Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Plasma concentrations of amino acids in rainbow trout (Oncorhynchus mykiss) in response to nutritional… Tantikitti, Chutima 1994

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

Item Metadata

Download

Media
831-ubc_1994-893796.pdf [ 5.17MB ]
Metadata
JSON: 831-1.0088016.json
JSON-LD: 831-1.0088016-ld.json
RDF/XML (Pretty): 831-1.0088016-rdf.xml
RDF/JSON: 831-1.0088016-rdf.json
Turtle: 831-1.0088016-turtle.txt
N-Triples: 831-1.0088016-rdf-ntriples.txt
Original Record: 831-1.0088016-source.json
Full Text
831-1.0088016-fulltext.txt
Citation
831-1.0088016.ris

Full Text

PLASMA CONCENTRATIONS OF AMINO ACIDS IN RAINBOW TROUT(ONCORIIYNCHUS MYKISS) IN RESPONSE TO NUTRITIONAL AND FEEDINGMANAGEMENTbyCHUTIMA TANTIKITTIB.Ed. (Biol.), Srrnakharinwirot University, 1979M.Sc. (Mar. Biol.), Chulalongkorn University, 1982A THESIS SUBMITTED iN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ANIMAL SCIENCEWe accept this thesis as confonningto the required standardTHE UNIVERSfl’Y OF BRITISH COLUMBIAFEBRUARY 1994© Chutima Tantikitti, 1994In presenting this thesis in partial fulfilment of the requirements for an advanced degree at TheUniversity of British Columbia, I agree that the Library shall make it freely available for reference andstudy. I further agree that permission for extensive copying of this thesis for scholarly purposes may begranted by the Head of my Department or by his or her representatives. It is understood that copyingor publication of this thesis for financial gain shall not be allowed without my written permission.Department of Animal ScienceThe University of British ColumbiaVancouver, CanadaDate: 25 February, 1994ABSTRACTThis study considered a number of dietary factors that may alter the balanceand constancy of amino acid concentrations in the plasma of rainbow trout. Fishgiven five equal feedings per day had relatively constant plasma amino acidconcentrations and deposited more protein and less lipid than did fish fed the sameration at one feeding per day. The rate at which amino acids from the single mealfeeding entered the circulation was evidently in excess of the rate at which thoseamino acids could be utilized for immediate protein synthesis with the result that ahigher proportion of them was catabolized and the carbon skeletons employed inlipid synthesis. Supplementation of dietary protein with lysine and methiorune inthe free form resulted in more rapid appearance of these amino acids in the plasmathan occurred with the intact dietary protein alone. A surge of plasma arginine,alanine, histidine, and lysine concentrations observed at 36 li after the fish were feddiets containing a mixture of protein sources suggested delayed digestion ofparticular proteins. Isonitrogenous substitution of free glycine for that supplied bygelatin delayed the time at which plasma glycine peaked postprandially comparedwith the response to the gelatin-containing diet. When a diet was supplementedwith a mixture of free essential amino acids, elevated concentrations remained inthe plasma and muscle pools as long as 26 h after feeding, indicating that dietarysupplements of free amino acids may remain available for protein synthesis evenwhen the fish are fed once daily. Plasma concentrations of free amino acids in fishfed different concentrations of dietary lipid indicated that dietary lipid at 24% of thediet had no effect of the lipid on digestion of protein or absorption of amino acids.Postprandial concentrations of plasma amino acids in fish fed fish meal that hadIIAbstractbeen subjected to heat treatment showed that the predominant nutritional effect ofprotein denaturation was a reduction in availability of threonine and histidine. Inconclusion, the responses of plasma amino acid concentrations to different dietaryconditions observed in this study indicate that they provide a useful tool forinvestigating the effects of various nutritional factors on protein metabolism in fish.mTABLE OF CONTENTS11Table of Contents ivList of Tables viiiList of Figures xiiList of Appendices xivAcknowledgement xvChapter 1 11Chapter 2Plasma Free Amino Acids 3Protein Sources in Salmonid Diets 102.2.1 Present Status of the Use of Protein Sources in Salmonid Diets 102.2.2 Restrictions on the Uses of Other Protein Sources as Fish MealReplacers 132.3 Supplementation and Utilization of Amino Acids 142.4 Factors Affecting Protein Utilization 162.4.1 Size of Fish 162.4.2 Composition of Diets 162.4.3 Feeding Practice 182.4.4 Gastric Evacuation Time 19Chapter 3Experiment 1 20Patterns and Concentrations of Free Amino Acids in the Plasma of Rainbow TroutFed Fish Meal as the Principal Source of Dietary Protein in Comparison with(1) a Mixture of Protein Sources and (2) a Mixture of Protein Sources PlusSelected Amino Acids 203.1 Introduction 203.2 Experiment 1.1: Effects of Diets and Feeding Frequency on Growth andChanges in Plasma Amino Acid Concentrations in Rainbow Trout 213.2.1 Materials and Methods 213.2.1.1 Diets 213.2.1.2 Fish and Rearing Conditions 22Literature Review.2.12.2lvTable of Contents3.2.1.3 Design of Feeding Trial 243.2.1.4 Sampling Procedure 253.2.1.5 Proximate Analysis 263.2.1.6 Amino Acid Analysis 263.2.1.7 Statistical Analysis 273.2.2 Results 283.2.2.1 Comparison Between Amino Acid Composition ofExperimental Diets and Requirements 283.2.2.2 Responses of Fish to Experimental Diets and FeedingFrequency 283.2.2.3 Carcass Composition 323.2.2.4 Gastrointestinal Contents 423.2.2.5 Plasma Amino Acid Proffles 423.2.2.6 Effects of Supplementary Lysine and Methionine 613.2.2.7 Plasma Taurine Concentrations 613.2.2.8 Relationship Between Plasma and Dietary Amino Acids.. 623.3 Experiment 1.2: Dietary Effects on Plasma Concentrations of Amino AcidsMonitored Over a 120-Hour Period Following Diet Consumption 653.3.1 Materials and Methods 653.3.1.1 Diets 653.3.1.2 Fish 653.3.1.3 Sampling Procedure 673.3.1.4 Amino Acid Analysis 683.3.1.5 Statistical Analysis 683.3.2 Results 683.3.2.1 Amino Acid Compositions of Experimental Diets 683.3.2.2 Responses of Fish to Experimental Diets 713.3.2.3 Plasma Amino Acid Proffle 713.3.2.4 The Effects of Dietary Amino Acid Supplementation onPlasma Amino Acids 843.3.2.5 Plasma Taurine Concentrations 843.3.2.6 Relationship Between Plasma and Dietary Amino Acids.. 853.4 Discussion 88Chapter 4 100Experiment 2 100Patterns and Concentrations of Free Amino Acids in the Plasma of Rainbow TroutFed Diets Containing Protein From a Mixture of Different Protein SourcesWith and Without Dietary Supplements of Selected Amino Acids 1004.1 Introduction 1004.2 Materials and Methods 1014.2.1 Diets 1014.2.2 Facilities 1034.2.3 Fish 1034.2.4 Sampling Procedure 1034.2.5 Amino Acid Analysis of Plasma and Muscle 104VTable of Contents4.2.6 Statistical Analysis 1054.3 Results 1054.3.1 Amino Acid Composition of Experimental Diets 1054.3.2 Initial Weight and Feeding Behavior 1074.3.3. Plasma Amino Acid Profile in Fish Fed Experimental Diets forSeven Days 1084.3.4. Relationship Between the Concentrations of Free Amino Acids inPlasma and Muscle 1204.3.5 Comparison Between Concentrations of Plasma Amino Acids inFish in Experiment 1 (Freshwater) and Experiment 2 (Seawater)1304.4 Discussion 132Chapter 5 139Experiment 3 139Patterns and Concentrations of Free Amino Acids in the Plasma of Rainbow TroutFed Fish Meal as the Principal Source of Protein in Diets Containing 6% or24% of Lipid 1395.1 Introduction 1395.2 Materials and Methods 1405.2.1 Diets 1405.2.2 Fish 1405.2.3 Sampling Procedure 1415.3 Results 1435.3.1 Amino Acid Compositions of Experimental Diets 1435.3.2 Diet Acceptance 1445.3.3 Plasma Amino Acid Profile 1445.4 Discussion 155Chapter 6 159Experiment 4 159Examination of Patterns and Concentrations of Free Amino Acids in the Plasma ofRainbow Trout as Criteria of Amino Acid Availability from Herring MealsSubjected to Heat Treatment 1596.1 Introduction 1596.2 Materials and Methods 1606.2.1 Test Protein Sources 1606.2.2 Fish 1606.2.3 Diets 1616.2.4 Sampling Procedure 1636.2.5 Pepsin Digestibility of Fish Meal 1636.2.6 Amino Acid Analysis 1656.2.7 Statistical Analysis 165viTable of Contents6.3 Results 1656.3.1 Pepsin Digestibility of Fish Meal 1656.3.2 Responses of Fish to Experimental Diets 1696.3.3 Plasma Amino Acid Profile 1696.4 Discussion 185Chapter 7 190Conclusions 190Bibliography 195Appendices 212VIILIST OF TABLESTable 1.1. Ingredient (air-dry basis) and proximate composition (dry matter basis)of diets used in Experiment 1.1 23Table 1.2. Amino acid composition of experimental diets and the NRC requirementvalues 29Table 1.3. Initial weight, final weight, body weight gain and feed consumption ofrainbow trout of different sizes fed different diets either once or five times dailyover a 26-day period in Experiment 1.1 33Table 1.4. Whole body proximate composition of rainbow trout of different sizesfed different diets either once or five times daily over a 26-day period inExperiment 1.1 34Table 1.5. Specific growth rates, feed conversion efficiency, productive proteinvalue and energy efficiency of rainbow trout of different sizes fed different dietseither once daily or five times daily over a 26-day period in Experiment 1.1 35Table 1.6. Mean initial weight, final weight, weight gain, and feed consumption ofrainbow trout of different sizes fed different diets either once or five tunes dailyover a 26-day period in Experiment 1.1 36Table 1.7. Mean initial weight, final weight, body weight gain and feed consumptionof rainbow trout fed different diets according to feeding regime over a 26-dayperiod in Experiment 1.1 37Table 1.8. Mean specific growth rate, feed conversion efficiency, productive proteinvalue, and energy efficiency of rainbow trout of different sizes fed different dietseither once or five times daily over a 26-day period in Experiment 1.1 38Table 1.9. Mean specific growth rates, feed conversion efficiency, productiveprotein value and energy efficiency of fish fed different diets according tofeeding regimes over a 26-day period in Experiment 1.1 39Table 1.10. Mean carcass composition of rainbow trout of different sizes feddifferent diets either once or five times daily over a 26-day period inExperiment 1.1 40Table 1.11. Mean carcass composition of rainbow trout fed different diets accordingto feeding regimes over a 26-day period in Experiment 1.1 41Table 1.12. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 1 (fish meal based diet) in Experiment 1.1. Fish fedonce daily to satiation 48wilList of TablesTable 1.13. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 2 (amino acid deficient diet) in Experiment 1.1. Fishfed once daily to satiation 49Table 1.14. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 3 (supplemented with lysine, methionine, andtryptophan) in Experiment 1.1. Fish fed once daily to satiation 50Table 1.15. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 1 (fish meal based diet) in Experiment 1.1. Fish fed fivetimes daily 51Table 1.16. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 2 (amino acid deficient diet) in Experiment 1.1. Fishfed five times daily 52Table 1.17. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 3 (supplemented with lysine, methionine, andtryptophan) in Experiment 1.1. Fish fed five times daily 53Table 1.18. Distribution of plasma amino acids at 9 h postprandial in fish feddifferent diets once daily in Experiment 1.1 60Table 1.19. Plasma taurine, methionine, and cystine concentrations in trout feddifferent diets according to feeding regimes in Experiment 1.1 63Table 1.20. Ingredient (air-dry basis) and proximate composition (dry matter basis)of diets used in Experiment 1.2 66Table 1.21. Amino acid composition of experimental diets in Experiment 1.2 69Table 1.22. Body weight gain and feed consumption of rainbow trout over a 10-dayperiod in Experiment 1.2 70Table 1.23. Specific growth rates and feed conversion efficiencies for rainbow troutfed different diets in Experiment 1.2 70Table 1.24. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 1 (fish meal based diet) in Experiment 1.2 76Table 1.25. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 2 (amino acid deficient diet) in Experiment 1.2 77Table 1.26. Concentrations of plasma amino acids in rainbow trout at differenttimes after feeding diet 3 (diet 2 supplemented with isoleucine, methionine,lysine and tryptophan) in Experiment 1.2 78ixList of TablesTable 1.27. Concentrations of plasma taurine, methionine, and cystine in rainbowtrout at different times after feeding experimental diets in Experiment 1.2 86Table 2.1. Ingredient and proximate composition of diets employed in Experiment2 102Table 2.2. Amino acid composition of experimental diets employed in Experiment 2106Table 2.3. Mean initial weights of fish in different experimental treatmentsemployed in Experiment 2 107Table 2.4. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 1 (fish meal, corn gluten meal, soy protein) on day 7 inExperiment 2 111Table 2.5. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 2 (fish meal, corn gluten meal, soy protein, and gelatin) onday 7 in Experiment 2 112Table 2.6. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 3 (fish meal, corn gluten meal, soy protein supplemented withglycine) on day 7 in Experiment 2 113Table 2.7. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 4 (fish meal, corn gluten meal, soy protein supplemented witharginine, histidine, lysine, methionine, and threonine) on day 7 in Experiment 2.114Table 2.8. Concentrations of plasma amino acids in rainbow trout at 26 and 36 hafter feeding experimental diets on day 17 in Experiment 2 123Table 2.9. Relative concentrations of plasma free amino acids (expressed as apercentage) in rainbow trout at 26 and 36 h after feeding experimental diets onday 17 in Experiment 2 124Table 2.10. Concentrations of muscle free amino acids in rainbow trout at 26 and 36h after feeding experimental diets on day 17 in Experiment 2 125Table 2.11. Relative concentrations of muscle free amino acids (expressed as apercentage) in rainbow trout at 26 and 36 h after feeding expenmental diets onday 17 in Experiment 2 126Table 2.12. Correlation coefficients of concentrations between plasma and musclefree amino acids in Experiment 2 127xList of TablesTable 2.13. Plasma concentrations of branched-chain amino acids, lysine, andthreonine, and total concentrations of essential amino acids, non-essentialamino acids, and total amino acids in fish fed diet 2 sampled at different timesafter feeding in 131Table 3.1. Ingredient and proximate composition (air-dry basis) of diets used inExperiment 3 142Table 3.2. Amino acid composition of experimental diets used in Experiment 31 143Table 3.3. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 1 (6% lipid) in Experiment 3 147Table 3.4 Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 2 (24% lipid) in Experiment 3 148Table 4.1. Proximate composition of low-temperature dried herring meal 160Table 4.2. Ingredient (air-dry basis) and proximate (dry matter basis) composition ofdiets used in Experiment 4 162Table 4.3. Pepsin digestibility of protein in low temperature herring meal heated at127°C for different periods of time in Experiment 4 166Table 4.4. Body weight gains and feed consumptions of rainbow trout over a 16-dayperiod in Experiment 4 168Table 4.5. Specific growth rates, and feed conversion efficiencies for rainbow troutfed different diets in Experiment 4 168Table 4.6. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 1 (control) in Experiment 4 173Table 4.7. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 2 (45 mm-heated fish meal) in Experiment 4 174Table 4.8. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 3 (90 nun-heated fish meal) in Experiment 4 175Table 4.9. Concentrations of plasma amino acids in rainbow trout at different timesafter feeding diet 3 (180 mm-heated fish meal) in Experiment 4 176Table 4.10. Comparisons of peak concentrations of plasma essential amino acids infish fed diets containing fish meal heated for different periods of time 183xlLIST OF FIGURESFigure 1.1. Mean wet weight (± SE) of gastrointestinal contents of rainbow trout atdifferent times after feeding in Experiment 1.1 43Figure 1.2. Total concentrations of plasma amino acids (essential, non-essential,and total amino acids) determined at different times postprandial for rainbowtrout fed once daily and five times daily in Experiment 1.1 54Figure 1.3. Plasma concentrations of amino acids in rainbow trout fed differentdiets according to different feeding regimes in Experiment 1.1 55Figure 1.4. Plasma taurine, and methionine + cystine concentrations in rainbowtrout fed different diets according to different feeding regimes in Experiment1.1 64Figure 1.5. Total concentrations of plasma amino acids (essential, non-essential,and total amino acids) in rainbow trout determined at different times afterfeeding in Experiment 1.2 79Figure 1.6. Plasma concentrations of amino acids in rainbow trout determined atdifferent times after feeding experimental diets in Experiment 1.2 80Figure 1.7. Plasma taurine, and methionine + cystine concentrations in rainbowtrout fed different diets in Experiment 1.2 87Figure 2.1. Total concentration of plasma amino acids (essential, non-essential, andtotal amino acids) in rainbow trout determined at different times after feedingexperimental diets on day in Experiment 2 115Figure 2.2. Plasma concentrations of amino acids in rainbow trout determined atdifferent times after feeding experimental diets in Experiment 2 116Figure 2.3. Plasma concentrations of supplementary amino acids in rainbow troutdetermined at 26 h (a), and 36 h (b) after feeding the experimental diets on day17 in Experiment 2 128Figure 2.4. Muscle concentrations of supplementary amino acids in rainbow troutdetermined at 26 h (a), and 36 h (b) after feeding the experimental diets on day17 in Experiment 2 129Figure 3.1. Total concentrations of plasma amino acids (essential, non-essential,and total amino acids) in rainbow trout determined at different times afterfeeding in Experiment 3 149Figure 3.2. Plasma concentrations of amino acids in rainbow trout fed differentdiets in Experiment 3 150)Q1List ofFiguresFigure 3.3. Percentage increases of plasma amino acids at peak time in comparisonwith the concentrations at 36 h after feeding in rainbow trout fed two differentdiets in Experiment 3 154Figure 4.1. Pepsin digestibility of protein in fish meal heated for different periods oftime in Experiment 4 167Figure 4.2. Total concentrations of plasma amino acids (essential, non-essential,and total amino acids) in rainbow trout determined at different times afterfeeding diets containing herring meal heated for different periods of time inExperiment 4 177Figure 4.3. Plasma concentrations of amino acids in rainbow trout fed dietscontaining herring meal heated for different periods of time in Experiment 4178Figure 4.4. Percentage changes in plasma amino acid concentrations in rainbowtrout fed heated fish meal diets relative to the control (diet 1) in Experiment 4...184xliiLIST OF APPENDICESAppendix 1. ANOVA of initial weight, final weight, weight gain, and feedconsumption of rainbow trout in Experiment 1.1 212Appendix 2. ANOVA of specffic growth rate (SGR), feed efficiency, productiveprotein value (PPV), and energy efficiency in Experiment 1.1 212Appendix 3. ANOVA of protein gain and lipid gain of rainbow trout in Experiment1.1 213Appendix 4. ANOVA of dry matter, protein, lipid and ash content of rainbow troutin Experiment 1.1 213Appendix 5. ANOVA of specific growth rate (SGR), and feed conversion efficiencyin Experiment 1.2 213Appendix 6. ANOVA of pepsin digestibility in Experiment 4 214Appendix 7. ANOVA of initial weight, final weight, weight gain, and feedconsumption of rainbow trout in Experiment 4 214Appendix 8. ANOVA of specific growth rate (SGR), and feed conversion efficiencyin Experiment 4 214xivACKNOWLEDGEMENTI would like to express my gratitude to Prof. B.E. March for her continuedsupport, guidance, commitment, and patience she has shown throughout this study.My appreciation also goes to Dr. D.A. Higgs, Dr. D. Beames, Dr. 3. Thompson, andDr. K. Cheng for their contribution to this thesis.I would also like to extend my sincere gratitude to Carol MacMillan for herassistance with my experimental work and freindship. I would like to thank CarolMazur and Ellen Teng for their assistance with sampling the fish. To RyszardPuchala, Gilles Galzi, and Maureen Evans, their technical assistance with aminoacid analysis is highly acknowledged. The valuable advice on statistics from RickWhite is greatly appreciated. Thanks also go to members of the faculty and studentsin the Department of Animal Science, particularly Marina VonKyselink and ShellyMacDonald, for their support and encouragement.I would also like to thank Dr. Reungchai Tanskul and IDRC (InternationalDevelopment Research Centre) for their initiation of my Ph.D. study.Very special thanks are due to John Rosene and Amonrat Sermwatanakulfor their genuine support during my writing. The most important people aremembers of my family and the Helms family who have had faith and immeasurableinfluence on my education.xvCHAPTER 1INTRODUCTIONFree amino acids in plasma of animals are derived from dietary protein,catabolism of tissue proteins, and synthesis. At the same time free amino acids areremoved from the plasma for synthesis of tissue protein, or other nitrogenouscompounds, and degradation. Consequently, the size of the plasma pool of eachamino acid at any time is the result of the balance between input and removal(Munro, 1970). Numerous studies with animals of various species including fishhave demonstrated a marked rise of plasma amino acids following consumption ofprotein (McLaughlan and Morrison, 1968; Eggum, 1972; Nose, 1972; Yamada et a!.,1982; Walton and Wilson, 1986; Lyndon et a!., 1993). Furthermore, theconcentrations of essential amino acids in plasma are positively correlated with theirdietary concentrations during the absorptive period. The above relationshipsuggests that studies of the amino acid pattern appearing in the plasma during andfollowing protein digestion might give an indication of rates and extent ofproteolytic release of the amino acids from dietary proteins.As with other animals, the rate at which different amino acids are absorbedfrom the digestive tract and enter the circulation have an important bearing on therate of protein synthesis in fish. The inferior growth rate of rainbow trout observedby Cowey et a!., (1992) when fish were fed diets containing large quantities ofsupplementary free amino acids was assumed by these investigators to be due to thedifference in uptake rates into plasma of free amino acids compared with aminoacids derived from digestion of intact protein in the diet. In an experiment in whichfish were fed a purified diet containing free amino acids , but no source of intact1Chapter 1protein, there was more rapid appearance and a higher concentration of free aminoacids in the plasma (Murai et a!., 1987). It was considered that the rapid uptake ofamino acids into the plasma enhanced the activities of amino acid-catabolizingenzymes leading to increased amino acid degradation and decreased proteinsynthesis (Cowey and Luquet, 1983).From a practical point of view, fish diets often contain a combination ofdifferent protein sources. Sometimes supplementary amino acids are added toimprove the amino acid profile of the diet (Hardy, 1989). It has been welldocumented that different protein sources vary in their digestibility andconsequently in the efficiency of absorption of amino acids from the gastrointestinaltract. Under this circumstance, the rate at which free amino acids are absorbed intothe circulation from the hydrolysis of different dietary proteins in thegastrointestinal tract and from supplementary amino acids may be different.Furthermore, although a formulated diet may have an amino acid profile that meetsthe requirements of the fish, the availability of amino acids to the fish may bereduced by factors that affect digestibility of the protein.The present study evolved from the above observations. It was devoted toinvestigating the responses of plasma amino acid concentrations in rainbow troutunder a variety of dietary treatments. The plasma concentrations of individualamino acids were monitored following the feeding of 1) diets contaimng differentprotein sources, 2) diets containing different protein sources supplemented with freeamino acids, 3) experimental diets at two feeding frequencies, 4) diets containingdifferent lipid concentrations, and 5) diets containing experimental fish mealssubjected to over-heating.2CHAPTER 2LITERATURE REVIEW2.1 PLASMA. FREE AMINO ACIDSThe release and absorption of free amino acids from ingested proteins in fishfollows similar processes to those in other animals. Protein consumed in the diet ishydrolyzed enzymatically in the gastrointestinal tract, and the final products of thedigestion, i.e. free amino acids, are absorbed into the circulation via the portal bloodstream (Nissen, 1992). A proportion of amino acids liberated in the intestinallumen is also incorporated into proteins of mucosal cells. Moreover, some aminoacids may be metabolized within the epithelium of small intestine, for example thetransamination of glutamic acid and aspartic acid with pyruvate results in lowrecoveries of these amino acids from the mucosal cells (Munro and Crim, 1980).After entering the circulation, free amino acids are subjected to a series ofmetabolic reactions which can be grouped into three categories (Munro and Crim,1980). First, part of the free amino acids is incorporated into tissue proteins.Because of tissue protein catabolism, these amino acids eventually return to the freepool after a variable length of time and become available for reutilization. Second,part of the free amino acids undergoes catabolic reactions. This leads to loss of thecarbon skeleton as CO2 or its conversion to fat and/or glucose, while the nitrogen isexcreted as ammonia in fish. Third, some amino acids are used for synthesis of N-containing compounds, such as purine bases, hormones, nucleic acids, andneurotransmitters. In addition, some nonessential amino acids are synthesized inthe body using amino groups derived from other amino acids, and carbon skeletons3Chapter 2from intermediary metabolism (Munro and Crim, 1980; Cowey and Walton, 1989).Consequently, the size of the pooi of each free amino acid varies depending uponthe rate of entry and removal of respective amino acids into and from the pooi(Munro, 1970). The pattern of plasma amino acids has been shown to relate to thatof the ingested protein in many animals (Longenecker and Hause, 1959; Hill andOlsen, 1963; McLaughlan and Iliman, 1967; Munro, 1970; Young and Scrimshaw,1972). On this basis, amino acids present in greatest abundance in the diet shouldcause the largest increases in plasma concentrations. This relationship has beenconfirmed by several researchers (Eggum, 1972; Hagemeister et a!., 1990). Someinvestigators, on the other hand, have failed to establish a relationship between therelative abundance of dietary amino acids and their increase in plasma during theabsorptive period. For example, Nasset et a!., (1963) could not find any relationshipbetween amino acid composition of the diets and plasma free amino acids in dogs.Despite this contradiction, Johnson and Anderson (1982) were able to qualitativelyand quantitatively relate plasma amino acids to dietary amino acids. They usedretrospective analysis of plasma amino acid data from a number of studies in whichprotein quantity and quality of the diet were varied. The level of each plasmaamino acid in male weanling rats was examined in relation to their intake fromgluten, casein, synthetic amino acid, or zein diets. They found that levels of mostamino acids in the plasma were predictable if both the concentration of the aminoacid in the diet and chronic level of protein intake were known.Based on the above relationship, the alterations of plasma amino acidconcentrations have been employed as an indicator for various purposes in proteinnutrition. Plasma amino acid levels have been investigated in relation to the4Chapter 2limiting amino acid in the diet (McLaughlan and Morrison, 1968; Sarwar et a!.,1983), amino acid requirement (Harper, 1977), amino acid imbalance (Peng andHarper, 1970; Peng et a!., 1972), and absorption and availability of amino acids(Young and Scrimshaw, 1972; Ostrowski, 1977).Investigations have also been conducted on the relationship between thestate of body protein metabolism and plasma amino acid concentrations (Young andScrimshaw, 1972). For example, Galibois et a!., (1987) have attempted to determinethe relationship between protein synthesis rate and concomitant plasma amino acidlevels in rats. The rats were fed with four types of proteins, i.e. beef rapeseed flour,casein, and soybean. Plasma amino acid concentrations in the portal vein and theaorta, and hepatic ribosome aggregation were investigated at different times duringthe active eating period. Each protein source was found to generate differentvariations in portal and aortic plasma amino acid levels. Furthermore, proportionsof essential amino acids in portal and aortic plasma were characteristic of theprotein fed and tended to remain constant. Although no direct relationship wasdetected between the concentrations and pattern of plasma amino acids, andribosome aggregation, these two parameters were both affected by dietary protein.Most of the studies in fish on plasma amino acid levels have been limited to afew species. The investigations have been conducted mostly in rainbow trout, carp,and channel catfish (commercially important species). Some studies have also beencarried out in Atlantic salmon, coho salmon, sea bass, tilapia, and goldfish (Cowey etat., 1962; Carrillo et a!., 1980; Yamada et a!., 1982; Thebault, 1985; Ogata and Arai,1985). Studies with fish have generally shown similar results to those with mammalsin that the plasma amino acid concentrations, particularly essential amino acids,5Chapter 2were positively correlated with their dietary concentrations (Nose, 1972; Plakas eta!., 1980; Dabrowski, 1982; Wilson et aL, 1985; Walton and Wilson, 1986; Blasco eta!., 1991; Lyndon et al., 1993). Concentrations of non-essential amino acids inplasma, however, do not show such correlation, presumably because these aminoacids undergo extensive interconversion and metabolism within the body (Coweyand Walton, 1989).Interesting interspecies differences have been noted in the chronology of theappearance of essential amino acids in the plasma of fish following the provision ofa protein containing diet. In rainbow trout, several studies have indicated that afterconsumption of diets containing intact proteins, such as casein or a commercialpelleted diet, plasma amino acids rose soon after feeding and attained peaks at 6 hor between 12-24 h post-feeding depending on experimental conditions such aswater temperature, salinity, fish size, duration of starvation, and types of diets(Nose, 1972; Kaushik and Luquet, 197Th; Walton and Wilson, 1986). Yamada et at.,(1981a), on the other hand, reported different results. They force-fed rainbow trouta casein diet, and observed a lag period until plasma amino acids started to rise at12 h postprandial. Peaks of amino acid concentrations occurred between 24-36 hafter feeding. An explanation for the 12 h lag period observed by Yamada et a!.,(1981a) might conceivably reside in the stress effect on the digestive physiologyinduced by the forced-feeding protocol (Ash, 1985). While this might be true, Muraiet a!., (1987) also force-fed rainbow trout a diet containing the same type of protein,but did not observe any lag period. Walton and Wilson (1986) suggested that theperiod of starvation before the time of feeding was possibly the cause of the delay ofstomach emptying resulting in the delay of digestive and absorptive processes.6Chapter 2The times when most amino acids reached peaks after meal consumption inother species of fish have differed from those of rainbow trout. In carp, tilapia andsea bass, most amino acids have been reported to rise within 2 h and reached peaksbetween 4-6 h depending upon the species (Plakas et a!., 1980; Yamada et a!., 1982;Thebault, 1985). Differences in the appearance of amino acids in rainbow trout(cold water species) and the species mentioned above (warm water species) weredue to temperature and morphology of the digestive tract particularly in the case ofcarp which is an agastric species. Regarding the latter reason, when compared withthe rainbow trout, digestion of casein in carp is very rapid. Ash (1985) postulatedthat in agastric species the ingested protein will become immediately subjected tothe action of proteolytic enzymes and transport systems present within the anteriorsmall intestine.When different species of fish were fed diets containing synthetic amino acidsin comparison with those containing intact protein, a similar phenomenon on theappearance of plasma free amino acids was observed. The plasma amino acids infish fed free amino acid diets rose and reached their peaks earlier than those in fishfed intact protein diets (Plaskas et at., 1980; Murai et a!., 1987; Yamada et a?., 1982).Moreover, the examination of stomach contents in rainbow trout by Yamada et a!.,(1981b) revealed that fish fed an amino acid mixture emptied the stomach morerapidly than fish fed a casein diet. Plakas et a!., (1980) also observed that not onlywere free amino acids in carp that were fed amino acids absorbed at a faster rate,but there were also variations in the response of the plasma amino acids comparedwith when they were fed intact protein.7Chapter 2The above finding has provided useful information for better understandingof amino acid utilization in fish. For example, it has been reported that fish ofseveral species fail to utilize an aniino acid diet or show inferior growth compared tofish fed a diet containing intact protein. In the light of this finding severalresearchers concluded that the inability of fish to utilize a diet containing free aminoacid effectively was due to faster absorption and catabolism of dietary free aminoacids before amino acids from intact dietary protein would be available for proteinsynthesis (Ash, 1985).Concentrations of plasma amino acids have been used as a parameter todetermine amino acid requirements in several species of fish such as channel catfish,rainbow trout, and sea-bass. The success of this technique has varied. For example,Harding et a!., (1977) and Wilson et a!., (1977) successfully used plasma free aminoacid levels to determine the requirements for lysine and methionine in channelcatfish. They found that plasma concentrations of these free amino acids increasedsignificantly once the requirement of the amino acid was exceeded on the basis ofmaximum growth. Kaushik et al., (1988) and Cho et al., (1992), also attempted torelate the plasma concentrations of arginine to growth when estimating the argininerequirement of rainbow trout. The results obtained from both studies, however,indicated that the patterns of change in plasma argiriine levels did not provide themeans for estimation of the arginine requirement.Assessment of protein quality has been attempted on the basis of the plasmaamino acid profile following ingestion of a test protein. For instance, Ogata et a!.,(1986) fed carp diets containing various ratios of casein to gelatin and comparedfree amino acid concentrations in the plasma with dietary amino acid composition8Chapter 2as predictors of growth response to the diets. They concluded that performance ofcarp can be estimated more precisely from plasma free amino acid level than fromthe dietary amino acid levels. A similar finding was found in the same species offish by Murai et a!., (1989a) when they fed diets containing soy flour supplementedwith methioriine.Lyndon et a!., (1993) measured the changes in the free amino acid pools ofvarious tissues, including plasma, of the cod after a single meal, and endeavored tointerpret the results in relation to protein synthesis. They found that the patterns ofplasma amino acids were similar to those in rainbow trout. They suggested from theresults on tissue pools of free amino acids together with the results found in otherstudies on plasma amino acids that the changes in the concentration of someessential amino acids, particularly tryptophan, have a role in stimulation of proteinsynthesis. This is because tryptophan, which was consistently found in the lowestlevel in all the pools, showed a significant increase between 12-24 h post-feeding,this range in time coincided with the time when the rate of protein synthesis wasmaximum. Their conclusion was based on the assumption that protein synthesis canonly proceed if all amino acids are simultaneously present in the tissue. The rate ofprotein synthesis will be limited by the least abundant essential amino acid presentin the precursor pool.However, the interpretation of plasma amino acids in relation to proteinstatus is a difficult task. This is because qualitative and quantitative changes in theplasma free amino acid proffle are determined by a number of factors. Theseinclude 1) amino acid composition, digestibility and quantity of protein consumed 2)composition of remaining food constituents 3) rate of stomach emptying 4) rate of9Chapter 2absorption of amino acids 5) extent to which the gastrointestinal mucosal cellsmetabolizes various amino acids 6) rate of amino acid removal from the bloodwhich is influenced by numerous metabolic processes associated with amino acidmetabolism 7) time of sampling after giving the test meal 8) endogenous protein inthe gastrointestinal tract (to a small and temporary extent) and 9) age and strain ofanimals (Young and Scrimshaw, 1972; Simon, 1989; Munro and Portugal, 1972).2.2 PROTEIN SOURCES IN SALMONID DIETS2.2.1 Present Status of the Use of Protein Sources in Salmonid DietsFish meal is the major dietary protein source in salmonid diets, ranging from25-60% of commercial diets (Murai, 1992), and there have been many attempts topartly or completely replace fish meal protein with alternative protein sources.Protein sources that have been studied in salmonids are from animals and their byproducts, plants and their by-products, and unconventional protein sources. Theanimal and animal by-product protein sources include locally available fish meal,meat and bone meal, blood meal, poultry-by-product meal, hydrolyzed feather meal,and, dried milk by-product (Tacon and Jackson, 1985). The plant protein sourcesinclude soybean meal and concentrate, rapeseed meal and concentrate, canolameal, cotton seed meal, corn gluten meal, field bean meal, wheat germ meal, broadbean meal, sunflower meal, and sweet lupin meal (Higgs, et a!., 1979 and 1991;Tacon and Jackson, 1985; Fowler and Burrows, 1971). The unconventional proteinsources include single cell protein (yeast, bacteria, blue green algae), fry larvae, krillmeal, leaf protein concentrate, and dried domestic sewage (Tacon and Jackson,10Chapter 21985; Cowey et a!., 1971; Rosenlund, 1986).Among the proteins from animals and their by-products, poultry-by-productshave been reported to have the most potential, even though the results fromdifferent laboratories are still contradictory. Gropp et a!., (1979) reported that fishmeal in practical trout diets could be replaced entirely by a mixture of poultry byproduct meal and hydrolyzed feather meal on an isonitrogenous basis if the essentialamino acid deficiencies were corrected. The results from studies of Tiews et a!.,(1979) corresponded well with the work of Gropp et a!., (1979) in rainbow trout.Higgs et a!., (1979) were unsuccessful in their attempt to completely replace herringmeal protein with poultry by-product meal in diets for coho salmon. A mixture ofpoultry by-product meal with hydrolyzed feather meal, however, was found tosuccessfully replace up to 75% of herring meal in the diet for coho salmon withoutany reduction in growth rate. Fowler (1991) attempted to partially replace fish mealwith poultry by-product meal in diets for chinook salmon. He found that the growthrates and feed efficiencies of chinook fed a diet containing 20% of poultry byproduct meal (replaced 50% of fish meal protein) were not different from those fedthe diet without poultry by-product meal. Feather meal, usually considered to be aninferior source of protein for fish can be included in salmonid diets by replacing upto 50% of fish meal (Tiews et al., 1979; Higgs et a!., 1979; Fowler, 1990). Thedietary combination of meat and bone meal and blood meal was mentioned byTacon and Jackson (1985) to be successfully employed to replace up to 50% of fishmeal protein.Soybean meal would appear to have the most potential of the plant proteinsources available as a partial substitute for fish meal in salmonid diets although11Chapter 2results from many studies have been variable. Reinitz (1980) found that fish fed adiet with 65% soybean meal and no herring meal grew at an acceptable rate andremained in good health. He, therefore, concluded that soybean meal, although notnutritionally equal to herring meal, could be used as the primary source of proteinin trout diets. Partial replacement of soybean meal in salmonid diets was reportedby several researchers to substitute for 75% of fish meal without any adverse results(Higgs et a!., 1979; Alexis et a!., 1985; Smith et a!., 1988). In other studies, however,the inclusion of soybean meal in salmonid diets at various concentrations has beenshown to reduce growth rate and feed efficiency (Fowler, 1980; Dabrowski et a!.,1989; Murai et a!., 1989a). Murai (1992) pointed out that there are differencesamong species in the utilization of soybean meal and that rainbow trout are able toutilize this protein source more efficiently than chum salmon. The results of Fowler(1980) support this finding because he found that the growth of chinook and cohosalmon fed the same diet as that fed to rainbow trout was depressed and there wasconcomitant high mortality. Krogdahl (1991) has reported that feeding rainbowtrout with soybean products induces histological and chemical changes in theabsorptive cells of the intestinal mucosa which may, in turn, alter the digestiveprocess.Other plant protein sources having a potential for partial replacement of fishmeal are canola meal, cotton seed meal, and corn gluten meal (Gropp et a!., 1979;Higgs et a!., 1979, 1983; Fowler, 1980; March, 1991). The substitution levels arementioned to be between 5-15% of diets (Tacon and Jackson, 1985). Higgs et a!.,(1991), however, suggested that undephytinized rapeseed protein concentrate cancomprise about 38% of dietary protein (fish meal only 11% of diet) without12Chapter 2adversely affecting growth rate, appetite, feed efficiency and protein utilization oftrout.2.2.2 Restrictions on the Uses of Other Protein Sources as Fish Meal ReplacersTwo of the main factors responsible for the differences in the resultsobtained from many experiments, in which fish meal has been either partially ortotally replaced in practical fish diets, are related to amino acid balances and/orprotein digestibility. Most of protein sources used as replacement for fish meal haveimbalanced amino acid patterns. Many protein sources are deficient in one or moreamino acids according to the review of Tacon and Jackson (1985). For example, theconcentrations of the sulfur-containing amino acids and lysine are low in all nonanimal-proteins when compared with fish muscle protein (Rosenlund, 1986). Theexception is rapeseed protein concentrate which has an amino acid proffle similar tothat of fish meal.Digestibility among various protein sources varies in terms of the rate ofdigestion and proportions of amino acids released. The digestibility coefficientsvary from 37-97% depending upon the source of protein (NRC, 1981, 1983). Thiswide range of digestibility coefficients of different protein sources is due to variousfactors: the structure of constituent proteins in products such as animal proteinmeals, processing conditions, high fiber contents in plant products, and the presenceof antinutritional factors (Tacon and Jackson, 1985). In some protein sources, theamino acid profile may be as good as that of fish meal protein, but the availability ofsome amino acids may be poor. Rosenlund (1986), for instance, found a differencein results obtained from feeding rainbow trout two types of fish meal. Rainbow13Chapter 2trout fed a diet based on low temperature-dried fish meal showed a higher capacityfor protein synthesis than did fish fed a diet based on regular processed fish meal.He concluded that low temperature-dried fish meal had better bio-availability ofamino acids and that heat treatment during processing may have led to chemicalchanges with resultant reductions in protein digestibility.2.3 SUPPLEMENTATION AND UTILIZATION OF AMINO ACIDSSupplementation of proteins with amino acids is often suggested as a meansof improving the utilization of diets formulated with proteins that are deficient inone or more essential amino acids (NRC, 1981, 1983; Cowey, 1979). The efficacy ofdietary supplementation with free amino acids for fish, including salmonids, is stillinconclusive. Many researchers have shown promising results with single ormultiple supplementation of amino acids to the diets for rainbow trout, Atlanticsalmon, chinook salmon, channel catfish, and carp (Rumsey and Ketola, 1975;Dabrowska and Wojno, 1977; Gropp et a!., 1979; Higgs et a!., 1983; Tacon et a!.,1983; Abel et al., 1984). Fowler (1980) on the other hand found no benefit fromsupplementing soybean-fish meal based diets with methionine. The growth rates ofchinook and coho salmon were decreased by methionine supplementation of thediet. Aoe et al., (1970) and Dean and Robinette (1983) found similar results withcarp and catfish to those obtained by Fowler (1980) with salmon when methioninewas used as a dietary supplement.According to Lovell (1989), fish do not utilize dietary crystalline amino acidsas well as chickens or swine. Generally, fish fed diets containing large amounts offree amino acids will show lower growth rates than those obtained when all the14Chapter 2protein in the diet is supplied as high quality intact protein (Cowey, 1992).Furthermore, the ability to utilize free amino acids varies within and betweenspecies. Carp used in various experiments, for instance, showed variable resultswhen diets supplemented with free amino acids were tested (Murai, 1989a; Aoe etal., 1970). Rainbow trout appear to use synthetic amino acids more efficiently thancarp and catfish (Cowey, 1979; Wilson, 1989). It is suggested that if a dietcontaining free amino acids is neutralized or adjusted to pH 6.5-6.7 for carp and pH6.0-8.0 for catfish, the utilization of the diet will be improved (Nose et al., 1974;Wilson et al., 1977). Dean and Robinette (1983), however, found no effect whencatfish were fed a diet supplemented with amino acids and pH was about 5.9.Supplementation of free amino acids at excess levels might adversely affect fishgrowth. Murai (1989a), for instance, found depression of growth and feed efficiencyin carp fed a diet supplemented with 0.50% methionine (130% above requirements)as compared with the addition of 0.25% methiomne. He suggested that the higherlevel of methionine is toxic to fish and that fish might be much more sensitive tomethionine toxicity than land animals when it is added in the free form. Feedingthree to four times the requirement of methiomne has been shown to suppress foodintake and to cause near-cessation of growth in land animals (Benevenga and Steel,1984).15Chapter 22.4 FACTORS AFFECTING PROTEIN UTILIZATIONApart from the amino acid balance and digestibility of proteins there areseveral other factors that affect protein utilization in fish. These include size of fish,composition of diets, feeding practice, and gastric evacuation time (Steffens, 1981;Pfeffer, 1982).2.4.1 Size of FishThe ability of fish to utilize protein varies with the size of fish. Small fish(immature fish) seem to utilize proteins less efficiently than large fish (mature fish).A possible important reason may be lower enzyme activities in small fish (Steffens,1981). Kitamikado and Tachino (1960) found that the proteolytic activity of anextract from the pyloric caeca and intestine of rainbow trout was low in the earlystages (body weight less than 4 grams) and increased with growth of the fish.2.4.2 Composition of DietsDietary fiber has been the subject of studies regarding its influence onprotein utilization in fish. Its presence might prevent nutrients from becomingavailable to the fish (Davies, 1985). It has been found that rainbow trout fed dietscontaining 10 and 20% of cellulose (alpha-floc) displayed poorer growth than fishfed a control diet without added cellulose (Hilton et al., 1983). In contrast to thesefindings, Davies (1985) found that inclusion of 15% and 20% of cellulose in diets forrainbow trout gave better protein efficiency ratios and net nitrogen utilization valuesthan the control diet containing no cellulose. This positive effect of dietary fibermay have been due to stimulation of cell turnover in the intestinal mucosa and16Chapter 2induction of enzyme secretions which may, in turn, improve protein utilization(Davies, 1985; Simon, 1989). Digestible carbohydrate in diets may also beimportant as a protein-sparing component. Ufodike and Matty (1989) showed that20-30% inclusion of corn and potato meal in rainbow trout diet resulted in betterprotein digestibility and growth performance than that obtained with the controldiet. The authors concluded that the improved growth response was due toutilization of carbohydrate energy in place of energy from protein. Beamish andThomas (1984), on the other hand, found that the apparent digestibility of proteinvaried inversely with the concentration of dietary starch (10-30%) in rainbow trout.Dietary lipid influences protein utilization through its protein-sparing action.Increases in dietary lipid to a certain level in fish diets have been shown to promotegrowth in several studies (Atherton and Aitken, 1970; Lee and Putnam, 1973; Adronet a!., 1976; Watanabe et al., 1979; Gropp et a!., 1982; De Silva et al., 1991). Higueraet a!., (1977) fed rainbow trout with diets containing 6.7%-20.9% of lipid and founda higher biological value (BV) for the diet containing 18% lipid than for the dietcontaining 6.7% lipid. He suggested that the higher BV obtained with the elevatedconcentration of dietary lipid was due to availability of calories from the lipid whichspared the catabolism of amino acids for energy purposes. The optimum level ofdietary lipid varies according to the dietary concentration of protein. Watanabe eta!., (1979), for instance, found maximum nitrogen retention with a ratio of 35%protein to 15-20% lipid in rainbow trout. A similar ratio of 36% protein and 16%lipid was suggested by Cho and Kaushik (1990) using rainbow trout in their study.Davies (1989) found that the optimum dietary levels of crude protein and lipid forgrowing juvenile trout were 40% and 12-14%, respectively. Reinitz and Hitzel17Chapter 2(1980), however, obtained maximum nitrogen retention in rainbow trout with dietscontaining 26% protein and 11-17% lipid.Inclusion of lipid at high levels in the diets of animals other than fish hasbeen found to delay gastric evacuation (Church and Pond, 1988). In the case of fish,Windell et al., (1972) suggested that dietary lipid levels of 15% or higher may inhibitgastric motility in rainbow trout. Jobling (1980) found that low-energy diets wereevacuated from the stomach of plaice more rapidly than those with a higher energycontent. Lie et al., (1988) fed young cod diets containing different levels of dietaryprotein and fat. They found that the digestibilities of protein and fat were reducedwhen the fish were fed an imbalanced diet containing 27% protein energy and 61%fat energy. They concluded that the reduction in protein digestibifity was due to anoverloading of the digestive system with fat. Higuera et a!., (1977), on the otherhand, reported that dietary lipid levels did not affect protein digestibility in rainbowtrout. Furthermore, Wilson et a!., (1985) did not find any differences in the serumpatterns of free amino acids of channel catfish fed diets with varying ratios ofprotein to energy.2.4.3 Feeding PracticeFeeding techniques that have been used, either in laboratory experiments oron fish farms include feeding at a restricted level per day and, feeding to satiation byhand or automatic feeder once or more times daily, or through the use of demandfeeders (Tacon and Cowey, 1985; Rumsey, 1991). Feeding at a restricted levelprevents the fish from realizing their maximum growth potential for a given diet.Grayton and Beamish (1977) found lower specific growth rates and gross food18Chapter 2conversion efficiencies when rainbow trout were fed a restricted daily ration of 2%of wet body weight as compared to satiation feeding. Feeding to satiation may,depending upon the nature of the diet, size of fish, and temperature, have anadvantage over restricted feeding. For instance, Grayton and Beamish (1977) foundthat rainbow trout fed to satiation two times/day exhibited maximum growth rate.Andrews and Page (1975) also obtained the maximum growth and feed efficiencywhen catfish were hand-fed to satiation two times per day. Anderson (1988)reported maximum protein utilization in the luderrick, a marine herbivorous fish,fed ad libitum.2.4.4 Gastric Evacuation TimeGastric evacuation time plays an important role in digestion and absorptionof nutrients in the digestive tract. The period of time during which digestiveenzymes may attack their substrates depends on the rate of passage of digesta andthis is in turn influenced by various factors. These factors include watertemperature, fish size, the amount of food eaten, feeding frequency, dietcompositions, type of food, and stress (Talbot, 1985). The effects of these factors ongastric evacuation time are still open to question. The interaction of these factorson gastric evacuation and digestion further increases the complexity of the system asa whole.19CHAPTER 3EXPERIMENT 1PAfERNS AND CONCENTRATIONS OF FREE AMINO ACIDS IN THEPLASMA OF RAINBOW TROUT FED FISH MEAL AS THE PRINCIPALSOURCE OF DIETARY PROTEIN IN COMPARISON WITH (1) A MIXTURE OFPROTEIN SOURCES AND (2) A MIXTURE OF PROTEIN SOURCES PLUSSELECTED AMINO ACIDS3.1 INTRODUCTIONStudies in several species of fish have shown that growth rate and feedefficiency of fish fed amino acid supplemented diets were poorer than when theywere fed a complete protein meal (Aoe et al., 1970; Wilson et a!., 1978; Walton eta!., 1986). A possible explanation for these observations was that free amino acidsadded as supplements to diets, may be rapidly absorbed and metabolized beforeother amino acids derived from digestion of dietary protein reach the sites ofprotein synthesis. Studies with rainbow trout have demonstrated that when aminoacid-based diets are fed to the fish there is more rapid appearance and higherconcentrations of free amino acids in the plasma (Yamada et al., 1981). Similarly,when a mixture of protein is consumed, not all of amino acids from the proteins arenecessarily available for absorption at the same time as different proteins vary in therates at which they are hydrolyzed by the digestive enzymes. In both situations,some essential amino acids may not be available in tissues in time to complementother amino acids so that a balanced mixture is available for protein synthesis. Anyamino acids that cannot be utilized for protein synthesis because of some limitation20Chapter 3of one or more amino acids will be rapidly oxidized or converted to glucose andfatty acids.The importance of similar rates of absorption of amino acids from thedigestive tract on the profile of free amino acids in the plasma, which are availablefor protein synthesis at any one time, is a question that has not been given muchconsideration in salmonid nutrition.Experiment 1.1 was, therefore, designed to investigate the changes inconcentrations of free amino acids in the plasma and the growth responses ofrainbow trout fed (1) diets containing different protein sources, (2) diets with andwithout supplementation of free amino acids, and (3) experimental diets fed eitheronce or five times daily. Experiment 1.2, was similar to Experiment 1.1 except thatthe amino acid profiles were monitored over a longer period following feeding.32 EXPERIMENT 1.1: EFFECTS OF DIETS AND FEEDING FREQUENCY ONGROWfH AND CHANGES IN PLASMA AMINO ACIDCONCENTRATIONS IN RAINBOW TROUT3.2.1 MAThRIALS AND METHODS3.2.1.1 DietsThe compositions of the three diets formulated for this experiment areshown in Table 1.1. Diet 1 contained herring meal as the principal source ofprotein. Diet 2 contained herring meal, soybean protein concentrate, gelatin, andcorn gluten meal as the protein sources. Diet 3 contained the same proportions of21Chapter 3protein from the respective sources as were used in diet 2 but was supplementedwith lysine, methionine and tryptophan to levels equivalent to those of diet 1.Tabulated concentrations of amino acids in feed ingredients (NRC, 1981) were usedto estimate the dietary concentrations of amino acids. The protein concentration ofeach diet was calculated to be approximately 35% (air-dry), with additional nitrogenin diet 3 from the supplementary amino acids (equivalent to approximately 1.3% ofprotein).3.2.1.2 Fish and Rearing ConditionsRainbow trout used in this experiment ranged in size from 16-3 1 g. Theywere divided into three size groups which had average weights of 25.9 ± 2.4,21.5 ± 1.4, and 18.5 ± 1.5 g (mean ± SD) and these were referred to as large,medium, and small, respectively. The reason for sorting the fish was to reduce sizevariation and thus minimize establishment of feeding hierarchies. Fish from each ofthe size categories were distributed at random into six 150 L tanks with 50 fish/tank(eighteen tanks total). The tanks and facilities were located at the UBC aquariumfacifities. The water supply was dechlorinated Vancouver city water. Watertemperature was maintained at 13°-14°C by means of a heat exchanger unit. Waterflow to each tank was 2 L/min, and dissolved oxygen was 8 ppm. The photoperiodwas 24 h. Twelve fish from the same stock (mean weight ± SD = 16.19 ± 0.83)were killed and frozen for subsequent determination of initial whole body proximatecomposition. The feeding trial was conducted during November-December, 1989.22Chapter 3Table 1.1. Ingredient (air-dry basis) and proximate composition (dry matter basis) of diets used inExperiment 1.1Ingredient Diet 1 Diet 2 Diet 3g/kg g/kg g/kgHerring meal çwhole steam-dried) 423.5 141.2 141.2Ground wheat 300.0 300.0 300.0Gelatin- 117.4 117.4Corn gluten meal- 77.1 77.1Soybean protein concentrate- 59.1 59.1Sardine 0112 93.0 118.3 118.3Bone meal 16.0 38.0 38.0Dextrin 107.5 88.9 88.9Premi,? 4.o 4o.o io.oCalcium lignosulphonate 20.0 20.0 20.0L-lysine-- 7.5DL-methionine-- 4.0L-tryptophan-- 1.5Total 1000.0 1000.0 1013.0Proximate analysis4Crude protein (%) 36.2 39.0 39.6Ether-extractable lipid (%) [5.8 15.8 15.8Ash (%) 8.0 6.4 6.1Gross energy (kcal/kg) 5288 5364 5461tAutoclaved at 121°C for 1.5 h2Stabied with 0.05% ethoxyquinThe premix supplied the following per kg of diet as fed (except for diet 3 in which the percentage ofeach will be proportionally lower): thianiin HC1, 67.3 mg; riboflavin, 104.2 mg; niacin, 400 mg; biotin,5 mg; folic acid, 25 mg; pyridoxine HCI, 60.8 mg; cyanocobalamine, 0.1 mg; D-cakium pantothenate,218.3 mg; ascorbic acid, 1500 mg; choline chloride, 4000 mg; inositol, 2000 mg; menadione, 30 mgvitamin A, 10,000 IU; vitamin D3, 1000 IU; vitamin E, 1000 IU; Mg (as MgSO4),380 mg; Mn (asMnSO4JI2O), 17 mg; Zn (as ZnO), 50 mg; Fe (as FeSO4.7H20),85 mg; Cu (as CuSO4.5H20),2mg; Co (as CoC1.6H20),0.003 mg; K (asK2S04),895 mg; I (as K103), 5 mg; NaC1 (as NaCI), 2836mg; F (as NaF), 4.5 mg; Se (asNa2SeO.5H0),0.10 mg.4me values for crude protein, ether-extractable lipid, and ash were obtained by proximate analyses.The values for gross energy were estimated by ascribing 5.65 kcal/g crude protein, 9.5 kcal/g crudelipid, 4.0 kcal/g carbohydrate (Alexis eta?., 1985)23Chapter 33.2.1.3 Design of Feeding TrialThe experiment consisted of six treatments (i.e. three dietary treatments, andtwo feeding regimes). Each experimental treatment was assigned to a tank of large,medium, and small size fish. The design of the experiment was, therefore, asfollows:r Large— Fed once daily F MediumSmallThree experimental diets— LargeFed five times daily Medium— SmallFish that were fed once daily were fed at 09:30 hours until satiation. Thefeeding of fish five times daily was done at intervals of 3 h from 06:30 to 18:30 hours.The amount of feed fed to these fish was controlled relative to the average foodconsumption of the fish in the first group in the previous 2 days. The daily rationwas divided equally among the five feedings. The exception was the first 2 dayswhen fish in both feeding regimes were fed to satiation. The total amount of foodconsumed by both groups was averaged, with respect to the dietary treatment and24Chapter 3size of fish, to determine the amount of feed for day-three feeding of the fish thatwere fed five times daily. The groups of fish that were fed once daily were locatedin the inner section of the experimental room in order that they would not bedisturbed by the feeding of the groups five times daily.Fish were gradually accustomed to experimental diets over a four-day periodby mixing increasing amounts of the respective diets with the commercial diet(EWOS) that had been fed to the fish during the pre-experimental period. Theexperimental period was 26 days following acceptance of the experimental diets.The amount of food given to the fish was recorded daily to determine feedefficiency. There was no mortality during the feeding trial. After 26 days, the fishwere sampled according to the following protocols for determination of plasmaamino acid concentrations, gastrointestinal contents, and carcass composition.3.2.1.4 Sampling ProcedureBlood was withdrawn from five fish from each tank at each sampling time,i.e. 15 fish were sampled per dietary treatment under each feeding regime. The fishthat were fed once daily were bled at 0, 3, 9, and 15 h after feeding, and fish fed fivetimes daily were sampled at 0, 3, and 9 h after the first morning feeding. Thefeeding for the frequently-fed fish was continued during the sampling period. Thereason for limiting the sampling times to three for the latter group of fish wasbecause of the likelihood that disturbance of the fish would affect food intake anddigestion. The fish to be sampled were anesthetized with 0.01% tricainemethanesulfonate (MS-222), and weighed. Blood was collected from the caudalvein artery complex into heparinized tubes by severing the caudal peduncle. The25Chapter 3reason for sampling blood from the caudal vein complex was to obtain samplesrepresentative of the systemic circulation.Blood samples from five fish were pooled per tank and centrifuged at 780xgfor 15 nun. The resultant plasma was collected and kept at -70°C pending analysisfor amino acids. After the blood samples were taken, any remaining food wasstripped from the digestive tracts of the fish and the carcasses were frozen forsubsequent proximate analyses. The contents of the gastrointestinal tracts wereweighed.3.2.1.5 Proximate AnalysisThe proximate analyses of formulated diets and fish were conductedaccording to AOAC methods (AOAC, 1984). The analyses for each pooled sampleof five fish for each replicate tank were performed in triplicate. The formulateddiets were also subjected to acid hydrolysis for amino acid determination.32.1.6 Amino Acid AnalysisPlasma samples that were kept at -70°C were thawed and deproteinized bythe addition of 10% trichioroacetic acid (1:1 v/v), followed by vortexing for 2 s, andcentrifugation at 10,000xg for 5 miii at -4°C. The deproteinized plasma was pipettedinto 20 mL test tubes, and 3 mL of diethyl ether were added for each mL of plasma.Lipid was extracted into the ether by vortexung, and the ether layer was pipetted off.The deproteinized, defatted plasma was filtered through a 0.22 j.m polycarbonatemembrane filter. The ifitered plasma samples were kept at -70°C until amino acidanalyses could be performed. Plasma amino acid concentrations were determined26Chapter 3using ion exchange chromatography (Beckman Amino Acid Analyzer Model 6300).Identification and quantification of individual amino acids were accomplished withthe use of external standards obtained from Sigma (Amino acid standard solution,catalog number A2908). The procedure did not detect tryptophan. In addition,threonine peaks were often eluted with serine. As a result, the concentrations ofthreonine in some samples were calculated from the area under the co-eluted peakaccording to Beckman instruction (Beckman, 1982).3.2.1.7 Statistical AnalysisAn analysis of variance with size classes of fish as blocks was perfonned usinga factorial model to examine the effects of feeding frequency and of dietarytreatment on fish weight, specific growth rate, feed consumption, feed efficiency,protein gain, productive protein value, lipid gain, energy efficiency, and bodyproximate composition of fish. In the case of the data for protein gain, lipid gain,dry matter, and proximate composition of the fish, the interaction terms werepooled with the error term because the interactions were not significant. To testdifferences between treatment means, the Tukey HSD test was employed (Zar,1984). Correlations were determined on the relationship between theconcentrations of plasma and dietary amino acids. The statistical analyses wereperformed using Systat (Wilkinson, 1990).27Chapter 33.2.2 RESULTS3.2.2.1 Comparison Between Amino Acid Composition of Experimental Diets andRequirementsThe amino acid compositions of the experimental diets are shown in Table1.2. Diet 1 (fish meal based diet) contained similar proportions of essential andnon-essential amino acids with the ratio of EAA/NEAA of 1.0. Diets 2 and 3contained lower concentrations of essential amino acids and higher concentrationsof non-essential amino acids. The ratios of EAA/NEAA in diet 2 and 3 were 0.7and 0.8, respectively.The comparisons between amino acid concentrations in the experimentaldiets and the requirement values recommended by the NRC (1981) and recentlyreported values for rainbow trout are shown in Table 1.2. It was found that theconcentrations of essential amino acids present in diets 1 and 3 exceeded therequirement levels, expressed as % protein.3.2.2.2 Responses of Fish to Experimental Diets and Feeding FrequencyThe data for initial weight, final weight, weight gain, and feed consumptionof fish of the three different sizes subjected to the different treatments are providedin Table 1.3. Carcass composition of fish of different sizes subjected to differenttreatments is given in Table 1.4. The data in Table 1.3 and 1.4 were used tocalculate different indices shown in Table 1.5. Data were subjected to analysis ofvariance and the significance of the treatment effects is shown in Appendices 1, 2,and3.28Chapter 3Table 1.2. Amino acid composition of experimental diets and the NRC requirement valuesExperimental diet1 RequirementAmino acid Diet 1 Diet 2 Diet 3 Salmon2 Troutg/l6gNArg 6.4 5.7 6.3 6.0 205•9a-fHis 2.2 1.8 22 1.8lie 3.8 2.9 2.9 2.2Leu 7.1 6.6 6.8 3.9Lys 5.9 4.0 5.7 5.0Met 2.5 1.6 2.5 -Met+Cys 3.5 2.5 3.3 4.0Phe 3.7 3.4 3.4 -Phe+Tyr 6.4 5.6 5.4 5.1Thr 4.1 3.0 3.0 22Trp3--- 0.5 051.41Val 4.6 3.5 3.5 3.2Ala 5.5 6.4 6.3Asp 8.7 8.1 8.1Glu 14.5 14.5 14.6Gly 5.9 10.1 10.0Pro 4.9 8.0 7.9Ser 4.1 3.9 3.9Tau 1.0 0.7 0.7TAA4 87.3 86.5 90.0EAA 43.9 35.5 39.2NEAA 43.5 51.0 50.8EAA/NEAA 1.0 0.7 0.81 Diet 1 = fish meal based diet, Diet 2 = fish meal, soybean protein concentrate, corn gluten meal, andgelatin, Diet 3 = Diet 2 supplemented with methionine, lysine, and tryptophan.2W (1981)Tryptophan was not detected in the diets by the procedure used.‘Exc1uding asparagine, glutamine, tryptopan, taurine.a‘he values are cited from Mural (1992); The values are cited from Wilson (1989) C Ketola (1983);The values are cited from Steffens (I989); e The value is from Cho et al., (1989); ‘value is fromOgino (1980); g ialton et al., (1982); R1umsey et al., (1983); ‘Kim et al.,(1992); Poston andRumsey (1983); Walton et al., (1984b); Kim et al., (1987)29Chapter 3Mean initial weight, final weight, weight gain, and feed consumption of fishof different sizes, and treatments are shown in Table 1.6. The averaged values ofthe mentioned parameters were also tabulated for fish fed different diets accordingto feeding regimes as shown in Table 1.7. As expected, fish in different sizecategories (large, medium, and small) were significantly (P < 0.05) different inweights (initial weight, final weight, and weight gain). With similar mean initialweights, fish fed diet 1 (herring meal-based diet) had greater final weight and weightgain (P < 0.05) than those of fish fed either diet 2 (amino acid deficient diet) or diet3 (diet 2 supplemented with lysine, methioriine, and tryptophan), regardless of sizeand feeding frequency. Moreover, feeding fish either once or five times daily didnot affect final weight or weight gain of the fish fed different diets (P >0.05).In general, larger fish consumed more feed than small fish (Table 1.3).Although it was assumed that the amount of food consumed by the groups of fishfed once daily and five times daily would be similar, the results showed that fish fedfive times daily consumed less feed (P <0.0002). There was also a significantinteraction between diet and size of fish. Medium and small size fish had lower feedconsumption when fed diets 2 and 3 as compared to diet 1, but large fish consumedless of diet 1 compared to diets 2 and 3. The variation in feed consumption amongthe three size groups was minimum for diet 1 and maximum for diet 3.Mean specific growth rates, efficiency of feed conversion, productive proteinvalues (PPV = gain in body nitrogen/nitrogen intake), and energy efficiency of fishof different sizes, and on different treatments are shown in Table 1.8. The averagevalues for these parameters were also tabulated for fish fed different diets accordingto feeding regimes as shown in Table 1.9. No significant (P > 0.05) differences in30Chapter 3specific growth rate, feed efficiency, or energy efficiency were found amongdifferent sizes of fish. The specific growth rate of fish of different size categories,however, showed the trend that small size fish grew faster than medium size, orlarge size fish (Table 1.8). Regardless of size of fish and feeding frequency, fish feddiet 1 showed higher (P < 0.05) specffic growth rate, feed efficiency, and energyefficiency than fish fed either diet 2 or diet 3 (Table 1.9). Fish fed either once dailyor five times daily in the present experiment showed similar growth rate, feedconversion efficiency, and energy efficiency (P > 0.05)(Table 1.8). The difference infeed consumption between these two groups as mentioned in the previousparagraph was mainly due to very small variation of the data.In general, protein gain and productive protein values (PPV) for fish fed diet1 were significantly higher than those for fish fed diets 2 and 3. A significantinteraction between diets and sizes of fish, however, was observed (P < 0.05) for thePPV values (Table 1.5). The PPV values for fish of different sizes fed either diet 2or diet 3 were similar. Large fish fed diet 1, however, had significantly higher PPVvalue than those for medium and small fish fed the same diet. Furthermore, thePPV values for fish that were fed five times daily were slightly but significantly(P <0.05) better than for fish fed once daily (Table 1.9). Data in Table 1.5 indicatethat only fish fed diet 3, regardless of the size, had improved PPV when fed fivetimes daily, although the improvement was not statistically significant. Protein gainsof fish that were fed experimental diets at two different feeding regimes weresimilar (P >0.05). Total lipid gain of fish varied with the size, diet, and feedingfrequency as shown in Table 1.8.31Chapter 33.2.2.3 Carcass CompositionMean whole body proximate composition of fish of different sizes feddifferent diets according to feeding regimes are provided in Table 1.10. Theaverage values for carcass composition of fish were also tabulated for fish feddifferent diets according to feeding regimes as shown in Table 1.11. The analyses offish carcasses showed that the body compositions of fish of the three sizes weresimilar (P > 0.05). Furthermore, no significant differences (P> 0.05) were found inthe concentrations of body protein, lipid and ash among fish fed diets 1, 2 and 3.The dry matter content of fish fed diet 1 under both feeding regimes, nevertheless,was slightly but significantly higher than those of fish fed diet 2 and diet 3 (P <0.05).Although feeding fish at different frequencies did not have any effect on growth rateor feed efficiency, significant effects were detected in the dry matter, and carcassprotein and lipid concentrations. Regardless of diet and size of fish, the dry matterand lipid concentrations in fish that were fed five times daily were significantly lowerthan in fish that were fed once daily (P <0.05). Moreover, protein concentrations infish that were fed five times daily were significantly higher than in fish fed once daily(P < 0.05). Ash concentrations of fish fed once or five times daily were similar(P>0.05).32Chapter 3Table 1.3. Initial weight, final weight, body weight gain and feed consumption of rainbow trout ofdifferent sizes fed different diets either once or five times daily over a 26-day period inExperiment 1.1Feeding Diet1 Size2 Initial Final Weight Feedregime weight weight gain consumption(g/flsh) (g/lish) (g/fish) (g/flsh/day)Once daily1 L 25.6 50.6 25.0 1•5b3M 21.3 47.2 25.9S 18.6 39.6 21.02 L 24.8 48.0 23.3M 22.1 40.1 17.9S 19.8 35.4 16.6 097de3 L 26.0 48.2 22.2 127aM 21.0 39.0 18.0 l.0’7S 18.1 34.6 16.6 0•92eFive times daily1 L 26.5 52.0 25.6 Ll3bM 21.8 44.7 22.95 18.3 38.6 20.3 098d2 L 26.5 49.1 22.6M 24.6 40.0 18.4 103cdS 18.6 36.3 17.7 095de3 L 26.0 49.2 23.3 125aM 21.3 40.4 19.1 105CS 18.4 34.3 15.9 089e1Diet 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.2 L = large, M = medium, S = small, each value represents a mean of 50 fish/tank.Superscript letters in the same column within the same feeding regime refer to Tukey HSD test (Zar,1984) when the interaction between diets and sizes of fish were detected. Values followed by thesame letters are not significantly different (P>0.05). Where there are no superscript letter, nointeraction among the means was detected.33Chapter 3Table 1.4. Whole body proximate composition of rainbow trout of different sizes fed different dietseither once or five times daily over a 26-day period in Experiment 1.1Feeding Diet1 Size2 Dry Protein Lipid Ashregime matter(%) (% of dry matter)Initial 20.12 54.03 31.01 ND3Once daily1 L 30.12 51.27 36.30 7.54M 29.86 49.58 37.50 7.41S 29.37 50.38 36.45 8.452 L 28.48 50.94 3530 7.91M 29.09 49.68 37.00 7.58S 29.40 51.33 34.05 7.563 L 28.94 49.81 37.70 7.48M 29.07 50.94 36.10 7.38S 28.70 50.84 33.60 7.52Five times daily1 L 29.92 51.32 37.60 7.39M 29.41 50.57 36.00 7.21S 29.08 52.14 33.50 7.542 L 29.08 50.56 35.00 7.48M 28.38 51.10 34.30 7.72S 27.60 51.87 29.51 7.733 L 28.60 51.53 28.55 7.35M 28.30 52.91 32.90 8.02S 28.71 51.86 34.65 7.381Diet 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.= large, M = medium, S = small, each value represents a pooi sample of five fish/tank.determinedNo interaction among means in the same column was detected.34Chapter 3Table 1.5. Specific growth rates, feed conversion efficiency, productive protein value and energyefficiency of rainbow trout of different sizes fed different diets either once daily or live timesdaily over a 26-day period in Experiment 1.1Feeding Diet1 Size2 Specffic Feed Protein Productive Lipid Energyregime growth rate3 efficiency4 gain protein value5 gain efficiency6(g/tank) (g/tank)Once daily1 L 2.62 0.82 250.9 05aS 194.9 0.46M 3.06 0.86 233.9 0471b 1973 0.44S 2.90 0.80 191.9 0440b 1535 0.402 L 2.54 0.72 212.8 O375 171.7 0.36M 2.29 0.68 171.9 OL358’ 147.4 0.37S 2.23 0.66 165.3 O.369’ 118.3 0.343 L 2.37 0.67 208.1 OL3525 0.36M 2.38 0.64 173.9 349 138.1 0.34S 2.49 0.69 151.0 O.35l 107.9 0.32Five times daily1 L 2.59 0.87 250.1 0516a 205.5 0.4M 2.76 0.80 217.0 0•451b 170.3 0.41S 2.87 0.79 186.6 126.2 0.372 L 2.37 0.72 214.7 0.386 165.0 0.36M 2.37 0.69 173.5 0367C 127.3 0.34S 2.57 0.72 165.8 O379 93.4 0.303 L 2.45 0.71 219.6 OL3Tf 118.2 0.30M 2.46 0.70 188.3 0L3&C 122.1 0.33S 2.40 0.68 153.7 O3lO 111.7 0.341Diet 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3diet 2 supplemented with lysine, methionine, and tryptophan.= large, M = medium, S = small.= (h1W2InW1)*100/(T2T1).= Gain in body weight/feed consumption.= Gain in body nitrogen/nitrogen intake.6 Gain in body energy/GE intake, (body energy was estimated on the basis of carcass gains inprotein and lipid using caloric value of 5.7 and 9.5 kcal/g respectively according to March et at.,1985).7Each value represents a mean of 50 fish/tank, except for protein and lipid gain.8 Superscript letters in the same column within the same feeding regime refer to Tukey HSD test (Zar,1984) when the interaction between diets and sizes of fish were detected. Values followed by thesame letters are not significantly different (P>0.05). Where there is no superscript letter, nointeraction among the means was detected.35Chapter 3Table 1.6. Mean initial weight, final weight, weight gain, and feed consumption of rainbow trout ofdifferent sizes fed different diets either once or five times daily over a 26-day period inExperiment 1.1Main effect Initial Final Weight Feedweight weight gain consumptionSize1 (g) (g) (g/fish) (g/fish/day)L 25.9 ± 04a 49.6 ± 0•3a 23.7 ± 0•4a 1.207 ±M 21.5 ± 41.9 ± 20.4 ± 0•4b 1.077 ±S 18.5 ± 04c 36.5 ± 03c 18.0 ± 04C 0.953 ±Diet21 22.0 ± 04a ± (J3a 23.5 ± 04a 1.090 ±2 22.0 ± 04a 415 ± 03b 19.5 ± 0•4b 1.071 ± 0.017I3 21.8 ± 04a 41.0 ± 03b 19.2 ± 1.075 ±Feeding frequency3Once daily 21.8 ± 03a 42.5 ± 03a 20.71 ± 036a 1.090 ±Five times daily 22.1 ± 0•3a 42.8 ± 0•3a 20.70 ± 036a 1.068 ± 0•17b= large, M = medium, S = small, each value is a mean of six values (n = 6).2 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3diet 2 supplemented with lysine, methionine, and tryptoph, each value is a mean of six values(n= 6).3Each value is a mean of nine values (n= 9).letters in the same column within a main effect refer to Tukey HSD test (Zar, 1984).Values followed by the same letters are not significantly different (P >0.05).36Chapter 3Table 1.7. Mean initial weight, final weight, body weight gain and feed consumption of rainbow troutfed different diets according to feeding regime over a 26-day period in Experiment 1.1Feeding Diet1 Initial Final Weight Feedregime weight weight gain consumption(g) (g) (g/fish) (g/fish/day)Once daily1 21.82 45.8 24.0 1.1032 22.2 41.1 19.3 1.0803 21.7 40.6 18.9 1.087Five times daily1 22.2 45.1 22.9 1.0772 22.2 41.8 19.6 1.0633 21.9 41.5 19.6 1.063Pooled SEM 0.5 0.7 0.7 0.002tDiet 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.2 value is a mean of three values for three sizes of fish, each contains 50 fish, (n=3). Nointeraction among means in the same column was detected.37Chapter 3Table 1.8. Mean specific growth rate, feed conversion efficiency, productive protein value, and energyefficiency of rainbow trout of different sizes fed different diets either once or live times daily overa 26-day period in Experiment 1.1Main effect Specific Feed Protein Productive Lipid Energygrowth rate1 efficiency2 gain protein value3 gain efficiency4(g/tank) (g/tank)Size5L 2.49 ± a 0.75 ± 01a 226.0 ± 19a 0.419 ± 3a 172.6 ± 84a 0.39 ±M 2.55 ± a 0.73 ± •01a 193.0 ± 0.397 ± 150.4 ± 84b 0.37 ± 01aS 2.57 ± a 0.72 ± 01a 169.0 ± l.9’ 0.392 ± 3b 118.5 ± 8.4 0.35 ±Diet61 2.80 ± 06a8 0.82 ± •01a 221.7 ± 19a 0.472 ± 003a 174.6 ± 84a 0.43 ±2 2.40 ± 0.70 ± 184.0 ± 19b 0.372 ± 3b 137.2 ± 84b 0.35 ± 01b3 2.43 ± 0.68 ± 01b 182.4 ± 19b 0.364 ± 3b 129.7 ± 8.4 0.33 ± 01bFeeding regime7Once daily 2.54 ± 05a 0.73 ± 01a 195.5 ± 15a 0.397 ± )2a 1566 ± 68a 0.38 ± 01aFive times 2.54 ± 05a 0.74 ± 01a 196.6 ± 15a 0.408 ± 2b 137.8 ± 68b o. ± 01adaily1= (lnW2lnW1)*100/(T2.T1).2 Gain in body weight/feed consumption.= Gain in body nitrogen/nitrogen intake.= Gain in body energy/GE intake, (body energy was estimated on the basis of carcass gains hiprotein and lipid using caloric value of 5.7 and 9.5 kcal/g respectively according to March et al.,1985).5L = large, M medium, S = small, each value is a mean of six values (n= 6).6 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan, each value is a mean of six values(n= 6).Each value is a mean of nine values (n= 9).8 Superscript letters in the same column within a main effect refer to Tukey HSD test (Zar, 1984).Values followed by the same letters are not significantly different (P >0.05).38Chapter 3Table 1.9. Mean specific growth rates, feed conversion efficiency, productive protein value and energyefficiency of fish fed different diets according to feeding regimes over a 26-day period inExperiment 1.1Feeding Diet5 Specific Feed Protein Productive Lipid Energyregime growth rate1 efficiency2 gain protein value3 gain efficiency4(g/50 fish) (g/50 fish)Once daily1 2.866 0.83 225.53 0.474 181.90 0.432 2.35 0.69 183.32 0.367 145.82 0.363 2.41 0.67 177.64 0.351 142.02 0.34Five times daily1 2.74 0.82 217.88 0.470 167.33 0.422 2.44 0.71 184.65 0.377 128.57 0.333 2.44 0.70 187.21 0.377 117.34 0.32Pooled SEM 0.08 0.01 2.65 0.004 11.82 0.011= (1nW2lnW1)*100/(T2T1).2= Gain in body weight/feed consumption.= Gain in body nitrogen/nitrogen intake.= Gain in body energy/GE intake, (body energy was estimated on the basis of carcass gains inprotein and lipid using caloric value of 5.7 and 9.5 kcal/g respectively).1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.6 value is a mean of three values for three sizes of fish, each contains 50 fish, (n= 3). Nointeraction among means in the same column was detected.39Chapter 3TabLe 1.10. Mean carcass compositions of rainbow trout of different sizes fed different diets eitheronce or five times daily over a 26-day period in Experiment 1.1Main effect Dry matter Protein Lipid Ash(%) (% of dry matter)Size1L 29.19 ± 5090 ± 031a 35.08 ± 085a 7.51 ± 014aM 29.02 ± 018a 50.79 ± 031a 3561 ± 085a 736 ± 014aS 28.81 ± 018a 5144) ± 031a 33.63 ± 085a 775 ±Diet21 29.63 ± 018a 50.88 ± 031a 36.20 ± 085a ± 13a2 28.67 ± 0.181) 50.91 ± 031a 34.16 ± 085a ±3 28.72 ± 018b 51.31 ± 031a 33.85 ± 085a 7.48 ± 013aFeeding frequency3Once daily 29.22 ± 0•15a 50.53 ± 0•25a 35.98 ± 069a 7.65 ±Five times daily 28.79 ± 015b 51.54 ± 0•25b 33.56 ± 0•69b 7.43 ±= large, M = medium, S = small, each value is a mean of six values (n=6).2 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan, each value is a mean of six values(n= 6).3Each value is a mean of nine values (n= 9).4Superscript letters in the same column within a main effect refer to Tukey HSD test (Zar, 1984).Values followed by the same letters are not significantly different (P >0.05).40Chapter 3Table 1.11. Mean carcass composition of rainbow trout fed different diets according to feedingregimes over a 26-day period in Experiment 1.1Feeding Diet1 Dry Matter Protein Lipid Ashregime(%) (% of dry matter)Once daily1 29.782 50.41 36.75 7.812 28.99 50.65 35.45 7.693 28.90 50.53 35.80 7.47Five times daily1 29.47 51.34 35.70 7.382 28.35 51.17 32.94 7.513 28.53 52.10 32.03 7.51Pooled SEM 0.24 0.43 1.27 0.20‘Diet 1 = fish meal based diet; Diet 2 = diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.2 value is a mean of three mean values for three sizes of fish, each was analysed from a poolsample of five fish, (n = 3). No interaction among means in the same column was detected.41Chapter 33.2.2.4 Gastrointestinal ContentsThe average wet weights of the gastrointestinal contents from the fish thatwere fed once daily and five times daily are compared graphically in Figure 1.1. Thefish that were fed once daily showed a gradual decline in the amount of gut contentsbetween 3 and 15 h after feeding. The rate of feed passage in fish fed the differentexperimental diets appeared to be similar. As expected, the fish that were fed fivetimes daily showed less variation in the amounts of gut contents over theexperimental period.322.5 Plasma Amino Acid ProfilesThe concentrations of individual free amino acids in the plasma expressed asmol/mL and as percentage composition determined at different times after mealconsumption are shown in Tables 1.12-1.17. The concentrations of total amino acids(TAA), total essential amino acids (EAA), total non-essential (NEAA), and ratiosof EAA/NEAA in the plasma of fish fed the different diets are also tabulated inthese tables. The total essential amino acids included argimine, cystine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine andvaline. Since the procedure used in analysing plasma amino acids did not detecttryptophan, it was not reported anywhere. The changes in plasma concentrations oftotal essential amino acids, total nonessential amino acids, and total amino acids areshown in Figure 1.2. The changes in plasma concentrations of individual free aminoacids at different times after meal consumption are depicted in Figure 1.3. It shouldbe mentioned that the fish fed diets once daily had been without feed for 24 h atzero-time i.e. just before feeding. The fish fed five times daily, on the other hand,were without feed for only 12 h at zero-time.424Ia)•1-CSC-)6j 12 15Hats after initial feedingC Diet 2Figure 1.1. Mean wet weight (± SE) of gastrointestinal contents of rainbow trout atdifferent times after feeding in Experiment 1.1; arrows indicate the time of feeding.Each value is a mean of three pooled samples of five fish (n=3).Fed once daily3210of 3 6 g 12Hare after feedingDiet 1 C Diet 2 Diet 31534-’-CIIred five times daily2I0— Diet 1 Diet 343Chapter 3Plasma Total Amino Acids (TAA), Total Essential Amino Acids (EAA) and TotalNon-Essential Amino Acids (NEAA)As shown in Figure 1.2, feeding regimen affected the patterns of plasmaEAA, NEAA, and TAA concentrations in fish fed different diets over the samplingperiod. In fish that were fed once daily, plasma EAA, NEAA, and TAAconcentrations peaked differently depending upon the nature of the diet. PlasmaEAA concentrations in fish that were fed diets 1, 2, 3 followed a similar pattern inthat they started to rise soon after feeding and reached a peak 9 h postprandial(Figure 1.2a). The patterns of NEAA and TAA concentrations in fish fed diets 2and 3 were similar. In both treatments, the concentrations of amino acids remainedelevated and had not declined before the fish were re-fed at 24 h after the previousfeeding. Plasma TAA concentrations in fish that were fed diet 1 resembled thechanges of EAA whereas those of NEAA showed only a small increase afterfeeding. Unlike the fish that were fed once daily, plasma EAA, NEAA., and TAAconcentrations of fish fed five times daily, as would be expected, variedcomparatively little throughout the sampling period regardless of the diet.The peak concentration of plasma EAA at 9 h after feeding in fish fed diet 1once daily was approximately 125% higher than that in fish fed diet 2, and 75%higher than that in fish fed diet 3 (Figure 1.2a). Interestingly, the plasma EAAconcentrations in fish fed the different diets five times daily, however, showed onlysmall differences (Figure 1.2a). Distinctively, plasma NEAA concentrations in fishfed diet 2 and 3 were more than two-fold higher than those of fish fed diet 1 underboth feeding regimes. As a result, the patterns and levels of TAA concentrations infish fed diet 2 and 3 resembled the NEAA patterns and concentrations while, thepatterns and levels of TAA concentrations in fish fed diet 1 resembled those of44Chapter 3EAA concentrations (Figure 1.2b and 1.2c).The ratio of plasma EAA/NEAA concentrations in fish fed the differentdiets revealed the differences in the concentrations of non-essential amino acidsbetween diet 1 and those of diet 2 and 3 (Tables 1.12-1.17). The ratios of plasmaEAA/NEAA in fish fed diet 1 were between 0.78- 1.02 and 0.74-0.8 1 in fish fed oncedaily and five times daily, respectively (Table 1.12 and 1.15). The ratios of plasmaEAA/NEAA of fish fed diet 2 were lower than those of fish fed diet 1 rangingbetween 0.24-0.36 and 0.25-0.3 1 in fish fed once daily and five times daily,respectively (Table 1.13 and 1.16). The ratios of plasma EAA/NEAA in fish feddiet 3 were between 0.34-0.45 in fish fed once daily, and 0.35-0.40 in fish fed fivetimes daily (Table 1.14 and 1.17). The ratios were higher than those of fish fed diet2 regardless of feeding schedule, and lower than those in fish fed diet 1.Plasma Concentrations of Individual Amino Acids in Fish Fed Different Diets andon Different Feeding RegimesEssential Amino AcidsIn fish fed once daily, patterns of several individual amino acids in fish feddiets 1 and 3 seemed to follow the overall patterns of EAA concentrations. As withplasma EAA patterns, fish fed diet 1 once daily showed fluctuation of most essentialamino acids namely histidine, isoleucine, leucine, lysine, methionine + cystine,valine, and threonine over time, i.e. increasing after meal consumption andgradually decreasing subsequently. Plasma concentrations of arginine,phenylalanine, and tyrosine did not, however, show any changes. In fish fed diet 3,plasma concentrations of isoleucine, leucine, lysine, methionine + cystine, and valine45Chapter 3showed similar patterns to that of the overall change in EAA concentrations. Theremaining amino acids, however, stayed relatively constant. Plasma concentrationsof most essential amino acids in fish fed diet 2 showed only slight increases orremained constant in the period following the meal. There were only two essentialamino acids, leucine, and valine, that showed a fluctuation similar to the overallpattern of the plasma EAA (Figure 1.3a-1.3i).In fish fed five times daily, levels of most essential amino acids in fish fed thethree different diets tended to remain fairly stable throughout the sampling periodin agreement with the overall pattern of EAA concentrations (Figures 1.3a-1.3e,1.3i, and l.3p). Exceptions were plasma concentrations of methionine + cystine, andvaline. The levels of plasma valine dropped at 3 h and rose at 9 h after feeding. Incontrast, the levels of plasma methionine + cystine started to rise at 3 h after feedingand thereafter remained relatively stable (Figures 1.3f-1.3g).The most abundant essential amino acid in the plasma was found to differ amongthe fish that were fed different diets. Valine concentrations were the highest in fishfed diet 1 or 2 under both feeding regimes. The average relative concentrations forthis amino acid calculated as a percentage of total amino acids were 11.3% and9.6% for fish fed diet 1 once daily and five times daily (Tables 1.12 and 1.15); and4.9% and 4.5% for fish fed diet 2 once daily and five times daily, respectively(Tables 1.13 and 1.16). In contrast, the fish that were fed diet 3 (which wassupplemented with lysine) once daily or five times daily had lysine as the mostabundant essential amino acid in the plasma constituting 5.4 and 5.0% of TAArespectively (Table 1.14 and 1.17).46Chapter 3Non-Essential Amino AcidsThe patterns of individual non-essential amino acids in plasma of fish feddifferent diets once daily resembled the overall pattern of NEAA (Figures 1.3j-o,1.2b), except for the levels of glycine in fish fed diets 2 and 3. Plasma glycine inthese fish started to rise after feeding and peaked at 15 h. This was different fromthe NEAA pattern which showed a drop at 3 h after feeding.The patterns of individual non-essential amino acids in fish fed diets 1 and 3five times daily were similar to the overall pattern of NEAA. Some non-essentialamino acids in fish fed diet 2, however, differed from the overall pattern of NEAA.Plasma alanine, proline, and aspartic acid concentrations dropped at 3 h afterfeeding (Figures 1.3j-o, 1.2b).The concentrations of alanine, aspartic acid, glycine, proline and serine infish fed diets 2 and 3 were considerably higher than those of fish fed the diet 1 undereither feeding regime. Differences between fish fed diet 2 and 3 were also found inthe levels of the above mentioned amino acids. The levels of these amino acids infish that were fed diet 2 were higher than those in fish fed diet 3 under both feedingregimes (Tables 1.12-1.17 and Figures 1.3j-p).Glycine, of the non-essential amino acids, generally represented the highestconcentrations, between 27.6-38.8%, of the total plasma amino acids dependingupon the diets and feeding regimes. Serine was the second most abundant nonessential amino acid, constituting about 11.7-14.7% of total plasma amino acidsdepending upon the diet and feeding regime (Tables 1.12-1.17, and Table 1.18).47Table1.12.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafterfeedingdiet1(fishmeal baseddiet)inExperiment1.1.Fishfedoncedailytosatiation.AminoHoursafterfeedingacid03915Non-essentialEssentialX±SEM1X±SEMX±SEMX±SEM(niol/mL)%2(Lmo1/mL)%(Lmo1/mL)%(Lmo1/mL)Arg0.125±0.0032.50.159±0.0052.50.146±0.0112.10.136±0.024Cys0.016±0.0020.30.026±0.0020.40.030±0.0070.40.023±0.001His0326±0.0236.50.383±0.0185.90.428±0.0416.30.369±0.009lIe0.175±0.0173.50.269±0.0204.10.302±0.0554.40.173±0.033Leu0.307±0.0336.10.476±0.0417.30.568±0.1108.30297±0.040Lys0.267±0.0145.30.343±0.0105.30.337±0.0404.90.261±0.010Met0.165±0.0073.30.232±0.0123.60.264±0.0143.90.227±0.015Phe0.095±0.0041.90.124±0.0091.90.129±0.0021.90.122±0.004Thr0.218±0.0184.40244±0.0443.80.292±0.0834.30205±0.023Tyr0.027±0.0100.50.083±0.0051.30.061±0.0200.90.053±0.014Val0.567±0.04811.30.715±0.05611.00.881±0.14812.90.526±0.042Ala0.423±0.0228.50.530±0.034820.535±0.0347.80391±0.006Asp0.075±0.0041.50.093±0.0051.40.090±0.0161.30.099±0.008Glu0.105±0.0022.10.175±0.0242.70.181±0.0232.70.167±0.020Gly1.452±0.07629.01.718±0.10226.51.728±0.10125.31.690±0.198Pro0.081±0.0101.60.162±0.0062.50.157±0.0982.30.122±0.022Ser0.582±0.02511.60.758±0.04611.80.780±0.02014.40.657±0.027TAA35.005±0.1316.434±0.3906.822±0.6235.477±0257EAA42.286±0.1193.053±0.1753.443±0.4402.391±0.065NEAA52.718±0.0773.380±0.2123.378±0.1853.086±0252EAA/NEAA0.840.901.020.782.5 0.4 6.7 3.1 5.4 4.7 4.1 2.2 3.7 1.0 9.5 7.1 1.8 3.0 30.6 2.212.01Meanofthreepoolsfplasma(fivefish/pool)andstandarderrorofthemean(n=3).2of totalaminoacids.essential aminoacids.Totalnon-essentialaminoacids3Totalaminoacids.4TotalTable1.13.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimeafterfeedingdiet2(aminoaciddeficientdiet)inExperiment1.1.Fishfedoncedailytosatiation.AminoHoursafterfeedingacid03915Non-essential3Totalaminoacids.4TotalEssentialArg CysHislie LenLysMetPheThrTyrValX±SEM1X±SEMX±SEMX±SEM(I.Lmol/mL)%2(mol/mL)%(I.mo1/mL)%(.tmo1/mL)0.118±0.0031.30.155±0.0311.80.118±0.0331.20.134±0.0090.012±0.0010.10.032±0.0120.40.026±0.0050.30.021±0.0030.346±0.0203.70.348±0.0574.00.346±0.0183.50.261±0.1120.101±0.0161.10.177±0.0252.10.193±0.0311.90.153±0.0100.251±0.0452.70.335±0.0463.90.399±0.0584.00.305±0.0060.223±0.0172.40.255±0.0343.00237±0.0222.40.236±0.0040.070±0.0030.80.126±0.0211.50.130±0.0231.30.118±0.0210.103±0.0011.10.123±0.0111.40.137±0.0161.40.129±0.0170.175±0.0131.90.175±0.0462.00200±0.0342.00.202±0.0270.032±0.0020.40.080±0.0120.90.050±0.0050.50.040±0.0180.378±0.0514.10.483±0.0715.60.568±0.0645.70.448±0.034Ala1.014±0.09110.90.717±0.1018.30.935±0.0489.40.953±0.024Asp0.816±0.0448.80.669±0.0847.80.802±0.0848.10.892±0.036Glu0.163±0.0171.80.164±0.0161.90.188±0.0141.90.180±0.016Gly2.780±0.56629.83.215±0.18237.33.579±0.22236.04.100±0.141Pro1.155±0.17912.40.461±0.0125.40.653±0.0976.60.606±0.116Ser1.584±0.15617.01.096±0.11412.71.393±0.23714.01.559±0.087TAA39.318±0.8758.610±0.8039.953±0.73810.340±0.200EAA41.808±0.1502.289±0.3382.404±02062.047±0.147NEAA57.510±0.7356.321±0.4847.550±0.5838.289±0.334EAA/NEAA0.240.360.320.25% 1.3 0.2 2.8 1.5 2.9 2.3 1.1 1.22.00.44.39.28.6 1.7 36.9 5.9 15.11 Meanofthreepoolsfplasma(fivefish/pool)andstandarderrorofthemean(n= 3).2oftotalaminoacids.essentialaminoacids.Totalnon-essentialaminoacidsAminoHoursafterfeedingacid03915Non-essentialTAA3 EAA4 NEAA5 EAA/NEAA9.014±0.5102.303±0.1196.711±0.3950.348.405±0.8002.621±0.1625.784±0.6560.459.260±0.7232.755±02706.506±0.4540.429.491±0.3812.526±0.0496.964±0.3610.36aminoacids.‘TotalTable1.14.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimeafterfeedingdiet3(supplementedwithlysine,methionine,andtryptophan)inExperiment1.1.Fishfedoncedailytosatiation.EssentialX±SEM1X±SEMX±SEMX±SEM(.Lmol/mL)%2(mol/mL)%(I.mol/mL)%(j.mo1/mL)%Arg0.122±0.0151.40.166±0.0141.90.164±0.0171.80.149±0.0131.6Cys0.027±0.002030.024±0.0020.30.030±0.0030.30.033±0.0010.4His0.321±0.0233.60.336±0.0404.00.340±0.0243.70.357±0.0123.8lie0.107±0.0081.20.169±0.0192.00.166±0.0241.80.142±0.0161.5Len0.256±0.0132.80.310±0.0223.70.358±0.0473.90.313±0.0263.3Lys0.509±0.0235.70.517±0.0456.20.488±0.0385.30.439±0.0284.6Met0.274±0.0143.00.340±0.0184.00.355±0.0293.80.330±0.0183.5Phe0.098±0.0041.10.130±0.0071.50.114±0.0101.20.120±0.0071.3Thr0.171±0.0101.90.140±0.0211.70.186±0.0232.00.168±0.0301.8Tyr0.024±0.002030.058±0.0030.70.039±0.0110.40.044±0.0170.5Vai0.394±0.0204.40.431±0.0275.10.515±0.0635.60.432±0.0274.6Ala0.863±0.0459.60.707±0.0608.40.775±0.0268.40.895±0.0559.4Asp0.730±0.0148.10.601±0.1107.20.627±0.0316.80.689±0.0617.3Glu0.159±0.0131.80.163±0.0151.90.175±0.0141.90.153±0.0141.6Gly2.947±0.14032.72.971±0.33535.33.262±0.12735.23.498±0.16036.9Pro0.847±0.1339.40.353±0.0264.20.568±0.2296.10.615±0.0736.5Set1.167±0.10412.90.989±0.15611.81.099±0.06011.91.114±0.06011.71Meanofthreepoolsfplasma(fivefish/pool)andstandarderrorof themean(n=3).2of totalaminoacids.essentialaminoacids.‘Totalnon-essentialaminoacidsTable1.15.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafterfeedingdiet1(fishmealbaseddiet)inExperiment1.1.FishfedStimesdaily1.AminoHoursafterfeedingacid039X±SEM2%3X±SEM%X±SEM%(mol/mL)(mo1/mL)(I.Llnol/mL)EssentialMg0.111±0.0022.10.154±0.0182.90.130±0.0162.5Cys0.016±0.0030.30.034±0.0080.60.026±0.0070.5His0.333±0.0296.40.334±0.0066.20.310±0.0335.9lie0.171±0.0383.30.173±0.0163.20.174±0.0223.3Leu0.314±0.0776.00.284±0.014530.307±0.0305.9Lys0.268±0.0225.10.280±0.013520235±0.0234.5Met0.171±0.009330225±0.021420.206±0.0353.9Phe0.106±0.0092.00.116±0.026220.118±0.00423Thr0.242±0.0344.60.155±0.0072.90.163±0.0143.1Tyr0.045±0.0070.90.070±0.036130.079±0.01515Vai0.566±0.11010.80.472±0.0188.70.488±0.044)9.3Non-essentialAla0.412±0.0477.90.403±0.0247.50.398±0.0447.6Asp0.090±0.0151.70.096±0.0051.80.134±0.0512.6Glu0.146±0.0262.80.162±0.0123.00.139±0.0082.7Gly1.565±0.08730.01.712±0.01831.71.645±0.20731.4Pro0.106±0.0232.00.042±0.0420.80.050±0.0511.0Ser0559±0.05010.70.693±0.02612.80.642±0.051122TAA45222±05405.485±0.1725.244±0.458EAA52.344±0.3332298±0.1262.234±0.193NEAA62.878±0.2333.108±0.0463.008±0349EAA/NEAA0.810.740.74ITotaldailyintakeof feedwasequaltotheamountconsumedbythefishfedoncedaily, foreachrespectivediet.Meanofthrepoolsofplasma(fivefish/popl)andstandarderrorofthemean(n=3).%of totalaminoacids.aminoacids.‘Totalessentialaminoacids.°Totalnon-essentialaminoacidsTable1.16.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafterfeedingdiet2(aminoaciddeficientdiet)inExperiment1.1.Fishfed5timesdaily1.AminoHoursafterfeedingacid039X±SEM%3X±SEM%X±SEM%(.tmol/mL)(.tmol/mL)(mo1/mL)EssentialArg0.124±0.0131.20.165±0.0171.60.143±0.0151.4Cys0.017±0.0000.20.032±0.0060.30.046±0.0120.5His0.342±0.0153.20.361±0.0043.50.361±0.0163.6lie0.138±0.0191.30.135±0.0221.30.177±0.0171.8Leu0.335±0.0463.20.265±0.0212.50.347±0.0373.4Lys0.226±0.0122.10.236±0.0062.30.225±0.0302.2Met0.083±0.0070.80.115±0.0111.10.148±0.0151.5Phe0.120±0.0091.10.129±0.0061.20.136±0.0101.4Thr0.211±0.0362.00.192±0.0411.80.198±0.0202.0Tyr0.058±0.0120.60.090±0.0160.90.094±0.0060.9Val0.490±0.0704.60.403±0.0153.90.496±0.0444.9Non-essentialAla0.965±0.1249.10.892±0.0108.60.828±0.1008.2Asp0.900±0.0158.50.808±0.0317.80.846±0.0698.4Glu0.179±0.0111.70.192±0.0121.90.176±0.0201.7Gly3.847±0.21136.54.400±0.22642.23.806±0.24237.6Pro1.051±0.12610.00.505±0.0674.90.550±0.1665.4Ser1.469±0.05313.91.501±0.05114.41.534±0.24515.2TAA410.533±0.46410.419±0.28210.111±1.030BAA52.143±0.1612.125±0.0602.370±0.195NEAA68.411±0.3438.298±0.3077.740±0.835EAA/NEAA0.250.260.311Totaldailyintakeoffeedwasequaltotheamountconsumedbythefishfedoncedaily,foreachrespectivediet.2Meanofthreppoolsofplasma(fivefish/poil)andstandarderrorofthemean(n=3).%of totalaminoacids.aminoacids.Totalessentialaminoacids.Totalnon-essentialaminoacidsTable1.17.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafter feedingdiet3(supplementedwithlysine,methionine,andtryptophan)inExperiment1.1.Fishftd5timesdaily1.AminoHoursafterfeedingacid039X±SEM2%3X±SEM%X±SEM%(tmol/mL)(tmol/mL)(mol/mL)EssentialArg0.132±0.0121.40.156±0.0061.80.158±0.0181.8Cys0.027±0.0020.30.095±0.0471.10.037±0.0070.4His0.312±0.0173.30337±0.0213.90.335±0.0363.8Ile0.120±0.0071.30.132±0.0091.50.128±0.0171.4Leu0.297±0.0183.10254±0.0083.00.262±0.0292.9Lys0.457±0.0114.80.469±0.0425.40.421±0.0324.7Met0.321±0.0053.40.294±0.0063.40.336±0.0553.8Phe0.113±0.0081.20.125±0.0081.40.118±0.01313Thr0.199±0.0122.10.157±0.0301.80.170±0.0241.9Tyr0.040±0.0080.40.045±0.0120.50.040±0.0050.4Val0.446±0.0304.70.44)1±0.0134.60.385±0.0334.3Non-essentialAla0.797±0.0668.40.697±0.0818.00.765±0.0788.6Asp0.790±0.0178.30.668±0.0197.70.757±0.0468.5Glu0.200±0.0172.10.183±0.0182.10.170±0.0171.9Gly3388±0.16635.63.259±0.06437.43381±0.49337.9Pro0.683±0.0867.20.396±0.0664.50.327±0.0473.7Ser1.189±0.03112.51.057±0.01812.11.139±0.15712.8TAA49.510±0.1078.725±03458.926±1.000EAA52.464±0.0762.466±0.0382387±0.193NEAA67.046±0.0686.260±0.3096.541±0.812EAA/NEAA0350.400.361Totaldailyintakeoffeedwasequaltotheamountconsumedbythefishfedoncedaily,foreachrespectivediet.of threepoolsofplasma(fivefish/pool) andstandarderrorofthemean(n= 3).%of totalaminoacids.4Totalaminoacids.Totalessentialaminoacids.6non-essentialaminoacids(a)TotalessentialaminoacidsE—B--dietl+diet2.:z.,:----*diet31fedoncedailyfedfivetimesdaily00369121503691215(b)Totalnon-essentialaminoacids—.-(c)Totalaminoacids1010.+*EE--.3++.3+i::*j :‘2fedoncedailyfedfivetimesdaily2fedoncedailyfedfivetimesdaily0110iiIII03691215036912150369121503691215HoursafterfeedingHoursafterfeedingFigure1.2.Total concentrationsof plasmaaminoacids(essential,non-essential,andtotalaminoacids)determinedatdifferent timespostprandialforrainbowtroutfedoncedailyandfivetimesdailyinExperiment1.1.Diet1=fishmealbaseddiet, diet2=dietdeficientinmethioriine,lysine,andtryptophan, diet 3=diet2supplementedwithmethionine,lysine,andtryptophan.Eachpointrepresentsameanofthreepoolsofplasma,fivefish/pool, (n=3).Arrowsindicatethetimeof feeding.110.80.60.40.2(b)Histidinefedoncedailyfedfivetimesdaily(1,(J’(a)Arginine0.8EfedoncedailyfedfivetimesdailyC.20.6—9—dieti0.4diet20 o-*-diet3(U0.2 U36912153691215(c)LysineE0.8fedoncedailytedfivetimesdailyC.20.6C0.4+,0.2(U aC369121536Hoursafterfeeding0-J E 0 E C 0 (U I-. C 0 C 0 0 (U E (U (U 0-j E 0 E C 0369121536912159121536912153691215HoursafterfeedingFigure1.3.Plasmaconcentrationsofaminoacidsinrainbowtroutfeddifferent dietsaccordingtodifferentfeedingregimesinExperiment 1.1.Diet1=fishmeal baseddiet,diet2=dietdeficientinmethionine,lysine,andtryptophan,diet3diet2supplementedwithmethionine,lysine,andtryptophan.Eachpoint representsameanof threepoolsofplasma,fivefish/pool,(n =3).1-J E 0 E C.20.60 I C a)0.40 C) E0.20 -J E 0.8E C.20.6I 4- C 00.40 C) 0 E ,0.2Ca 0(e)Isoleucinefedfivetimesdaily—B—diet1+diet2-*-diet3fedoncedaily-J E 0 E C 0 Ca I C a)0.40 00.2*C’,36912153691215(g)Methionine+Cystine36912153691215fedoncedaily-*fedfivetimesdaily*-J E0.8E C 0 C S 0 C 0 0 (a E Ca Ca 036912153691215Hoursafterfeeding36912153691215HoursafterfeedingFigure1.3.(Continued)1.60)Alanine1.4E1.2Cl0 C0.60.40.2 0fedoncedaily+.*-___fedfivetimesdaily•+-U(I)Threonine-J E0.8fedonceadayfedfivetimesadayC.20.6—6—dieti0.4+diet21.4(k)Asparticacid1.2fedoncedailyfedfivetimesdailyCl0 I--I-+C-*0.6•*-C)0.40.2-fl__--J E 0 E C 0 I C C.) C 0 C) 0 E 0 0 0369121536912151.b(I)Glutamicacid1.41.2fedoncedailyfedfivetimesdaily0.80.60.40.2.36912153691215Hoursafterfeeding36912153691215HoursafterfeedingFigure1.3.(Continued)-J E 0 E C 0 C 0 C) C 0 0 E 0 0 01.61.4E1.2 10 Cs j::0.8C a) C) C0.60 00.40.2 0-j E 0 E C 0 C a, 0 C 0 0 Cs E Cs (5 05-J E 0 E C.2CCs 4- C a, 0 C) Cs E 0, Cs 0(m)+.--*fedoncedailyGlycine+fedfivetimesdaily3691215fedoncedaily+----(n)Proline fedfivetimesdaily*-0036912151.81.61.41.2 10.80.60.40.2(0)+++.____-*--fedoncedailySerine-F-+-- fivetimesdaily0.80.60.40.236912153691215(p)Tyrosinefedoncedailyfedfivetimesdaily—El—diet1+diet2-*-diet3V“369121536912iS36912153691215HoursafterfeedingHoursafterfeedingFigure1.3.(Continued)Chapter 3Distribution of Plasma Essential Amino AcidsThe data regarding the percentages of individual amino acids in the plasmashown in Tables 1.12-1.17 indicated that the pattern of the distribution of essentialamino acids in fish over the sampling times was fairly consistent with respect to thediet fed. The data from Tables 1.12-1.14 on the percentage of essential amino acidsrelative to the total amino acids at 9 h postprandial in fish fed once daily wererearranged according to their order of abundance, and presented in Table 1.18. Thereason why the plasma amino acids at 9 h postprandial were chosen was becauseplasma concentration of most amino acids attained their peaks at this time andwould be expected to reflect the proffle of dietary amino acids. Differences betweenthe pattern of distribution of plasma amino acids in fish fed diet 1 and those of fishfed diet 2 and diet 3 were apparent. The order of abundance of plasma valine,leucine, histidine, lysine, tyrosine, and cystine in fish fed diet 2 corresponded wellwith those of fish fed diet 1. The order of plasma threonine, isoleucine,phenylalanine, methionine, and arginine in fish fed the former diet, however, did notagree with the fish fed the latter diet. With the supplementation of free lysine andmethionine in diet 3, fish fed this diet showed a very noticeable deviation of thepattern for lysine, leucine, methionine, histidine, threoriine, and isoleucine from thatof fish fed diet 1.59Chapter 3Table 1.18. Distribution of plasma amino acids at 9 h postprandial in fish fed different diets oncedaily In Expenment 1.1Diet 1 Diet 2 Diet 3Order2 AA %3 AA % AA %Essential1 Vai 12.9 Val 5.7 Val 5.62 Leu 8.3 Leu 4.0 Lys 5.33 His 6.3 His 3.5 Leu 3.94 Lys 4.9 Lys 2.4 Met 3.85 lie 4.4 Thr 2.0 His 3.76 Thr 4.3 lie 1.9 Thr 2.07 Met 3.9 Phe 1.4 lie 1.88 Arg 2.1 Met 1.3 Arg 1.89 Phe 1.9 Arg 1.2 Phe 1.210 Tyr 0.9 Tyr 0.5 Tyr 0.411 Cys 0.4 Cys 0.3 Cys 03Non-essential1 Gly 25.3 Giy 36.0 Gly 35.22 Ser 14.4 Ser 14.0 Ser 11.93 Ala 7.8 Ala 9.4 Ala 8.44 Gin 2.7 Asp 8.1 Asp 6.85 Pro 2.3 Pro 6.6 Pro 6.16 Asp 1.3 Gin 1.9 Glu 1.91 = fish meal based diet; Diet 2 = diet deficient in iysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with iysine, methionine, and tryptophan.acids listed in order of concentrations in the plasma.3Percent of total plasma amino acid concentrations.60Chapter 33.2.2.6 Effects of Supplementary Lysine and MethiomneSupplementary lysine and methionine caused a pronounced increase in theirconcentrations in plasma of fish fed diet 3 compared with the concentrations in fishfed diet 2. The levels of these two amino acids in fish fed diet 3 under both feedingregimes increased after feeding, and were maintained at a considerably higher leveluntil 24 h after the first feeding. The peak plasma concentration of lysine in fish feddiet 3 once daily was approximately 1.5 and 2.0 times greater than in fish fed diet 1and diet 2, respectively. Similarly, peak concentrations of plasmamethioriine + cystine in fish fed diet 3 once daily was approximately 1.3 and 2.5 foldgreater than those in fish fed diet 1 and diet 2, respectively. The plasmaconcentrations of lysine and methionine + cystine in fish fed diet 3 five times dailybehaved in a similar maimer to those observed in the fish fed once daily (Figure 1.3cand 1.3g).3.2.2.7 Plasma Taurine ConcentrationsPlasma taurine concentrations in fish varied with dietary treatment (Table1.19, and Figure 1.4). Fish fed diet 1 had higher taurine concentrations than fish feddiet 2 or diet 3 under both feeding regimes. Taurine concentrations in fish fed diet3 were higher than those in fish fed diet 2 either once daily or five times daily. Thefluctuations of plasma taurine concentrations as compared to those of other aminoacids were interesting. With once daily feeding, fish fed the different diets showedsimilar patterns in plasma taurine fluctuation over time. The concentrations werenoticeably high at the time of feeding. The concentrations then gradually declined,and subsequently rose again after 9 h. These changes were the reverse of the61Chapter 3changes in methionine concentrations (Figure 1.4). A similar trend up to 9 h post-feeding were also observed in the fish that were fed five times daily.3.2.2.8 Relationship Between Plasma and Dietary Amino AcidsThe comparison between dietary (as j.mo1/16g N) and plasma amino acids(as .tmo1/ml of plasma) was made using the concentrations of plasma amino acidsmeasured at 9 h after feeding in fish fed once daily (Table 1.12-1.14). Most aminoacids attained peaks at this time. The analysis did not include glutamic acid andaspartic acid because their dietary concentrations also include their amide(glutamine and asparagine), whereas plasma concentrations do not. Significantcorrelation coefficients (P < 0.05) between the levels of amino acids in the plasmaand those in the diets were obtained (diet 1, r=0.737; diet 2, r=0.915; diet 3,r = 0.890).62Chapter 3Table 1.19. Plasma taurine, methionine, and cystine concentrations in trout fed different dietsaccording to feeding regimes in Experiment 1.1Hour Fed once daily Fed five time dailyafterfeeding Diet 1 Diet 2 Diet 3 Diet 1 Diet 2 Diet 3(.Lmo1/mL ± SEM) (pmol/mL ± SEM)Taurine0 0.472 ± 0.0842 0.241 ± 0.033 0.329 ± 0.014 0.633 ± 0.046 0.252 ± 0.020 0.391 ± 0.0333 0.450 ± 0.051 0.161 ± 0.044 0.268 ± 0.032 0.447 ± 0.034 0.170 ± 0.017 0.285 ± 0.0339 0.325 ± 0.157 0.119 ± 0.050 0.201 ± 0.089 0.331 ± 0.053 0.126 ± 0.026 0.258 ± 0.04915 0.475 ± 0.056 0.165 ± 0.014 0.306 ± 0.030Methionine0 0.165 ± 0.007 0.070 ± 0.003 0.274 ± 0.014 0.171 ± 0.009 0.083 ± 0.007 0.321 ± 0.0053 0.232 ± 0.012 0.126 ± 0.021 0.340 ± 0.018 0.225 ± 0.021 0.115 ± 0.011 0.294 ± 0.0069 0.264 ± 0.014 0.130 ± 0.023 0.355 ± 0.029 0.206 ± 0.035 0.148 ± 0.015 0.336 ± 0.05515 0.227 ± 0.015 0.118 ± 0.021 0.330 ± 0.018Cystine0 0.016 ± 0.002 0.012 ± 0.001 0.027 ± 0.002 0.016 ± 0.003 0.017 ± 0.000 0.027 ± 0.0023 0.026 ± 0.002 0.032 ± 0.012 0.024 ± 0.002 0.034 ± 0.008 0.032 ± 0.006 0.095 ± 0.0479 0.030 ± 0.007 0.026 ± 0.005 0.030 ± 0.003 0.026 ± 0.007 0.046 ± 0.012 0.037 ± 0.00715 0.023 ± 0.001 0.021 ± 0.003 0.033 ± 0.0011Diet 1 = fish meal based diet; Diet 2 diet deficient in lysine, methionine, and tryptophan; Diet 3= diet 2 supplemented with lysine, methionine, and tryptophan.2values shown are means of three pools of plasma, five fish/pool, (n= 3).63-JE0E0Ca,CC00a,Ea,a,0-JE0EC0I4-Ca,0C0C,a,E0)a,0Figure 1.4. Plasma taurine, and methioriine + cystine concentrations in rainbowtrout fed different diets according to different feeding regimes in Experiment 1.1.Diet 1 = fish meal based diet, diet 2 = diet deficient in methionine, lysine, andtryptophan, diet 3 = diet 2 supplemented with methionine, lysine, and tryptophan.Each data point is a mean of three pools of plasma, five fish/pool, (n=3).Chapter 3Methionine+Cystine10.80.60.40.2fed once daily*fed five times daily—8— diet 1+ diet2-*- diet 33 6 9 12 15 3 6 9 12 1511.41.20.80.60.40.2Taurine• fed once daily fed five times daily3 6 9 12 15 3 6 9 12 15Hours after feeding64Chapter 33.3 EXPERIMENT 1.2: DIETARY EFFECTS ON PLASMA CONCENTRATIONSOF AMINO ACIDS MONITORED OVER A 120-HOUR PERIODFOLLOWING DIET CONSUMPTION3.3.1 MATERIALS AN]) METHODS3.3.1.1 DietsThe ingredient and proximate compositions of the three diets formulated forthis experiment are shown in Table 1.20. The ingredients used in the diets weresimilar to those in experiment 1 except that anchovy meal was used in place ofherring meal in all three diets. Diet 3 was supplemented with isoleucine in additionto lysine, methionine, and tryptophan. As with the latter three amino acids,isoleucine was added to mimic the concentration of that calculated to be present indiet 1. The addition of isoleucine was considered advisable because the calculatedvalues of the four supplemental amino acids in diet 3 appeared to be lower thanfound in the control diet. The formulated diets were analyzed for proximatecomposition and subjected to acid hydrolysis for later determination of amino acidcompositions of the diets according to AOAC methods (AOAC, 1984).3.3.1.2 FishRainbow trout with a mean weight of 180.4 ± 16.0 g (mean ± SD) were usedin this experiment. They were distributed randomly into fifteen 150 L tanks locatedat the UBC aquarium facilities. Each tank contained ten fish. Each dietarytreatment was assigned at random to five tanks of fish.65Chapter 3Table 1.20. Ingredient (air-dry basis) and proximate composition (dry matter basis) of diets used inExperiment 1.2Ingredient Diet 1 Diet 2 Diet 3g/kg g/kg g/kgAnchovy meal who1e-steam dried) 460.00 153.20 153.20Ground wheat 300.00 300.00 300.00Gelatin- 102.00 102.00Corn gluten meal- 79.00 79.00Soybean protein concentrate- 60.20 60.20Sardine oil2 93.00 118.00 118.00Bone meal 9.00 38.00 38.00Dextrin 88.00 100.00 87.00Premix3 30.00 30.00 30.00Calcium lignosulphonate 20.00 20.00 20.00L-lysine-- 730DL-methionine-- 4.00L-tryptophan-- 1.70L-isoleucine-- 3.50Total 1000.00 1000.40 1003.70Proximate analysis4Crude protein (%) 39.0 39.0 403Ether-extractable lipid (%) 15.5 16.0 16.2Ash (%) 9.4 7.1 7.1Gross energy (kcal/kg) 5082 5220 52381Autoclaved at 121°C for 1.5 h.with 0.05% ethoxyquin.premix supplied the following per kg of diet as fed (except for diet in which the percentage ofeach will be proportionally lower): thiamin HC1, 67.3 mg; riboflavin, 104.2 mg; niacin, 400 mg; biotin,5 mg; folic acid, 25 mg; pyridoxine HC1, 60.8 mg; cyanocobalamine, 0.1 mg; D-calcium pantothenate,200 mg; ascorbic acid, 1500 mg; choline chloride, 4000 mg; inositol, 2000 mg; menadione 30 mg;vitamin A, 10,000 IU; vitamin D3, 300 IU; vitamin E, 1000 IU; Mg (as MgSO4),380 mg; Mn (asMnSO4.H20),17 mg; Zn (as ZnO), 50 mg; Fe (as FeSO4.7H20),85 mg; Cu (as CuSO4.5H20),2mg; Co (as CoC1.6H20),0.003 mg; K (asK2S04),895 mg I (as K103), 5 mg NaC1 (as NaC1),2836 mg; F (as NaP), 4.5 mg; Se (asNaSeO3.5H0),0.10 mg.values for crude protein, ether-extractable lipid, and ash were obtained by proximate analyses.The values for gross energy were estimated by ascribing 5.65 kcal/g crude protein, 9.5 kcal/g crudelipid, 4.0 kcal/g carbohydrate (Alexis et al., 1985).66Chapter 3To acclimate the fish to the experimental diets, each experimental diet wasgradually mixed with the commercial diet (EWOS) to which the fish wereaccustomed (from 50:50% to 100:0%), and was given to the fish in the respectivetanks once daily. After 5 days of acclimation, i.e. when they fully accepted theexperimental diets, fish in each tank were fed the designated diets for tenconsecutive days. Fish were fed once daily until satiation in the morning. Satiationwas determined when the fish stopped taking pellets. The amounts of food fed tothe fish were recorded daily.During the experiment, the water supply was dechlorinated Vancouver citywater. Temperature was maintained between 13.0°C and 13.5°C by control of theheat exchanger unit. Water flow to each tank was 2 L/min, and dissolved oxygenwas 8 ppm. Photoperiod was 24 h. There was no mortality.3.3.1.3 Sampling ProcedureFollowing the final feeding at 10:00 hour, fish were bled at 3, 12, 24, 36, 48,72, and 120 h post-feeding. Six fish were taken from one replicate tank for eachdietary treatment at 3, 12, 24, 36, and 48 h sampling times. Fish that were sampledin the last two sampling times (72, and 120 h) were taken randomly from allreplicated tanks with respect to the dietary treatments. The fish that were sampledwere anesthetized with 0.01% tricaine-methanesulfonate (MS-222), and weighed.Blood was withdrawn from the caudal vein/dorsal aorta into 4 mL heparinizedvacutainers. Blood samples were immediately centrifuged at 780xg for 15 mm. Theresultant plasma samples from three fish were pooled (i.e. there were two plasmasamples/dietary treatment at each sampling time). The plasma samples were keptat -70°C for subsequent analyses.67Chapter 33.3.1.4 Amino Acid AnalysisThe procedures for the preparation of plasma for analysis and thedetermination of concentrations of free amino acids were the same as described forExperiment 1.1.3.3.1.5 Statistical AnalysisThe data on growth of fish were subjected to one-way analysis of variance.The analysis was performed using Systat (Wilkinson, 1990). To test the significanceof differences between the treatment means, the Tukey FTSD test was employed(Zar, 1984).3.3.2 RESULTS3.3.2.1 Amino Acid Compositions of Experimental DietsThe amino acid compositions of the experimental diets are shown in Table1.21. As with the results of analyses conducted on the diets in Experiment 1.1, theessential amino acid levels in diet 1 were higher than those in diet 2 or diet 3. Theproportion of EAA to NEAA in diet 1 was close to 1. Diets 2 and 3, on the otherhand, contained a higher proportion of non-essential amino acids. Theconcentration of arginine in diet 1 was markedly lower than the concentration ofthis amino acid in diet 1 formulated for Experiment 1.1.68Chapter 3Table 121. Amino acid composition of experimental diets in Experiment 1.2Experimental diet’Amino acid Diet 1 Diet 2 Diet 3g/l6gNMg 4.8 5.3 5.0His 2.7 3.0 2.6lie 3.6 2.9 3.7Leu 6.6 6.7 6.5Lys 6.4 4.4 5.9Met 2.3 1.5 2.3Cys 1.0 0.9 0.8Phe 3.9 3.6 3.6Tyr 2.6 2.3 1.9Thr 3.8 2.9 2.9Va1 4.3 3.5 3.4Ma 5.4 6.0 6.0Asp 8.2 7.8 7.6Glu 13.3 14.1 14.0Gly 5.7 9.5 9.1Pro 4.5 7.3 7.3Ser 3.7 3.8 3.7Tan 1.2 0.8 0.7TAA2 82.8 85.3 86.5BAA 42.1 36.9 38.8NEAA 40.7 4&4 47.7EAA/NEAA 1.03 0.76 0.811Diet 1 = fish meal based diet; Diet 2 = amino acid deficient diet; Diet 3 = diet 2 supplementedwith isoleucine, lysine, methionine, and tryptophan.2exc1ug asparagine, glutamine, and taurine, and tryptophan was not detected by the procedureused.69Chapter 3Table 122. Body weight gain and feed consumption of rainbow trout over a 10-day period inExperiment 12Diet1 Initial Final Weight Feedweight weight gain consumption(g) (g) (g/flsh) (g/flsh/day)1 182.0 ± 1.32 224.9 ± 3.9 42.9 ± 4.9 4.1 ± 0.202 179.9 ± 4.1 218.7 ± 5.9 38.8 ± 7.7 3.8 ± 0.033 179.5 ± 1.9 221.9 ± 3.0 42.4 ± 12 3.8 ± 0.171 Diet 1 = fish meal based diet; Diet 2 = amino acid deficient diet; Diet 3 = diet 2 supplementedwith isoleucine, lysine, methionine, and tryptophan.2 The values shown are means ± SEM of 5 replicated tanks, 10 fish/tank, (n = 5).Table 1.23. Specific growth rates and feed conversion efficiencies for rainbow trout fed different dietsin Experiment 1.2Diet1 Specific growth rate2 Feed efficiency3%1 2•12a4 104a2 195a 104a3 212aPooled SEM 0.19 0.091Diet 1 = fish meal based diet; Diet 2 = amino acid deficient diet; Diet 3 = diet 2 supplementedwith isoleucine, lysine, methionine, and tryptophan.2= (lnW2lnW1)*100/(T2T1)= Gain in body weight/feed consumption4The values shown are means ± SEM of 5 replicated tanks, 10 fish/tank, (n= 5). Values within the samecolumn with the same superscript were not significantly different (Tukey HSD, P >0.05).70Chapter 33.32.2 Responses of Fish to Experimental DietsThe initial weights, final weights, weight gains and feed consumptions of fishfed the different diets are provided in Table 1.22. Values for specific growth ratesand feed efficiencies of fish are shown in Table 1.23. The data were subjected toanalysis of variance and the results of the analyses are shown in Appendix 5. Therewere no significant dietary effects on the specific growth rate or feed efficiency.3.3.2.3 Plasma Amino Acid ProfileThe free amino acid concentrations in the plasma of fish expressed asj.Lmol/mL and their percentages as determined at different times after mealconsumption are shown in Table 1.24-1.26. The levels of total amino acids (TAA),total essential amino acids (EAA), total non-essential amino acids (NEAA), andratios of EAA/NEAA in plasma of fish fed the different diets are also tabulated inthe tables indicated above. The changes in plasma concentrations of EAA, NEAA,and TAA are shown in Figure 1.5. The changes in plasma concentrations ofindividual amino acids at different times after meal consumption are depicted inFigure 1.6. It should be noted that the concentrations of plasma amino acids in fishfed different diets at 24 h were used to represent the concentrations of plasmaamino acids at 0 h, that is, at the time of feeding.Plasma Total Essential Amino Acids (EAA), Total Non-Essential Amino Acids(NEAA), and Total Amino Acids (TAA)The patterns of plasma EAA concentrations in fish that were fed diets 1, 2,and 3 differed from each other during the absorptive period (3-24 h postprandial) asshown in Figure 1.5a. The plasma EAA concentrations in fish fed diet 1 rose from 371Chapter 3h and peaked between 12-24 h postprandial, after which the concentrations fellsharply at 36 h. Fish fed diet 3 showed an increase in the concentrations of EAAafter feeding and peaked at 12 h. The level then dropped at 24 h postprandial. Incontrast, the level of EAA in fish fed diet 2 fluctuated very little from 3 h to 36 hafter feeding. After 36 h, the concentrations of EAA in fish fed all diets declinedfrom 36 h to reach their lowest level at 48 h. After this time the plasma EAAconcentrations increased until 120 h when the experiment was terminated (Figure1.5a).The patterns of plasma NEAA and TAA in fish in the different dietarytreatment groups displayed a similar trend with respect to the diets fed. Fish thatwere fed diet 2 and diet 3 showed plasma NEAA and TAA peaks at 12 h afterfeeding, thereafter the levels gradually declined to the lowest level at 48 h afterfeeding. After this time concentrations of TAA slightly increased, whereas theconcentrations of NEAA slightly decreased. The fluctuations in the concentrationsof NEAA and TAA in fish fed diet 1 were similar in that they peaked at 24 h, anddeclined to the lowest concentrations at 48 h after which the level of both groupsincreased (Figure 1.5b and, 1.5c).During the absorptive period, the magnitude of the increase in plasmaconcentrations of EAA in fish fed the different diets differed in the following order,diet 1 > diet 3 > diet 2 (Figure 1.5 a). The concentrations of EAA in fish fed diet 1at peak (12 h) was approximately 10% and 70% greater than those of fish fed diet 3and diet 2 respectively. The concentrations of EAA in fish on the different dietarytreatments during the post-absorptive period were similar. The concentrations ofNEAA in fish fed diet 2 and 3 were similar, and were maintained at concentrations72Chapter 3higher than those in fish fed diet 1 throughout the sampling period, except at 120 hpost-feeding (Figure 1.5b). The highest concentrations (at 12 h) of NEAA in fishfed diets 2 and 3 were about double the highest level of NEAA in fish fed diet 1.The differences in the concentrations of TAA between fish fed diet 2 and 3 andthose in fish fed diet 1 corresponded with those for NEAA (Figure 1.5c).The ratio of EAA/NEAA in fish fed the different diets changed over time,showing the influence of diets on the plasma pattern of amino acids. The ratios infish fed diet 1 ranged between 0.55 and 1.08 depending upon the sampling time,being highest at 12 h and lowest at 36 h postprandial. A ratio of 1 was obtained at120 h postprandial (Table 1.24). In contrast to the response of fish fed diet 1, theratios in fish fed diet 2 were lower. The ratios of EAA/NEAA in fish of this groupwere between 0.28 and 0.72, being highest at 120 h and lowest at 12 h postprandial(Table 1.25). Fish fed diet 3 displayed a similar trend to that seen for fish fed diet 2,with ratios between 0.36 and 0.86 with the highest ratio at 120 h and lowest at 24 hpostprandial (Table 1.26).Plasma Concentrations of Individual Amino Acids in Fish Fed Different DietsEssential Amino AcidsFish fed diet 1 displayed some variations among the individual plasmaessential amino acids from the overall pattern of EAA, and could be grouped intothree groups. The first group included histidine, lysine, methionine + cystine,phenylalanine, and threonine. These amino acids had their peaks at 24 h afterfeeding, after which time concentrations gradually subsided and reached minimumlevels at either 36 or 48 h depending upon the particular amino acids (Figures 1.6b-73Chapter 3d, 1.6g, and L6i). The second group were the branched-chain amino acids,isoleucine, leucine, and valine, which attained their peak concentrations at 12 lifollowed by moderate declines to the fasting level at 36 h postprandial (Figures 1.6e-f, and 1.6h). The last group contained tyrosine and arginine. The concentrations oftyrosine were constant throughout the sampling period (Figures L6a). Theconcentrations of arginine were constant during the absorptive period and droppedat 48 h postprandial (Figures L6a).Most essential amino acids in fish fed diets 2 and 3 followed similar patternsto the overall pattern of EAA for the respective diet. Exceptions werephenylalanine and tyrosine which stayed constant over the test period. Very strikingpatterns of changes in the plasma concentrations of arginine, histidine, and lysine infish fed diets 2 and 3 were observed during 24-48 h postprandial. The levels of theseamino acids, after being constant or attaining moderate peaks followed by decreasesat 24 h, showed increases at 36 h and dropped at 48 h postprandial (Figure 1.6a-1.6c).Those essential amino acids that were present in greatest amounts variedwith the time of sampling, and with diets. Fish that were fed diets 1 and 2 hadvaline as the most abundant essential amino acid during the absorptive period (3-24h), histidine at 36 h, and histidine and valine at 48 h post-feeding. At 72 h, histidine,lysine, and valine were present in great amounts in fish fed diet 1, and only lysineand valine in fish fed diet 2. Lysine and valine concentrations dominated in plasmaof fish fed diet 1 and 2 at 120 h postprandial (Table 1.24 and 1.25). Fish fed diet 3,however, showed a variation in the amino acids present in the greatestconcentrations throughout the sampling period. Leucine and valine, present in74Chapter 3similar amounts, represented the largest proportion of plasma amino acids at 3 hpostprandial, methionine during 12-24 h, lysine at 36 h postprandial. Histidine,lysine, and valine concentrations were predominant in the plasma at 48-72 hpostprandial. Lysine, followed by valine, were the most abundant amino acids at120 h postprandial (Table 1.26).Non-Essential Amino AcidsIt should be recognized that the plasma concentrations of proline throughoutthe sampling period in fish fed diet 1 were too low to be detected by the amino acidanalyzer. As a result, they do not appear in Figure 1.6n.The plasma patterns of most non-essential amino acids in fish fed diet 1 weresimilar to the overall pattern of their NEAA. Glycine, proline, and serine in fishdiets 2 and 3 resembled the overall pattern of their NEAA (Figures 1.6m-o, andFigure 1.5b). The concentrations of glutamic acid in these fish remained stable.The changes in the concentrations of alanine and aspartic acid in fish fed thesediets, however, differed from the overall pattern of NEAA. Plasma alanine in thesefish had two peaks, one at 12 h, and the other at 36 h postprandial (Figure 1.6j).The peak at 36 h was similar to those observed for the fluctuations of arginine,histidine, and lysine in fish fed the same diets. While plasma aspartic acid in fish feddiet 3 resembled that of NEAA, the concentration of this amino acid in fish fed diet2 peaked differently from the overall pattern.Of the non-essential amino acids, glycine was found as the most abundant,and serine the second most abundant in the plasma of the fish fed each of the diets.75Table1.24.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferent timesafterfeedingdiet1(fishmeal baseddiet)inExperiment1.2AminoHoursafterfeedingacid31224364872120X±SEMI%2X±SEM%X±SUM%X±SUM%X±SUM%X±SUM%X±SUM%(.LmoI/mL)(/mo1/mL)(gtLmoI/mL)(IAmol/mL)(.Lmo1/mL)(.Lmoi/mL)(gumol/mL)EssentialArgO.234±.0184.40.220±.0143.60.189±.0392.80.157±.056340.057±.0191.70.092±.00422O.151±.02832Cys0.038±.0010.70.055±.0060.90.055±.0020.80.016±.0160.40.072±.0712.20.075±.0001.80.073±.0081.6His0.326±.0066.20.334±.0085.50.381±.0325.70.307±.0196.60214±.0396.50.301±.0677.30.339±.0047.2Tie0.208±.0003.90.320±.0215.30.247±.0253.70.097±.0212.10.098±.0533.00.120±.0282.90.191±.0194.1Lcu0.350±.0036.60.543±.0489.00.419±.0306.30.156±.0023.40.150±.0774.50.199±.0424.80.294±.0246.3Lys0.285±.0355.40.297±.0224.90.353±.0055.30.279±.0076.00.144±.0494.40.289±.0487.00.401±.0368.6Met0.170±.0053.20.222±.0073.70.268±.0404.00.111±.0002.40.103±.0353.10.092±AI002.20.104±.0022.2Phe0.189±.0273.60.183±.0313.00.226±.0193.40.128±.0412.80.162±.0444.90.109±.0182.70.131±.0002.8Thr0.193±.0143.70.200±.0183.30.277±.0384.20.116±.0312.50.068±.0132.10.119±.0322.90.148±.0163.2Tyr0.072±.0191.40.087±.0001.40.096±.0211.40.053±.0061.1O.086±.0442.60.024±.0120.60.064±.00614Val0.466±.0008.80.684±.03411.30.582±.0538.70.223±.0324.80.212±.1056.40.299±.0497.30.451±.0409.6Non-essentialAla0.593±.0711120.568±.0489.40.605±.0529.10.596±.09612.80.366±.06311.10.433±.10610.50.554±.07711.8Aspt130.00.120±.0432.00.072±.0091.10.077±.0041.70.086±.0262.60.097±.0072.40.059±.0091.3Glu0.318±.0486.00.356±.0035.90.413±.0466.20.335±.0227.20.225±.0696.80.249±.0186.00.166±.0133.6Gly1.154±.07421.81.139±.00218.81.624±.13424.41.269±.09227.30.755±.21322.80.894±20221.70.735±.12115.7ProtrtrtrtrtrtrtrSer0.691±.01513.10.730±.02312.10.860±.08912.90.737±.06115.80509±.18815.40.725±.14717.60.829±.02317.7TAA45.286±.1506.058±.1146.668±.8914.657±.0093.307±1.3924.118±.7614.687±.161EAA2330±.0283.144±.1303.093±.2931.643±.0211.367±.S101.720±.2642.344±.159NEAA62.756±.1782.914±.1163.575±.3133.014±.0131.940±.5642.398±.4802.343±.002EAA/NBAA0.921.080.870.550.700.711.00‘Meanoftwopoolsofplasma(threefish/pool)andstandarderrorofthemean(n=2).2oftotalaminoacids.3presentinavexysmall amount4Totalaminoacids.Total essentialaminoacids.6Totalnon-essentialaminoacidsTable1.25.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafterfeedingdiet2(aminoaciddeficientdiet)inExperiment1.2AminoHoursafterfeedingacid31224364872120X±SEMI%2X±SEM%X±SEM%X±SEM%X±SEM%X±SEM%X±SEM%(i.Lmol/mL)(.Lmo1/mL)(.Lmo1/mL)(J.Lmol/mL)(j.Lmol/mL)(J.Lmol/mL)(bLmol/mL)EssentialArg0.204±.0273.20.209±.0152.40.214±.0423.00.255±.0284.30.138±0.623.00.199±.0083.90.175±.0523.7Cystr0.041±.0100.50.035±.0130.50.005±.0050.1tr0.053±.0061.00.046±.0011.0His0.251±.0393.90.254±.0003.00.243±.0403.40.313±.0195.30.239±.0265.20.280±.0095.50.260±.0055.5Ile0.118±.0031.80.127±.0151.50.099±.0001.40.098±.0051.70.090±.0162.00.153±.0243.00.147±.0093.1Leu0.230±.0153.60.315±.0353.70.261±.0023.60.197±.0033.40.170±.0183.7O.251±.0354.90.245±.03152Lys0204±.0273.20.147±.0021.70.134±.0331.90.238±.0264.10.136±.0223.00.327±.0056.40.346±.0587.3Met0.110±.0311.70.096±.0061.10.078±.0091.10.073±.0051.20.070±.0161.50.092±.0011.80.072±.0011.5Phe0.187±.0072.90.190±.0212.20.197±.0242.70.154±.0052.60.138±.0193.00.157±.0063.10.140±.0193.0Thr0.081±.0031.30.117±.0121.40.088±.0251.20.076±.0161.3O.062±.0231.30.070±.0134.30.109±.0162.3Tyr0.061±.0031.00.042±.0420.50.050±.0110.70.047±.0100.80.043±.0160.90.065±.0041.30.064±.0231.4Val0.270±.0324.20.347±.0274.00.268±.0063.70.243±.0084.10229±.0325.00.350±.0376.90.367±.0157.8Non-essentialAla0.785±.12412.30.864±.21510.00.678±.1659.40.881±.02615.00.645±.09814.00.598±.00411.70.586±.050124Asp0.271±.0484.20.337±.0293.90.335±.0834.70.076±.0481.30.163±.0843.50224±.024440.053±.0311.1Glu0.196±.0053.10.322±.0253.70.266±.0053.70.314±.0375.40.222±.0404.80.250±.0044.90.186±.0183.9Gly2.142±.40333.43.189±.11937.12.531±.93435.21.680±.32328.61.392±.20930.11.227±.02824.11.163±.06424.6Pro0.429±.0006.70.864±.51910.00.634±.3518.80.289±.0004.90.197±.0004.3trtrSer0.868±.14013.11.143±.10513.31.089±.24215.10.928±.04715.80.688±.06214.90.796±.06715.60.764±.027162TAA46.406±1.0388.604±1.1587.196±1.7385.866±.5934.621±.3875.091±.1994.721±.340BAA51.715±.1131.884±.0131.664±.1161.699±.0401.314±.2501.996±.1401.969±.150NEAA64.691±.9256.719±.9635.532±1.7824.168±.5533.307±.0253.094±.0622.752±.191EAA/NEAA0.370.280.300.410.400.660.721Meanandstandarderrorofthemean(ii=2).2oftotalaminoacids.inaverysmallamountTotalaminoacids.Totalessentialaminoacids.6Totalnon-essential aminoacidsTable1.26.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafterfeedingdiet3(diet2supplementedwithisoleucine,methionine,lysine,andtryptophan)inExperiment1.2AminoHoursafterfeedingacid31224364872120X±SEM1%2X±SEM%X±SEM%X±SEM%X±SEM%X±SEM%X±SEM%(Lmol/mL)(4LLmoi/mL)(Lmoi/mL)(Lmo1/mL)(.Lmol/mL)(iLmoi/mL)(JLmoi/mL)EssentialArg0.223±.0163.30.196±.0592.10.155±.0022.30.178±.0012.70.127±.0412.80.201±.009420.302±.0246.3O.043±.0010.60.068±.0000.70.040±.0000.6O.049±.0010.8O.043±,0061.00.058±.0011.20.062±.0071.3His0.206±.0203.00.256±.0012.70.211±.0103.10.301±.0114.60.227±.0115.00.271±.0035.70.248±.0585.2lie0.231±.0243.40.266±.0192.80.111±.0031.60.109±.0021.70.084±.0011.90.115±.O112.40.156±.0153.2Leu0.385±.0275.60.401±.0414.20.217±.0053.20.196±.0163.00.151±.0133.30.191±.0234.00260±.0235.4Lys0.319±.0904.70.324±.0223.40.187±.0042.70.329±.0405.00.216±.0394.80.290±.0466.1O.455±.0339.4Met0.266±.0403.90.508±.0235.40.367±.0475.40.228±.0173.50.156±.0333.40.100±.0812.10.079±.0061.6Phe0.174±.0062.50.208±.O012.2O.180±.0032.60.142±.0252.10.107±.0362.40.136±.0062.90.094±.0171.9ThrO.081±.001120.149±.0011.60.099±.0201.40.093±.0071.40.052±.0061.10.064±.0021.30.138±.0132.9Tyr0.059±.0060.90.047±.0070.50.045±.0040.70.031±.0300.5O.037±.0000.80.064±.0051.30.025±.0160.5Val0.386±.0195.70.407±.0284.30.206±.0023.00.236±.0023.60.210±.0034.60.278±.0295.90.409±.0298.5Non-essentialAla0.712±.07310.40.775±.0628.20.690±.13910.10.856±.17712.90.652±.00914.40.520±.08111.00.518±.15510.7Asp0.237±.0233.50.414±.0594.40.362±.0085.30.324±.0134.90.123±.0182.70.218±.0174.60.121±.0052.5Glu0.212±.0233.10.290±.0053.10.305±.0094.50.323±.0164.90295±.0266.50.250±.0045.30.195±.0274.0Gly2.013±.26929.53.118±.00133.02.286±.04333.41.985±28130.01.386±.39030.71.238±.20826.10.960±30819.9Pro0.496±.0167.30.801±.0668.50.381±.0555.60.314±.0064.8t?trtrSer0.793±.06511.61.230±.01013.00.994±.05414.50.920±.01313.90.673±.09514.80.752±.04615.90.804±.13616.7TAA46.835±.7049.458±2126.837±.1756.612±.1254.538±.5214.744±.1354.826 ±.820EAA52.370±.2362.831±.0241.818±.0801.890±.0271.392±.0701.767±.1012.230±.189NEAA64.464±.4696.627±.0535.018±.0944.722±.0973.146±.4842.977±.0352.597±.631EAA/NEAA0.530.430.360.400.440.590.861 Meanoftwopoolsofplasma(threefish/pool)andstandarderrorofthemean(n=2).2oftotalaminoacids.presentinaveiysmallamountTotalaminoacids.Totalessentialaminoacids.6Totalnon-essentialaminoacids-J E 0 E a 0 Cu I., C a, U C 0 0 Cu E 0, Cu 0-JEE10o0 E aCCo0CuCu‘-6CC0 o0CCo0o0CuCuCuCu0IIIII3122436486072120HoursafterfeedingFigure1.5.Totalconcentrationsofplasmaaminoacids(essential,non-essential,andtotalaminoacids)inrainbowtroutdeterminedatdifferenttimesafterfeedinginExperiment1.2.Diet1=fishmeal baseddiet,diet2=aminoaciddeficientdiet,diet3=diet2supplementedwithisoleucine,methionine,lysine,andtryptophan.Eachpointrepresentsameanof twopoolsofplasma,threefish/pool,(n=2).12(b)Totalnon-essentialaminoacids..,3122436486072Hoursafterfeeding120Figure1.6.PlasmaconcentrationsofaminoacidsinrainbowtroutdeterminedatdifferenttimesafterfeedingexperimentaldietsinExperiment1.2.Diet1=fishmealbaseddiet,diet2=aminoaciddeficientdiet,diet3=diet2supplementedwithisoleucine,methionine,lysine,andtryptophan.Eachpointrepresentsameanof twopoolsofplasma,threefish/pool,(n=2).0.80.60.40.2 C(a)Arginine—6—diet1+diet2--*---diet32:6o120a’-J E 0 E C 0 4-. Ca I a) C.) C 0 C) E 0 -J E 0 E C 0 I- 4- C a) 0 C 0 C) ca E 000-j E 0 E C 0 I 4- C a) C) C 0 C.) Ca E 0 a) a--J E 0 E 0 I(b)Histidine0.80.6 :----0II3122436486072120(d)Phenylalanine0.80.60.4o.24i-*31224364860Hoursafterfeeding7212031224364860Hoursafterfeeding72120a -J E.- 0 E C 0 I C 0 C 0 0 E 0. -J E 0.8.20.6I- 4- C o0.4C 0 0 E0.20 0 0-J E o0.8E C 0 I...1 C a) 0 C 0 0 E 0 0-J E 0 E C 0 4- 0 4- C 0 C 0 0 0 E 0.10.80.60.40.2 V(e)Isoleucine—&-diet1+diet2--*--diet33122436486072”O00(g)Methionine+CystineHoursafterfeeding31224364860Hoursafterfeeding72120Figure1.6.(Continued)1-J E 0 E C 0 I(j)Alanine+.——-‘._fSSs312243648006072120-j 2 O.8.20.6(0 I C 0 0 C 0 0-J E 0.8.20.6CO I- 4- C C)0.4C 0 C) CO 20.20) CO0-j 2 0 E C 0773122436486072120(k)Asparticacid0.80.60.4-.02+CVh//1Hoursafterfeeding(I)Glutamicacid31224364860Hoursafterfeeding72120Figure1.6.(Continued)1-J E 0 E C 0 L.. C a) 0 C 0 0 a) 0 a)-J E 0 F z. C 0 I 4- C a) 0 C 0 C) a) E 0 a) 0-J F 0 F C 0 a) L.. 4- C 0 C 0 0 F 0 0 0-J F 0 F C 0 0 4- C a) 0 C 0 0 E Co a) 000(n)Proline±0.8/“+O.6,‘‘0.40.2+03122436486072120(p) Tyrosine0.8—El—diet10.6-f-diet2-*--diet30.40.23122436486072”120Hoursafterfeeding31224364860Hoursafterfeeding72120Figure1.6.(Continued)Chapter 33.3.2.4 The Effects of Dietary Amino Acid Supplementation on Plasma Amino AcidsDietary supplements of lysine and methionine resulted in pronouncedincreases in their plasma concentrations in fish fed diet 3 compared with those feddiet 2 (Figures 1.6c, and 1.6g). The plasma concentrations of lysine in fish fed diet 3at 3 and 12 h were very much higher than those in fish fed diet 2. The concentrationdropped at 24 h postprandial. The level was, however, maintained above that of fishfed diet 2 at the same sampling time and beyond. The elevation ofmethionine + cystine concentrations in fish fed diet 3 was remarkable. Theconcentration at the peak (12 h) was about double and five-fold higher than theconcentrations in fish fed diet 1 and 2, respectively (Figure 1.6g). Furthermore, theaddition of isoleucine in diet 3 caused an elevation in its plasma concentrations infish fed this diet. Interestingly, leucine and valine also showed increases in theirconcentrations in a similar manner to isoleucine in fish fed this diet. In comparisonwith plasma concentrations of branched-chain amino acids in fish fed diet 2, theconcentrations of these amino acids in fish fed diet 3 were very much higherbetween 3 and 12 h postprandial. The concentrations subsequently declined to thesame levels as those in fish fed diet 2 at 24 h postprandial (Figures 1.6e-f, and 1.6h).3.3.2.5 Plasma Taurine ConcentrationsPlasma taurine concentrations in fish fed the different diets are showntogether with methionine and cystine concentrations in Table 1.27. The changes inplasma taurine, and methionine + cystine concentrations are illustrated graphicallyin Figure 1.7. Plasma taurine concentrations were the highest in fish fed diet 1.Furthermore, the concentrations in fish fed diet 3 were generally higher than those84Chapter 3in fish fed 2. As shown in Figure 1.7, the concentrations of taurine in fish fed diets 1and 3 rose from 3 h postprandial, and peaked at 36 h postprandial after which theconcentrations declined at 48 h. Plasma taurine concentrations in fish fed diet 2,however, showed a different pattern of fluctuation in that they peaked twice, once at12 h postprandial and the other at 36 h. The concentrations dropped at 48 h, androse again at 72- 120 h after the last feeding. The pattern of the fluctuation oftaurine was similar to the results observed in Experiment 1.1 in that theconcentrations of taurine tended to rise when methionine + cystine concentrationsstarted to decline.3.3.2.6 Relationship Between Plasma and Dietary Amino AcidsA correlation analysis between plasma (as mol/mi of plasma) and dietaryamino acids (as tmol/16g N)was performed. The values for plasma amino acidsused in the analysis were the concentrations at 12 h postprandial when most aminoacids attained their peaks (Table 1.24-1.26). Significant correlation coefficients(P <0.05) between the levels of amino acids in the plasma and those in the diets(except for glutamic acid and aspartic acid) were obtained (diet 1, r = 0.845; diet 2,r=0.929; diet 3, r=0.893).85Table1.27.Concentrationsofplasmataurine,methionine,andcystineinrainbowtrout atdifferenttimesafterfeedingexperimentaldietsinExperiment12AminoHoursafterfeedingacid31224364872120MeanX±SEM2X±SEMX±SEMX±SEMX±SEMX±SEMX±SEMX±SEM(j.mo1/mL)(umol/mL)(jLmol/mL)(mo1/mL)(mo1/mL)(jLmo1/mL)(Jimol/mL)(mol/mL)TaurineDiet11.193±.1111.397±.1371.612±.2641.672±.1291.178±.2631.152±.0311.106±.0561.367±.120Diet20.718±.1071249±.0651.055±.1321.266±.1500.909±.1541.092±.0741.209±.1131.071±.100Diet 30.773±.0021.176±.1421291±.1681.538±.0031.258±.0911.202±.0681.466±.1381.243±.130MethionineDiet10.170±.0050.222±.0070.268±.0400.111±.0000.103±.0350.092±.0000.104±.0020153±.036Diet20.110±.0310.096±.0060.078±.0090.073±.0050.070±.0160.092±.0010.072±.0010.084±.008Diet30.266±.0400.508±.0230.367±.0470.228±.0170.156±.0330.100±.0810.079±.0060.243±.080CystineDiet10.038±.0010.055±.0060.055±.0020.016±.0160.072±.0710.075±.0000.073±.0080.055±.011Diet2tr30.041±.0100.035±.0130.005±.005tr0.053±.0060.046±.0010.036±.021Diet30.043±.0010.068±.0000.040±.0000.049±.0010.043±.0060.058±.0010.062±.0070.052±.0061Diet1=fishmealbaseddiet;Diet2=aminoaciddeficientdiet;Diet 3=diet2supplementedwithisoleucine,lysine,methionine,andtryptophan.2valuerepresentsameanof twopoolsofplasma,threefish/pool,andstandarderrorofthemean(n= 2).PresentinaverysmallamountChapter 3Methioriine+Cystine0.83—B—dietl0.6 ± diet 2--*--diet3o0.:4.-Jg Taurine0.5Ea)00I I I I /1 I3 12 24 36 48 60 72 120Hours after feedingFigure 1.7. Plasma taurine, and methionine + cystine concentrations in rainbowtrout fed different diets in Experiment 1.2. Diet 1 = fish meal based diet, diet 2= amino acid deficient diet, diet 3 = diet 2 supplemented with isoleucine,methionine, lysine, and tryptophan. Each points represents a mean of two poolsof plasma, three fish/pool, (n=2).87Chapter 33.4 DISCUSSIONIn Experiment 1.1, the fish were sorted initially into three groups based onbody weight to reduce the error term in analysis of variance of the data. Anotheradvantage of segregating the fish on the basis of body weight was the reduction inthe variation in feed intake of individual fish due to the feeding hierarchy thatnormally occurs when fish of a large size range are in the same tank. The results forfinal weight, weight gain, and feed consumption showed good consistency within thesame size category of fish. Although large and medium-sized fish fed diet 1 (fishmeal based diet) consumed similar amounts of food, this did not affect thecomparison between the results obtained from fish that were fed the control dietand test diets. Furthermore, growth rate, feed efficiency, and energy efficiencyshowed that fish of different sizes responded to experimental diets and feedingfrequency in a similar manner.The results for growth rate, feed efficiency, protein gain, productive proteinvalue, and energy efficiency showed that fish fed diet 1 (fish meal based diet) hadbetter growth, and protein and energy utilization than noted for fish fed either diet 2(amino acid deficient diet) or diet 3 (diet 2 supplemented with methionine, lysine,and tryptophan). Furthermore, fish that were fed diet 3 showed comparable growthto that of fish that were fed diet 2. Dietary supplementation with free amino acidsin diet 3 was, therefore, ineffective in improving growth and nutrient utilization ofrainbow trout in comparison with fish fed diet 2 which was similar but notsupplemented with amino acids in Experiment 1.1.It has been theorized that fish do not efficiently use supplemental aminoacids when fed only once a day because crystalline amino acids may be absorbed88Chapter 3from the intestine and oxidized before amino acids derived from protein digestionare available for absorption (Robinson, 1992). The feeding of fish five times daily inthis experiment showed apparent constancy in the supply of amino acids derivedfrom both intact dietary protein and supplementary free amino acids to the plasma,and ultimately to the tissue sites of protein synthesis. Growth of fish fed diet 3 (dietsupplemented with lysine, methionine, and tryptophan) five times per day inExperiment 1.1, however, was not improved significantly. The results from thisstudy were different from those found in carp by Yamada et a!., (1981b) who foundthat the growth rate of carp fed an amino acid diet dramatically improved when thenumber of feedings increased. This might be explained by a species difference, andthe nature of diets used in the experiments. Carp is a stomachiess fish, and theretention time of food in the digestive tract, particularly of purified diets such as anamino acid mixture is, therefore, shorter than that in salmonids, e.g. rainbow trout.Continuous provision of feed to carp when fed a free amino acid mixture is,consequently, necessary.The body composition of fish fed five times daily in this experiment,nevertheless, was significantly higher in protein concentration and lower in drymatter and lipid concentrations than those in fish fed once daily. Although, proteingain by the two groups of fish was similar, the lipid content in fish fed the differentdiets was reduced when they were fed five times daily. These differences wereobvious, particularly in fish that were fed diet 3, which showed a 3% increase inprotein concentration and a 10% reduction in lipid concentration. A higherproductive protein value (PPV) for fish fed five times daily was also observed. Thisobservation was similar to that reported in rats. Cohn (1963) summarized the89Chapter 3results of several studies carried out on the effects of feeding frequency on bodycomposition of rats. He observed that the body of meal-fed rats contained more fatbut less protein and water than that of rats fed ad libitum, and vice versa. Feedingfish five times per days in the present experiment appeared to be a factor in theregulation of the intermediary metabolism of dietary protein. In fish fed five timesdaily, the load of amino acids derived from ingested protein was used for proteinsynthesis more efficiently. By contrast, when the same amount of food was fed oncedaily, the load of amino acids derived from protein digestion seemed to exceed thecapacity of the fish for protein synthesis. Excess amino acids in this group of fishwere evidently catabolized and the reduced carbon skeletons of the amino acidswere converted to fat. The results corresponded with those reported for rats byCohn et at., (1963).Steffens (1981) stated that a reliable indication of protein utilization in fishby means of growth rate will be reached only if the experiment lasts for at least 2-3months with a two-week adaptation period. Kaushik et at., (1988) found in theirexperiment that the response of growth rate and feed conversion ratio to thesupplementation of arginine in the diet was only demonstrated in rainbow troutduring the last 3 weeks of their experiment which lasted 9 weeks. Rumsey andKetola (1975), on the other hand, concluded from studies with rainbow trout andother fish that experiments of 2-6 weeks duration appeared to be adequate in thestudies of protein and amino acids metabolism. March et al., (1985) alsodemonstrated in their study that a short term bioassay of 21 days was sufficient toevaluate dietary protein quality in rainbow trout previously selected for uniformityof size. The experimental period of 27 days in Experiment 1.1, therefore, was likely90Chapter 3adequate.A possible explanation for the lack of response to either the amino acidsupplementation or feeding frequency treatment was an imbalance in the aminoacid profile of diet 3. Rumsey and Ketola (1975) and Ketola (1982) did not find anyimprovement in growth of rainbow trout when a diet containing soybean meal wassupplemented with a single amino acid or several combinations of amino acids, forexample, histidine + methionine + lysine. They obtained better results when themixtures of several amino acids were added to the diet to simulate the concentrationof essential amino acids in trout eggs or isolated fish protein. They concluded thatdiets containing soybean meal had disproportionate levels of amino acids, thuscausing a reduction in growth rate. The same situation of amino acid imbalancemay have occurred in the present experiment.Diet 3 in this experiment was supplemented with methionine, lysine, andtryptophan to the levels present in the control diet (fish meal based diet).Comparisons between amino acid concentrations in this diet, the requirementvalues for juvenile salmon as recommended by the NRC (1981), and more recentlyreported values for rainbow trout revealed that the concentrations of all essentialamino acids in the diets were above the requirements (Table 1.2). There was noassurance, however, that all the amino acids were totally available to the fish.Moreover, the balance of dietary essential amino acids may not have been optimal.This was revealed by the results on the distribution of essential amino acids in theplasma of fish fed the different diets (Table 1.18). The pattern of plasma essentialamino acids in fish fed either diet 2 or 3 deviated from the pattern in fish fed thefish meal based diet.91Chapter 3The results of Experiment 1.2 showed that growth of fish tended to beimproved when they were fed diet 3 which contained supplementary isoleucine,lysine, methionine, and tryptophan. The inclusion of isoleucine in this diet inExperiment 1.2 may have adjusted the balance of amino acids. A decisiveconclusion could not be drawn from this because the improvement in growth wasnot statistically significant. Furthermore, the sizes of fish in Experiment 1.2 differedfrom those in Experiment 1.1. The average weight of rainbow trout in Experiment1.1 was 18-21 g whereas that in Experiment 1.2 was 180 g. Larger fish may utilizeamino acids supplemented in the free form in diets more efficiently than smallerfish. Variations in growth response results due to fish size have been observed inother laboratories (Dabrowski, 1986; Murai et a!., 1989b).The ratio of essential to non-essential amino acids either in the diets or theplasma is important in relation to protein quality and utilization in mammals(Young and Zamora, 1968; Hartog and Pol, 1972; Fujita et a!., 1981; Mercer et a!.,1989). Noteworthy results on the ratio between plasma total essential to total nonessential amino acids were also found in Experiments 1.1 and 1.2. During theabsorptive period, fish fed diet 1 (control diet) had a ratio of close to 1 in fish fedfive times daily, and a ratio of 1 in fish fed once daily to satiation. Plasma ratios ofessential to non-essential amino acids in fish fed diet 2 or 3, on the other hand, wereconsiderably lower. This reflected the amino acid composition of the diets as theratio of EAA to NEAA in the different diets also agreed with the ratios in theplasma of fish fed respective diets. The high levels of plasma non-essential aminoacids in fish fed diets 2 and 3 resulted in higher levels of total plasma amino acidsthan in fish fed diet 1 in both Experiment 1.1 and 1.2 (Figures 1.2 and 1.5).92Chapter 3The differences between the plasma concentrations of essential, and nonessential amino acids in fish fed the different diets had disappeared 48-72 hpostprandial. The increase in the concentrations of most essential amino acids inthe plasma by 120 h after feeding indicated the input of free amino acids from thecatabolism of tissue protein. The increased ratios of plasma EAA/NEAA in thefish on the different dietary treatment groups at this sampling time, particularlythose of fish fed diet 2 and 3 (ratio of approximately 1), suggested the contributionof amino acids from the catabolism of the body protein.The time at which plasma amino acids reached their peaks in fish fed diet 1in Experiment 1.2 differed from the results in Experiment 1.1. Amino acidconcentrations in fish fed this diet in Experiment 1.2 peaked between 12 and 24 hpostprandial, whereas those for fish fed diet 1 in Experiment 1.1 peaked at 9 h andhad declined at 15 h postprandial. The difference between the two experimentscould possibly have been due to the differences in size of the fish in the respectiveexperiments. A greater amount of food consumed by the larger fish in thisexperiment was probably evacuated at a slower rate than the food ingested by thesmaller fish in Experiment 1.1 resulting in the delay of the appearance of free aminoacids in the plasma. Nose (1972) found similar results on the time course of plasmaamino acid concentrations when rainbow trout weighing 150 g were fed acommercial diet. In his study, most free amino acids began to increase immediatelyafter feeding and attained maximum concentrations between 12 and 24 h afterfeeding. Another factor which might affect gastric evacuation and digestion in fishwas the sampling method. Fish were sampled from the same tank at every samplingtime in Experiment 1.1, whereas fish were taken from different tanks in Experiment93Chapter 31.2. The disturbance associated with catching the fish in Experiment 1.1 may havecaused stress to the remaining fish that were left in the tanks and sampled at thenext sampling time.In Experiment Li, the elevations of the plasma amino acids lysine andmethionine in fish fed diet 3 were appreciably higher (Figures 1.3c and 1.3g) than infish fed the other two diets. Although, the amounts of both amino acids wereincreased in diet 3 to equal the concentrations in diet 1, the plasma concentrationsin fish fed diet 3 (fed once and five times daily) were approximately double those offish fed diet 1. The same phenomenon was observed in Experiment 1.2 andindicated that synthetic lysine and methionine were effectively absorbed from thedigestive tract of fish fed diet 3. A rise of plasma concentrations in response todietary supplementation with free amino acids has been reported in other studies infish (Barash, 1984; Murai et a!., 1989a). In the present study, plasma methionine infish fed diet 3 was maintained at a level higher than in fish fed diets 1 and 2 until 36h after feeding. Although the concentrations of plasma lysine in fish fed diet 3dropped abruptly at 24 h after feeding in Experiment 1.2, lysine concentration wasmaintained at a higher level than in fish fed diet 2 until 48 h after feeding.There have been studies, both in fish and in other animals, showing thatamino acid oxidation is influenced by the level of amino acid in that the rate ofoxidation increases with increments of amino acids, and a surplus of amino acidswill be oxidized rapidly if they are not used for protein synthesis (Brett and Zala,1975; Walton et a!., 1982; Harper, 1983; Young et a!., 1985; Kim et a!., 1992).Thebault (1985), for example, found in sea-bass that the level of plasma methioninein response to dietary supplementation reached a peak and then returned to the94Chapter 3pre-feeding level sooner than was the case with other essential amino acids. Heconcluded, therefore, that supplementary free methionine was absorbed anddegraded faster than methionine originating from dietary protein. The continuedelevation of plasma methionine and lysine until 36 h after feeding in the presentstudy is contradictory to the above observation. It is possible that the enzymesresponsible for oxidation of these amino acids were overloaded (Harper et al., 1970)so that oxidation of lysine and methionine was not rapid and high plasmaconcentrations were maintained for a relatively long period.The patterns of change in the concentrations of plasma branched-chainamino acids in Experiment 1.2 were very interesting. Diet 3 in this experiment wassupplemented with isoleucine to raise the concentration to that present in diet 1.Not only was the level of plasma isoleucine in fish fed diet 3 elevated over thatoccurring with diet 2, but the concentrations of leucine and valine were alsoincreased. A similar behaviour of branched-chain amino acids was reported in astudy of channel catfish fed diets containing increasing amounts of isoleucine(Wilson et al., 1980). Although an antagonistic interaction between branched-chainamino acids has been extensively documented for other animals (Harper, 1964;Harper, et al., 1970; Harper et a!., 1984; Block, 1989), little information is availablefor fish. The evidence for the antagonistic interaction among these amino acidsbased on growth data was observed by Chance et al., (1964) in chinook salmon, andWilson et a!., (1980) in channel catfish. Choo et aL, (1991) did not find such aninteraction in rainbow trout when the fish were fed diets containing excess leucine.Plasma concentrations of isoleucine and valine were not affected by the changes ofdietary leucine concentrations. The fish fed a high leucine diet (13.4% of diet),95Chapter 3however, exhibited gross lesions (scoliosis, and deformed opercula). Choo et al.,(1991) attributed these signs to “toxicity” of excess dietary leucine. The parallelalteration of leucine and valine to that of isoleucine found in Experiment 1.2suggested an antagonism among these amino acids in rainbow trout.A significant phenomenon regarding the changes of the plasmaconcentrations of arginine, histidine, lysine and alanine in fish fed diets 2 and 3 wasobserved in Experiment 1.2. These plasma amino acids in fish fed the respectivediets exhibited similar patterns of change in that they either remained constant orpeaked during 3-12 h postprandial and then rose at 36 h. Diets 2 and 3 in thisexperiment were both formulated with the same proportions of protein sources.The surges in the concentrations of arginine, histidine, lysine, and alanine at 36 hcould be interpreted as the result of delayed digestion of particular proteins.Although, the data on the composition of amino acids in the dietary protein wasinformative as to the level of each amino acid which was supplied in the diet, itcould not be ascertained when the constituent amino acids would be released,absorbed and available for protein synthesis following diet consumption.As with other studies on rainbow trout, glycine was found in the highestconcentration among plasma non-essential amino acids in Experiments 1.1 and 1.2(Nose, 1972; Gras et al., 1982; Walton and Wilson, 1986). The magnitude of theconcentrations, however, depends on the nature of the food eaten. For example,fish fed diets 2 or 3 in both experiments had more than double the concentrations ofplasma glycine relative to fish fed diet 1. This was a result of gelatin in diet 2 and 3which is rich in glycine. Likewise, Hill et a!., (1961) observed a rise in plasmaglycine concentration in chickens fed a diet containing 10% glycine. Alanine,96Chapter 3aspartic acid, proline, and serine concentrations in fish fed diets 2 or 3 were alsovery high. The higher levels of alanine and proline in fish fed these two diets wereundoubtedly a result of gelatin in the diet (Tables 1.2, and 1.20).The higher concentrations of plasma serine in fish fed diets 2 or 3 than in fishfed diet 1 are assumed to be a cosequence of metabolism of plasma glycine in theformer fish. Glycine and serine are readily interconvertible, and glycine can becondensed to yield serine mostly for gluconeogenesis (Bender, 1985). As withmammals under normal conditions, the major pathway of glycine catabolism isprobably via the glycine cleavage system, whereby the carboxyl carbon is released asCO2 and methylene carbon asN5°-methylenetetrahydrofolate, and the NH4 isreleased (Walton and Cowey, 1981). In the present experiment, the plasmaconcentrations of glycine were unusually high. The conversion of glycine to senile isa control mechanism for maintaining of the balance of the amino acids in theplasma of the fish (Schepartz, 1973). Since the formation of one molecule of serinefrom glycine requires two molecules of glycine, this pathway is probably an effectiveway to reduce the excess glycine in the circulation of fish. Serine formed from thisreaction is the substrate for gluconeogenesis (Bender, 1985; Walton and Cowey,1982).Aspartic acid is usually low in fish plasma (Dabrowski, 1982). The elevatedconcentrations of plasma aspartic acid in this study were possibly the result of animbalance of amino acids in diets 2 and 3 as well. The mechanism involved in therise of this amino acid is not clear.97Chapter 3The high level of plasma taurine observed in the present study has also beenreported in various fish (Nose, 1972; Wilson and Poe; 1974 Plaskas et a!., 1980;Walton and Wilson, 1986). The origin and metabolism of taurine in fish has notbeen studied intensively. In mammals and some other animals, taurine issynthesized mainly in the liver from sulfur amino acids (cysteine). Evidence for thebiosynthesis of taurine in fish is limited. The source of taurine has been, therefore,believed to be from diets (Sakaguchi et a!., 1988; van Waarde, 1988). The results onthe concentrations of plasma taurine in the present study were in accord with thisconclusion in that fish fed diet 1 (fish meal based diet, see Table 1.2) had asignificantly higher plasma taurine concentration than those of fish fed diets 2 or 3.The results from several studies, however, have shown that fish may synthesizetaurine since the level of plasma taurine is significantly increased when the fish arefed a diet containing excess methionine (Murai et al., 1989a; Cowey et a!., 1992).Yokoyama and Nakazoe (1989) also found an induction of cysteine dioxygeneseactivity, the enzyme which catabolizes the first step reaction in taurine formation, inthe liver of rainbow trout when the fish were fed a diet containing excess sulfuramino acids. The concentrations of plasma taurine in fish fed diet 3 (methioninesupplemented diet) were higher than those in fish fed diet 2 which was notsupplemented with methionine. Furthermore, plasma taurine concentrations werelow when the plasma concentrations of methionine were increasing, but rose whenplasma methionine declined (Table 1.19). Degradation of methionine to taurine isthereby suggested.98Chapter 3In conclusion, the results on plasma amino acids indicated that they werepositively correlated with the dietary amino acids concentrations. Dietarysupplements of free amino acids were absorbed more rapidly, and they weremaintained in the plasma at levels higher than those in the diet containing similaringredients but without amino acid supplementation until 36 h after feeding. Thisfinding indicated that free amino acids added to the diets were stifi available totissues for protein synthesis at least at the time of routine feeding. Dietssupplemented with amino acids in this study did not improve the growth of rainbowtrout even when the fish were fed more frequently. This could be explained by anunidentified imbalance of amino acids present in the diets. Feeding fish five timesdaily when the fish were pair-fed to the control group fed once daily did not haveany beneficial effects on growth or feed conversion efficiency. The results on thebody composition of the fish, however, showed that feeding fish more frequentlyaffected the intermediary metabolism in the fish. The carcasses of fish fed fivetimes daily contained higher protein concentrations and lower lipid concentrations.The high concentrations of senile observed in the present study indicatedthat the excess of glycine absorbed from the diet was converted to serine. Also, theresults found in Experiment 1.2 suggested that different proteins from variousprotein sources in diets were digested at different rates judging from the time ofappearance of amino acids in the plasma. This could be useful information forconsideration when diets containing different kinds of proteins are formulated.99CHAPTER 4EXPERIMENT 2PATTERNS AND CONCENTRATIONS OF FREE AMINO ACIDS IN THEPLASMA OF RAINBOW TROUT FED DIETS CONTAINING PROTEIN FROM AMIXTURE OF DIFFERENT PROTEIN SOURCES WITH AND WITHOUTDIETARY SUPPLEMENTS OF SELECTED AMINO ACIDS4.1 INTRODUCTIONThe results from Experiment 1 showed that plasma concentrations of glycinein fish fed diets 2 and 3 (containing gelatin) were strikingly high. Plasma amino acidprofiles in response to a diet formulated to be the same as diet 2 and the dietcontaining synthetic glycine were, therefore, investigated in the present experiment.Assuming that the crystalline amino acids were in a readily available form, the rateat which they enter the circulation should be faster than the rate at which the intactproteins were digested and the amino acids absorbed into the circulation.The amino acid pattern of fish carcass protein has been claimed to be a goodquick reference for formulation of fish diets (Aral, 1981; Ketola, 1982; Ogata et a!.,1983; Wilson and Poe, 1985). Fish fed diets supplemented with amino acids tosimulate the pattern of fish whole body protein have shown better growth and feedefficiency than those fed diets with suboptimal amino acid balance. Plasma aminoacid proffles in response to a diet supplemented with several essential amino acidsto simulate the amino acid pattern of fish carcass protein were, therefore,investigated in the present experiment.100Chapter 44.2 MAtERIALS AND METHODS4.2.1 DietsThe compositions of the four diets formulated for this experiment are shownin Table 2.1. Diet 1 (control diet) contained herring meal, corn gluten meal, andsoybean protein concentrate as the main protein sources. The concentrations ofessential amino acids in the control diet, which were calculated from tabulatedvalues (NRC 1981), were equivalent to or higher than the stated requirements ofrainbow trout (Table 1.2, Experiment 1). Diet 2 in this experiment was formulatedto be similar to diet 2 in Experiment 1, i.e. the diet containing herring meal, corngluten meal, soybean protein concentrate, and gelatin as principal protein sources.Diet 3 was formulated to have herring meal, corn gluten meal, and soybean proteinconcentrate as predominant protein sources. The amounts of protein provided byherring meal, corn gluten meal, and soybean protein concentrate were in the sameproportion as in diet 2, i.e. 2 herring meal: 1 corn gluten meal: 1 soybean proteinconcentrate. Glycine was added to diet 3 in an amount comparable to that suppliedby gelatin in diet 2. This enables comparison of the speed by which glycine wasabsorbed following digestion of gelatin by fish fed diet 2. Diet 4 was formulatedwith herring meal, corn gluten meal, and soybean protein concentrate as majorsources of protein with the proportions of each identical to those in diet 3. This dietwas supplemented with arginine, histidine, lysine, methionine, and threonine tosimulate the amino acid profile of trout muscle which was reported by Wilson andCowey (1985). The formulated diets were analyzed for protein and amino acidconcentrations as described previously.101Chapter 4Table 2.1. Ingredient and proximate composition of diets employed in Experiment 2.Ingredient Diet 1 Diet 2 Diet 3 Diet 4g/kg g/kg g/kg g/kgHerring meal (whole steam-dried) 231.89 155.00 215.38 215.38Corn gluten meal 124.16 83.00 115.32 115.32Soya protein concentrate 90.04 60.00 83.36 83.63Ground wheat1 300.00 300.00 300.00 300.00Gelatin- 102.00--Herring oil2 105.52 114.74 107.50 107.50Dextrin 64.89 100.26 72.94 72.91Bone meal 13.50 15.00 13.50 13.50Calcium lignosulfonate 30.00 30.00 30.00 30.00Premix3 40.00 40.00 40.00 40.00L-Arginine-- 2.63L-Histicline (HC1.H20)-- 3.78L-lysine HC1-- 14.28DL-Methionine-- 0.98L-Threonine-- 4.16L-Glycine- 22.00Total 1000.00 1000.00 1000.00 1004.07Proximate analysis4Protein (%) 38.2 38.5 38.7 38.7Ether-extractable lipid (%) 14.0 14.0 14.0 14.0Ash (%) 5.4 4.4 5.1 5.1Gross energy (kcal/kg) 4833 4880 4788 48121Autoclaved at 121°C for 1.5 h25tabili2d with 0.05% ethoxyquin3The premix supplied the following per kg of the diet as fed (except for diet 4 in which the percentageof each will be proportionally lower): thiamine HC1, 60 mg; riboflavin, 100 mg niacin, 400 mg;biotin, 5 mg; folic acid, 25 mg; pyridoxine HC1, 50 mg; cyanocobalamine, 0.1 mg; D-calciumpanthothenate, 200 mg; ascorbic acid, 1500 mg; choline chloride, 4000 mg; inositol, 2000 mg;vitamin K, 30 mg; vitamin A, 10,000 1U; vitamin D3, 1000 IU; vitamin E, 1000 IU; Mg (as MgSO4),380 mg; Mn (as MnSO4.5H20),30 mg; Zn (as ZnO), 70 mg; Fe (as FeSO4.7H20),85 mg; Cu (asCu504.5H20),2 mg; Co (as CoCL6H2O), 0.003 mg; K (as KH2PO4),895 mg; P (as KH2PO4),2070 mg; I (as K103), 5 mg; F (as NaF), 4.5 mg; Se (asNaSeO3.50),0.10 mg.4The values for dietary protein were from determination (on dry matter basis), and for ether-extractable, ash, and gross energy were from the calculation (air-dry basis) using analysis values ofeach ingredient. The values for gross energy were estimated by ascribing 5.65 kcal/g crude protein,9.5 kcal/g crude lipid, 4.0 kcal/g carbohydrate (Alexis et aL, 1985).102Chapter 44.2.2 FacilitiesTwelve 200 L tanks designated as A-L were set up at the UBC aquariumfacility at the West Vancouver Laboratory of Fisheries and Oceans, Canada. Theywere indoor tanks, and supplied with seawater (32 ppt). The water temperatureranged from 9.0-11.0°C, and dissolved oxygen was 8.5 ppm during the experiment.Photoperiod was 14 h. Mortality of the fish was 8% over the entire experiment.4.2.3 FishRainbow trout that were already acclimated to seawater, were used in thisexperiment. The fish were distributed randomly so that each of the 12 tanks had 15-16 fish. The average initial weights of each group were in the range of 237.9 ± 9.3 gto 250.1 ± 20.1 g (mean ± SE). The four experimental, diets were then assignedrandomly to the tanks with three replicate tanks of fish per diet.Before the experiment began, the fish were accustomed to the diets bygradually substituting the test diets for the commercial diet (EWOS) which hadbeen fed previously. The fish accepted the test diets after 3 days. Thereafter, fishwere daily fed to satiation their prescribed diets for 17 days. Records of daily feedconsumption were maintained.4.2.4 Sampling ProcedureFish sampling was done at two different times.1. After the fish had been on their respective diets for 7 days, blood waswithdrawn at different times after feeding for amino acid analyses. On the samplingday, the fish were fed to satiation. Then at 3, 9, 15, and 24 h after feeding, four fish103Chapter 4from each dietary treatment were caught and anesthetized with 0.01 % tricainemethanesulfonate (MS-222). To avoid disturbance of fish in other tanks, the fishwere taken from one of the replicate groups for each dietary treatment at eachsampling time. The exception was the last sampling time when they were randomlytaken from all replicated tanks with respect to the dietary treatment. The fish wereweighed and blood was taken with heparinized vacutainers from the caudal vessels.Blood was centrifuged at 750xg for 10 mm and plasma samples from two fish fromthe same treatment were pooled i.e. two pooled samples/dietary treatment. Theplasma was frozen immediately in liquid nitrogen, and stored at -70°C forsubsequent analyses.2. Ten days after the first sampling, the fish were sampled a second time.After feeding fish to satiation on day 17 at 08:00 hour, blood was withdrawn fromfour fish per dietary treatment 26 and 36 h later by the procedure described for theprevious sampling. After bleeding, a piece of white muscle from the area below thedorsal fin of each fish was excised immediately. The time required to take themuscle sample was less than 10 s. Pieces of muscle were frozen immediately inliquid nitrogen, and held at -70°C until analyzed for free amino acids.4.2.5 Amino Acid Analysis of Plasma and MusclePlasma samples were treated as described previously for Experiment 1.1.Muscle samples were extracted for free amino acid analysis after homogenizationwith trichloroacetic acid to deproteinize tissue protein. Five grams of the musclefrom each fish were homogenized for 10 mm with 10 % (v/v) trichloroacetic acid inthe ratio of 1/3 (w/v) using a Virtis No. 45 homogenizer. The homogenization was104Chapter 4done at low temperature by immersing the homogenizing cup in an ice bath duringthe homogenization. The homogenate was centrifuged at 20,000xg (at 4°C) for 20mm. The supernatant was decanted into a 50 mL test tube and further treated inthe same way as described for plasma samples.4.2.6 Statistical AnalysisCorrelation analyses were applied to test the relationships between plasmaand muscle free amino acids using Systat (Wilkinson, 1990).4.3 RESULTS4.3.1 Amino Acid Composition of Experimental DietsThe amino acid composition of the experimental diets as determined on acidhydrolysates is shown in Table 2.2. The concentrations of respective essential aminoacids in diets 1, 3, and 4 were higher than those in diet 2. Diets 1 and 4 containedhigher proportions of essential than non-essential amino acids. The ratios ofEAA/NEAA were 1.1 and 1.2 in diets 1 and 4, respectively. The ratio ofEAA/NEAA in diet 3 was 1.0. Diet 2 had the lowest concentration of essentialamino acids and the highest concentration of nonessential amino acids with the ratioof EAA/NEAA of 0.8.105Chapter 4Table 22. Amino acid composition of experimental diets employed in Experiment 2Experimental diet1Diet 1 Diet 2 Diet 3 Diet 4g/l6gNArg 9.3 8.3 8.3 8.7His 2.9 2.7 3.4 3.1lie 4.0 3.2 3.4 3.8Leu 8.5 7.5 8.5 8.7Lys 5.3 4.7 5.3 7.9Met 2.2 1.7 2.0 2.3Cys 1.4 1.1 1.4 1.4Phe 4.9 3.9 4.8 4.8Tyr 3.4 2.4 3.3 3.3Thr 3.6 3.2 3.4 4.5Va! 4.7 3.8 4.4 4.5Ala 5.8 6.6 5.2 5.4Asp 8.3 8.3 7.6 7.7Glu 17.5 16.1 15.9 16.6Gly 5.1 10.4 10.6 4.7Pro 6.1 8.4 5.4 4.3Ser 4.6 4.3 4.3 5.7TAA2 97.4 96.5 97.1 97.1EAA3 50.2 42.4 48.1 52.8NEAA’4 47.2 54.1 49.0 44.4EAA/NEAA 1.1 0.8 1.0 1.21The main protein sources were as follows: diet 1 = fish meal, corn gluten meal, and soybean proteinconcentrate; diet 2 = fish meal, corn gluten meal, soybean protein concentrate, and gelatin; diet 3 =fish meal, corn gluten meal, soybean protein concentrate, and glycine; diet 4 = fish meal, corn glutenmeal, and soybean protein concentrate, and supplemented with arginine, histidine, lysine, methionine,and threonine.2 amino acids.Total essential amino acids, except tryptophan which was not detected by the procedure used.non-essential amino acids.106Chapter 44.3.2 Initial Weight and Feeding BehaviorThe mean initial weights of the fish used in the experiment are shown inTable 2.3. Comparisons of the growth of the fish fed the different diets were notjustifiable because fish were sampled at different times from the groups.Feed consumption per fish varied with dietary treatment and feeding period.In general, food consumption of fish decreased between period 1 and 3. It wasobserved that fish fed diet 3 (glycine supplemented diet) had a poor appetite.Tabie 2.3. Mean initial weights of fish in different experimental treatments employed in Experiment 2Diet Initial weight(g)1 243.3 ± 3.012 250.1 ± 20.13 237.9 ± 9.34 250.0 ± 5.51Mean of three replicate tanks (15-16 fish/tank) and standard error of the mean, (n= 3).107Chapter 44.3.3. Plasma Amino Acid Profile in Fish Fed Experimental Diets for Seven DaysData on plasma amino acid concentrations in fish at 3, 9, 15, and 24 h afterfeeding different experimental diets for 7 days (first sampling) are shown in Tables2.4-2.7. The levels of total amino acids (TAA), total essential amino acids (EAA),total non-essential amino acids (NEAA), and ratios of EAA/NEAA of fish fed thedifferent diets are also tabulated in these tables. The changes in plasmaconcentrations of total essential amino acids, total non-essential amino acids, andtotal amino acids are shown in Figure 2.1. The changes in plasma concentrations ofindividual amino acids at different times after meal consumption are depicted inFigure 2.2. It should be noted that concentrations of amino acids at 24 hpostprandial were applied to 0 h values in all figures presented here. Furthermore,values for amino acids at 36 h were from the second sampling at which time the fishhad been fed for 17 days.Plasma Total Amino Acids (TAA), Total Essential Amino Acids (EAA), Total NonEssential Amino Acids (NEAA)Plasma EAA, NEAA, TAA concentrations in fish fed the different dietscould be placed into two groups according to their patterns of change over thesampling time. One group included the fish whose EAA, NEAA, and TAA in theplasma reached their highest concentrations at 9 h postprandial. The other groupcontained those fish whose amino acid concentrations rose more slowly and peakedlater at 15 h postprandial. The former group included fish that were fed diets 2 and4, and the latter group included fish that were fed diets 1 and 3. There was a furtherdifference between fish that were fed diets 2 and 4 in that the levels of EAA,108Chapter 4NEAA, and TAA in fish fed diet 4 abruptly decreased after reaching the peak,whereas the levels of amino acids tended to plateau between 9 and 15 h in fish feddiet 2 (Figure 2.1).As shown in Figure 2.la, concentrations of plasma EAA in fish fed diets 1, 3,and 4 were generally similar and were higher than those in fish fed diet 2.Differences between the concentrations of NEAA in fish fed diets 2 and 3 ascompared to fish fed diets 1 and 4 were prominent. The NEAA concentrations inthe fish fed the former diets were more than double those of the fish fed the latterdiets (Figure 2.lb and Tables 2.4-2.7). As expected, the patterns and magnitude ofchanges in TAA in fish fed diets 2 and 3 (Figure 2. lc) resembled the changes ofNEAA.Plasma ratios of EAA/NEAA in fish fed diets 1 and 4 were noticeably higherthan those in fish fed diets 2 and 3, and higher than in fish fed any diet in theprevious experiments (Tables 2.4-2.7).Plasma Concentrations of Individual Amino Acids in Fish Fed Different DietsEssential Amino AcidsThe changes in the concentrations of most plasma essential amino acids,particularly branched-chain amino acids, in fish fed the different diets were similarto the overall pattern of EAA with respect to the diets. In fish fed diets 2 and 4,most essential amino acids started to rise and reached their highest concentrationsat 9 h postprandial, whereas those in fish fed diets 1 and 3 started to rise and peakedlater at 15 h after feeding. Some deviation from this pattern was observed in the109Chapter 4changes in the levels of lysine, arginine, and methionine in fish fed diet 4. Theseplasma amino acids increased as soon as 3 h after feeding.Non-Essential Amino AcidsPostprandial changes in the concentrations of individual non-essential aminoacids in the plasma of fish fed diets 1 and 4 resembled the overall pattern of NEAA(Figures 2.2j-o and 2.lb). Interesting results were observed in the changes inglycine, alanine, and serine concentrations in fish fed diets 2 and 3. The plasmaconcentrations of glycine in fish fed diet 2, which contained gelatin, a protein rich inglycine, increased more rapidly than in fish fed diet 3, which contained free glycine.Furthermore, elevated concentrations of this amino acid were maintained until 24 hafter feeding in fish fed diet 2 (contained gelatin) whereas the levels abruptlydropped after 15 h in fish fed diet 3 (contained free glycine but no gelatin). Whileincreases in plasma alanine, and proline concentrations in fish fed diet 2 reflectedthe levels in the diet, increases in plasma serine concentrations generally did not butrather corresponded to the changes in glycine concentrations. Moreover, plasmasenile concentrations in fish fed diet 3 increased to similar levels to those in fish feddiet 2 (Figure 2.2o). A similar trend was observed for plasma alanine in fish fed diet3 (Figure 2.2j).Irrespective of whether the diets were supplemented with gelatin or glycine,glycine and senile were the non-essential amino acids present in largest amounts inthe plasma of the fish (Tables 2.4-2.7).110Non-essentialTable2.4.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafterfeedingdiet1(fishmeal,cornglutenmeal,soyprotein)onday7inExperiment 2.AminoHoursafter feedingacid391524X±SEM1%2X±SEM%X±SEM%X±SEM(j.tmol/mL)(j.mol/mL)(tmol/mL)(mo1/niL)EssentialMg0339±0.0025.70.256±0.0043.80.353±0.0374.20.252±0.041Cys0.028±0.0020.50.048±0.0020.70.051±0.0060.60.035±0.005His0360±0.0166.00.352±0.0025.30.368±0.0214.40.340±0.045lie0.381±0.0926.40.420±0.0586.30.638±0.0357.60.460±0.003Leu0.800±0.20213.40.916±0.10913.81.445±0.06317.21.175±0.045Lys0341±0.0635.70297±0.0184.50.321±0.0493.80.249±0.062Met0.092±0.0401.50.198±0.0053.00.154±0.0221.80.113±0.044Phe0243±0.0104.10288±0.0054.30.280±0.0103.30.243±0.032Thr0.155±0.0182.60.197±0.0273.00.320±0.0283.80222±0.012Tyr0.166±0.0212.80.180±0.0072.70.185±0.0132.20.170±0.033Val0.773±0.15913.00.816±0.10812.31.284±0.05215.30.950±0.005Ala0.484±0.0238.10.585±0.0718.80.484±0.0545.80.358±0.035Asp0.032±0.0070.50.054±0.0010.80.036±0.0000.40.045±0.024Glu0.178±0.0243.00.185±0.0022.80.306±0.0183.70.196±0.048Gly0.675±0.13711.30.770±0.12111.60.957±0.11311.40.628±0.100Pro0213±0.0233.60.277±0.0124.20.374±0.0284.50.216±0.034Ser0.698±0.01111.70.816±0.00212.30.832±0.0079.90.574±0.087TAA35.960±0.4476.654±0.0968.390±0.3436.225±0.560EAA43.680±0.6053.969±02785.399±0.2324.209±0.232NEAA52.280±0.1582.685±0.1822.991±0.1132.016±0.328EAA/NEAA1.611.481.802.09% 4.1 0.6 5.5 7.418.9 4.0 1.8 3.93.62.715.3 5.80.73.110.1 3.5 9.21Meanof twopoolsof5plasma(two fish/pool) andstandarderrorofthemean(n=2).2oftotalaminoacids.3Totalaminoacids.4rotalessential aminoacids.Total non-essential aminoacids.Table2.5.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferent timesafterfeedingdiet2(fishmeal,cornglutenmeal,soyprotein,gelatin)onday7inExperiment 2.AminoHoursafterfeedingacid391524Non-essential3rotalaminoacids.4TotalEssentialX±SEM1%2X±SEM%X%X±SEM%(mol/mL)(j.Lmol/mL)(jmol/mL)(.mol/mL)Mg0247±0.0703.40.261±0.0652.30.3383.00.271±0.0293.0Cys0.037±0.0080.50.051±0.0100.50.0710.60.041±0.0050.5His0.352±0.0364.80.413±0.0333.60.4353.90370±0.0194.0fle0242±0.0023.30.406±0.0753.60.3823.40313±0.0733.4Leu0.534±0.0347.30.897±0.1417.90.8817.90.837±0.1469.1Lys0.298±0.0404.10.368±0.0473202982.70.195±0.0512.1Met0.107±0.0031.50.176±0.0231.60.148130.104±0.0161.1Phe0220±0.0013.00293±0.0352.602482.20.234±0.0012.6Thr0.153±0.0482.10.357±0.0733.10.4153.70279±0.0023.0Tyr0.110±0.0101.50.161±0.0221.40.1171.10.115±0.0071.3Val0.561±0.0147.70.870±0.1167.60.8297.40.767±0.1058.4Ala0.663±0.0649.00.990±0.2698.70.9458.40.635±0.0127.0Asp0.151±0.0202.10239±0.0342.10.260230.190±0.0032.1Glu0.230±0.0282.80229±0.0082.00.3012.70.198±0.0052.2Gly1.779±0.22624.33.401±0.17829.83.11627.82.797±0.12830.6Pro0.942±0.28212.80.896±0.2257.91.16910.40.841±0.19292Ser0.737±0.04810.01391±0.22112.21.24611.10.962±0.01210.5TAA37337±0.59011.400±1.44411.1979.148±0.531EAA’12.861±0.1054.253±0.5104.1623.525±0.465NEAA54.476±0.4857.147±0.9357.045.623±0.066EAA/NEAA0.510.600.600.63‘Meanoftwopools ofplasma(twofish/pool) andstandarderrorof themean(n=2).2of totalaminoacids.essential aminoacids.‘Total non-essentialaminoacids.Non-essentialTAA3 EAA4 NEAA5 EAA/NEAA6.358±1.4333.002±0.7453.337±0.6890.906.420±0.2592.734±0.1343.686±0.1240.7411.955±0.0775.320±0.3296.635±0.2520.808.665±2.5804.809±0.8803.856±1.7021.24aminoacids.Table2.6.Concentrationsof plasmaaminoacidsinrainbowtrout atdifferent timesafterfeedingdiet3(fishmeal,coni glutenmeal,soyprotein,supplementedwithglycine)onday7inExperiment2.EssentialAminoHoursafter feedingacid391524X±SEM1%2X±SEM%X±SEM%X±SEM%(mol/mL)(.tmo1/mL)(tmo1/mL)(Lmo1/mL)Arg0216±0.0843.40.241±0.0573.80.287±0.0062.40.204±0.1262.4Cys0.026±0.0260.40.046±0.0040.70.072±0.0100.60.051±0.0120.6His0.322±0.0515.10.280±0.0244.40.470±0.0013.90338±0.0523.9lie0.303±0.0844.80.240±0.0263.70.568±0.0534.80.564±0.1056.5Leu0.651±0.18710.20.491±0.0567.71.326±0.12111.11.442±0.11716.6Lys0.254±0.0504.00.229±0.0133.60.263±0.0062.20.209±0.0752.4Met0.113±0.0211.80.129±0.0032.00.199±0.0161.70.182±0.0462.1Phe0213±0.004330.250±0.0083.90.287±0.0212.40.242±0.0422.8Thr0.149±0.034230.169±0.0732.60.504±0.0164.20.257±0.1383.0Tyr0.114±0.0201.80.144±0.0022.30.193±0.0171.60.175±0.0262.0Val0.641±0.18310.10.515±0.0418.01.150±0.1079.61.145±0.14013.2Ala0.480±0.0877.60.536±0.0308.40.766±0.0066.40.629±0.2767.3Asp0.058±0.0050.90.053±0.0090.80.065±0.0220.60.028±0.0050.3Gin0.143±0.034230.148±0.0192.30.274±0.0162.30.194±0.0372.2Gly1.788±0.39628.11.929±0.02530.13.611±0.25930.21.936±0.99922.3Pro0.156±0.0382.50.191±0.0193.00.451±0.0113.80.216±0.0682.5Ser0.731±0.12811.50.826±0.06112.91.468±0.00412.30.853±0.3879.8‘Meanof twopoolsofplasma(twofish/pool) andstandarderrorofthemean(n=2).2oftotalaminoacids.essential aminoacids.‘Totalnon-essential aminoacids.Non-essential3Totaiaminoacids.4TotalTable2.7.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafter feedingdiet4(fishmeal,cornglutenmeal,soyprotein,supplementedwitharginine, bistidine,lysine,methionine,andthreonine)onday7inExperiment 2.EssentialAminoHoursafter feedingacid391524X±SEM%X±SEM%X±SEM1%2X±SEM(mol/mL)(mol/mL)(j.mol/mL)(mol/mL)Arg0.323±0.0416.40.330±0.0064.20.341±0.0725.20.178±0.085Cys0.029±0.0000.60.053±0.0060.70.040±0.0060.60.025±0.002His0.344±0.0166.80.440±0.0085.60.367±0.0165.60.332±0.003lie0.307±0.0416.10.535±0.0496.80.495±0.0497.50.524±0.008Len0.665±0.06713.11.120±0.10314.11.165±0.123[7.71.216±0.022Lys0.561±0.01611.10.739±0.123930.484±0.1347.40.355±0.008Met0.145±0.0072.90.268±0.0143.40.177±0.0152.70.090±0.002Phe0.232±0.0034.60.329±0.0294.20.230±0.0053.50.238±0.003Thr0.205±0.0084.10.409±0.0865.20.286±0.0494.30.271±0.004Tyr0.155±0.0153.10.224±0.0312.80.163±0.0212.50.152±0.002Val0.638±0.10612.60.978±0.10312.30.952±0.09514.51.083±0.020Ala0.279±0.0145.50.521±0.1596.60.415±0.0886.30.271±0.007Asp0.040±0.0180.80.051±0.0030.60.023±0.0020.40.028±0.001Gin0.166±0.0143.30.200±0.0082.50234±0.0193.60.178±0.005Gly0.434±0.1148.60.588±0.1417.40.474±0.066720.446±0.009Pro0.108±0.0032.10348±0.0774.40.213±0.0293.20.166±0.007Ser0.435±0.0208.60.792±0.23110.00.522±0.0267.90.414±0.009TAA35.065±0.0117.925±0.6596.582±0.7835.970±0.028BAA’13.604±0.1385.425±0.0464.700±0.5544.460±0.011NEAA51.461±0.1492.500±0.6131.882±0.2291.510±0.038EAA/NEAA2.472.172.502.95% 3.0 0.4 5.6 8.8 20.4 6.0 1.54.0 4.5 2.6 18.1 45 0.5 3.0 75 2.8 6.91Meanof twopoolsof5plasma(twofish/pool) andstandarderrorofthemean(n=2).2oftotalaminoacids.essentialaminoacids.Total non-essentialaminoacids.(a)Totalessentialaminoacids-J E 0 E .100 4_Ba, 4- C a,BC.) C 0 a, E a,2a,-J E 0 E C 0 a, I C 0 C 0 0 a, E a, a, 012•-€--diet1+diet2-*--diet3—&-diet43691215243636912152436Hour8afterfeedingFigure2.1.Totalconcentrationofplasmaaminoacids(essential,non-essential,andtotalaminoacids)inrainbowtroutdeterminedatdifferenttimesafterfeedingexperimentaldietsonday7inExperiment2.Diet1=fishmeal,cornglutenmeal,soyprotein;diet2=fishmeal,cornglutenmeal,soyprotein,gelatin;diet 3=fishmeal,cornglutenmeal,soyprotein,andglycine;diet4=fishmeal,cornglutenmeal,soyprotein,supplementedwitharginine,histidine,lysine,methionine,andthreonine.Eachpointrepresentsameanoftwopooisofplasma,twofish/pool,(n=2).1-J E 0 E C 0 I C a, C, C 0 C.) Ca E 0) as 0-J E 0 E C 0 I-. C a) C, C 0 0 Ca E-J E 0 F C 0 Ca C 0, 0 C 0 0 CO F 0) CO0.80.60.40.2(a)Arginine—E—diet1diet2-*-diet3—es-diet4U(b)Histidine0.8E =. C 00.6.‘-36912152436C’369121524361-0.80.60.40.2(d)Phenylalanine36912152436Hoursafterfeeding369121524Hoursafterfeeding36Figure22.PlasmaconcentrationsofaminoacidsinrainbowtroutdeterminedatdifferenttimesafterfeedingexperimentaldietsinExperiment2.Diet1=fishmeal,cornglutenmeal,soyprotein;diet2=fishmeal,cornglutenmeal,soyprotein,gelatin;diet 3=fishmeal,cornglutenmeal,soyprotein,andglycine;diet4=fishmeal,cornglutenmeal,soyprotein,supplementedwitharginine,histidine,lysine,methionine,andthreonine.Eachpointsrepresentsameanoftwopoolsofplasma,twofish/pool,(n=2).-J E 0 E Cl0 !08aa_J EE-S.3EEaaCC00.6.2—w 6.CC0.4CC00o0 ECo0CII36912152436Hoursafterfeeding36912152436(g)Methionine+Cystine36912152436HoursafterfeedingFigure2.2.(Continued)(000.II.0Plasmaconcentration(pmol/mL)p811Plasmaconcentration(ijmol/mL)ppppof•).ctI(0OiC.) C)C.) C)Plasmaconcentration(pmol/mL)C,)C)Plasmaconcentration(ijmol/mL)(00-.101(D0.(01.4-J E 0 E3C 0 L2C 0 0 C 0 0 E 0, 0-J E 30.8E C o0.64-. I-.1 C0.4C 0 C) C’, 0(m)Glycine+-,++-J E1.20 E10.8 0.60 C 0 o0.40, E co0.20,036912152436-J E1.-S 0 E C 0 I(n)Proline36912152436(p)Tyrosine—B-diet1+diet2-*-diet3—&-diet436912152436Hoursafterfeeding36912152436Hoursafterfeeding0Figure2.2.(Continued)Chapter 44.3.4. Relationship Between the Concentrations of Free Amino Acids in Plasma andMuscleThe concentrations of plasma free amino acids in blood samples collected at26 and 36 h after feeding the experimental diets for 17 days are shown in Table 2.8.Their relative concentrations, expressed as percentage of total amino acids, arecalculated and shown in Table 2.9. The levels of muscle free amino acids and theirrelative concentrations, as percentage of total amino acids, at the same samplingtime are shown in Tables 2.10 and 2.11, respectively.It was observed that the total concentrations of free amino acids in themuscle were several fold higher than in the plasma at all sampling times (Tables 2.8and 2.10). Comparison of plasma and muscle TAA concentrations in fish feddifferent diets revealed that whereas plasma TAA concentrations in fish decreasedbetween 26 and 36 h after feeding, the muscle TAA concentrations were morestable. Muscle EAA concentrations in fish fed diet 4 which containedsupplementary amino acids were higher than in fish fed other diets, particularly at26 h postprandial (approximately 30% higher). Furthermore, muscle NEAAconcentrations in fish fed diet 3 containing free glycine were higher than in fish fedother diets, and approximately 35% higher than in fish fed diet 2 which containedsimilar amounts of dietary glycine regardless of sampling time.Although the total free amino acid concentrations in muscle were higherthan in plasma, concentrations of branched-chain amino acids in plasma wereroughly double those in the muscle (Tables 2.8 and 2.10).The levels of most free essential amino acids in muscle were positivelycorrelated with the plasma levels in fish fed the experimental diets (Table 2.12).120Chapter 4Exceptions were arginine, histidine, and threonine. With respect to non-essentialamino acids, significant correlations between the levels in plasma and muscle weredetected only in the case of aspartic acid and proline. The results on the levels ofglycine in plasma and muscle of fish fed diets 2 and 3 were interesting. While theglycine concentrations in plasma of fish fed diets 2 and 3 were equally high at 26 and36 h postprandial, the muscle levels of this amino acid in fish fed diet 2 wereremarkably lower than in fish fed diet 3 (Tables 2.8 and 2.10). A similarphenomenon was also observed for the levels of senile between the same groups offish at 26 h postprandial.Since diet 4 was supplemented with arginine, histidine, lysine, methionine,and threonine, the comparison between the levels of these amino acids in muscleand the plasma free pool of these fish and those of fish fed diets 1, 2 and 3 between26 and 36 h postprandial were depicted graphically (Figures 2.3 and 2.4). Theplasma concentrations of lysine, methionine, and threonine in fish fed diet 4 at 26 hafter feeding were higher than those in fish fed diet 3, which contained similarproportions of dietary ingredients. In muscle, the levels of histidine, lysine andthreonine in fish fed diet 4 were prominently higher than those in fish fed diet 3 aswell as diets 1 and 2 at 26 h after feeding (Figure 2.4). The higher concentrations ofthese amino acids in muscle of fish fed diet 4 than in fish fed diet 3 were alsoobserved at 36 h after feeding. In plasma, on the other hand, neither free argininenor histidine, and in muscle neither free arguffine nor methionine were appreciablydifferent among fish fed different diets.121Chapter 4Regardless of the differences in the levels of individual amino acids due todietary differences, the amino acids that characterized the plasma and muscle freeamino acids differed. In plasma, the branched-chain amino acids and glycine werethe predominant essential and non-essential amino acids, respectively (Table 2.9).In muscle, histidine characterized the essential amino acid pool making upapproximately 15-24% of TAA concentrations, and glycine dominated the nonessential amino acid pooi, constituting 46.6-59.8% of TAA concentrations (Table2.11).122Table2.8.Concentrationsof plasmaaminoacidsinrainbowtrout at26and36hafterfeedingtheexperimental diets1onday17inExperiment 2.Ammo26hafterfeeding36hafterfeedingacidDiet 1Diet 2Diet3Diet4Diet1Diet 2Diet 3Diet4(jtmol/mL)2________(jAmol/mL)EssentialMg0.112±.0050.133±.0400.123±.0080.179±.0730.128±.0070.165±.0110.142±.0530.176±.002Cys0.036±.0000.047±.0070.044±.0020.042±.0040.028 ±.0080.022±.0050.032±.0060.015±.015His0332±.0260.371±.0150.308±.0260.337±.0020.194±.0080.228±.0050.236±.0200.237±.025lie0300±.0600.319±.0120.306±.0950.400±.0130.136±.0210.147±.038O.190±.0240.129±.054Leu0.787±.1810.784±.0040.751±.147O.999±.0510.350±.0470.422±.1150.503±.0910.327±.153Lys0.185±.0150.236±.0140.217±.0680.379±.0510.114±.0040.082±.0090.115±.0010.156±.030Met0.175±.0240.149±.0050.160±.0020.226±.0100.069±.007O.075±.0100.093±.0260.108±.013Phe0.205 ±.0110.212±.0080.236 ±.0060.236 ±.0080.185 ±.0140.141 ±.0020.203±.0220.161±.006Thr0.241±.002O.315±.0360.207±.0510.382±.0610.115±.0250.098±.0170.115±.0270.157±.037Tyr0.142±.0050.095±.0060.179±.0070.166±.0170.118±.0350.044±.0040.142±.0200.096±.026Val0.703±.1390.817±.0440.701±.1590.825±.0250.288±.0410.476±.0980.460±.0880.300±.124Non-essentialAla0319±.0560.738±.2010.414±.0510370±.0450.517±.0160.439±.0870.380±.1220.172±.061Asp0.035±.0100.193±.0640.059±.0020.021±.0000.024±.0080.119±.0210.031±.0100.020±.005Gin0.142±.0300.209±.0050.187±.0350.144±.0040.094±.0010.110±.0180.156±.0400.084±.020Gly0.624±.0532.047±.1832.393±.2760.641±.0820.608±.1760.726±.1660.94±.2030.347±.113ProO.194±.0040.749±.0840.225±.0560.193±.0230.097±.015O.366±.1070.119±.0330.050±.019Ser0.542±.0100.943±.1410.787±.1200.539±.0140.468±.0200.4i2±.0850.433±.0490302±.071TAA45.075±.5528.359±.6557.296 ±1.1086.081±.0253533±.0824.123 ±.7594.299±.8332.837±.770EAA53.219 ±.4183.479±.0123.232±.5674.173 ±.0671.724±.1211.902±.2752.232±.3761.862±.481NEAA61.856±.1344.880±.6684.065±.5401.908±.0431.809±.2032.221±.4842.067±.4570.975±.289EAA/NEAA1.70.70.82.20.950.861.081.911Diet1containedfishmeal,cornglutenmeal,soyproteinconcentrate,diet2containedbasalingredientsasindiet1andgelatin, diet 3containedbasalingredientsasindiet1plusglycine,diet4containedthesameingredientsasindiet3butsupplementedwitharginine,histidine,lysine,ethionine, andthreonine.Meansoftwopooisofplasma(twofish/pool) andstandarderrorof themeans(n=2).present intraceamount.aminoacids.Totalessential aminoacids.6Totalnon-essential aminoacids.Table2.9.Relativeconcentrationsofplasmafreeaminoacids(expressed asapercentage) inrainbowtroutat26and36hafterfeedingtheexperimentaldiets1onday17inExperiment 2.Amino26hafter feeding36hafterfeedingacidDiet 1Diet 2Diet 3Diet4Diet1Diet 2Diet 3Diet 4EssentialMg2.21.61.83.03.64.03.36.2Cys0.70.60.60.70.80.50.70.5His6.54.44.25.55.55.55.58.4lie5.93.84.26.63.83.64.44.6Leu15.59.410.316.49.910.211.711.5Lys3.62.83.06.23.22.02.75.5Met3.51.82.23.72.01.82.23.8Phe4.02.53.23.95.23.44.75.7Thr4.73.82.86.33.22.42.75.5Tyr2.81.12.52.73.31.13.33.4Val13.99.89.613.68.211.610.710.6Non-essentialAla6.38.85.76.114.610.68.86.1Asp0.72.30.80.40.72.90.70.7Glu2.82.52.62.42.72.73.63.0Gly12.324.532.810.517.217.622.112.2Pro3.89.03.13.22.88.92.81.7Ser10.711.310.88.913.311.210.110.71Diet 1containedfishmeal,cornglutenmeal,soyproteinconcentrate,diet2containedbasal ingredientsasindiet1andgelatin, diet 3containedbasalingredientsasindiet1piusglycine,diet4containedthesameingredientsasindiet3but supplementedwitharginine,histidine,iysine,methionine, andthreonine.Table2.10.Concentrationsofmusclefreeaminoacidsinrainbowtrout at26and36hafterfeedingtheexperimentaldiets1onday17inExperiment2.Amino26hafterfeeding36hafter feedingacidDiet 1Diet2Diet3Diet4Diet1Diet 2Diet 3Diet4SEM(mol/gwetweight)2______SEM(jmol/gwetweight)EssentialMg0370±.0600.415±.0490244±.0760.311±.0350.134±.0420.422±.0450.301±.0200.242±.013cystr3tr0.128 ±.0000.155 ±.0000.041 ±.041ErtrErHis5.749±4606.389±.2576.119±.5537.587±.7337.223±.9527.091±.2906.661±.4507.603±.601lie0.135±.0400.119±.0150.148±.0350.158 ±.0600.059±.0280.096±.0230.135±.0150.063±.025Leu0.472±.1270.312±.0360.347±.0540.454±.1S10.189±.0490.244±.0470.300±.0440.179±.045Lys0.109±.0250.128±.048O.169±.0850.384±.1080.091±.0910.048±.0200.038±.0190.149±.037Met0.218±.035O.178±.0240.197±.0350.235±.0070.088±.0060.121±.0210.159±.0320.177±.019Phe0.187±.0000.128±.0000.201±.0720.259±.0000.081±.082tr0.121±.087trThr0.820±.1510.524±.0720.598±.0921.190±.0820.466±.0480.344±.2110.905±.0651.062±.111TyrtrEr0.144±.0990.146±.0000.090±.090tr0.090±.090trVal0.287±.1010.371±.0290.360±.0870.356±.1310.138±.0370.301±.0520.278±.0490.175±.053Non-essentialAla1.876±.3801.953±2071.841±.3591.465±.2712.280±.6142.159±.34.82.378±.3600.956±.136Asp0.483±.0520.693±.1930.493±.1280.285±.0950.296±.0990.865±.0660.507±.0690.281±.040Glu1.186±.1491.074±.0810.886±.0701.065±.1640.930±.1061.068±.1341.021±.1030.998±.189Gly19551±2.23215.075±1.70024.880±2.43017.363±1.22319.891±1.28413.461±.73224.042±1.25917.527±2.256Pro1.711±.3403.523±.5911397±.1890.971±.2140.892±.1303.969±.6800.950±.1480.594±.111Ser2.596±.3081.495±.1003.340±.6751.486±.1561.430±.1321.839±.2522.398±.4780.966±.057TAA435.750±2.63432.377±1.82841.491±4.13333.870±1.73534.320±1.85632.226±1.47840.284±2.22230.972±1.989EAA58.347±.9608.564±2578.655±.79411.235±.7318.600±.5428.865±.4618.988±.6369.650±.716NEAA627.403±2.42023.813±1.22732.837±3.42022.635±1.86625.719±1.55123.361±1.22731.296±1.95021.322±2.331EAA/NEAA0.300.350.260.500.330.380290.451Diet 1containedfishmeal,cornglutenmeal,soyproteinconcentrate,diet2containedbasal ingredientsasindiet1andgelatin, diet 3containedbasalingredientsasindiet1plusglycine,diet4containedthesameingredients asindiet3andsupplementedwitharginine, histidine,lysine,methionine,andthreonine.2Meoffoprsamples(fourfish)andstandarderrorofthemeans(n=4).presentintraceamount.aminoacids.Totalessentialaminoacids.“Totalnon-essentialaminoacids.Table2.11.Relativeconcentrationsofmusclefreeaminoacids(expressed asapercentage)inrainbowtroutat 26and36hafter feedingexperimental diets1onday17inExperiment 2.Amino26hafter feeding36hafterfeedingacidDiet1Diet2Diet 3Diet 4Diet1Diet2Diet 3Diet 4EssentialMg1.01.30.60.90.4130.70.8Cys000.30.50.1000His16.119.714.922.421.022.016.524.5Ile0.40.40.40.50.2030.302Leu1.31.00.8130.60.80.70.6Lys030.40.41.10.30.10.10.5Met0.60.60.50.70.30.40.40.6Phe0.50.40.50.80.20030Thr2.31.61.43.51.41.72.23.4Tyr000.30.40300.20Vat0.81.10.91.10.40.90.70.6Non-essentialAla5.26.04.4436.66.75.93.1Asp1.42.11.20.80.92.71.30.9Glu3.33.32.13.12.73.32.53.2Gly54.746.659.851358.041.859.756.6Pro4.810.93.42.92.61232.41.9Ser734.68.04.4425.76.03.11Diet1containedfishmeal,cornglutenmeal,soyproteinconcentrate,diet2containedbasal ingredientsasindiet1andgelatin,diet 3containedbasalingredientsasindiet1plusglycine,diet4containedthesameingredients asindiet3 butsupplementedwitharginine,histidine,lysine,methionine,andthreonine.Chapter 4Table 2.12. Correlation coefficients1of concentrations between plasma and muscle free amino acidsin Experiment 2Amino acid Correlation coefficientsArg 0.080His-0.475ile 0.845*Leu 0.906*Lys 0.937*Met 0.924*Phe 0.914*Thr 0.436Val 0.899*Ala 0.560Asp 0.795*Glu 0.064Gly 0.301Pro 0.830*Ser 0.3521 The values used in the calculation were the means of each dietary treatment at 26 and 36 h (n = 8).The coefficients with an asterisk were significant at P <0.05.127— diet 1 diet 2 diet 3 diet 4diet 1 diet2 diet 3 diet 4Figure 2.3. Plasma concentrations of supplementary amino acids in rainbow troutdetermined at 26 h (a), and 36 h (b) after feeding the experimental diets on day17 in Experiment 2. Diet 1 = fish meal, corn gluten meal, soy protein; diet 2 =fish meal, corn gluten meal, soy protein, gelatin; diet 3 = fish meal, corn glutenmeal, soy protein, and glycine; diet 4 fish meal, corn gluten meal, soy protein,supplemented with arginine, histidine, lysine, methionine, and threonine. Eachpoint represents a mean of two pools of plasma, two fish/pool, (n = 2).(a)1.000.900.800.700.600.500.40:::0.100.0026 hIIArg His Lys Met TlAmino acid(b)I1.000.900.800.700.600.500.400.300200.100.0036 hHis Lys MetAmino acid]Arg128Amino aciddiet 1 diet 2 diet 3 diet 4Figure 2.4. Muscle concentrations of supplementary amino acids in rainbow troutdetermined at 26 h (a), and 36 h (b) after feeding the experimental diets on day17 in Experiment 2. Diet 1 = fish meal, corn $luten meal, soy protein; diet 2 =fish meal, corn gluten meal, soy protein, gelatm; diet 3 = fish meal, corn glutenmeal, soy protem, and 1ycine; diet 4 = fish meal, corn gluten meal, soy protein,supplemented with argmine, histidine, lysine, methionine, and threonine. Eachpoint represents a mean of four samples from four fish (n=4).(a) 26h2I010987654320SI-sIIAmino acidArg Lys Met Ttw Hisdiet 1 diet 2 diet 3(b) 36 h21diet 41098765432L 0Lys Met Th His129Chapter 44.3.5 Comparison Between Concentrations of Plasma Amino Acids in Fish inExperiment 1 (Freshwater) and Experiment 2 (Seawater)Diet 2 used in Experiment 1 and 2 was similar in composition. The aminoacid compositions of diet 2 in the two experiments were also similar except theconcentrations of particularly arginine and leucine in the diet used in Experiment 2were higher than those for the diet used in Experiment 1 (Table 1.2, 1.21, and 2.2).The results of the plasma concentrations of total essential (EAA), total nonessential (NEAA), and total amino acids (TAA) in fish fed this diet that weresampled at the same intervals after feeding in Experiments 1.1 and 2 are presentedin Table 2.13.The levels of plasma TAA between the two experiments did not markedlydiffer. The concentrations of plasma EAA in fish in Experiment 2, however, wereconsistently higher than those in fish in Experiment 1.1. Comparison of theconcentrations of individual essential amino acids in fish between the twoexperiments revealed that arginine, the branched-chain amino acids, lysine, andthreonine were responsible for the higher levels of EAA detected in fish inExperiment 2. The higher concentrations in the plasma, however, corresponded tothe dietary levels of these amino acids in the latter experiment.130Table2.13.Plasmaconcentrationsofbranched-chainaminoacids,lysineandthreonineandtotal concentrationsofessentialaminoacids,nonessentialaminoacids,andtotalaminoacidsinfishfeddiet2sampledatdifferenttimesafterfeedinginExperiment 1.1andExperiment2AminoOh3h9hIShacidExp.1.1Exp.2Exp.1.1Exp.2Exp.1.1Exp.2Exp.1.1Exp.2X±SEM1X±SEMX±SEMX±SEMX±SEMX±SEMX±SEMX±SEM(J.Lmol/mL)(mo1/mL)(I.Lmol/mL)(iLmol/mL)(mo1/mL)(mol/mL)(mo1/mL)(mol/mL)lie0.101±.0160.313±.0730.177±.0250.242±.0020.193±.0310.406±.0750.153±.010O382Leu0.251±.045O.837±.1460.335±.0460.534±.034O.399±.0580.897±.141O.305±.0060.881Val0.378±.051O.767±.1050.43±.O710.561±.014O.568±.064O.870±.116O.448±.0340.829Lys0.223±.0170.195±.0510255±.0340.298±.040O.237±.022O.368±.047O.236±.0040.298ThrO.175±.0130.279±.002O.175±.046O.153±.048O.200±.034O.357±.073O.202±.0270.415TAA49.318±.8759.148±.5318.610±.8037.337±.5909.953±.73811.400±1.4441O.340±.20611.197EAA51.808±.1503.525±.4652.289±.3382.861±.1052.404±2064.253±.51O2.047±.1474.162NEAA67.510±.7355.623±.0606.321±.4844.476±.4857.550±.5837.147±.9358.289±.3347.040valueinExperiment1.1representsameanofthreepoolsofplasma(fivefish/pool) andstandarderrorofthemean(n= 3).2valueinExperiment2repreentsameanof twopoolsofplasma(twofish/pool),andstandarderrorof themean(n=2).3Onlyonepoolofplasma.4Totalaminoacids.Totalessentialaminoacids.non-essentialaminoacidsChapter 44.4 DISCUSSIONPlasma concentrations of essential and non-essential amino acids in fish feddiets 2 and 4 started to increase, and they reached peaks earlier than observed infish fed diets 1 and 3. More interestingly, plasma glycine in fish fed diet 2(contained gelatin) increased sooner following meal consumption than in fish feddiet 3 (contained free glycine). This finding negated the hypothesis that glycinesupplied in the free form in the diet would be absorbed more rapidly than when fedas a constituent of protein. It may be inferred from the more rapid increases inother plasma amino acids after consumption of diet 2 compared to diet 3 thatgelatin present in the diet was rapidly hydrolyzed. In relation to this finding, gelatinpresent in the diet possibly has favorable effects on the digestive process in general.This finding was actually supported by the studies of Boge et al., (1981) who foundthat glycine uptake by rainbow trout was more rapid from glycyiglycine (a dipeptide)than from the equivalent free glycine, especially at a high concentration. A similarphenomenon that has been observed in mammals (Matthews, 1973).The great increases in the concentrations of plasma alanine, glycine, andproline in fish fed diet 2 between 9 and 15 h after feeding corresponded to the highlevels of these amino acids in diet 2 which originated from gelatin (Table 2.2).Plasma serine concentrations in fish fed diet 2 also rose to a great extent. Thisfinding was similar to the results of Experiment 1 and is consistent with theformation of serine from glycine. The increases in the levels of plasma serine andalanine in fish fed diet 3 did not agree with the amino acid composition of this diet.Moreover, the concentrations of these two plasma amino acids were many foldhigher than the concentrations observed in fish fed diet 4 which contained similar132Chapter 4proportions of feed ingredients. Diet 3 was supplemented with free glycine, whereasdiet 4 was supplemented with free arginine, histidine, lysine, methionine, andthreonine. The increased concentrations of plasma serine and alanine in fish feddiet 3 clearly resulted from the effects of the inclusion of glycine in this diet. Therise in the levels of these two amino acids indicated that excess plasma glycine infish fed diet 3 was converted to serine and alanine. In general, serine is catabolizedby three routes which are initiated by, (1) serine dehydratase which involves thenon-oxidative deamination of serine to pyruvate, (2) serine pyruvate transaminase inwhich serine is transaminated with pyruvate to form hydroxypyruvate and alanine,and (3) serine hydroxymethyltransferase which is the reversed reaction of theglycine cleavage system to form glycine (Walton and Cowey, 1982; Bender, 1985).All three enzymes have been detected in rainbow trout liver and kidney (Waltonand Cowey, 1981). Cowey and Walton (1989) suggested that thehydroxymethyltransferase is probably the main route of serine catabolism inrainbow trout as it is in mammals. The high plasma concentrations of glycine andalanine in rainbow trout in the present experiment indicated that serine wascatabolized via serine pyruvate transaminase. This finding is in agreement with thehigh levels and low Km of this transaminase in rainbow trout liver as reported byWalton and Cowey (1981). Cowey and Walton (1989) stressed that this pathway islikely important during gluconeogenesis from serine. Dehydratase has been foundat a very low level in rainbow trout tissues and, seemingly, does not have animportant role in serine metabolism in this species.The pattern of changes observed in concentrations of glycine and serine inplasma of fish fed diet 3 seemed to be followed, as well, in the muscle of the fish fed133Chapter 4this diet. In comparison with the concentrations of free amino acids in the muscle offish fed other diets, fish fed diet 3 had higher levels of both glycine and serine at 26and 36 h postprandial. The results of the present study showed that excess amountsof these two amino acids in the circulation were absorbed and retained in muscle(Christensen, 1964). The accumulation of serine, glycine, and alanine has similarlybeen observed in muscle of carp fed a diet supplemented with a mixture of nonessential amino acids (Murai et a!., 1989a).The high levels of serine and glycine, gluconeogenic amino acids, in plasmaand muscle of fish fed diet 3 suggested the possibility that the capacity of the fish forcatabolism of glycine was exceeded. The depression of appetite observed with thisgroup of fish might reflect a toxic effect of excess glycine in diet 3. The reduction offeed intake in these fish was possibly a natural mechanism to reduce the influx ofglycine (Harper et a!., 1970). Fauconneau, (1988) also observed a decrease involuntary food intake of rainbow trout fed a diet singly supplemented with alanine,aspartic acid, or glutamic acid. The negative effects of supplementary glycine werealso observed in the studies of Todd et a!., (1967). They found that chinook salmonfed a diet supplemented with 10% glycine had higher liver glycogen than fish fed thecontrol diet. Hughes (1985) also found that rainbow trout and lake trout showedinferior growth rate and feed conversion efficiency when they were fed a dietsupplemented with 15% glycine (as % of diet) in comparison with 15 % glutamicacid.Comparisons of the concentrations of plasma amino acids in fish fed thedifferent diets showed that plasma essential amino acids in fish fed diet 1 and 4 werehigh. The total essential amino acid concentrations in plasma of fish fed diets 1 and134Chapter 44 reached 63.4 and 68.6% of TAA at 26 h postprandial (day 17 sampling). Theconcentrations of essential amino acids in fish fed diets 1 and 4 later decreased tothe levels lower than those in diet 2 and 3 at 36 h. This suggested that thecirculating amino acids in fish fed diets 1 and 4 were removed more rapidly from theplasma pool and were associated with faster protein synthesis than in fish fed diet 2and 3.Higher free histidine, lysine, and threonine concentrations in muscle of fishfed diet 4 than in fish fed other diets were responsible for the higher muscleconcentrations of total essential amino acids, particularly at 26 h postprandial. Thisimplied that the concentrations of histidine, lysine, and threonine in the circulationwere in excess of those required by the fish. Under this circumstance, muscle tissuesabsorbed these amino acids and acted as a temporary storage reservoir. In fact, theaccumulation of the aforementioned amino acids in the muscle of fish in the presentexperiment suggested that these amino acids were still available to the fish at 26 and36 h after feeding. The results contradicted the belief that a rapid and greatincrease of plasma amino acids will lead to rapid catabolism and that the excessamino acids accordingly are not available for protein synthesis over a protractedperiod (Cowey, 1980). The evidence for an enhanced rate of amino acid catabolismwith increased levels of particular amino acids in the plasma has been reported byothers (Walton et a!., 1984a; Cowey and Walton, 1988; Kim et a!., 1992) in rainbowtrout. The results of different studies regarding the enzyme activity responsible foramino acid degradation in fish are, however, confusing. For instance, Cowey et al.,(1981) studied the effects of quality and quantity of dietary protein on activity ofurocase, histidine deanilnase, AMP deaminase, and serine pyruvate transaminase in135Chapter 4rainbow trout. Changes in enzyme activity were detected only with serine pyruvatetransaminase. The results contradicted those found for carp (Sakaguchi and Kawai,1970). Kim et al., (1992) recently reported that liver bistidine deaminase activity inrainbow trout was higher in fish fed a commercial diet containing 50% protein thanin fish fed diets containing 10 or 35% protein. Although it is well understood thatother animals adapt to changes in dietary proteins concentration through alterationsin the activity of enzymes, the mechanism which controls protein metabolism in fishis still unclear. The results of the present study, however, suggested thatsupplementary amino acids were still available to the fish until at least 26 h afterfeeding based on routine feeding, once daily.In contrast to histidine, lysine, and threonine, the levels of arginine andmethionine in the muscle of fish fed diet 4 did not markedly differ from those in fishfed diet 3 or the other diets. The plasma concentrations of these two amino acids inthe fish that were fed diet 4 increased to higher levels than those in fish fed diet 3(contained similar proportions of ingredients) on day 7 after feeding (firstsampling). The concentrations decreased to levels similar to those in fish fed diet 3at 36 h after feeding. Thus, the concentrations of both arginine and methionine inthe circulation of fish fed diet 4 likely met the requirements and consequently noexcess accumulated in the muscle pooi.The high concentrations of free amino acids, particularly non-essential aminoacids, observed in muscle relative to plasma in the present experiment agree withthe results found in channel catfish (Wilson and Poe, 1974), rainbow trout (Kaushikand Luquet, 1977a; Kaushik and Luquet, 1979), carp (Ogata, 1986), Atlantic cod(Lyndon et a!., 1993), and Atlantic salmon (Espe et a!., 1993). The concentrations of136Chapter 4most essential amino acids in muscle were found to be correlated with those inplasma (six amino acids out of nine) whereas the relationships between muscle andplasma non-essential amino acids were inconsistent. This suggests conservation ofessential amino acids for protein synthesis, and a variable role of non-essentialamino acids in metabolism.Lower concentrations of branched-chain amino acids in the muscle than inthe plasma suggested that these amino acids are catabolized in the muscle of fish asin mammals. High activity of branched-chain amino acid aminotransferase has beenfound in kidney and red muscle of rainbow trout (Teigland and Klungsøyr, 1983).Branched-chain keto acid dehydrogenase, the enzyme in the second step of theircatabolic pathway, has also been reported in red muscle in the same species of fish(Christiansen and Klungsøyr, 1987). Furthermore, Hughes et a!., (1983) foundbranched-chain amino acid aminotransferase in five tissues of lake trout fingerlings.The activity of the enzyme varied among tissues as follows: posterior kidney>skeletal muscle> gill> liver> anterior kidney.Histidine has been reported in many studies as the most abundant essentialamino acid in the muscle pool of carp and rainbow trout (Kaushik and Luquet,1977a; Medale et al., 1987; Van der Boon et a!., 1989). A similar phenomenon wasfound in the present experiment. Histidine is believed to have an important role asa buffer which relates to the iniidazol group of histidine (Suyama et al., 1986; Vander Boon et a!., 1989). The predominance of histidine in the muscle tissue pool is,however, characteristic of only a few species. Wilson and Poe (1974) did not findeither histidine or a derivative of histidine as the most abundant free essentialamino acid in the muscle of channel catfish. Furthermore, Lyndon et al., (1993)137Chapter 4found that arginine was the most abundant free amino acid in the white muscletissue of Atlantic cod. The presence of glycine as the predominant free amino acidin fish tissue seems to be a common phenomenon. Jüss (1980) suggested, on thebasis of its low molecular weight, that glycine is the most suitable amino acid for useas an osmotic effector.Comparisons between the concentrations of plasma amino acids in fish feddiet 2 in the present experiment (seawater), and Experiment 1 (freshwater) showedthat there was no marked difference in the levels of TAA. The levels of branched-chain amino acids, arginine, lysine, and threonine in fish fed diet 2 in the presentexperiment were higher than those in fish in Experiment 1. Their increases,however, corresponded to the levels supplied in the diet. Plasma amino acidconcentrations in fish did not seem to be affected by salinity of water in the presentexperiment.In considering the above results, no guidance was found to support thehypothesis that free glycine is absorbed at a faster rate than glycine derived fromdigestion of intact protein. Furthermore, excess glycine in fish fed diet 3 may haveexerted a toxic effect. Fish fed the diet supplemented with arginine, histidine,lysine, methionine, and threonine to simulate the concentrations in fish muscleprotein showed higher concentrations of these amino acids in plasma and musclefree pooi. The concentrations of some of these amino acids in the muscle werehigher than those in muscle of fish fed the diet containing similar ingredients until atleast 26 h postprandial indicating that these amino acids were still available in thetissue for protein synthesis at least at the time of feeding on the next day. Lastly, theresults did not suggest any effect of water salinity on plasma amino acidconcentrations in the fish.138CHAPTER 5EXPERIMENT 3PATfERNS AND CONCENTRATIONS OF FREE AMINO ACIDS IN THEPLASMA OF RAINBOW TROUT FED FISH MEAL AS THE PRINCIPALSOURCE OF PROTEIN IN DIETS CONTAINING 6% OR 24% OF LIPID5.1 INTRODUCTIONFish, like other animals, eat to satisfy energy requirements. If diets containinadequate amount of energy part of dietary protein will be potentially utilized as asource of energy (Walton, 1985). Dietary lipid and carbohydrates may to a limitedextent spare protein (Cho, 1985; Cho and Kaushik, 1990). The benificial effects ofthe incorporation of such protein-sparing nutrients have been found in severalspecies of fish (Gropps et al., 1982; Lie et a!., 1988; De Silva et a!., 1991). Theinclusion of dietary lipid at high levels have, however, been found to delay gastricevacuation in other animals (Gitler, 1964; Mclaughlan and Morrison, 1968).Windell et al., (1972) suggested that 15% or higher dietary lipid may reduce gastricmotility in rainbow trout.The results from the previous two experiments showed that plasmaconcentrations of amino acids increased after consumption and digestion of protein,and were correlated with the amino acid concentrations in the dietary protein. Thechanges in the plasma concentrations of different amino acids also indicated thatdifferent proteins were digested at different rates. The patterns of plasma freeamino acids were, therefore, used as indicators of the time required for proteindigestion, and the entrance of free amino acids into the circulation when dietscontained 6 or 24% lipid.139ChapterS5.2 MATERIALS AND METHODS5.2.1 DietsThe compositions of the two diets formulated for this experiment are shownin Table 3.1. Diets 1 and 2 were formulated to contain 6 and 24% lipid,respectively. The additional level of lipid in diet 2 was added at the expense ofground wheat in the formulation. The dietary concentration of protein wasmaintained by increasing the proportion of herring meal. Due to the mechanicalproblems associated with pelleting diets containing high concentrations of oil, partof the herring oil was added to the feed before pelleting and the remainder of theoil was sprayed on after pelleting. The protein concentration in both diets was 40%.522 FishRainbow trout weighing between 400 and 500 g were distributed randomlyinto eight 150 L tanks so that each tank contained five to seven fish. Each of thetwo experimental diets was then assigned randomly to four groups of fish (i.e. fourreplicate groups). The fish were transferred from the pre-experimental diet(EWOS) to the test diets over a period of one week. The feeding of theexperimental diets was then continued for another week. The fish were fed oncedaily to satiation every morning.The study was conducted at the UBC aquarium facilities, and theexperimental conditions were similar to those described for experiments 1.1 and 1.2except water temperature was 15°C and photoperiod was 14 h.140Chapter 552.3 Sampling ProcedureAt the conclusion of the experiment, blood samples were taken from the fishat 6, 12, 24, and 36 h after the last feeding. At each sampling time, two fish fromone replicate tanks on each of the respective diets were caught, and anesthetizedwith 0.01% tricaine-methanesulfonate (MS-222). The procedures for bloodsampling and for treatment of blood and plasma samples were the same asdescribed in the previous experiments except analyses were conducted on bloodsamples from individual fish.141Chapter 5Table 3.1. Ingredient and proximate composition (air-dry basis) of diets used in Experiment 3Ingredient Diet 1 Diet 2g/kg g/kgHerring meal vho1e steam-dried) 44550 495.00Ground wheat 474.50 237.00Herring oil2 10.00 190.00Calcium monophosphate.H0 10.00 18.00Calcium lignosuiphonate 20.00 20.00Premi? 40.00 40.00Total 1000.00 1000.00Calculated analysis4Crude protein (%) 40.00 40.00Ether-extractable lipid (%) 6.00 24.00Ash (%) 7.56 8.65Gross energy 4390 53591Autoclaved at 120°C for 1.5 hours.2Stabed with 0.05% ethoxyquinThe premix supplied the following per kg of diet as fed: thiamin HC1, 67.3 mg; riboflavin, 104.2 mg;niacin, 400 mg; biotin, 5 mg; folic acid, 25 mg; pyridoxine HC1, 60.8 mg; cyanocobalamine, 0.1 mg; Dcalcium pantothenate, 2183 mg; ascorbic acid, 1500 mg; choline chloride, 7680.5 mg; inositol, 2000mg; menadione 30 mg; vitamin A, 10,000 IU; vitamin D3, 1000 IU; vitamin E, 1000 IU; Mg (asMgSO4),380 mg; Mn (as MnSO4.H20),17 mg; Zn (as ZnO), 50 mg; Fe (as FeSO4.7H20),85 mg;Cu (as CuSO4.5H20),2 mg; Co (as CoCI.6H20),0.003 mg; K (asK2S04),895 mg; I (as K103), 5mg; NaC1 (as NaC1), 2830 mg; F (as NaP), 4.5 mg; Se (asNa2SeO.5H0),0.10 mg.values for crude protein, ether-extractable lipid, and ash were calculated using values from NRC(1984). The values for gross energy were estimated by ascribing 5.65 kcal/g crude protein, 9.5kcal/g crude lipid, 4.0 kcal/g carbohydrate (Alexis et al., 1985)142Chapter 55.3 RESULTS5.3.1 Amino Acid Compositions of Experimental DietsThe calculated (NRC, 1984) amino acid composition of two diets is shown inTable 3.2. Although, the proportions of fish meal and ground wheat in the two dietsdiffered slightly, the concentrations of most amino acids were similar, except lysine.Table 3.2. Amino acid composition of experimental diets used in Experiment 31Experimental diet2Amino acid Diet 1 Diet 2g/16g NArg 6.2 6.4His 2.2 2.3lie 4.4 4.4Leu 7.2 7.3Lys 6.9 7.4Met 2.6 2.7ys Li 1.1Phe 4.1 3.9Thr 3.9 4.0Tyr 3.1 3.1Val 5.7 5.9EAA 47.4 48.51 Calculated from values given in NRC handbook (1984).2 1 contained 6% lipid, diet 2 contained 24% lipid (as-fed basis).143Chapter 55.3.2 Diet AcceptanceFish fed the diet containing 6% lipid had poor appetite, whereas those fedthe diet with 24% lipid had excellent appetite. Feed consumption of fish fed the lowlipid diet was about two-thirds that of fish fed 24% lipid diet during the entirefeeding trial and on the sampling day.5.3.3 Plasma Amino Acid ProfileThe plasma free amino acid concentrations expressed as Ihmol/mL and aspercentage of the total plasma free amino acids as determined at different timesafter meal consumption are presented in Tables 3.3-3.4. The concentrations of totalamino acids (TAA), total essential amino acids (EAA), total non-essential aminoacids (NEAA), and ratios of EAA/NEAA in fish fed the two diets are also includedin these tables. The changes in plasma concentrations of total essential amino acids,total non-essential amino acids, and total amino acids following meal consumptionare displayed in Figure 3.1. Alterations in plasma concentrations of individualamino acids at different times postprandial are also presented graphically in Figure3.2. The plasma amino acid concentrations at 24 h postprandial were used torepresent the amino acid concentrations at the time of feeding (0 h).Plasma Total Essential Amino Acids (EAA), Total Non-Essential Amino Acids(NEAA), and Total Amino Acids (TAA)The patterns of plasma EAA concentrations in fish fed the diets containing6% and 24% lipid were similar (Figure 3.la). EAA concentrations peaked between12 and 24 h after feeding. Subsequently, they gradually declined to lower levels at36 h. The concentrations of EAA in fish fed the high lipid diet were consistently144Chapter 5higher than those in fish fed the low lipid diet. Plasma NEAA concentrations in fishfed the two diets did not follow the same pattern. At the time of feeding, plasmaNEAA concentrations in fish fed the diet containing 6% lipid had not declined fromthe last feeding 24 h previously. The level declined at 6 h after which it rose andpeaked at 24 h postprandial. Plasma NEAA concentrations in fish fed the dietcontaining 24% lipid, on the other hand, increased after feeding and peaked as earlyas 6 h and then gradually diminished to the same level as that in fish fed the 6%lipid diet at 36 h postprandial (Figure 3.lb). In the case of TAA, the levels in fishthat were fed the high lipid diet were fairly consistent from the time of feeding until24 h postprandial (Fig 3.lc), whereas the level in fish fed the 6% lipid dietfluctuated and showed a clear peak at 24 h postprandial. Furthermore, the TAA infish fed the 24% lipid diet was higher than that in fish fed the 6% lipid dietthroughout the sampling period. The levels of TAA in both groups of fishapproached similar concentrations by 36 h after feeding.Plasma Concentrations of Individual Amino Acids in Fish Fed Different DietsEssential Amino AcidsAlthough there were similarities in the fluctuations of plasma EAA betweenfish fed high and low lipid content diets, there were considerable differences in thepatterns of some of the individual essential amino acids between fish fed the twodiets. While plasma branched-chain amino acids in fish fed the 6% lipid dietpeaked at 12 h, the concentrations of the same amino acids in fish fed the 24% lipiddiet did not peak until 24 h postprandial (Figures 3.2e-f, and 3.2h). Plasma arginine,lysine, and phenylalanine in fish fed the former diet, on the other hand, peaked at 24145ChapterSh, and in fish fed the latter peaked earlier between 6-12 h (Figures 3.2a, and 3.2c-d).The similar trends were noted for fluctuations in the levels of the remaining plasmaessential amino acids in fish fed the two diets.Non-Essential Amino AcidsAlarilne, glycine, and serine, which contributed the most to the total for nonessential amino acid concentrations in the plasma (Tables 3.3-3.4), were largelyresponsible for the pattern of plasma NEAA in fish fed the two different diets(Figures 3.2j,m,o and 3.lb). The distinction in the fluctuations of these amino acidsbetween the two groups of fish was very pronounced. For example, plasma senileconcentrations in fish fed the 6% lipid diet were lowest at 6 h whereas those for fishfed the 24% lipid diet were highest. At 24 h, the opposite trend was seen. Dietarytreatment did not influence the patterns for the other non-essential amino acids.Differences in the Percent Increases of Amino acids in Fish Fed Two Diets ofDifferent Lipid contentThe differences in the concentrations of plasma amino acids at the peak andat 36 h postprandial in fish fed the two different diets are presented in Figure 3.3.Most plasma amino acids, particularly essential amino acids, in fish fed the 24%lipid diet showed greater increases in concentration compared to those in fish fedthe 6% lipid diet.146Table3.3.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafterfeedingdiet1(6%lipid)inExperiment 3.Non-essentialAminoHoursafter feedingacid6122436EssentialArgCysHislie LeuLysMetPheThrTyrValX±SEMI%2)(±SEM%X±SEM%X±SEM(moi/mL)(mol/mL)(mol/mL)(mol/mL)0.259±0.0226.80.378±0.0017.90.458±0.0158.80.272±0.0130.015±0.0110.40.030±0.0030.60.060±0.019120.028±0.0100.169±0.0034.40.181±0.0073.80.225±0.016430.144±0.0200.248±0.0126.50.286±0.0135.90.230±0.0624.40.090±0.0080.440±0.02111.50.513±0.02610.70.359±0.0846.90.148±0.0120.139±0.0073.60.231±0.0264.80.450±0.1458.60.180±0.0480.164±0.0074.30.192±0.0124.00.160±0.0253.10.078±0.0130.069±0.0121.80.068±0.0071.40.123±0.0362.40.051±0.0210.230±0.0156.00.358±0.0187.40.258±0.0245.00.138±0.0290.025±0.0030.70.012±0.0030.20.058±0.0071.10.046±0.0240.544)±0.01614.10.624±0.03413.00.505±0.1009.70.213±0.020Ala0.209±0.0125.50.285±0.0035.90.475±0.0779.10.327±0.014Asp0.068±0.0061.80.074±0.0071.50.044±0.0110.80.037±0.014Glu0.152±0.0094.00.101±0.0172.10.089±0.0171.70.056±0.016Gly0.539±0.06314.10.739±0.10815.40.975±0.18518.70.768±0.148Pro0.113±0.0003.00.162±0.0053.40.063±0.0121.20.025±0.018Ser0.444±0.00811.60.574±0.00311.90.679±0.01613.00.470±0.036TAA33.823±0.0104.808±0.2425.211±0.2893.071±0.350EAA42.298±0.0652.873±0.1392.886±0.5301.388±0.103NEAA51.525±0.0551.935±0.1032.325±0.2411.683±0.246EAA/NE.AA1.511.481.240.82% 8.9 0.9 4.7 2.9 4.8 5.9 2.5 1.74.5 1.5 6.9 10.6 1.2 1.8 25.0 0.8 1531Thealuesaremeansoftwofishandstandarderrorof themeans(n=2).2of totalaminoacids.aminoacids.4Totalessential aminoacids.Total non-essentialaminoacidsTable3.4Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafterfeedingdiet2(24%lipid)inExperiment 3.EssentialNon-essentialX±SEM1%2(.tmoi/mL)X±SEM%(I.Lmol/mL)X±SEM(tmol/mL)X±SEM(j.Lmol/niL)AminoHoursafterfeedingacid6122436Mg0.314±0.0575.30.468±0.0257.90396±0.0047.00322±0.0499.6Cys0.027±0.0080.50.039±0.0010.70.060±0.0031.10.059±0.0051.8His0.216±0.0093.70.231±0.0223.90.290±0.0355.10.176±0.0075.3lie0242±0.0204.10.264±0.0434.40.317±0.0665.60.082±0.0182.5Leu0.415±0.0377.00.466±0.0767.80.572±0.13810.10.124±0.0283.7Lys0.349±0.0485.90.334±0.0195.60297±0.0185.20253±0.0257.6Met0.234±0.0104.00.299±0.0165.00238±0.0014.20.102±0.0293.0Phe0.142±0.0312.40.160±0.0252.70.095±0.0211.70.065±0.0311.9Thr0.446±0.0287.60.529±0.0988.90.478±0.0728.40.184±0.0125.5Tyr0.089±0.0131.50.054±0.0220.90.057±0.0271.00.012±0.0000.4Val0.565±0.0399.60.620±0.08110.40.764±021213.50205±0.0486.1Ala0.413±0.0937.00.306±0.0305.20.252±0.0134.40.308±0.0699.2Asp0.079±0.0041.30.087±0.0031.50.090±0.0111.60.029±0.0090.9Glu0.281±0.0684.80.122±0.0082.10.174±0.0103.10.120±0.0103.6Giy1.206±0.08220.41.127±0.01819.01.038±0.02518.30.863±0.06825.8Pro0.144±0.0202.40.152±0.0312.60.127±0.0242.20.043±0.00313Ser0.736±0.03012.50.681±0.07211.50.430±0.0947.60399±0.05111.9TAA35.898±0.1565.939±0.5885.675±0.4273346±0.002EAA’13.039±0.1403.464±0.4253.564±0.4711.584±0.046NEAA52.859±02952.475±0.1632.111±0.0441.762±0.047EAA/NEAA1.061.401.690.901Theyaiuesaremeansoftwofishandstandarderrorofthemeans(n=2).2of totalaminoacids.3Totalaminoacids.4Totalessentialaminoacids.Totalnon-essentialaminoacids.(a)Totalessentialaminoacids8 7—8—6%lipid6+24%lipid(U.1 C a) 0400369122436(b)Totalnon-essentialaminoacids(c)Totalaminoacids.37-37CC.26.26iI--IIIIII‘03691224360369122436HoursafterfeedingHoursafterfeedingFigure3.1.Totalconcentrationsofplasmaaminoacids(essential,non-essential,andtotalaminoacids)inrainbowtroutdeterminedatdifferenttimesafterfeedinginExperiment 3.Diet1=6%lipid,diet2=24%lipid.Eachpointrepresentsameanoftwofish(n=2).110.80.6OA 0.2(a)Arginine—6--6%lipid+24%lipid0.80.60.40.23-J E 0 2 C 0 a, C 0 0 C 0 0 a, 2 0. -j 2 0 E C 0 i(b)Histidine691224Lu36-J 2 0 E C 0 L.. C a, 0 C 0 0 a, 2 a, a, 0-J 2 S. 0 2 C 0 .1 C a, 0 C 0 0 a, E a, a, 0.369122436.0 1-0.80.60.40.2 0(d)Phenylalanine3691224Hoursafterfeeding363691224HoursafterfeedingFigure3.2.PlasmaconcentrationsofaminoacidsinrainbowtroutfeddifferentdietsinExperiment 3.Diet 1=6%lipid,diet224%lipid.Eachpointrepresent ameanof twofish(n=2).361E.-.. B.3 C 0 Ca 4- C a, 0 C 0 0 Ca B. a.IBBEUi.3CC0o Ca4-4- ca)a)0oCC0oC)0CaCaB£ 0Figure3.2.(Continued)-J B 0 E C 0 L.. C a) 0 C 0 C.) B 0(e)Isoleucine0.8—s--6%lipid0.6+24%lipid(g) Methionine÷Cystine0.60.40.23691224Hoursafterfeeding360369122436Hoursafterfeeding-J E E C o0.6C0.4C 0 C)-J E 50.8E C o0.6.1-i Cu I-.1 C0.4C 0 0 0) CC 0-J E 0 E C 0.1 cC I C C) C) C 0 C) cC E Cu CC a..-J E 0 E C 0 C a) 0 C 0 0 C) E 0) CC(i)Threonine0.8—H—6%lipid+24%lipid0.6------------E-:+3691224360369121(k)Asparticacid(I)Glutamicacid0.80.60.4--12-0.:HoursafterfeedingHoursafterfeeding2436Figure3.2.(Continued)-j E 0 E C 0 L.. C C, C 0 0 2 0 0 0-j 2 0 2 C 0 I.1 C 0 C) C 0 0 (a E 0, (a 0-J 2 0 E C 0 1 L.. C 0 C 0 C) (a 2.2G0 (a 0-J 2 0 E C 0 Ca C C.) C 0 0 Ca 2 0) 010.80.60.4369122436(n)Proline(p)Tyrosine0.80.60.40.23691224Hoursafterfeeding36Figure3.2.(Continued)3691224Hoursafterfeeding36Chapter 5700600 Diet 1 (6%)El Diet 2 (24%)C;g 500I..4-400‘ 3004-co200__1L__Arg Cys His lie Leu Lys Met Phe Thr Tyr Val Ala Asp Giu Gly Pro SerFigure 3.3. Percentage increases of plasma amino acids at peak time in comparisonwith the concentrations at 36 h after feeding in rainbow trout fed two differentdiets in Experiment 3. Diet 1 = 6% lipid, diet 2 = 24% lipid.154Chapter 55.4 DISCUSSIONIt is well known that animals eat to satisfy their energy need. This has beendemonstrated in fish by several researchers (Cho et a!., 1976; Jobling, 1980; Jobling,1983; Wilson et a!., 1985; Davies, 1989). The results on feed consumption in thepresent experiment, however, did not agree with this general trend since feedconsumption declined when the diet contained only 6% lipid. This was believed tobe due to the low palatability of the diet. It was observed that fish refused to acceptthe 6% lipid diet early in the acclimation period, whereas fish that were fed the 24%lipid diet were eager to consume food within 1-2 days. Beamish and Medland(1986) also observed in their studies, that large rainbow trout (250-500 g) fed dietslow in energy were unable to consume the same amount of food as those fed diets ofhigher energy content. Moreover, the higher level of ground wheat in the 6% lipiddiet in the present experiment may have reduced diet palatability.Inclusion of lipid at high levels (>15% of diet) in fish diets has been shownto improve fish growth by its protein sparing action (Takeuchi et a!., 1978; Beamishand Thomas, 1984). Windell et aL, (1972) stated that a high level of dietary lipidmight reduce gastric motility. This would directly affect the rate of digestibility ofother food components; protein being the most important. The temporal changes inthe levels of plasma essential amino acids in fish fed diet 1 (6% lipid), paralleledthose of fish fed diet 2 (24% lipid) in the present experiment. Thus, the protein inthe two diets was probably digested at similar rate.The constancy of the plasma TAA concentrations, and the consistently higherlevels of plasma EAA concentrations in fish fed the high lipid diet than in fish fedthe low lipid diet possibly reflected the higher food intake in the former group of155ChapterSfish. Although the temporal changes in plasma EAA concentrations in the twogroups of fish were similar, there were differences observed in the patterns ofarginine, lysine, phenylalanine, and branched-chain amino acids. While plasmaarginine, lysine and phenylalanine concentrations in fish fed the 6% lipid diet werevery low at 6 h and did not reach their peaks until 24 h after feeding, the changes inthe concentrations of the same amino acids in fish fed the 24% lipid diet were inreverse order. From this it may be inferred that the amounts and the rate at whichamino acids were absorbed in fish fed the low lipid diet were lower than in fish fedthe high lipid diet. The increase of these amino acids at 6 h in fish that were fed thehigh lipid diet, on the other hand, showed that the availability of these amino acidsfor protein synthesis in the peripheral tissues was greater. Wilson et a!., (1985),however, did not find any differences in the patterns for serum amino acids incatfish fed diets containing dissimilar protein to energy ratios.Branched-chain amino acid (BCAA) metabolism mainly occurs in the muscleof salmonids (Hughes et a!., 1983). Therefore, changes in the concentration ofplasma BCAA will reflect their availability from the digested food for proteinsynthesis. Differences in the pattern of change of this group of amino acids in theplasma of fish fed low and high lipid diets were noted. Both groups of fish exhibitedincreases in plasma BCAA concentrations after feeding. In fish that were fed thelow lipid diet, BCAA concentrations reached their peaks earlier at 12 h, while theconcentrations of these amino acids in fish that were fed the high lipid dietcontinued to increase and reached higher peaks later at 24 h after feeding. Thisagain suggested that the availability of these amino acids to the fish was higher infish fed the high lipid diet.156ChapterSThe different levels of dietary lipid also appeared to affect the metabolism ofother amino acids in the present experiment. The differences in the time ofappearance of alanine, glycine, and serine in the plasma between the two groups offish were very pronounced. Alanine, senile, and glycine have been shown to beincorporated into glucose or glycogen in various fish species (Nagai and Ikeda, 1973;French et al., 1981; Walton and Cowey, 1982; Moon et a!., 1985). The sharpreduction in the plasma levels of these amino acids after 6 h in fish fed the high lipiddiet suggest that they were being catabolized at a faster rate than in fish fed the lowlipid diet. With a higher level of food intake of the same dietary proteinconcentration (40%) than fish fed the 6% lipid diet, fish fed the 24% lipid dietwould have had an adequate supply of energy for metabolism. The carbon skeletonsderived from the transamination of the preceding amino acids probably servedmainly as substrates for glycogenesis or lipogenesis instead of being oxidized forenergy. Higuera et al., (1977) found increased activity of glucose-6-phosphatase, anenzyme converting glucose-6-phosphate to glucose, in rainbow trout fed a high lipiddiet. Furthermore, the reduction in the concentrations of alanine, glycine, andserine at a faster rate than that of essential amino acids in fish fed the high lipid dietin the present experiment suggests that the fish conserved the essential amino acidsfor protein synthesis. This finding supports results of several studies which haveshown that rainbow trout utilize amino acids more efficiently for protein synthesiswhen diets contain high levels of lipid or energy (Atherton and Aitken, 1970; Leeand Putnam, 1973; Higuera et a!., 1977; Gropp et al., 1982).157ChapterSA confounding factor that might have affected the appearance andconcentrations of plasma amino acids in fish fed the 6% lipid diet was the higherproportion of ground wheat in this diet. The higher level of this ingredient with itshigh content of carbohydrate could have reduced protein digestibility and aminoacid absorption. Comparisons of the patterns of change of plasma amino acidconcentrations in fish fed the 6% lipid diet in the present experiment with those forfish that were fed the fish meal based diet in Experiment 1.2 (large fish) showed thatthe patterns were similar. The inclusion of ground wheat at a high level (47% of thediet) in the 6% lipid diet in the present experiment did not seem to have affectedthe digestibifity of the dietary protein.In conclusion, as far as the appearance of amino acids in the plasma of fishwas concerned, a higher concentration of dietary lipid in the present experiment didnot appear to delay protein digestion. The higher levels of plasma amino acids infish fed the high lipid diet than in fish the low lipid diet suggested that the supply ofamino acids in the circulation was adequate for protein synthesis. The rapidremoval of alanine, glycine, and serine (glucogenic amino acids) from the plasma ofthe former group of fish suggests that excess amino acids were converted to otherintermediates in metabolism.158CHAPTER 6EXPERIMENT 4EXAMINATION OF PATFERNS AND CONCENTRATIONS OF FREE AMINOACIDS IN THE PLASMA OF RAINBOW TROUT AS CRITERIA OF AMINOACID AVAILABILITY FROM HERRING MEALS SUBJECTED TO HEATTREATMENT6.1 INTRODUCTIONHigh quality proteins are those that are well digested and contain all theessential amino acids necessary for protein synthesis in the proportions that supportgood growth (Stahman and Woldegiorgis, 1974). A protein source with a wellbalanced amino acid pattern such as fish meal, therefore, does not always providesufficient amino acids for maximum protein synthesis and growth of animals if thedigestibility of that protein is poor. Heat damage during processing of the feedstuffis one of several factors that alters the digestibility of dietary protein. The aminoacid analysis of heat-treated proteins may represent the potential nutritive qualitybut, their solubility and digestibility are usually altered.In order to investigate the effect of heat treatment on digestibility of fishmeal, temporal changes in plasma amino acid concentrations were followed inrainbow trout fed diets in which herring meal had been subjected to heattreatments. To corroborate the data regarding plasma amino acid patterns in fishon the different dietary treatments, the experimental fish meals were also analyzedfor the in vitro pepsin digestibility of their proteins.159Chapter 66.2 MATERIALS AND METHODS6.2.1 Test Protein SourcesA commercial herring meal processed at low temperature (courtesy ofMoore Clarke Co) was used as the control protein source in this experiment. Theproximate analysis of the meal is shown in Table 4.1.Table 4.1. Proximate composition of low-temperature dried herring meal% of dry matterProtein 76.3Ether-extract 10.5Ash 13.1Crude fiber <1.06.2.2 FishRainbow trout used in this experiment ranged in weight from 163.9-199.0 g.They were distributed randomly (11 fish per tank) into sixteen 150 L tanks locatedat the UBC aquarium facility. Each dietary treatment was assigned at random tofour tanks of fish. The fish were fed the experimental diets to satiation once daily at10:00 hour for 16 days. The daily amounts of food consumed were recorded. Thewater supply was dechlorinated Vancouver city water. During the experiment,temperature varied between 8.5 and 9.5°C. Water flow rate was 2 L/min, anddissolved oxygen was 8 ppm. Photoperiod was 14 h.160Chapter 66.2.3 DietsFour diets were formulated. The control diet, diet 1, contained untreatedlow-temperature dried herring meal. Diets 2, 3, and 4 contained low-temperaturedried herring meal that was further heated for different periods of time. The lowtemperature herring meal was weighed out for diets 2, 3, and 4 into the enamel pans(1.5 kg each) and autoclaved at 127°C for 45, 90, and 180 minutes, respectively. Alldiets were formulated to provide 35% protein, and all other ingredients were thesame as shown in Table 4.2.161Chapter 6Table 4.2. Ingredient (air-dry basis) and proximate (dry matter basis) composition of diets used inExperiment 4.Ingredientg/kgHerring meal1 394.00Ground wheat2 300.00Dextrin 140.00Herring 81.00Calcium monophosphate 15.00Calcium lignosulfonate 30.00Premix4 ‘10.00Total 1000.00Proximate analysis5Crude protein (%) 36.5Ether-extractable lipid (%) 13.4Ash (%) 7.01An equal amount of herring meal which was heat-treated at different time intervals was substitutedfor low temperature dried herring meal in the basal dietat 121°C for 1.5 h3Stabilized with 0.05% ethoxyquinpremix supplied the following per kg of diet as fed: thiamin HC1, 67.3 mg; riboflavin, 104.2 mg;niacin, 400 mg; biotin, 5 mg; folic acid, 25 mg; pyridoxine HC1, 60.8 mg; cyanocobalamine, 0.1 mg; Dcalcium pantothenate, 218.3 mg; ascorbic acid, 1500 mg; choline chloride, 4000 mg; inositol, 2000 mg;menadione 30 mg; vitamin A, 10,000 IU; vitamin D3, 1000 IU; vitamin E, 1000 IU; Mg (as MgSO4),380 mg; Mn (as MnSO4.H20),17 mg; Zn (as ZnO), 50 mg; Fe (as FeSO4.7H20),85 mg; Cu (asCuSO4.5H20),2 mg; Co (as CoC1.6H20),0.003 mg; K (asK2S04),895 mg; I (as K103), 5 mg;NaCl (as NaC1), 2836 mg; F (as NaP), 4.5 mg; Sc (asNa2SeO3.5H0),0.10 mg.values were averaged from the analyses of four diets.162Chapter 662.4 Sampling ProcedureFish were bled at 3, 6, 12, 18, and 24 h on day 16 after the morning feeding ofthe experimental diets. At each sampling time (except the last sampling time), fourfish from one of the replicate tanks for diets 1, 2, 3, and 4 were sampled. At the lastsampling time, fish were taken from the first of the respective replicate tankssampled at 3 h. This procedure was followed to minimize the stress associated withsampling since only one of the replicate tanks for each treatment was subjected todisturbance. The fish were anesthetized with 0.01% tricaine-methanesulfonate(MS-222) and weighed. The blood was withdrawn from the caudal vein arterycomplex into 4 mL heparinized vacutainers, and was immediately centrifuged at750xg for 15 mm. The resultant plasma from two fish was pooled, i.e. two plasmasamples/tank. The plasma samples were frozen immediately in liquid nitrogen.The plasma was deproteinized soon after blood collection as described in theprevious experiments. The deproteinized plasma was kept at -70°C for subsequentanalysis for free amino acids.6.2.5 Pepsin Digestibility of Fish MealThe effect of the heat-treatment on digestibility of the fish meal protein wasassessed by the in vitro pepsin digestibility test. The procedure was similar to theAOAC (1984) method except the concentration of pepsin (activity 1:10,000) in thedigestion mixture was reduced to 0.00005% to increase the sensitivity of the test(March and Biely, 1967; Johnston and Coon, 1978; March and Hickling, 1982; Hanand Parsons, 1991; Anderson et a!., 1993). Moreover, the incubated temperaturewas 35°C.163Chapter 6Prior to the pepsin digestibility test, samples (of the fish meal from each heattreatment and the control fish meal were extracted with diethyl ether using theGoldfisch apparatus. The pepsin digestibility test was then performed as follows.Samples of approximately 0.3 g of the defatted fish meals were placed in thedigestion bottles. The digestion bottles and the end-over-end agitator employedwere as described in the AOAC (1984) procedure. A total of 150 niL of 0.00005%pepsin solution1was added to the bottles. The bottles were capped, clamped to theagitator, and incubated with constant agitation at 35°C for 16 h. The digestibilityassay was performed in duplicate with a sample of each fish meal incubated in HC1solution without pepsin to ascertain the amount of protein solubilized in HC1without the action of the enzyme.At the end of the incubation, the insoluble residue in each bottle was filteredand dried (AOAC, 1984). Protein present in the insoluble residue was thendetermined according to the Kjeldahl procedure. The calculation of % indigestibleprotein was based on the ether-extracted sample weight. The results represented %indigestible protein in the sample. The calculation of percent digestibility of fishmeal protein was based on percent crude protein present in the HC1-insolubleresidue.The 0.00005% pepsin solution was prepared as follows: One g of pepsin (activity 1:10,000) wasdissolved in 100 mL 0.075N IIC1. One hundred L of the stock solution was then diluted to 2000 mLwith freshly prepared 0.075N HC1 in a volumetric flask.164Chapter 66.2.6 Amino Acid AnalysisThe determination of plasma amino acid concentrations was conducted asdescribed in the previous experiments.6.2.7 Statistical AnalysisThe data for initial and final weights, weight gain, and feed consumption offish were subjected to one way analysis of variance. The arcsin transformation wasapplied to the percentage values for pepsin digestibility prior to analysis of variance.The analysis was performed using Systat (Wilkinson, 1990).6.3 RESULTS6.3.1 Pepsin Digestibility of Fish MealThe values obtained for the pepsin solubilization of protein present in fishmeal heated at 127°C for different periods of time are given in Table 4.3 and Figure4.1. These data were tested statistically by analysis of variance, and the results weretabulated in Appendix 6.The values for percent digestibility of fish meal protein clearly showed thedamaging effects of heat treatment. As fish meal protein was subjected to heattreatment for increasing periods of time, the percent of pepsin digestibilitysignificantly decreased (P <0.05). When fish meal proteins were incubated with HC1alone, percentages of protein left in the residue of control fish meal and in the fishmeals that were heated for 45 and 90 nun were similar (81.0-81.9% of crudeprotein). The HC1-insoluble protein of fish meal heated for 180 mm was, however,lower (77.2% of crude protein).165Chapter 6Table 4.3. Pepsin digestibffity of protein in low temperature herring meal heated at 127°C fordifferent periods of time in Experiment 4.Duration of heat treatment2 (miii)0 45 90 180% of crude protein not 14.9 23.5 29.2 45.5solubilized by pepsin% of crude protein not 81.4 81.9 81.0 77.2solubilized by HC1 alone% of crude protein not 18.3 28.7 36.0 59.0solubilized by pepsinbased on the HC1-insoluble residue% of crude protein 817a4 71.3 b 64.0 C 41.0 dsolubilized by pepsinbased on the HC1-insoluble residue1 Pepsin concentration = 0.00005% (activity 1:10,000)at 127°C.3flach value is a mean of two pepsin digestibility tests (n = 2).within the same row with the same superscript were not significantly different (Tukey HSDtest, P>0.05).166Chapter 6>a)iFigure 4.1. Pepsin digestibility of protein in fish meal heated for different periods oftime in Experiment 4. Percent digestibility was calculated on the basis of theprotein not solubilized by HC1 alone.60045 mm 90 mm 180 mmDuration of heat treatment167Chapter 6Table 4.4. Body weight gains and feed consumptions of rainbow trout over a 16-day period inExperiment 4.Diet1 Initial Final Weight Feedweight weight gain consumption(g) (g) (g/fish) (g/fish/day)1 192.2 ± 4.82 237.1 ± 6.7 44.9 ± 4.8 3.8 ± 0.352 180.2 ± 6.5 216.8 ± 4.8 36.6 ± 3.1 3.1 ± 0.113 179.6 ± 5.8 212.2 ± 10.4 32.6 ± 5.1 3.1 ± 0.214 193.7 ± 1.4 227.8 ± 5.9 34.1 ± 5.7 3.1 ± 0.281Djet 1 = unheated fish meal (control diet); Diet 2 45 mm-heated fish meal diet; Diet 3 = 90 miii-heated fish meal diet; Diet 4 = 180 mm-heated fish meal diet.2value represents a mean of four tanks, eleven fish/tank, (ii = 4). No significant differenceamong means in the same column was detected.Table 4.5. Specific growth rates, and feed conversion efficiencies for rainbow trout fed different dietsin Experiment 4.Diet1 Specific Growth Rate2 Feed Efficiency3%1 1.31 0.742 1.16 0.743 1.03 0.674 1.01 0.69Pooled SEM 0.135 0.0741 Diet 1 = unheated fish meal (control diet); Diet 2 45 min heated fish meal diet; Diet 3 = 90 mmheated fish meal diet; Diet 4 = 180 miii heated fish meal diet.2= (InW2InW1)*100/(T2T1)= Gain in body weight/feed consumptionvalue represents a mean of four tanks, eleven fish/tank, (n= 4). No significant differenceamong means in the same column was detected.168Chapter 66.3.2 Responses of Fish to Experimental DietsData for initial and final weights, weight gains and feed consumptions of fishfed the different diets are provided in Table 4.4. Values for specific growth ratesand feed efficiencies of the fish are given in Table 4.5. These data were subjected toone-way analysis of variance, and the statistical results are shown in Appendix 7-8.Dietary treatment did not have a significant effect on any of the parametersmentioned above.6.3.3 Plasma Amino Acid ProfileThe free amino acid levels in plasma expressed as mol/mL and aspercentages of the total plasma amino acids of fish as determined at different timespostprandial are shown in Table 4.6-4.9. The levels of total amino acids (TAA),total essential amino acids (EAA), total non-essential amino acids (NEAA), and theratios of EAA/NEAA of fish fed different diets were also calculated and included inthese tables. The fluctuations in plasma concentrations of total essential aminoacids, total non-essential amino acids, and total amino acids are displayed in Figure4.2. Furthermore, the changes in plasma concentrations of individual amino acids atdifferent times postprandial are illustrated in Figure 4.3. Assuming that the plasmaamino acid concentrations at 24 h after meal consumption represented theconcentrations at the time of feeding when the fish were fed once daily, plasmaamino acid concentrations at 24 h were used to represent the concentrations at 0 h(feeding time).169Chapter 6Plasma Total Essential Amino Acid (EAA), Total Non-Essential Amino Acids(NEAA), and Total Amino Acids (TAA)As shown in Figure 4.2a, the fluctuations in plasma concentrations of EAA infish fed diets 1, 2, and 3 did not differ markedly from each other. Theconcentrations increased after feeding and peaked only once, between 6-12 hdepending upon the diets. The pattern for changes of plasma EAA concentrationsin fish fed diet 4 (fish meal heated for 180 mm) differed notably from those of fishfed the other three diets. Plasma EAA concentrations in fish of this group peakedtwice, once at 3 h and again at 18 h postprandial. The magnitude of the fluctuationsof plasma NEAA in fish fed different diets was less than those noted for the EAAconcentrations. With this in mind, differences in the alterations of plasma NEAA infish fed diets 1, 2 and 3, and that in fish fed diet 4 were noticeable. Theconcentrations of NEAA in fish fed the former three diets increased and peakedbetween 6-12 h after feeding where they gradually subsided (Figure 4.2b). In thefish that were fed diet 4, the concentrations increased at 3 h, and then the levelsdeclined, and remained low until 24 h after feeding.Plasma Concentrations of Individual Amino Acids in Fish Fed Different DietsEssential Amino AcidsThe fluctuations of most individual plasma essential amino acids in fish feddiets 1, 2 and 3 followed the overall patterns of EAA in that the levels increasedafter feeding and reached peak values between 6 and 12 h depending upon theamino acid under consideration (Figure 4.3). Some variations from this pattern infish fed these three diets were found with respect to changes in the concentrations ofbranched-chain amino acids. The levels of these amino acids in fish fed diet 1170Chapter 6increased from the time of feeding and stayed at a high level between 6-12 h afterfeeding. The levels of the same group of amino acids in fish fed diet 2, on the otherhand, rose from the time of feeding and reached the peak at 6 h after which time theconcentrations dropped sharply. In the fish that were fed diet 3, the concentrationsof branched-chain amino acids remained low at 3 h after which time they increasedand peaked between 6 and 12 h after feeding. The plasma concentrations ofarginine, phenylalanine, and tyrosine in fish fed diets 1, 2, and 3 fluctuated onlyslightly (Figures 4.3a,d,p).The fluctuations in the plasma concentrations of isoleucine, leucine, lysine,methionine + cystine, and valine in fish fed diet 4 were similar to those of EAA withrespect to the same diet, i.e they peaked twice, once at 3 h and again at 18 h afterfeeding. Plasma concentrations of arginine, histidine, phenylalanine, threonine, andtyrosine, however, showed very small variations (Figures 4.3 a-i).Non-Essential Amino AcidsAs shown in Figures 4.3j,1,n,o, the alterations in the levels of plasma alanine,glutarnic acid, proline, and serine in fish fed diets 1, 2 and 3 were comparable to theoverall pattern of NEAA with respect to the diets, i.e. peak values were foundbetween 6 and 12 h after feeding, and then the levels gradually subsided. Plasmaglycine concentrations in fish fed the foregoing three diets, nevertheless, behaveddifferently from each other over the sampling period. The responses of plasma nonessential amino acids in fish fed diet 4 were interesting. While the changes inplasma alanine and serine concentrations showed two peaks, one at 3 h and theother at 18 h, resembling those for EAA, plasma glycine concentrations fluctuated171Chapter 6from time to time during the sampling period. Plasma concentrations of asparticacid, glutamic acid, and proline remained low and invariable. Moreover, the datafrom Tables 4.6-4.9 indicated that levels of most non-essential amino acids,particularly glutamic acid, in fish fed diet 4 were notably lower than in fish fed theother three diets.172Table4.6.Concentrationsof plasmaaminoacidsinrainbowtroutatdifferenttimesafter feedingdiet1(control)inExperiment4.EssentialNon-essentialX±SEM1%2(.tmol/mL)X±SEM%X±SEM%X±SEMAminoHoursafter feedingacid36121824%X±SEM(I.Lmol/mL)(iLmol/mL)(jmol/mL)Mg0.207±0.0114.90.159±0.0032.70.186±0.0083.70.189±0.0074.7Cys0.018±0.0010.40.035±0.0090.60.044±0.0270.90.017±0.0010.4His0.227±0.0285.40.349±0.0365.90.272±0.0315.40255±0.0116.4lie0.203±0.0034.80.397±0.1056.70.360±0.144720.198±0.0055.0Leu0.356±0.0098.50.694±0.18711.80.666±0.24S13.30.375±0.0019.4Lys0.338±0.0328.00.356±0.0296.10.252±0.0685.00.192±0.0134.8Met0.128±0.0133.00.217±0.0103.70.175±0.0343.50.162±0.0034.1Phe0.137±0.0093.30.119±0.0032.00.167±0.051330.156±0.0303.9Thr0.244±0.0225.80.339±0.0025.80.275±0.1235.50252±0.0626.3Tyr0.080±0.0041.90.110±0.0081.90.087±0.0161.80.070±0.0061.8Val0.447±0.01210.60.888±0.20615.10.812±0.27516.20.529±0.00513.2Ala0.435±0.01510.30.469±0.0178.00.461±0.1309.20.356±0.0278.9Asp0.044±0.0041.10.042±0.0010.70.041±0.0150.80.044±0.0061.1Glu0.083±0.0222.00.149±0.0132.50.134±0.0352.70.0%±0.0072.4Gly0.681±0.10016.20.770±0.18113.10.518±0.00910.30.656±0.01316.4Pro0.101±0.0002.40.117±0.0282.00.112±0.0562.20.072±0.0191.8trSet0.475±0.05111.30.667±0.01011.50.444±0.0718.90375±0.0509.4TAA44.203±0.3465.877±0.5895.007±1.3423.995±0.013EAA52.385±0.1043.663±0.7783.297±1.0262395±0.017NEAA61.819±0.2432.214±0.1891.710±0.3161.600±0.005EAA/NEAA1311.651.931.501.144niol/mL)0.170±0.004tr3 0.205±0.0050.088±0.0170.159±0.0300.133±0.0200.093±0.0080.169±0.0030.129±0.0300.057±0.0010.260±0.0310.300±0.0680.035±0.0020.088±0.0110.523±0.017% 62 7.5 3.2 5.8 4.8 3.4 62 4.7 2.1 9.5 10.9 13 32 19.10.333±0.06612.12.743±0.3861.462±0.1871.281±0.1991Meanof twopoolsofp1asma{two fish/pool)andstandarderrorofthemean(n=2).2of totalaminoacids.3Presentinaverysmallamount.4rotalaminoacids.Totalessentialaminoacids.6non-essential aminoacids.Table4.7.Concentrationsofplasmaaminoaddsinrainbowtrout atdifferent timesafterfeedingdiet2(45mm-heatedfishmeal)inExperiment 4.Non-essentialAminoHoursafterfeedingacid36121824EssentialArg CysHislie LeuLysMetPheThrTyrValX±SEM1%2X±SEM%X±SEM%X±SEM%X±SEM(j.mo1/mL)(mol/mL)(j.Lmol/mL)4Lmol/mL)(mol/mL)0.261±0.019530.149±0.0022.50.223±0.0294.70.174±0.0024.60.163±0.0080.020±0.0010.40.021±0.0060.40.037±0.0150.70.016±0.0000.40.014±0.0020.246±0.0025.00.296±0.0085.10.315±0.0396.40.209±0.0175.50.265±0.0020.263±0.0035.30.393±0.0716.70242±0.0624.90.182±0.0564.80.184±0.0040.456±0.007920.703±0.12312.00.439±0.1318.90.345±0.1109.00.338±0.0040.342±0.0196.90.352±0.0256.00.226±0.0344.60.175±0.0134.60.161±0.0420.159±0.0043.20.214±0.0213.60.133±0.0302.70.188±0.0044.90.163±0.0170.183±0.0283.70.114±0.0062.00.179±0.0373.60.157±0.0304.10.105±0.0090.245±0.0185.00.297±0.0265.10.264±0.0095.40.241±0.0016.30.239±0.0590.113±0.010230.097±0.0101.70.102±0.0112.10.084±0.016220.073±0.0080.565±0.00611.40.869±0.14314.80.555±0.16211.30.468±0.10212.20.492±0.000Ala0.461±0.0489.30.552±0.0439.40.461±0.0809.30.304±0.0127.90.290±0.037Asp0.054±0.0011.10.058±0.0021.00.055±0.0021.10.033±0.0030.90.034±0.005Gin0.101±0.0092.10.116±0.0032.00.107±0.0012.20.080±0.0112.10.095±0.005Gly0.751±0.00615.20.857±0.03914.60.897±0.20018.20.696±0.0711820.792±0.221Pro0.126±0.0042.50.158±0.0332.70.087±0.0071.80.052±0.0051.40.050±0.003Ser0.592±0.02912.00.618±0.05710.50597±0.13312.10.429±0.05511.20.452±0.058TAA34.938±02305.864±0.8614.919±0.1573.833±0.1233.910±0.662EAA42.853±0.1443.505±0.6242.715±0.2602.239±0.2712.197±0.197NEAA52.085±0.0862.359±0.2372.204±0.4171.594±0.1481.713±0.466EAA/NEAA1.371.491.231.401.28% 4.2 0.4 6.8 4.7 8.7 4.1 4.2 2.7 6.1 1.912.6 7.4 0.9 2.4 20.3 1.3 11.61Meanoftwopoolsofplasma(two fish/pool) andstandarderrorofthemean(n=2).2oftotalaminoacids.3Totalaminoacids.4Totalessential aminoacids.‘Totalnon-essential aminoacids.Table4.8.Concentrationsofplasmaaminoacidsinrainbowtroutatdifferenttimesafter feedingdiet3(90mm-heatedfishmeal)inExperiment4.Non-essentialEssentialAminoHoursafterfeedingacid36121824%X±SEM%X±SEM%X±SEM(ILmol/mL)(mol/mL)(mol/mL)X±SEM1%2X±SEM(mol/mL)(pmol/mL)Arg0.223±0.0095.70.197±0.0073.70.194±0.0143.70.170±0.0124.40.146±0.008Cys0.024±0.0050.60.038±0.0000.70.034±0.0060.70.022±0.0010.60.018±0.000His0.193±0.0164.90.283±0.0095.20.286±0.020550.235±0.0036.10.225±0.013lie0.175±0.0354.50324±0.0106.00.355±0.0396.80.174±0.0304.50.192±0.007Leu0.315±0.0688.10581±0.02410.80.647±0.06612.30.316±0.0568.20.353±0.013Lys0.211±0.0295.40.347±0.0076.40.271±0.062520.199±0.0035.20.180±0.005Met0.131±0.0093.30.208±0.0093.90.180±0.0463.40.180±0.0264.70.148±0.008Phe0201±0.0045.10.149±0.0072.80.146±0.0122.80.177±0.0184.60.084±0.012Thr0.144±0.0323.70.294±0.030550.281±0.0185.40.206±0.0265.40.224±0.009Tyr0.097±0.0012.50.106±0.0112.00.111±0.0222.10.130±0.0493.40.058±0.041Val0.402±0.08310.30.730±0.03913.50.771±0.06614.70.428±0.03711.20.497±0.036Ala0393±0.03610.10.501±0.031930.432±0.0768.20315±0.0178.20.339±0.024Asp0.033±0.0010.90.050±0.0030.90.037±0.0100.70.044±0.0021.20.044±0.003Glu0.071±0.0021.80.108±0.0072.00.102±0.0112.00.088±0.0052.30.099±0.005Gly0.778±0.08919.90.741±0.00213.70.686±0.02813.10.642±0.05216.70.580±0.078Pro0.053±0.0091.40.144±0.0012.70.130±0.046250.079±0.0032.10.053±0.002Ser0.461±0.02511.80.596±0.06511.00.583±0.04811.10.428±0.02311.20.339±0.004TAA33.905±0.3625397±0.1185.246±0.3983.833±0.1443.579±0.162EAA42.116±03923.257±0.6343.276±0.2782.237±0.2462.125±0.003NEAA51.789±0.0342.140±0.0171.970±0.1201596±0.1021.454±0.160EAA/NEAA1.181521.661.401.46% 4.1 0.5 63 5.4 9.9 5.0 4.1 2.4 63 1.6 13.9 9.5 12 2.816.2 1.5 951Meanof twopoolsofpiasma(two fish/pool)andstandarderrorofthemean(n=2).2oftotalaminoacids.3Totalaminoacids.4Totalessential aminoacids.‘Totalnon-essential aminoacids.Table4.9.Concentrationsofplasmaaminoacidsinrainbowtrout atdifferenttimesafter feedingdiet4(180 mm-heatedfishmeal)inExperiment 4.Non-essentialtMeanoftwopoolsof5plasma(two fish/pool)andstandarderrorofthemean(n=2).essentialaminoacids.Total non-essentialaminoacids.EssentialAminoHoursafterfeedingacid36121824X±SEM1%2X±SEM%X±SEM%X±SEM%X±SEM(mol/mL)(Lmol/mL)(.mo1/mL)(#mol/mL)(mol/mL)Arg0.212±0.0044.40.168±0.0023.80215±0.0136.10.197±0.0054.20.139±0.002Cys0.075±0.0301.60.036±0.0020.80.051±0.0291.50.041±0.0060.90.057±0.001His0.241±0.0065.00.253±0.0055.70223±0.0036.30.244±0.019520.204±0.018lIe0.270±0.0225.60.274±0.0356.10.139±0.0023.90.300±0.0436.40.167±0.024Len0.466±0.0399.70.482±0.05710.80233±0.0046.60.544±0.08411.60.295±0.043Lys0.306±0.0016.40.333±0.0247.50205±0.0165.80.268±0.0095.70.250±0.070Met0.180±0.0173.70.152±0.0203.40.119±0.0383.40.219±0.0174.70.155±0.004Phe0.143±0.0043.00.110±0.0032.50.139±0.0043.90.136±0.0032.90.118±0.023Thr0.240±0.0535.00.199±0.0174.50.154±0.0054.30.224±0.0194.80.221±0.039Tyr0.116±0.0162.40.093±0.0172.10.119±0.0153.30.113±0.0272.40.076±0.005Val0.583±0.03212.10.594±0.05313.30326±0.0069.20.684±0.08914.60.417±0.044Ala0.451±0.0019.40.425±0.0639.50318±0.1059.00.434±0.0189.20340±0.087Asp0.035±0.0060.70.045±0.0001.00.037±0.0041.10.038±0.0010.80.051±0.004Glu0.080±0.0081.70.078±0.0081.80.067±0.0011.90.083±0.0111.80.086±0.030Gly0.726±0.01615.10.634±0.06414.20.666±0.04218.80.584±0.04012.50.634±0.117Pro0.123±0.0152.60.100±0.0222.20.079±0.0062.20.100±0.0272.10.111±0.028Ser0.555±0.00311.60.485±0.08010.90.457±0.02612.90.485±0.01010.30397±0.061TAA34.802±0.2624.461±0.6703.547±0.0094.694±0.2763.718±0.776EAA42.832±0.1962.694±0.3331.923±0.0012.970±0.2502.099±0314NEAA51.970±0.0661.767±0.3371.624±0.0081.724±0.0271.619±0.463EAA/NEAA1.441.521.181.72130% 3.7 1.5 5.5 4.5 7.9 6.7 4.2 3.2 5.9 2.0 11.2 9.1 1.42.3 17.0 3.0 10.72of totalaminoacids.3Totaianilnoacids.4Total-J E 0 0 Ca I G) C.) C 0 0 E a.C-JE£ 0E6ECCo0CaCaCCa) o0CCo0 0CaCaEECa CaCa a.IIIIIU36912182436HoursafterfeedingFigure4.2.Totalconcentrationsofplasmaaminoacids(essential,non-essential,andtotalaminoacids)inrainbowtrout determinedatdifferenttimesafterfeedingdietscontainingherringmealheatedfordifferent periodsoftimeinExperiment4.Diet1=control,diet2=45mm-heatedfishmeal,diet3=90mm-heatedfishmeal,diet4=180mm-heatedfishmeal.Eachpointsrepresentsameanoftwopoolsof plasma,twofish/pool,(n=2).36912182436(b)Totalnon-essentialaminoacidsII—36912182436Hoursafterfeeding11Figure4.3.Plasmaconcentrationsofaminoacidsinrainbowtroutfeddietscontainingherringmealheatedfordifferent periodsoftimeinExperiment 4.Diet1=control, diet2=45mmheatedfishmeal,diet3=90mmheatedfishmeal,diet4=180mmheatedfishmeal.Eachpoint representsameanof twopoolsofplasma,twofish/pool,(n=2).0.80.60.40.2(a)Arginine—s—diet1-I-diet2-*-diet3—&diet4-J 0 E C 0 4- La C a) 0 C 0 0 C-0.80.60.40.2(b)Histidine‘S-J E..-. 0 C 0 Lu I. 4- C a) C.) C 0 0 Lu E 0 C--J E 0 E C 0 I3 6912243636912182436(d)Phenylalanine30.8E . 10.6 0.4C 0 036912182436Hoursafterfeeding369121824Hoursafterfeeding3622EE 0EE33 C.2°—a.CC00C.)C)CCo0o0CuCuEE0)033EE.-.•3EE33CC.20.6.2 4-CuCu—4-CC040 0CCo0o0o2CO•Eco0CO0II36912182436Hoursafterfeeding36912182436(g)Methionine+Cystine36912182436HoursafterfeedingFigure4.3.(Continued)Figure4.3.(Continued)-J E 0.8E o0.6I0.4C 0 0 0) 0..36(i)Threonine—fe—diet1+diet2-*-diet3--diet40IIII 36-J E 0.8E $0.6 0.4C 0 00.29121824-J E 0 C 0 4-. I C a) 0 C 0 C.) E U) Ce 0 E0.8.20.6:i C)0.4 C 0 C) E0.2 C’) 0..3691218(k)Asparticacid2436(I)Glutamicacid‘I, Ce a-3691224Hoursafterfeeding36369121824Hoursafterfeeding361-0.80.60.40.2-J E 0 E C 0 Ca I a, C) C 0 0 Ca 0 -J E 0 E C 0 I- •1 C a) 0 C 0 C) E 0, Ca(n)Proline3691218240036-j E 0 E C 0 L.. C 0 C 0 0 Ca E 0 Ca-J E 0 E C 0 I- 4- C a) C) C 0 C, Ca 0 Ca 036912182436(p)Tyrosine1-0.80.60.40.236912182436Hoursafterfeeding369121824Hoursafterfeeding36Figure4.3.(Continued)Chapter 6Percentage Changes in Plasma Essential Amino Acid Concentrations in Fish FedDifferent DietsThe maximum levels of individual essential amino acids in the plasma of fishfed the different diets were taken from Tables 4.6-4.9, and listed in Table 4.10. Thepercentage changes in plasma essential amino acids in fish fed the different diets ascompared to those in fish fed the control diet were then calculated. The results arepresented in Table 4.10, and Figure 4.4. It was found that the levels of most plasmaessential amino acids in fish fed diets 2, 3, and 4 that contained fish meal subjectedto heat treatment were lower than those in fish fed the control meal. The extent towhich they differed from the control fish varied with the periods of heating. In mostcases, the longer the heating period the lower the peak concentration reached aftermeal consumption. The greatest negative percentage changes of most essentialamino acids occurred in fish fed diet 4 (the most severe heat-treatment). Theplasma concentrations of histidine, threonine, branched-chain amino acids, andphenylalanine in fish fed this diet were remarkably lower than those in the fish fedthe control diet. The largest relative change occurred in the plasma level ofthreonine, and second largest in the level of bistidine. Plasma methionine andcystine concentrations in fish fed diet 4, however, did not seem to be depressed.182Chapter 6Table 4.10. Comparisons of peak concentrations of plasma essential amino acids in fish fed diets1containing fish meal heated for different periods of time.Amino Diet 1 Diet 2 Diet 3 Diet 4 (BA)*10O (C.A)*100 (D.A)*100acid (A) (B) (C) (D) A A Aj.mo1/mL % % %Arg 0.207(3h) 0261(3h) 0.223(3h) 0.215(12h) 26.1 7.7 3.9Cys 0.044(12h) 0.037(12h) 0.038(6h) 0.057(24h) -15.9 -13.6 29.5His 0.349(6h) 0.315(12h) 0.286(12h) 0.253(6h) -9.7 -18.1 -27.5lie 0.397(6h) 0.393(6h) 0.355(12h) 0.300(18h) -1.0 -10.6 -24.4Leu 0.694(6h) 0.703(6h) 0.647(12h) 0.544(18h) 1.3 -6.8 -21.6Lys 0.356(6h) 0.352(6h) 0.347(6h) 0333(6h) -1.1 -2.5-6.5Met 0.217(6h) 0.214(6h) 0.208(6h) 0.219(18h) -1.4 -4.1 0.9Phe 0.167(12) 0.183(3h) 0.201(3h) 0.143(3h) 9.6 20.4 -14.4Thr 0.339(6h) 0.297(6h) 0.294(6h) 0.240(3h) -12.4 -13.3 -29.2Tyr 0.110(6h) 0.113(3h) 0.130(18h) 0.119(12h) 2.7 18.2 8.2Val 0.888(6h) 0.869(6h) 0.771(12h) 0.684(18h) -2.1 -13.2 -23.01 = unheated fish meal (control); diet 2 = 45 mm-heated fish meal; diet 3 = 90 mm-heatedfish meal; diet 4 = 180 mm-heated fish meal.2Mm plasma concentrations of different amino acids and the sampling time (in parenthesis) atwhich the peak was observed.183Chapter 6-10-20-30-40Amino acidsFigure 4.4. Percentage changes in plasma amino acid concentrations in rainbowtrout fed heated fish meal diets relative to the control (diet 1) in Experiment 4.Diet 2 = 45 mm-heated fish meal, diet 3 = 90 mm-heated fish meal, diet 4 =180 mm-heated fish meal.4030.; 20C,o 10C0 flJDiet 2Diet 3Diet 4II I I I I IArg Cys His lie Leu Lys Met Phe Thr Tyr Val184Chapter 66.4 DISCUSSIONAlthough heat processing may cause little or no change in the amino acidcomposition of a food protein, it may profoundly influence the course of itsdigestion (Ford and Salter, 1966). The steric hindrance of the structure of proteinscaused by the peptide cross-linkages formed during heating prevents the access ofproteolytic enzymes, and this may cause marked changes in the susceptibifities ofdifferent amino acids to enzymic liberation (Ford, 1973). Consequently, the rateand proportion at which different amino acids are released from protein during thecourse of digestion will be different. Effects of heat treatment on the patterns ofchange of plasma amino acids in fish fed experimental diets in the presentexperiment were observed. Although, most plasma free amino acids in fish fed diets1 (control), 2 (45 mm-heated fish meal), and 3 (90 mn-heated fish meal) exhibitedsimilar patterns of change, some differences were found in the plasmaconcentrations of branched-chain amino acids. These amino acids in fish fed diet 1increased and reached their peaks at 6 h at which time they plateaued or graduallydeclined. Those in fish fed diet 2 sharply rose to their highest level at the same timeas above, but suddenly subsided. The levels in fish fed diet 3 showed a lag periodbetween the time of feeding and 3 h, after which they increased and attained theirpeak values at 12 h after feeding. The dissimilarity in the time of appearance in theplasma of these amino acids was attributed to the difference in the rate of digestionand absorption of these amino acids in fish. In contrast to the pattern of plasmaamino acids observed with fish fed diets 1, 2, and 3 which peaked only once, those infish fed diet 4 (contained the most severely heated fish meal) exhibited two peaks,one at 3 h and the other at 18 h. This pattern of plasma amino acids possibly185Chapter 6reflected the damaging effects of heat treatment on the digestibility of proteinpresent in the over heated fish meal used in diet 4. The denaturation of protein byheat treatment may accelerate the solubility and digestion of some proteins in fishmeal causing the rise of plasma amino acid concentrations at 3 h after feeding. Onthe other hand, peptide cross-linkages, which form as a result of heat treatment, areresistant to hydrolysis by the proteases of the gut and release of free amino acids forabsorption is slower. As a consequence, the faster rate of uptake by the tissues,compared with the absorption rate from the gut, likely accounted for the decline inthe concentrations of most amino acids in fish fed diet 4 at 12 h after feeding.When the maximum levels of each of the essential amino acids in fish fed thedifferent diets were listed together with their peak time in Table 4.10, it was foundthat the degree of synchronization of essential amino acids varied with dietarytreatment. Most plasma essential amino acids in fish fed diet 1 peaked at 6 and 12h, whereas those in fish fed diet 2 peaked at 3, 6 and 12 h. Peak times for fish feddiet 3 were 3, 6, 12, and 18 h, and those for fish fed diet 4 were 3, 6, 12, 18, and 24 hpost-feeding. The foregoing findings provide evidence that different amino acids inthe heated-damaged protein were released at widely different rates during thedigestive process (Ford, 1973).The percentage changes in plasma amino acid concentrations in fish fed thediets containing over heated fish meals compared to those in fish fed the diet withthe control meal revealed, further, that the maximum levels of most plasma aminoacids in fish fed diets containing heated fish meal were lower (Figure 4.4).Moreover, the degree of the reduction in the plasma levels of amino acids clearlydepended on the heating time. These results suggested that the availabifity of186Chapter 6amino acids to the fish was depressed when the fish were fed diets containing heatedfish meal. In accordance with this conclusion, Anderson et at., (1992) found thatavailabffities of amino acids to Atlantic salmon smolts from low temperature fishmeal were significantly higher than from steam-dried fish meal. Growth of juvenilechinook salmon has also been shown to be depressed when the fish were fed a dietcontaining herring meal processed at 120-150°C (McCallum, 1985; McCallum andHiggs, 1989).Among plasma amino acids in fish fed diets containing heated fish meal, theamino acids that showed the largest negative changes as compared to those in fishfed the control diet were threonine and histidine. Indeed, these amino acidsappeared to be the most adversely affected by the heat treatments. This resultdisagrees with the general finding that lysine and the sulfur amino acids are themost vulnerable amino acids in fish meal protein during processing or storage(Waibel and Carpenter, 1972; El-Lakany and March, 1974; Opstvedt et a!., 1984;Plakas et a!., 1985; Plakas et a!., 1988). The reduction in the level of plasmathreonine in fish fed heated fish meal diets in the present experiment, however,supports the chemical score of fish meal protein (Tacon and Jackson, 1985). Taconand Jackson (1985) calculated chemical scores based on mean essential amino acidrequirements of rainbow trout and carp, and listed threonine as the first limitingamino acid in herring meal. The result relating to decrease of plasma histidine wassimilar to those noted for chickens. Smith and Scott (1965), for instance, found thatchicks fed 2 h heated fish meal showed the greatest reduction in plasma histidine ascompared to those fed unheated fish meal. Bjarnason and Carpenter (1970) studiedthe chemical changes in pure protein, and found that at high temperature (145°C)187Chapter 6histidine, serine, and threonine were prone to destruction as well. Although, lysineis most prone to damage by heat treatment, the availability of this amino acid in theplasma in relation to the requirement of the fish was stifi better than that ofthreomne and histidine in the present experiment.There were noticeable effects of heat treatment on reduction of plasmaglutamic acid levels in fish fed diet 4. The concentrations of this amino acid in fishfed diet 4 were consistently the lowest. The possible causes of the lowconcentrations of glutamic acid may have been decreased digestibility due to (1) anester link between the carboxyl group of glutamic acid and hydroxyl group of ahydroxy-amino acid (threonine and serine), or (2) an imide link between glutamicacid and glutamine or asparagine (Ford, 1973).The above results on the plasma amino acid profiles supported the in vitropepsin digestibility results for the fish meals. Graded reductions of proteindigestibility in herring meal subjected to heat treatments for 45, 90, and 180 mm inthe present experiment were observed. These findings were in accord with thosefound by March and Hickling (1982). Moreover, Miyazono and Inoue (1989)reported high correlations between pepsin digestibility values of fish mealincorporated into diets for rainbow trout and growth rate and feed efficiency values.This trend appeared similarly in the present experiment. Fish fed diets containingherring meal with a low percentage of pepsin-digestible protein, in comparison withthose fed the control fish meal, showed depressed growth rate and feed efficiency.The depression was not, however, statistically significant, because of the variabilityamong replicate groups.188Chapter 6The magnitude and patterns of change of plasma amino acids in fish fed theexperimental diets in the present study suggested that the biological availability ofamino acids to the fish varied inversely with the severity of heating of the fish meal.The least available amino acids to the fish appeared to be threonine and histidine.Although, this finding was contradictory to the results found in vitro by otherinvestigators, it may indicate the true availability of amino acids in relation to therequirements of the fish.189CHAPTER 7CONCLUSIONSAn attempt was made in this thesis to investigate the changes in the patternsof plasma amino acids in rainbow trout in relation to dietary effects, feedingregimen, and processing conditions for the protein sources used in fish diets. Theresults of the studies provided a better understanding of protein nutrition in fish.The results of growth studies in Experiment 1.1 demonstrated that fish fed adiet containing fish meal as the principal protein source had growth rates superior tothose of fish fed diets containing a mixture of fish meal, soybean proteinconcentrate, corn gluten meal, and gelatin, either supplemented or unsupplementedwith free lysine, methionine, and tryptophan. Growth rates of the fish fed the testdiets were not improved when feeding frequency was increased from once to fivetimes daily. Although statistically insignificant, the results of Experiment 1.2demonstrated that the growth rate of fish tended to improve when the diet was alsosupplemented with isoleucine. The inferior growth rates of fish fed experimentaldiets in comparison with those fed the control may have been due to amino acidimbalances in the diets used in Experiment 1.1. Comparison between the aminoacid composition of diets and the requirement values indicated that the levels of allessential amino acids in diet 3 (supplemented with lysine, methionine, andtryptophan) were equivalent to or above the requirement values. Thisdemonstrated that chemical analyses can only be used as guide when formulating adiet for fish. The true availability of amino acids to the fish for maximum growthdepends on both the amino acid balance of the diet and the digestibility of theprotein sources.190ConclusionsFeeding fish five times per day in the present experiment, nevertheless,resulted in better utilization of absorbed amino acids for protein synthesis. Carcasscomposition of fish in this group, particularly of fish that were fed a dietsupplemented with free essential amino acids, contained higher concentrations ofprotein and lower lipid than fish fed once daily. In comparison with fish that werefed once daily to satiation, fish fed five times daily showed lower but more constantconcentrations of amino acids in the plasma. According to the substraterelationship of amino acids and catabolizing enzymes, the rate at which plasmaamino acids were catabolized in fish that were fed five times daily was lower than infish that were fed once daily. Amino acids in the free pooi were utilized moreefficiently in the former group of fish for protein synthesis than in the latter group.The effect of frequent feeding on body composition was greater than the effect onover all body weight gain.When the fish were fed a diet containing free glycine in Experiment 2,plasma concentrations of glycine started to rise and peaked later than in fish fed adiet containing gelatin (a rich source of glycine). This finding was contradictory tothe expectations, and showed that amino acids supplemented in free form do notalways cause an immediate increase in plasma concentrations.The results of Experiments 1 and 2 also showed that the peak plasmaconcentrations of specific amino acids in trout following ingestion of dietssupplemented with amino acids in the free form were as much as double those offish fed diets containing similar amounts of the amino acids but derived solely fromintact protein. This finding is similar to those of other studies in fish and otheranimals. Clearance of the higher concentrations of amino acids in the plasma of fish191Conclusionsfed the diets supplemented with free amino acids was not completed until 24 to 36 hafter feeding. Some of the amino acids that were in excess (histidine, lysine, andthreonine) had accumulated in the muscle tissue pooi at 26 and 36 h after feeding inExperiment 2. This finding suggests that the amino acids are still available to fishwhen they are fed a diet supplemented with free essential amino acids to satiationonce daily. Although amino acid toxicity has often been reported with regard toessential amino acids, the present results suggested that excess glycine may also betoxic. Elevated concentrations of glycine in both plasma and muscle pools wereaccompanied by reductions in feed intake. Non-essential amino acids are frequentlyused as substitutes for intact protein when formulating isonitrogenous diets. Theabove finding suggests that caution should be exercised when glycine is used for thispurpose, although the mechanism of the observed toxicity is unknown.The peaks of plasma arginine, alanine, histidine and lysine concentrations at36 h postprandial in fish fed diets containing similar ingredients in Experiment 1.2indicates that the rates of digestion of proteins from some sources may be slowerthan from others. Apart from the amino acid proffle of the diet, this factor shouldbe borne in mind when feeding fish diets containing different protein sources.A high level of dietary lipid (24%) did not alter the rate of absorption ofessential amino acids into the plasma. There was, however, a considerabledifference in the pattern of plasma non-essential amino acid concentrations. Therapid reduction in the concentrations of plasma non-essential amino acids afterfeeding fish the high lipid diet provides evidence of conversion of these amino acidsto intermediates in gluconeogenesis and lipogenesis.192ConclusionsWhen fish were fed diets containing fish meal that had been subjected toheat treatment, plasma amino acid profiles and in vitro pepsin digestibility valuesindicated that the longer the duration of heating, the lower the protein digestibility.Moreover, the plasma amino acid profiles in fish fed the diet containing fish mealwhich was heated for the longest time (180 mm) indicated slow release of aminoacids from dietary protein. The least available amino acids to the fish appeared tobe threonine and histidine. The plasma amino acid profile appears to be a goodindicator, and can thus be used for assessment of protein quality in productssubjected to heating during manufacture.The changes in the plasma concentrations of branched-chain amino acids inevery experiment were noteworthy in their resemblance to one another. Elevatedconcentrations of plasma leucine and valine as a result of addition of isoleucine inthe diet were also observed which might indicate an antagonistic effect of isoleucineon degradative enzymes. Since these amino acids share the same enzymes in thefirst two steps of the degradation, the increase in the concentration of any one of thebranched-chain amino acids can be mutually competitive to the others. Themechanism of inhibitory effects was not, however, explored in the present studies.Higher concentrations of plasma taurine in fish fed a diet supplemented withmethionine than in fish fed the basal diet were observed. In addition, theconcentrations of taurine increased when methionine concentrations in the plasmadecreased. This observation demonstrated the existence of degradation pathwaysfrom methionine to taurine in rainbow trout.A very prominent phenomenon that was observed in every experiment wasthe change in plasma non-essential amino acid concentrations. The patterns of193Conclusionschange of these amino acids indicated their roles in intermediary metabolism. Anexample was the relationship among plasma glycine, alanine and serineconcentrations in Experiments 1, 2 and 3. The dramatic increase of serine in theplasma of fish that were fed a diet containing a high concentration of glycine revealsthat serine possibly play an important role as an effective glucogenic amino acid.Further studies in relation to the metabolic role of non-essential amino acids will beuseful for better understanding the metabolism of amino acids, and they will providemore scientifically based information for fish diet formulation.The results of the studies indicate that plasma amino acid concentrations aresensitive to nutritional changes and they can be used to evaluate the proteinnutritive value of formulated diets for fish.194BIBLIOGRAPHYAbel, H., Becker, K., Meske, C., and Friedrich, W., 1984. Possibilities of using heat-treated full-fat soybeans in carp feeding. Aquaculture, 42: 97-108.Adron, 3. W., Blair, A., Cowey, C. B., and Shanks, A. M., 1976. Effects of dietaryenergy levels and dietary energy source on growth, feed conversion and bodycomposition of turbot (Scophthalmus Maximus L.). Aquaculture, 7: 125-132.Alexis, M. N., Papoutsoglou, E. P., and Theochari, V., 1985. Formulation ofpractical diets for rainbow trout (Salmo gairdneri) made by partial orcomplete substitution of fish meal by poultry by-products and certain plantproducts. Aquaculture, 50: 61-73.Anderson, 3. S., Lall, S. P., Anderson, D. M., and Chandrasoma, J., 1992. Apparentand true availability of amino acids from common feed ingredients forAtlantic salmon (Salmo salar) reared in sea water . Aquaculture, 108: 111-124.Anderson, J. S., Lall, S. P., Anderson, D. M., and McNiven, M. A., 1993. Evaluationof protein quality in fish meals by chemical and biological assays.Aquaculture, 115: 305-325.Anderson, T. A., 1988. The effect of feeding frequency on utilization of algalnutrients by marine herbivore, luderrick, Girella tricuspidata (Quoy andGaimard). J. Fish Biol., 32: 911-921.Andrews, J. W., and Page, J. W., 1975. The effects of frequency of feeding on cultureof catfish. Trans. Amer. Fish. Soc., 3 17-321.Aoe, H., Masuda, I., Abe, I., Saito, T., Toyoda, T., and Kitamura, S., 1970. Nutritionof protein in young carp-I. Nutritive value of free amino acids. Bull. Jpn. Soc.Sci. Fish., 36: 407-413.Arai, S., 1981. A purified test diet for coho salmon, Oncorhynchus kisutch, fry. Bull.Jpn. Soc. Sci. Fish., 47: 547-550.Ash, R., 1985. Protein digestion and absorption. In: C. B. Cowey, A. M. Mackie, andJ. G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London,69-93.Association of Official Analytical Chemists (AOAC), 1984. Official Methods ofAnalysis. Association of Official Analytical Chemists, Washington, D.C.Atherton, W. D., and Aitken, A., 1970. Growth, nitrogen metabolism and fatmetabolism in Salmo gairdneri, Rich. Comp. Biochem. Physiol., 36: 719-747.195BibliographyBarash, H., 1984. The influence of the lysine level in the diet on nitrogen excretionand on the concentration of ammonia and free amino acids in the plasma ofrainbow trout (Salmo gairdneri). Nutr. Rep. Tnt., 29: 283-289.Beamish, F. W. H., and Medland, T. E., 1986. Protein sparing effects in largerainbow trout, Salmo gairdneri. Aquaculture, 55:35-42.Beamish, F. W. H., and Thomas, E., 1984. Effects of dietary protein and lipid onnitrogen losses in rainbow trout, Salmo gairdneri. Aquaculture, 41: 359-371.Beckman, 1982. The System 6300 High Performance Amino Acid Analyzer:Instruction Manual. Beckman Instruments Inc., USA.Bender, D. A., 1985. Amino Acid Metabolism. John Wiley & Sons Ltd., GreatBritain, 263Benevenga, N. J., and Steele, R. D., 1984. Adverse effects of excessive consumptionof amino acids. Ann. Rev. Nutr., 4: 157-18 1.Bjarnason, J., and Carpenter, K. J., 1970. Mechanisms of heat damage in proteins. 2.Chemical changes in pure proteins. Br. J. Nutr., 24: 3 13-329.Blasco, 3., Fernandez, J., and Gutierrez, J., 1991. The effects of starvation andfeeding on plasma amino acid levels in carp, Cyprinus carpio., 1758. J. FishBiol., 38: 587-598.Block, K. P., 1989. Interactions amon leucine, isoleucine, and valine with specialreference to the branched-cham amino acid antagonism. In: M. Friedman(Editor), Absorption and Utilization of Amino Acids. CRC Press, Florida,229-244.Bogé G., Rigal, A., and Pérés, G., 1981. Rates of in vivo intestinal absorption ofglycine and glycyiglycine by rainbow trout (Salmo gairdneri R). Comp.Biochem. Physiol., 69A: 455-459.Brett, J. R., and Zala, C. A., 1975. Daily pattern of nitrogen excretion and oxygenconsumption of sockeye salmon (Oncorhychus nerka) under controlledconditions. J. Fish. Res. Board Can., 32: 2479-2486.Carrillo, M., Zanuy, S., and Herrera, E., 1980. Daily rhythms of amino acid levels inthe plasma of goldfish (Carassius auratus). Comp. Biochem. Physiol., 67A:581-586.Chance, R. E., Mertz, E. T., and Halver, J. E., 1964. Isoleucine, leucine, valine andphenylalanine requirements of chinook salmon and interrelationshipbetween isoleucine and leucine for growth. 3. Nutr., 83: 177-185.196BibliographyCho, C. Y., and Kauskik, S. J., 1985. Effects of protein intake on metabolizable andnet energy values of fish diets. In: C. B. Cowey, A. M. Mackie, and J. G. Bell(Editors), Nutrition and Feeding in Fish. Academic Press, London, 95-117.Cho, C. Y., and Kaushik, S. 3., 1990. Nutritional energetics in fish: energy andprotein utilization in rainbow trout (Salmo gairdneri). World Rev. Nutr. Diet.,61: 132-172.Cho, C. Y., Slinger, S. J., and Bayley, H. S., 1976. Influence of level and type ofdietary protein and of level of feeding on feed utilization by rainbow trout. J.Nutr., 106: 1547-1556.Cho, C. Y., Kaushik, S. J., and Woodward, B., 1989. Dietary arginine requirementfor rainbow trout (Salmo gairdneri). FASEB J., 3: A649, 2459.Cho, C. Y., Kaushik, S. J., and Woodward, B., 1992. Dietary arginine requirement ofyoung rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol., 102A:211-216.Choo, P., Smith, T. K., Cho, C. Y., and Ferguson, H. W., 1991. Dietary excesses ofleucine influence growth and body composition of rainbow trout. J. Nutr.,121: 1932-1939.Christensen, H. N., 1964. Free amino acids and peptides in tissues. In: H. N. Munro,and J. B. Allison (Editors), Mammalian Protein Metabolism. AcademicPress, New York, 105-124.Christiansen, D. C., and Klungsøyr, L., 1987. Metabolic utilization of nutrients andthe effects of insulin in fish. Comp. Biochem. Physiol., 88B: 701-7 11.Church, D. C., and Pond, W. G., 1988. Basic Animal Nutrition and Feeding. JohnWiley & Sons. Inc., New York, 472Cohn, C., 1963. Feeding frequency and body composition. Ann. N. Y. Acad. Sci.,110: 395-409.Cohn, C., Joseph, D., Bell, L., and Oler, A., 1963. Feeding frequency and proteinmetabolism. Am. J. Physiol., 205: 71-78.Cowey, C. B., 1979. Protein and amino acid requirements of finfish. In: WorldSymposium on Finfish Nutrition and Fishfeed Technology, J.E. Halver, andK. Tiews (Editors). Heenemann, Hamburg, 3-16.Cowey, C. B., 1980. Protein metabolism in fish. In: P. J. Buttery, and D. B. Lindsey(Editors), Protein Deposition in Animals. Butterworths, London, 271-288.Cowey, C. B., 1992. Nutrition: estimating requirements of rainbow trout.Aquaculture, 100: 177-189.197BibliographyCowey, C. B., and Luquet, P., 1983. Physiological basis of protein requirements offishes. Critical analysis of allowances. In: IVth Tnt. Symp. Protein Metabolismand Nutrition, Ed. INRA Pubi., Clermont-Ferrand (France), 365-384.Cowey, C. B., and Walton, M. J., 1988. Studies on the uptake of(14C) amino acidsderived from both dietary(14C) protein and dietary(14C) amino acids byrainbow trout, Salmo gairdneri Richardson. 3. Fish Biol., 33: 293-305.Cowey, C. B., and Walton, M. J., 1989. Intermediary metabolism. In: I. E. Halver(Editor), Fish Nutrition. Academic Press, San Diego, 260-330.Cowey, C. B., Daisey, K. W., and Parry, G., 1962. Study of amino acids, free or ascomponents of protein, and of some B vitamins in the tissues of the Atlanticsalmon, Salmo salar, during spawning migration. Comp. Biochem. Physiol., 7:29-38.Cowey, C. B., Pope, J. A., Adron, J. W., and Blair, A., 1971. Studies on the nutritionof marine flatfish. Growth of the plaice Pleuronectesplatessa on dietscontaining proteins derived from plants and other sources. Mar. Biol., 10:145-153.Cowey, C. B., Cooke, D. 3., Matty, A. J., and Adron, J. W., 1981. Effects of quantityand quality of dietary protein on certain enzyme activities in rainbow trout. J.Nutr., 111: 336-345.Cowey, C. B., Cho, C. Y., Sivak, J. G., Weerheim, J. A., and Stuart, D. D., 1992.Methionine intake in rainbow trout (Oncorhynchus mykiss), relationship tocataract formation and the metabolism of methionine. J. Nutr., 122: 1154-1163.Dabrowska, H., and Wojno, T., 1977. Studies on the utilization by rainbow trout(Salmo gairdneri Rich.) of feed mixtures containing soya bean meal and anaddition of amino acids. Aquaculture, 10: 297-3 10.Dabrowski, K. R., 1982. Postprandial distribution of free amino acids betweenplasma and erythrocytes of common carp (Cyprinus carpio L.). Comp.Biochem. physiol., 72A: 753-763.Dabrowski, K. R., 1986. Ontogenetical aspects of nutritional requirements in fish.Comp. Biochem. Physiol., 85A: 639-655.Dabrowski, K. R., Poczyczynski, P., Köck, G., and Berker, B., 1989. Effect ofpartially and totally replacing fish meal protein by soybean meal protein ongrowth, food utilization and proteolytic enzyme activities in rainbow trout(Salmo gairdneri). New in vivo test for exocrine pancreatic secretion.Aquaculture, 77: 29-49.198BibliographyDavies, S. J., 1985. The role of dietary fibre in fish nutrition. In: J.F.Muir, andR.J.Roberts (Editors), Recent Advances in Aquaculture. Westview Press,Colorado, 219-249.Davies, S. 3., 1989. Comparative performance of juvenile rainbow trout, Salmogairdneri Richardson,, fed to satiation with simulated ‘standard’ and ‘highenergy’ diet formulations. Aquacult. Fish. Manage., 20: 407-4 16.De Silva, S. S., Gunasekera, R. M., and Shim, K. F., 1991. Interactions of varyingdietary protein and lipid levels in young red tilapia: evidence of proteinsparing. Aquaculture, 95: 305-3 18.Dean, J. C., and Robinette, H. R., 1983. Effect of high-lysine corns and lysinesupplements on growth of fingerling catfish fed practical diets. Prog. Fish-Cult., 45: 225-227.Eggum, B. 0., 1972. The levels of blood amino acids and blood urea as indicators ofprotein quality. In: J. W. G. Porter, and B. A. Rolls (Editors), Proteins inHuman Nutrition. Academic Press, London, 317-327.El-Lakany, S., and March, B. E., 1974. A comparison of chemical changes in freeze-dried herring meals and a lipid-protein model system. J. Sci. Food. Agric., 25:889-897.Espe, M., Lied, E., and Torrissen, K. R., 1993. Changes in plasma and muscle freeamino acids in Atlantic salmon (Salmo salar) during absorption of dietscontaining different amounts of hydrolysed cod muscle protein. Comp.Biochem. Physiol., 105A: 555-562.Fauconneau, B., 1988. Partial substitution of protein by a single amino acid or anorganic acid in rainbow trout diets. Aquaculture, 70: 97-106.Ford, 3. E., 1973. Some effects of processing on nutritive value. In: 3. W. G. Porter,and B. A. Polls (Editors), Proteins in Human Nutrition. Academic Press,London, 5 15-529.Ford, J. E., and Salter, D. N., 1966. Analysis of enzymically digested food proteins bysephadex-gel filtration. Br. J. Nutr., 20: 843-860.Fowler, L. G., 1980. Substitution of soybean and cotton seed products for fish mealin diets fed to chinook and coho salmon. Prog. Fish-Cult., 42: 87-9 1.Fowler, L. G., 1990. Feather meal as a dietary protein source during parr-smolttransformation in fall chinook salmon. Aquaculture, 89: 301-3 14.Fowler, L. G., 1991. Poultry by-product meal as a dietary protein source in fallchinook salmon diets. Aquaculture, 99: 309-321.199BibliographyFowler, L. G., and Burrows, R. E., 1971. The Abernathy salmon diet. Prog. Fish-Cult., 33: 67-75.French, C. J., Monimsen, T. P., and Hachachka, P. W., 1981. Amino acid utifizationin isolated hepatocytes from rainbow trout. Eur. J. Biochem., 113: 311-3 17.Fujita, Y., Yamamoto, T., Rikimaru, T., Ebisawa, H., and Indue, G., 1981. Effect ofquality and quantity of dietary protein on free amino acids in plasma andtissues of adult rats. J. Nutr. Sci. Vitaminol., 27: 129-147.Galibois, 1., Parent, G., and Savoie, L., 1987. Effects of dietary proteins on time-dependent changes in plasma amino acid levels and on hver protein synthesisin rats. J. Nutr., 117: 2027-2035.Gitler, C., 1964. Protein digestion and absorption in nonruminants. In: H. N. Munroand J. B. Allison (Editors), Mammalian Protein Metabolism. AcademicPress, New York, 35-69.Gras, J., Gudefin, Y., Chagny, F., and Perrier, H., 1982. Free amino acids andnirihydrin-positive substances in fish-Il. Cardio-respiratory system: plasma,erythrocytes, heart and gills of the rainbow trout (Salmo gairdneriiRichardson). Comp. Biochem. Physiol., 73B: 845-847.Grayton, B. D., and Beamish, F. W. H., 1977. Effects of feeding frequency on foodintake, growth and body composition of rainbow trout (Salmo gairdneri).Aquadulture, 11: 159-172.Gropp, J., Koops, H., Tiews, K., and Beck, H., 1979. Replacement of fish meal introut feeds by other feedstuffs. In: T. V. R. Pillay, and Wm. Dill (Editors),Advances in Aquaculture. Fishing News Books Ltd., Great Britain, 596-601.Gropp, J., Schwalb-Bühling, A., Koops, H., and Tiews, K., 1982. On the proteinsparing effect of dietary lipids in pellet feeds for rainbow trout (Salmogairdneri). Arch. Fischereiwiss., 33: 79-89.Hagemeister, H., Scholz-Ahrens, K. E., and Schulte-Coerne, H., 1990. Plasma aminoacids and cholesterol following consumption of dietary casein or soy proteinin minipigs. J. Nutr., 120: 1305-13 11.Han, Y., and Parsons, C. M., 1991. Protein and amino acid quality of feather meals.Poult. Sci., 70: 8 12-822.Harding, D. E., Allen, 0. W., JR., and Wilson, R. P., 1977. Sulfur amino acidrequirement of channel catfish : L-methionine and L-cystine. J. Nutr., 107:2031-2035.Hardy, R. W., 1989. Diet preparation. In: J. E. Halver (Editor), Fish Nutrition.Academic Press, California, 476-548.200BibliographyHarper, A. E., 1964. Amino acid toxicities and imbalances. In: H. N. Munro, and J.B. Allison (Editors), Mammalian Protein Metabolism. Academic Press, NewYork, 87-131.Harper, A. E., 1977. Animal models : plasma amino acids and body protein status.In: Symposium on Clinical Nutrition Update: Amino Acids, H. L. Greene, M.A. Holhday, and H. N. Munro (Editors). American Medical Association,Chicago,llhinois, 111-115.Harper, A. E., 1983. Some recent development in the study of amino acidmetabolism. Proc. Nutr. Soc., 42: 437-449.Harper, A. E., Benevenga, N. J., and Wohlheuter, R. M., 1970. Effects of ingestionof disproportionate amounts of amino acids. Physiol. Rev., 50: 428-558.Harper, A. E., Miller, R. H., and Block, K. P., 1984. Branched-chain amino acidmetabolism. Ann. Rev. Nutr., 4: 409-454.Hartog, C. D., and Pol, G., 1972. D. Assays based on measurements of plasma-freeamino acids. In: E. 3. Bigwood (Editor), Protein and Amino Acid Function.353-361.Higgs, D. A., Teskeredzic, Z., and Dosanjh, B., 1991. Nutritive value ofundephytiid and dephytinized rapeseed protein concentrate for rainbowtrout. In: 20” Fish Feed and Nutrition Workshop, Cornell University, Ithaca,NY., Abstract.Riggs, D. A., Markert, 3. R., Macquarrie, D. W., Mcbride, J. R., Dosanjh, B. S.,Nichols, C., and Hoskins, G., 1979. Development of practical dry diets forcoho salmon, Oncorhynchus kisutch, using poultry-by-product meal, feathermeal, soybean meal and rapeseed meal as major protein sources. In: WorldSymposium on Finfish and Fishfeed Technology, 3. E. Halver, and K. Tiews(Editors). Heeneman, Berlin, Hamburg, 191-218.Higgs, D. A., Fagerlund, U. H. M., Mcbride, 3. R., Plotnikoff, M. D., Dosanjh, B. S.,Markert, 3. R., and Davidson, J., 1983. Protein quality of altex canola mealfor juvenile chinook salmon (Oncorhynchus tshawytscha) considering dietaryprotein and 3,5,3’-triiodo-L-thyronine content. Aquaculture, 34: 213-238.Higuera, M. D. L., Murillo, A., Varela, G., and Zamora, S., 1977. The influence ofhigh dietary fat levels on protein utilization by the trout, (Salmo gairdnerii).Comp. Biochem. Physiol., 56A: 37-4 1.Hill, D. C., Indoo, E. M. M., and Olsen, E. M., 1961. Influence of dietary zein on theconcentration of amino acids in the plasma of chicks. J. Nutr., 74: 16-23.Hill, D. C., and Olsen, E. M., 1963. Effect of addition of imbalanced amino acidmixture to a low protein diet, on weight gains and plasma amino acids ofchicks. 3. Nutr., 79: 296-302.201BibliographyHilton, J. W., Atkinson, J. L., and Slinger, S. J., 1983. Effect of increased dietaryfiber on the growth of rainbow trout, Salmo gafrdneri. Can. J. Fish. Aquat.Sd., 40: 8 1-84.Hughes, S. 0., 1985. Evaluation of Glutamic acid and glycine as sources ofnonessential amino acids for lake trout (Salvelinus namaycush) and rainbowtrout (Salmo gairdneri). Comp. Biochem. Physiol., 81A: 669-671.Hughes, S. G., Rumsey, G. L., and Nesheim, M. C., 1983. Branched-chain aminoacid aminotransferase activity in the tissues of lake trout. Comp. Biochem.Physiol., 76B: 429-431.Jobling, M., 1980. Gastric evacuation in plaice, Pleuronectesplatessa L., effects ofdietary energy level and food composition. J. Fish. Biol., 17: 187-196.Jobling, M., 1983. A short review and critique of methodology used in fish growthand nutrition studies. J. Fish. Biol., 23: 685-703.Johnson, D. J., and Anderson, G. H., 1982. Prediction of plasma amino acidconcentration from diet amino acid content. Am. J. Physiol., 243: R99-R103.Johnston, J., and Coon, C. N., 1978. The use of varying levels of pepsin for pepsindigestion studies with animal proteins. Poult. Sci., 58: 1271-1273.Jürss, K, 1980. The effects of changes in external salinity on the free amino acidsand two aminotransferases of white muscle from fasted Salmo gairdneriRichardson. Comp. Biochem. Physiol., 65A: 501-504.Kaushik, S., and Luquet, P., 1977a. Endogenous nitrogen loss from the muscle ofrainbow trout starved in fresh water and sea water. Ann. Hydrobiol., 8: 129-134.Kaushik, S., and Luquet, P., 197Th. Study of free amino acids in rainbow trout inrelation to salinity changes. I. Blood free amino acids during starvation. Ann.Hydrobiol., 8: 135-144.Kaushik, S. 3., and Luquet, P., 1979. Influence of dietary amino acid patterns on thefree amino acid contents of blood and muscle of rainbow trout (Salmogairdnerii R). Comp. Biochem. Physiol., 64B: 175-180.Kaushik, S. J., Fauconneau, B., Terrier, L., and Gras, J., 1988. Arginine requirementand status assessed by different biochemical indices in rainbow trout (Salmogairdneri K). Aquaculture, 70: 75-95.Ketola, H. G., 1982. Amino acid nutrition of fishes : requirements andsupplementation of diets. Comp. Biochem. Physiol., 73B: 17-24.Ketola, H. 0., 1983. Requirement for dietary lysine and arginine by fry of rainbowtrout. J. Anim. Sci., 56: 101-107.202BibliographyKim, K. I., Grimshaw, T. W., Kayes, T. B., and Ainundson, C. H., 1992. Effect offasting or feeding diets containing different levels of protein or amino acidson the activities of the liver amino acid-degrading enzymes and amino acidoxidation in rainbow trout (Oncorhynchus mykiss). Aquaculture, 107: 89-105.Kim, K. I., Kayes, T. B., and Amundson, C. H., 1987. Effects of dietary tryptophanlevels on growth, feed/gain, carcass composition and liver glutamatedehydrogenase activity in rainbow trout (Sahno gairdneri). Comp. Biochem.Physiol., 88B: 737-74 1.Kim, K. I., Kayes, T. B., and Amundson, C. H., 1992. Requirements for sulfur aminoacids and utilization of D-methionine by rainbow trout (Oncorhynchusmykiss). Aquaculture, 101: 95-103.Kitamikado, M., and Tachino, S., 1960. Studies on digestive enzymes of rainbowtrout-Il Proteases. Bull. Jpn. Soc. Sci. Fish., 26: 685-690.Krogdahl, A., 1991. Srbean products may reduce fat digestibility in Atlanticsalmon. In: 20 Fish Feed and Nutrition Workshop, Cornell University,Ithaca, NY., Abstract.Lee, D. J., and Putnam, G. B., 1973. The response of rainbow trout to varyingprotein/energy ratio in a test diet. J. Nutr., 103: 916-921.Lie, 0., Lied, E., and Lambertsen, G., 1988. Feed optimization in Atlantic cod(Gadus morhua) : fat versus protein content in the feed. Aquaculture, 69:333-34 1.Lovell, T., 1989. Nutrition and Feeding of fish. Van Nostrand Reinhold, New York,260Longenecker, 3. B., and Hause, N. L., 1959. Relationship between plasma aminoacids and composition of the ingested protein. Arch. Biochem. Biophys., 84:46-59.Lyndon, A. R., Davidson, I., and Houlihan, D. F., 1993. Changes in tissue andplasma free amino acid concentrations after feeding in Atlantic cod. FishPhysiol. Biochem., 10: 365-375.March, B. E., 1991. Concentrates from canola meal as protein sources in salmoniddiets. In: 20t Fish Feed and Nutrition Workshop, Cornell University, Ithaca,NY., Abstract.March, B. E., and Biely, 3., 1967. Nutritional evaluation of fishmeals for poultryfeeding. Cornell Nutr. Conf. Feed Mfrs., 133-135.March, B. E., and Hickling, D. R., 1982. Assessment of heat damage to proteindigestibility from fish meals by in vitro pepsin solubilization at differenttemperatures. Can. J. Anim. Sci., 62: 657-660.203BibliographyMarch, B. E., Macmillan, C., and Ming, F., 1985. Techniques for evaluation ofdietary protein quality for the rainbow trout (Salmo gairdneri). Aquaculture,47: 275-292.Matthews, D.M., 1973. Protein absorption. In: J.C. Somogyi and I. Macdonald(Editors), The Gut and Nutrition. Proceedings of the Twelfth Symposium ofthe Group of European Nutritionists, Cambridge, July 8-10, 1973, 28-4 1.McCallum, I., 1985. Qualitative and Quantitative Aspects of the Protein Nutrition ofJuvenile Chinook Salmon (Oncorhynchus tshai’ytscha). Ph.D Thesis, TheUniversity of British Columbia.McCallum, I., and Higgs, D.A., 1989. An assesment of processing effects on thenutritive value of marine protein sources for juvenile chinook salmon(Oncorhynchus tshawytscha). Aquaculture, 77: 181-200.Mclaughlan, J. M., and Iliman, W. I., 1967. Use of free plasma amino acid levels forestimating amino acid requirements of the growing rat. J. Nutr., 93: 21-24.McLaughlan, J. M., and Morrison, A. B., 1968. Dietary factors affecting plasmaamino acid concentrations. In: J. H. Leathem (Editor), Protein Nutrition andFree Amino Acid Patterns. Rutgers University Press, New Jersey, 3-18.Medale, F., Parent, 3. P., and Vellas, F., 1987. Responses to prolonged hypoxia byrainbow trout (Salmo gairdneri). I. Free amino acids and proteins in plasma,liver, and white muscle. Fish Physiol. Biochem., 3: 183-189.Mercer, L. P., Dodds, S. J., and Smith, D. I., 1989. Dispensable, indispensable, andconditionally indispensable amino acid ratios in the diet. In: M. Friedman(Editor), Absorption and Utilization of Amino Acids. CRC Press Inc.,Florida, 1-14.Miyazono, I., and Inoue, Y., 1989. The relationship between the growth of rainbowtrout and pepsin digestibility of brown fish meal. In: The Current Status ofFish Nutrition in Aquaculture, M. Takeda, and T. Watanabe (Editors). Toba,Japan, 373-378.Moon, T. W., Walsh, P. J., and Mommsen, T. P., 1985. Fish hepatocytes: a modelmetabolic system. Can. 3. Fish. Aquat. Sci., 42: 1772-1782.Munro, H. N., 1970. Free amino acid pools and their role in regulation. In: H. N.Munro (Editor), Mammalian Protein Metabolism. Academic Press, NewYork, 299-388.Munro, H. N., and Portugal, F. H., 1972. B. Free Amino Acid Pools. In: E. J.Bigwood (Editor), Protein and Amino Acid Functions. Pergamon Press,Oxford, 197-213.204BibliographyMunro, H. N., and Crim, M. C., 1980. Protein and Amino Acids. In: R. S. Goodhart,and M. E. Shils (Editors), Modern Nutrition in Health and Disease. Lea &Febiger, Philadelphia, 5 1-97.Murai, T., 1992. Protein nutrition of rainbow trout. Aquaculture, 100: 19 1-207.Murai, T., Daozun, W., and Ogata, H., 1989a. Supplementation of methionine to soyflour diets for fingerling carp, Cyprinus carpio. Aquacuhure, 77: 373-385.Murai, T., Ogata, H., Villaneda, A., and Watanabe, T., 1989b. Utilization of soyflour by fingerling rainbow trout having different body size. Bull. Jpn. Soc.Sci. Fish., 55: 1067-1073.Murai, T., Ogata, H., Hirasawa, Y., Akiyama, T., and Nose, T., 1987. Portalabsorption and hepatic uptake of amino acids in rainbow trout force-fedcomplete diets containing casein or crystalline amino acids. Bull. Jpn. Soc.Sci. Fish., 53: 1847-1859.Nagai, M., and Ikeda, S., 1973. Carbohydrate metabolism in fish- Effects of dietarycomposition on metabolism of acetate-U-14Cand L-alanine-U-14Cin carp.Bull. Jpn. Soc. Sci. Fish., 39: 633-643.Nasset, E.S., Ganapathy, S.N., Goldsmith, D.P.J., 1963. Amino acids in dog bloodand gut contents after feeding zein. J. Nutr. 343-347.National Research Council (NRC), 1981. Nutrient Requirements of Cold WaterFishes. National Academy of Sciences, Washington. D.C., 83National Research Council (NRC), 1983. Nutrient Requirements of WarmwaterFishes and Sheilfishes. National Academy of Sciences, Washington, D.C., 102National Research Council (NRC), 1984. Nutrient Requirements of Poultry.National Academy of Sciences, Washington. D.C., 71Nissen, S., 1992. Modern Methods in Protein Nutrition and Metabolism. AcademicPress, San Diego, 157-159Nose, T., 1972. Changes in pattern of free amino acid in rainbow trout after feeding.Bull. Freshwater Fish. Res. Lab., 22: 137-144.Nose, T., Arai, S., and Lee, D. L., 1974. A note on amino acids essential for youngcarp. Bull. Jpn. Soc. Sci. Fish., 40: 903-908.Ogata, H., 1986. Correlations of essential amino acid patterns between the dietaryprotein and the blood, hepatopancreas, or skeletal muscle in carp. Bull. Jpn.Soc. Sci. Fish., 52: 307-3 12.205BibliographyOgata, H., and Arai, S., 1985. Comparison of free amino acid contents in plasma,whole blood and erythrocytes of carp, coho salmon, rainbow trout, andchannel catfish. Bull. Jpn. Soc. Sci. Fish., 51: 1181-1186.Ogata, H., Arai, S., and Nose, T., 1983. Growth response of cherry salmonOncorhychus masou and amago salmon 0. rhodurua fry fed purified dietssupplemented with amino acids. Bull. Jpn. Soc. Sci. Fish., 49: 138 1-1385.Ogata, H., Murai, T., and Hfrasawa, Y., 1986. Assessment of performance using freeamino acid levels in plasma of carp. Bull. Jpn. Soc. Sci. Fish., 52: 1071-1075.Ogino, C., 1980. Requirements of carp and rainbow trout for essential amino acids.Bull. Jpn. Soc. Sci. Fish., 46: 171-174.Opstvedt, J., Miller, R., Hardy, W., and Spinelli, J., 1984. Heat-induced changes insuithydryl groups and disulfide bonds in fish protein and their effect onprotein and amino acid digestibility in rainbow trout (Salmo gairdneri). J.Agric. Food. Chem., 32: 929-935.Ostrowski, H. T., 1977. Analysis for availability of amino acid supplements in foodsand feeds : biochemical and nutritional implications. In: Symposium onImprovement of Protein Nutritive Quality of Foods and Feeds, Plenum Press,New York, Chicago, 497-547.Peng, Y., and Harper, A. E., 1970. Amino acid balance and food intake: effect ofdifferent dietary amino acid patterns on the plasma amino acid pattern ofrats. 3. Nutr., 100: 429-437.Peng, Y., Tews, J. K., and Harper, A. E., 1972. Amino acid imbalance, proteinintake, and changes in rat brain and plasma amino acids. Am. J. Physiol., 222:314-321.Pfeffer, E., 1982. Utilization of dietary protein by salmonid fish. Comp. Biochem.Physiol., 73B: 5 1-57.Plakas, S. M., Lee, T. C., and Wolke, R. E., 1988. Bioavailability of lysine in maillardbrowned protein as determined by plasma lysine response in rainbow trout(Salmo gairdneri). 3. Nutr., 118: 19-22.Plakas, S. M., Katayama, T., Tanaka, Y., and Deshiniaru, 0., 1980. Changes in thelevels of circulating plasma amino acids of carp (Cyprinus carpio) afterfeeding a protein and an amino acid diet of similar composition.Aquaculture, 21: 307-322.Plakas, S. M., Lee, T. C., Wolke, R. E., and Meade, T. L., 1985. Effect of maillardbrowning reaction on protein utilization and plasma amino acid response byrainbow trout (Salmo gairdneri). J. Nutr., 115: 1589-1599.206BibliographyPoston, H. A., and Rumsey, G. L., 1983. Factors affecting dietary requirement anddeficiency signs of L-tryptophan in rainbow trout. J. Nutr., 113: 2568-2577.Reinitz, G., 1980. Soybean meal as a substitute for herring meal in practical diets forrainbow trout. Prog. Fish-Cult., 42: 103-106.Reinitz, G., and Hitzel, F., 1980. Formulation of practical diets for rainbow troutbased on desired performance and body composition. Aquaculture, 19: 243-252.Robinson, E. H., 1992. Use of supplemental lysine in catfish feeds. AquacultureMagazine., May/June: 94-96.Rosenlund, G., 1986. Comparisons between unconventional proteins and fish mealas a dietary nitrogen source for rainbow trout (Salmo gafrdneri): effects on invitro muscle protein synthesis. Fiskeridir. Skr. Ser. Ernaer., 2: 193-200.Rumsey, G. L., 1991. Effects of feeding methods and dietary amino acid levels ongrowth response. In: 20th Fish Feed and Nutrition Workshop, CornellUniversity, Ithaca, New York,Rumsey, G. L., and Ketola, H. G., 1975. Amino acid supplementation of casein indiets of Atlantic salmon (Salmo salar) fry and of soybean meal for rainbowtrout (Salmo gairdneri) fingerlings. J. Fish. Res. Board Can., 3: 422-426.Rumsey, G. L., Page, J. W., and Scott, M. L., 1983. Methionine and cystinerequirements of rainbow trout. Prog. Fish-Cult., 45: 139-143.Sakaguchi, M., and Kawai, A., 1970. Histidine metabolism in fish. V. The effect ofprotein-deficiency and fasting on the activity of histidine deaminase andurocase in carp liver. Bull. Jpn. Soc. Sci. Fish., 36: 783-787.Sakaguchi, M., Murata, M., Daikoku, T., and Arai, S., 1988. Effects of dietarytaurine on tissue taurine and free amino acid levels of the chum salmon,Oncorhynchus keta, reared in freshwater and seawater environments. Comp.Biochem. Physiol., 89A: 437-442.Sarwar, G., Peace, R. W., and Botting, H. G., 1983. Validity of rat plasma aminoacids in predicting first limiting amino acids in protein mixtures. Nutr. Rep.Tnt., 28: 613-620.Shepartz, B., 1973. Regulation of Amino Acid Metabolism in Mammals. Saunder,Philadelphia, 205Simon, 0., 1989. Metabolism of proteins and amino acids. In: H. D. Bock, B. 0.Eggum, A. G. Low, 0. Simon, and T. Zebrowska (Editors), ProteinMetabolism in Farm Animals. Oxford University Press, Berlin, 273-366.207BibliographySmith, R. E., and Scott, H. M., 1965. Use of free amino acid concentrations in bloodplasma in evaluating the amino acid adequacy of intact proteins for chickgrowth. I. Free amino acid patterns of blood plasma of chicks fed unheatedand heated fishmeal proteins. J. Nutr., 86: 37-44.Smith, R. R., Kincaid, H. L., Regenstein, J. M., and Rumsey, G. L., 1988. Growth,carcass composition, and taste of rainbow trout of different strains fed dietscontaining primarily plant or animal protein. Aquaculture, 70: 309-321.Stahmann, M.A., and Woldegiorgis, G., 1974. Enzymatic methods for protein qualitydetermination. In: M. Friedman (Editor), Protein Nutritional Quality ofFoods and Feeds. Part 1: Assay Methods-Biological, Biochemical, andChemical. Marcel Dekker, Inc., New York, 211-234.Steffens, W., 1981. Protein utilization by rainbow trout (Salmo gairdneri) and carp(Cyprinus carpio): a brief review. Aquaculture, 23: 337-345.Steffens, W., 1989. Principles of Fish Nutrition. Ellis Horwood Limited, WestSussex, England, 384Suyama, M., Hirano, T., and Suzuki, T., 1986. Buffering capacity of free histidineand its related dipeptides in white and dark muscle of yellowfin tuna. Bull.Jpn. Soc. Sd. Fish., 52: 2171-2175.Tacon, A. 0. J., and Cowey, C. B., 1985. Protein and Amino Acid Requirements. In:P. Tytler, and P. Calow (Editors), Fish Energetics: New perspectives. TheJohns Hopkins University Press, Baltimore, Maryland, 155-184.Tacon, A. G. J., and Jackson, A. 1., 1985. Utilisation of conventional andunconventional protein sources in practical fish feeds. In: C. B. Cowey, A. M.Mackie, and J. G. Bell (Editors), Nutrition and Feeding in Fish. AcademicPress, London, 119-145.Tacon, A. G. J., Haaster, 3. V., and Featherstone, P. B., 1983. Studies on theutilization of full-fat soybean and solvent extracted soybean meal in acomplete diet for rainbow trout. Bull. Jpn. Soc. Sci. Fish., 49: 1437-1443.Takeuchi, T., Watanabe, T., and Ogino, C., 1978. Optimum ratio of protein to lipidsin diets of rainbow trout. Bull. Jpn. Soc. Sci. Fish., 44: 683-688.Talbot, C., 1985. Laboratory methods in fish feeding and nutritional studies. In: P.Tytler, and P. Calow (Editors), Fish Energetics: New Perspectives. The JohnHopkins University Press, Maryland, 125-154.Teigland, M., and Klungsøyr, L., 1983. Accumulation of aipha-keto-isocaproate fromleucine in homogenates of tissues from rainbow trout (Salmo gairdnerii) andrat. An improved method for determination of branched chain keto acids.Comp. Biochem. Physiol., 75B: 703-705.208BibliographyThebault, H., 1985. Plasma essential amino acid changes in sea-bass (Dicentrarchuslabrax) after feeding diets deficient and supplemented in L-methionine.Comp. Biochem. Physiol., 82A: 233-237.Tiews, K., Koops, H., Gropp, 3., and Beck, H., 1979. Compilation of fish meal-freediets obtained in rainbow trout (Salmo gairdneri) feeding experiments atHamburg (1970-1977/78). In: World Symposium on Finfish Nutrition andFishfeed Technology, 3. E. Halver and K. Tiews (Editors). Hamburg, 219-228.Todd, W. R., Laastuen, L. E., and Thomas, A. E., 1967. Effect of amino acidimbalance on liver glycogen levels in young salmon. Comp. Biochem.Physiol., 23A: 43 1-435.Ufodike, E. B. C., and Matty, A. J., 1989. Effect of potato and corn meal on proteinand carbohydrate digestibility by rainbow trout. Prog. Fish-Cult., 51: 113-114.Van der Boon, 3., Van den Thillart, G. E. E. J. M., and Addirilc, A. D. F., 1989. Freeamino acid profiles of aerobic (red) and anaerobic (white) skeletal muscle ofthe cyprinid fish, Carassius auratus L. (goldfish). Comp. Biochem. Physiol.,94A: 809-8 12.Van Waarde, A., 1988. Biochemistry of non-protein nitrogenous compounds in fishincluding the use of amino acids for anaerobic energy production. Comp.Biochem. Physiol., 207-228.Vifiamar, D. 991. Soybean products as a replacement for fish meal in trout feed.In: 2O’ Fish Feed and Nutrition Workshop, Cornell University, Ithaca, NY.,Abstract.Waibel, P. E., and Carpenter, K. 3., 1972. Mechanisms of heat damage in proteins. 3.Studies with -(gamma-L-glutamyl)-L-lysine. Br. J. Nutr., 27: 509-5 15.Walton, M. 3., 1985. Aspects of amino acid metabolism in teleost fish. In: C. B.Cowey, A. M. Mackie, and 3. G. Bell (Editors), Nutrition and Feeding inFish. Academic Press, London, 47-68.Walton, M. J., and Cowey, C. B., 1981. Distribution and some kinetic properties ofserine catabolizing enzymes in rainbow trout Salmo gairdneri. Comp.Biochem. Physiol., 68B: 147-150.Walton, M. J., and Cowey, C. B., 1982. Aspects of intermediary metabolism insalmonid fish. Comp. Biochem. Physiol., 73B: 59-79.Walton, M. J., and Wilson, R. P., 1986. Postprandial changes in plasma and liverfree amino acids of rainbow trout fed complete diets containing casein.Aquaculture, 51: 105-115.209BibliographyWalton, M. I., Cowey, C. B., and Adron, J. W., 1982. Methionine metabolism inrainbow trout fed diets of differing methionine and cystine content. 3. Nutr.,112: 1525-1535.Walton, M. J., Cowey, C. B., and Adron, J. W., 1984a. The effect of dietary lysinelevels on growth and metabolism of rainbow trout (Salmo gafrdneri). Br. 3.Nutr., 52: 115-122.Walton, M. J., Coloso, R. M., Cowey, C. B., Adron, J. W., and Knox, D., 1984b. Theeffects of dietary tryptophan levels on growth and metabolism of rainbowtrout (Salmo gairdneri). Br. J. Nutr., 51: 279-287.Walton, M. J., Adron, 3. W., Coloso, R. M., and Cowey, C. B., 1986. Dietaryrequirements of rainbow trout for tryptophan, lysine and arginine asdetermined by growth and biochemical measurements. Fish Physiol.Biochem., 2: 161-169.Watanabe, T., Takeuchi, T., and Ogino, C., 1979. Studies on the sparing effect oflipids on dietary protein in rainbow trout (Salmo gairdneri). In: WorldSymposium on Finfish Nutrition and Fish Feed Technology, J. E. Halver andK. Tiews (Editors). Hamburg, 113-125.Wilkinson, L., 1990. Systat:The System for Statistics. Evanston, IL:Systat, Inc.Wilson, R. P., 1989. Amino acids and proteins. In: J. E. Halver (Editor), FishNutrition. Academic Press, California, 112-147.Wilson, R. P., and Poe, W. E., 1974. Nitrogen metabolism in channel catfish,Ictaluruspunctatus-llI. Relative pooi sizes of free amino acids and relatedcompounds in various tissues of the catfish. Comp. Biochem. Physiol., 48B:545-556.Wilson, R. P., and Cowey, C. B., 1985. Amino acid composition of whole body tissueof rainbow trout and Atlantic salmon. Aquaculture, 48: 373-376.Wilson, R. P., and Poe, W. E., 1985. Relationship of whole body and egg essentialamino acid patterns to amino acid requirement patterns in channel catfish,Ictaluruspuntatus. Comp. Biochem. Physiol., 80B: 385-388.Wilson, R. P., Harding, D. E., and Garling, D. L. J., 1977. Effect of dietary pH onamino acid utilization and the lysine requirement of fingerling channelcatfish. J. Nutr., 107: 166-170.Wilson, R. P., Poe, W. E., and Robinson, E. H., 1980. Leucine, isoleucine, valine andhistidine requirements of fingerling channel catfish. 3. Nutr., 110: 627-633.Wilson, R. P., Gatlin III, D. M., and Poe, W. E., 1985. Postprandial changes inserum amino acids of channel catfish fed diets containing different levels ofprotein and energy. Aquaculture, 49: 101-110.210BibliographyWilson, R. P., Allen, 0. W., Robinson, E. H., and Poe, W. E., 1978. Tryptophan andthreonine requirements of fingerling channel catfish. J. Nutr., 108: 1595-1599.Windell, 3. T., Hubbard, J. D., and Horak, D. L., 1972. Rate of gastric evacuation inrainbow trout fed three pelleted diets. Prog. Fish-Cult., 34: 156-159.Yamada, S., Tanaka, T., and Katayama, T., 1981a. Feeding experiment with carp fryfed an amino acid diet by increasing the number of feedings per day. Bull.Jpn. Soc. Sci. Fish., 47: 1247.Yamada, S., Simpson, K. L., Tanaka, Y., and Katayama, T., 198 lb. Plasma aminoacid changes in rainbow trout Salmo gairdneri force-fed casein and acorresponding amino acid mixture. Bull. Jpn. Soc. Sci. Fish., 47: 1035-1040.Yamada, S., Tanaka, Y., Katayama, T., Sameshima, M., and Simpson, K. L., 1982.Plasma amino acid changes in Tilapia nilotica fed a casein and correspondingfree amino acid diet. Bull. Jpn. Soc. Sci. Fish., 48: 1783-1787.Yokoyama, M., and Nakazoe, J., 1989. Induction of cysteine dioxygenase activity inrainbow trout liver by dietary sulfur amino acids. In: The Current Status ofFish Nutrition in Aquaculture, M. Takeda, and T. Watanabe (Editors). Toba,Japan, 367-372.Young, V. R., and Zamora, J., 1968. Effects of altering the proportion of essential tononessential amino acids on growth and plasma amino acid levels in the rat.J. Nutr., 96: 21-27.Young, V. R., and Scrimshaw, N. S., 1972. The Nutritional significance of plasmaand urinary amino acids. In: E. J. Bigwood (Editor), Protein and Amino AcidFunctions. Pergamon Press, Oxford, Great Britain, 54 1-568.Young, V. R., Meridith, C., Hoerr, R., Bier, D. M., and Matthews, D. E., 1985.Amino acid kinetics in relation to protein and amino acid requirements: Theprimary importance of amino acid oxidation. In: J. S. Garrow, D. Holliday,and L. Libbey (Editors), Substrate and Energy Metabolism. 119-134.Zar, 3. H., 1984. Biostatistical Analysis. Prentice-Hall, Inc., New Jersey, 718211APPENDICESAppendix 1. ANOVA of initial weight, final weight, weight gain, and feed consumption of rainbow troutin Experiment 1.1Initial weight Final weight Weight gain Feed consumptionSource df Mean P Mean P Mean P Mean Psquare square square squareDiet (D) 2 0.013 0.978 35.641 0.001* 33.897 0.004* 0.00057 0.00214*Feeding 1 1.227 0.213 0.339 0.525 0.000 0.985 0.00222 0.00022*frequency (F)Size (S) 2 4.801 0.036’ 262.618 0.000* 49.847 0.002* 0.09629 0.00001*D*F 2 0.267 0.652 1.059 0.324 1.209 0.435 0.00004 0.17361D*S 4 0.956 0.308 1.303 0.119 2.551 0.234 0.00821 0.00001*F*S 2 0.977 0.285 2.562 0.268 0.329 0.768 0.00002 0.30864Error 4 0.560 0.700 1.169 0.00001Total 17* Significantly differentAppendix 2. ANOVA of specific growth rate (SGR), feed efficiency, productive protein value (PPV), andenergy efficiency in Experiment 1.1SGR Feed efficiency PPV Energy efficiencySource df Mean P Mean P Mean P Mean Psquare square square squareDiet (D) 2 0.306 0.015* 0.036 0.004* 0.0218 0.001* 0.016 0.007*Feeding 1 0.001 0.952 0.001 0.396 0.0005 0.040* 0.001 0.229frequency (F)Size (S) 2 0.012 0.608 0.001 0.406 0.0013 0.007* 0.003 0.119D*F 2 0.016 0.522 0.001 0.653 0.0003 0.067 0.000 0.947D*S 4 0.027 0.414 0.000 0.824 0.0082 0.013* 0.001 0.388F*S 2 0.007 0.743 0.000 0.807 0.00001 0.801 0.000 0.947Error 4 0.085 0.005 0.00006 0.001Total 17* Significantly different212AppendicesAppendix 3. ANOVA of protein gain and lipid gain of rainbow trout in Experiment 1.1Protein gain Lipid gainSource df Mean P Mean Psquare squareDiet (D) 2 2968.14 0.001* 3475.61 0.001*Feeding 1 5.26 0.783 1595.56 0.013*frequency (F)Size (S) 2 4910.46 0.001* 4432.57 0.001*Error 12 66.28 188.49Total 17* Significantly differentAppendix 4. ANOVA of dry matter, protein, lipid and ash content of rainbow trout in Experiment 1.1Dry matter Protein Lipid AshSource df Mean P Mean P Mean P Mean Psquare square square squareDiet (D) 2 1.736 0.002* 0.356 0.532 9.544 0.156 0.019 0.828Feeding 1 0.867 0.042* 4.611 0.012* 26.541 0.030* 0.207 0.177frequency (F)Size (S) 2 0.217 0.307 0.630 0.341 6.323 0.273 0.253 0.123Error 12 0.167 0.534 4374 0.110Total 17* Significantly differentAppendix S. ANOVA of specific growth rate (SGR), and feed conversion efficiency in Experiment 1.2SGR Feed efficiencySource df Mean Square P Mean Square PDiet 2 0.0476 0.28 0.0107 0.29Error 12 0.1732 0.0370Total 14213AppendicesAppendix 6. ANOVA of pepsin digestibility in Experiment 4.Pepsin digestibilitySource df Mean Square PDiet 3 215.744 0.000*Error 4 0.284Total 7Significantly differentAppendix 7. ANOVA of initial weight, final weight, weight gain, and feed consumption of rainbow troutin Experiment 4.Initial weight Final weight Weight gain Feed consumptionSource df Mean P Mean P Mean P Mean Psquare square square squareDiet 3 229.206 0.130 502.917 0.120 120.253 0313 0.522 0.164Error 12 100.116 210.591 90.981 0.258Total 15Appendix 8. ANOVA of specific growth rate (SGR), and feed conversion efficiency in Experiment 4SGR Feed efficiencySource df Mean Square P Mean Square PDiet 3 0.079 0.392 0.007 0.814Error 12 0.073 0.022Total 15214

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0088016/manifest

Comment

Related Items