s t u d i e d i n t e s t i n a l d i s a c c h a r i d a s e s i n the c h i c k . The a c t i v i t i e s o f m a l t a s e , s u c r a s e , and l a c t a s e a l l i n c r e a s e d from one t o f o r t y - t h r e e days o f age, but t h e l a c t a s e a c t i v i t y was con-s i d e r a b l y l o w e r t h a n t h a t o f m a l t a s e o r s u c r a s e . The newly h a t c h e d c h i c k may be l i k e n e d t o t h e weaned mammal i n t h a t most o f t h e c a r b o h y d r a t e i n i t s d i e t i s i n t h e f o r m o f s t a r c h . T h i s , a c c o r d i n g t o S i d d o n s , would e x p l a i n t h e h i g h m a l t a s e a c t i v i t y , w h i l e t h e h i g h s u c r a s e may be due t o non-s p e c i f i c « X - g l y c o s i d a s e s . These enzymes are c a p a b l e o f hydro-l y z i n g b o t h m a l t o s e and s u c r o s e t o some e x t e n t ( 7 ) . I n t h e dog, l a c t a s e a c t i v i t y was h i g h e s t i n the young a n i m a l and d e c r e a s e d t o t h e l e v e l f o u n d i n t h e a d u l t dog by t w e n t y - n i n e t o s i x t y - o n e days o f age. Sucrase and m a l t a s e a c t i v i t i e s were l o w e r i n young t h a n a d u l t a n i m a l s ( 9 5 ) . Rubino e t a l . ( 8 4 ) , reporting on intestinal disaccharidases in adult and suckling rats, noted that sucrase was absent at birth but -activity appeared at fifteen or sixteen days and developed rapidly to adult levels around the twentieth day. Maltase was present at very low levels at birth, increasing with the appearance of sucrase at sixteen days. Lactase was very high at birth and diminished more gradually than previous reports had indicated (67) to adult levels. Siddons (6*7) found lactase act i v i t i e s to be highest in the four day old calf, decreasing up to forty-three days but changing l i t t l e between forty-three and 1 4 4 days. A further decrease bet-ween the 1 1 4 day old calves and the adults was noted. Again the decrease was more marked in the distal sections of the small intestine. Maltase was very much lower than the lac-tase activity and did not change with age. Sucrase was not detected in any of the calves or in the adults. Huber et a l . ( 6 1 ) also found that intestinal maltase of the calf was i n -dependent of age. According to Siddons ( 8 7 ) , conflicting reports of other workers indicating increasing activity with age, could be due to an increase in the total intestinal size. The absence of sucrase in the calf as well as the adult cow and sheep has been reported by several workers. Calves are unable to u t i l i z e fructose without getting severe diarrhoea (93). This i s in sharp contrast to results reported for pigs, where high levels of sucrase were present after two weeks of age (9), and sucrose was well uti l i z e d at ten days of age ( 4 1 ) . •*-y 3. Adaptation of Disaccharidases The observed change in lactase activity corres-ponding to a decreased lactose intake at weaning, has led many workers to investigate the causal relationship. Wein-land (94) in 1899 reported that lactase remained high even after weaning i f lactose v/as added to the diet. PIiminer (77) reviewed early experiments on lactase adaptability in 1906 and concluded that the intestine was incapable of adapting to feedstuffs. More recently several workers have failed to prevent the decrease in lactase at weaning by feeding lactose (5) . Doell and Kretchmer (37) in 1962 were unable to prevent the lactase decrease in infant rats by intraperitoneal administrations of lactose. However, the experiments of Koldovsky (67) have shov/n that the diet can have some effect on lactase levels, although i t i s not the only decisive factor. Rats were weaned to a standard diet or to an experimental diet containing either lactose or glucose plus galactose as the only carbohydrate source. The rats fed the lactose diet had a higher intestinal lactase activity at nineteen or twenty days of age than the rats fed the glucose galactose mixture. In both control and experi-mental animals, there was a f a l l in activity between the fifteenth and nineteenth day, but the f a l l v/as less pro-nounced in the lactose fed group than in the group fed glucose plus galactose, which showed similar activity to the normally fed animals. Koldovsky therefore concluded that under certain conditions the diet could have an effect on the usually occurring changes in lactase activity, but that other factors must be involved since the f a l l in a c t i -vity also occurs i f the lactose diet i s fed. Siddons (6*7) reported no marked differences in the carbohydrase ac t i v i t i e s of the intestinal mucosa of four month old calves that had been fed solely on milk, and calves of the same age that had been given a concentrate-hay diet from six weeks of age. Although Fischer (45) reported large amounts of lactase activity in lactose fed rats, this was due to an i n -crease in weight of the intestinal mucosa; the specific activity did not increase. Recent work in both rat and man has shown that dietary carbohydrate can play a role in the regulation of sucrase and maltase a c t i v i t i e s , and this i s reflected by chan ges in rates of sucrose hydrolysis in vivo. Both sucrose and 1 fructose feeding produced an increase in intestinal sucrase activity within two to five days, a time comparable to that required for crypt cells to migrate up to the v i l l u s tip (82) This suggested that change in disaccharidase act i v i t i e s was due primarily to an effect on the crypt c e l l . Feeding of glucose did not produce an increase in the enzymes and since sucrose is hydrolyzed to glucose and fructose, i t was suggested that fructose was the only sugar in the diet that could increase sucrase or maltase a c t i v i -ties ( 5 1 ) . Reddy et a l . ( B l ) , i n 1968, studied the effect of dietary carbohydrates on certain enzymes i n germ-free and conventional r a t s . In a l l treatments, germ-free rats showed higher disaccharidase l e v e l s than conventional rats, and changes i n maltase and•sucrase l e v e l s r e s u l t i n g from feeding the respective disaccharides, were shown to occur independently of the i n t e s t i n a l microflora. Since diet alone was not able to account f o r the normal changes seen i n i n t e s t i n a l enzyme a c t i v i t i e s , other factors were investigated. Cortisone caused precocious development of sucrase i n developing rat inte s t i n e when administered to three and nine day old rats, but i t did not appear to influence the a c t i v i t y of lactase. This suggested that cortisone did not act solely by hastening the normal maturation process. Deren et a l • (36) adrenalectomized adult rats and found sucrase and maltase l e v e l s comparable to those of con-t r o l s . Steroid administration did not s i g n i f i c a n t l y increase t h e i r a c t i v i t i e s . The response to sucrose feeding was simi-l a r i n both control and adrenalectomized rats, i n d i c a t i n g the absence of s t e r o i d a l control on sucrase and maltase a c t i v i t y i n the adult animal. In contrast to the experiments of Doell and Kretch-mer (39), who found no e f f e c t of cortisone on lactase a c t i v i t y , Koldovsky (67) was able to demonstrate a d e f i n i t e e f f e c t . He explained the discrepancy on the basis of e a r l i e r work i n 12-which he showed that adrenalectomy and the application of cortisone both had had very irregular effects on rats aged five to nine days, the approximate age of Doell's rats. • It i s generally accepted now that adrenalectomy of the young developing animal results in the maintenance of enzymatic activity at a level that corresponds to a younger developmental stage. In other words, i t prevents the usual rise in sucrase and maltase, and inhibits the decrease in lactase activity (67). C. DIGESTION OF POLYSACCHARIDES 1 . Digestion of Starch Ingested.starch is composed of two types of glu-cose polymers, amylose and amylopectin. Salivary and pan-creatic 0<-arnylases attack the interior 1 ,4-<Xgluccsidic bonds, apparently at random, but have l i t t l e or no speci-f i c i t y for the outermost links of native or partially digested amylose. Therefore, the end products of amylase action on amylose are maltose and maltotriose. Maltotetrose, having a single interior l , ^ . - ^ link, i s the smallest subs-trate which can be rapidly hydrolyzed by ©(-amylase. Amylopectin has a 1 , 6 - c * branching point approxi-mately every twenty-five glucose units along the chain, in addition to the l , 4 - ° < l i n k e d straight chain of glucose molecules. Again, the ©t-amylases are capable of hydrolyzing only the interior 1 , 4 - ° < -glucose bonds, show no specificity for the 1 ,6 - c x . branching points and possess markedly decreased activity against the 1 , 4 bonds adjacent to these branching points ( 5 1 ) . Therefore, the f i n a l products of amylopectin digestion are maltose, maltotriose and a mixture of limit dextrins. The smallest <x-dextrin i s a pentasaccharide. Because of the low or absent specificity of CK-amy-lase. for the outermost 1 , 4 links, no glucose i s formed under physiological conditions of relatively short exposure of sub-strate to enzyme in the intestine. Also, contrary to the previous view, isomaltose i s not formed at a l l ( 8 6 ) . The biosynthesis of pancreatic amylase is under alimentary and hormonal control (86). Fasting reduces the levels of amylase activity, while animals on a high glucose or starch diet produce much more pancreatic amylase than controls on a protein rich diet (74) • Semenza (8"6) suggests that the stimulus for amylase synthesis i s insulin, a theory supported by the fact that alloxan-diabetic animals produce less amylase than normal until treated with insulin. The chain of events in this case would be: Absorption of glucose—>hyperglycemia—& insulin secretion —> increased amylase synthesis (80). The fact that pancreatic amylase i s secreted into the intestinal lumen and that i t s activity i s high in intes-t i n a l fluids has long been accepted as evidence that the enzyme acts on starch while both are free in the intestinal cavity. However, Ugolev and co-workers (92) found that pan-creatic amylase can be adsorbed onto the intestinal mucosal surface and act at this site. As a result of the work of Dahlqvist and Thompson (29), i t i s known that i n t r i n s i c and adsorbed pancreatic amylase are present in intestinal mucosal homogenates. Dahlqvist (29) separated two amylases from the mucosa, one that could not be distinguished from pancreatic amylase and was probably adsorbed amylase, and the other having quite different properties. The latter attacked maltose as well as starch, with glucose as the end product, and was similar to an amylase found in other organs such as the l i v e r and kidney. The results indicated that although i n t e s t i n a l mucosa does possess amylase a c t i v i t y , the bulk of digestion probably occurs by the action of enzyme free within the i n t e s t i n a l f l u i d (27). I t i s dubious whether mucosal membrane digestion of starch i s ever of physiolo-g i c a l importance (51).. Amylase a c t i v i t y i n s a l i v a and i n the pancreas i s low i n suckling animals during the f i r s t few post-natal days (67). In the young dog, i t decreases about the second week and. then again at about the s i x t h week when weaning occurs. S i m i l a r l y i n the rat, a c t i v i t y increases between the second and t h i r d week. The percentage increase in a c t i v i t y varies with d i f f e r e n t reports and t h i s i s pro-bably due to considerable amylase adaptation to the amount of amylose i n the diet (95). Cunningham (25) showed that the newborn p i g l e t can deal well with dissolved starch, but not with rough starch. By f i v e days of age, digestion of either was equally e f f i -c i ent, although addition of amylase did not a f f e c t the digestion of rough starch i n newborn p i g l e t s . De Laey (35) reported that the amylase a c t i v i t y i n washings of the small i n t e s t i n e increases l i n e a r l y with age i n the rat, while the so-called contact digestion described by Ugolev (92) decreases somewhat with age. The a p p l i c a t i o n of 1 mg of cortisone per 1 0 0 g body weight per day f o r four days increased pancreatic amy-lase a c t i v i t y i n suckling rats, but did not have an e f f e c t on rats during the weaning period. Low amylase a c t i v i t i e s i n most suckling mammals are understandable from the point of view of the food con-sumed during t h i s period. Cunningham's experiments (25) indicate that the change i n amylase a c t i v i t y occurring during weaning might not only be quantitative but also q u a l i t a t i v e . 2. Digestion of Cellulose. The d i g e s t i b i l i t y of cellulo s e and hemicellulose by nonruminant herbivores has received r e l a t i v e l y l i t t l e a ttention. There are, however, several recent reports i n -d i c a t i n g that c e l l u l o s e i s p a r t i a l l y digested by those animals possessing ceca and large i n t e s t i n e s capable of b a c t e r i a l fermentation. Conrad et a l . (20), i n 1958, found as much as 50$ of the cel l u l o s e from various sources fed to rats was digested and the products were absorbed and meta-bolized. This was based on the observation that about 50$ of the r a d i o a c t i v i t y of the dietary c e l l u l o s e appeared as .radioactive carbon dioxide i n the expired a i r . Evidence that the degradation of ce l l u l o s e i s by i n t e s t i n a l micro-organisms came from the addition of s u l f a t h a l i d i n e i n pig rations (47). The s u l f a drug decreased the d i g e s t i b i l i t y of cel l u l o s e from about 50$ to 38"$. In work with both guinea pigs (76) and rabbits (60), gum arabic, a hemicellulose, was at l e a s t 90$ digested when fed at 15$ and 20$ l e v e l s respec-t i v e l y . However, c e l l u f l o u r was e s s e n t i a l l y undigested by guinea pigs. The d i g e s t i b i l i t y of cel l u l o s e in non- l i g n i -f i e d or d e - l i g n i f i e d feedstuffs fed to pigs was similar to that found i n sheep, i . e . 7^-90$ (23), whereas i n l i g n i f i e d feedstuffs c e l l u l o s e d i g e s t i b i l i t y was much lower. However, there i s a considerable v a r i a t i o n i n the f i b r e d i g e ' s t i b i l i t i e : reported by d i f f e r e n t workers, and much i n d i v i d u a l v a r i a t i o n , e s p e c i a l l y i n rabbits and pigs. The a b i l i t y of pigs of simi-l a r weight to digest p u r i f i e d forms of cellulose often varies with a standard deviation of ± 5$. The l e v e l of f i b r e has l i t t l e e f f e c t on i t s d i g e s t i b i l i t y when composing up to 30$ of the r a t i o n , but i t has a s i g n i f i c a n t e f f e c t on the diges-t i b i l i t y of other nutrients, t h e i r . d i g e s t i b i l i t y decreasing as the f i b r e increases, the more di g e s t i b l e the f i b r e the less the decrease. To determine the role of the cecum i n cel l u l o s e breakdown, the d i g e s t i b i l i t y of various nutrients v/as com-pared i n whole and cecectomizad pigs eight to twenty-eight weeks old (71). Crude f i b r e was,in some cases, more e f f i -c i e n t l y digested by the in t a c t pig, but the large i n t e s t i n e seemed to be a more important s i t e of b a c t e r i a l fermentation than the cecum. Cellulose was p a r t i a l l y digested by rats whether the cecum was present or-not, although cecectomy decreased the digestion of cel l u l o s e from 37$ to 24$ ( 9 6 ) . In d i g e s t i b i l i t y studies with most rodents, cop-rophagy i s an important consideration. The rec y c l i n g of feces permits the remaining unabsorbed products from de-graded c e l l u l o s e to be absorbed in the upper sections of the tract (96). Rabbits have been reported to excrete both hard and soft feces, the soft feces being almost completely consumed and being rich in nitrogen, B vitamins and most of the dietary minerals (90). Yoshida et a l . (9$) reported that germ-free rabbits did not consume any feces, and he suggested that coprophagy depended on bacterial products in the feces, such as volatile fatty acids or amines, giving off a characteristic odour. He also concluded that intes-t i n a l microbes, even without the enhancing effect of copro-phagy, aid in the digestion of carbohydrates by the rabbit, and that reingestion of fecal carbohydrates, crude fat and protein might improve the quality of the total nutrient i n -take. The absorbed products of cellulose digestion in most herbivores appear to be the lower volatile fatty acids. Elsden and co-workers (43), in 1946, found large quantities of volatile fatty acids present in the cecum and colon of seven ruminant and non-ruminant species, and Barcroft et a l . (11) showed that these acids were absorbed from the organs in which they were produced. Alexander (4) found that the volatile fatty acid content of the cecum and colon of the rabbit was considerable, while only a trace was found in the stomach. A similar situation was reported in the guinea pig (57). Evidence of some microbial activity in the stomach was obtained from the production of la c t i c acid when food homogenate was incubated with gastric contents. This in vitro production was prevented by p e n i c i l l i n and chloram-phenicol, and also when, gastric mucosa was used instead of the contents ( 4 ) . Volatile fatty acid production of a pony fed hay was only slightly less than when the same pony was fed grass and oats; whereas a sheep fed grass produced nearly twice the amount of fatty acid than when fed hay alone ( 3 ) . This might be due to the more soluble carbohydrate being digested and absorbed in the ileum of the horse, while in the sheep, a l l the food was fermented in the rumen. Annison et a l . ( 6 ) , in 1 9 6 7 , found acetic, propionic, and butyric acids to be the major end products of fermentation in the digestive tract of fowl, and the ceaca were the main sites of their formation. Johnson and McBee ( 6 3 ) found the average proportion of these acids in •the porcupine cecum to be 7 4 $ acetic, 1 2 $ propionic, and 1 4 $ butyric. 88$ of the acids absorbed were from the cecum, and 1 2 $ from the large intestine. Similar concentrations of volatile fatty acids were found in the kangaroo fore stomach and the guinea pig cecum ( 5 7 ) . Although l i t t l e information i s available concern-ing the nutritional contribution of volatile fatty acids in monogastric animals, at least nine different species have been reported to contain volatile fatty acids in their cecum ( 9 7 ) . These are l i s t e d in Table II. In langur mon-keys, gastric VFA reportedly contributed energy in excess of that required for maintenance, and in porcupines the cecal VFA supplied 1 6 $ to 3 3 $ of their requirements ( 6 3 ) . Yang et a l . (97) measured the rate of disappearance of VFA from the cecum of rats k i l l e d at various intervals after feeding, and concluded that the contribution of the acids to the rat's energy metabolism was 4 . 7 $ of the caloric i n -take. This amounted to 9*4$ of the energy required for maintenance. Further support indicating that VFA are energy sources for rats, came from the appearance of radio-active carbon dioxide in the expired air when radioactive VFA were placed in the cecum ( 9 7 ) . TABLE I I CONCENTRATIONS OF VFA IN THE CECA OF SEVERAL SPECIES OF MONOGASTRIC ANIMALS (YANG, (97)) Approximate Species Age or Weight Diet A c e t i c P r o p i o n i c B u t y r i c References me q/g dry content Chicken 17 weeks Standard l a y e r 0.42 0.21 0.007 Annison et a l . feed (6) (5) Guinea p i g Adult (?) Standard com- -0.38 a c e t i c e q u i v a l e n t - Hagen and merc i a l (?) Robinson (52) Hamster . Adult (?) Standard com- — p r e s e n t — Hoover et aL(5#) m e r c i a l (?) Horse 530 Kg Grass, straw, 0.75 0.17 0.05 Elsden et al.(43) mangolds P i g 9 weeks Bran 0.40 0.15 0.04 Elsden et al . (43 ) P i g 84 Kg Barley, whey, 0.72 0.51 0.10 F r i e n d et al.(48) bran P i g 6 weeks Casein, soy, 0.31 0.41 0.04 Hendricks et a l . ce r e l o s e , c e l - (56) l u l o s e , f a t Porcupine 10 Kg Natur a l w i l d 0.38 0.06 0.07 Johnson and vegetation McBee (63) Rabbit Adult (?) Bran, oats, 0.26 0.03 0.03 Elsden et al . ( 4 3 ) mangolds Rat Adult (?) Bran, oats, hay 0.37 0.15 0.14 Elsden et- a l . (43) Rat 487 g Natural g r a i n 0.50 0.07 0.15 Yang et a l . (96) mix I l l GENERAL METHODS A. MATERIALS 1 . Experimental Animals The chinchilla used for a l l studies were Chin-c h i l l a lanigera from the University of British Columbia herd. They were obtained from various ranches, but had been kept under the same conditions of feeding and housing for nine months prior to experimentation. 2. Feeding A l l animals were fed ad l i b , once a day a com-mercial pelleted chinchilla ration (National Feeds, Abbots-ford, B.C.), and a l f a l f a hay. The pellets provided 1 5 $ protein on analysis and 8 2 0 Kcal. per pound of metaboliz-able energy. The pellet ingredients are li s t e d in Appendix 1 . 3. Housing The chinchilla were kept in 1 " x hn galvanized wire mesh cages, 1 1 " x 1 5 " x 1 8 " , which were suspended over shavings. A dust bath, Blue Cloud Chinchilla Dust, was pro-vided twice weekly, and small wooden blocks for the animals to gnaw on were placed in a l l the cages. 4. Records A polygamous breeding system was used whereby one male had access to eight females. Breeding records were kept on a l l animals and daily weights from birth to' weaning were recorded. After weaning at eight weeks, the animals were weighed weekly. The average growth of ten animals is tabulated and graphed in Appendix 2. B. METHODS 1. Organ Weights After an animal v/as k i l l e d , the l i v e r , heart, lungs, spleen, kidneys, adrenal glands, and pancreas, were removed and rinsed free from adhering tissue and blood, blotted gently and weighed. The weights were recorded as per cent of liv e body weight in Appendix 3. 2. pH of Digesta The pH of the contents of the stomach, small i n -testine, cecum and large intestine was measured immediately after death. The digesta were rinsed out with d i s t i l l e d water and the pH was read on a Radiometer, type PHM 28. & Large and small colons I V . EXPERIMENTAL PART A- - ENZYME STUDIES 1 . D i s a c c h a r i d a s e s A s t u d y was made o f t h e change i n a c t i v i t y o f i n -t e s t i n a l s u c r a s e , m a l t a s e , and l a c t a s e , f r o m b i r t h t o a d u l t -hood i n t h e c h i n c h i l l a , (a) Methods and M a t e r i a l s The g l u c o s e - o x i d a s e method o f D a h l q v i s t ( 3 0 ) was used t o determine g l u c o s e l i b e r a t e d f r om d i s a c c h a r i d e s . T h i s i s a m o d i f i e d p r o c e d u r e i n which t r i s ( h y d r o x y m e t h y l ) aminomethane ( T r i s ) i s i n c o r p o r a t e d t o i n h i b i t contaminant d i s a c c h a r i d a s e s p r e s e n t i n commercial g l u c o s e - o x i d a s e r e -age n t s . ( i ) M a t e r i a l s : S u b s t r a t e s o l u t i o n s - The s u b s t r a t e s used were r e -agent grade m a l t o s e , s u c r o s e , and l a c t o s e . A O.O56M s o l u t i o n o f each d i s a c c h a r i d e was p r e p a r e d i n O.IM maleate b u f f e r o f optimum pH. The optimum pH o f l a c t a s e a c t i v i t y was below p H 5 « 0 , so a sodium a c e t a t e b u f f e r was used i n s t e a d . T oluene, about 1 ml p e r 1 0 0 ml s u b s t r a t e s o l u t i o n , was added as a p r e s e r v a t i v e and t h e s o l u t i o n s were t h e n s t o r e d i n t h e r e -f r i g e r a t o r . . 3 5 Tris-glucose oxidase (TGQ) reagent - A commercial glucose oxidase reagent, "Glucostat", from Vforthington Bio-chemical Co. was used i n the following way. A 0.5M Tris-HCl buffer of pH7.0 and a detergent solution, consisting of 10ml of t r i t o n - X 100 (Sigma Chemical Co.) i n 40ml of 95$ ethanol were prepared. The contents of the chromagen and Glucostat v i a l s were dissolved in detergent solution and T r i s buffer respectively, combined and d i l u t e d with T r i s buffer to 100ml. The reagent was kept at 5°C and used within forty-eight hours of i t s preparation. Standard glucose solution - A 10$ solution of glu-cose i n d i s t i l l e d v/ater was prepared, and 0.27$ benzoic acid was added as a preservative. A standard curve was prepared using 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 rnl of the glucose solu t i o n . These tubes contained 0, 10, 20, 30, 40 and 50 mg respectively of glucose. ( i i ) Methods: Preparation of homogenates - The animals were k i l l e d with ether and the stomach, small i n t e s t i n e , cecum and large i n t e s t i n e were immediately cut out and kept i n i c e . The digesta were rinsed out v/ith d i s t i l l e d water and saved f o r pH determinations. The small i n t e s t i n e was then rinsed again with i c e - c o l d physiological saline, and divided into three equal lengths. The fat and mesentery were removed from the outside and the i n t e s t i n e was weighed and homogenized with p u r i f i e d sand using a mortar and pestle. The disac-charidases were extracted with four volumbes of 0.15M NaCl and centrifuged f o r ten minutes at 4,000 r.p.m. i n a So r v a l l RC2-B centrifuge. The supernatant was froz.en u n t i l assayed. Homogenates from the stomach, cecum and large i n -testine were prepared i n a s i m i l a r manner. To determine a c t i v i t y in the contents of the digestive t r a c t , as much as possible of the digesta was removed without disturbing the mucosa, and an equal volume of saline was added. Homogenation and centrifugation were as f o r the tissue preparations. Enzyme assay - 0.1ml of the diluted enzyme solution plus an equal volume of the appropriate substrate s o l u t i o n were incubated i n a water bath at 37°C f o r one hour. A small drop of toluene was added as a preservative. After incuba-t i o n , 0.3ml of d i s t i l l e d water was added and the tube immersed i n b o i l i n g water f o r two minutes to stop the reaction. For the determination of glucose, 0.5ml of the solu-tion was mixed with'3.0ml of the TG0 reagent. The tube was again incubated f o r one hour at 37°C. The colour was measured i n a Spectronic 20 (Bausch and Lomb) spectrophotometer at 420 mu, against a reagent blank. The disaccharidase a c t i v i t y of the preparation ana-lyzed was obtained by the following formula (Dahlqvist, I 9 6 4 ) . Disaccharidase activity =• a x d units/rnl n x 540 1 where a = )xg glucose liberated in sixty minutes; d = dilution factor of the enzyme solution used for mixing with the substrate; n = number of glucose molecules per mole-cule of disaccharide (for maltose, n - 2 ; for sucrose and lactose, n = 1 ) ; unit definition: 1 unit'of disaccharidase hydro-lyses 1 ^irnole disaccharide per minute under the incubation conditions used. To evaluate the activity of the intestinal homo-genates, the disaccharidase units were then calculated per g protein. Protein determination - Protein was determined using the method of Lowry et a l . (72). The standard curve was prepared using bovine serum albumen. Optimum pH determination - The pH for optimum activity of each enzyme was determined over the range of pH 3.6 to pH 9.0. The buffers used were: pH 3»6 - 4.8 sodium acetate, pH 5.0 - 7.0 t r i s maleate, pH 8.0 - 9.0 sodium barbital. (b) Results and Discussion In preliminary experiments, i t was found that homo-genates of the small intestine from adult chinchilla readily hydrolyzed maltose and sucrose, and hydrolyzed lactose slowly. Homogenates of the walls of the stomach, cecum and large i n -testine did not have any disaccharidase activity, and neither did the stomach contents. This was as expected, since the disaccharidases are known to be secreted from the crypts of Lieberkuhn which are found only in the small intestinal mucosa (53). However, digesta from the small intestine con-tained very active maltase and sucrase, as i s shown in Table III. This high activity i n the intestinal contents does not coincide with results reported in other animals (86) nor with the presently held theories concerning the site of disacchari-dase activity (75). The enzymes act intracellularly, and there i s l i t t l e diffusion into the lumen of the small intes-tine. The preparative technique of gently squeezing out the contents could have removed some of the mucosa as well, which v/ould increase the apparent activity of the contents. Maltase and sucrase activity in the cecal contents was 3$.5$ and 10.5$ respectively of that in the small intes-tinal contents, and in the large intestine activity of maltase and sucrase was 57.2 and 3.5$. Although present in low levels in the small intestinal mucosa of adult animals, lactase could not be detected in contents of the small intestine, cecum or large intestine. 3 9 TABLE I I I A c t i v i t y of Maltase, Sucrase and Lactase  in the Alimentary Tract of the C h i n c h i l l a Enzyme a c t i v i t y (units/mg protein) t r a c t assayed & „ S.l 3.M. f o r 3 ani ma Is Maltase • Sucrase Lactase Small i n t e s t i n a l wall -proximal 3 5 . 4 5 _ 5 . 7 D 1 3 . 9 1 - 1 . 7 1 5 . 1 5 _ 1.701 -middle . 7 7 . 2 7 - 1 7 . 3 ° 27.40 _ 9 . 5 1 6 . 9 9 _ 1.1°" - d i s t a l 22.02 _ 2.6D 7 . 1 6 _ 0.81 5 . 2 8 _ 1.6* Small i n t e s t i n a l contents 1 2 7 - 9 1 - 1 5 . 5 b 39.95 - 14.2 b — Cecal contents 4 9 . 2 5 - 6 . 7 a 4 . 1 7 - 4 . 2 a — Large i n t e s t i n a l contents 7 3 . 2 3 - 1 0 . 4 a 3 . 3 8 _ 3 . 4 a to CD to -p c o o * No a c t i v i t y of maltase, sucrase or lactase was found i n the stomach, cecal or large i n t e s t i n a l walls, or in the stomach contents. ^ Means f o r each enzyme within region or contents having common superscripts are not s i g n i f i c a n t l y d i f f e r e n t (P<.05). 40 Since starch and simple sugar fermenting organisms have been found in the cecum of the horse (62), an animal similar in i t s digestive anatomy to the chinchilla, and also since the magnitude of.disaccharidase activity attributed to the small intestinal contents has been questioned, i t seemed possible that the maltase activity in the cecum and large i n -testine might be of bacterial origin, and not necessarily due to indigenous enzymes washed through with digesta from the small intestine. Having found that the disaccharidases exerted most of their activity in the small intestine, the distribution of the enzymes was noted in the proximal, middle and distal sec-tions of the small intestinal wall. These areas would roughly correspond to the duodenum and upper jejunum, the lower jeju-num and upper ileum, and the lower ileum. Three adult animals were used and the results in Table III indicate activity of a l l three enzymes i s highest in the middle section, i.e. the lower jejunum and upper ileum. A non-uniform distribution of disaccharidases was observed in other animals by Malhotra and Ph i l l i p (73)• In the dog, guinea pig and pig, the enzymes were concentrated in the jejunum and to some extent in the upper ileum, while the lower ileum always had less than 15$ of the enzyme activity. Activities of lactase, sucrase, and maltase in dogs, studied by Welsh et a l . (96), were highest in the proximal and middle sections of the small intestine. As the disaccharidases exert their activity intracellularly ( 7 5 ) , i t has been suggested by Siddons (8$) that the loca-tion of optimum activity along the small intestine may re-f l e c t the site of absorption of the disaccharides. -The average pH of the intestinal contents of animals of a l l ages was 7 .1 , a higher pH than that of optimum activity of the disaccharidases studied. This i s not too surprising since i t i s known that the optimum pH of a particular enzyme in vitro i s not necessarily the same pH at which i t functions physiologically. The pH did not change significantly with age in the stomach, small intes-tine, cecum or large intestine, and was 3*4, 7*1, 7«9> and 7 . 9 respectively. The pH optima for sucrase, maltase and lactase in homogenates of the small intestinal wall were 6.4> 6 . 0 , and 4 . 6 respectively. The activity of each enzyme relative to the maximum activity at 100$ has been plotted versus pH in Figure 3 . The values for sucrase and maltase are in agree-ment with the range found in other animals, but the lactase pH optimum i s comparatively low. In the dog, sucrase and maltase are optimum at pH 6 . 0 ( 9 6 ) , in pigs, pH 6.5 ( 2 6 ) , and in man, pH 6 . 0 ( 7 ) . In rats the optimum for both en-zymes is pH 5-3 ( 3 4 ) . The optimum pH for lactase has been reported from pH 5 . 0 in dogs (96) to pH 6 . 0 in pigs and rats ( 2 6 ) . The 4 . 6 value obtained v/ith chinchilla i s therefore lower than most others reported in the literature, although Siddons, in 1969, (88) found an intestinal lactase of pH 3*6 in the chick. Of the 3 @-galactosidases separated by Gray and Santiago (49), one had a pH optimum at 4.5, but i t was found only in lysosomes. It would therefore have a dubious role in the digestion of dietary lactose, since intact disa-ccharide i s considered incapable of quantitively entering the absorptive c e l l , a requirement i f i t is to come in con-tact with lysosomal enzymes. Gray suggested the enzyme of importance in the mucosal digestion of dietary lactose had a pH optimum of 6.0. The in vitro activity at pH 4.6 found in the chinchilla would appear to be indicative of a lack of the pH 6.0 enzyme in the adult animal. However, assays of intestinal homogenates of animals two hours, one week and -two weeks of age also resulted in an active lactase with a pH optimum at 4.6, indicating lack of the pH 6.0 enzyme in the young animal as well. Since lactose was being hydro-lyzed by small intestinal homogenates of the young chinchill i t seems reasonable to conclude that the lactase of physio-logical importance in this animal does have optimum activity at pH 4.6. Changes in lactase, sucrase, and maltase levels were observed from birth to maturity in the chinchilla, animals nine months and older being classified as mature. The results are plotted in Figure 4. Sucrase was absent at birth and remained negligible until two weeks of age, at x x Lactase o o Maltase o o Sucrase 0 4.0 5.0 6.0 7.0 8.0 9.0 PH FIGURE 3 - Optimum pH of Intestinal Lactase, Sucrase and Maltase In the Chinchilla which time i t i n c r e a s e d slowly, and reached a d u l t l e v e l s and s t a r t e d to i n c r e a s e f a i r l y r a p i d l y between t h r e e and f i v e weeks, r e a c h i n g a d u l t l e v e l s between twelve and s i x t e e n weeks of age. Lactase was the most a c t i v e o f the t h r e e enzymes at b i r t h , and i n c r e a s e d t o a peak of a c t i v i t y at two weeks. I t then dropped s h a r p l y at three weeks to the low l e v e l s found i n the a d u l t animal. The changes i n a c t i v i t y are r e f l e c t e d i n the nature of the d i g e s t a i n the stomach, and changes i n the r e l a t i v e s i z e s o f the d i g e s t i v e organs, e s p e c i a l l y the cecum. At b i r t h , the cecum i s no n - e x i s t e n t ' a s such; by seven days, t h e r e i s a f u s e d f o l d i n the l a r g e i n t e s t i n e ; and by f o u r -teen days, t h e r e i s a marked i n c r e a s e i n s i z e and s a c c u l a -t i o n s i n t h i s p o r t i o n of the gut. The cecum i s comparable 'in s i z e and complexity to t h a t of the a d u l t c h i n c h i l l a by f i v e weeks of age. Also at f i v e weeks, the stomach contents were composed of at l e a s t 9 0 $ green f e e d and the remainder was milk, t h i s being a c o n s i d e r a b l e i n c r e a s e over the r e l a -t i v e amounts i n a two week o l d animal, and c o i n c i d i n g w i t h the.decrease i n l a c t a s e a c t i v i t y a t t h i s time. Young c h i n -c h i l l a were observed to eat p e l l e t s a t two or th r e e days o f age and green f e e d was found i n the stomach i n smal l amounts as e a r l y as one v/eek a f t e r b i r t h . Maltase was present a t t h i s time, but sucrase was n e g l i g i b l e . Although sucrase does i n c r e a s e between s i x and twelve weeks of age, i t r e -mains f a i r l y low when compared to maltase, even i n a d u l t FIGURE 4 - Intestinal Lactase, Sucrase and Maltase Activity as a Function of Age in the Chinchilla. 46 Specific activity a n i m a l s . S i n c e t h e enzyme i s not p r e s e n t i n l a r g e amounts i n t h e a d u l t c h i n c h i l l a w h i l e the d i e t o f p e l l e t s and hay c o n t a i n s c o n s i d e r a b l e s u c r o s e , i t would suggest some m i c r o -b i a l f e r m e n t a t i o n o f s u c r o s e i n t h e cecum and l a r g e i n t e s -t i n e . I t was v e r i f i e d i n t h e second p a r t o f t h i s s t u d y t h a t a c t i v e f e r m e n t a t i o n does t a k e p l a c e i n t h e s e a r e a s w i t h t h e p r o d u c t i o n o f v o l a t i l e f a t t y a c i d s . A l s o , i n t h e cow, s i m p l e s u g a r s , as w e l l as complex p o l y s a c c h a r i d e s , are f e r -mented i n t h e rumen, and Huber ( 6 1 ) has r e p o r t e d an absence o f i n d i g e n o u s s u c r a s e i n t h e a d u l t i n t e s t i n e o f t h e s e a n i m a l s . 2. Amylase The oc-amylase a c t i v i t y o f t h e pancreas and i n t e s -t i n e was measured a t d i f f e r e n t s t a g e s o f growth i n t h e c h i n -c h i l l a . (a) Methods and M a t e r i a l s A c t i v i t y was measured by c o l o r i m e t r i c d e t e r m i n a t i o n o f t h e r e d u c i n g groups produced from s t a r c h . The assay p r o -cedure was a m o d i f i c a t i o n o f s e v e r a l r e p o r t e d methods ( 1 4 , 4 6 ) u s i n g t h e 3 , 5 - d i n i t r o s a l i c y l a t e r e a g e n t o f Sumner ( 8 9 ) . ( i ) M a t e r i a l s : A 1% s o l u t i o n o f s o l u b l e s t a r c h ( J . T. Baker, r e -agent) was made i n 0.04M T r i s maleate b u f f e r , pH 6 . 9 * The s t a r c h was d i s s o l v e d by b o i l i n g f o r t e n m i n u t e s . An a l k a l i n e s o l u t i o n o f d i n i t r o s a l i c y l i c a c i d was p r e p a r e d a c c o r d i n g t o F i s c h e r and S t e i n (46). ( i i ) Methods: Preparation of.homogenates - The animals were euthanized with ether and the entire digestive tract and pancreas were removed. The pancreas was homogenized with nine volumes 0.01M calcium chloride in a mortar and pestle, and the homogenate was centrifuged at 15,000 r.p.m. for ten minutes. The uppermost clear layer of the supernatant was frozen and assayed for amylase within forty-eight hours. The contents of the stomach, small intestine, cecum and large intestine were each well mixed with one volume of 0.15M sodium chloride, and centrifuged at 4,000 r.p.m. for ten minutes. The stomach, cecal and large i n -testinal walls were rinsed free of any remaining digesta and homogenized with four volumes 0.15M sodium chloride. They were centrifuged at 4,000 r.p.m. for ten minutes and the supernatants were assayed. The small intestinal wall was i n i t i a l l y prepared in the same way, but the unlikely high values for amylase activity suggested contamination with pancreatic amylase. Therefore, before homogenizing the.small intestine, i t was rinsed well with a cold Ifo starch solution followed by 0.15M sodium chloride to remove as much as possible of any intraluminal amylase. Enzyme assay -1.0 ml of starch and 1.0 ml of enzyme were incubated at 37°G for fifteen minutes. The enzyme was-diluted so as not to cause the liberation of more than 1 .5 mg of maltose per ml under the above conditions. This implied a dilution of 1:200 to 1:500 for the pancreatic-homogenate, and 1:20 for the homogenates of digesta and intestinal wall. The reaction was stopped by the addition of 2ml dinitrosalicylate reagent, and the tubes were boiled, for five minutes. They were then diluted six times and the concentration of 3-amino, 5-nitrosalicylic acid formed was determined on a Spectronic 20 at 570mu. A standard curve was prepared using maltose (range 0.1 - 1.5rng/ml). One unit of amylase activity was defined as that amount of enzyme releasing reducing groups corresponding to lmg of maltose per minute at 37°G. The specific activity i s expressed as units per rng protein. Protein determination - Protein v/as again deter-mined by the method of Lowry et a l . (72) using bovine serum albumen as standard. (b) Results and Discussion Pancreatic amylase v/as negligible at birth, but increased f a i r l y rapidly with age and remained at a relatively constant level after twelve weeks (Figure 7). L'ucosal amy-lase v/as present in very low levels, but considerable activity was found in intestinal contents. This i s in agreement with the intraluminal breakdown of starch. When the intestine was prepared for assay by gently squeezing out the contents and rinsing with physiological saline, considerable activity was found in the intestinal wall. Since i t has been demonstrated that pancreatic amylase although active on starch in the lumen i t s e l f , also adheres to the intestinal c e l l wall, i t was thought that much of the activity attributed to the mucosa of the intestine was probably pancreatic amylase. To verify this and also to see i f the intestine i t s e l f was a site of amylase production, the intestine was rinsed several times with a 1 $ starch solution and then again with physio-logical saline before i t was homogenized and assayed. This resulted in a marked decrease in amylase activity. At the present time, the importance of this mucosal amylase in the breakdown of dietary starch is not known ( 2 9 ) . The activity recovered from the pancreas was ten to twenty times greater than that of the intestinal contents. Homogenates of the stomach, cecal, and large intestinal walls had no amylase activity, but both the cecal and large intestinal contents had almost 5 0 $ of the activity of the contents of the small intestine (Table IV). This suggested a bacterial amylase in these areas, although i t could also be pancreatic amylase remaining in the digesta. Starch fermenting organisms have been found in the feces of horses and pigs, and in the cecum of the horse ( 6 2 ) . TABLE IV Amylase Activity in the Chinchilla Homogenate assayed Specific activity Pancreas 1 4 . 5 9 ± 3 . 1 5 Small intestinal contents 0 . 9 4 - 0 . 0 1 Cecal contents . 0 . 5 2 ± 0 . 0 5 Large intestinal contents 0 . 5 7 i 0 . 1 1 *Means it 3.E.M. for three adult chinchilla. Specific activity = units/rug protein Age in weeks FIGURE 5- Pancreatic Amylase as a Function of Age in the Chinchilla PART B - DIGESTIBILITY STUDIES 1. Time of Passage and Site of Cellulose Digestion This study was conducted f i r s t , to determine the time of passage of feedstuffs through the digestive tract of the chinchilla; and second, to determine the cellulose diges-t i b i l i t y of a commercial chinchilla pellet and the dominant sites of cellulose digestion, i.e. the stomach, cecum or large intestine, (a) Methods and Materials: (i) Time of passage - Feed was removed, from four adult female chinchilla for twenty-four hours before the start of the experiment. They were then given the basal pelleted ration to which 2% chromic oxide had been added. After twenty-four hours, the intake of marked feed was re-corded and the regular ration of pellets and hay was resumed. Fecal samples were collected at intervals of 1 8 , 24, 3 0 . 5 , 3 5 , 4 3 , and 56 hours after feeding and were ana-lyzed for chromic oxide by the method of Schurch et a l . ( 8 5 ) . ( i i ) Cellulose digestion - The same four adult females that were used for the time of passage determination were used again for this experiment. They were fed the pel-lets " containing 2$ chromic oxide for a two-week preliminary period. No hay was fed and water was freely available. Daily feed intake was recorded and a representative sample of feces was collected daily for six days. Pooled fecal samples from each animal were then assayed for cellulose by the method of Crampton and Maynard (21) and chromic oxide by the method of Schiirch et a l . ( 8 5 ) . At the end of the collection period, the animals were k i l l e d with ether and the contents of the stomach, cecum, and large intestine were removed quantitatively and weighed. Saturated mercuric chloride was added to prevent further microbial activity and sodium hydroxide (l.ON) to prevent volatilization of the short chain acids. D i s t i l l e d water was added to double the original volume of digesta and the contents were centrifuged at 7 , 0 0 0 r.p.m. for twenty minutes. Total volatile fatty acids were measured in the supernatants and cellulose and chromic oxide were determined in the dried precipitates. For the determination of v o l a t i l fatty acids, 5ml of the supernatant plus 2ml of ION sulfuric acid were steam d i s t i l l e d and titrated against standardized sodium hydroxide to the phenolphthalein endpoint. By comparing the ratios of cellulose to chromic oxide in the feed, stomach, cecum, large intestine, and fece the cellulose d i g e s t i b i l i t i e s in the various organs were de-termined. (b) Results and Discussion (1) Time of passage - Time of passage of feedstuff through the digestive tract i s related to the a b i l i t y of the animal to u t i l i z e carbohydrates, microbial breakdown of corn-plex polysaccharides taking longer than intestinal enzyme breakdown of those more readily available. Since the chin-c h i l l a has a large functioning cecum and she can digest cellulose (69), i t was expected to have a relatively long time of passage. The results are indicated in Table V and Figure 6. Chromic oxide was f i r s t noted in the feces eighteen hours after feeding the marked feed, and trace amounts were found at f i f t y - f i v e hours. The bulk of the chromic oxide, expressed as a per cent of the total'chromic oxide ingested, was excreted thirty to forty-five hours after feeding with an average time of thirty-five hours. The early appearance of chromic oxide in the feces was probably due to the animal's having been starved for a day prior to the feeding of the marked ration. The above average feed consumption at this time could have caused an i n i t i a l increase in the rate of passage. It was necessary to starve the animals to guarantee they would a l l start eat-ing as soon as the marked feed was put before them. Chin-c h i l l a are basically nocturnal, nibbling at their feed intermittently during the day but eating mainly in the early evening and at night. Also, they defecate l i t t l e or not at a l l in the day time which made i n i t i a l attempts to visually record the f i r s t appearance of marked feces unsuccessful. As expected, the time of passage in the chinchilla is comparable to that of the horse and guinea pig, both these animals also possessing large ceca and the a b i l i t y to digest cellulose. Vander Koot et al'. (93) found the rate of passage in horses on a chopped a l f a l f a hay diet to be greatest at thirty-six hours and forty-eight hours, with 83/0 of the chromic oxide fed being excreted after the f i r s t forty-eight hours. Individual animal variation was quite large and they therefore recommended a collection period of at least four days in digestion t r i a l s with horses. From the results obtained in this study with chin-c h i l l a , i t was concluded that the collection period in the following di g e s t i b i l i t y t r i a l s should also be at least four days. A somewhat longer period would probably be necessary when hay was included in the ration. Any further comparisons of the time of jjassage in chinchilla with that of other animals i s complicated by di f -ferences in the rations fed, the age of the animals used, the feeding levels applied and the experimental technique. ( i i ) Site of cellulose digestion - The per cent cellulose digestion in the stomach, cecum and large intes-tine and the volatile fatty acids in these areas are re-ported in Table VI. The results indicate that most cellulos digestion in the chinchilla takes place in the cecum and large intestine, while the total cellulose d i g e s t i b i l i t y of a commercial chinchilla pellet containing 18.7$ cellulose i s 53.9$. Evidence from studies with other monogastric herbi-vores indicates that the products of cellulose fermentation TABLE V Time of Passage of a Pelleted Feed Through The Digestive Tract of the Chinchilla 24 hr. feed Time after feeding (hrs) Animal No. Consumption (g) 18 3 0 . 5 3 5 4 3 5 6 1 2 1 C r 2 0 3 (mg) 9 2 6 2 6 1 6 0 C r 2 0 3 % 1/43 4 . 1 3 4.13 2 . 5 4 23 C r 2 0 3 (mg) 15 2 6 35 2 4 C r 2 0 3 % 2.17 3.77 5.07 3-48 0 0 0 16 C r 2 0 3 (mg) 0 16 21 12 0 C r 2 0 3 fo 0 3.33 4.38 2.50 0 10 20 30 40 50 60 Time after feeding (hours) FIGURE 6 _ Time of Passage of a Pelleted Feed through the Chinchilla Digestive Tract are volatile organic acids (43)• The relatively high vola-t i l e fatty acid levels in the same areas where cellulose digestion occurred were therefore to be expected, and sup-port the observation that the cecum and large intestine are the main sites of cellulose breakdown. The validity of the values for cellulose digesti-b i l i t y found in the stomach i s doubtful. Not only i s there considerable variation between animals, but also the low pH of the stomach contents is well below the optimum for sur-vival of cellulose fermenting bacteria ( 6 2 ) . The apparent digestion of cellulose as well as the presence of volatile fatty acids in the stomach could have been due to coprophagy. Chinchilla practise coprophagy to a considerable extent, as do most rodents, and measurable amounts of volatile fatty acids have been found in the feces. Germ-free rabbits do not consume any feces, and i t has therefore been suggested that coprophagy depends on bacterial products such as vola-t i l e fatty acids giving off a characteristic odour in the feces. The practise of coprophagy i s important in allowing the breakdown and absorption of some products of cellulose digestion which would otherwise be wasted. The chinchilla large intestine has a greater volume than does the cecum and yet contains less total volatile fatty acid. This could be due to an increased absorption of acids in this area. TABLE VI Cellulose Digestibility and Volatile Fatty Acid Production in the Alimentary Tract of Chinchilla Fed a Commercial Chinchilla Pellet Figures for cellulose are expressed as per cent of the total cellulose digestion, and VFA is in total milli-equivalents. Animal No. Stomach Cecum Large Intestine Total** 2 4 cellulose VFA 8 . 0 3 0 . 0 1 2 4 8 . 6 0.272 4 8 . 8 0 . 1 5 3 5 9 . 1 2 6 cellulose VFA 0 . 0 0 7 4 0 . 0 0 . 3 5 3 4 3 . 5 0 . 2 9 0 5 3 . 5 1 1 cellulose VFA 1 7 . 5 0 . 0 2 4 4 2 . 5 0 . 3 2 3 4 5 . 9 0 . 2 5 0 4 9 . 1 Mean _ S.E.M. cellulose 8 . 5 2 - 2 . 1 2 4 3 - 7 - 1 . 4 7 Z k 6 . 1 _ 0 . 8 3 5 3 . 9 - 1 . 6 7 VFA 0 . 0 1 4 - . 0 0 3 0 . 3 1 6 - . 0 1 4 0 . 2 3 1 - . 0 2 4 ^cellulose digestion = cellulose cone, in feces x CrgO^ in feed x cellulose cone, in feed C r 2 ° 3 i n i " e c e s 2. Production of Volatile Fatty Acids This experiment was conducted to extend the results obtained in the previous t r i a l , and also to determine the effect on dig e s t i b i l i t y of adding a l f a l f a hay to the ration. Having found that volatile fatty acids are present in signi-ficant amounts, i t v/as decided to determine the relative distribution of the major acids throughout the chinchilla digestive tract, (a) Methods and Materials: Four adult animals were placed in metabolism cages for a ten day adjustment period. They v/ere fed al f a l f a hay and chinchilla pellets ad libitum. They v/ere. then fed pel-lets only for a preliminary period of seven days and a collec-tion period of six days. Fecal output and feed intake was recorded daily. Total dry matter dige s t i b i l i t y and cellulose d i g e s t i b i l i t y v/ere then determined. Ad libitum feeding of hay and pellets was resumed for a second preliminary period of seven days, after which daily consumption of hay and pellets was recorded and total feces collected. The animals were k i l l e d after the six-day collection period and the contents of the stomach, cecum, large intestine, and rectum were removed and analysed as be-fore for cellulose and total volatile fatty acids. The dis-t i l l a t e from the total VFA determination was made alkaline with l.ON NaOH and dried over low heat. The VFA salts were removed and purified by the method of Ross ( 8 3 ) in prepara-tion for gas chromatographic analysis. A Hicrotec (Model 2 , 0 0 0 MF) gas chromatograph f i t t e d with a hydrogen flame ionization detector was used. The columns used and the operation of the machine are described by Ranta ( 7 9 ) ' The peak areas and their relation to the concentration of the individual acids were analyzed by the method of Baumgardt (12). (b) Results and Discussion Again, the bulk of cellulose digestion, as reflected by the volatile fatty acid content, takes place in the cecum and large intestine (Table VII). Acetic, propionic and buty-r i c acids expressed per gram of dry matter are higher in the cecum, although the large intestine has been demonstrated by other workers to be of equal importance in the digestion of cellulose. Yang et a l . ( 9 7 ) found that rats could digest cellulose whether the cecum was present or not, although cecectomy decreased the cellulose digestion from 3 7 $ to 25$. Considerable absorption of volatile fatty acid took place in the large intestine as was indicated by a marked de-crease in concentration of a l l the acids in the rectum as compared with those in the cecum. Absorption was also appa-rent in the rectum, there being very low levels of VFA excreted in the feces. However, the fecal values might be slightly low,- since some VFA w i l l have been volatilized be-fore the feces v/ere analyzed. Also, the "rectum" in this experiment consisted of the pelvic colon as well as the true rectum, so that part of the VFA contributed to this area should really be included with that of the large i n -testine. There was no measurable titratable acidity in the stomach, (titration against 0.0243N NaOH), although there were trace amounts of acids present which showed up on the gas chromatogram and are included in Table VII. The apparent dry matter and cellulose d i g e s t i b i l i -ties of a commercial chinchilla pellet, with and without al f a l f a hay are recorded in Table VIII. There v/ere no sig-nificant changes In body weight on either ration for the four week duration of the experiment, and nor were there any outward, signs of digestive upsets as determined by erratic feed intake and appearance and quantity of feces. The slight increase in dry matter di g e s t i b i l i t y of pellets alone would therefore suggest that hay i s not necessary in the ration. However, there are many other factors to consider when the feeding regime i s continued for more than a short time. Impaction i s very common in chinchilla v/ith i n s u f f i -cient bulk ( 6 9 ) , and nibbling at the hay also provides a distraction from fur chewing. This i s one of the main pro-blems facing the chinchilla rancher and i s generally believed to be caused by stress factors such as boredom. TABLE VII VOLATILE FATTY ACID (VFA) IN THE DIGESTIVE TRACTS AND FECES OF FOUR ADULT CHINCHILLA FED ALFALFA HAY AND A COMMERCIAL CHINCHILLA PELLET Acid Stomach . Cecum Large Intestine Rectum Feces ;aequiv^ 182.4 121.7 44.9 3.66 Acetic molar % 84.6 72.2 72.2 67.0 87.2 /lequiv 27.5 20.5 11.3 . 0.45 Propionic molar % 9 . 6 2 11.3 . 12.1 16.0 10.7 Iso-butyric ;aequiv 2.16 . 4.61 • 1.07 0.01 molar fo 5.81 1.02 2.73 1.58 0.25 ^iequiv 35.8 22.1 8.30 0.07 Butyric molar % 14.1 13.1 12.1 1.88 ;aequiv . 1.77 3.55 . 1.20 Iso-valeric molar fo 0.51 2.11 1.36 ;aequiv 1.59 1 . 2 8 0.99 Valeric molar fo O . 6 9 0.76 1.49 ± micro-equivalents per gram dry matter. TABLE VIII DRY MATTER AND CELLULOSE DIGESTIBILITY OF A COMMERCIAL CHINCHILLA PELLET WITH AND WITHOUT ALFALFA HAY Pellets Hay and Pellets Aver, daily feed 18.7 27.1 consumption (g)^ 11.9 15«2 Aver, daily cellulose 3*2 6.85 consumption (g) 4.2 2.6 fo cellulose digestion 47.5 4$«9 fo dry matter digestion 63.4 57.9 •average of four adult females. I t i s apparent that f o r the purposes of most diges-tion t r i a l s with c h i n c h i l l a , the removal of hay from the rat i o n does not have a detrimental physical e f f e c t , although the n u t r i t i o n a l effect would be dependent on the remaining ration components. SUMMARY The conclusions that can be drav/n from this study are summarized below. 1. Maltase, sucrase and lactase activity were found in homogenates of the small intestinal wall and contents of adult chinchilla, while-only sucrase and maltase activity were found in the cecal and large intestinal contents. Ko disaccharidase activity was found in the walls of the sto-mach, cecum or large intestine. 2. Assuming that the disaccharidase activities in intestinal homogenates reflect the ability of the animal to u t i l i z e various sugars, the young preweaned chinchilla i s readily able to u t i l i z e lactose, can u t i l i z e maltose to a lesser extent, and possesses limited a b i l i t y to u t i l i z e sucrose. Between four and six weeks of age, there i s a marked decrease in lactase activity and an increase in the activi t i e s of sucrase and maltase. By twelve weeks of age, maltase has attained levels found in the adult animal, hav-ing increased to five times the levels measured in the new-born animal. Sucrase also has reached levels found in the adult by twelve weeks of age, but the increase from birth i s far less than that noted in maltase. Lactase i s present in very low levels from six weeks of age onwards. 3 . The disaccharidases v:ere non-uhiformly.'distributed along the small, intestine. Activity of maltase was s i g n i f i -cantly greater in the lower jejunum and upper ileum than in the remainder of the small intestine. A similar trend was observed with sucrase and lactase ac t i v i t i e s . 4 . The optimum pH for in vitro activity of intestinal lactase, sucrase and maltase v/as pH 4 . 6 , 6 . 4 and 6 . 0 , res-pectively. The optimum pH for lactase activity was lower than that reported in most other animals. 5 . Levels of pancreatic amylase indicate that until four weeks of age, the young chinchilla has a very limited a b i l i t y to u t i l i z e starch. Amylase increases f a i r l y steadily with age from negligible levels at birth. 6 . The time of passage of a pelleted ration through the digestive tract of chinchilla, as indicated by the time of excretion of the highest concentration of chromic."oxide was 3 5 to 4 0 hours after feeding. Traces of marker remained 5 5 hours after feeding. 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VII - A P P E N D I C E S Appendix 1 CHINCHILLA PELLETS Ingredients Ground Wheat Ground Corn Ground Barley Soybean Heal ( 4 $ . 5 $ ) Sun-cured Alfalfa Ground Beet Pulp D i s t i l l e r ' s Solubles Defluorinated Phosphate Dried Whey Iodized Salt Molasses Durabond Vitamin Mix^ ^Vitamin Mix Vitamin A Vitamin D3 Vitamin E Riboflavin Calcium Pantothenate Niacin Vitamin B-12 Choline Chloride DL. Methionine Vitamin K. Oleandomycin Zinc oxide Potassium Iodide Manganese oxide Made up to 5 lbs. v/ith v/he Amounts (lbs) 3 0 0 100 100 268 6 0 0 100 200 50 17 10 200 50 5 2,000 4,000,000 units. 1,000,000 units 5,000 units 4 grams 8 grams 18 grams 6 milligrams 227 grams 5 5 4 grams 1 gram 2 grams 60 grams 0 . 4 5 grams 80 grams middlings. APPENDIX 2 - Growth Curve of 6 Female and 8 Male Chinchilla lanigera Appendix 2 - GROWTH OF CHINCHILLA LANIGERA Age (days) Average 'Weight ± S.E.l-i. No. of Animals* 1 49.6 + 0.9 1 0 1 4 86.5 + 2.2 9 ...28 128.0 + 3.6. 1 0 42 186.3 + 4.3 10 56 233.6 + 4.1 10 70 275.0 + 3 . 8 1 0 84 307.5 + 4.3 1 0 98 329.0 + 5.7 10 1 1 2 359.0 + 9.6 6 126 378.0 + 3.6 10 1 4 0 393.2 + 9 . 2 8 1 5 4 415.6 + 12.7 10 168 426.6 + 12.2 1 0 182 • 439.4 + 10.4 8 1 9 6 472.0 + 15.1 4 252 (adult) 524.4 + 7.9 7 •When 10 animals were used, they consisted of 6 females and. 4 males. Appendix 3 - ORGAN WEIGHTS A3 PER CENT OF LIVE -BODY WEIGHT AT VARIOUS AGES IM THE CHINCHILLA Age (weeks) li v e r kidneys Organ Weight {% lungs heart of body wt.) spleen adrenals pancreas 1 3.63 0.88 1.21 0.47 0 . 0 3 2 4.86 1.21 0.77 0.40 0 . 2 1 0 . 0 2 0 . 1 6 3 4 . 4 1 0.94 0.63 0 . 1 8 0 . 0 2 3.83 1.11 0.63 . 0 . 2 0 6 3.20 0.95 0.43 0 . 1 5 0 . 0 3 0 . 5 2 6 4.56 0.90 0.67 0 . 3 3 • 0 . 1 7 0 . 0 3 6 3.67 •1.06 0 . 5 2 0 . 4 1 0 . 1 5 0 . 0 2 0 . 2 8 7 4.10 1.03 0 . 4 9 0 . 3 4 0.18 0 . 0 2 0 . 3 0 7. 3.55 0.93 0 . 5 1 0 . 3 7 0 . 1 2 0 . 0 4 8 3 . 9 4 1.0 O . 4 8 0 . 3 1 0 . 1 3 0 . 0 4 11 3.71 0.92 0 . 5 3 0 . 3 1 0 . 1 2 0 . 0 2 0 . 3 2 12 4.08 0.95 0 . 4 2 0 . 3 4 0 . 1 0 0 . 0 3 0 . 2 0 adult 3.35 0.77. 0 . 4 2 0 . 3 3 0 . 1 0 0 . 0 2 0 . 4 1 (aver, of 5 animals) 


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