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Gastric evacuation rates in Rainbow Trout (Oncorhynchus mykiss) fed different diets McDonald, Shelley Marie 1996

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GASTRIC EVACUATION RATES IN RAINBOW TROUT (Oncorb.vncb.us mvkiss) FED DIFFERENT DIETS BY SHELLEY MARIE MCDONALD B . S c . The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1978 A T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f 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 a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA MAY 1996 © S h e l l e y M a r i e M c D o n a l d , 1996 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 or her 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, Canada Date DE-6 (2/88) 1 1 ABSTRACT The passage time of ingested food through the digestive t r a c t a f f e c t s the appropriate timing of meals and the amount of feed that can be consumed. The rate of digestion of dietary components and the e f f i c i e n c y with which nutrients are absorbed may also be affected by passage time. The present study examined the effects of d i f f e r e n t dietary concentrations of f i b r e , l i p i d and g e l a t i n on the rate of food passage i n rainbow trout reared i n fresh and s a l t water. Different ingredients were added to diets i n two separate experiments. Feed conversion rates for these diets were determined. The e f f e c t of these ingredients on the rate of passage was determined through timed dissections of the stomach and i n t e s t i n e of rainbow trout. The contents were weighed and, i n experiment 2 , the water content determined. In experiment 1, feeding t r i a l s involving three experimental diets and one control diet were conducted. The control diet contained 8% l i p i d and no f i b r e additions. The experimental diets were: a 20% l i p i d diet, 12% pectin diet and a 12% c e l l u l o s e d i e t . The pectin diet resulted in poor feed consumption and growth, i n d i c a t i n g that an attribute of the diet, taste or texture, was d i s l i k e d by the f i s h . The high l i p i d diet had the best feed conversion while the diet containing c e l l u l o s e resulted in poor feed conversion. In Experiment 2, both a fresh water and a s a l t water location I l l were used. Four d i f f e r e n t dietary treatments were imposed on f i s h i n the fresh water locati o n : a basal diet (control); the basal diet containing 10% g e l a t i n ; the basal diet containing an additional %10 of o i l ; and, a basal diet containing 10% c e l l u l o s e and an additional 8% of o i l . At the s a l t water location, diets were si m i l a r except the omission of the diet containing g e l a t i n . The ef f e c t s of the dietary additions on the movement of ingesta through the stomach and intestine were determined for each diet at each s i t e . The percentages of water i n the ingesta i n the stomachs were compared to determine i f there were differences when f i s h are grown i n a saltwater environment as compared to when they are grown i n a freshwater environment. In both experiment 1 and experiment 2 , the addition of ce l l u l o s e to the basal diet, (Diet 4) increased the rate of passage during the i n i t i a l 12 hours a f t e r feeding. Consumption of the diet containing cellose was greater than that of the other experimental d i e t s . An increased consumption of diets with a c e l l u l o s e addition concurs with the findings reported i n the l i t e r a t u r e . The increased evacuation rate, however, i s contrary to previous findings. The high l i p i d diets evacuated slowly for the f i r s t 6 hours and then rapidly after that time. There was a s i g n i f i c a n t l y higher water concentration i n the stomach contents i n the f i s h reared i n s a l t water than i n the f i s h reared i n fresh water. The drinking of s a l t water by f i s h affects the rate of evacuation from the stomach, decreasing the time the ingesta takes to pass through the g a s t r o i n t e s t i n a l t r a c t . i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS , X 1.0 INTRODUCTION 1 1.1 LITERATURE REVIEW 3 1.1.1 History of Rainbow Trout Culture 3 1.1.2 Abiotic Factors 3 1.1.3 Feed Requirements 5 1.1.4 Physical Anatomy Of The Digestive Tract . . . . 6 1.1.4.1 Mouth and Oesophagus .6 1.1.4.2 Stomach And Associated Gastric Glands . . . 7 1.1.4.3 Intestine 8 1.1.4.4 Associated Glands and Organs 10 1.1.5 Carbohydrates and Fibre i n Fi s h Diets . . . . 11 1.1.6 Digestion and Absorption of V Nutrients 14 1.1.6.1 Protein Digestion.and Absorption 14 1.1.6.2 L i p i d Digestion and Absorption 15 1.1.6.3 Carbohydrate Digestion and Absorption . . 16 1.1.7 Food Passage Time Through the Gastrointestinal Tract 17 1.1.7.1 Characteristics of Feed Which Affe c t Passage Rates . . '. . 20 1.1.7.2 Methods of Measurement of Passage Time . . 25 2.0 Experiment 1 . . ' . . 26 2.1 Introduction 26 2.2 Materials and Methods 26 2.2.1 Experimental Design 26 2.2.2 Experimental Fish . "'. 27 2.2.3 Diet Preparation and Composition 28 2.2.4 Experimental Protocol 28 2.3 Results 31 2.3.2 Evacuation of the Stomach Contents Over Time 35 2.3.3 Evacuation of In t e s t i n a l Contents Over Time 39 2.4 Discussion 41 3.0 Experiment 2 45 3.1 Introduction 45 3.2 Materials and Methods 45 v i 3.2.2 Experimental Fish 46 3.2.3 Diet Preparation and Composition 47 3.2.4 Experimental Protocol 47 3.3 Results 52 3.3.1 Fresh Water F a c i l i t y 52 3.3.1.2 Evacuation of the Stomach Contents Over Time . 55 3.3.1.3 Evacuation of I n t e s t i n a l Contents Over Time 59 3.3.2 Salt Water F a c i l i t y 62 3.3.2.1 Evacuation of the Stomach Contents Over Time 64 3.3.2.2 Evacuation of I n t e s t i n a l Contents Over Time 67 3.4 Comparison of Water content of Stomach Contents of Fish Reared i n Fresh Water Versus F i s h Reared i n Salt Water 70 3.4 Discussion 74 4.0 CONCLUSIONS 77 REFERENCES 81 APPENDICES 87 v i i TABLE 1 TABLE 2 TABLE 3 TABLE 4: TABLE 5: TABLE 6 TABLE 7 TABLE 8 TABLE 9 TABLE 10: TABLE 11: TABLE 12: TABLE 13: TABLE 14: TABLE 15: LIST OF TABLES DIET FORMULATION FOR EXPERIMENT 1 29 VITAMIN AND MINERAL PREMIX FOR EXPERIMENT 1 30 RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 1 32 EXPERIMENT 1, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN FRESH WATER, 34 EXPERIMENT 1, STOMACH CONTENTS OF FISH GROWN IN FRESH WATER (Average Wet weights of stomach contents As % Body Weight) AT DIFFERENT TIMES POST PRANDIAL 35 EXPERIMENT 1, INTESTINAL CONTENTS OF FISH GROWN IN FRESH WATER (Average Wet weights of i n t e s t i n a l contents As % Body Weight) AT DIFFERENT TIMES POST PRANDIAL 39 DIET FORMULATION FOR EXPERIMENT 2 49 VITAMIN AND MINERAL PREMIX FOR EXPERIMENT 2 51 RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 2 .... 53 EXPERIMENT 2, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN FRESH WATER 54 STOMACH CONTENTS OF FISH GROWN IN FRESH WATER (Average dry weights of stomach contents as % Body Weight) AT DIFFERENT TIMES POST PRANDIAL 55 INTESTINAL CONTENTS OF FISH GROWN IN FRESH WATER (Average dry weights of stomach contents as a % Body Weight) AT DIFFERENT TIMES POST PRANDIAL 59 RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 3 DIFFERENT DIETS IN SALT WATER 61 EXPERIMENT 2, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN SALT WATER 62 STOMACH CONTENTS OF FISH GROWN IN SALT WATER (Average dry weights of stomach contents as % Body Weight) AT DIFFERENT TIMES POST PRANDIAL 63 v i i i TABLE 16: INTESTINAL CONTENTS OF FISH GROWN IN SALT WATER (Average dry weights of stomach contents as % Body Weight) AT DIFFERENT TIMES POST PRANDIAL ..67 TABLE 17: THE WEIGHT AND % WATER CONTENT OF INGESTA IN THE STOMACHS OF RAINBOW TROUT REARED IN FRESH WATER 71 TABLE 18: THE WEIGHT AND % WATER CONTENT OF INGESTA IN THE . STOMACHS OF RAINBOW TROUT REARED IN SALT WATER 73 ix LIST OF FIGURES FIGURE 1: STOMACH CONTENTS OF RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER ...38 FIGURE 2: INTESTINE CONTENTS OF RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER 40 FIGURE 3: STOMACH CONTENTS OF RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 2 57 FIGURE 4: INTESTINE CONTENTS OF RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 2. . . .60 FIGURE 5: STOMACH CONTENTS OF RAINBOW TROUT REARED ON 3 DIFFERENT DIETS IN SALT WATER, EXPERIMENT 2 65 FIGURE 6: INTESTINE CONTENTS OF RAINBOW TROUT REARED ON 3 DIFFERENT DIETS IN SALT WATER, EXPERIMENT 2 69 FIGURE 7: % WATER OF STOMACH CONTENTS OF RAINBOW TROUT REARED IN FRESH AND SALT WATER, EXPERIMENT 2 72 X ACKNOWLEDGEMENTS I would l i k e to express my appreciation to a l l those who helped me during my experimental work and thesis preparation. In pa r t i c u l a r , I am grateful to my advisor Dr. B. March for her knowledgeable advice, guidance and patience during the course of th i s research. Special appreciation i s extended to those individuals who endured the long days and nights of sampling around the clock. These include, e s p e c i a l l y Chutima T a n t i k i t t i , Bridget Ennevor, Richard Henly, Darlene McCauley and Carol McMillan. Their friendships have sustained me. 1 1.0 INTRODUCTION • -Salmonids are the subject of intensive c u l t u r i n g i n B r i t i s h Columbia and other parts of the world. Culturing can be a t o o l for enhancement and management of natural stocks as well as a major source of food production. The major goal of aquaculture i s to raise f i s h to a marketable size i n the most economically feasible way. Feed costs can represent up to f i f t y percent of aquaculture costs; therefore, methods of reducing feed expenditure have been the focus of extensive research. Formulated balanced, diets are created to s a t i s f y the n u t r i t i o n a l requirements of growing f i s h . Salmonids have high dietary protein requirements which have t r a d i t i o n a l l y been supplied by f i s h meal, the most expensive component of the feed. Protein i s required for growth and as an energy source. To minimize the u t i l i z a t i o n of protein as an energy source, other dietary additions are used to reduce the amount of dietary protein, thus reducing the cost of feeds. Experiments involving the additions Of'higher proportions of l i p i d s , plant proteins and carbohydrates to diets have been conducted by many researchers. The e f f e c t of the ingredients on the f i s h e s ' growth and u t i l i z a t i o n e f f i c i e n c y has been examined and are considered within t h i s research. The e f f e c t of these 2 ingredients on the passage of feed through the digestive t r a c t has been studied to some degree, but further information concerning these e f f e c t s and t h e i r impact on nutrient u t i l i z a t i o n i s required. The rate of passage i s influenced by several factors, some of which include feed c h a r a c t e r i s t i c s , water temperature, stress on the animal, and feeding frequency. The passage time can affe c t the fi s h ' s a b i l i t y to u t i l i z e ingredients of the d i e t . The rate of passage of feed can affe c t the nutrient absorption and u t i l i z a t i o n . I f ingredients within the diets have an adverse e f f e c t on absorption, t h i s may resu l t i n poor growth rates and feed conversion. It would therefore not be recommended to add such ingredients to the diets i n any quantity. The objective of t h i s study was to examine the ef f e c t s that d i f f e r e n t dietary additives have on the ga s t r i c evacuation time and feed conversion i n rainbow trout (Oncorhynchus mvkiss) . An additional objective was to examine the e f f e c t of water consumption when f i s h are reared i n a marine environment on the water content of the stomach contents and rate of passage of feed. 3 1.1 LITERATURE REVIEW 1.1.1 History of Rainbow Trout Culture A f t e r carp, Rainbow trout have the longest history i n f i s h culture. They are farmed on a l l continents and i n many countries under various environmental conditions. Records of eyed trout eggs being transferred for culturing from wild stock dates back to 1874 (Gall and Crandell, 1990) . Soon afte r these i n i t i a l egg transfers, many private and government agencies developed hatcheries for producing rainbow trout for recreational f i s h i n g (Gall and Crandell, 1990). 1.1.2 Abiotic Factors A b i o t i c factors can affe c t both the metabolic rate and the processing rate of food i n f i s h . Salmonids are defined as poikilotherms, which means t h e i r body temperatures and metabolic rates are governed by the surrounding water temperature. The optimum temperature for maximum growth rates i n salmonids i s between 12 and 17 °C, depending on the species (Brett, 1979) . Colder temperatures such as 5 °C result i n lower feed requirements and reduced growth rates due to a decrease i n biochemical reactions r e s u l t i n g i n less food being required. In colder temperatures i t 4 has also been noted that protein assimilation of salmonids i s impaired i f c e l l u l o s e i s incorporated i n the d i e t . The impairment has att r i b u t e d to the need for more nutrients required for maintenance and therefore less going into growth (Brett, 1979) . Temperature also affects the l e v e l of dissolved oxygen i n the water. Oxygen leve l s should be maintained with at least 5 - 7 ppm dissolved oxygen (Logan and Johnston, 1990) . Flow rates i n pens and ponds are important to consider, mainly i n r e l a t i o n to oxygen tension. An adequate flow rate w i l l f l u s h away toxic metabolites produced by the f i s h which can demand oxygen and change the pH of the surrounding environment (Logan and Johnston, 1990). Photoperiod length has also been found to have a high c o r r e l a t i o n with growth. An increased day length stimulates endocrine a c t i v i t y i n spring, an e f f e c t that i s enhanced by the r i s i n g temperature of the water. The increase i n growth i s accompanied by an increase i n consumption of feed (Brett, 1979). Rainbow trout have been found to be able to withstand salt water when introduced to f u l l strength sea water after reaching a minimum size, but the response depends on the s t r a i n of trout and the location i t came from (Gall and Crandell, 1990). Stocking densities must be considered when culturing rainbow trout. Densities impact growth and feed e f f i c i e n c y and can affect water q u a l i t y . The potential for disease also increases with increased stocking density (Logan and Johnston, 1990) . 5 •1.1.3 Feed Requirements Feed i s the largest component i n the cost of trout production, often accounting for over 50% of the operating costs (Logan and Johnston, 1990). It i s estimated that up to 5-10% of the feed given to the f i s h often remains uneaten, f a l l i n g through the pens or dropping to the bottom of the tank or pond. Trout w i l l eat off the bottom of a cement pond but seldom o f f the bottom of an earth pond (Logan and Johnston, 1990) . The search for the most cost e f f e c t i v e method of feeding trout to a t t a i n t h e i r f i n a l or marketable weight has been the subject of extensive research. Since Salmonids are carnivorous, the major portion of t h e i r protein requirements i s normally provided as animal protein. In the wild, t h i s consists of zooplankton, invertebrate and vertebrate sea l i f e . H i s t o r i c a l l y , most of the protein i n aquaculture diets has been provided by f i s h meals. Fish meal has been the single most expensive component of salmonid diets (Hilton and Slinger, 1981) . Salmonids are t r a d i t i o n a l l y fed complete diets, both dry and moist, containing two to four times the levels of protein t y p i c a l of diets for non-carnivorous, warm blooded animals. Reliance on f i s h meal as the p r i n c i p a l protein source for f i s h feeds, the increased demand for high quality meal, and the decreasing a v a i l a b i l i t y of these meals has resulted i n alternative sources of protein being investigated. Research into alternative sources of protein has resulted in the assessment and determination of the 6 e f f i c i e n c y of digestion, the available energy contained i n f i s h feeds and the feed intakes (Nose, 1978). Research has shown that f i s h eat to s a t i s f y t h e i r c a l o r i c requirements. When a diet i s d i l u t e d with an in d i g e s t i b l e component, the f i s h compensates for the decreased c a l o r i c content by eating a greater amount of food per day (Grove et a l . , 1978). Also, diets d i l u t e d with these " i n e r t " materials are evacuated from the stomach more rapidly with the rate of emptying increasing with the increasing d i l u t i o n (Jobling, 1981) . Excessive l e v e l s of f i l l e r may d i l u t e the food to the extent that the f i s h cannot eat enough to s a t i s f y t h e i r needs (Buhler & Halver, 1961/ Hilton et a l . , 1983) . To help decrease the feed cost of intensive culturing, researchers need to obtain precise information on n u t r i t i o n a l requirements and components of the diets and t h e i r e f f e c t s on the fi s h ' s a b i l i t y to u t i l i z e them. This w i l l r e s u l t i n diets that can be t a i l o r e d for maximum growth, and an economically p r a c t i c a l diet can be manufactured. 1.1.4 Physical Anatomy Of The Digestive Tract 1.1.4.1 Mouth and Oesophagus As predators, salmonids have well developed o r a l grasping and holding mechanisms. They have teeth both on the jaws and other surfaces of the mouth. The tongue has sensory taste buds and there 7 are mucous c e l l s scattered throughout the inner l i n i n g of the mouth (Reviewed by Iwama, 1989). The oesophagus i s short, wide and muscular. It functions i n transport and taste of food. In salmonids, the oesophagus terminates i n a cardiac sphincter or valve which separates i t from the stomach (Fange and Grove, 1979) . 1.1.4.2 Stomach And Associated Gastric Glands The stomach i s a U-shaped distendable organ which can accommodate large meals. It i s l i n e d with e p i t h e l i a l t i s s u e and has secretory c e l l s which secrete hydrochloric acid (HC1) and pepsinogen and are stimulated by the distension of the stomach. It i s comprised of two sections, a descending or cardiac portion and an ascending or p y l o r i c portion. The cardiac region contains a muscularis mucosa, consisting of an inner c i r c u l a r and an outer longitudinal layer of smooth muscles (Fange and Grove, 1979) . The g a s t r i c gland c e l l s are active both i n acid production and i n the synthesis of pepsinogen. The stomach culminates i n the p y l o r i c sphincter which i s a ring of muscle formed from a thickening of the c i r c u l a r smooth muscle layer, which controls the passage and size of food p a r t i c l e s passing from the stomach to the i n t e s t i n e . Mucous membranes provide l u b r i c a t i o n to aid i n the passage of food. Movement of digesta through the system i s controlled by p e r i s t a l t i c waves of muscle contraction (Fang and Grove, 1979) . Under s t r e s s f u l situations these muscle contractions are i n h i b i t e d , which 8 indicates that the p e r i s t a l s i s i s under nervous control (Reviewed by Iwama, 1989). The distention of the stomach stimulates the secretion of digestive enzymes into the stomach. Gastric glands, made up of one type of c e l l only, contain abundant secretory granules, the c e l l s being active i n both acid production and the synthesis of pepsinogen (Fange and Grove, 1979) . The pH of the stomach i s aci d i c , between 2 to 4 pH units. Pepsin a c t i v i t y i s dependent on pH and temperature, while HC1 secretion i s dependent on temperature and meal size (Reviewed by Iwama, 1989) . 1.1.4.3 Intestine The in t e s t i n e i s a simple tube which has digestive glands and extensive blood and lymph vessels (Reviewed by Iwama, 1989) . The surface area i s increased by folds of the mucosa, but the length i s less than the body length. The anterior end of the intestine has b l i n d tubes, c a l l e d p y l o r i c ceca which vary i n number between 1 and 1000, depending on the species of t e l e o s t . In salmonids, ceca are very well developed and the numbers vary between 30 and 80. The ceca resemble the inte s t i n e i n structure with a well developed muscularis consisting of c i r c u l a r muscle f i b r e s . The ceca f i l l with digesta and are discharged by a n t i - p e r i s t a l t i c action, forcing the digesta down the i n t e s t i n e . The material forced into the ceca already contains digestive enzymes secreted into the i n t e s t i n e . Digestion continues i n the ceca where a large 9 part of the digestion and absorption occurs due to the increased surface area. Between the ceca, . basophilic pancreatic exocrine gland c e l l s may be present i n the connective t i s s u e . Also i n t h i s anterior region of the intestine, b i l e produced i n the l i v e r enters from the b i l e ducts. The inner epithelium of the intestine contains goblet c e l l s which secrete mucus which aids i n the passage of ingesta along the i n t e s t i n e . The surface of the intestine contains longitudinal folds which'increases the absorptive surface area. The e p i t h e l i a l c e l l s have a brush border of m i c r o v i l l i which further increase the absorptive surface (Fange and Grove, 1979) . Lipase, which breaks down t r i a c y l g l y c e r o l s into mono-glycerides and di-glycerides, i s released by the pancreas into the i n t e s t i n e and i s present in the digesta i n the p y l o r i c ceca. Absorption of the fat occurs i n the anterior portion of the i n t e s t i n e (Reviewed by Iwama, 1989). However, as stated previously, absorption may occur further along the intestine under heavy l i p i d loading. The terminal part of the in t e s t i n e i s d i f f e r e n t i a t e d by an area i n which the rectum i s wider and i s separated from the i n t e s t i n e by an i l e o - r e c t a l valve formed of smooth muscle. The mucosal epithelium i s r i c h l y endowed with goblet c e l l s and contains a brush border of m i c r o v i l l i for increased absorptive capacity. The r e c t a l gland i s believed to secrete s a l t ions into the lumen to aid i n osmoregulation i n high s a l t environments (Fange and Grove, 1979) . Mucous c e l l s are present i n the rectum. The vent i s the terminal location of the urinary and i n t e s t i n a l t r a c t s (Reviewed by 10 Iwama, 1989) . 1.1.4.4 Associated Glands and Organs The l i v e r i s a two-lobed organ with a variety of functions. The hepatocytes contain numerous mitochondria, endoplasmic reticulum, g o l g i v apparatus, lysosomes and peroxisomes as well as l i p i d and glycogen deposits. The l i v e r produces b i l e . The excess b i l e i s stored i n the g a l l bladder and the b i l e duct opens into the anterior intestinev B i l e s a l t s a i d i n fat digestion through emulsification and aid i n adjustment of the pH i n the in t e s t i n e to the desired neutral or basic pH (Reviewed by Iwama, 1989) . The pancreas i s diffuse i n salmonids, and i s scattered amongst, and associated with, f a t t y material around the p y l o r i c ceca. It secretes not only i n s u l i n and glucagon i n response to blood concentrations of amino acids and glucose, but also digestive enzymes through the pancreatic duct into the anterior i n t e s t i n e . Pancreatic enzymes include trypsin, chymotrypsin, lipase, amylase, carboxypeptidases, and elastase (Fange and Grove, 1979). Bicarbonate, secreted by the pancreas, increases the pH of the egesta from the stomach. The pancreatic enzymes are most active at neutral to basic pH. 11 1.1.5 Carbohydrates and Fibre i n Fish Diets Salmonid f i s h , such as rainbow trout, are mainly carnivorous and can not u t i l i z e carbohydrates e f f i c i e n t l y (Shiau et a l . , 1988) . Carbohydrates, as alternate energy sources, have been shown to be used with low e f f i c i e n c y due to i n s u f f i c i e n t enzymatic break-down in the digestive t r a c t , i n s u f f i c i e n t absorption of the end products of digestion and i n s u f f i c i e n t metabolization of absorbed monosaccharides. Complex carbohydrates must be broken down to simple sugars before the nutrients can be absorbed. Trout have been found to be able to u t i l i z e alternate energy sources such as d i g e s t i b l e carbohydrates, sucrose and g e l a t i n i z e d starch (Peiper and Pf e f f e r , 1979). Hilton and Atkinson (1982) found that trout maintained on high carbohydrate diets had poor glucose tolerance and that the provision of diets with d i g e s t i b l e carbohydrates i n excess of 140 g.kg - 1 resulted i n s i g n i f i c a n t growth depression. The reported tolerance of rainbow trout for glucose varies among experiments. Buhler and Halver (1961) found that Chinook f i n g e r l i n g s could toler a t e 48% carbohydrate diets without enlarged glycogen-rich l i v e r s on mortality, as long as t h e i r diet was n u t r i t i o n a l l y adequate. Divergent results may be due to the difference i n the age, size of f i s h , water temperature, type of carbohydrate, and method of feeding, i . e . single versus multiple meals (Hilton and Slinger, 1981) . D i g e s t i b i l i t y of starch i s poor in salmonids. Even though 12 cooking or heating starch improves i t s d i g e s t i b i l i t y , the f i s h ' s a b i l i t y to u t i l i z e glucose i s s t i l l l i m i t e d . High glucose diets can r e s u l t i n larger l i v e r s and high l i v e r glycogen l e v e l s , and can cause decreased growth and increased m o r t a l i t i e s , e s p e c i a l l y i n cold water temperatures (Hilton and Slinger, 1981). However, the f i s h ' s a b i l i t y to u t i l i z e carbohydrates may depend on the type of carbohydrate and i t s effect on g a s t r i c m o t i l i t y . F i s h with simple stomachs have l i m i t e d microbial action i n the gut due to the small capacity of the digestive system, the short food retention time and the low temperature. Fish have a very l i m i t e d a b i l i t y to u t i l i z e f i b r e (Smith, 1978). It has been the practice i n f i s h feed formulation for experimental studies to adjust nutrient balance by introducing f i b r e , a s f i l l e r or as a s t a b i l i z e r for p e l l e t formation i n high density d i e t s . Also, the use of carbohydrates which contain varying amounts of f i b r e i s common (Buddington and Hilton, 1988). Fibre has been thought to be i n d i g e s t i b l e and therefore of no consequence to the c a l o r i c content or nutrient value of the d i e t . However, f i b r e may affect the a v a i l a b i l i t y of other nutrients by a l t e r i n g the rate of passage of digesta through the g a s t r o i n t e s t i n a l t r a c t (Hilton and Atkinson, 1982) . Dietary f i b r e includes i n d i g e s t i b l e plant materials such as c e l l u l o s e , l i g n i n and other complex carbohydrates. Fibre can be divided into viscous or water soluble polysaccharides such as guar gum, pectin, and carboxymethyl c e l l u l o s e and non-viscous or water insoluble polysaccharides such as alpha-cellulose. The use of 13 binders such as alginates, which have a water-binding e f f e c t i n the inte s t i n e , may also have an a n t i - n u t r i t i o n a l e f f e c t (Storebakken and Austreng, 1987). Dietary f i b r e affects the movement of nutrients along the ga s t r o i n t e s t i n a l t r a c t and l i k e l y influences the nutrient absorption (Shiau et a l . , 1988) . Juvenile rainbow trout reared on 10 to 20% c e l l u l o s e diets adapted to increased dietary f i b e r by increased feed consumption, increased g a s t r i c evacuation time and increased stomach volume. Gastric emptying time was much faster on high f i b e r diets (Hilton et a l . , 1983). Fibre has been shown to in t e r f e r e with the absorption of many nutrients such as bivalent metals, fats and amino acids (Southgate & Durin, 1970) . With rats, f i b r e has been shown to a l t e r g a s t r o i n t e s t i n a l mucin quantities and to bind to the mucin which can a f f e c t nutrient absorption (Satchithanandam et a l . , 1990) . Both dietary f i b r e and f i b r e i s o l a t e s have been found to sequester b i l e s a l t s and bind f a t t y acids thereby decreasing the a v a i l a b i l i t y of cholesterol and the by-products of t r i a c y l g l y c e r o l digestion (Vahouny et a l . , 1981) . The practice of including f i b r e and carbohydrates as f i l l e r s and binders i n the diet may have a negative ef f e c t on the nutrient uptake, thereby a f f e c t i n g the growth of f i s h . Viscous polysaccharides such as guar gum and pectin appear to slow gastric emptying (Leeds, 1982) . 14 1.1.6 Digestion and Absorption of Nutrients 1.1.6.1 Protein Digestion and Absorption Food intake distends the stomach which stimulates the release of HC1 and pepsinogen. This brings the pH of the stomach to between 2 and 4. Pepsin i s the major acid protease which i s secreted by the g a s t r i c gland c e l l s and i s active at the pH l e v e l s created by the HC1. Pepsin i s the f i r s t enzyme to cleave peptide linkages i n the dietary proteins within the feed (Fange and Grove, 1979). The release of HC1 and pepsinogen i s stimulated by the hormone gastrin. The d i s t e n t i o n of the stomach stimulates the vagal nerve which i n turn activates secretion of gastrin from the c e l l s i n the stomach (March, 1990) . The digestion of protein begins i n the stomach with the endopeptidase a c t i v i t y breaking down the protein into a slurry (Fange and Grove, 1979). As the digesta passes from the stomach into the int e s t i n e , the presence of t h i s a c i d i c mixture stimulates the secretion of the hormone se c r e t i n . This in turn stimulates the secretion of pancreatic juices with bicarbonate compounds to increase the pH to a neutral or basic l e v e l . The i n t e s t i n a l digestion of proteins continues with the release of t r y p s i n and chymotrypsin which further s p l i t the polypeptides. Carboxypeptidases and peptiadases in the i n t e s t i n e further break down the bonds of the protein. Enzymes such as elastase and collagenase may attack s p e c i f i c protein bonds. Elastase, which i s activated by tr y p s i n i s active in !5 breaking peptide bonds in the protein e l a s t i n (Fange and Grove,1979). The proteins continue to break down u n t i l they are low molecular peptides and individual amino acids. The amino acids are absorbed against concentration gradients, coupled with inorganic ions (Fange and Grove, 1979) . Peptides are most l i k e l y to be absorbed by pinocytosis. 1.1.6.2 L i p i d Digestion and Absorption Li p i d s i n f i s h diets are hydrolysed by lipases and phospholipases and are subsequently used either as an energy source, stored as fat deposits, or incorporated into phospholipids in tissues (National Research Council, 1973). Lipases released by the pancreas are the major factor i n fat digestion (Fange and Grove, 1979). Lipase i s secreted into the upper i n t e s t i n e and p y l o r i c ceca where a great deal of l i p i d digestion and absorption occurs (Fange and Grove, 1979). Lipids are not soluble i n water. Lipases hydrolyse neutral fat into diglycerides, monoglycerides, glycer o l and free f a t t y acids (Fange and Grove, 1979) . Lipids are emulsified and s o l u b i l i z e d p r i o r to hydrolysis with the aid of b i l e s a l t s released with the b i l e . The b i l e s a l t s and l i p i d s form micelles which then can be absorbed. Next, the l i p i d i s formed into chylomicron or very low density lipoprotein p a r t i c l e s and absorbed into the lymph 16 (Fange and Grove, 1979) . This mainly occurs i n the proximal i n t e s t i n e or i n the p y l o r i c caeca. I f there i s excessive dietary l i p i d , the absorption could extend to the d i s t a l i n t e s t i n e (Leger, 1985). Lipids are slow to be absorbed, and t h i s may depend on the g a s t r i c emptying and i n t e s t i n a l t r a n s i t rates as well as on the temporary storage of l i p i d s i n the enterocyte (Leger, 1985). 1.1.6.3 Carbohydrate Digestion and Absorption Salmonids are not e f f i c i e n t users of carbohydrates; however, trout do have the necessary enzymes to s p l i t disaccharides and starches into monosaccharides for absorption. The p y l o r i c caeca and upper in t e s t i n e contain digestive enzymes such as maltase, sucrase, lactase and amylase. Carbohydrates are absorbed as simple sugars. The trout's a b i l i t y to use carbohydrates as energy i s l i m i t e d (National Research Council, 1973). Carbohydrates are used.in commercial feeds because they are generally inexpensive and they aid i n s t a b i l i z i n g and binding the p e l l e t s (Hemre et a l . , 1989). In addition, carbohydrates may supply up to 20 % of the available c a l o r i e s with a protein-sparing e f f e c t . . Cereal grains, which are often i n diets for mammals, contain starch; however, starch i s poorly digested by salmonids. The d i g e s t i b i l i t y of starch can be improved by heat processing and the end product of starch digestion i s glucose. In an experiment with rainbow trout, glucose gave an absorption of 90%. There was a rapid increase i n blood glucose which took 24 hours to return to 17 i n i t i a l l e v e l s (Hemre et a l . , 1989) . The i n a b i l i t y of rainbow trout to regulate glucose i n the blood may be p a r t l y due to a lack of glucose phosphorylating capacity (Cowey et a l . , 1977) Carbohydrates can also a f f e c t the glycogen content i n the l i v e r . Large loading of starch i n the diet can res u l t i n enlarged l i v e r s with high l i v e r glycogen l e v e l s , depressed growth and increased m o r t a l i t i e s (Hilton and Slinger, 1981) . Fibre, such as ce l l u l o s e , has a very low d i g e s t i b i l i t y and can adversely af f e c t the absorption of other nutrients as well, as indicated previously. 1.1.7 Food Passage Time Through the Gastrointestinal Tract Gastric evacuation rates are altered by metabolic rate changes which change with water temperature, oxygen l e v e l , l i g h t intensity and stress. Other factors, which may influence the rate of passage, include the frequency of feeding and the c h a r a c t e r i s t i c s of the feed. Evacuation time i s dependent on the amount of food i n the stomach, the v a r i a t i o n i n the food or prey size, and meal size (Mills et al.,1984) . These factors also influence the rate of feed intake and determine the stomach volume. Gastric evacuation in sockeye salmon was shown to take approximately twice as long when water temperatures were decreased from 23 . °C to 10 °C (Cho, 1990). E l l i o t (1991) found that evacuation rates i n brown trout increased with temperature. In juvenile chum salmon grown under d i f f e r e n t temperature regimes, 18 8,10,12, 14 and 16 °C, the dry weight of the stomach contents decreased exponentially over time (Koshiishi, 1980) . Koshiishi also found that rates were not s i g n i f i c a n t l y affected by temperatures between 8 and 12 °C i n small f i s h (0.7 g) but larger f i s h (2 g) needed a shorter time for evacuation as temperature increased. Rogers and Burley (1991) found that smallmouth bass, on a diet of juvenile salmon, had an increase i n t o t a l evacuation rates with an increase i n water temperature. Similar results were found i n rainbow trout (Fauconneau et a l . , 1983) . Robb (1990) found that i n whiting, larger f i s h eliminated meals at a faster rate than smaller f i s h with meals of a given size following a l i n e a r evacuation model. However, larger meals eliminated faster grams per hour but took longer to evacuate by vir t u e of t h e i r s i z e . J o l l i v e t et al.(1988) had s i m i l a r results with turbot i n that a smaller d a i l y ration (0.5% of the test animal's body weight) evacuated more rapidly that larger rations. Santos and Jobling (1991), contrary to Robb's findings, found that cod of d i f f e r e n t size fed fixed proportions of t h e i r body weight evacuated at a constant rate independent of t h e i r body size. Garber (1983) found that in yellow perch, g a s t r i c evacuation rates were not affected by the size of the f i s h or by the dietary moisture. The meal size resulted i n variations i n the t o t a l evacuation rate, although the differences were not proportional to the changes i n meal s i z e . Nagata (1989) found that a square root model described g a s t r i c evacuation i n juvenile masu salmon most cl o s e l y . Smithe et a l . (1989) found that walleye pollack fed a 19 range of meal sizes, 0.5% to 2.5% of the t e s t animals' body weight, evacuated food as a negative exponential function of time. An increased i n the amount of food eaten at one time resulted i n a decrease i n the t o t a l evacuation rate but an increase i n the amount of food evacuated. Smithe et a l . (1989) also found that an increase i n temperature resulted i n an increased evacuation rate as a l i n e a r function when pollack were grown i n temperatures ranging from 3 to 9 °C. Ryer and Boehlert (1983) also found that evacuation rates were temperature dependant with more rapid evacuation occurring with increased temperature;. and that the evacuation rate i s p o s i t i v e l y correlated to gut content, the rate slowing with decreasing gut content. This may allow for increased e f f i c i e n c y for assimilation during periods when there is. l i t t l e food available. Models to describe g a s t r i c evacuation rates are- varied. Jobling (1981, 1986) suggested that evacuation of large prey follow a l i n e a r or square root curve. Ruggerone (1989) found that a exponential curve model f i t t e d o v e r a l l evacuation more clo s e l y . Robb (1990) indicated that a l i n e a r evacuation model gave a good description of the data for whiting. Meal size can a f f e c t the rate of digestion. The rate of digestion i s proportional to surface area of the food bolus. Enzymes attack the bolus at the surface; therefore, a smaller meal w i l l be digested more quickly. Larger f i s h w i l l digest a meal that i s 1% of i t s body weight at a greater rate than a smaller f i s h ; but, the time required for complete digestion w i l l be longer (Fange 20 and Grove, 1979) . An increase i n meal siz e (as a percentage of body weight) increases both the residence time of food i n the stomach and the rate of evacuation (Talbot et a l . , 1984) . Periods of starvation and s t r e s s f u l procedures, such as force feeding af f e c t the feeding and digestive processes i n f i s h . Forced feeding of tes t animals usually decreases the rate of evacuation of the meal from the stomach when compared with f i s h that fed ad l i b (Fange and Grove, 1979) . Fish that have been deprived of food for some time p r i o r to feeding show a slower g a s t r i c emptying rate; only 50 to 68% of the rates found i n a c t i v e l y feeding f i s h (Fange and Grove, 1979) . Talbot et a l . (1984) found that periods of starvation affected the evacuation rate independent of the effects of meal siz e and f i s h s i z e . F i s h that were prevented from ingesting further meals had a slower evacuation rate. Fish that were starved p r i o r to the feeding t r i a l consumed a larger meal (as a percentage of body weight) than f i s h that were not. Therefore, the rate of passage w i l l not be representative of f i s h under growing conditions with a normal feeding schedule. Fish that are stressed due to handling or disease w i l l also have altered passage times. 1.1.7.1 Characteristics of Feed Which Affect Passage Rates The type of food w i l l affect the g a s t r i c emptying rate. D i g e s t i b i l i t y of the food w i l l a f f e c t the speed of g a s t r i c emptying and w i l l also determine the length of time post-prandial before the 21 decrease of the stomach contents occurs. Fish compensate by increasing t h e i r intake when t h e i r diet i s d i l u t e d with material that reduces the c a l o r i c content (Grove et a l . . 1978) . Before f i b r e and l i p i d s are incorporated into a diet to bulk, bind or add c a l o r i c content to the diet, consideration should f i r s t be given to the effects that these additions may have 'on the passage of the feed through the digestive t r a c t and the r e s u l t i n g e f f e c t on absorption of nutrients. Li p i d s P r a c t i c a l trout diets normally contain between 6 and 14% crude fat (Hilton and Slinger, 1981) . To maintain an adequate l e v e l of energy and the required concentrations of omega-3 and omega-6 fa t t y acids i n the diet, ingredients are supplemented with animal, marine or vegetable f a t s . Diets with 15 to 20% l i p i d levels are b e n e f i c i a l , the higher l e v e l of l i p i d s having a protein-sparing e f f e c t . This allows the quantity of protein required i n the diet to be decreased, providing an economic advantage. However, high l i p i d l e v e l s can cause d i f f i c u l t y i n p e l l e t i n g and crumbling of the diet and a potential for oxidation of the l i p i d s (Hilton and Slinger, 1981) . The eff e c t of higher l i p i d s incorporated into the diets must be considered when formulating the d i e t . The l e v e l of protein combined with the l i p i d l e v e l s also must be considered. Bromley (1980) found that turbot grown on high l i p i d diets with r e s t r i c t e d amounts of feeds had a reduced growth i n weight and 22 length due to the decreased amount of protein i n the diet, even though there was a high l e v e l of l i p i d . Therefore, adequate protein f o r growth i s required before l i p i d can have a protein-sparing e f f e c t . Diets with increased fat levels decrease g a s t r i c evacuation rates i n rainbow trout. The presence of fat i n food may delay emptying by a release from the i n t e s t i n a l wall of a hormone similar to enterogastrone which, in mammals, i n h i b i t s g a s t r i c m o t i l i t y (Fange and Grove, 1979) . Fibre F i s h adjust t h e i r dietary energy intake to d i g e s t i b l e energy le v e l s i n the d i e t . It has been found that f i s h that have been starved and then resume feeding have decreased evacuation rates ( E l l i o t t , 1991). Diets d i l u t e d with i n e r t material are evacuated more rapidly as increased d i l u t i o n speeds up the g a s t r i c emptying time (Jobling, 1981) . Trout, l i k e turbot, w i l l increase t h e i r feed intake to p a r t l y offset a decreased energy supply (Grove et a l . , 1985). Hil t o n et a l . (1983) found that rainbow trout had a faster g a s t r i c emptying time on high f i b r e diets versus control or basal die t s ; however, there was no apparent difference between diets of 10 and 20% f i b r e . Hilton et a l . (1983) suggested that a rapid g a s t r i c evacuation i s a physiological adjustment of trout designed to permit increased consumption of low energy d i e t s . In contrast, Fange and Grove (1979) found decreased evacuation rates with less 23 d i g e s t i b l e foodstuffs; the d i g e s t i b i l i t y of food s t u f f s a f f e c t i n g not only the rate of g a s t r i c evacuation but also the time after eating before stomach contents begin to leave the stomach. These res u l t s can both be true i n that g a s t r i c emptying times vary with such factors as species, f i s h size, temperature and food type. The method of acquiring the passage information can a f f e c t the r e s u l t s . Additions of bran, c e l l u l o s e or guar gum to a meal for humans caused g a s t r i c evacuation to take longer (Eastwood and Brydon, 1984). Fibre may l i m i t nutrient intake and w i l l increase faecal waste production (NRC, 1983). Carbohydrate g e l l i n g agents, such as quar gum, pectin, and carrageenin may delay g a s t r i c emptying or impair absorption within the small in t e s t i n e due to the altered d i f f u s i o n rate or by binding with f i b r e constituents (Elsenhans et a l . , 1980) . Pectin has been found to impair i n t e s t i n a l absorption and reduce postprandial blood glucose and i n s u l i n concentrations i n rats (Elsenhans et a l . , 1984). Moisture Fish grown i n s a l t water have been found to drink water continuously while f i s h i n fresh water drink very l i t t l e (Fange and Groves, 1979). Marine f i s h swallow water to between 5 to 12% of t h e i r body weight d a i l y (Fange and Groves, 1979). In sea raised trout, a syndrome of distended, w a t e r - f i l l e d stomachs have been found to occur where f i s h are grown i n water of high s a l i n i t y (sea l e v e l and higher), and low temperatures. Fish displaying t h i s 24 syndrome have, amongst other symptoms, distended stomachs that are approximately 2 to 6 times the normal stomach size, with a higher moisture l e v e l of stomach content. High l i p i d l e v e l s , above 25%, i n the diet appear to cause a higher frequency of stomach distention. The accumulation of l i p i d i n the stomach can be expected to influence digestion, absorption and metabolism (Staurnes et a l . , 1990). Garber (1983) found i n yellow perch that evacuation rates were not affected by dietary moisture. However, i f there are ingredients within the diet, such as guar gum, which have greater water holding capacities, the evacuation rates may be affected by the dietary moisture. Binders Binders, such as alginates, are used i n f i s h diets to reduce wastage from moist and wet feeds. It i s important to know how the binders act i n the g a s t r o i n t e s t i n a l t r a c t i n order to use them e f f e c t i v e l y . When bran, ce l l u l o s e and guar gum were added to human diets, guar gum was found to have the longest passage time, which may be related to i t s greater water-holding capacity. More viscous meals are emptied more slowly from the stomach (Eastwood and Brydon, 1984) . Storebakken and Austreng (1987) added alginates to moist diets, at levels up to 5% of the dry weight of the feed. They found that the water-binding e f f e c t of the alginates i n the in t e s t i n e was evident by the increased water content of the faeces. 25 1.1.7.2 Methods of Measurement of Passage Time Methodology of measuring g a s t r i c emptying rates can be complicated by the handling of the test animals. A variety of techniques has been used to study the passage time of digesta. One method i s to k i l l f i s h after they have been fed a diet to determine the extent of stomach emptying over time. The degree of breakdown has been measured v i s u a l l y , by volume with both wet and dry weights and by c a l o r i c and biochemical analysis of the residue. Other studies have induced vomiting and used stomach pumps to c o l l e c t the residue (Fange and Grove, 1979) . Methodology of c o l l e c t i n g measurements of gas t r i c emptying rates can be complicated by the handling of the test animals. X-radiography has been used on trout during digestion. A l a b e l l e d isotope can be incorporated into the food to monitor food m o t i l i t y by X-ray techniques (Grove et a l . , 1978). Other techniques have included incorporating dye markers into the d i e t . Digestion rates have been estimated by co r r e l a t i n g the time of feeding to the appearance of the dye i n faeces; however, there can be a problem with f i s h that ingest large meals. The dye can appear i n the faeces while part of the o r i g i n a l meal i s s t i l l in the stomach. This technique i s also based on the premise that the inert marker passes through the digestive t r a c t at the same rate as the rest of the meal (Fange and Grove, 1979) . As discussed previously, d i f f e r e n t types of materials within the diet can affect the passage time. Markers must be non-absorbable, not i r r i t a t e the 26 gut and not stream i n the digestive t r a c t . 2.0 Experiment 1 2.1 Introduction Experiment 1 consisted of feeding t r i a l s involving three experimental diets and one control diet as described above. The ef f e c t on g a s t r i c emptying was determined through a time series d i s s e c t i o n . The diet additions included: c e l l u l o s e , a water insoluble f i b r e which i s commonly used to bulk up the die t ; pectin, a water soluble f i b r e which could be used as a binder and add bulk to the die t ; and, an increased l e v e l of l i p i d as an additional energy source. 2.2 Materials and Methods 2.2.1 Experimental Design Experiment 1 was c a r r i e d out over a 2 6-day period, during which time the f i s h were fed to s a t i a t i o n once d a i l y i n the morning. The f i s h were weighed p r i o r to the s t a r t of the experiment and again at the end of the experiment. The feed consumption per tank was measured d a i l y and on completion of the experiment. The fresh water f a c i l i t y consisted of 16 fresh water tanks of 27 approximately 150 L each. Each tank had a central standpipe sleeve to allow for water c i r c u l a t i o n and flushing. Vancouver c i t y water was dechlorinated by a sodium thiosulphate i n j e c t i o n system. A heat exchanger maintained the water at temperatures between 6.3 and 7.0 °C. The oxygen levels were between 6.4 to 10.8 ppm. The passage through the g a s t r o i n t e s t i n a l t r a c t was determined by dissec t i o n of the stomach and. i n t e s t i n e at timed inter v a l s following feeding. The f i s h were fed on the f i n a l day of the experiment and the contents of the stomach and int e s t i n e were removed and weighed at 1, 6, 12, 18 and 24 hours post-prandial. The f i s h were anaesthetized and weighed. They were then decapitated, opened, and the stomach and i n t e s t i n a l contents removed separately and weighed. 2 .2 .2 Experimental Fish There were 144 f i s h which were anaesthetized, weighed and sorted; f i s h with outlying weights were removed i n an attempt to standardize the population. The f i s h were weighed p r i o r to the star t of the experiment before the feeding regime began and again at the end, after f i n a l feeding to determine the weight gain. At the beginning of the experiment the f i s h weighed between 200 and 440 g. The f i s h were randomly d i s t r i b u t e d into 16 tanks with 9 f i s h per tank. The mean f i s h weight per tank was 280.4 g +, 23.77 g. The f i s h were treated for f i n and t a i l rot 1 week p r i o r to sorting. 28 There were four re p l i c a t e s of each d i e t . The diets were assigned to the tanks randomly to eliminate location bias. The tanks had an i n i t i a l stocking density of 280.41 gram mean weight i n 150 l i t r e s of water to equal 1.87 g/1. 2.2.3 Diet Preparation and Composition The diets were prepared i n the laboratory of the Department of Animal Science at the University of B r i t i s h Columbia. Vitamin and mineral premixes were compiled i n the required amounts before addition to the di e t s . Herring o i l was used to coat the diets to bring them to the desired l i p i d l e v e l s . The compositions of the diets and the vitamin and mineral premixes are presented i n Tables 1 and 2, respectively. 2.2.4 Experimental Protocol The f i s h were fed a commercial diet f o r the f i r s t week. For 4 days p r i o r to the experiment, increasing amounts of the experimental d i e t s - were introduced. By the fourth day the experimental diets were the only source of nutrients. 29 TABLE 1: DIET FORMULATION FOR EXPERIMENT 1 BASAL DIET INGREDIENTS g/kg Diet g Protein/ kg of Diet g L i p i d / kg of Diet HERRING MEAL 500 367.60 46.20 GROUND WHEAT 300 43.17 5.58 OIL - SARDINE 50 50 .00 CALCIUM MONO-PHOSPHATE 10 PREMIX 30 CALCIUM LIGNOSE SULPHONATE 30 WHEY 30 3.60 0.24 FISH SOLUBLES 50 15.75 3.90 DIET % OF BASAL % INGREDIENT OF DIET % MOISTURE DIET 1 100 0.0 13.4 DIET 2 88 12.0 FISH OIL 11.8 DIET 3 88 12.0 PECTIN 11.8 DIET A 88 12.0 CELLULOSE 11.8 TABLE 2: VITAMIN AND MINERAL PREMIX FOR EXPERIMENT 1 VITAMIN mg/kg fed Thiamine HC1 60.00 Riboflavin 100.00 Niacin 400.00 B i o t i n 5.00 F o l i c Acid 25.00 Pyridoxine.HC1 50.00 V i t . B 1 2 - Cyanocobalamine 0.10 D-Calcium Pantothenate 200.00 V i t . C - Ascorbic Acid 1500.00 Choline Chloride (60%) 4000.00 In o s i t o l 2000.00 V i t . A Retinyl Palmitate 10000.00 V i t . D3 - C h o l e c a l c i f e r o l 300.00 V i t . E - DL-<*:-tocopherol Acetate 1000 .00 V i t . K Menadione 30.00 MINERALS mg/kg fed Magnesium as MgS04 380.00 Manganese as MnS04.5H20 30.00 Zinc as ZnO 70.00 Iron as FeSO„.7H20 85.00 Copper as CuS04.5H20 2.00 Cobalt as CoCl.6H20 0.003 Iodine as KI0 3 5.00 Fluorine as NaF 4.50 Selenium as Na2Se03.5H20 0.10 31 The f i s h were fed once d a i l y to s a t i a t i o n on the experimental diets for 26 days. After feeding on the 26th day f i v e f i s h per diet were selected at 1, 6, 12 and 18 hours post prandi a l l y . At 24 hours, the remaining f i s h were sampled which resulted i n four or f i v e f i s h per d i e t 1 . The f i s h were anaesthetized immediately after s e l e c t i o n to minimize ingesta l o s s . The f i s h were k i l l e d , the contents of the stomach removed and weighed. The intestine was also dissected and the contents removed and weighed. F i s h which had not fed were recorded as empty. A l l r e s u l t s were analyzed using Analysis of Variance (ANOVA) using SYSTAT (Wilkinson, 1989), with differences between means tested at P< 0.05, using Tukey's multiple range t e s t . Analysis of Variance was ca r r i e d out on the data to determine i f there were any s i g n i f i c a n t tank e f f e c t s . Since none was shown, the subsequent analysis was c a r r i e d out on the pooled data for the f i s h i n the tanks. 2.3 Results The i n i t i a l and f i n a l weights of f i s h , feed u t i l i z a t i o n and growth are summarized i n Table 4. The stomach contents and i n t e s t i n a l contents over the passage time are i n d i v i d u a l l y shown in Appendix 1 and 2. For detailed results of stomach and i n t e s t i n a l contents by diet see Appendix 3. 1 A portion of the f i s h were used i n another experiment or blood sampling. 32 2.3.1 Feed Consumption and Weight Gain There was no s i g n i f i c a n t difference i n the i n i t i a l weights of the f i s h between the d i f f e r e n t d i e t s . There was a s i g n i f i c a n t difference between diets for the amount of feed eaten and for the weight gain i n conjunction with the amount of feed eaten. The results are summarized i n Table 3. TABLE 3: RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 1 DIETS AMOUNT OF DIET EATEN AS FED PER FISH (g) FISH WEIGHT (COVARIATE OF AMOUNT OF DIET EATEN AS FED) (g) DIET 1 BASAL 72.2A + 13.5 51.7A + 18.8 DIET 2 HIGH LIPID 65.5 M +6.4 49.8A + 11.0 DIET 3 PECTIN 35.0B + 8.3 18.3B ± 1 1 . 6 DIET 4 CELLULOSE 73.5 A + 12.4 38. l c ± 17.3 SAMPLE NUMBERS N=16 N=16 NB: Values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Tukey's test was used to test for differences between the means. The basal diet (Diet 1) and the c e l l u l o s e diet (Diet 4) at P = 0.006, indicated that there was a higher weight gain i n the f i s h fed on the basal diet; the high l i p i d diet (Diet 2) and the ce l l u l o s e d i e t (Diet 4) at P = 0.001 indicated that there was a higher weight gain for the f i s h fed Diet 2; and, between the pectin diet (Diet 3) and the ce l l u l o s e diet (Diet 4) at P = 0.025 33 indicated that there was a higher weight gain i n the f i s h fed on the c e l l u l o s e d i e t . The quantities of feed eaten and weight gained by diet and tank are l i s t e d i n Table 4. The pectin diet (Diet 3) was consumed less and resulted i n poorer weight gain than a l l other diets. There was a s i g n i f i c a n t difference i n weight gain for the high l i p i d d i e t (Diet 2) compared to the c e l l u l o s e diet when the amount of feed eaten i s considered. Diet 2 had the lowest feed-to-weight gain r a t i o and therefore a better feed conversion. The cel l u l o s e diet (Diet 4) was s i g n i f i c a n t l y d i f f e r e n t than a l l other diets for weight gain when the feed per f i s h eaten i s considered. The f i s h fed t h i s diet gained less weight than f i s h fed either the basal diet or the high l i p i d diet but more than those fed the pectin diet.. 34 TABLE 4: EXPERIMENT 1, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN FRESH WATER (9 FISH/TANK) DIET TANK # FEED EATEN (g) FEED /FISH (g) MEAN I N I T I A L F I S H WEIGHT (g) MEAN FINAL F I S H WEIGHT (g) WEIGHT GAIN (g) BASAL 15 698.7 77.6 279.8 343.5 63.7 11 740.3 82.3 310.6 374.1 63.5 13 718.9 79.9 310.8 371.0 60.2 441.0 49.0 270.1 289.4 19.3 MEAN SD 649 121 72.2 13.5 292. 18. 344.5 34.0 51.7 18.8 HIGH L I P I D 2 16 684.9 7 6.1 256.2 324.4 68.2 14 584.1 64.9 260.7 308.9 531.1 59.0 286.9 327.8 48.2 40.9 559.6 62.2 293.5 335.4 41.9 MEAN SD PECTIN 3 589. 58. 65.5 6.4 274. 16. 324.1 9.6 10 341.8 38.0 285.9 284.6 397.8 44.2 253.9 278.3 529.8 58.9 292.3 323.5 402.3 44.7 264.4 281.9 49.8 11.0 0.0* 24.5 31.2 17.5 MEAN SD 417. 68. 35.0 8.3 274. 15. 292.1 18.3 18.3 11.6 CELLULOSE 4 801.2 89.0 302.0 361.7 59.6 718.0 79.8 ' 296.2 346.6 50.4 500.3 55.6 220.6 239.6 19.0 12 625.0 69.4 302.6 326.0 23.5 MEAN SD 661. 111. 73.5 12.4 280. 34. 318.5 47.3 38.1 17.3 NB: * = A NEGATIVE WEIGHT RECORDED AS ZERO. WHERE THE FISH LOST WEIGHT IN THIS TANK, 35 2 . 3 . 2 Evacuation of the Stomach Contents Over Time The data from the stomach contents as a percentage of f i s h weight for the f i s h fed on the d i f f e r e n t diets are summarized i n Table 5 . In Figure 1, there appears to be a difference at 12 hours between Diet 3 and the rest of the di e t s . However, Analysis of Variance showed no s i g n i f i c a n t differences (P=0.108). TABLE 5: EXPERIMENT 1, STOMACH CONTENTS OF FISH GROWN IN FRESH WATER (Average Wet Weights of stomach contents As % body Weight) AT DIFFERENT TIMES POST PRANDIAL HOURS DIET 1 DIET 2 DIET 3 DIET 4 POST CONTROL 12% ADDED 12% ADDED 12% ADDED FEEDING LIPID PECTIN CELLULOSE 1 MEAN 2 .49 2.13 2.95* 3.34 SD 0.90 1.03 0 .77 1.80 6 MEAN 2 .57* 1.33 2.04 2 .28 SD 0 .57 0.87 1.13 1.28 12 MEAN 0 . 81* 0 .53* 1.87* 0.61 SD 0.43 0.69 0.71 0.45 18 MEAN 1.46* 0.83* 1.22 0.65 SD 0.66 0.66 0.31 0.28 24 MEAN 0.28* A C 0.43 A C 0.03** B 1.23 c SD 0.19 0.28 0 .01 0.52 *= One f i s h was empty, data removed from mean ** = Three f i s h were empty, data removed from mean. The mean represents only 2 f i s h . NB 1: When both the stomach and the int e s t i n e were empty of contents, i t was assumed that the f i s h did not eat and therefore i t was not included i n the data. After 12 hours, i f the int e s t i n e had contents, the stomach contents were included i n the mean even i f the stomach was empty. Stomach contents were recorded as zero. NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . 36 As indicated i n Table 5 , over 50% of the digesta was evacuated by 12 hours with the exception of Diet 3 , the pectin d i e t . This took up to 18 hours before 50% of the feed was evacuated as seen i n Figure 1. The c e l l u l o s e diet (Diet 4) was the diet with largest quantity of ingested material (as determined by the 1 hour diss e c t i o n s ) , and the contents l e f t the stomach at a fa s t e r rate than any of the other d i e t s . Figure 1, shows that both the c e l l u l o s e diet (Diet 4) and the high l i p i d diet (Diet 2) evacuated to 50% of the i n i t i a l f i l l by approximately 8 .25 hours. The control diet (Diet 1) took 9 hours to evacuate to 50% of the i n i t i a l contents and the pectin diet took 15 .75 hours to evacuate to 50% of the i n i t i a l quantity. The change i n stomach f i l l from 1 hour sampling to 12 hours sampling can be used to give a rate for the contents evacuated per hour. The c e l l u l o s e diet evacuated the stomach the quickest, and the pectin diet remained i n the stomach the longest. The d i f f e r e n t diets were evacuated at: control diet (Diet 1") 0 .153 g/h; the high l i p i d (Diet 2) 0 .146 g/h; the pectin diet (Diet 3) 0 .098 g/h; and, the c e l l u l o s e diet 0 .248 g/h. When the data were s t a t i s t i c a l l y analyzed, no s i g n i f i c a n t difference among responses to the diets were found except for samples taken at 24 hours (P=0 .018 ) . Tukey's te s t showed differences between the control diet (Diet 1) and the pectin diet (Diet 3) , and Diet 3 and the c e l l u l o s e diet (Diet 4) at P=0.043 and P=0 .029 , respectively. The c e l l u l o s e diet (Diet 4) took longer to f u l l y evacuate from 37 the stomach than did either the control diet (Diet 1) or the pectin diet (Diet 3) . However, afte r 12 hours, most of the stomachs were empty. The major movement of stomach contents occurred p r i o r to 12 hours with the exception of the pectin diet (Diet 3). The sample size f o r Diet 3 at 24 hours was very l i m i t e d (only two f i s h ) . There i s a large v a r i a t i o n i n data due to the differences of feed consumption for i n d i v i d u a l f i s h . Larger numbers of experimental f i s h would be needed to reduce the v a r i a t i o n . The passage time for each f i s h sampled i s represented graphically i n Appendix 1. LL E f > 39 2.3.3 Evacuation of In t e s t i n a l Contents Over Time The data for the passage of digesta through the intestine i s shown i n Table 6. TABLE 6: EXPERIMENT 1, INTESTINAL CONTENTS OF FISH GROWN IN FRESH WATER (Average Wet Weights of i n t e s t i n a l contents as % body Weight) AT DIFFERENT TIMES POST PRANDIAL . HOURS DIET 1 DIET 2 DIET 3 DIET 4 POST CONTROL 12% ADDED 12% ADDED 12% ADDED FEEDING LIPID PECTIN CELLULOSE 1 MEAN 1.10 1.15 1.17* 3.55 SD 0 .38 0.30 0.47 0.68 6 MEAN 4.33* 3.36 1.72 2.94 SD 1.68 1.33 0.93 1.62 12 MEAN 1.98 3.19 1.71* 3.22* SD 1.10 1.53 0.71 2.17 18 MEAN 4 . 67 0.82* 1.05 1.22 SD 1.57 0.28 0.61 0.31 24 MEAN 0.98 0.90 0 . 98 1.34 SD 0 .52 0.56 0.32 0.30 *= One f i s h was empty, data removed from mean NB 1: When both the stomach and the inte s t i n e were empty, i t was assumed that the f i s h did not eat and therefore i t was not included in the data. There were no s i g n i f i c a n t differences found between the amounts of i n t e s t i n a l contents of f i s h reared on the diff e r e n t experimental diets at any time. 40 o DC o < LU * Q o £ cc (/> LU W z u j § LU LL > H - I g Q (/> U «r UU o ^ E Z Q ± PHI </> oc UJ < I— UJ Z CC CM UJ DC D O <fr CO CM i -% sjuajuoo |eu|isaiu| £ Q co E O ^ 111 I O CM 1-LU UJ Q a + f _l O CC z h- i-z o o UJ o a. 1 - CO H h-LU LLI Q a ) a ¥ c © o 0> a (0 o> (8 W c © 4 ^ c o o « c w *J © £ — o s ° i I f * 41 2 . 4 Discussion A l l data indicated a large range of values, making comparisons and conclusions d i f f i c u l t . The f i s h population i n i t i a l l y was diverse, and the poor selection of available f i s h from which to select a standardized population created a test group of f i s h with a wider v a r i a t i o n i n weight than desired. There were some signs of continued f i n and t a i l rot which may have resulted i n reduced feeding and therefore bias the r e s u l t s . For i n d i v i d u a l Diet results see Appendices 1 and 2 . I n t e s t i n a l contents were d i f f i c u l t to analyze because the i n i t i a l dissections represented the residue of meals eaten p r i o r to the f i n a l feeding. The diets w i l l a f f e c t the amount of i n t e s t i n a l contents found i n the early dissections and the time at which the f i n a l meal was f u l l y represented i n the i n t e s t i n e was hard to determine. The f i s h fed the pectin diet (Diet 3) had the lowest feed consumption i n d i c a t i n g that the f i s h preferred the other experimental diets to the pectin d i e t . This may have been due to a texture or flavour caused by the pectin. The c e l l u l o s e diet (Diet 4) resulted i n poorer feed conversion (feed/weight gain), than did the control or basal diet (Diet 1) or the high l i p i d diet (Diet 2 ) . There was no s i g n i f i c a n t difference between the basal diet (Diet 1) and the high l i p i d diet (Diet 2) with regard to the amount 42 of feed eaten and the weight gain. The only s i g n i f i c a n t difference found for evacuation rates from the stomach was between the basal diet (Diet 1) and the ce l l u l o s e diet (Diet 4). The quantity of feed i n the stomach was greater for Diet 4 at 1 hour than for a l l other d i e t s . The ce l l u l o s e diet (Diet 4) and the high l i p i d diet (Diet 2) both evacuate to 50% of the i n i t i a l content by 8.25 hours and the control basal diet (Diet 1) took 9 hours. The pectin d i e t (Diet 3) appeared to take the longest to evacuate stomach contents to 50% of the i n i t i a l f i l l , taking 15.75 hours. The c e l l u l o s e diet (Diet 4) had the fastest rate of evacuation, evacuating at 0.248 g/h, whereas the pectin diet (Diet 3) was the slowest at 0.098 g/h. There was no s i g n i f i c a n t difference between diets and passage time for the i n t e s t i n a l contents. The expected hypothesis that high l i p i d diets would have decreased g a s t r i c evacuation rates i n f i s h was suggested by Windell et a l . (1969) . High l i p i d diets have decreased g a s t r i c evacuation rates i n other species. In t h i s experiment, the high l i p i d diet (Diet 2) appeared to have evacuated to 50% of the i n i t i a l f i l l i n the approximately the same length of time as the control basal diet (Diet 1) and the ce l l u l o s e diet (Diet 4). Fish increase t h e i r food intake by increasing meal size to part l y o f f s e t decreased energy supply (Grove et al.-, 1985). The f i s h ate more of the c e l l u l o s e diet (Diet 4) than of the other d i e t s . There also was a greater quantity eaten i n one feeding. These res u l t s would agree with the research quoted by Grove et a l . 43 (1985). F i s h fed diets containing components of low d i g e s t i b i l i t y have decreased evacuation rates according to Fange and Grove (1979). On the other hand Jobling (1981) reported that i f diets are dil u t e d with i n e r t material, they w i l l be evacuated more rapidly, according to which, the diet with c e l l u l o s e (Diet 4), i n the present experiment should have evacuated more slowly. The pectin diet (Diet 3) could be expected to evacuate more rapidly than Diet 1, the basal control d i e t . However, there was no s i g n i f i c a n t difference noted between these d i e t s . Diet 3 took longer (15.75 hours) to evacuate to 50% of the i n i t i a l f i l l content, than did either the control diet (Diet 1) , the high l i p i d diet (Diet 2) or the c e l l u l o s e diet (Diet 4). The c e l l u l o s e diet was evacuated at the fastest rate, 0.248 g/h while the pectin diet was evacuated at 0.098 g/h. Both Diets 3 and 4 had f i b r e additions. Fibre can be divided into viscous or water-soluble polysaccharides such as guar gum, pectin, and carboxymethyl c e l l u l o s e and into non-viscous or water insoluble polysaccharides such as alpha-cellulose. As stated previously, the addition of f i b r e has an e f f e c t on the movement of nutrients along the ga s t r o i n t e s t i n a l t r a c t , l i k e l y influencing the nutrient absorption (Shiau et a l . , 1988) . The addition of f i b r e to the d i e t has been shown to in t e r f e r e with the absorption of many nutrients such as bivalent metals and with other nutrients such as fats and amino acids by decreasing t h e i r absorption (Southgate and Durin, 1970). Both dietary f i b r e and f i b r e i s o l a t e s have been 44 found to sequester b i l e s a l t s and bind f a t t y acids, thereby decreasing the a v a i l a b i l i t y of cholesterol and by-products of t r i g l y c e r i d e digestion (Vahouny et a l . , 1981) . Barrow pigs fed semi-purified diets containing 17% crude protein had one of the diets substituted with a diet i n which 7.5% of the cornstarch was replaced with an equal portion of pectin. This resulted i n the d i g e s t i b i l i t y of protein being 21.4% higher i n the diets where pectin was not added (Anonymous, 1992). The data for the passage rates for experimental diets with f i b r e additions were c o n f l i c t i n g . The water-soluble f i b r e pectin slowed the rate of passage and the water insoluble f i b r e c e l l u l o s e had a higher evacuation rate to 12 hours than the other experimental d i e t s . The quantity of water within the stomach contents could be due to the pectin i n t e r a c t i n g with the water, r e s u l t i n g i n a high water content of the stomach contents for the pectin diet . When wet weights were used, meal size had a marked e f f e c t on evacuation rates compared to when dry weights of stomach contents ( E l l i o t , 1991) . Wet weight measurements appeared to have a much higher evacuation rate than dry weights, suggesting that the moisture of the bolus affected evacuation measurements ( E l l i o t , 1991). However, the present experiment findings could be suspect due to the large v a r i a t i o n in f i s h size and the poor health of the f i s h r e s u l t i n g i n a high variation of results for the experiment. 45 3 . 0 Experiment 2 3.1 Introduction In experiment 1, when wet weights of stomach and i n t e s t i n a l contents were used to determine passage times, the results were highly variable; therefore, for experiment 2, both wet weights and dry weights of stomach and i n t e s t i n a l contents were calculated for a more accurate estimate of evacuation rates. In Experiment 2, both a fresh water location and a s a l t water location were used. The f i s h were four d i f f e r e n t diets i n the fresh water location; a control basal diet (Diet 1); a diet with g e l a t i n added (Diet 2); a diet with a high l i p i d l e v e l (Diet 3); and a diet with a c e l l u l o s e addition coupled with a l i p i d l e v e l of 19.9% (Diet 4) . In the s a l t water location only Diets 1, 3 and 4 were used due to r e s t r i c t e d space. Gastric evacuation rates were compared for the d i f f e r e n t diets at both locations. Also, the water content of the ingesta i n the stomachs were determined and compared between the locations. 3.2 Materials and Methods This experiment consisted of two locations being compared simultaneously. There were 24 fresh water tanks and 18 sea water tanks used. A continuous flow of water was used i n each f a c i l i t y 46 to maintain the oxygen l e v e l s . A i r stones were necessary at the fresh water f a c i l i t y to maintain oxygen leve l s at 11 to 12.2 ppm. Temperatures at the fresh water f a c i l i t y were between 8.2 and 9.5 °C with an average of 8.57 °C. The s a l t water f a c i l i t y had water temperatures which ranged from 8.5 to 8.7 °C with an average of 8.52 °C. Oxygen leve l s at the s a l t water f a c i l i t y were between 8 and 11 ppm. 3.2.2 Experimental Fish Rainbow trout were transported to the fresh water f a c i l i t y using a transport tank supplied with oxygen. They were placed into tanks i n the fresh water f a c i l i t y to be sorted. A l l f i s h were weighed and sorted; f i s h outside the average f i s h size or with abnormalities were removed to spare tanks. The 424 f i s h remaining weighed between 134 grams and 247 grams. The f i s h were then d i s t r i b u t e d randomly, 10 f i s h per tank r e s u l t i n g i n six replicates for each of the dietary treatments. There were 240 f i s h at the fresh water f a c i l i t y and 184 f i s h at the s a l t water f a c i l i t y . In fresh water there was 183.15 grams mean weight of f i s h per tank in 150 l i t r e s of water to equal a stocking density of 1.22 g/1. In sa l t water, there was 178.96 grams mean weight of f i s h per tank in 150 l i t r e tanks for a density of 1.19 g/1. The f i s h allocated to the s a l t water f a c i l i t y were transported i n a f i s h transport tank with oxygen supplied. A small amount of Ma r i n i l was used to t r a n q u i l i z e the fish, to reduce the stress of 47 transport. The transport took 40 minutes and upon a r r i v a l at the sa l t water f a c i l i t y the f i s h were introduced immediately into s a l t water tanks. 3.2.3 Diet Preparation and Composition The diets were formulated i n the laboratory of the Department of Animal Science of The University of B r i t i s h Columbia (Table 7) . Vitamin and mineral premixes were compiled i n the required amounts before adding to the diets (Table 8). After p e l l e t i n g , herring o i l was sprayed on to coat the diets to bring the diets up to the desired l i p i d l e v e l s . 3.2.4 Experimental Protocol The f i s h were fed on a commercial diet for the f i r s t week. During four days p r i o r to the experiment increasing amounts of the experimental diets were introduced u n t i l the fourth day when the experimental diets were 100% of the diets fed. The f i s h in' both f a c i l i t i e s were fed to s a t i a t i o n once dai l y i n the morning. The f i s h were fed the diets for 14 days. On the 14th day, the f i s h were f i r s t fed, then two f i s h from each tank at each time period were randomly selected, placed i n a M a r i n i l bath to t r a n q u i l i z e them, thus reducing the chance of stress induced vomiting. The f i s h were then placed i n an anesthetic bath to immobilize them (Marinil bath mixture and anesthetic mixture 48 described i n Appendix 13). They were then removed from the bath, weighed and i n s t a n t l y frozen whole i n l i q u i d nitrogen. They were lab e l l e d , bagged and stored i n a freezer for l a t e r analysis. This procedure was c a r r i e d out at time i n t e r v a l s post-feeding. The time in t e r v a l s selected were 1, 6, 12, 18 and 24 hours. When analyzing the stomach and i n t e s t i n a l contents of the test f i s h , the frozen f i s h were s l i g h t l y thawed using a microwave oven, for 2 to 4 minutes, depending on the size of the f i s h . Thawing was just s u f f i c i e n t to allow a scalpel or scissors to be inserted into the r e c t a l passage of the f i s h . The body wall was then cut to allow access to the digestive t r a c t which was s t i l l frozen. 49 TABLE 7 : DIET FORMULATION FOR EXPERIMENT 2 BASAL DIET INGREDIENTS q/kq DIET DIET 2 INGREDIENTS q /kg DIET a PROTEIN/ kg DIET g PROTEIN/ kg DIET q LIPID/ ADDED kg DIET LATER q* Herring Meal 263 .88 175 .24 30 .35 Corn Gluten Meal 141 .29 85 .85 0 .90 Soy Protein 102 .46 86 .80 0 .41 Ground Wheat 341 .37 50 .39 7 .58 Herring O i l 81.00 . 8 1 . 0 0 Premix 40 .00 Calcium Lignose Sulphonate 30 .00 TOTAL 1000.00 398 .75 120 .24 37 .34 q LIPID/ ADDED kg DIET LATER g* Herring Meal 231 .89 154 .00 26 .67 Corn Gluten Meal 124 .07 . 75 .39 0 .79 Soy Protein 90 .22 76 .44 • .0 .36 Ground Wheat 298 .26 44 .02 6 .62 Herring O i l 85 .55 85 .55 Premix 40 .00 Calcium Lignose Sulphonate 30 .00 Gelatin 100 .00 100 .00 TOTAL 1000.00 449 .85 119 .99 33 .54 *= o i l sprayed on l a t e r to coat the p e l l e t s 50 TABLE 7: CONTINUED DIET FORMULATION FOR EXPERIMENTS DIET 3 INGREDIENTS g/kg DIET q PROTEIN/ kg DIET q LIPID/ ADDED kg DIET LATER g* Herring Meal 231.8 9 Corn Gluten Meal 124.07 Soy Protein 90.22 Ground Wheat 298.26 Herring O i l 85.55 Premix 40.00 Calcium Lignose Sulphonate 30.00 Herring O i l 100.00 TOTAL 1000.00 154.00 75.39 76.44 44 .02 349.85 26.67 0.79 0.36 6.62 85.55 100.00 219.99 128.35 DIET 4 INGREDIENTS q/kq DIET q PROTEIN/ kg DIET q LIPID/ mm kg DIET LATER a* Herring Meal 208.70 114.31 24.00 Corn Gluten Meal 111.67 55.96 0.71 Soy Protein 81.20 56.74 0.32 Ground Wheat 2 68.43 32.68 5.96 Herring O i l 77.00 77.00 106.7 Premix 36.00 Calcium Lignose Sulphonate 27.00 Herring O i l 90.00 90.00 Alpha-cellulose 100.00 TOTAL 1000.00 259.68 197.99 *=oil sprayed on l a t e r to coat the p e l l e t s A l l moisture l e v e l s were calculated and moisture lev e l s of the diet brought up to 18% moisture. The portion of the o i l added l a t e r was reserved from the quantity of the diet to sprayed on to coat the p e l l e t s . Diets 1, 3, and 4 were made i n quantities of 15 kg. Diet 2 was made to 7.5 kg. 51 TABLE 8: Vitamin and Mineral Premix for Experiment 2 Vitamin mq/kq fed Thiamine HC1 60.00 Riboflavin 100.00 Niacin 400.00 B i o t i n 5.00 F o l i c Acid 25.00 Pyridoxine.HCl 50.00 V i t . B 1 2 - Cyanocobalamine 0.10 D-Calcium Pantothenate 200.00 V i t . C - Ascorbic Acid 1500.00 Choline Chloride (60%) 4000.00 In o s i t o l 2000.00 V i t . A - Retinyl Palmitate 10000.00 V i t . D3 - C h o l e c a l c i f e r o l 300.00 V i t . E - DL -oc-tocopherol Acetate 1000.00 V i t . K - Menadione 30.00 Minerals mq/kq fed Magnesium as MgSO„ Manganese as MnS04.5H20 (75%) Zinc as ZnO Iron as FeS04.7H20 Copper as CuS04.5H20 Cobalt as CoCl.6H20 Iodine as KI0 3 Fluorine as NaF Selenium as Na2Se03.5H20 380.00 30.00 70.00 85.00 2.00 0 .003 5.00 4.50 0.10 Phosphorus (as Potassium Phosphate) 2070.00 52 The stomach wall peeled away from the bolus cleanly, allowing the bolus to be weighed in pre-dried weigh dishes. The contents were then dried to a constant weight i n a drying oven at 80 °C for 72 hours. The same procedure was followed for the i n t e s t i n a l contents. The p y l o r i c caeca were squeezed out into weighing dishes and a l l i n t e s t i n a l contents removed v i a dissection, weighed and then dried i n a hot-air drying ovens for 72 hours. Analyses of the data was done using Lotus and SYSTAT. S t a t i s t i c a l analyses included Analysis of Variance using SYSTAT (Wilkinson, 1989). Differences between means were tested at P < 0.05, using Tukey's multiple range t e s t . Analysis of Variance was c a r r i e d out to determine i f there were tank e f f e c t s . Since none was shown, the subsequent analyses were ca r r i e d out on the pooled data. 3.3 Results 3.3.1 Fresh Water F a c i l i t y 3.3.1.1 Feed Consumption and Weight Gain There were no s i g n i f i c a n t differences i n the amounts of feed consumed of the d i f f e r e n t test d i e t s . There were s i g n i f i c a n t differences i n the amounts of weight gained per f i s h for the d i f f e r e n t dietary treatments. There was s i g n i f i c a n t l y more weight 53 gained by the f i s h fed the high l i p i d diet (Diet 3) than either the basal (Diet 1) or the gelatin (Diet 2) d i e t s . Although there was a higher weight gain for the high l i p i d diet (Diet 3) than for the cellulose/high l i p i d diet (Diet 4), the difference was not s i g n i f i c a n t (Table 9) . TABLE 9: RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 4 DIFFERENT DIETS IN FRESH WATER, EXPERIMENT 2 , DIETS FISH WEIGHT GAIN (COVARIATE OF AMOUNT OF DIET EATEN) MEAN (g) FEED(AS FED)/ WEIGHT GAIN DIET 1 BASAL 3 8 .0 A + 10.1 0.92A* +0.16 DIET 2 GELATIN 3 8 .9 A + 8 .5 0.92A* +0.16 DIET 3 HIGH LIPID 49.2B + 8 .3 0.70B* + 0.06 DIET 4 CELLULOSE/ HIGH LIPID 40.7AB +11.3 0 .85 A B * + 0.00 SAMPLE NUMBERS N=24 N=24 NB: * = OVERALL SIGNIFICANCE WAS BELOW 5% BUT THE DIFFERENCE BETWEEN DIETS WAS NOT SIGNIFICANT AT P<0.05 BUT WAS AT P < 0 . 1 0 . Mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . There were no s i g n i f i c a n t differences between the weights of the f i s h for each diet before the start of the experiment. Table 10 provides a l l feed and weight information. 54 TABLE 10: EXPERIMENT 2, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN FRESH WATER (10 FISH/TANK) DIET TANK » FEED EATEN (g) FEED / F I S H (g) MEAN I N I T I A L F I S H WEIGHT (g) MEAN F INAL F ISH WEIGHT (g) WEIGHT GAIN (g) F E E D / WEIGHT GAIN BASAL 1 14 3 3 3 . 0 3 3 . 3 1 7 4 . 5 2 2 0 . 3 45 .8 0 .73 1 23 264 .8 2 6 . 5 179 2 1 5 . 0 3 6 . 0 0 .74 1 20 2 3 5 . 2 2 3 . 5 1 8 4 . 5 2 1 2 . 3 2 7 . 8 0 .85 1 3 449 .7 45 .0 1 9 2 . 9 2 3 8 . 3 45 .4 0 . 9 9 1 4 544 .8 5 4 . 5 1 8 0 . 8 231 .1 5 0 . 3 1 .08 1 11 2 6 0 . 4 2 6 . 0 1 7 7 . 3 2 0 0 . 0 2 2 . 7 1 .15 MEAN SD 3 4 8 . 0 1 1 3 . 0 3 4 . 8 1 1 . 3 1 8 1 . 5 5 . 9 2 1 9 . 5 1 2 . 5 3 8 . 0 10 .1 0 .92 0 . 1 6 GELATIN 2 10 3 7 3 . 2 3 7 . 3 1 8 3 . 4 2 2 9 . 6 4 6 . 2 0.81 2 2 3 5 0 . 0 3 5 . 0 1 8 3 . 8 227 .1 4 3 . 3 0.81 2 3 3 5 1 . 0 35 .1 1 6 7 . 7 210 .1 4 2 . 4 0 .83 2 1 3 9 6 . 9 3 9 . 7 1 7 6 . 4 2 2 3 . 6 4 7 . 2 0 .84 2 13 2 5 6 . 4 2 5 . 6 1 9 5 . 6 221 .1 2 5 . 5 1.01 2 16 3 5 4 . 4 3 5 . 4 1 7 0 . 3 1 9 9 . 0 2 8 . 7 1.23 MEAN SD 3 4 7 . 0 4 3 . 7 3 4 . 7 4 .4 1 7 9 . 5 9.4 2 1 8 . 4 1 0 . 6 3 8 . 9 8 . 5 0 .92 0 . 1 6 HIGH L IP ID 3 9 213 .1 2 1 . 3 1 7 4 . 8 2 0 9 . 7 3 4 . 9 0 .61 3 19 3 0 5 . 5 3 0 . 5 1 9 9 . 6 2 4 6 . 5 4 6 . 9 0 .65 3 18 312 . 9 3 1 . 3 178.1 2 2 2 . 6 4 4 . 5 0 .70 3 22 3 9 0 . 3 3 9 . 0 186 .4 2 3 9 . 5 53 .1 0 .73 3 6 412 .0 4 1 . 2 1 9 4 . 7 2 5 0 . 0 5 5 . 3 0 .75 3 5 466 .5 4 6 . 6 1 9 7 . 0 2 5 7 . 5 6 0 . 5 0 .77 MEAN SD 3 5 0 . 0 82 .8 3 5 . 0 8 . 3 188 .4 9 .4 2 3 7 . 6 1 6 . 5 4 9 . 2 8 . 3 0 .70 0 . 0 6 C E L L U L O S E / H I G H L IP ID 4 21 291 .7 2 9 . 2 1 9 4 . 7 2 3 3 . 5 3 8 . 8 0 .75 4 0 3 2 7 . 0 3 2 . 7 1 7 0 . 6 2 1 3 . 6 4 3 . 0 0 . 7 6 4 15 5 1 0 . 3 5 1 . 0 1 7 5 . 6 2 3 8 . 2 6 2 . 6 0 .82 4 7 2 9 1 . 3 29 .1 1 8 2 . 0 2 1 7 . 6 3 5 . 6 0 .82 4 12 2 2 5 . 7 2 2 . 6 189 .4 2 1 4 . 4 2 5 . 0 0 .90 4 17 4 2 1 . 2 42 .1 1 8 6 . 6 2 2 5 . 7 3 9 . 1 1.08 MEAN SD 3 4 4 . 5 94.4 34 .4 9.4 183 .1 8 .2 2 2 3 . 8 9.4 4 0 . 7 1 1 . 3 0 .85 0.11 3.3.1.2 Evacuation of the Stomach Contents Over Time The data from the stomach contents as a percentage of f i s h 55 weight for the f i s h fed on the d i f f e r e n t diets are summarized i n Table 11. TABLE 11: STOMACH CONTENTS OF FISH GROWN IN FRESH WATER (Average Dry Weights of stomach contents As % body Weight) AT DIFFERENT TIMES POST PRANDIAL HOURS POST FEEDING 12 18 24 MEAN SD N MEAN SD N MEAN SD N MEAN SD N MEAN SD N DIET 1 CONTROL 2.07 0.70 (9) 1.82A 0 . 67 (9) 1.48 0 . 69 (11) 1.12 0.61 (8) 0.78 0.64 (9) DIET 2 GELATIN 2.32 0.40 (9) 1.89AB 0.47 (9) 1.67 0.76 (9) 1.48 0 .51 (10) 0.94 0 .53 (10) DIET 3 HIGH LIPID 2.28 0.93 (8) 2.33B 0.69 (9) 1.68 0.58 (7) 1.50 0.80 (ID 1.09 0.58 (11) DIET 4 CELLULOSE 2.71 0.49 (7) 2.45B 0.45 (8) 1.51 0.48 (10) 1.85 1.04 (11) 1.12 0 .43 (10) NB 1: When both the stomach and the in t e s t i n e were empty of contents, i t was assumed that the f i s h did not eat and therefore i t was not included i n the data. After 12 hours, i f the in t e s t i n e had contents, the stomach contents were included i n the mean even i f the stomach was empty. Stomach contents were recorded as zero. NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . There was a s i g n i f i c a n t difference (P=0.014, N=39) between the quantity of contents at 6 hour. Using. Tukey 1s mean comparison there were s i g n i f i c a n t differences between the control diet (Diet 1) and the high l i p i d diet (Diet 3) at P=0.049; and, between the control diet (Diet 1) and the c e l l u l o s e / l i p i d diet (Diet 4) at P=0.027. Fish fed the high l i p i d diet (Diet 3) and the 56 c e l l u l o s e / l i p i d diet (Diet 4) had larger quantities of feed s t i l l present i n the stomach than did the control diet (Diet 1) or the ge l a t i n diet (Diet 2) by 24 hours. In Figure 3, the slopes of the l i n e s describing the weight of the stomach contents were v i s u a l l y compared. The c e l l u l o s e diet (Diet 4) had a steeper slope from 1 hour to 12 hours than the other three d i e t s . For Diet 4, a larger quantity of the diet had been consumed as evidenced by the weight of stomach contents aft e r 1 hour, than was the case with the other d i e t s . By 12 hours, the stomach contents i n f i s h fed Diet 4 had dropped to the same quantity as was the case with the control diet (Diet 1). and was less than i n the f i s h fed the high l i p i d or g e l a t i n d i e t s . It was, therefore, apparent that gastric evacuation for Diet 4 was faster to 12 hours than for the other three experimental d i e t s . In comparing the quantity of the stomach contents from 1 to 12 hours for the d i f f e r e n t diets, the basal diet (Diet 1) evacuated at 0.054 g/h, the g e l a t i n diet (Diet 2) at 0.59 g/h, the high l i p i d diet (Diet 3) at 0.055 g/h and the cellulose/high l i p i d diet (Diet 4) at 0.109 g/h. The cellulose/high l i p i d diet evacuated at a faster rate than did the other experimental d i e t s . After 12 hours the va r i a t i o n in data for Diets 3 and 4 was high making any further analysis of the curves d i f f i c u l t . An estimate of the time i t takes to reach 50% evacuation was car r i e d out on the graph i n Figure 3. Using the dry weights of the CC LU < D 0 CC H 0 CO co Z I -CE O LL H 0 z CO CC Z LL 5 Hi LL 1. 0 •* jy 1 0 z 0 < H LU CO DC • • CO UJ CC D 0 LL o E • UJ CO z LO D < -J -J _J LU UJ 0 0 CM H H LU UJ O Q — T -I < CO < m • E • x 0 1 co IJJ LiJ Q 5 C ffi o v. 0 a n co a c Q c 0 o £ u c w i { o ^ O 0) o LU > 0 58 stomach contents, evacuation times to reach 50% were estimated as follows: the control diet (Diet 1) approximately 20 hours; the gelatine diet (Diet 2), 22.5 hours; the high l i p i d diet (Diet 3) > 24 hours; and, the c e l l u l o s e / l i p i d diet (Diet 4), 23 hours. 3.3.1.3 Evacuation of I n t e s t i n a l Contents Over Time Table 12 presents the data of the dry weight i n t e s t i n a l contents of the f i s h in fresh water over the time series. At 1 hour there was a s i g n i f i c a n t difference between the diets (P=0.030, N=33) . Using Tukey 1 s test for mean comparison, there was a s i g n i f i c a n t difference between the g e l a t i n diet (Diet 2) and the cellulose/high l i p i d diet (Diet 4) at P=0.020. There was also s i g n i f i c a n t difference at 18 hours (P=0.008, N=44). There was a s i g n i f i c a n t difference between the basal diet (Diet 1) and the high l i p i d diet (Diet 3), P=0.006. There were no other s i g n i f i c a n t differences between i n t e s t i n a l contents over time. Figure 4 graphically presents the results demonstrating that the differences were not c l e a r l y defined. As stated previously, i n t e s t i n a l contents were d i f f i c u l t to analyze because the i n i t i a l dissections represented the residual of meals eaten p r i o r to the f i n a l feeding. 59 TABLE 12: INTESTINAL CONTENTS OF FISH GROWN IN FRESH WATER (Average Dry Weights of i n t e s t i n a l contents As % body Weight) AT DIFFERENT TIMES POST PRANDIAL HOURS DIET 1 . DIET 2 DIET 3 DIET 4 POST CONTROL GELATIN HIGH CELLULOSE FEEDING LIPID 1 MEAN 0 .50 M 0.40A 0.52 M 0.59B SD 0 .10 0.07 0.16 0.11 N (9) (9) (8) (7) 6 MEAN 0.54 0.48 0.53 0.59 ' SD 0.09 0.10 0.10 0.05 N (9) (9) (9) (8) 12 MEAN 0.50 0 .53 0.51 0 .57 SD 0.15 0.17 0.16 0.17 N (11) (9) (7) (10) 18 MEAN 0.56A 0.63^ 0.65B 0.57AB SD 0.23 0 .05 0.12 0.14 N (8) (10) (ID (ID 24 MEAN 0.62 0 .50 0.60 0.65 SD 0.19 0.14 0.10 0.13 N (9) (10) (ID (10) NB 1 When both the stomach and the xntestme contents, i t was assumed that the f i s h did not was not included i n the data. After 12 hours, contents, the stomach contents were included were empty of eat and therefore i t i f the int e s t i n e had in the mean even i f the stomach was empty. NB .2: Mean values s i g n i f i c a n t l y (P<0.05) Stomach contents were recorded as zero, with unlike superscript l e t t e r s were dif f e r e n t according to Tukey's t e s t . 60 if) LU O DC O CD Z < DC Q O Z D C PI? O «t UJ O ^ DC < ° z Z Q — 0)5 ui < |_ UJ z oc • • 111 DC D g L L CM CO CM v> c © C o o (0 c © <0 CO • O i CM • O < UJ 0 CM I-UJ 5 + E < (0 < C D Q Q. -I UJ (0 O UJ O l-UJ Q o 0. _J X o X 1- (0 H I-LJJ UJ Q O © > T J 0 c © o o a a w (9 n c c o O o c w CM 2 Z £ 4) P> LU $ 61 3.3.2 Salt Water F a c i l i t y 3.3.2.1 Feed Consumption and Weight Gain There was no s i g n i f i c a n t difference i n the amount of the •various diets consumed. There was a s i g n i f i c a n t difference i n the amount of weight the f i s h gained at P < 0.10. There was s i g n i f i c a n t l y more weight gained by f i s h fed the control diet (Diet 1) than for the high l i p i d diet (Diet 3) . There was no s i g n i f i c a n t difference between the cellulose/high l i p i d diet (Diet 4) and Diets 1 or 3. There was a s i g n i f i c a n t difference at P < 0.10 between weight gains when a COVARIATE of amount of diet eaten was considered between Diets 1 and 2 as well. TABLE 13: RELATIONSHIP OF FEED CONSUMPTION TO BODY WEIGHT GAIN IN RAINBOW TROUT REARED ON 3 DIFFERENT DIETS IN SALT WATER, EXPERIMENT 2 DIETS FISH WEIGHT GAIN MEAN (g) FEED (AS FED) / WEIGHT GAIN DIET 1 BASAL 37.1A + 4.7 0.84 + 0.07 DIET 3 HIGH LIPID 30.7B + 2.3 0.94 + 0.09 DIET 4 CELLULOSE/ HIGH LIPID 33.2AB +4.2 1.01 + 0.22 OVERALL P=0.056* SAMPLE NUMBERS N=18 N=18 NB: Values with unlike superscript l e t t e r s were s i g n i t i c a n t i y (P<0.10) d i f f e r e n t according to Tukey's te s t but not s i g n i f i c a n t at P<0.05. 62 There were no s i g n i f i c a n t differences between the weights of the f i s h for each diet before the sta r t of the experiment. Table 14 provides a l l feed and weight information. TABLE 14: EXPERIMENT 2, FEED CONSUMPTION AND WEIGHT GAINS OF RAINBOW TROUT REARED IN SALT WATER (10 FISH/TANK) DIET TANK FEED EATEN (g) FEED /FISH (g) MEAN INITIAL FISH WEIGHT (g) MEAN FINAL FISH WEIGHT (g) WEIGHT GAIN (g) FEED/ WEIGHT GAIN BASAL 1 B 291.0 29.1 178.7 216.3 37.6 0.77 1 I 344.0 34.4 184.5 228.4 43.9 0.78 1 L 283.0 28.3 177.6 211.3 33.7 0.84 1 D 361.0 32.8 198.4 241.1 42.6 0.77 1 0 291.0 29.1 170.6 203.0 32.4 0.90 1 P 308.0 30.8 174.5 207.0 32.5 0.95 MEAN SD 313.0 29.3 30.7 2.2 180.7 9.0 217.8 13.1 37.1 4.7 0.84 0.07 HIGH LIPID 3 G 268.0 26.8 159.6 194.0 34.4 0.78 3 J 298.0 29.8 175.7 208.1 32.4 0.92 3 R 278.0 27.8 172.2 201.4 29.2 0.95 3 c 281.0 28.1 187.7 215.5 27.8 1.01 3 Q 322.0 29.3 167.3 198.9 31.6 0.93 3 M 307.0 30.7 180.1 209.0 28.9 1.06 MEAN SD 292.3 18.5 28.7 1.3 173.8 9.0 204.5 7.1 30.7 2.3 0.94 0.09 CELLULOSE/HIGH LIPID 4 N 291.0 29.1 182.8 224.7 41.9 0.69 4 A 301.0 27.4 180.4 214.0 33.6 0.81 4 E 307.0 27.9 176.8 207.4 30.6 0.91 4 K 351.0 35.1 183.8 214.1 30.3 1.16 4 H 345.0 34.5 178.3 207.9 29.6 1.17 4 F 441.0 44.1 192.2• 225.5 33.2 1.33 MEAN SD 339.0 50.0 33.0 5.8 182.4 5.0 215.6 7.2 33.2 4.2 1.01 0.22 63 3.3.2.1 Evacuation of the Stomach Contents Over Time Table 15 summarizes weights of the stomach contents at the various timed inte r v a l s for the s a l t water location. They are presented as a percentage of body weight for comparison purposes.. TABLE 15: STOMACH CONTENTS OF FISH GROWN IN SALT WATER (Average Dry Weights of stomach contents As % body Weight) AT DIFFERENT TIMES POST PRANDIAL HOURS DIET 1 DIET 3 DIET 4 POST CONTROL HIGH CELLULOSE FEEDING LIPID (%) (%). (%) 1 MEAN 1.53 1.47 1.38 SD 0 .43 0.64 0.61 N (12) (12) (12) 6 MEAN 0.97 1.42 0.98 SD 0.43 0.62 0 .52 N (12) (11)- -- (12) 12 MEAN 0.84 0.81 0.54 SD 0.39 0.52 0.34 N (12) (11) (12) 18 MEAN 0.65 0.50 0.59 SD 0.28 0.41 0.45 N (12) (12) (12) 24 MEAN 0.43AB 0.28A 0.53B SD 0.33 0.18 0.30 N (12) (12) (10) NB 1: . When both the stomach and the int e s t i n e were empty or contents, i t was assumed that the f i s h did not eat and therefore i t was not included i n the data. After 12 hours, i f the inte s t i n e had contents, the stomach contents were included i n the mean even i f the stomach was empty. Stomach contents were recorded as zero. NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) di f f e r e n t according to Tukey's t e s t . After 24 hours, there was a s i g n i f i c a n t difference between diets in 64 the weight of the stomach contents (P=0.048, N=34). Using Tukey's test for mean comparison, there was a s i g n i f i c a n t difference between the high l i p i d diet (Diet 3 ) .and the c e l l u l o s e diet (Diet 4) , P = 0.038; a higher quantity of Diet 4 remained i n the stomach. There were no other s i g n i f i c a n t differences between the diets for any other time period. Figure 5, i l l u s t r a t e s the rates of ga s t r i c evacuation of the f i s h i n s a l t water fed the d i f f e r e n t d i e t s . The control diet and the diet containing c e l l u l o s e were si m i l a r i n the rates at which they l e f t the stomach. The high l i p i d diet (Diet 3) d i d not leave the stomach u n t i l a f t e r 6 hours. In comparing the quantity of stomach contents from 1 to 12 hours for the d i f f e r e n t diets, the basal diet (Diet 1) evacuated at 0.063 g/h, the high l i p i d diet (Diet 3) at 0.060 g/h and the cellulose/high l i p i d diet (Diet 4) at 0.076 g/h. This indicated that the cellulose/high l i p i d diet evacuated faster for the f i r s t 12 hours aft e r feeding. 65 66 The times to evacuate 50% of the stomach contents were also compared to determine i f the c e l l u l o s e / l i p i d diet did evacuate fa s t e r . According to figure 5 , 50% of the c e l l u l o s e / l i p i d diet (Diet 4) had l e f t the stomach by 10 .25 hours. This was faster than either the high l i p i d diet (Diet 3 ) , at approximately 13 .25 hours, or the control diet (Diet 1) at 14 .25 hours. 3 . 3 . 2 . 2 Evacuation of I n t e s t i n a l Contents Over Time Table 16 presents the data of the dry weight i n t e s t i n a l contents of the f i s h i n s a l t water over the time seri e s . At 24 hours there was a s i g n i f i c a n t difference between diets (P=0.015,N=34) . Using Tukey's test for mean comparison, there was a s i g n i f i c a n t difference between the control diet (Diet 1) and the cellulose/high l i p i d diet (Diet 4 ) , P=0.070; and, between the high l i p i d diet (Diet 3) and the cellulose/high l i p i d diet (Diet 4) at P=0.018. There was no other s i g n i f i c a n t difference between diets throughout the 24 hours. 67 TABLE 16: INTESTINAL CONTENTS OF FISH GROWN IN SALT WATER (Average Dry Weights of i n t e s t i n a l contents as % body Weight) At DIFFERENT TIMES POSTPRANDIAL HOURS DIET 1 DIET 3 DIET 4 POST CONTROL HIGH CELLULOSE FEEDING LIPID % % % 1 MEAN 0 .57 0.62 0.56 SD 0 .14 0.10 0.21 N (12) (12) (12) 6 MEAN 0 . 62 0.64 0.67 . SD 0.20 0.18 0.26 N (12) (ID (12) 12 MEAN 0.71 0.78 0.71 SD 0 .23 0 .35 0 .24 N (12) (11) (12) 18 MEAN 0 .54 0.54 0.62 SD 0 .20 0 .25 0.20 N (12) (12) (12) 24 MEAN 0.43A 0.49AB 0.69B SD 0 .17 0 .14 0.28 N (12) (12) (10) NB 1 When both contents, i t was assumed that the f i s h did not eat and therefore i t was not included i n the data. After 12 hours, i f the inte s t i n e had contents, the stomach contents were included i n the mean even i f the stomach was empty. Stomach contents were recorded as zero. NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . NB 3: SD = 1 standard deviation N = sample size Figure 6 i l l u s t r a t e s the results graphically. The i n t e s t i n a l contents for the d i f f e r e n t diets were si m i l a r i n quantity u n t i l 18 hours at which time the alpha-cellulose diet was retained i n the inte s t i n e longer. The high l i p i d diet was also retained i n the int e s t i n e longer than the control diet, but not s i g n i f i c a n t l y longer. 68 At 12 hours, the diets had increased to the maximum quantity within the i n t e s t i n e . This corresponds to the time i t took for the stomach to evacuate approximately 50% of the i n i t i a l f i l l . Therefore at 12 hours, 50% or more of the meal had passed through the stomach into the i n t e s t i n e . After 12 hours, the feed passed through the int e s t i n e at d i f f e r e n t rates depending on the ingredients of the d i e t . 70 3.4 Comparison of Water content of Stomach Contents of Fish Reared i n Fresh Water Versus Fish Reared i n Salt Water Tables 17 and 18 and Figure 7 compare the water content of the ingesta i n the stomach of f i s h raised i n fresh water to that of f i s h raised i n s a l t water. Water content i n the ingesta i n the stomach of f i s h raised i n s a l t water was consistently higher than that of the f i s h in fresh water. In the f i s h raised i n fresh water, there was a s i g n i f i c a n t difference of water content i n the stomach contents at 1 hour between a l l d i e t s . At a l l other time periods there was no s i g n i f i c a n t difference, P< 0.05. As seen i n Table 17 and Figure 7, the c e l l u l o s e diet (Diet 4) had a higher percentage of water than the other two diets at 1 hour. There was no s i g n i f i c a n t difference between the other d i e t s . 71 TABLE 17: THE WEIGHT AND % WATER CONTENT OF INGESTA IN THE STOMACH OF RAINBOW TROUT RAISED IN FRESH WATER, EXPERIMENT 2 TIME OF SAMPLING DIET 1 CONTROL (g) DIET 1 CONTROL (%) DIET 3 HIGH LIPID (g) DIET 3 HIGH LIPID (%) DIET 4 CELLULOSE (g) DIET 4 CELLULOSE (%) 1 HOUR MEAN SD N 6.40 4.24 .9 67.38A 6.85 4.86 8 66.19A 5.45 4.69 7 75.48B 6 HOURS MEAN SD N 7.41 3.23 9 68 .20 7.45 4.22 9 68 .15 6.96 4.40 8 72.22 12 HOURS MEAN SD N 7.86 3.28 11 69.62 10 .27 3.36 7 68 .85 8.04 1.67 10 68.64 18 HOURS MEAN SD N 6.34 3.07 8 73.28 9.24 5.84 11 70 .16 9.88 4 .21 11 70.91 24 HOURS MEAN SD N 6.18 3.34 9 78 .18 6.35 2.50 11 72.75 6.19 1.62 10 71.79 NB 1: SD= STANDARD DEVIATION N= NUMBER OF FISH IN SAMPLE NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) di f f e r e n t according to Tukey's t e s t . II * = > 73 At the s a l t water location, there was no s i g n i f i c a n t difference between water content of the ingesta i n the stomachs of f i s h fed the experimental diets at 1 and 6 hours. At 12 hours there was a s i g n i f i c a n t difference between the control diet (Diet 1) and the cellulose/high l i p i d d iet (Diet 4); and between the l i p i d diet (Diet 3) and the cellulose/high l i p i d diet (Diet 4) . The stomach contents of the f i s h that were fed the c e l l u l o s e diet had an increasingly higher water content than the stomach contents of the f i s h fed the other two diets, u n t i l at 12 hours, the percentage of water i n the stomach contents of f i s h that have eatjen diet 4 has increased to be higher than water l e v e l s of stomach contents of f i s h fed Diets 1 or 3. TABLE 18: THE WEIGHT AND % WATER CONTENT OF INGESTA IN THE STOMACH OF RAINBOW TROUT RAISED IN SALT WATER, EXPERIMENT 2 TIME OF . SAMPLING DIET 1 CONTROL (g) DIET 1 CONTROL (%) DIET 3 HIGH LIPID (g) DIET 3 HIGH LIPID (%) DIET 4 CELLULOSE (g) DIET 4 CELLULOSE (g) 1 HOUR MEAN SD N 7.90 3.24 12 74.38 5.70 3.31 12 69.16 7.27 2.93 12 71.27 6 HOURS MEAN SD N 7.97 1.93 12 78.25 7.79 2.64 11 73.37 7.37 2.91 12 79.08 12 HOURS MEAN SD N 6.24 1.97 12 78.93* 6.86 2.80 11 80.46A 7.65 3.46 12 86.34B 18 HOURS MEAN SD N 6.40 3.81 12 77.99 4.40 1.88 12 84.48 6.05 4.11 12 81.64 24 HOURS MEAN SD N 3.58 2.31 12 78.73 3.59 2.59 12 84.82 4.57 1.73 10 78.88 NB 1: SD= STANDARD DEVIATION N= NUMBER OF FISH IN SAMPLE NB 2: Mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . 74 3.4 Discussion Fish fed on the high l i p i d diet had a s i g n i f i c a n t l y better feed conversion rate than the f i s h fed on the other experimental d i e t s . The duration of the experiment was short and therefore, further assessment of growth and feeding e f f e c t s were not s i g n i f i c a n t . As seen by the stomach contents of the f i s h i n fresh water, the high l i p i d diet (Diet 3) and cellulose/high l i p i d diet (Diet 4) took longer to leave the stomach than either the basal diet or g e l a t i n d i e t . The cellulose/high l i p i d d i et, however l e f t the stomach at a faster rate for the f i r s t 12 hours aft e r feeding. Similar results were seen i n Experiment 1 with the c e l l u l o s e diet evacuating more rapidly i n the f i r s t 12 hours than did the other experimental d i e t s . The i n t e s t i n a l contents of the f i s h i n Experiment 2 showed a gradual increase u n t i l 18 hours, except i n the f i s h fed the c e l l u l o s e diet i n which the amount of i n t e s t i n a l contents remained f a i r l y constant. At 24 hours, there was a s i g n i f i c a n t difference i n the passage time for i n t e s t i n a l contents between the high l i p i d diet (Diet 3) and the cellulose/high l i p i d d iet (Diet 4) ; however, the high v a r i a t i o n i n data makes a determination at 24 hours very d i f f i c u l t . The f i s h i n s a l t water that were fed the high l i p i d diet (Diet 3) d i d not show movement of ingesta from the stomach during the f i r s t six hours a f t e r feeding. This finding agrees with that for the f i s h i n fresh water. At 24 hours, the amount of the 75 cellulose/high l i p i d diet (Diet 4) l e f t i n the stomach was almost 50% higher than that of the l i p i d alone. In Figure 5, there was again, a steep slope to the curve for the c e l l u l o s e / l i p i d diet stomach content supporting the findings i n the fresh water f a c i l i t y and Experiment 1 that the c e l l u l o s e / l i p i d diet evacuated from the stomach more rapidly i n the f i r s t 12 hours a f t e r feeding than d i d the other experimental d i e t s . This indicates that the c e l l u l o s e had accelerated the passage time of digesta. In contrast, Fange and Grove (1979) stated that additions of i n d i g e s t i b l e materials decreases the g a s t r i c evacuation rate. The c e l l u l o s e diets i n both Experiments 1 and 2 had a faster evacuation rate u n t i l 12 hours a f t e r which the remaining stomach contents took a long time to evacuate. The remaining content may have been mainly c e l l u l o s e which was i n d i g e s t i b l e and therefore not moving out the stomach quickly. Analysis of the stomach contents for c e l l u l o s e would be required to determine i f t h i s i s the case. In fresh water, f i s h that were fed the cellulose/high l i p i d d i e t (Diet 4) had less water i n t h e i r stomach contents at 1 hour, the water l e v e l increasing steadily, u n t i l at 18 hours, the percentage of water i n the stomach contents of f i s h that have eaten Diet 4 was higher than Diets 1 or 3 as seen i n Figure 7. In the s a l t water, stomach contents had an increasing l e v e l of water u n t i l approximately 12 hours aft e r which the amount of water l e v e l l e d o f f . This may have resulted from the f i s h continuing to drink during the hours after the feeding. In fresh water the percent of water of the stomach content increased s l i g h t l y , which 76 could have been a result of the water ingested while feeding or water coming out of the tissues into the stomach. There was no steady increase i n water i n the stomach contents as was seen i n the stomach contents of f i s h i n s a l t water. The stomach contents of f i s h reared i n fresh water having eaten the high l i p i d diet (Diet 3) had a low percentage of water when compared to the stomach contents of f i s h fed the other experimental d i e t s . In s a l t water, the stomach contents of f i s h fed Diet 3 had a lower percentage of moisture for the f i r s t 12 hours but then increased rapidly i n water content a f t e r 12 hours when over 50% of the stomach contents have passed through into the in t e s t i n e . The e f f e c t of moisture on the stomach contents has not been well documented. Its ef f e c t on the rate of passage could a l t e r the rate of feeding and absorption of nutrients, and therefore should be further investigated. 4.0 CONCLUSIONS In Experiment 1 the f i s h fed the c e l l u l o s e diet had poor feed conversion compared with f i s h fed the other d i e t s . This result would be anticipated since the concentration of available nutrients was reduced by the c e l l u l o s e i n the d i e t . The f i s h ate more of the diet due to the decreased energy supply i n the d i e t . In Experiment 2, there was a shorter period of experimentation with c o n f l i c t i n g 77 r e s u l t s between the fresh water and s a l t water s i t e s . In fresh water, the f i s h fed the high l i p i d diet had a better feed conversion than did the f i s h fed the basal d i e t . In s a l t water, f i s h fed the basal diet had a better feed conversion than did the f i s h fed the high l i p i d d i e t . A longer study i n which growth, and feed:gain r a t i o s were obtained, could determine i f the decrease i n protein l e v e l s , with an addition of c e l l u l o s e , impairs growth i f the energy l e v e l of the diet i s maintained through high l i p i d l e v e l s . Diet 3, Experiment 1, the diet containing an addition of a water soluble f i b r e (pectin) did not show any s i g n i f i c a n t difference i n evacuation times; however, when the graphs of the stomach contents are compared (Figure 1) the pectin diet took longer to evacuate to 50% of the i n i t i a l f i l l than did the ce l l u l o s e diet (Diet 4) , the high l i p i d diet (Diet 2) or the control diet (Diet 1). Pectin appeared to slow the gas t r i c evacuation rate down; however, i n Experiment 1, only wet weights were used, therefore the larger quantities within the stomachs could have been due to a large percentage of water. It was noted that the pectin diet resulted i n poorer growth and feed r a t i o conversions. Fibres such as pectin have been found to increase the v i s c o s i t y of digesta, thereby lowering the digestion or absorption of c e r t a i n nutrients (Storebakken and Austreng, 1987) . It would therefore re s u l t in decreased growth and feed conversion r a t i o s . In Experiment 2, the high l i p i d content diet was found to evacuate rapidly a f t e r the i n i t i a l 6 hours. Lipids have been c i t e d 78 as slowing evacuation . rates but the results of these two experiments disagreed with the l i t e r a t u r e findings. The c e l l u l o s e / high l i p i d diet (Diet 4) evacuated rapi d l y for the f i r s t 12 hours aft e r which the rate of evacuation declined. The remaining contents stayed i n the stomach s i g n i f i c a n t l y longer than the other d i e t s . Further analysis of the stomach contents at 12, 18 and 24 hours could have determined i f there was a higher percentage of c e l l u l o s e within the remaining contents. The c e l l u l o s e portion of the diet may have remained longer within the stomach, r e s u l t i n g i n a slower evacuation rate o v e r a l l . Cellulose has been found to impair protein u t i l i z a t i o n from the diet (Fange and Grove, 1 9 7 9 ). However, with the addition of l i p i d , the energy required for metabolic maintenance may be adequate to sustain maximum growth i f s u f f i c i e n t protein and e s s e n t i a l amino acids are provided within the d i e t . The cellulose/high l i p i d d i et, although having a high l i p i d content, had a poor feed conversion when compared to the other experimental diets, i n d i c a t i n g that the c e l l u l o s e may have impaired protein assimilation and therefore growth even when there was available energy i n the form of l i p i d . The energy l e v e l of Diet 4, was maintained by a high l e v e l of l i p i d , but the f i s h ate more of t h i s diet than the other diets when consumption and the quantities of stomach f i l l at 1 hour were compared. There was a s i g n i f i c a n t difference i n the water content of the stomach contents of f i s h fed i n fresh water versus f i s h fed i n s a l t water. There was more water found i n the stomach contents of f i s h 79 reared i n s a l t water. Fish drink water while i n s a l t water which resulted i n a s i g n i f i c a n t difference i n moisture levels i n the ingesta i n the stomach. The water content a f f e c t s the f l u i d i t y of the stomach contents, contents evacuating.faster when there i s a higher water content of the ingesta. 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"Effects of Pre-and Post-Prandial Starvation on Meal Size and Evacuation rate of juvenile A t l a n t i c Salmon, Salmo Salar L." J. F i s h B i o l . 23:551 - 560. Vahouny, G.V., Tombes, R., Cassidy, M.M., Kritchevsky, D. and Gallo, L.L., 1981. "Dietary f i b e r s VI: Binding of f a t t y acids and monolein from mixed micelles containing b i l e s a l t s and l e c i t h i n " . Proceedings of the Society for Experimental Biology and Medicine, 166:12-16. Watanabe, T. 1982. " L i p i d N u t r i t i o n In Fish".Comp.Biochem. Physiol. 73B:3 - 15. 85 Wilkinson, L. 1989. SYSTAT: The System for S t a t i s t i c s . Evanston, IL: Systat, Inc. Woodward, B. 1994. "Dietary Vitamin Requirements of Cultured Young Fish, with Emphasis on Quantitative Estimates For Salmonids". Aquaculture 124:133 - 168. 86 APPENDICES 87 APPENDIX 1: Experiment 1: Stomach Contents Of Fish Reared i n Fresh Water (Wet weight (g) as % Body Weight). 88 FIGURE 1A: Experiment 1: Stomach Contents Of F i s h Reared on a Diet 1 i n Fresh Water (Wet weight (g) as % Body Weight). Stomach Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water •5-o ca o i35 3.5 2.5 1.5 0.5 0 I 4 2 6 | 12 I 16 10 14 20 I 24 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL DIET 89 2LGUHE 2A: Experiment 1: Stomach Contents Of F i s h Reared on Diet 21,in? Fresh Water (Wet weight (g) as % Body Weight) . •I. •uc. JSC-; •W-.m Stomach Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water Time (h) mi- EACH DATA POINT REPRESENTS 1 FISH MET: 2 = HIGH LIPID DIET 90 FIGURE 3A: Experiment 1: Stomach Contents Of F i s h Reared on a Diet 3 i n Fresh Water (Wet weight (g) as % Body Weight). Stomach Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water • f r o 03 a o 55 3.5 L 2.5 1.5 0.5 [] []' I 12 I 10 14 Time (h) 16 20 18 24 22 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 =PECTIN DIET 91 FIGURE 4A: Experiment 1: Stomach Contents Of F i s h Reared on a Diet 4 i n Fresh Water (Wet weight (g) as % Body Weight). •6> o ca Stomach Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water a n u [] 12 16 • [] • 20 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE DIET 92 APPENDIX 2: Experiment 1: I n t e s t i n a l Contents Of F i s h Reared i n Fresh Water (Wet weight of contents as a % Body Weight) 93 FIGURE 2A: Experiment 1: I n t e s t i n a l Contents Of F i s h Reared on a Diet 1 i n Fresh Water (Wet weight (g) as % Body Weight). 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Intestinal Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water • [] 12 16 20 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL DIET 94 FIGURE 2B: Experiment 1: I n t e s t i n a l Contents Of F i s h Reared on Diet 2 i n Fresh Water (Wet weight (g) as % Body Weight) 6 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Intestinal Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water [] [] — i — 12 16 10 14 a 18 20 24 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 2 = HIGH LIPID DIET 95 FIGURE 2C: Experiment 1: I n t e s t i n a l Contents Of F i s h Reared on Diet 3 i n Fresh Water (Wet weight (g) as % Body Weight). 2.2 2.1 2 1.9 1.8 1.7 _ 1.6 _ 1.5 _ 1.4 _ 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 _ 0.5 _ 0.4 0.3 0.2 _ 0.1 0 Intestinal Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water cp [] [] [] 12 16 20 —i— 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 = PECTIN DIET 96 FIGURE 2D: Experiment 1: I n t e s t i n a l Contents Of F i s h Reared on Di e t 4 i n Fresh Water (Wet weight (g) as % Body Weight). Intestinal Contents (As a % Body Wt) Of Rainbow Trout Reared in Fresh Water •a o EQ 20 | 24 18 . 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE DIET 97 APPENDIX 3: S t a t i s t i c a l Results Experiment 1 Fresh Water ANOVA R e s u l t s : Stomach Contents Time P e r i o d Between Die t s 1 P = 0.537 2 P = 0.210 3 P = 0.108 4 P = 0.168 5 P = 0.018 TUKEY HSD MULTIPLE COMPARISON FOR TIME PERIOD 5 Between Die t s 1 and 4: P = 0.043 Between Die t s 3 and 4: P = 0.029 I n t e s t i n a l Contents Time P e r i o d Between D i e t s 1 p = 0.967 2 P = 0.231 3 P = 0.104 4 P = 0.547 5 P = 0.629 No s i g n i f i c a n t d i f f e r e n c e between d i e t s during any time p e r i o d . To t e s t f o r Tank E f f e c t s and ANOVA was done. Results were P= 0.085. APPENDIX 4: EXPERIMENT 1, FRESH WATER, RAW DATA OF STOMACH AND INTESTINAL CONTENTS WET WEIGHTS TIME Dnrri DIET 1 DIET 2 DIET 2 DIET 3 DIET 3 DIET 4 DIET 4 ftSTWBT WNTWBT %STWBT %INTWET %STWET WNTWET fcSTWET WNTWET OVER BODY OVER BODY OVER BODY OVER BODY OVERBODY OVER BODY OVER BODY OVER BODY 3.42 1.28 0.53 0.83 0.00 0.00 6.15 0.86 1 3.66 0.38 2.51 0.87 1.64 0.55 3.29 0.96 1.84 1.41 1.73 1.08 3.41 1.80 4.19 0.95 1 1.37 1.08 2.27 1.39 3.14 1.40 2.25 1.02 1 2.15 1.35 3.60 1.59 3.61 0.93 0.83 1.47 MEAN Z487 1.099 2.128 1.153 ^360 0.935 3.339 1.055 SD 0.896 0.375 1.003 0297 1.367 0.628 1.795 0215 6 0.00 0.00 1.42 0.76 2.64 0.79 3.27 1.01 6 2.25 1.25 0.57 0.77 1.70 0.63 3.82 0.72 6 3.40 1.80 0.68 1.02 0.00 0.04 4.16 1.23 6 1.87 0.80 2.95 2.04 3.15 1.12 0.00 0.13 6 2.76 1.17 1.02 1.17 2.73 1.01 0.16 1.43 MEAN 2.058 1.004 1.328 1.152 2.043 0.717 2.282 0.905 SD 1.150 0.595 0.864 0.470 1.126 0.381 1.820 0.455 12 0.00 0.07 0.00 1.22 0.00 0 1.39 0.85 12 0.48 0.71 0.60 0.45 2.13 0.38 0.74 1.03 12 1.42 0.56 0.12 0.62 0.91 0.97 0.00 0 12 0.36 0.71 0.11 0.53 1.58 0.23 0.00 0.04 12 0.99 0.56 1.80 1.52 2.84 0.46 0.29 1.59 MEAN 0.650 0.523 0.527 0.868 1.493 0.408 0.485 0.702 SD 0.501 0.237 0.671 0.424 0.980 0.322 0.529 0.608 18 0.93 1.45 0.36 1.16 0.00 2.03 1.17 1.10 18 2.26 1.46 1.41 1.43 1.70 1.24 0.85 1.77 18 0.00 1.39 0.00 0.09 0.00 0.16 0.85 1.33 18 1.48 1.01 1.56 1.30 0.92 0.87 0.40 0.88 18 2.62 0.99 0.00 0.14 0.58 0.96 0.00 1.01 MEAN 1.458 1.260 0.666 0.824 0.640 1.052 0.654 1.217 SD 0.939 0.214 0.683 0.585 0.636 0.605 0.409 0.312 24 0.39 1.21 0.54 0.82 0.00 1.45 0.50 1.44 24 0.00 0.02 0.80 0.35 0.08 0.88 1.56 0.88 24 0.47 1.00 0.02 0.60 0.00 0.56 1.86 1.70 24 0.02 1.08 0.34 1.83 0.05 1.04 0.98 1.32 24 0.49 1.59 24 MEAN 0.275 0.979 0.426 0.901 0.033 0.982 1.226 1.338 SD 0.220 0.520 0284 0.561 0.034 0.319 0.522 0.297 • tataUbMa nice (SD) ol vm di ta.Partifakiaf nn i body, dttcfiwnMi o§ tonehdeorflsc odfl mis radot 99 APPENDIX 5: Experiment 2: Stomach Contents O f Rainbow Trout Reared i n Fresh Water 100 FIGURE 5A: Experiment 2: Stomach Contents Of F i s h Reared on Diet 1 i n Fresh Water (Dry weight (g) as % Body Weight). •6-o 03 3. 8 Stomach Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water 3.4 3.2 3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 a [] 12 16 10 14 18 Time (h) 20 22 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL DIET 101 FIGURE 5B: Experiment 2: Stomach Contents Of F i s h Reared on Diet 2 i n Fresh Water (Dry weight (g) as % Body Weight). Stomach Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water •fr o 03 S o 55 3.2 3 2.8 2.6 |_ 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 n [] 12 [] [] -1— 16 20 n 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 2 = GELATIN DIET 102 FIGURE 5C: Experiment 2: Stomach Contents Of F i s h Reared on Diet 3 i n Fresh Water (Dry weight (g) as % Body Weight). Stomach Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water o CO E Q 3.5 L 2.5 2 L 1.5 0.5 • 5 -i 1 — 0 | 4 2 6 [] C ] I 12 I 10 14 Time (h) 16 20 18 22 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 = HIGH LIPID DIET 103 FIGURE 5D: Experiment 2: Stomach Contents Of Fish Reared on Diet 4 i n Fresh Water (Dry weight (g) as % Body Weight). fr o m 6 s: § co 'o Stomach Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water 3.5 L 2.5 1.5 0.5 12 16 10 14 18 Time (h) 20 24 22 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE DIET 104 A P P E N D I X 6: E x p e r i m e n t 2: I n t e s t i n a l C o n t e n t s Of R a i n b o w T r o u t R e a r e d i n F r e s h W a t e r 105 FIGURE 6A: Experiment 2: I n t e s t i n a l Contents Of F i s h Reared on Di e t 1 i n Fresh Water (Dry weight (g) as % Body Weight). fr o pa Intestinal Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 9 12 16 20 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL DIET 106 FIGURE 6B: Experiment 2: I n t e s t i n a l Contents Of F i s h Reared on Die t 2 i n Fresh Water (Dry weight' (g) as % Body Weight). Intestinal Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water fr o 03 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 —I 1 1 1— | 12 I 16 10 14 20 | 24 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 2 = GELATIN DIET 107 FIGURE 6C: Experiment 2: I n t e s t i n a l Contents Of F i s h Reared on Diet 3 i n Fresh Water (Dry weight (g) as % Body Weight). fr o P3 Intestinal Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 10 12 I 16 14 20 18 Time (h) 22 • E3 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 = HIGH LIPID 108 FIGURE 6D: Experiment 2: In t e s t i n a l Contents Of Fish Reared on Diet 4 i n Fresh Water (Dry weight (g) as % Body Weight). o pa o Intestinal Contents (As a % of Body Wt) of Rainbow Trout Reared in Fresh Water 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 [] [] -. $ r I 12 I 10 14 Time (h) — i — 16 20 18 22 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE/HIGH LIPID A P P E N D I X 7 : S t a t i s t i c a l R e s u l t s E x p e r i m e n t 2 F r e s h W a t e r ANOVA R e s u l t s : S t o m a c h C o n t e n t s T i m e P e r i o d B e t w e e n D i e t s 1 P = 0 . 3 5 4 2 P = 0 . 0 1 4 * 3 P = 0 . 8 7 7 4 P = 0 . 3 1 8 5 • P = 0 . 6 1 6 T U K E Y HSD M U L T I P L E COMPARISON B e t w e e n D i e t s 1 a n d 3 : P = 0 . 0 4 3 B e t w e e n D i e t s 1 a n d 4 : P = 0 . 0 2 9 I n t e s t i n a l C o n t e n t s T i m e P e r i o d B e t w e e n D i e t s 1 P '= 0 . 0 3 0 * 2 P = 0 . 4 4 1 3 P = 0 . 8 2 5 4 P = 0 . 5 0 5 5 P = 0 . 3 3 2 T U K E Y HSD M U L T I P L E COMPARISON B e t w e e n D i e t s 2 a n d 4 : P = 0 . 0 2 0 To t e s t f o r T a n k E f f e c t s a n d ANOVA was d o n e . R e s u l t s w e r e 0 . 2 3 3 110 I APPENDIX 8 : EXPERIMENT 2 .STOMACH AND INTESTINAL CONTENTS OF RAINBOW TROUT REARED IM PRESH WATER DOT 1 A M P U N O TANK RSHWT STOMACH SnanckOoam laud . 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0 1.791 0.834 0.386 4.570 3.738 1.730 2 3 231 1.374 0.882 5.904 4.330 1.874 0.954 0 .413 5 0 0 9 4.055 1.755 M E A N 221 3.381 1.363 9.557 6.173 2.520 0 .993 0.407 5.045 4.052 1.673 S O 4 8 2 5 0 3 0.960 8.506 4.041 1.485 0.611 0 2 4 6 2 0 6 4 £ 3 5 6 0.943 2 1 2 8 2 8.402 2 2 7 0 19.447 13.045 4.626 1.887 0.669 9.928 8.042 2 8 5 2 1 2 2 3 3.833 1.719 12.415 8.582 3 8 4 8 0.995 0.446 1 2 4 1 5 11.420 5 1 2 1 2 143 0.000 0.000 M T 0.000 0.000 0.000 0.000 0 2 9 0 0 2 9 0 0 2 0 3 2 3 0 3 7.313 2.414 23.960 16.647 5.494 1.600 0.528 8.191 6.591 2 1 7 5 8 172 0.000 0.000 0.025 0.025 0 .015 0.000 0.000 0 .355 0 .355 0 2 0 8 8 2 2 3 3 5 5 0 1.578 10.947 7.397 3 2 8 8 1.189 0.520 5.638 4.469 1.986 1 0 176 3.547 2 0 1 5 10.802 7 2 5 5 4.122 0.466 0 2 6 5 2 0 3 6 1.570 0.892 1 0 260 6.331 2 .435 19.185 1 2 8 5 4 4.944 1 2 4 7 0.480 6.101 4.854 1.887 1 3 224 4 .853 2.077 1 4 2 5 4 9.601 4 2 8 8 1.167 0.521 5 7 6 8 4.601 2 0 5 4 1 3 173 0.000 0.000 M T 0.000 0.000 0.000 0.000 0.523 0 .523 0.302 1 6 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18.404 15.245 5.031 2 2 194 3.945 2 0 3 3 10.666 6.723 3 4 6 6 0.833 0.429 4 .083 3 2 5 0 1.673 2 2 211 0.000 0.000 0.076 . 0.078 0.036 0.000 0.000 0 .419 0 .419 0.199 M E A N 2 3 3 4.387 1.744 1 1 2 1 8 8.829 2 .733 1.000 0.401 3.997 4 .907 1.952 8 0 S 4 3 3 .033 1.172 7.555 4 .533 1.780 0.637 0 2 4 9 4.353 3 9 0 4 1 2 8 9 4 0 2 3 8 5.308 2 2 4 9 14.170 - 8.862 3.755 1 2 9 2 0.548 6.372 5.080 2 1 5 2 0 2 3 3 6 2 3 9 2.447 18.326 10.067 3.956 1.526 0.598 7.395 5.869 2 3 0 2 7 2 1 4 7.153 3.342 17.926 10.773 5.034 1 2 8 5 0.591 5.987 4 .722 2 2 0 6 7 142 0.000 0.000 0.120 0.120 0.085 0.000 0.000 0.424 0.424 0 2 9 9 1 2 102 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.600 0.600 0 .313 1 2 243 5 3 5 6 2.204 1 3 7 1 2 8.358 3.439 1.669 0.687 8 2 5 3 6.584 2 7 0 9 1 3 234 6 .013 2.368 1 8 2 1 8 1 0 2 0 2 4 .016 1.314 0.517 8 2 9 8 4.984 1.962 1 3 2 3 3 3.934 £ 5 4 7 14.371 8.437 3 6 2 1 1.454 0.624 7.389 5 .913 2 5 3 8 1 7 173 0.000 0.000 0.050 0.050 0.029 0.000 0.000 0.121 0.121 0.070 1 7 2 3 3 6.581 2 8 0 1 18.805 1 2 2 2 4 5 2 0 2 1.311 0.558 8.653 5.342 2 2 7 3 21 269 4.487 1.688 11.949 7.482 2.774 1.818 0.601 7 2 2 9 5.611 2 0 8 8 21 170 0.000 0.000 0.031 0.031 0.018 0.000 0.000 0.494 0.494 0 2 9 1 M E A N 2 1 8 3.923 1.833 10.308 8.384 2 8 6 1 0.954 0.394 4.768 3 8 1 2 1.600 S O S 3 8 2.845 1 2 1 4 7.461 4 .833 1.957 0.685 0 2 8 1 3 .135 2 .452 0.970 112 I APPENDIX 9: EXPERIMENT 2 .STOMACH A N D INTESTINAL CONTENTS Or R A I N B O W TROUT REARED IN rftESH WATER ant M I O U N O T A N K F D B W T STOMACH S] n a i a d Iouaoul In •tfad Gntcntt I Bonsai O m a n D i a j u n Warn Dry T W B ODtnmro ( A j i « a f (WtWakbd 3 uuiicll Cbousa (Aaa«of (W«W«%ta) h l i a in in ia ta og • CO CO 8c* Wl) CO Worn Dry (Aaa«of CO Body W0 CO Wa in Dry CAiaKof CO BodrW» CO Body wo 1 12 3 231 3.418 1.382 11.491 8.072 3216 1.672 0.686 8207 7235 £883 3 228 3.354 1.471 9257 5.803 £568 1258 0.596 8.840 7.481 3281 4 289 6.082 2261 18.561 13.478 5.011 1.823 0.678 10.549 8.726 3244 4 247 6221 £519 18.423 12202 4.840 1.627 0.858 8260 7.633 3090 11 227 5.628 £478 15990 10.384 4.568 1.037 0.457 6.477 5.440 2298 11 211 1.671 0.792 3.441 3.770 1.787 0435 0206 £163 1.728 OS18 14 243 3.506 1.443 11.369 8.063 3218 1228 0.547 8.114 8.766 £793 14 286 3.008 1283 13221 10213 3.877 1.017 0282 8219 3203 £181 20 168 0.000 0.000 0.000 0.000 0.000 0.000 0.000 £158 £158 1200 20 228 1.661 0.733 6.108 4.448 1268 0.734 0225 3.025 4290 1288 23 190 0.749 0.394 3.919 3.170 1.668 0.648 0.448 6222 3275 £829 23 283 £602 0.932 9228 6.628 2.500 1.545 . 0583 9204 7.760 £928 M E A N SOS 232 3223 1280 10.528 7201 £933 1.119 0.462 8.888 5268 £470 30 1.897 0.782 5.788 3220 1.440 0.524 0.198 £828 £121 0.751 2 1 283 3.537 1.335 12.534 8.997 3.385 1.738 0.855 9.394 7.658 £890 1 190 4.190 2205 14.786 10.596 5.577 1.343 0.707 7.687 8.344 3339 2 270 5.441 £015 20.138 14.697 5443 1.813 0708 10.773 8.860 3281 2 231 2.704 1.171 10208 7.605 3292 1.303 0.584 8245 7.042 3.049 8 207 4204 £078 15310 11206 3.414 0.929 0.448 5885 4.956 £394 8 260 6.947 £872 18.588 12.642 4.862 1.825 0.702 8.737 6.812 £658 10 228 5.601 £446 16.881 11280 4.926 0.995 0.435 4.573 3.580 1.563 10 213 4.687 2205 16.438 11.741 5512 1.162 0.545 5274 4.112 1.931 13 173 0.567 0.338 1.764 1.177 0.672 0249 0.142 1289 1.040 0.594 13 279 8.001 £151 21.596 15.595 5.590 1.645 0.590 9.025 7280 £845 IS 203 0.595 0293 3.471 £876 1.417 0.566 0279 4.158 3.593 1.770 18 198 2296 1.171 8.820 6.525 3.329 1249 0.637 7.191 5.943 3.032 M E A N 227 33 3.908 1.673 13.466 8.578 4.119 1243 0.534 8.861 5.818 £429 SOS 1.955 0.763 6.081 4204 1.630 0.485 0.172 £561 £122 0.786 3 3 193 0.000 0 000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 3 273 4.055 1.483 13.418 9.383 3.430 1.057 0.387 11.507 10.450 3.828 8 330 8.324 £522 22.914 14.591 4.421 1.858 0.594 8.448 6.489 1.868 8 291 3.592 1.922 18.794 13202 4.537 1.847 0.635 11.605 9.758 3.353 9 139 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 8 198 2.003 1.011 7.387 5365 £719 0.429 0216 £060 1.632 0.824 18 130 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.966 1.966 1.311 18 178 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.692 1.692 0.981 19 312 5.153 1.652 13.017 8.865 3.162 2235 0.716 12.576 10241 3214 18 289 8.861 £338 20.360 13.369 4.471 1292 0.432 9202 7.911 £646 22 176 233 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 22 2.142 0.847 8235 8093 £408 1.514 0.588 8.704 7.190 £842 M E A N 234 £835 0.981 8.844 3.989 £086 0.861 0298 5847 4.788 1.754 SOS 63 £833 0.941 8.374 3.676 1.879 0.647 0280 4.877 4.108 1.361 4 0 246 3.794 1.542 12255 8.461 3.440 1.934 0.788 11.138 9202 3.740 0 140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 7 177 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.525 0.325 0297 7 238 3664 1.420 9.568 5803 £289 1249 0.484 5.852 4.803 1.784 12 221 4.542 £055 12.724 8.182 1702 1248 0.565 3.879 4.431 £005 12 261 5.411 £073 15525 10.114 3.873 0.882 0.338 4.803 3.921 1.502 13 221 4.693 £123 14.928 10.238 4.832 1.854 0.884 10.922 8.969 4.058 13 236 4.063 1.367 12.381 8288 • 3.241 '1.753 0.685 9.798 8.046 3.143 17 243 328 £823 1.133 11225 8.400 3.428 1.507 0.615 8.536 7.029 £869 17 3.448 1.051 1£138 8.687 £848 1.848 0.583 10279 6.431 £570 21 276 1.481 0.536 5.875 4.395 1.582 1.146 0.415 7.502 6.358 £303 21 212 3.412 1.609. 11.123 7.711 3637 0.781 0.368 5759 4.978 £348 M E A N 237 1111 1263 8.810 6.699 £707 1.192 0.475 6.733 5241 £218 SDS 46 1.679 0.713 4.975 3.382 1.424 0.651 0263 3.573 £938 1.172 113 I A P P E N D I X 8: E X P E R I M E N T 2 .STOMACH ANO INTESTINALCONTENTSOr HAINBoWTftOln* B E A R E D IN fflESH WATER DIET SAUTUNO TANK FISHWT STOMACH a Stoanca 1 DlffiftQBB buaaoi b T o m CONTJLVn ( A l l » o t (WoWttjW) Sn mamWakjbt Warn B r CBftaBa <Aj«»of (W«w«Kta) I ttiannConto) WaraDiy 08 » It) (*> CD VamDqr CAii*af Ct) BoojWQ Ct) Wa TJ Dry ( A i t « a t CD Bosrwt) Co Boor Wt) 1 18 3 150 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.853 1.853 1235 3 260 4.737 1.822 17.787 13.050 3.018 1.644 0.832 8.888 8225 1164 4 258 5422 £ 0 8 4 12201 8.879 £ 8 5 6 1.720 0.664 10244 1624 1330 £ 8 8 3 4 218 3.056 1.402 8.103 5047 £ 3 1 5 2282 1.047 1567 6285 11 163 0.000 0.000 0.000 0.000 0.000 0.000 1.708 1.709 1.048 11 152 0.000 0.000 0.000 0.000 0.000 0.000 1.740 1.740 1.145 14 161 0.153 0.085 £ 0 0 4 1.851 1.023 0.654 0.382 4.108 1451 1.907 14 228 1.705 0.748 6.804 5.099 2237 1.418 0.621 8274 8.858 1052 20 173 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 20 212 1.634 0.771 7.794 6.160 £ 9 0 8 0.633 0299 4.706 4.073 1.821 23 203 1289 0788 6.086 4.487 £ 2 1 0 1.137 0.560 6.988 5.851 £ 8 8 2 23 215 2.759 1283 10.873 8.114 1774 0.633 0294 4.708 4.076 1.896 M E A N 201 1.755 0.748 5979 4224 1.843 1.124 0.373 5247 4.404 £ 0 3 8 S O S 38 1.825 0.728 5557 3.898 1.591 0.668 0.325 1378 £ 6 7 6 1.001 2 1 237 4.061 1.714 15.194 11.133 4.697 1.586 0.669 8.988 7.402 1123 1 238 . 4.465 1.876 16.816 12.451 5232 1.313 0.636 8.121 7.608 1187 2 242 £ 8 0 7 1.160 11.154 8.348 1449 1.738 0.719 10.969 9230 3.814 2 159 0.000 0.000 MT 0.000 0.000 0.000 MT 0.000 0.000 8 188 3.020 1.606 12.355 8.335 4.965 1.094 0.582 6.714 5.620 £ 9 9 0 8 241 £ 5 2 3 1.047 8.395 8.872 £ 8 5 1 1.643 0.682 10.364 8.721 1618 10 236 £ 8 1 8 1.194 12.064 8246 1918 1.500 0.638 8.973 7.473 1166 1178 10 215 5.936 £ 7 6 2 20.055 14.117 6.586 1.335 0.821 6.169 6.834 13 187 0.000 0.000 M T 0.000 0.000 0.000 1.957 1.957 1.046 13 280 £ 8 7 7 1.063 12.649 8.672 3.454 1.690 0.604 10241 8.551 1054 18 257 £ 8 7 0 1.156 12.548 9.578 1727 1.315 0.589 8.845 7.330 £ 8 5 2 18 187 £ 3 0 4 1232 11.448 8.145 4.690 0.994 0.531 8.818 5.923 1167 M E A N 222 £ 8 2 3 1234 11.148 8.323 1648 1.461 0.522 7.605 8.387 £ 7 6 7 S O S 34 1.589 0.721 5680 4.134 1.888 0236 0238 3223 £ 8 4 8 1.056 3 S 280 3.437 1228 18.094 14.657 5234 1.656 0.581 8.826 8.170 £ 9 1 6 3 281 £ 1 5 2 0.768 8.822 7.770 £ 7 6 5 1.486 0.528 11.615 10.129 1605 8 285 3.643 1235 25.811 22.169 7.515 £ 5 1 7 0.853 10.628 8.111 £ 7 4 9 6 303 8.817 £ 8 2 4 12.674 1757 1232 1.815 0.595 11909 1 £ 0 9 4 1965 8 231 £ 6 7 2 1243 8.854 7.082 1068 1.738 0.751 10.439 1703 1767 8 228 0.812 0.355 5028 4214 1.840 1.347 0.568 8.981 7.634 3233 18 226 4.413 1.954 1 £ 7 7 2 8257 3.698 1205 0.533 6.845 5.841 £ 4 9 6 18 318 3082 0.969 11.034 7.852 £ 5 0 1 1.640 0.578 11264 9.445 £ 9 7 0 19 155 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 19 338 8.832 £ 0 5 1 21.315 14.383 4255 £ 8 0 0 0.858 15086 12.188 1805 22 268 £ 4 8 1 0.937 8.663 8.172 £ 3 2 0 1.494 0.582 8.899 1405 1160 22 268 7.522 £ 8 2 8 21.928 14.407 5.418 1.856 0.688 11.027 9.171 1448 M E A N 266 3.856 1.374 13099 9243 1320 1.654 0.595 9.961 1307 1001 S D S 48 £ 5 7 4 0.867 7.147 3.838 1.964 0.676 0211 1628 1049 0.996 4 0 208 5.979 £ 8 6 1 18.317 12.338 5803 1.088 0.521 7.611 6.523 1121 0 226 3.027 1.338 1 £ 3 2 S 8.301 4.116 1.413 0.625 8.876 7.483 1302 7 200 5.837 £ 8 1 8 17.148 11.511 5.755 0.877 0.438 4.992 4.116 £ 0 5 8 7 240 £ 5 0 0 1.042 8.939 6.439 £ 6 8 3 1.036 0.433 6278 5241 £ 1 8 4 12 264 8.599 3257 27.817 19218 7279 1.831 0.693 10.403 8.572 3247 12 210 1235 0.588 8.472 5237 £ 4 9 4 1.926 0.917 10.567 8.641 4.115 13 295 6286 £ 1 3 1 17.862 11.576 1924 1.154 0.391 6.661 5.506 1.867 15 208 7276 3.532 21.543 14267 8.926 1.379 0.668 8309 6.930 1364 17 143 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 17 270 £ 1 3 0 0.789 7.712 5.582 £ 0 6 7 1.508 0.558 8.109 6.801 £ 4 4 5 21 228 1.942 0.848 7.315 5273 £ 3 4 6 1.156 0.505 6258 5.101 £ 2 2 7 21 273 3.164 1.159 10.969 7.803 £ 6 5 9 1.423 0.521 1569 7.148 £ 6 1 7 M E A N 230 3.981 1.897 13035 8.034 1863 1233 0.523 7218 5.987 £ 5 4 6 S O S 40 £ 5 6 5 1.122 7.388 4.867 £ 1 1 3 0.476 0208 £ 8 9 8 2231 0.998 114 I APPENDIX 8 : EXPERIMENT 2,STOMACH AND INTESTINAL CONTENTS OF RAlNBOW TH6Uf REARED IH PHEsH WATER O C T lAkDUMO TANK FBHWT STOMACH a DtfbraBM Uaafca b m a i l t OMini I natal C m Dtfflnoco WaaDrjr TUB OQMTBNTi (Aa.Kof (WaWokkQ 3 nsach Wi%ti WanDrr Cbatatf (Ai>*°r (Waw«*o t MokdOaaa 0 4 • <D <•> Bob) WI) (D Worn Dry (tot* at <l> Boo} WO <D WanDqr (A»l«of (D Body WO Bo* W 0 1 2 4 3 2 6 8 1 2 0 0 0 . 4 5 1 5 7 9 9 4 . 5 9 9 1 . 7 2 9 1 . 6 2 8 0 . 6 1 1 1 0 2 5 1 6 6 2 4 3 2 4 2 3 3 1 8 5 8 7 1 1 . 8 4 6 1 9 2 9 6 1 3 . 5 2 5 4 2 5 3 3 0 6 5 0 . 9 6 4 1 5 1 3 2 1 2 0 6 6 2 7 9 3 4 2 7 2 1 . 4 2 9 0 . 5 2 3 6 7 8 8 5 2 5 9 1 . 9 7 0 1 2 0 4 0 2 5 3 9 . 4 5 2 7 2 4 0 2 2 2 2 4 2 7 9 2 . 5 4 4 0 . 9 1 2 6 2 9 9 6 4 5 5 2 2 1 4 1 . 7 9 7 0 . 6 4 4 1 0 . 0 9 3 6 2 9 6 2 9 7 3 11 1 7 1 0 . 0 3 4 0 . 0 2 0 1 . 6 3 0 1 2 9 6 0 . 8 3 3 0 2 2 9 0 . 1 9 2 3 . 1 0 4 2 7 7 3 1 2 2 3 11 2 3 5 2 2 4 0 0 2 5 3 8 . 9 4 3 6 . 7 0 2 2 8 5 2 1 .581 0 . 6 6 4 9 2 0 4 7 . 9 4 3 3 2 8 0 1 4 1 5 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 2 0 0 0 . 0 0 0 0 . 0 0 0 1 4 2 0 2 2 . 4 4 5 1 2 1 0 9 . 5 5 3 7 . 1 0 8 3 5 1 0 1 2 7 0 0 . 7 7 7 9 . 4 3 0 7 2 6 0 3 2 9 1 2 0 1 5 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 , 0 . 0 0 0 0 . 0 0 0 1 2 1 6 1 2 1 6 1 2 1 0 2 0 2 2 2 3 8 7 2 1 . 7 4 4 1 2 . 0 4 5 8 . 1 7 3 3 6 6 1 1 2 9 0 0 . 6 3 0 6 4 6 7 7 . 0 6 6 3 . 1 8 4 2 3 1 8 4 0 2 6 1 0 . 1 4 2 2 2 2 3 2 0 8 2 1 . 1 2 0 1 . 0 1 8 0 . 5 5 3 5 9 1 4 4 2 9 6 2 6 6 1 2 3 1 6 6 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 M E A N 8 0 S 2 1 8 1 . 6 5 6 0 . 8 5 0 6 2 9 0 4 . 8 3 2 1 2 6 4 1 . 1 5 6 0 . 4 6 6 6 . 9 3 0 5 7 7 4 2 4 0 7 5 4 1 . 7 6 8 0 . 6 5 1 3 6 7 6 3 9 3 8 1 . 4 3 8 0 . 8 9 0 0 2 1 7 4 . 5 4 4 3 . 6 7 0 1 2 1 2 1 1 5 4 0 . 0 0 0 0 . 0 0 0 M T 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 1 2 1 1 1 2 1 1 1 . 1 7 8 2 1 2 2 5 1 . 3 1 8 0 . 5 8 3 9 . 6 7 3 8 2 5 9 3 7 1 5 1 .601 0 . 7 1 2 8 . 5 7 9 6 . 9 7 8 2 1 0 1 2 1 6 9 1 .911 1 .131 7 . 5 9 0 5 . 8 7 9 3 2 6 0 0 . 9 3 1 0 . 5 5 1 6 . 5 4 8 5 . 6 1 7 2 3 2 4 2 2 9 0 3 6 9 5 1 2 7 4 1 8 . 9 5 4 1 3 2 5 9 4 . 5 7 2 1 2 0 3 0 . 5 1 8 7 . 7 9 2 6 2 8 9 2 1 6 9 8 1 6 4 0 . 0 0 0 0 . 0 0 0 M T 0 . 0 0 0 0 . 0 0 0 0 2 2 8 0 . 1 3 9 2 6 1 0 2 3 8 2 1 . 453 8 2 2 1 2 1 4 0 0 . 9 6 8 9 . 6 2 6 7 . 4 8 8 3 3 8 7 1 2 0 2 0 . 5 4 4 6 0 5 0 6 . 6 4 8 2 0 9 9 1 0 2 1 5 1 . 8 8 5 0 . 7 8 4 7 2 7 6 5 . 5 9 2 2 . 6 0 1 1 2 3 6 0 . 5 7 5 6 6 3 8 7 . 4 0 0 2 4 4 2 1 0 2 7 8 5 . 361 1 . 9 2 8 1 9 . 4 0 9 1 4 . 0 4 8 5 . 0 5 3 1 2 3 1 0 . 4 4 3 6 4 9 1 7 2 6 0 2 8 1 1 1 3 2 7 9 4 2 4 9 1 . 5 2 3 1 5 . 5 2 1 1 1 2 7 2 4 . 0 4 0 1 . 7 1 2 0 . 8 1 4 3 2 3 9 1 2 2 7 0 2 4 7 1 3 2 7 9 2 1 2 8 0 . 7 6 3 1 0 . 5 3 7 8 . 4 0 9 3 0 1 4 1 . 6 2 3 0 . 5 8 2 3 1 5 5 1 . 5 3 2 0 2 4 9 1 8 1 9 9 0 . 0 61 0 . 0 3 1 1 . 7 3 9 1 . 6 7 8 0 . 8 4 3 0 . 8 1 8 0 . 4 1 1 5 3 5 5 4 . 5 3 7 2 2 6 0 1 8 1 8 1 0 . 8 7 2 0 . 3 7 1 4 2 2 7 3 . 6 5 5 2 0 2 0 0 . 7 8 0 0 . 4 3 1 5 4 4 6 4 . 6 6 8 2 5 7 8 M E A N 2 2 1 1 . 9 3 5 0 . 7 8 0 6 . 5 5 4 6 . 6 2 0 2 7 1 7 1 . 0 7 2 0 . 4 6 0 5 8 0 9 4 . 7 3 7 2 1 9 4 8 0 8 4 8 1 . 6 8 6 0 . 3 9 6 6 1 6 8 4 . 5 7 0 1 . 6 1 7 0 . 5 2 3 0 . 1 9 4 2 4 5 9 2 2 5 2 0 . 9 9 2 3 S 2 3 9 1 . 1 4 3 0 . 4 7 8 4 . 6 0 9 3 6 6 6 1 . 5 3 4 1 . 6 8 8 0 . 7 0 5 8 . 4 5 8 8 . 7 7 2 2 8 3 4 S 2 6 8 1 . 5 4 6 0 . 5 7 7 5 6 7 5 4 . 1 2 9 1 .541 1 . 5 6 9 0 . 5 8 6 9 . 1 4 5 7 . 5 7 5 2 8 2 7 8 1 7 8 1 2 9 8 0 . 7 8 5 5 . 6 8 8 4 2 9 0 2 . 4 1 0 0 . 9 0 6 0 . 5 0 9 6 2 2 3 5 . 3 1 7 2 9 6 7 6 2 1 8 3 9 0 7 1 . 7 9 2 1 2 5 8 8 8 . 6 81 3 . 9 8 2 1 . 1 7 4 0 . 5 3 8 7 . 4 7 6 6 . 3 0 3 2 8 9 1 8 2 1 6 0 . 9 4 8 0 . 4 3 9 5 2 8 2 4 . 3 3 4 2 0 0 7 1 . 1 3 3 0 . 5 2 5 7 2 1 5 6 0 8 0 2 8 1 5 1 8 2 4 7 2 9 4 9 1 . 1 9 4 1 0 . 4 9 8 7 . 5 4 8 3 0 5 6 1 . 7 6 4 0 . 7 2 2 1 0 2 8 5 6 6 0 0 2 4 8 2 1 8 2 3 9 2 0 2 9 0 . 8 4 9 7 . 7 8 2 5 7 3 2 2 4 0 7 1 2 2 8 0 . 7 6 5 1 0 . 1 7 9 8 . 3 5 1 2 4 9 4 1 9 1 5 8 0 . 0 0 0 0 . 0 0 0 1 7 2 5 4 1 7 2 5 4 1 0 . 9 2 0 0 . 0 0 0 0 . 6 8 3 9 . 8 8 3 6 1 2 8 1 9 2 6 1 5 . 6 6 0 2 1 6 9 7 1 2 4 1 0 . 4 7 6 1 .731 0 . 4 9 0 0 . 1 8 8 2 2 2 1 8 3 . 7 8 7 1 . 7 2 9 1 2 . 4 1 3 8 . 6 2 6 3 9 3 9 1 . 1 2 5 0 2 1 4 6 . 9 0 9 5 7 8 4 2 8 4 1 2 2 2 4 6 2 1 3 0 0 . 8 6 6 8 . 9 9 8 4 . 8 6 8 1 . 9 7 9 1 . 6 1 7 0 . 6 5 7 8 . 9 0 0 7 2 8 2 2 9 6 0 M E A N 2 2 6 2 3 1 8 0 . 9 8 9 8 . 0 9 0 6 . 9 1 5 3 2 7 7 1 . 4 0 6 0 . 5 4 5 7 . 8 4 8 8 . 5 8 7 2 0 2 2 S O S 3 2 1 . 5 5 0 0 . 6 3 3 4 2 3 0 3 8 8 3 2 6 4 9 0 2 0 9 0 . 1 9 6 2 3 3 3 2 2 9 6 1 2 9 3 4 0 2 3 7 1 . 4 0 2 0 . 5 9 2 7 . 1 6 6 5 7 6 3 2 4 3 2 1 . 8 4 7 0 . 7 7 9 1 0 . 0 3 5 6 1 6 8 2 4 5 5 0 2 1 3 2 5 4 9 1 . 1 0 6 5 7 3 1 3 . 1 6 3 1 . 4 9 4 1 . 4 5 3 0 . 6 8 2 9 . 4 9 7 8 . 0 4 4 2 7 7 7 7 2 5 9 2 2 1 5 0 . 8 9 4 8 . 8 8 5 6 3 7 0 2 5 3 7 1 . 8 7 9 0 . 7 2 5 1 0 . 8 7 6 6 9 9 8 2 4 7 4 7 2 4 8 2 0 1 4 0 . 8 1 2 7 . 0 9 1 5 0 7 7 2 0 4 7 1 . 4 8 2 0 . 5 9 8 3 4 1 4 6 . 9 3 2 2 7 9 5 1 2 2 1 4 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 2 1 2 2 2 1 2 2 0 . 9 91 1 2 1 7 3 3 . 1 9 1 1 . 8 4 5 1 0 . 4 1 9 7 2 2 8 4 . 1 7 8 0 . 8 0 2 0 . 4 6 3 6 . 1 1 8 3 2 1 5 2 0 7 2 1 3 1 9 8 2 4 7 8 1 2 5 1 9 2 5 9 6 7 8 1 3 4 2 5 1 2 0 9 0 . 6 1 1 8 . 9 4 4 5 7 3 5 2 6 9 7 1 3 2 2 5 4 . 1 0 3 1 2 2 3 1 3 2 2 7 9 2 2 5 4 . 1 0 0 1 . 6 4 3 0 . 7 3 0 9 . 9 6 9 6 3 4 6 2 7 0 9 1 7 2 1 7 2 5 4 6 1 . 1 7 4 9 . 6 1 7 7 . 0 6 9 3 2 5 8 1 . 6 7 7 0 . 7 7 3 6 . 8 4 7 7 . 1 7 0 3 2 0 4 1 7 2 1 6 1 .021 0 . 4 7 2 3 2 4 1 4 2 2 1 1 . 9 5 4 0 . 8 0 2 0 . 3 7 1 5 . 7 9 8 4 . 9 9 6 2 3 1 3 2 1 1 6 3 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 1 .711 1 .711 1 . 0 5 0 2 1 2 4 4 2 6 3 1 1 . 1 6 0 9 . 8 0 3 6 . 7 7 2 2 7 7 6 1 . 8 1 4 0 . 7 4 3 9 . 6 6 0 7 . 8 4 6 3 2 1 6 M E A N 2 1 7 2 . 0 3 8 0 . 9 3 5 7 . 1 9 3 5 1 5 7 2 3 5 0 1 .461 0 . 5 4 0 7 . 5 0 1 6 2 8 3 2 8 3 8 S O S 2 8 1 . 1 8 2 0 . 5 7 7 3 6 3 3 2 . 7 4 0 1 .311 0 2 8 2 0 2 6 9 2 9 2 8 2 2 9 3 0 . 9 0 0 115 APPENDIX 9: Experiment 2: Stomach Contents Of Rainbow Trout Reared i n S a l t Water' 116 FIGURE 9A: Experiment 2: Stomach Contents Of F i s h Reared on Diet 1 i n S a l t Water (Dry weight (g) as % Body Weight). o 03 Stomach Contents (As a % of Body Wt) 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 of Rainbow Trout Reared in Salt Water 12 i— 16 20 10 14 18 Time (h) 22 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL 117 FIGURE 9B: Experiment 2: Stomach Contents Of F i s h Reared on Diet 3 i n S a l t Water (Dry weight (g) as % Body Weight). o pa Stomach Contents (As a % of Body Wt) 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 of Rainbow Trout Reared in Salt Water • []• [] [] 8 I 12 I 10 14 Time (h) —i— 16 20 18 22 • [] 24 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 = HIGH LIPID 118 FIGURE 9C: Experiment 2: Stomach Contents Of F i s h Reared on Diet 4 i n S a l t Water (Dry weight (g) as % Body Weight). o pa o 55 o Stomach Contents (As a % of Body Wt) 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 of Rainbow Trout Reared in Salt Water u 12 —i— 16 • 20 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE/HIGH LIPID 119 APPENDIX 10: E x p e r i m e n t 2: I n t e s t i n a l C o n t e n t s Of R a i n b o w T r o u t R e a r e d i n S a l t W a t e r . 120 FIGURE 10A: Experiment 2: Stomach Contents Of F i s h Reared on Diet 1 i n S a l t Water (Dry weight (g) as % Body Weight). o m Intestinal Contents (as a % of Body Wt) 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 [] of Rainbow Trout reared in Salt Water [] a n n I 12 | 16 | 20 | 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 1 = CONTROL DIET FIGURE 10B: Experiment 2: Stomach Contents Of F i s h Reared on Diet 3 i n S a l t Water (Dry weight (g) as % Body Weight). 121 o 03 Intestinal Contents (As a % of Body Wt) 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 U 0 of Rainbow Trout Reared in Salt Water 12 16 20 10 14 18 Time (h) • 24 22 NB: EACH DATA POINT REPRESENTS 1 FISH DIET 3 = HIGH LIPID 122 FIGURE IOC: Experiment 2 : Stomach Contents Of F i s h Reared on Diet 4 i n S a l t Water (Dry weight (g) as % Body Weight). Intestinal Contents (As a % of Body Wt) of Rainbow Trout Reared in Salt Water •5-o m o 1.4 1.3 1.2 -1.1 -1 -0.9 a 0.8 [] 0.7 [] 0.6 — [] 0.5 0.4 -[3 n 0.3 0.2 § 0.1 0 ~1— 12 9 16 20 • [] • [] [] 24 10 14 18 22 Time (h) NB: EACH DATA POINT REPRESENTS 1 FISH DIET 4 = CELLULOSE/HIGH LIPID 123 APPENDIX 11: Experiment 2, Salt Water, S t a t i s t i c a l Results ANOVA Results: Stomach Contents Time Period Between Diets 1 • p = 0.819 2 P = 0.096 3 P = 0.188 4 P = 0.641 5 P = 0.048* TUKEY HSD MULTIPLE COMPARISON Between Diets 3 and 4: P = 0.038 I n t e s t i n a l Contents Time Period Between Diets 1 P = 0.675 2 P = 0.849 3 P = 0.800 4 P = 0.665 5 P = 0.015 TUKEY HSD MULTIPLE COMPARISON Between Diets 1 and 4: P = 0.070 Between Diets 3 and 4: P = 0.018 To test for Tank Effects and ANOVA was done. Results were P= 0.2096 124 I APPENDIX 12: EXPERIMENT 2. STOMACH AND INTESTINAL CONTENTS OF RAINBOW TROUT REARED IN SALT WATER! D R Y W T . W B T W T DIFFBRHN03 DIFFERENCE DD7FERBNCB DIFFERENCE WBTWl D R Y W T . D IET S A M P L I N G T A N K PISH S T O M A C H S T O M A C H W B T W T W B T W T W B T W T WBTWT INTESTINAL INTESTINAL mm • W E I G H T CONTENTS CONTENTS V S D R Y S T M V S D R Y S T M V S D R Y V S D R Y INT CONTENTS CONTENTS JhJ JpJ Jfll JgJ (g) ( H B O D Y W T ) INTESTOIAL( I ) Q B O D Y W D (q) (B) 1 B 228 3-968 12.546 8378 3.762 6.868 2324 7363 1.196 1 B 224 1.772 3311 1339 0.687 9.071 4.049 10254 1.184 0 243 3.679 11.099 7.420 3.054 6390 2376 8.124 1.135 CONTROL 0 303 &750 10.114 7384 2.430 7.777 2367 9302 1324 1 221 4.893 17.965 13.072 5315 5.441 2.462 6292 0351 1 241 4.107 16.426 11.319 4.696 9274 3348 10.980 1.708 L 234 4.358 14.038 9.681 4.137 8.749 3.739 10.589 1.839 L 170 0.000 1.764 1.764 1338 3.791 2230 3.791 0300 O 188 2292 8.005 5.713 3.071 6359 3.741 7333 0374 0 207 2.482 8348 6.065 2330 5389 2.700 8377 0388 P 200 3380 11.893 8214 4.157 8356 4278 10233 1377 P 182 3.485 10.790 7304 4.013 5.086 2.794 6.146 1360 MEAN 220 3.397 10.458 7344 3324 6396 3.184 8.174 1285 STD 34 0.916 4.447 3236 1.411 1.684 0.669 2.104 0325 C 210 0.000 0.000 0.000 6317 3.008 6317 0300 3 C 224 1.847 6.669 4.822 2.153 7.163 3.198 8300 1.137 G 199 4.380 13.308 8.948 4.497 6.470 3251 7.699 1229 HIGH G 240 2.417 8257 5.840 2.433 8.497 3.540 9.632 1.135 OIL J 144 0.000 0.000 0.000 3324 2.447 3324 J 255 3.421 10.140 6.719 2.635 10.347 4.057 12264 1318 M 197 0.871 5256 4385 2226 7.179 3.644 8256 1.076 M 211 1.796 5.430 3.634 1.722 6289 3.028 7332 1.142 Q 238 4.732 12.528 7.796 3276 9.159 3.848 10.883 1.725 Q 201 4.675 14268 9392 4.772 7.167 3366 8.634 1.467 R 178 3.818 11.635 7.817 4.441 7.701 4376 8398 1297 R 261 4.178 13.092 8.914 3.415 8310 3.184 9390 1380 MEAN 213 3211 8382 5.706 3.157 7352 3.429 8.494 1371 STD 32 1.307 4.777 3.132 1.042 1.636 0.499 2.132 0274 A 289 4381 14.232 9.650 3.339 7325 2.604 9266 1.741 A 202 4300 15.965 11.464 5.675 6313 3274 7.799 1.185 4 E 281 2.771 8.859 6.088 2333 9.180 3317 11.154 1374 E 212 2.156 7313 5357 2327 6.090 2.873 7302 1212 ALPHA- F 256 4369 15.880 11.011 4318 8.042 3.154 9.195 1.152 CELL F 312 4324 12.457 7333 2343 11.365 3.643 14.047 2.682 H 196 2.077 7.140 5.063 2397 4312 2314 4391 0.479 H 220 1.784 6.854 5.070 2305 8.835 4318 10288 1.453 K 168 0.402 3288 2.886 1.738 5294 3.189 5.944 0350 K 159 3.420 10.072 6.651 4.183 4272 2.687 4393 0.722 N 138 1.193 5335 4.142 3.001 3.958 2367 4311 0356 N 282 6.080 17.988 11.906 4222 10.870 3.855 13.467 2396 MEAN 224 3.197 10.465 7288 3232 7213 3.166 8363 1350 STD 53 1.655 4343 2.935 1.095 2.411 0301 3.127 0.742 125 I APPENDIX 12: EXPERIMENT12. STOMACH AND INTEST INAL CONTENTS OF RAINBOW TROUT REARED IN SALT WATERI DRY wT. WBTWT DIFFERENCE DIFFERENCE DIFFERS! ICS DIFFERENCE WBTWl DRYWT. DDT SAMPUNO TANK FISH STOMACH STOMACH WBTWT WBTWT WBTWT WBTWT INTESTINAL INTBSTINAL TIME # WEIGHT CONTENTS CONTENTS VS DRY STM VS DRY STM VS DRY VS DRY INT CONTENTS CONTENTS (h) (g) (8) (g) (g) (VBODYWT) INTBSTINAL(fJ (*BODYWT) (g) (g) 1 6 B 230 0253 1&696 12.443 5.410 10297 4.477 11547 0250 B 231 3285 11.420 8.134 3521 9560 4.138 11.048 1.486 0 249 2.024 11.185 9.161 3579 8559 3557 9536 1577 CONTROL 0 208 2227 9.005 6.778 3259 6547 3.148 7535 0588 1 212 2519 11.399 8.480 4500 5526 2512 6211 0585 1 278 1348 8248 6599 2519 9.129 3508 10.828 1599 L 275 1.425 7.055 5.831 2547 9.109 3512 10.884 1.775 L 242 1577 8531 6.754 2.791 9270 3530 11.064 1.794 O 185 1.745 9252 7.507 4.058 4.492 2.428 6237 1.745 O 243 3577 12.911 9534 3541 8.983 3.697 10.985 2.002 P 196 1.877 7.078 5201 2554 8.914 3528 8.190 1276 P 224 3.684 13.559 9.875 4.409 7.972 3559 9559 1587 MEAN 231 2203 10.178 7.975 3.499 7596 3.441 9.418 1.422 STD 27 . 0559 2.199 1556 0511 1.724 0564 1.802 0.475 C 224 3.430 12.696 9266 4.137 9.150 4.085 10.547 1597 3 C 240 3.123 11.420 8297 3.457 9207 3536 11.048 1539 G 183 3.308 12.511 9205 5.647 4.636 2544 5586 0.750 HIGH G 182 0.981 4.077 3.098 1.701 3.912 2.149 4547 0535 OIL J 195 0.897 4.013 3.116 1598 5.734 2541 6.732 0598 J 195 3.149 11.017 7.868 4.035 5.935 3.043 7.009 1.075 M 233 2.842 11.562 8.720 3.742 11.499 4.935 13.843 2544 M 232 1518 7.460 5.842 2518 9.846 4244 12.062 2216 Q 196 4285 15247 10.962 5593 6565 3549 7.795 1230 O 138 0.000 0.000 0.000 0.000 2.698 1584 2.698 0.000 R 207 4.711 12.557 7.846 3.790 4568 2.400 6.152 1.184 R 214 3.877 15.351 11.474 5562 6517 2552 7271 0554 MEAN 201 2.929 9.826 7.141 3.465 6.706 3230 7.949 1557 STD 29 1208 4.644 3520 1.667 2549 0556 3.134 0514 A 230 3239 11.708 8.469 3.682 6554 2.763 8.031 1.677 A 231 3501 14.660 10.759 4.858 7242 3.135 8.696 1.453 4 E 190 1.694 10.018 8.324 4581 5.170 2.721 7549 2.179 - E 245 1.672 6513 5.141 2.098 6.559 2.677 8.138 1580 ALPHA- F 222 2215 13.404 11.190 5.040 8.644 3594 10.656 2.011 CELL F 198 3.692 12.824 9.132 4.612 5.482 2.768 6.644 1.163 H 170 0.680 3.760 3.080 1512 3577 1.987 3.762 0585 H 209 1.198 6294 5.096 2.438 8587 4.108 10.180 1594 K 230 2299 8567 6.669 2599 10.371 4509 12.460 2.089 K 258 3.793 13.485 9.692 3.757 7255 2.812 9.177 1.922 N 222 1.635 10.839 9204 4.146 7546 3509 8.668 1522 N 169 0.070 1.716 1.646 0.974 2.640 1562 2.923 0283 MEAN 215 2.174 9541 7.367 3575 6588 3.020 8.057 1.471 STD 27 1206 3.948 2.913 1250 2.105 0508 2582 0589 126 [APPENDIX! 12: EXPERIMENT 2. STOMACH AND INTESTINAL CONTENTS OF RAINBOW TROUT REARED IN SALT WATER) DRYWT. WBTWT 3D7FERENCB DIFFERENCE DIFFERS •JOB DIFFERENCE WBTWl DRYWT. DIET SAMSUNG TANK FISH STOMACH STOMACH WBTWT WBTWT WBTWT WBTWT INTESTINAL INTESTINAL TUB * WEfOHT CONTENTS CONTENTS VS DRY STM VS DRY STM VS DRY VS DRY INT CONTENTS CONTENTS (h) (s) (g) (g) la) C*BODYWT) INTESTINAL (l) (•BODYWT) (s) (g) DIET 12 B 182 1.104 5.719 4315 2336 8.147 4.476 9372 1325 1 B 211 0383 3.003 2.640 1251 6.175 2327 7353 1.178 D 290 1.748 8.769 7.021 2.421 10.325 3360 12.150 1325 CONTROL D 224 1.465 6281 4216 2.150 7377 3361 9323 1346 1 232 1.938 9233 7295 3.144 10.681 4304 12.888 2206 1 207 3.070 12.132 9.062 4378 8.484 4389 9339 1.475 L 199 1386 5.143 4.056 2338 8.495 4269 10.171 1378 L 196 2.411 10388 8.176 4.172 5322 2362 5332 0210 O 167 0358 6.193 5335 3.194 5238 3.136 6358 0320 O 190 2.778 11.512 8.736 4398 6231 3279 7.067 0336 P 242 2306 10.407 7301 3265 10.328 4287 13.097 2.771 P 200 1325 7.014 5.189 2395 8392 4.196 9334 1.442 MEAN 212 1.762 7399 6237 2378 7356 3.744 9.474 1318 STD 31 0.791 2.732 1374 0373 1.861 0337 2.409 0388 C 222 0380 4318 3.738 1.684 7228 3258 8.755 1327 3 C 197 1.101 5.472 4371 2219 7375 3.998 9367 1.491 G 144 0.000 0.000 0.000 0.000 2.622 1.821 2.722 0.100 HIGH G 259 3310 13.783 10273 3.968 2.928 1.131 5.081 2.153 OIL J 272 2.982 12.188 9205 3384 10.568 3.885 12.798 2230 J 182 1.814 8.474 6.660 3.660 8.179 4.494 9385 1305 M 219 3390 14.333 10.743 4.905 9230 4214 11.743 2314 M 210 0.054 2.997 2.943 1.401 5313 2.625 6.136 0.623 Q 170 1.961 7.822 5.662 3330 6.102 3389 7357 1255 O 202 2.190 9358 7.168 3348 7.479 3.703 8.499 1.020 R 165 0.710 4.623 3.913 2372 14.058 8319 18.378 2321 R 214 2.481 13252 10.770 5.033 10.932 5.108 13.196 2264 MEAN 205 1307 8.035 6287 2359 7.726 3362 9335 1.609 STD 36 1.138 4.484 3319 1.413 3.134 1.786 3.848 0.720 A 245 1.408 11.310 9302 4.042 9300 3378 10.747 1247 DIET A 195 0304 6.428 5322 2332 6.695 3.433 7.141 0.446 4 E 194 0307 2302 2.195 1.131 5.187 2.674 5323 0.637 E 202 0.861 5363 4.702 2328 7375 3.651 8.976 1.601 ALPHA- F 333 2.843 14.787 11.944 3387 16.105 4336 20.005 3.899 CELL F 209 0326 8.132 7.606 3.639 8399 4.114 10.206 1.807 H 280 0.620 7326 6.706 2.395 2.789 0.996 5.111 2321 H 270 1.797 10.301 8304 3.150 10.819 4.007 1&694 1374 K 233 1371 9377 7305 3221 8265 3347 10295 2331 K 212 0209 2.452 2244 1.058 8374 4.044 10.150 1377 N 300 1.752 14.403 12.650 4217 10.903 3.634 12960 2.057 N 195 2392 14.883 12291 6303 7355 4.028 9315 1.660 MEAN 239 1307 8.955 7.648 3.159 8356 3370 10.302 1.746 STD 45 0338 4.198 3.463 1357 3.143 0318 3.725 0337 127 I APPENDIX 12: EXPERIMENT 2. STOMACH AND INTESTINAL CONTENTS OF RAINBOW TROUT REARED IN SALT WATER! DRYWT. WBTWT I HFFBRBNCB DIFFERENCE DIFFERENCE DIFFERENCE WBTWt DRYWT. DIET SAMPUNO TANK FISH STOMACH STOMACH WBTWT WBTWT WBTWT WBTWT INTESTINAL INTESTINAL TTMB * WEIGHT CONTENTS CONTENTS VS DRY STM VS DRY STM VS DRY VS DRY INT CONTENTS CONTENTS (h) (g) (g) (fl) (g) (*BODYWT) INTESTINAL CD (SfcBODY WT) (g) (g) DIET 18 B 290 1578 14.625 12.747 4596 11.306 3598 13576 2.070 1 B 139 0505 2.189 1564 1.413 3520 2532 3.765 0545 D 260 2508 4.039 1531 0589 1566 0502 2317 1551 CONTROL D 209 1590 2.796 1506 0.721 1554 0.744 2567 1.413 I 217 1591 7528 5537 2590 5582 2572 8516 0534 I 233 0.779 7519 6540 2507 8575 3594 9.481 1.106 L 221 2.429 12.422 9593 4522 9535 4560 11587 1552 L 172 1.709 16.063 14.354 8545 5544 3.456 6.781 0337 O 1S8 0542 4.046 3504 2518 5.482 3.469 6503 0522 O 222 1.632 10.169 8537 3545 8.087 3543 9.762 1375 P 215 1554 5527 3.773 1.755 5524 2.430 6.790 1568 P 196 0567 7.408 8542 3538 8.734 4.458 9562 0.827 MEAN 211 1.424 7.826 6.402 3.053 6551 2580 7.434 1.183 STD 40 0.684 4.410 4.123 2.030 2554 1509 3552 0532 C 201 1.685 7.839 6.154 3.062 9566 4.610 11.218 1551 3 C 195 1.733 6543 5.110 2.620 6581 3529 8.013 1.132 G 210 2583 11.809 8.826 4503 9508 4528 11565 1.756 HIGH G 179 1511 7.882 6.370 3559 7.743 4526 9.405 1.662 OIL J 188 0.724 4.090 3.366 1510 4376 2.622 5.717 0541 J 179 0.331 2552 2520 1.408 5550 2.989 5591 0541 M 245 0565 3.664 3599 1546 6.775 2.765 7519 1.045 M 162 0.062 2538 2576 1.405 3558 2.073 3.630 0572 O 255 0576 4573 4.197 1.846 11.449 4.490 12.952 1503 0 155 0.177 2540 2.163 1595 3.935 2539 4.494 0558 R 223 1.421 5.676 4555 1508 8556 3.837 10.027 1.470 R 198 0588 4509 4521 2.132 4.809 2.429 5533 0524 MEAN 199 0.996 5593 4596 2508 8.876 3.395 7.980 1.105 STD 29 0538 2.682 1.881 0.911 2394 0.895 2584 0537 A 246 1516 4.673 3.157 1583 4538 1566 8.878 2.042 DIET A 154 0598 7537 6.839 4.441 10.092 6553 11.101 1.009 4 E 174 0583 3.625 3.042 1.748 5.427 3.119 6586 0560 E 180 0.499 3515 2.716 1509 6.199 3.444 7557 1.058 ALPHA- F 269 1552 5514 3.962 1.473 13.437 4595 15.842 2.405 CELL F 243 1506 6.420 4.914 2.022 10.670 4391 12.615 1545 H 223 0.987 8.154 5.167 2517 7578 3564 8.608 1530 H 193 0593 5544 4.952 2566 6.732 3.488 7.637 0.905 K 184 0541 2.444 1.903 1.034 6.891 3.745 7.155 0564 K 280 0.729 8578 7.648 2.732 9558 3.413 11.314 1.757 N 257 3.740 15.103 11.362 4.421 1Z144 4.725 14505 2.061 N 247 4.100 21.021 16.921 6551 8.426 3.411 9.694 1568 MEAN 221 1579 7.427 6.049 2.700 8.474 3376 9383 1.409 STD 40 1500 5.163 4.113 1.645 2.604 1.110 3.003 0.608 128 I APPENDIX] 12: EXPERIMENT12. STOMACH AND INTESTINAL CONTENTS OF RAINBOW TROUT REARED IN SALT WATER] DRYWT. WBTWT DIFFERBNCB DIFFERBNCB DIFFEREh ICB DIFFERBNCB WBTWl DRYWT. DIET SAMFUNQ TANK FISH STOMACH STOMACH WBTWT WBTWT WBTWT WBTWT INTESTINAL INTESTINAL TTJffl • WEIGHT CONTENTS CONTENTS VS DRY STM VS DRY STM VSDRY VSDRY INT CONTENTS CONTENTS (h) (0) (g) (g) (g) CtBODYWT) INTESTINAL (l) (••BODY WT) (g) (g) DIET 24 B 275 0.796 3.000 2204 0302 6339 2323 8300 1361 1 B 153 0202 1.000 0.798 0322 2.438 1393 3.000 0362 D 250 0392 2.000 1308 0323 5346 2378 7300 1354 CONTROL D 1S6 1394 8300 6306 3371 4381 2388 8300 1319 0 220 2367 8300 5333 2351 6355 £752 7300 1.445 1 221 0.400 5300 5.100 2308 1352 0.702 2300 0348 1 224 0380 3300 2320 1336 5.180 2312 6300 1321 L 274 1.095 7300 6.405 2337 0.071 0.026 1300 0329 L 130 0.097 1.000 0303 0395 0.000 0.000 0.000 0.000 O 190 0.448 2.000 1352 0317 3268 1.720 4.000 0.732 O 282 1359 10.000 8.041 2351 7311 2399 9.000 1390 P 175 0.000 0.000 0.000 0.000 0.000 0.000 0.000 P 240 1324 4300 3.476 1.448 5.024 2.093 6.000 0378 MEAN 218 0396 4346 3.427 1.489 3.751 1.630 4.654 0378 STD 48 0.756 3207 2351 1.037 2.605 1.029 2.977 0390 C 261 0.433 2300 2.087 0.792 3.809 1.459 4300 0391 3 C 181 0.054 1.000 0.946 0323 4.088 2259 5.000 0.912 G 169 0.842 3.000 2.158 1277 2.803 1.658 3300 0.698 HIGH G 195 0.857 7.000 6.143 3.150 5237 2.686 6300 1263 OIL J 239 0.492 2300 2.008 0340 7.184 3.006 8300 1316 J 234 0256 1.000 0.744 0318 4371 2.124 6.000 1.030 M 172 0.181 2.000 1319 1.058 3.303 1.920 4.000 0.698 Q 252 1.618 12.000 10.382 4.120 6388 2335 7300 1.112 Q 195 0361 3.000 2.639 1353 5220 2.677 6300 1281 Q 188 0355 4300 3.945 2.098 4.185 2226 5.000 0215 R 203 0341 3.000 2.460 1212 6.427 3.166 8.000 1373 R 153 0.645 5.000 4355 2.847 2.475 1.617 3.000 0325 MEAN 204 0369 3.875 3308 1.632 4.674 2278 5.667 0.993 STD 34 0292 2.938 2377 1.125 1.431 0329 1.712 0307 A 199 1.654 7.000 5346 2.686 5279 2.653 6300 1221 A 214 2293 7300 5207 2.433 5222 2.440 6300 1278 DIET A 149 0.000 0.000 0.000 4 E 220 1330 4300 3.170 1.441 5395 2343 6300 0305 E 196 1387 5.000 3.413 1.742 4.127 2.105 6300 2373 ALPHA- F 218 1381 10.500 8319 4.091 7.804 3380 9300 1.696 CELL F 183 0.000 0.000 0.000 0.000 2335 F 189 0397 4300 3303 1307 4.622 2.446 5300 0.878 H 192 0299 2300 2201 1.146 4.721 2.459 5300 0.779 H 127 0.000 0.000 0.000 K 219 0338 5300 4.662 2.129 5.468 2.497 7.000 1332 K 200 0273 5300 4.627 2314 5.755 2377 7.000 1245 N 189 0.000 0.000 0.000 N 248 0.954 5.000 4.046 1.631 7.135 2.877 8300 1365 MEAN 198 1231 4.107 3228 2.152 5373 2.648 4.929 1.419 STD 29 0339 3.124 2313 0.785 1.067 0377 3272 0313 APPENDIX 13: MARINIL AND ANESTHETIC DOSAGE MARANIL DOSAGE 150 L tanks Dosage Desired = 0.25 ppm or .25 mg Maranil/L water Add to tank p r i o r to handling. ANESTHETIC DOSAGE 10 gallon container 1 g MS222 to 1 g bicarbonate of soda (buffer) Mix into 10 gallon container of water. Hold f i s h i n container u n t i l they are immobilized s u f f i c i e n t l y 

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