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Practical diet formulation for common carp (Cyprinus carpio) and walking catfish (Clarias macrocephalus)… Sermwatanakul, Amonrat 1993

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THE UNIVERSITY OF BRITISH COLUMBIAApril 1993PRACTICAL DIET FORMULATION FORCOMMON CARP (CYPRINUS CARPIO) ANDWALKING CATFISH (CLARIAS MACROCEPHALUS)LARVAE IN THAILANDbyAMONRAT SERMWATANAKULB. Sc. (Fisheries) Kasetsart University, Thailand, 1981M. Sc. (Environmental Sciences) Kasetsart University, Thailand, 1984A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDoctor of PhilosophyinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ANIMAL SCIENCESWe accept this thesis as conformingto the required standard© Amonrat Sermwatanakul, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of^All YY■^SThe University of British ColumbiaVancouver, CanadaDate Ar.^19^9-4DE-6 (2/88)ABSTRACTThere is a need for the development of formulated diets for fish larvae. Thepresent study was conducted in Thailand on common carp (Cyprinus carpio) and walkingcatfish (Clarias macrocephalus) larvae with the following objectives: 1) To assess thenutritive values of several available feed ingredients when used singly or in differentcombinations; 2) To formulate a cost effective practical diet, using locally availableingredients, to replace live foods in larval culture; 3) To determine the relative suitabilityof dry or moist forms of diets under conditions of local climate. Live Artemia nauplii,Moina, and practical diets formulated by the National Inland Fisheries Institute (NIFI),Thailand were used as reference diets for assessing the performance of fish fed theexperimental diets. Dietary particles size was adjusted to 125-250 pirn aftermeasurements of the larval mouth size between 13-18 days of age. Feeding trials wereconducted using many combinations of available ingredients and performance of the larvaewas based upon food acceptance, survival, and growth in length.Four diets: a moist and a dry diet of the experimental diets that had given superiorperformance, Artemia nauplii, and the NIFI 1 diet were used in a final feeding trialconducted on 1000 common carp larvae. The moist diet (now called Burirum 1) had thefollowing proximate analysis on dry weight basis: 40.4% crude protein, 15.6% crude fat,33.4% carbohydrate including fiber and 10.7% ash; and composition: 16.3% raw pork liver,8.1% coagulated cooked chicken diet, 16.3% whole chicken egg, 16.3% raw squid meal,26% frozen Artemia, 0.8% cooked peanut, 1.5% corn oil, 4.1% skim milk, a vitamin-mineralsupplement, 2% K-carrageenan. The four diets were not significantly different withrespect to their effects on larval survival. Mean survival was 67.7%. Larval body lengthresponses for Artemia nauplii., the moist diet, the dry diet and the NIFI 1 diet were 9.1,118.9, 8.4 and 8.2 mm, respectively. Dissolved oxygen, alkalinity and water hardness were8 mg/1, 620 ppm and 880 ppm. The water temperature in the rearing tanks ranged from25-29°C and water pH was 7.65.Three feeding trials with moist diets and four with dry diets were conducted onwalking catfish larvae. Four diets: a moist and a dry diet that had given superiorperformance, Artemia nauplii, and the NIFI 2 diet were used in a final feeding trialconducted on 1000 walking catfish larvae. Proximate analysis on dry weight basis of thedry diet (now called Burirum 2) was: 39.8% crude protein, 9.4% crude fat, 15.4%carbohydrate including fiber and 35.3% ash, and its composition was: 60% fish meal, 7.9%corn meal, 9.8% peanut meal, 7.9% rice bran, 4.6% corn oil with vitamin-mineral premixadded. Larval body length and survival indicated significantly different responses to thediets. Survival rates for Artemia nauplii, the dry diet, the NIFI 2 diet and the moist dietwere 65.6, 62.8, 56.3 and 48.5%, respectively. Body lengths in response to Artemianauplii, the moist diet, the dry diet and the NIFI 2 were 12.5, 12.0, 12.0 and 10.8 mm,respectively.It is concluded that appropriately formulated practical diets can satisfactorilyreplace live foods in larval culture of common carp and walking catfish. The moist dietwas more effective than the dry diet for common carp larvae whereas the dry diet wasmore effective than the moist diet for walking catfish larvae.111TABLE OF CONTENTSTABLE OF CONTENTS^ ivLIST OF TABLES viiiLIST OF FIGURES^ xiiACKNOWLEDGEMENTS xiiiCHAPTER 1^ 1GENERAL INTRODUCTION^CHAPTER 2^ 3LITERATURE REVIEW^ 32.1 Larval mortality 32.1.1 Mouth size 42.1.2 Larval digestive system^ 52.2 Availability of larval food 62.2.1 Live foods^ 72.2.2 Formulated diets  102.2.3 Combination of live foods and formulated diets^ 132.3 Selected species^  142.3.1 Common carp (Cyprinus carpio)^  142.3.2 Walking catfish (Clarias macrocephalus)^ 16CHAPTER 3^ 18GENERAL MATERIALS AND METHODS^ 183.1 Experimental site^  183.2 Diet preparation  183.2.1 Moina culture  183.2.2 Artemia culture^ 213.2.3 Moist diet 213.2.4 Dry diet^ 233.3 Source of larvae 243.3.1 Common carp larvae^  243.3.2 Walking catfish larvae 283.4 Experimental design^ 333.4.1 Larval feeding 333.4.2 Sampling 333.4.3 Data analysis^ 333.4.4 Criteria of larval feed acceptance^ 34ivCHAPTER 4^ 35EXPERIMENT 1: COMPARATIVE PERFORMANCE OF COMMON CARP ANDWALKING CATFISH LARVAE FED COMMONLY USED DIETS^354.1 Introduction^ 354.2 Materials and methods^ 364.3 Results and discussion 394.3.1 Mouth size 394.3.2 Larval feed-acceptance behavior^ 404.3.3 Dietary effects on larval body length 41CHAPTER 5^ 43DEVELOPMENT OF DIET FORMULATIONS FOR COMMON CARP LARVAE(EXPERIMENTS 2 TO 4)^ 43Introduction 43I. EXPERIMENT 2: MOIST DIETS FOR COMMON CARP LARVAE^ 44Experiment 2.1: Screening of acceptable dietary sources for common carp larvae ^ 44Materials and methods^ 45Results and discussion 47Experiments 2.2 to 2.10: Responses of common carp larvae to formulated diets ofdifferent composition^  53Experiment 2.2 56Materials and methods^ 56Results and discussion 59Experiment 2.3^ 62Materials and methods^ 62Results and discussion 65Experiment 2.4^ 66Materials and methods^ 66Results and discussion 67Experiment 2.5^ 72Materials and methods^ 72Results and discussion 75Experiment 2.6^ 76Materials and methods^ 76Results and discussion 79Experiment 2.7^ 80Materials and methods^ 80Results and discussion 83Experiment 2.8^ 84Materials and methods^ 84Results and discussion 87Experiment 2.9^ 88Materials and methods^ 88Results and discussion 91Experiment 2.10^  92Materials and methods^ 92Results and discussion 95II. EXPERIMENT 3: DRY DIETS FOR COMMON CARP LARVAE^ 96Experiments 3.1 to 3.5: Responses of common carp larvae to different dry dietformulations^ 96Experiment 3.1 99Materials and methods^ 99Results and discussion 102Experiment 3.2^  104Materials and methods^ 104Results and discussion 107Experiment 3.3^  108Materials and methods^ 108Results and discussion 111Experiment 3.4^  112Materials and methods^ 112Results and discussion 115Experiment 3.5^  116Materials and methods^ 116Results and discussion 119III. EXPERIMENT 4: RESPONSES OF COMMON CARP LARVAE FED SELECTEDFORMULATED DIETS, THE NIFI 1 DIET AND ARTEMIA NAUPLII^ 122Methods and materials^ 122Results and discussion 126Summary^ 129CHAPTER 6^ 131DEVELOPMENT OF DIET FORMULATIONS FOR WALKING CATFISH LARVAE(EXPERIMENTS 5 TO 7)^ 131Introduction  131I. EXPERIMENT 5: MOIST DIETS FOR WALKING CATFISH LARVAE^ 133Experiment 5.1: Screening of acceptable dietary sources forwalking catfish larvae^  133Materials and methods^ 133Results and discussion 134Experiments 5.2 and 5.3: Responses of walking catfish larvae to different moist dietformulations^ 137Experiment 5.2  137Materials and methods^ 137Results and discussion 141Experiment 5.3^  142Materials and methods^ 142Results and discussion 145viII. EXPERIMENT 6: DRY DIETS FOR WALKING CATFISH LARVAE^ 146Experiments 6.1 to 6.4: Response of walking catfish larvae to dry diets with differentformulations^ 146Experiment 6.1 146Materials and methods^ 146Results and discussion 149Experiment 6.2^  150Materials and methods^ 150Results and discussion 153Experiment 6.3^  154Materials and methods ^ 154Results and discussion 157Experiment 6.4^  158Materials and methods^ 158Results and discussion 161III. EXPERIMENT 7: RESPONSES OF WALKING CATFISH LARVAE FEDSELECTED FORMULATED DIETS, THE NIFI 2 DIET ANDARTEMIA NAUPLII^ 163Materials and methods^ 163Results and discussion 167CHAPTER 7^ 170OVERALL CONCLUSIONS^  170BIBLIOGRAPHY ^  172APPENDICES^Appendix 1 Appendix 2Appendix 3.Appendix 4.192Analysis of variance of the data from experiment 1 (Table 4.3)^ 192Analysis of variance of the data from experiment 4 (Table 5.33)^ 192Analysis of variance of the data from experiment 7 (Table 6.16)^ 193Composition of vitamin and mineral premix^ 194viiLIST OF TABLESTable 2.1 Effects of formulated diets on performance of some fish larvae^ 10Table 4.1 Composition of NIFI diets for common carp and walking catfish larvae inexperiment 1^ 37Table 4.2 Overall diet acceptance score responses of common carp and walking catfishlarvae for different diets in experiment 1^ 40Table 4.3 Mean values for final body lengths (mm) of common carp and walking catfishlarvae fed Artemia nauplii, Moina and NIFI diets from 3-18 days of agein experiment 1^ 41Table 5.1 Survivals and overall diet acceptance score responses for common carp larvaefed different food items in experiment 2.1^  48Table 5.2 Ingredient and nutrient compositions of diets fed to common carp larvae for 10days from 3-13 days of age in experiment 2.2^ 57Table 5.3 Percent survivals and overall diet acceptance score responses for common carplarvae fed microparticulate diets in experiment 2.2^ 59Table 5.4 Ingredient and nutrient compositions of diets fed to common carp larvae for 10days from 3-13 days of age in experiment 2.3^ 63Table 5.5 Percent survivals and overall diet acceptance score responses for common carplarvae fed microparticulate diets in experiment 2.3^ 65Table 5.6 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.4^ 68Table 5.7 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed different diets in experiment 2.4^ 70Table 5.8 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.5^ 73Table 5.9 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to moist diets in experiment 2.5^ 75Table 5.10 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.6^ 77Table 5.11 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.6^ 79viiiTable 5.12 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.7^ 81Table 5.13 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.7^ 83Table 5.14 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.8^ 85Table 5.15 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.8^ 87Table 5.16 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.9^ 89Table 5.17 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to moist diets in experiment 2.9^ 91Table 5.18 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 2.10^ 93Table 5.19 Final body lengths, percent survivals and overall diet acceptance scoreresponses of common carp larvae to moist diets in experiment 2.10^ 95Table 5.20 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 3.1^ 100Table 5.21 Survivals and overall diet acceptance score responses for common carp larvaefed different dry diets in experiment 3.1^ 102Table 5.22 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 3.2^ 105Table 5.23 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.2^ 107.Table 5.24 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 3.3^ 109Table 5.25 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.3^ 111Table 5.26 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 3.4^ 113Table 5.27 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.4^ 115ixTable 5.28 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 3.5^ 117Table 5.29 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to dry diets in experiment 3.5^ 120Table 5.30 Ingredient and nutrient compositions of diets fed to common carp larvae for 15days from 3-18 days of age in experiment 4^ 124Table 5.31 Proximate compositions of diets fed to common carp larvaein experiment 4^ 126Table 5.32 Proximate compositions of some ingredients used in diets for common carp andwalking catfish larvae^ 127Table 5.33 Mean values for final body lengths (mm) and percent survivals of commoncarp larvae in experiment 4^ 128Table 6.1 Survivals and overall diet acceptance score responses for walking catfish larvaefed different food items in experiment 5.1^  135Table 6.2 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 5.2^ 139Table 6.3 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed moist diets in experiment 5.2^ 141Table 6.4 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 5.3^ 143Table 6.5 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed moist diets in experiment 5.3^ 145Table 6.6 Ingredient and nutrient compositions of diets fed to walking catfish larvae for10 days from 3-13 days of age in experiment 6.1^ 147Table 6.7 Final body lengths and overall diet acceptance score responses of walkingcatfish larvae fed dry diets in experiment 6.1^ 149Table 6.8 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 6.2^ 151Table 6.9 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed dry diets in experiment 6.2^ 153Table 6.10 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 6.3^ 155Table 6.11 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed dry diets in experiment 6.3^ 157Table 6.12 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 6.4^ 159Table 6.13 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed diets in experiment 6.4^ 161Table 6.14 Ingredient and nutrient compositions of diets fed to walking catfish larvae for15 days from 3-18 days of age in experiment 7^ 165Table 6.15 Proximate composition of diets fed to walking catfish larvaein experiment 7^ 167Table 6.16 Mean values for final body lengths (mm) and percent survivals of walkingcatfish larvae in experiment 7^ 168xiLIST OF FIGURESFigure 3.1 Geographical distribution of some freshwater fisheries stations in Thailand ^ 19Figure 3.2 Cement tanks used for Moina culture and spawning ground of common carp atBurirum Freshwater Fisheries Station^ 20Figure 3.3 Glass containers for Artemia culture 20Figure 3.4 Burirum local market^ 22Figure 3.5 Common carp brood fish 25Figure 3.6 Earthen pond for rearing brood fish^ 27Figure 3.7 Spawning tank for common carp 27Figure 3.8 Walking catfish brood fish^ 30Figure 3.9 Hatching tank for walking catfish larvae^  32Figure 3.10 Experimental unit^ 32Figure 5.1 Body lengths of common carp larvae fed with different diets^ 130Figure 6.1 Body lengths of walking catfish larvae fed with different diets^ 169xiiACKNOWLEDGEMENTSDevelopment and completion of this thesis was possible only with enormousguidance from my supervisor, Dr. B.E. March. Her professional skills and kindness for mewill continue illuminating my ways. She is a great person.Special gratitude is extended to other members of my supervisory committee: Dr.R. Blair, Dr. D.A. Higgs, Dr. J.E. Halver, Dr. G.K. Iwama; university examiners Dr. R.M.Beames and Dr. J. Vanderstoep; and external examiner of the committee Dr. R.T. Lovellfor their taking time for reviewing this thesis and providing very valuable advice.Thanks go to Dr. P. Suraswadi, of the Department of Fisheries, Thailand whomanaged my pursuation of studies in Canada. Late Dr. W.E. Johnson very kindlyfamiliarized me with the topic. Mr. S. Sujaritvongsanon and in particular Mr. S. Trakuland Ms. S. Boonmee extended all their cooperation during my research conducted inThailand. CIDA under Northeast Fisheries Project (DOF/CIDA, 906/11415) took care ofthe monitory requirement; Ms. D.A. Turnbull, Mr. R. Schoenert, Ms. C. Beaudreault andother staff were always considerate for these needs. Fish Nutrition and Aquaculture DietsProjects (FAO, THA/891003) very generously provided the laboratory equipment.My Ph.D. accomplishment will be rewarding for my parents whose sole objectivehas always been educating their children. Continued understanding from my parents,sister and brothers kept my hopes alive. Mr. M.S. Bhatti will be recalled for his help. Imust mention Ven. Witchaysako Bhikkhu, Mr. S. Kribkratok, Ms. C. Tantikitti, and Mr.J.D. Rosene for their all the time being good friends. I will long remember beauties ofCanada and love of her people.CHAPTER 1GENERAL INTRODUCTIONThere is current concern for the development of aquaculture to help supply the foodrequirements of rapidly increasing populations in both developed and developing countries.In Thailand, freshwater aquaculture is an important source of inexpensive and abundantprotein for human consumption. The average per capita consumption of fish in Thailandwas 24.5 kg/year in 1985. This consumption, by the year 1995, is expected to reach 29.8kg/year (Tore11, 1984). The increased needs for fish protein must be supplied by expansionand improvement of the aquaculture industry.For cultivation purposes, fish larvae have been collected traditionally from naturalwaters. Water pollution and over-fishing have, however, reduced the availability of theselarvae. Artificial breeding of the major cultivated species such as carp and catfish hasbeen carried out successfully since 1956. The percent survival of the larvae has, however,often been poor (National Inland Fisheries Institute, 1985). This has resulted in wastedtime and effort and financial loss. Presently, seedfish supply still remains inadequate tomeet the demands of the local farmers. These problems need to be resolved.The feeding of fish larvae from the hatching stage until the end of the larval stagewith live foods is difficult, expensive and impractical for large scale operations (Kanazawaand Teshima, 1988). Hence, extensive research on the development of practical diets forlarval culture should be conducted. The present study was aimed at investigating theeffects of practical diet formulations on the performance of larval fish. The study wascarried out on the larvae of two fish species: common carp and walking catfish. Commoncarp was an excellent candidate for the study due to its ability to utilize a wide range ofnatural and formulated diets (Jhingran and Pullin, 1988). Also, the common carp is easy1to spawn in captivity (Alikunhi, 1966), and it is considered to be representative of othercarp species (Smith, 1980). Walking catfish is an important species in commercialfisheries of Thailand and it is representative of the Clarias species.In accordance with the recent policies of the Department of Fisheries in Thailand(Suraswadi, 1989 and 1991) to restore endangered freshwater fish species, several studiesof induced spawning of the aforementioned species have been conducted in the Departmentlaboratories. Also, Sujaritvongsanon and Sermwatanakul (1989) succeeded in inducingspawning in some endangered species such as sheatfish (Ompok bimaculatus), barb(Labiobabus spilopleura) and Jullien's mud carp (Cirrhinus jullieni). Unfortunately, themortality of these larvae was very high. This was probably because appropriate diets forthese larvae have not been developed. •The present study was carried out to investigatepossible formulations for practical diets applicable to the culture of common carp andwalking catfish larvae in Thailand.This thesis is composed of 7 chapters. A literature review of previous studies onlarval fish nutrition is presented in chapter 2. General materials and methods aredescribed in chapter 3. In chapter 4, the results of comparisons of conventional formulateddiets with live foods used for feeding common carp and walking catfish larvae areprovided. Selected feeds from experiments described in chapter 4 were used as controls forexperiments outlined in chapters 5 and 6. In chapter 5, results relating to comparisons ofthe nutritive values of test moist and dry formulated diets for common carp larvae aregiven. The best moist and dry diets were chosen to compare with the feeds selected fromchapter 4. Experimental procedures for the study of the nutritive value of test dietformulations for walking catfish larvae (chapter 6) were identical to those employed inchapter 5. The overall conclusions of the thesis are presented in the final chapter.2CHAPTER 2LITERATURE REVIEWThe life cycle of a fish from fertilization to death consists of five periods:embryonic, larval, juvenile, adult and senescent (Balon, 1975). Larval development in fishis completed in two stages. It begins with the prelarval development stage where theunabsorbed yolk sac provides an endogenous source of nutrition and ends when the yolk isutilized completely. This is followed by a postlarval development stage where nutritionmust be provided from an exogenous source and starts from first exogenous feeding andextends to the formation or ossification of the axial skeleton (Balon, 1975 and 1986). Thesecond stage is also called the stage of mouth feeding. The chronological sequence ofevents during the period of yolk utilization and concomitant development of internal andexternal organs has been reviewed by Stroband and Dabrowski (1981) and McFarlane etal. (1991). The successful transition from endogenous to exogenous sources of nutrition iscrucial for commercially efficient fish culture.2.1 LARVAL MORTALITYThe major causes of mortality during the early life stages of fish under naturalconditions are starvation and predation (May, 1974; 01la and Davis, 1990). Efficient fooduptake and assimilation by larvae are important factors to keep starvation at a minimumand these factors are dependent upon mouth size (Shirota, 1970; Hartmann, 1983) and thedevelopmental stage of the digestive enzyme system (Govini et al., 1986; Segner et al.,1989). Other factors influencing larval feeding behavior, and therefore larval survival,include water temperature and quality, salinity, and light color and intensity (Alderdice,31985; Chakrabarti and Jana, 1991). Of these factors, the development of mouth size andthe digestive enzyme system are reviewed below.2.1.1 Mouth sizeMouth size determines the size of prey that can be swallowed (Wankowski, 1979;Hoyle and Keast, 1988) and therefore this variable has considerable effect on the growthof larvae fed either live foods or formulated diets. Prey size increases with the mouth size(Hartman, 1958; Wong and Ward, 1972). Applegate (1981) observed that as muskellungefry (Esox masquinongy) grew, their mouth size increased and this was reflected by theincreased size of ingested organisms. Shirota (1970) measured the mouth gapes of 33species of marine and freshwater larval fish and correlated them with the sizes of ingestednatural foods and with larval growth rates. The study concluded that larvae with smallermouths grow more slowly than those with larger mouths. Mouth size can, therefore, be agood determinant of growth rate especially during the first few days after hatching. Adirect relationship between mouth size and body length has been noted in many specieslike walleye larvae Stizostedion vitrem (Mathias and Li, 1982) larval lake white fishCoregonus clupeaformis and lake herring Coregonus artedii (Arts and Evans, 1987). Wherephytoplankton and zooplankton in ponds provide exogenous nutrition, small mouth size orlarger sizes of plankton may result in poor survival (Woynarovich and Horvath, 1980).The larvae are then unable to feed on their prey.Mouth size varies greatly in cyprinid larvae. The ranges for ingestible food particlesizes for silver carp, grass carp and bighead carp have been estimated to be 50-90, 90-150, and 150-270 ixm, respectively (Dabrowski and Bardega, 1984) and these rangesreflect the mouth sizes of larvae of the respective species. Common carp larvae, right4from the beginning, can accept much larger food particles ranging from 0.3-0.4 mm(Dabrowski et al., 1983), whereas coregonid larvae accept food particles of 0.2 mm indiameter (Dabrowski et al., 1984). The body length of fish larvae is used as a parameterfor the measurement of growth (Hartmann, 1983).2.1.2 Larval digestive systemMost larval fish at the time of hatching are small and they possess an undevelopeddigestive system which cannot adapt to exogenous food. There are exceptions to thisgeneralization, such as the salmonids, which readily adapt to exogenous feeding shortlyafter hatching (Dabrowski, 1984a). The functional development of the digestive tract offish has been described for many species such as ayu Plecoglossus altivelis and pond smeltHypomesus olidus (Iwai, 1967a,b), whitefish Coregonus lavaretus (Bogdanova, 1972;Segner et al., 1989), juvenile grass carp Ctenopharyngodon idella (Stroband, 1977), larvaeof two cyprinid species Rutilus rutilus and Chalcaburnus chalcoides (Hinterleitner et al.,1989) and cod larvae Gadus morhua (Kjorsvik et al., 1991). Tanaka (1971) observed thatthe digestive system in first feeding larvae of 13 teleosts is stomachless in structure andmay be at an undifferentiated state of function. Subsequent studies on the development ofmorphological and physiological aspects of the digestive system of these larvae byStroband and Dabrowski (1981) classified them into three different groups based on theirgastrointestinal proteolytic enzyme activities.The first group is stomachless and the fish never develop pepsin digestion. Thisgroup consists mostly of cyprinid fish which have epithelial cells between the esophagusand the intestine that provide an area similar to the stomach epithelium (Labhart andZiswiler, 1979). Smallwood and Smallwood (1931) and Sriratanakul (1988) reported, in5common carp, that enormous goblet cells are present along the digestive tract whenfeeding commences two days after hatching whereas maltase and amylase activitiesdevelop during 7-10 days after hatching (Kawai and Ikeda, 1973b).The second group of fish have stomachless larvae which develop a stomach andpepsin digestion at the end of the larval period. In coregonid juveniles, for example, pepticdigestion cannot take place until 97 days of age (Mahr et al., 1983; Loewe and Eckmann,1988). In walking catfish larvae, the digestive tract and the oral mouth are completelydeveloped 3 days after hatching (Chandrasardula, 1989). In an African catfish (Clariaslazera), the first functional cells develop in the corpus of the stomach which contains atubular gland system secreting pepsinogen and HC1. The functional stomach is present incatfish juveniles when their body length nearly doubles after hatching (Stroband andKroon, 1981).Functional stomachs in salmonids, which have no larval period (Balon, 1975), areformed at the alevin stage i.e. a stage between hatching and feeding when the yolk sac isstill present. This allows them to adapt easily to formulated diets (Kawai and Ikeda,1973a; Lauff and Hofer, 1984). These advantages have prompted numerous investigatorsto explore the feasibility of using formulated diets for first feeding of the alevins.2.2 AVAILABILITY OF LARVAL FOODYolk provides an endogenous food source at the beginning of larval development.Subsequent transition to exogenous food sources, natural or formulated is critical for thesuccess of aquaculture (Smith, 1981; Krise and Meade, 1986; Meyer, 1987) and significantmortality is associated with unsuccessful transition (O'Connell and Raymond, 1970;Heinrich, 1981). Availability of suitable exogenous food, therefore, becomes more crucial6as the first feeding larvae are very sensitive to food deprivation and soon reach a pointwhere the harmful effects of starvation become irreversible (Blaxter and Ehrlich, 1974;Werner and Blaxter, 1980). Ingestion and digestion of food are the obvious indicators ofits acceptance by larvae. This acceptance is reported to be enhanced by making multiplechoices of food available (Zaika and Ostrovskaya, 1972). Exogenous food can be acompletely formulated diet, live food, or a combination of the two in various proportions.As complete success of aquaculture with formulated diets has yet to be achieved, largescale rearing of fish larvae currently requires provision of live foods that can be obtainedregularly and inexpensively. The making of an acceptable formulated diet for early ortransition stage larvae is an attractive area for scientific research.2.2.1 Live foodsLive food organisms for fish larvae include phytoplankton and zooplankton, as wellas microorganisms. Moina, Artemia nauplii and rotifers are the popular live foods offeredto growing fish larvae. Multiple species of these organisms are available around the worldand the nutritive value and efficiency of this diversified group of organisms have beenstudied extensively. Artemia can form cysts that are storable and will release freeswimming nauplii on incubation in saline water. These nauplii constitute an excellent foodsource for newborn fish larvae (Seale, 1933). The nutritional value of live foods for larvalfish has been well documented (van der Wind, 1979; Yurkowski and Tabachek, 1979;Watanabe et al., 1983; Leger et al., 1986). Many investigations have been focused at theculture of live food for feeding different species of fish at the larval stage (PathumthaniAquaculture Development Station, 1987; Pillay, 1990). Various reasons have been givenfor the suitability of live food species for fish larvae. It has been suggested, for instance,7that the prey themselves contain digestive enzymes, and that these enzymes help in thedigestion of the prey inside the larval gut. Dead Artemia nauplii inside the larval gut havebeen shown to have a good nutritive value and to contain digestive enzymes (Munilla-Moran et al., 1990). Another suggested reason for the general superiority of live foods isthat the prey often contain relatively high levels of free amino acids that are easilyabsorbed by the larvae (Dabrowski and Rusiecki, 1983; Holm and Walther, 1988).Techniques for nutrient enrichment of live prey have been used to increase theirnutritive value as larval foods. These have been applied with respect to: fatty acid,protein, vitamin and mineral content (Robin, 1985).Highly unsaturated fatty acids, especially eicosapentaenoic acid (20:5w3) anddocosahexaenoic acid (22:6w3) are essential fatty acids for membrane formation,osmoregulation and synthesis of prostaglandins in marine fish, crustaceans and molluscs.These fatty acids may not, however, be present in adequate amounts in live foods(Watanabe et al., 1983). Live foods such as rotifers and Artemia nauplii may be enrichedin essential fatty acids by adding these fatty acids to the culture media for theseorganisms. The feeding of microcapsules containing a high percentage of lipids to rotifersand Artemia nauplii has been shown to increase their nutritive value for marine larval fish(Walford and Lam, 1987). Enrichment of live foods has been used to improve growththrough the first week of larval development in various species i.e. sea bass larvae,Dicentrarchus labrax (Van Ballaer et al., 1985), Asian seabass, Lates calcarifer (Dhert etal., 1990), and dolphin larvae, Coryphaena hippurus (Ostrowski and Divakaran, 1990).The culture of live food is laborious and requires expensive equipment (Pourriot,1989). Success of culture also fluctuates with the environmental conditions.Unpredictable mortality of fish larvae frequently occurs due to undesirable variations in8the culture of live foods (Vanhaecke and Sorgeloos, 1983). The presence of theundigestible shell of Artemia nauplii may be harmful when ingested by larvae (Bruggemanet al., 1980). Bacterial and fungal spores may contaminate cyst shells and the infectionmay then be transferred to larvae (Wheeler et al., 1979). Artemia nauplii from differentgeographical origins may be very different in their nutritional value (Wickins, 1972; Becket al., 1980; Watanabe et al., 1983; Webster and Lovell, 1990a). In Thailand, Artemiacysts are imported and hence they are expensive (Tunsutapanit, 1985). This practiceraises the cost of fish and shrimp larval culture. Formulated diets, therefore, provide anopportunity for the replacement of live foods (Jones and Gabbott, 1976; Teshima et al.,1982; Kanazawa, 1986).The process of selective prey consumption by larvae is important in determiningthe size of live foods. The electivity index of Ivlev (1961) is often used to compare the sizedistribution of live foods in the larval gut to their distribution in the growing medium. Thisis explained by Zaika and Ostrovskaya (1972 and 1973), Talbot (1985) and Lazzaro(1987). Electivity is described by the following equation.Electivity (E) = r - pr + pwhere:r = the percentage of occurrence of food organisms in gut,p = the percentage of occurrence of food organisms in rearing tank.The value E ranges from -1 to +1 through 0. When E = 0, this indicates that nopreference is exhibited by larvae during ingestion and the food organisms are eaten in thesame proportion as they occur in the available food. A positive E value indicates apreference for a food type while a negative E value indicates an avoidance for the same.92.2.2 Formulated dietsMany fish larvae do not utilize formulated diets very well. Some of these effectsare shown in Table 2.1. Very short and simple digestive systems in developing larvaehave been considered as major contributing factors towards reduced utilization of non-living food at the time of transition from endogenous to exogenous feeding (Govini et al.,1986). Larval stages of different fish have been frequently indicated to have an essentialrequirement for a special kind of living food up to the stage of metamorphosis. Dabrowskaet al. (1979) fed common carp with a formulated diet supplemented with extracts of fishdigestive enzymes. They found an improvement in growth and survival but the resultswere not as good as those with natural food. Purely formulated diets, withoutsupplementation with live foods, also have been used with limited success for larvae ofcertain species such as milkfish Chanos chanos (Santiago et al., 1983) and silver carpAristichthys nobilis (Carlos, 1988).Table 2.1 Effects of formulated diets on performance of some fish larvaeResults^ StudiesHigh mortality in common carpPoor growth in common carpUnsuccessful in catfishPoor survival in catfishPoor growth in walleyeDabrowski and Poczyczynski, 1988Lubzens et al., 1984Knud-Hansen et al., 1990Verreth et al., 1987Nickum, 1978; Colesante et al., 1986Formulated diets, unlike live foods, can provide a constant and reliable source ofhigh quality food with the objective of high survival and satisfactory larval growth. Thephysical characteristics of food are important. Diets comprised of small andhomogeneously sized particles with stability towards storage are recommended. Food10should be water stable since larvae are highly sensitive to water pollution that may becaused by the presence of water-unstable feed (Chow, 1980; Pigott and Tucker, 1989).Optimum dietary particle sizes vary according to fish species and age and they may rangefrom 5-300 pm diameter. High nutritive value, palatability, convenience of preparation,transport and storage are also considerations. Larval dietary acceptance is not only afunction of food size but also texture, and flavor. Gunkel (1979) observed that coregonidlarvae spit out unacceptable diets after mouthing them. Final selection of the diet by solelarvae has been observed to occur in the mouth (Appelbaum et al., 1983). Odor receptorstructures present in the olfactory region of common carp larvae are functional at an earlystage and they have been hypothesized to have a role in selective food intake (Appelbaum,1980).Formulated diets may be classified according to moisture content. On this basis,diets may be either dry, moist or wet (New, 1987). Dry diets are made by drying feedsmade from moist ingredients, or from mixture of predominantly dry and small amount ofmoist ingredients, and they have moisture contents of 7-13%. These diets are generallyless expensive to manufacture, transport and store (Higgs, 1986) than moist or wet diets.Wet diets are made from high-moisture content ingredients and they have moistureconcentrations ranging from 45-70%. Moist diets are made by mixing wet, moist and dryraw materials and their moisture content varies from 18-45%.Larval formulated diets or microparticulate diets, based on manufacturing method,can be presented in microencapsulated, microbound and microcoated forms (Kanazawa,1986; Hardy, 1989a). Microencapsulated diets are made by encapsulating a solution,colloid or suspension of dietary ingredients with a membrane. Microbound diets areprepared by combining dietary ingredients with a binder such as carrageenan, agar, zein11or gelatin. Microcoated diets are made by coating a microbound diet with a water-insolublematerial such as cholesterol, lecithin, or nylon-protein.A balanced formulated diet must include an energy source and total protein withsufficient essential amino acids, total lipid with adequate essential fatty acids, vitaminsand minerals to support life and promote growth (Halver, 1979; Hardy, 1989b). Thenutrient compositions of feedstuffs, their market availabilities, digestibility coefficients,palatabilities and costs are important factors determining their use (March, 1990).Measurements of food intake, weight increment and digestibility are extremelydifficult at the larval stage of life history and this has limited the development of suitableformulated diets. Indeed, larval nutritional requirements have been extrapolated fromthose established for fry and juvenile fish for use in diet formulation. These methods arerecognized by NRC (1983) although they are not scientifically reliable for this purpose.Amino acid requirements of certain fish have been estimated from the essential amino acidcompositions of their whole body tissue and eggs (Wilson, 1989; Nose and Murai, 1990)and subsequently they have been used to formulate diets for fish like rainbow trout andAtlantic salmon (Wilson and Cowey, 1985), channel catfish, Ictarulus puntatus (Wilson andPoe, 1985), larval ayu, Plecoglossus altiveli (Kanazawa, 1986) and larval Dover sole, Soleasolea (Dendrinos and Thorpe, 1987).Little information is available on formulated diets and the nutritional requirementsof commercially important fish larvae in Thailand. The National Inland Fisheries Institute(NIFI) standard diets have been developed in an attempt to best meet the requirements forgrowth of the larvae. A formulated diet for larval carp species i.e. common carp and Thaicarp, is composed of 30% fish meal, 45% rice bran, 24% peanut meal and 1% premix andthis diet is dispensed in term of a powder (Sitasit et al., 1982).12Sitasit and Fedoruk (1981) modified the NIFI standard diet for walking catfishlarvae to the following composition 56% fish meal, 12% rice bran, 10% peanut meal, 10%starch, 10% fish oil, 1% guar gum and 1% of a vitamin-mineral premix. When this dietwas fed twice a day to 5-day old larvae, which were stocked in earthen ponds at a densityof 300/m2, the percent survival of the larvae was only 48%. Another practical diet is thecatfish standard larval diet No. 12 developed by the National Inland Fisheries Institute(NIFI). It is composed of 56% fish meal, 12% rice bran, 12% peanut meal, 14% starch, 4%fish oil, 1.6% vitamins and minerals and 0.4% binder (Sitasit et al., 1982). To date, noentirely satisfactory formulated larval diet has been developed.2.2.3 Combination of live foods and formulated dietsThis system of feeding can be conducted in two ways. In one method, larvae arefirst fed on live food and then a formulated diet is introduced gradually. This weaning overof foods may not be sudden and mixed food is offered for a short time before larvae becomeused to the formulated diet. In the second method, larvae are fed on a mixture of live andformulated foods from the beginning. The choice of method depends on the species of fish.Appelbaum (1985) reared Dover sole larvae (Solea solea) with best results when liveArtemia nauplii were provided for the first 10 days of feeding followed by a formulateddiet. Lindberg and Doroshov (1986) and Moksness (1991) suggested that a period of livefeeding and a period of weaning on mixed live and formulated diets were required for whitesturgeon (Acipenser transmontanus) and common wolffish (Anarhichas lupus).The schedule for feeding live foods and formulated diet has been established for theproduction of larvae (Watanabe, 1988). It varies according to the gape limitation of larvalfish and food selectivity as discussed previously. For example, three main steps have been13used in marine larval culture. Fish larvae, when they are first fed, initially select smallzooplankton such as rotifers (100-300 pm). As growth progresses, fish gradually adapt tolarger live foods such as Artemia nauplii (200-500 pm) and copepods (400-1,000 pm) andfinally to a formulated diet.2.3 SELECTED SPECIES2.3.1 Common carp (Cyprinus carpio)The common carp (Cyprinus carpio) belongs to the cyprinidae family and it has foursubspecies (Kirpitschnikov, 1976) i.e. C. c. carpio of the European, C. c. aralensis of themid-Asian region, C. c. haematopterus of the Amur-Chinese or Far Eastern region and C. c.viridivio of Vietnam. It is a native of the temperate regions of Asia, especially China andJapan. Common carp was introduced to Greece and Europe through Rome (Jhingran andPullin, 1988) and has been transplanted into many countries.Common carp eggs are 1.5-1.6 mm in diameter. The egg incubation perioddepends on water temperature and normally it is about 24 hours at 25°C. The bodylength of larvae at the hatching stage is 4-6 mm (Alikunhi, 1966). Mouth size is 570 pmat the onset of feeding which is about 2-3 days after hatching (Shirota, 1970). Commoncarp feed on zooplankton during the early larval stages. Adult common carp areomnivorous and detritivorous. It is a stomachless fish which is typical of many cyprinidsincluding goldfish, squawfish, minnows, dace, chubs and tench in North America (Smith,1980). The carp spawn throughout the year in tropical waters (Ling, 1977; NationalInland Fisheries Institute, 1981b) so that larvae are always available.14Several systems of breeding common carp have been developed under localconditions (Woynarovich and Horvath, 1980). In Thailand, common carp brood fish arereared in earthen ponds and breeding is induced in cement tanks. Water is continuouslysprayed into the tanks to simulate natural rain fall. Artificial aquatic weed, made fromnylon string, is suspended in the water to serve as a substrate for egg deposition.Spawning occurs 10-12 hours later. Eggs are taken to other cement tanks for hatching.The brood fish are then returned to the earthen ponds after spawning.Commercial production of common carp larvae currently relies almost completelyon the culture of live foods (Bryant and Matty, 1980; Kamler et al., 1990). Nauplii ofArtemia are the most successful and the most convenient first food for rearing cyprinid fishlarvae (Prolubnikov and Kokova, 1984). Dabrowski et al. (1978) showed that primefeeding for the first three days with live zooplankton before feeding a formulated diet,resulted in improved growth and survival in common carp larvae.So far attempts to rear larvae to the fry stage solely on formulated diets followingsuccessful artificial fertilization of common carp have had limited success (Szlaminska andPrzybyl, 1986; Kamler et al., 1990). Theoretically, it should not be difficult to produceformulated diets with similar chemical compositions to natural foods (van der Wind, 1979).Studies regarding the feeding of formulated diets to larvae have been conducted mainlywith reference to larval characteristics such as possible enzyme deficiencies (Dabrowska etal., 1979), specific nutritional needs (Dabrowski and Kaushik, 1982; Fluchter andRembold, 1986) and to the rearing systems employed for the larvae (Charlon and Bergot,1984).The nutritional requirements of common carp have been reviewed by NRC (1983)and Pillay (1990). The recommended dietary protein concentration for maximum growth15ranges from 25-38%. The amino acid requirements expressed as a percentage of proteinare: arginine 4.3, histidine 2.1, isoleucine 2.5, leucine 3.6, lysine 5.7, methionine pluscystine 3.1, phenylalanine plus tyrosine 6.5, threonine 3.9, tryptophan 0.8 and valine 3.6.Lipid requirement is 18% and the diet should contain at least 1% of the w3 and 1% w6series fatty acids (Watanabe et al., 1975; Takeuchi and Watanabe, 1977).2.3.2 Walking catfish (Clarias macrocephalus)Walking catfish belong to the family clariidae of which five species have been foundin Thailand. Clarias macrocephalus and Clarias batrachus, locally known as "pla duk oui"and "pla duk dan" are the only species of economic importance (Sidthimunka, 1972).They are distributed widely in both water and muddy areas (National InlandFisheries Institute, 1981a). Walking catfish larvae have been collected traditionally fromcanals and ditches where they breed in the comparative calm of the marginal shallowsabout 50 cm below the water surface. After capture, they are transferred into cementtanks for rearing (Sidthimunka et al., 1966). Pond breeding methods were developed byVanichakorn (1957) and Sidthimunka and Aguru (1959) to meet the increasing demand forwalking catfish larvae. The procedure involved making holes in pond banks and providingsome weeds for attachment of eggs. Tongsanga et al. (1963) and Sidthimunka et al. (1966)succeeded breeding C. macrocephalus artificially in small aquaria for commercialproduction of seed. However, the larval nursing operation is subject to high mortality.Mortality of up to 80-90 % can occur in larvae between 5-15 days of age due to improperdiet (Sitasit and Fedoruk, 1981) or infectious diseases (Areerat, 1987). Mortality can alsobe caused by poor water conditions. Larvae are very sensitive to low concentrations ofoxygen in the water (Carreon et al., 1976). Although the catfish has an accessory16respiratory organ (labyrinthine) to use atmospheric air directly (Ling, 1977), the organ isnot functional until the larvae are at least 10 days of age, (Carreon et al., 1976). Moinaprovides the major feed for catfish larvae in Thailand. The availability of Moina may belimited, especially during the rainy season (Taechajanta and Sitasit, 1981).Some information has been reported on the protein requirements of Clarias species.The protein requirement of 100 mg C. batrachus fry was found to be 30% by Chuapoehuk(1987), 35% according to Hasen et al. (1989) and 40% according to Khan and Jafri (1990).Cruz and Laudencia (1976) reported that further increases in the dietary protein contentdid not result in corresponding increases in growth of the fish.17CHAPTER 3GENERAL MATERIALS AND METHODS3.1 EXPERIMENTAL SITEThe experiments on the development and testing of practical diet formulations forcommon carp (Cyprinus carpio) and walking catfish (Clarias macrocephalus) larvae werecarried out during 1989-1990 at Burirum Freshwater Fisheries Station in BurirumProvince (Lat. 14° 55'; Long. 103° 02') of Thailand. The station is located 400 kmnortheast of Bangkok (Fig. 3.1). All of the aquaculture activities at the station use waterfrom underground and a nearby irrigation reservoir (Huai Jaurakay Mark). However,stored rain water was used in this study to control the experimental conditions.3.2 DIET PREPARATION3.2.1 Moina cultureMoina were cultured according to normal practice in a cement tank in which thewater was approximately 25-40 cm deep (Fig. 3.2). Continuous production was supportedby maintaining the water level and adding rice bran as a source of nutrients.Phytoplankton, especially Chlorella, was a major food source for Moina in the tank. After4-7 days, the Moina were harvested and rinsed with tap water before they were fed to thelarvae.18LAOSfrj•71. 1ENG RAI• TAX•UOORN•SAXON•NAKORK RAJSIMASUDAN •CHAINAT • •U8ONPHATHALUNGSONGPUK• CRIENG MAI• RICHIT•NAKORN $AwAN•KHORN KAEN•MAHA UR...4(Am• Burirum•(BANGKOKCAMBODIARAYONGHANOHA BURP• RACHUA8GULF OFTHAILANDMALAYSIATHAILAND• MARINE• BRACKISH WATER• FRESHWATERFigure 3.1 Geographical distribution of some freshwater fisheries stations in Thailand.•NONG KAI19Figure 3.2 Cement tanks used for Moina culture and spawning ground of common carp atBurirum Freshwater Fisheries Station.Figure 3.3 Glass containers for Artemia culture.203.2.2 Artemia cultureArtemia eggs were hatched according to the suppliers specifications (Pro 90 AlexBrand, Grade A from USA). Glass containers were used for incubation of the eggs (Fig.3.3). Eggs were hatched within 12-15 hours at 27±1°C and 30-35 ppt salinity. Atharvesting, the aeration system was turned off, and the glass containers were coveredwith dark paper except at the bottom. The nauplii then concentrated at the bottom andthe eggshells floated to the surface. The nauplii were collected by siphon, rinsed with freshwater and then fed to the larvae. Hatching debris were separated from the nauplii toprevent blockage of the digestive system in the larvae.3.2.3 Moist dietMost of the ingredients were chosen according to their availability from the localmarket (Fig. 3.4) and their tabulated nutrient compositions (Sidhu et al., 1970; Leung etal., 1972; Gallagher and Brown, 1975; Consumer and Food Economics Institute, 1979aand 1979b; Becker, 1981; Gohl, 1981; Consumer Nutrition Division, 1983; NRC, 1983;Patterson, 1983 and 1989; Nutrition Monitoring Division, 1984, 1987, 1989 and 1991;New, 1987; Allen, 1988; Kay, 1991). All the ingredient composition data were loaded intothe computer with Lotus 1-2-3 program release 2.2 for calculation of diet composition i.e.protein, lipid, carbohydrate, amino acid and fatty acid content. Carrageenan is aneffective binder for a microparticulate diet which supports growth of larval prawns,Penaeus japonicus (Koshio et al., 1989) and several species of fish (Kanazawa andTeshima, 1988) and this was used in the present study.21Figure 3.4 Burirum local market.22Fresh ingredients were each chopped into small pieces. Thereafter, each wasblended in a Mulinex blender and sieved using 53 pm diameter sieve size. Diet ingredientswere mixed and heated in a water bath at 85°C until the K-carrageenan dissolvedcompletely. The mixture was then cooled to room temperature and chopped to a particlesize of about 5x5x5 mm 3 for use as a moist diet. Portions of these mixed diets were driedovernight at room temperature (20°C), ground and sieved to the appropriate sizes: 125and 250 pm in diameter. The microparticulate diets were preserved under nitrogen toprevent oxidation and stored in the refrigerator until used. All diets were freshly preparedfor each experiment.3.2.4 Dry dietThe ingredients such as fish meal, peanut meal, soybean meal, rice bran meal etc.were ground to small particles to facilitate mixing. The ingredients were sieved through125 and 250 pm diameter mesh sieves. All dry ingredients were mixed thoroughly beforeadding oil, and the ingredients were then mixed again. Ingredients were hand mixed forfairly long duration to ensure adequate mixing. Prepared diets were kept in therefrigerator. The size of particles offered to fish was 125 pm during the first 10 days ofthe experiment and was increased to 250 pm after that. Larvae were fed twice a day.This feeding pattern mimics the conventional feeding pattern used for commercial larvalculture in Thailand.The proximate composition of the diets in experiments 4 and 7, and of theindividual ingredients were determined according to AOAC (1984) at the Fish NutritionSection, National Inland Fisheries Institute, Bangkok. Each sample was analysed in threereplicates. The crude protein contents (%) of ingredients and diets were determined by the23Kjeldahl technique. The percentage of total nitrogen in the sample was converted topercentage crude protein by multiplying by the empirical factor of 6.25 assuming, thatproteins contain an average of 16% nitrogen. Lipid content was measured by Soxhletextraction using petroleum ether. Moisture content was determined by drying samples toconstant weight at 95°C in an oven. Ash was determined by heating samples in a mufflefurnace for 3 hours at 600°C. Carbohydrate (nitrogen-free extract and fibre) wascalculated indirectly by difference following the quantitative analysis for moisture, protein,lipid, and ash content.3.3 SOURCE OF LARVAE3.3.1 Common carp larvaeThe common carp was chosen as a model species in this research because it hasworldwide distribution (Alikunhi, 1966). It constitutes a greater proportion of culturedfreshwater fish from aquaculture than any other species (FAO, 1991). Common carplarvae are planktivorous and they feed mainly on zooplanktonic organisms and later theybecome benthic feeders and omnivorous. Common carp will, therefore, provide a usefulmodel and the results can be applied to other endangered carp species.Generally, the sex of carp is difficult to distinguish except during the spawningseason. The female shows a soft and distended belly as well as a reddish rim around thegenital pore while the male shows a roughness of the pectoral fins and body. During thespawning period, male and female common carp brood fish (Fig. 3.5) of approximate age1.5-2 year and weight 250-400 g were chosen and taken from separate earthen ponds(Fig. 3.6).24Figure 3.5 Common carp brood fish.(A) Female (B) Male25A 50 m3 cement tank was prepared as the spawning ground for the common carp(Figs. 3.2 and 3.7). They were spawned in a cement tank with the ratio of male : femaleequal to 2:1. To protect the carp eggs from predators such as insects and tadpoles, thetank was cleaned and filled with fresh water on the day of spawning. Water was sprayedcontinuously into the tank to simulate natural rain-fall (Fig. 3.7). Artificial aquatic weedmade from nylon string was suspended in the water to serve as a substrate for eggdeposition. Spawning took place at dawn or in the early morning which was 10-12 hoursafter releasing brood fish into the cement tank. Eggs which stuck to substrate were takento fiberglass tanks with aerated water for hatching. The hatching period was 36-40 hoursat 27±1°C water temperature. During yolk absorption, common carp larvae weretransferred to a glass jar or aquarium containing aerated water (Fig. 3.10). At this time,dead larvae were replaced to maintain the number of experimental larvae. Every bodylength of common carp larvae at the beginning of the experiments was measured to be 4.9MM.26Figure 3.6 Earthen pond for rearing brood fish.Figure 3.7 Spawning tank for common carp.273.3.2 Walking catfish larvaeC. macrocephalus larvae were selected for this study because of the local higheconomic importance of the species (Kloke and Potaros, 1975). Catfish brood stock (1-2year of age, 150-250 g) were purchased from private farms in Bangkok, Burirum andnearby provinces. They were transported to Burirum Freshwater Fisheries Station forinduced breeding using a modification of the method outlined by Woynarovich and Horvath(1980) and Srisuwantach and Thangtrongpiros (1985).Breeding was induced by injection of gonadotrophic hormone. In this technique,gonadotrophic hormone for induced spawning is extracted from the pituitary glands(hypophysis) of some fish (donor) such as common carp, Chinese carp, Indian carp,walking catfish and striped catfish (Pangasius sutchi). This hormone is injected into bothmale and female brood fish (recipient).In this experiment, the pituitary glands were taken from walking catfish andstriped catfish. They were inexpensive ($0.04 each) and readily available from the localmarket of Burirum. The pituitary gland (3 mg each) came from approximately 1 kgcatfish which had been caught 2-3 hours prior to the experiment. The short time betweendeath and incision of the pituitary gland ensured that the tissue was still a reliable sourceof hormone activity. To excise the pituitary gland, the skulls were cut open with a knife.The brain was folded back with forceps and the pituitary gland under the mid-brain wastaken out. The glands were washed with 95% ethyl alcohol, preserved in fresh 95% ethylalcohol and stored in a refrigerator until needed.The pituitary glands of the donor fish were homogenized. Aliquots of distilledwater were added depending on the weight of recipient i.e. about 0.5 ml were added forfish weighing < 1 kg and 1 ml for fish weighing between 1 and 3 kg. Both males and28females were given an intramuscular injection of pituitary gland solution below the dorsalfin.Sex was distinguished by the shape of the genital papilla. That of the female isoval whereas the male's is pointed (Fig. 3.8). Female brood fish were tranquilized in 100-120 ppm quinaldine, and those with ripe eggs were selected. The belly of the sexuallymature female is soft and distended, and the rim around the vent is dark pink. Selectedmale and female brood fish were placed in separate containers before initiating artificialfertilization. Brood fish to be spawned were weighed. A system of two injections had beenevolved in which the first injection called a stimulating dose, consisted of 3 mg of pituitaryper kg of body weight of the recipient female, followed 12 hours later, by a second dose,called a resolving dose of 15 mg/kg body weight. In this manner, a total of 18 mg/kg bodyweight was injected into each female spawner. Males were injected with a single dose of 3mg/kg body weight at the same time as the first injection of the female.29Figure 3.8 Walking catfish brood fish.(A) Female (B) Male30Ovulation occurred 10-12 hours after the second injection. At this time, femaleswere wiped with a towel and pressed slightly at the abdomen to express the eggs. Thestripped eggs were collected in a dry container and wrapped with plastic film to preventdehydration. After each female was stripped, a male was killed. The testis was wrappedwith plastic net and squeezed strongly to obtain sperm into a separate dry container. Asmall amount of water was added to the milt. The milt and eggs were mixed, water wasadded and stirred as quickly as possible to assure high fertilization. Fertilized eggs werewashed with fresh water and the process was repeated three times. The eggs were thenready for incubation in hatchery tanks. Fertilized eggs were released onto the hatchingnet which was situated over the tanks and made from a 2-2.5 mm nylon mesh sieve whichmeasured about 1 x 1 m2. The eggs were sedimented and attached on the hatching netand incubated at 27±1°C with a constant flow of water and continuous aeration (Fig. 3.9).The hatching period was 19-26 hours. The hatched larvae fell through the hatching netand were retained at the bottom of the tanks. At 72 hours after hatching, the larvaeswam up and were transferred to glass jars or aquaria (Fig. 3.10). Every body length ofwalking catfish at the beginning of the experiments was measured to be 7.45 mm.31Figure 3.9 Hatching tank for walking catfish larvae.Figure 3.10 Experimental unit.323.4 EXPERIMENTAL DESIGN3.4.1 Larval feedingUpon yolk absorption, i.e. three days after hatching, fish larvae were placed intothe indoor aquaria (45x90x45 cm3) at a density of 1,000 larvae per aquarium. Theaquaria were covered with plastic lids. They were supplied with aerated, fresh water(27± 1°C). Continuous aeration maintained the concentration of dissolved oxygen at 8-9ppm.3.4.2 SamplingA random sample of 40 larvae was taken from each aquarium at the end of eachexperiment. Body lengths of larvae were measured from the tip of the snout to the end ofthe notochord using vernier calipers. Percent survival was also assessed at the end of theexperiment.3.4.3 Data analysisStatistical analyses of the results form each feeding trial were performed usingone-way analysis of variance (ANOVA). Tukey's HSD test with P=0.05 was used whereappropriate to detect significant differences among the means for body length in relation todiet treatment. For percent survival data, the arcsine transformation was used to achievehomogeneity of variance. Then the data were analyzed by ANOVA and Tukey's HSD test(Zar, 1984).33The University of British Columbia computing facilities were used for most of thedata compilation and the statistical analyses of the results. A computer program "Lotus1-2-3 release 2.2" was developed to facilitate formulation of the diet compositions.3.4.4 Criteria of larval feed acceptanceLarval feed acceptance responses were classified according to the following criteria:1. Approach: assessed by the degree to which the larvae swam towards the dietor were attracted by the diet.2. Ingestion: judged by the degree of food particle intake by the larvae.3. Acceptance: assessed by whether the larvae gained weight and werevigorously growing.4. Rejection: evaluated by whether the larvae died, lost weight or were weak andswam slowly.These response criteria, pertaining to the larvae fed the formulated diets, weresimultaneously compared to those of the fish fed with Artemia nauplii. Feeding responsefor each of the above criteria was given a score of 1 = poor, 2 = fair, 3 = good and 4= excellent.34CHAPTER 4EXPERIMENT 1: COMPARATIVE PERFORMANCE OF COMMON CARP ANDWALKING CATFISH LARVAE FED COMMONLY USED DIETS4.1 IntroductionCarps, catfish and tilapias are the main freshwater fish species produced bygovernment hatcheries in Thailand (Department of Fisheries, 1988). Their larvae arenursed in hatcheries for 15-20 days and thereafter they are stocked into rearing ponds ordistributed to farmers. Moina and Artemia nauplii are the commonly used foods for fishseed production. Provision of these foods is, however, limited.Moina is considered to be an excellent natural food for larval (PathumthaniAquaculture Development Station, 1987) and tropical aquarium fish (Shim, 1988)production. Moina originates from freshwaters and can only be collected in the earlymorning hours (National Inland Fisheries Institute, 1985), when it migrates to the surfaceas the light intensity increases and dissolved water-oxygen content decreases. Cultivationof Moina has been established to augment the supply from natural sources. Naturalproduction of Moina is constantly subject to fluctuations due to abrupt changes in theenvironment which may cause dramatic reductions in the rate and mode of itsreproduction. Artemia sauna has also been used as a food source in crustacean and fishhatcheries (Pillay, 1990; Vanhaecke et al., 1990). The culture of Artemia requires highmaintenance and extra facilities to obtain a consistent rate of production (Teshima et al.,1982) and the supply is consequently limited.There is an increasing trend to replace live or non-living foods with formulateddiets. Non-living foods such as hard-boiled chicken egg yolk, whole egg, or egg yolk plus35skim milk, followed by rice bran meal have been used during early larval rearing (Davyand Chouinard, 1981). The success rate for these foods has been low and often limited byhigh larval mortality and susceptibility to disease. These effects have been attributed tonutrient deficiencies or imbalances and/or to poor acceptance of the foods by larvae(Chuapoehuk and Boonyaratpalin, 1983). Various formulated diets have been developedand recommended by the National Inland Fisheries Institute (NIFI) of Thailand toovercome these problems. These diets have been adopted widely for larval feeding in thatcountry (Sitasit et al., 1982) but larval mortality is still high with their use (NationalInland Fisheries Institute, 1985).In this part of the study, the aim was to assess the effects of live foods Moina andArtemia nauplii and formulated NIFI diets on the growth performance and survival ofcommon carp and walking catfish larvae. The purpose was to select the best performingdiet as the reference diet for subsequent experiments.4.2 Materials and methodsMoina and Artemia were cultured as described in chapter 3. Two NIFI diets(Sitasit et al., 1982) differing in ingredient composition were prepared. The compositionsof the diets are shown in Table 4.1. Ingredients were ground and sieved through a 125 prnsieve mesh prior to mixing. The diets were kept refrigerated at 4°C until used.36Table 4.1 Composition of NIFI diets for common carp and walking catfish larvae inexperiment 1Ingredients (% as fed) DietsNIFI 1* NIFI 2**Fish meal 30.0 56.0Rice bran 45.0 12.0Peanut meal 24.0 12.0Alpha-starch 14.0Fish oil 4.0Wheat gluten 0.4Vitamins & mineralsa (NIFI) 1.0 1.6Total 100.0 100.0* NIFI 1 and ** NIFI 2 commonly used for common carp and walking catfish larvae, respectively.a The vitamin supplement supplied the following levels of nutrients/kg of dry diet: niacin, 100 mg; d-pantothenate, 24 mg; pyridoxine HC1, 20 mg; riboflavin, 8 mg; thiamine, 5 mg; Na-ascorbate, 500 mg; folicacid, 1 mg; choline chloride, 1400 mg; vitamin K, 4 mg; vitamin A, 12,000 IU; vitamin E, 100 mg; vitaminD3, 4,000 IU; BHT, 50 mg. The mineral supplement supplied the following nutrients/kg dry diet: Neel,3000 mg; MgSO4.7H20, 1400 mg; C6H5Fe07.H20, 200 mg; MnSO4.4H20, 250 mg; KI, 10 mg;Ca(H2PO4)2.2H20, 6000 mg; CuSO4, 10 mg; KC1, 1000 mg; ZnCO3, 130 mg.Common carp larvae were reared either on Artemia nauplii, Moina, or the NIFI 1diet whereas walking catfish larvae were reared on the same diets except on a differentrecommended formulation, NIFI 2, instead of the NIFI 1 diet. Each diet was fed to tworeplicates. Newly hatched larvae from each species, as described in chapter 3, were keptin the hatching tanks until two days of age. Yolks were almost completely absorbed bythat time. Exogenous feeding was started on the third day, immediately after the larvaewere transferred to 6 aquaria which measured 45x90x45 cm3 in which the water depthwas approximately 60 cm. Forty fish were measured initially for body length. A total of1,000 larvae were kept in each aquarium. A twice-a-day feeding schedule was maintaineduntil 18 days of age when the experiment was completed. At the end of the experiment, asample of 40 fish from each replicate group was measured for body length. The average37water temperature and dissolved oxygen content were kept at 27±1°C and 8-9 ppm,respectively for the entire period of the experiment. One-half to two-thirds of the water ineach aquarium was replaced daily.Five larvae were taken randomly from each aquarium on the first day ofexogenous feeding and fixed in 10% formalin. The mouth sizes of the larvae weremeasured under an ocular micrometer by manually spreading the jaws with forceps usingthe methods of Shirota (1970), Wankowski (1979), and Mathias and Li (1982). Tomeasure the mouth size of the larvae, lengths, in pm, of the upper jaw and lower jaw weremeasured. Assuming that the maximum width of the open mouth was 90 degree, themouth size was calculated using the equation (Swokowski, 1989).Mouth size (pm) = {(a)2 + (b)2 - 2ab cosTIY2Where:a = Length of upper jaw (pm);b = Length of lower jaw (pm);T = Angle between upper and lower jawFeed acceptance by larvae was recorded by observation. These methods havealready been described in chapter 3. Body length, in mm, from the tip of the snout to theend of the notochord was measured by vernier calipers on the third and eighteenth days ofage when the experiment was terminated.Statistical analysisStatistical analyses of the results for this feeding trial were performed usinganalysis of variance (ANOVA) as mentioned previously in section 3.4.3. Tukey's HSDtest was used to separate the significant mean differences of body length in response to theindividual diets. Differences were considered significant at a probability level of 95%.384.3 Results and discussion4.3.1 Mouth sizeMouth sizes of common carp and walking catfish larvae at the time of feedingcommencement were not significantly different and ranged between 425-450 pm. Themouth size for common carp is lower than that reported from the studies of Shirota (1970),who found that common carp larvae started to feed on exogenous diets when their mouthsize was about 570 pm. The difference can be explained on the basis of the different larvalbody lengths reported in the two studies (4.9 mm in this study and 6.6 mm in the reportedone).Dabrowski et al. (1983) assumed that carp larvae or fry should be able to ingestfood particle sizes as large as their mouth i.e. 400 pm in their experiment. Severalstudies, however, have reported that preferred food particles are in fact much smaller thanthose the animals are physically capable of ingesting (Knight, 1983; Mills et al., 1984).With the measured larval mouth size of 425-450 pm obtained from the study andpresuming that the optimum angle of the open jaw at the time of engulfing feed was 30degrees, the particle size of 125 pm was chosen for the NIFI diets. Larval performancesupported the selection of this particle size which was then adopted for the subsequentexperiments described in chapters 5 and 6. When food particle sizes were compared withbody length (4.9 mm in common carp and 7.7 mm in walking catfish) at the time of firstfeeding, the ratio of particle size to body length was 2.5 and 1.6%, respectively. Thisfindings agree with those of Hasen and Macintosh (1992) who reported an optimum rangefor this ratio of 1.7-3.7% for common carp larvae.394.3.2 Larval feed-acceptance behaviorAnemia nauplii were the most readily accepted diet among those tested for bothcommon carp and walking catfish larvae. These results are shown in Table 4.2.Table 4.2 Overall diet acceptance score responses of common carp and walking catfishlarvae for different diets in experiment 1Overall dietDiets^ acceptancescoreMoina 3Anemia nauplii 4NIFI 1 diet for common carp 2NIFI 2 diet for walking catfish 2* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentThe food intake observations revealed that all larvae continued to ingest Artemianauplii until their gut was almost completely filled and they became inactive. Thesignificance of Anemia nauplii as an excellent source of food for early stages of larval fishwas first documented by Seale (1933). Moreover, Appelbaum (1985) reported that newlyhatched Artemia nauplii constituted a highly nutritious diet for fish larvae. Indeed, theArtemia nauplii contained 47.2% protein, 19.3% lipid and 20.6% ash on a dry matter basis,and they had a suitable size range (200-400 pm) for larval ingestion. Recent studies havealso shown that Anemia nauplii are very well accepted by larval fish and crustaceans, andthey are used widely for larval culture (Watanabe et al., 1983). Knud-Hansen et al. (1990)suggested feeding newly hatched walking catfish (Clarias batrachus) with Artemia naupliiuntil day 8 of age followed by mixtures of Artemia nauplii and cladocerans from days 9 to16, and with cladocerans only from days 17 to 23.40The NIFI diets were least accepted by fish larvae as shown in Table 4.2. Thismay be due to their hard texture or to problems with their palatability. The diet particlessank to the bottom of the aquaria within 5-10 minutes of feeding while Artemia nauplii,being alive, remained suspended in the water for 45-60 minutes.4.3.3 Dietary effects on larval body lengthThe influence of diet treatment on the final mean body lengths of the larvae ispresented in Table 4.3 and Appendix 1.Table 4.3 Mean values for final body lengths (mm) of common carp and walking catfishlarvae fed Artemia nauplii, Moina and NIFI diets from 3-18 days of agein experiment 1Type of fish^ Mean* body length (mean + SD) Artemia^Moina^NIFI dietsCommon carp larvae^ 9.00+2.01b 8.70+ 1.8 9ab 8.23±1.24aWalking catfish larvae 13.91+2.16b 9.64+1.35a 8.98±1.69a* Mean values from duplicate tanks were each based on 40 fish.Common carp and walking catfish larvae (n=40 in each case) had body lengths on day 3 of 4.9 and 7.7 mm,respectively.Numbers with different superscripts are significantly different at the 5% level of significance.Results from the present study indicated that Artemia nauplii were the best foodsource for common carp and walking catfish larvae. The feeding of Artemia nauplii tocommon carp larvae resulted in a significantly greater body length at 18 days of agecompared to that for carp fed the NIFI 1 diet. A similar sequence for growth response41was noted in walking catfish and the feeding of Artemia nauplii significantly (P < 0.01)improved larval body length compared with Moina and the NIFI 2 diet.In conclusion, larvae fed on Artemia nauplii for 15 days after yolk absorptiondemonstrated the best performance in terms of food acceptance and growth in body lengthin both common carp and walking catfish. Based on the results obtained, Artemia naupliiwere selected accordingly as a reference diet in the following larval feeding studies.42CHAPTER 5DEVELOPMENT OF DIET FORMULATIONS FOR COMMON CARP LARVAE(EXPERIMENTS 2 TO 4)IntroductionIt has been difficult to adequately evaluate the nutritional requirements of fishlarvae (Kanazawa and Teshima, 1988). These larvae, therefore, have been fed frequentlyon live foods with relatively good success (Watanabe et al., 1983). Live foods, however, donot meet the demands of commercial aquaculture as their availability is limited and theyrequire facilities for consistent and safe production (Bautista et al., 1989). Hence, there isincreased need to develop nutritionally adequate, well balanced and cost-effectiveformulated diets. The development of formulated diet requires extensive experimentationto evaluate multiple food sources as possible dietary constituents. These evaluations willvary for individual fish species and type of rearing conditions i.e. freshwater, brackish orseawater. Recent extensive growth in aquaculture has widened the scope of these studiesfrom academic to commercial.On the basis of physical characteristics, formulated diets have been classified asmoist, microparticulate and dry as described previously in chapter 2. This part of thestudy was directed to examining the merits of these three different types of larval diets.Studies on moist and microparticulate diets are described in experiment 2 which wascomposed of a series of trials carried out to determine the effects of different formulateddiets on feeding response and body length of the larvae. This also included a trial toevaluate single food products for their dietary value. These sources of diets wereinvestigated in experiment 2.1. In the experiments 2.2 to 2.10, the acceptable food43ingredients were used to formulate compound diets. These ingredients were incorporatedinto the diets in variable proportions based on trial and error. Techniques were developedto incorporate various ingredients into suitable formulated diets. The dietary proportionsof the ingredients were modified according to the growth performance of larvae comparedwith the response to Artemia nauplii feeding. Experiment 3, the second part of thischapter, aimed to develop dry diets to compromise between nutritional and economicalfactors. Dry diets are easier to manufacture, transport and store than semi-moist or moistdiets. With a rationale similar to that employed for development of a moist diet, a seriesof experiments was carried out to develop a suitable dry diet for larvae. Experiment 4, thelast part of this chapter, was designed to compare the growth rates of common carp larvaefed on selected diets from experiments 2 and 3, as well as the NIFI 1 diet for common carplarvae and Artemia nauplii.I. EXPERIMENT 2: MOIST DIETS FOR COMMON CARP LARVAEEXPERIMENT 2.1: SCREENING OF ACCEPTABLE DIETARY SOURCES FORCOMMON CARP LARVAEAny feedstuff used as the sole source of nutrition usually does not contain adequateamounts of necessary nutrients required for optimum growth and performance of fishlarvae (Pillay, 1990). In commercial scale aquaculture, food ingredients are thereforecompounded in an effort to balance the diet in terms of essential nutrients. Because of thesmall size of fish larvae, efforts to evaluate their nutritional requirements have not beenentirely satisfactory (Appelbaum, 1985). Under natural conditions, larvae feed on avariety of foods. The purposes of experiment 2.1 were: (1) To evaluate several individual44products as potential diets for commercial scale aquaculture; (2) To determine percentsurvival and overall acceptance score responses of larvae fed food products immediatelyafter yolk absorption.Materials and methodsSixteen non-living food items (2.1.1 to 2.1.16) were tested singly for their nutritivevalue for common carp larvae. Two live foods (2.1.17 and 2.1.18), Artemia nauplii andMoina were fed as controls. Food items rich in protein, and reportedly in other nutrientswere chosen for this experiment and they have been listed below.Food items^ Food items2.1.1 Egg white meal2.1.2 Egg yolk meal2.1.3 Egg meal (white meal : yolk meal = 1: 1)2.1.4 Squid meal2.1.5 Dried Spirulina2.1.6 Dried Chlorella2.1.7 Skim milk2.1.8 Raw squid (Loligo sp.),(whole body excluding internal organs)2.1.9 Cooked chicken blood2.1.10 Raw chicken liver2.1.11 Cooked pork blood2.1.12 Raw pork liver2.1.13 Raw short neck clam, refuse shell2.1.14 Dried frozen Moina2.1.15 Dried frozen Artemia2.1.16 Dried frozen bloodworm2.1.17 Live Moina2.1.18 Artemia naupliiDiets 2.1.1 to 2.1.7 were commercial products and ready to use. Diet 2.1.8 wasprepared from fresh squid obtained from a local market with their viscera and penremoved. The purpose of the later was to minimize nutrient variability because of thepresence of the internal organs. The squid and diets 2.1.9 to 2.1.13 were each choppedwhen fresh into small pieces. Thereafter each was blended in a Mulinex blender, sievedusing a 53 pm diameter sieve size and then kept refrigerated until used. Fresh diets wereprepared every 2 or 3 days. Diets 2.1.14 to 2.1.16 were prepared from frozen Artemia,45Moina and bloodworms by grinding in a blender and then screening each through a 125 pmdiameter mesh sieve. They were mixed well with K-carrageenan, heated in a water bathat 85°C and dried overnight and then ground and sieved through a screen with a 125 prnmesh opening. Artemia nauplii and Moina were cultured as described in chapter 3. Theproducts that were found to support the growth of common carp larvae were selected foruse in the formulation of the moist and microparticulate diets in the experiments describedbelow.The research was conducted by feeding the products individually as the sole sourceof nutrition for 10 days; a trial duration regarded as sufficient by Szlaminska and Przybyl(1986). The common carp larvae which were used in these experiments were obtained byinduced spawning at Burirum Freshwater Fisheries Station as explained in chapter 3.Eggs from about 10 spawners were combined and transferred into the fiberglass tanks.During the first two days of life ten larvae were transferred into glass jars of 200 mlcapacity filled two-thirds with constantly aerated water. Each jar contained ten larvae.The small number of larvae made observations of feeding behavior easy and reliable.Dead larvae, if any, were replaced to maintain the original number (10) of experimentallarvae. Exogenous feeding was started on the third day after hatching. Larvae were fedtheir respective diets twice a day. Gentle aeration was used to keep the food particlessuspended without disturbing the larvae. Control diets were provided at 5-10 Artemianauplii or Moina organisms per ml of the culture medium. Other diets were fed inquantities nearly equal to or exceeding the number of control diet particles but not in suchquantities as to cause fouling of the water. It was not practical, however, to offer exactlysimilar amounts of diets as the diets differed in their moisture content, texture, andparticle size. Dead larvae, unconsumed food and feces were removed from the jars before46each feeding. Glass jars were cleaned daily and stored rain water was added to maintainthe water level. The percent survival of the larvae and the acceptability of the diets wereobserved and recorded twice a day.Results and discussionThe results of this experiment are provided in Table 5.1. Raw pork liver, squidmeal, live Moina and Artemia nauplii, as the control, supported the best survival. Thelarvae accepted Artemia nauplii readily. The non-living products appeared to be suitableas diet for common carp larvae with varying degrees of acceptability. The acceptance ofnon-living food could be attributable to imprinting of the larvae at initiation of exogenousfeeding by associative learning of pre and post ingestive stimuli (Lindberg and Doroshov,1986). The larvae used in this study were fed from initiation of exogenous feeding so thatno imprinting onto live food occurred. This finding agrees with that of Webster et al.(1991) who found that prepared diets were consumed readily by paddle fish (Polyodonspathula) when they were fed from initiation of exogenous feeding. By contrast, therefusal of white sturgeon (Acipenser transmontanus) to eat a prepared diet, when live foodis withdrawn, may have been caused by the larvae having imprinted onto live foods whichwere fed since initiation of exogenous feeding (Buddington and Doroshov, 1984).47Table 5.1 Survivals and overall diet acceptance score responses for common carp larvaefed different food items in experiment 2.1FooditemsLarvalsurvivalOverall dietacceptancescorenumber of fish(out of ten)2.1.1 Egg white meal 6 32.1.2 Egg yolk meal 6 32.1.3 Egg meal 8 42.1.4 Squid meal 10 42.1.5 Dried Spirulina 4 22.1.6 Dried Chlorella 8 22.1.7 Skim milk 9 32.1.8 Raw squid (Loligo sp.) 7 42.1.9 Cooked chicken blood 8 42.1.10 Raw chicken liver 8 32.1.11 Cooked pork blood 6 32.1.12 Raw pork liver 10 42.1.13 Raw short neck clam 6 32.1.14 Dried frozen Moina 5 22.1.15 Dried frozen Artemia 6 22.1.16 Dried frozen bloodworm 4 22.1.17 Live Moina 10 42.1.18 Artemia nauplii 10 4* Overall acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentNo mortality was observed for larvae fed squid meal, raw pork liver and thecontrol cLets Artemia nauplii and Moina. These diets also had the maximum overallacceptance scores. Maximum overall acceptance scores were also shown by larvae fed eggmeal, raw squid, and cooked chicken blood. The number of surviving larvae on the skimmilk diet was 9 out of 10 whereas on the egg meal, cooked chicken blood, dried Chlorellaand raw chicken liver diets 8 larvae survived. The poorer acceptance score of 3 for theskim milk diet might have been due to its powdery texture and this was reflected by aslightly lower survival for larvae fed this diet. The egg white and egg yolk meals resulted48in a survival of 6 out of 10 and each had an acceptance score of 3 which implies that thenutritional value of these diets for growing larvae may not be adequate. Lowestperformances in terms of survival and acceptance scores were recorded for larvaeingesting the dried forms of frozen Moina, Artemia and bloodworms.Visual observations revealed that the larvae had a consistent preference for thediets giving the highest acceptance scores. Attractiveness of food in terms of odor, textureand taste may be more important than nutritional value of the diet to small fish when theyfirst begin to feed as has been suggested by Lemm and Hendrix (1981). When these dietswere introduced to the larvae in the glass jars, ingestion took place within a few minutes.Food particles could be seen in the digestive tracts and the larvae grew continuously andwere vigorous compared to larvae fed the other diets.Raw pork liver gave a comparable performance to that obtained with the controldiets. This finding is consistent with that of Csengeri and Petitjean (1987) who showedthat fresh pork liver was as efficient as live foods when used as a starter diet for cyprinidlarvae. The efficiency of pork liver as a source of nutrition has been attributed to its highcontent of essential nutrients required for adequate larval growth (Dabrowski et al., 1983;Charlon and Bergot, 1984). The proximate composition of raw pork liver is 74.0% protein,12.8% lipid, 8.7% carbohydrate and 4.8% ash on a dry matter basis (Consumer NutritionDivision, 1983).Common carp larvae showed excellent acceptance of squid meal and raw squid.Larval survival on the raw squid diet, however, dropped to 70% compared with 100% onthe squid meal. The raw squid used in this study were eviscerated and that might explainthe difference in performance. Since squid meal is much more expensive than raw squid,the latter was used to replace squid meal in experiment 2.3. Raw squid is composed of4972.6% protein, 6.5% lipid, 14.4% carbohydrate and 6.5% ash on a dry matter basis (Panditand Magar, 1972; Nutrition Monitoring Division, 1987). It contains high levels of w3highly unsaturated fatty acids (Sugiyama et al., 1989) which are a requirement in larvalfish diets (Watanabe, 1988) and the squid seems to be palatable for fish as they are widelyused as lures in longline fishing (Asgard, 1987).Cooked chicken blood resulted in higher performance for the measured parametersin carp larvae than the cooked pork blood and therefore the former was selected for use asan ingredient in moist diets in the subsequent experiments (2.2 to 2.8). Degani andLevanon (1986) found that incorporation of chicken blood in a formulated diet improvedgrowth of eels (Anguilla anguilla). Von Schulz (1982a,b) found that fresh blood and bloodmeal gave similar results for rainbow trout when combined with the other dietary proteinsources i.e. feather meal, poultry meal. Digestibility of blood meal for fish can berelatively high (NRC, 1981; Asgard and Austreng, 1986). Blood meal has a high proteincontent and it is a rich source of valine, leucine and histidine but a very poor source ofmethionine and isoleucine. An excess amount of blood meal in the diet, therefore, maylead to an imbalance of dietary amino acids. Animals fed high dietary levels of blood mealsuffer from an isoleucine deficiency caused by an excess of dietary leucine because of theantagonistic effect of excess leucine on isoleucine (Chance et al., 1964; Tacon and Jackson,1985). Blood meal remains, however, an inexpensive protein supplement for aquaculturediets such as for salmonids (Fowler and Banks, 1976).Egg white and yolk meals, raw chicken liver, cooked pork blood and raw short neckclam exhibited intermediate acceptance. Although larvae survived on these diets theywere comparatively less vigorous than those fed the most acceptable diets. Whole eggmeal comprised of white and yolk supported better performance than any of the above50diets. This meal, however, is expensive and therefore may not be cost effective. As anequivalent alternative, interior egg contents were used as an ingredient in the formulationof the moist diet in subsequent experiments. Eggs being readily available and inexpensivein the local market may provide the basis for formulated diet development. In addition tohigh nutritive value, the adhesive quality of eggs can be useful for binding otheringredients. Eggs can also be stored conveniently for about 10 days at 18°C or for twomonths under refrigeration. Whole egg protein is a standard protein source which has anexcellent essential amino acid balance and a high nutritive value for fish larvae (Chow,1980; Fuze et al., 1985; Hardy, 1989b). The proximate composition of raw whole chickenegg is 47.6% protein, 45.3% lipid, 4.7% carbohydrate and 3.5% ash on a dry matter basis(Stadelman and Owen, 1977; Consumer and Food Economics Institute, 1979b). Uncookedegg white however, has an antinutritional factor called avidin that forms a complex withbiotin making the latter unavailable for intestinal absorption (Gyorgy et al., 1941; Bonjour,1991). Avidin in raw egg must, therefore, be inactivated by heat before eggs are fed tofish (Chow, 1980). To inactivate the avidin in raw chicken eggs so that they can be usedin diets, the egg may be heated in a water bath at 85-95°C for 15 min (Hempe andSavage, 1990).Larvae fed dried frozen Moina, Artemia, and bloodworms had the lowest dietacceptance scores and survivals. Larvae ingested these diets but they occasionally spatthem out with resultant poor food intake and performance. The rejection of the frozen foodmay be attributed to off flavors or odors that were postulated by Kentouri (1980) to beproduced by lipid peroxidation and to vitamin and protein denaturation caused by thethawing procedures used for these foods. A rapid loss of enzyme activity and leaching ofessential nutrients during thawing are probably the most important reasons why frozen51foods have proved to be unsuitable for larval rearing Grabner et a/.(1981). Frozen foods inthis experiment were lowest on the performance scale. The poor responses of larvae tothese foods may have been caused by the conditions under which the frozen foods werethawed. Temperature was not controlled in the laboratory and it was generally hot andhumid. These problems were avoided in subsequent experiments by grinding Artemia inthe frozen state. Since frozen Artemia is well documented for its high nutritive value, itsuse was continued in the experiments described below. Fresh Artemia is known to have ahigher nutritive value than frozen Artemia. A review by Leger et al. (1986), for instance,reported that final sturgeon weight was 1141% more than the initial weight after 35 dayswhen the fish were fed on live Artemia and only 75% when fed on dried Artemia. Bestsurvival and growth were accomplished with frozen Artemia in the initial feeding ofEuropean minnow larvae (Kestemont and Stalmans, 1992). In the case of a study byFluchter (1982) in which frozen Artemia had at least equivalent nutritive value to liveArtemia, the process of freezing may have increased the nutritive value by disrupting cells,leading to release of their contents. This may have facilitated food digestion. Free aminoacids leaked from dead or damaged zooplankton may also serve as feeding attractants forsalmon fry (Holm and Walther, 1988).52EXPERIMENTS 2.2 TO 2.10: RESPONSES OF COMMON CARP LARVAE TOFORMULATED DIETS OF DIFFERENT COMPOSITIONSeveral factors make it difficult to evaluate the nutritional requirements of fishlarvae (Watanabe, 1986; Pillay, 1990). Most of the current nutrient requirementrecommendations are extrapolations from studies on adult fish or they have been based onthe nutrient compositions of eggs. Two of the factors which make assessment of nutrientrequirements difficult include small larval size and the difficulty of estimating food intake.As an important parameter of performance, body length has been found to be directlyproportional to body weight (Alikunhi, 1966). The former parameter has been used in thisstudy since linear growth can be measured with better precision than somatic growth..The expansion of aquaculture on an intensive commercial scale requires the developmentof formulated diets that will improve survival and weight gain of growing larvae. This isparticularly true in countries such as Thailand where the supply of raw food materials isscarce and storage conditions affect supplies and quality. In this study, the different dietcompositions were kept nearly isonitrogenous and isocaloric since lipid and carbohydratecontent did not vary markedly between the diets. The relative efficacy of these dietsprovided the basis for additional changes in formulation to achieve further improvement ofperformance.In the absence of data on the nutrient requirements of larvae, diet formulations inthis study were based on recommendations of NRC (1977) for fry and fingerlings. Therecommended dietary protein levels for fry to fingerling carp ranged from 43-47%.Background experience in successful rearing of various fish larvae on formulated diets wasalso used to augment this information. Kanazawa and Teshima (1988) suggested thathigh nutritive value protein sources such as egg meal, skim milk and squid meal should be53used to formulate larval diets. Also dietary particle size was based on the size of Artemianauplii (160-1000 pm) and larval mouth sizes which were determined in experiment 1.The nutrient composition of natural prey on a dry matter basis was also considered whenformulating the test diets as suggested by Van der Wind (1979). Artemia nauplii arecomposed of 41.6-59.7% protein (Benijts et al., 1975). Yurkowski and Tabachek (1979)reported that most of the natural food for freshwater fish contains 10-20% lipid.A series of experiments was conducted on common carp larvae which were rearedfrom the time of yolk absorption to 10-15 days of age. Upon yolk absorption, i.e. two daysafter hatching, fish larvae were transferred from the hatching unit to the indoor aquaria(45x90x45 cm 3) so that each contained 1,000 larvae. At the end of each experiment, asample of 40 fish was taken for measurement of individual body lengths. The bodylengths of the larvae were measured from the tip of the snout to the end of the notochordusing vernier calipers. Percent survivals were recorded. The experimental diets werecompounded using various proportions of the foods that gave the best results with commoncarp larvae in experiment 2.1. The test diets varied in their contents of protein and otheressential nutrients. Experiments were conducted to find suitable proportions of ingredientsto promote the best performance for growing larvae. Raw pork liver, cooked chicken blood,raw whole chicken egg, raw squid, and frozen adult Artemia were the major dietaryingredients. Vitamins and minerals were added and skim milk, rice flour, corn oil, and abinder, K-carrageenan, were added to make microparticulate and moist diets. Allingredients were mixed thoroughly in a Mulinex blender for 5-10 minutes. Thehomogeneous mixture was heated in a water bath at 85°C until a pudding-like consistencywas obtained. The heat also served to inactivate the avidin in the chicken egg (Hempe andSavage, 1990) and to gelatinize the starch in the rice flour component of the diet.54Gelatinization of starch greatly improves its digestibility for fish (Bergot and Breque,1983). The moist diet was cooled to room temperature and then kept refrigerated.Portions of this diet were dried overnight at room temperature and sieved into the desiredparticle sizes of 125 and 250 pm to give the microparticulate diets. Larvae were fed twice-a-day with either Artemia nauplii as a control or with the experimental diets. Eachaquarium received 20,000-25,000 nauplii or 4 g of diet per day. These feeding amountswere assumed to be in excess since there was always diet or nauplii remaining in thetanks from the previous feeding. The aquaria were cleaned daily.55Experiment 2.2Materials and methodsNine microparticulate diets were compared in the first trial on common carp larvaewhich was conducted over a 10-day feeding period from July 27 to August 6, 1989. Thenutritional requirements of common carp larvae are not known but the dietary componentsand nutrient composition of the formulated diets were based on the nutritionalrequirements for the optimum growth of carp fry as suggested by NRC (1977). Therelative amounts of the essential amino acids and fatty acids were probably adequate toassure nutritional quality of the experimental diets.Details of diet preparation are described in chapter 3. Most of the ingredients usedin this experiment were purchased at the local market at Burirum, Thailand. Theingredient compositions are given in Table 5.2. The compositions of the vitamin andmineral supplements, which were used in the NIFI diets, were based on therecommendations of Sitasit et al. (1982). Larval survivals and overall diet acceptancescores were recorded.56Table 5.2 Ingredient and nutrient compositions of diets fed to common carp larvae for 10 days from 3-13 days of age in experiment 2.2Ingredients (% as fed)Diets2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9Wet mixRaw pork liver 11.6 11.4 23.2 10.8 11.9 11.8 11.7 11.6 11.6Cooked chicken blood 23.2 34.3 11.6 32.5 29.7 23.6 23.4 23.3 23.1Whole chicken egg 28.9 22.9 28.9 27.1 23.8 29.5 29.3 29.1 28.9Short neck clam meat 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3Soybean oil 2.0 2.0 2.0 1.8 2.0 0.7 1.3 2.0 2.5Dry mixSquid meal 16.4 14.3 16.4 13.5 16.8 17.7 17.6 17.4 17.4Skim milk 6.9 5.7 6.9 5.4 6.0 7.0 7.0 7.0 6.9Corn flour 7.2 5.7 7.2 5.4 6.0 5.9 5.9 5.8 5.8Vitamins & mineralsa 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.6 0.6K-carrageenan 2.9 2.8 2.9 2.7 2.9 2.9 2.9 2.9 2.9cn-.1 Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 49.2 45.6 49.9 44.6 47.8 48.3 48.7 49.0 49.4a The vitamin supplement supplied the following levels of nutrients/kg of dry diet: niacin, 100 mg; d-pantothenate, 24 mg; pyridoxine HC1, 20 mg; riboflavin, 8 mg; thiamine, 5 mg;Na-ascorbate, 500 mg; folic acid, 1 mg; choline chloride, 1400 mg; vitamin K, 4 mg; vitamin A, 12,000 11J; vitamin E, 100 mg; vitamin 133, 4,000111; BHT, 50 nig. The mineralsupplement supplied the following nutrients/kg dry diet: NaCI, 3000 mg; MgSO4.7H20, 1400 mg; C6113R07.1120, 200 mg; MuSO4.41120, 250 mg; KI, 10 mg; Ca(H2PO42.21120,6000 mg; Cu504, 10 mg; KC1, 1000 mg; ZnCO3, 130 mg.Table 5.2 (continued)Diets2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9Calculated nutrient levels of diets (% of DM)Protein 45.25 48.36 44.89 48.38 47.53 47.72 47.08 46.45 45.82Lipid 16.97 15.95 17.54 16.83 16.39 15.05 16.18 17.29 18.36Carbohydrate including fiber 31.49 29.27 31.48 28.44 29.58 30.57 30.17 29.77 29.41Ash 6.29 6.42 6.09 6.35 6.50 6.66 6.57 6.49 6.41Arginine 0.92 0.99 0.97 1.02 0.95 0.95 0.94 0.93 0.92Histidine 1.02 1.29 0.88 1.29 1.16 1.06 1.04 1.03 1.01Isoleucine 1.14 1.14 1.32 1.20 1.12 1.19 1.18 1.16 1.14Leucine 2.80 3.35 2.65 3.38 3.07 2.90 2.86 2.82 2.78Lysine 2.22 2.66 2.14 2.68 2.45 2.31 2.28 2.25 2.22Methionine 0.62 0.65 0.68 0.68 0.63 0.65 0.64 0.63 0.62Phenylalanine 1.53 1.82 1.45 1.85 1.66 1.58 1.56 1.54 1.52c..),oo Threonine 1.23 1.40 1.23 1.44 1.31 1.28 1.26 1.25 1.23Tryptophan 0.38 0.42 0.39 0.43 0.40 0.39 0.39 0.38 0.38Valine 1.95 2.34 1.84 2.37 2.14 2.03 2.00 1.97 1.95Fatty acidsC14:0 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01C16:0 2.29 2.06 2.36 2.35 2.05 2.08 2.19 2.30 2.40C18:0 0.80 0.75 0.95 0.82 0.74 0.72 0.76 0.80 0.84C18:1149 4.17 3.77 4.21 4.27 3.74 3.67 3.92 4.17 4.42C18:2w6 2.82 2.85 2.85 2.85 2.82 1.48 2.14 2.79 3.42C18:3w3 0.34 0.35 0.34 0.35 0.35 0.17 0.26 0.34 0.43C20:4w6 0.10 0.11 0.20 0.11 0.11 0.11 0.11 0.10 0.10C20:5w3C22:5w3 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01C22:6w3 0.01 0.01Results and discussionData related to the performance of the carp larvae fed the test diets are shown inTable 5.3. An acceptance score of 4 was noted for the larvae fed the control diet whereasother diets gave acceptance scores ranging from 2 to 3. Survival of the larvae fed themicroparticulate diet was lower than that of fish fed Artemia nauplii. It is difficult tocompare the results of the present experiment with those of other studies on carp larvaebecause of differences in rearing systems and the duration of the rearing period. Commoncarp larvae fed zooplankton ad libitum showed 90% survival after 7 days (Szlaminska andPrzybyl, 1986) and 75% survival after 14 days (Opuszynski et al., 1989). Dabrowski et al.(1983) reported 90% survival for carp larvae fed zooplankton while the highest survivalwas 60% when larvae were fed nine different test diets for 14 days.Table 5.3 Percent survivals and overall diet acceptance score responses for common carplarvae fed microparticulate diets in experiment 2.2DietsPercentSurvivalRelativesurvival(%)Overall dietacceptancescore2.2.1 47.3 66.3 2.02.2.2 51.2 71.8 2.02.2.3 65.7 92.2 3.02.2.4 58.4 81.9 2.02.2.5 52.7 73.9 2.02.2.6 44.5 62.4 2.02.2.7 49.1 68.9 2.02.2.8 42.1 59.1 2.02.2.9 46.3 64.9 2.0Artemia nauplii 71.3 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent.59In this study, fish fed diet 2.2.3 exhibited the best results in terms of survival anddiet acceptance. The amounts of pork liver and chicken blood and their proportions in thisdiet appeared to be suitable for good performance. Diets 2.2.1 and 2.2.3 varied only intheir content of cooked chicken blood and raw pork liver. The deleterious effects of theincreased level of chicken blood in diet 2.2.1 are evidently poor acceptance accompanied bypoor larval survival. All other diets (diets 2.2.2 and 2.2.4 to 2.2.9) in this experimentcontained a level of chicken blood which was 2 to 3 times more than in diet 2.2.3 and gavepoor results (Table 5.2). Diet 2.2.3 was therefore considered to have a balance ofnutrients which was more suitable to the needs of the growing larvae. Another dietaryingredient found to affect the performance of larvae was chicken egg. Its inclusion levelwas lower in diets 2.2.2 and 2.2.5 relative to the level in diet 2.2.3 of this experiment.Diets 2.2.2 and 2.2.5 gave poor performance. Hence, once again diet 2.2.3 was consideredto have the best balance of nutrients for the growing larvae.Blood is known to be high in protein, lysine and iron on a moisture-free basis. It isdeficient in isoleucine which creates an imbalance in the leucine/isoleucine ratio (New,1987). Whole egg, on the other hand, is unquestionably one of the most balanced foodsknown for man and animals (Chow, 1980). Its general availability, ease of preparationand feeding makes it a strong candidate for replacing expensive live foods like Artemianauplii in fish aquaculture. Egg white contains avidin, an antagonist of biotin, but theavidin can be deactivated by heating. Raw pork liver, when fed as the sole source ofnutrition, was shown to give performance similar to that of Artemia in experiment 2.1.Diet 2.2.3 supported similar larval performance to that observed for the control Artemiadiet, and it is noteworthy that diet 2.2.3 contained nearly twice the quantity of pork livercompared to all other experimental diets.60The reason for the efficiency of live food as the first diet of larval fish is not wellunderstood (Webster et al., 1991). Dabrowski (1979) proposed that initial digestion insome larval fish is carried out by the enzymes present in live food organisms. This impliesthat first feeding larval fish do not have the enzymes to digest formulated diets such ascoregonid fish (Stroband and Dabrowski, 1981). In the present study, however, the larvaedid grow on formulated diets which indicates that they had a complement of digestiveenzymes. A review by Dabrowski and Culver (1991) confirmed that cyprinid fishes, whichare stomachless throughout their life, have structurally and functionally differentiatedalimentary tracts at the time of first feeding. This information indicates that it should bepossible to rear common carp larvae entirely on formulated diets.61Experiment 2.3Materials and methodsThe results of experiment 2.2 showed that a high dietary content of pork liver isbeneficial for larval performance whereas a high dietary content of chicken blood isdeleterious. Five microparticulate diets containing 39-41% protein were, therefore,formulated in this experiment with greater amounts of pork liver and lesser concentrationsof chicken blood relative to what was used in most diets of experiment 2.2. FrozenArtemia were incorporated into the test diets. Diets 2.3.1 and 2.3.2 contained squid mealwhereas diets 2.3.3 to 2.3.5 contained raw squid. Whole chicken egg, corn oil and skimmilk were also used as dietary ingredients. The ingredient proportions and nutrient levelsin the diets are indicated in Table 5.4. The compositions of the vitamin and mineralsupplements used were based on the recommendations of Teshima et al. (1982). Theexperiment was conducted from August 6 to 16, 1989. A control group was fed Artemianauplii. Percent survivals were recorded and expressed in Table 5.5 relative to those forlarvae fed Artemia nauplii as a control. Overall diet acceptance scores were calculated atthe end of experiment as described in chapter 3.62Table 5.4 Ingredient and nutrient compositions of diets fed to common carp larvae for 10 days from 3-13 days of age in experiment 2.3Ingredients (% as fed)Diets2.3.1 2.3.2 2.3.3 2.3.4 2.3.5Wet mutRaw potic liver 20.7 20.9 18.4 19.6 20.6Cooked chicken blood 12.6 5.2 11.2 11.7 12.3Whole chicken egg 20.2 26.1 17.9 18.7 19.6Raw squid 22.4 18.7 14.7Frozen Artemia 13.1 13.6 11.7 12.2 12.8Com oil 1.9 1.9 1.8 1.7 1.8Dry mixSquid meal 12.6 13.0Skim milk 5.6 5.7 4.9 5.1 5.4Rice flour 6.1 6.3 5.4 5.6 5.9Water-soluble vitaminsbl 0.5 0.5 0.4 0.5 0.5cz Fat-soluble vitaminsb2 0.05 0.05 0.02 0.04 0.04ca Minerals1'3 4.1 4.2 3.6 3.7 3.9K-carrageenan 2.5 2.6 2.2 2.3 2.5Total 100.0 100.0 100.0 100.0 100.0Dry matter % 46.3 47.3 36.1 36.7 37.5bl The vitamin supplement supplied the following levels of nutrients/kg of dry diet: p-amino benzoic acid, 225.7 mg; biotin, 3.4 mg; inositol, 2266.7 tug; niacin, 453.3 mg; calciumpantothenate, 158.7 mg; pyridoxine HCI, 27 mg; riboflavin, 113.3 mg; thiamine Ha, 34 mg; cyanocobalamine, 0.05 mg; Na-ascorbate, 1133.3 mg; folic acid, 8.5 mg; cholinechloride, 4633.3 mg; b2menadione, 27 mg; beta-carotene, 56.7 mg; alpha-tocopherol, 226.7 mg; cholecalciferol, 5.7 mg. b3The mineral supplement supplied the followingnutrients/kg dry diet: NaCl, 4350 mg; MgSO4.7H20, 13700 mg; C6115Fe07.H20, 2970 mg; MnSO4.41120, 80 mg; KI, 15 mg; Ca(H2PO4)2.2H20, 13580 mg; NaH2PO4.2H20, 8720tug; KH2PO4, 23980 mg, C3H6Ca04, 32700 mg; AICI3.6H20, 15 mg; ZnSO4.7H20, 30 mg; CuC12, 10 mg; CoCl2, 100 mg.czTable 5.4 (continued)Diets2.3.1 2.3.2 2.3.3 2.3.4 2.3.5Calculated nutrient levels of diets (% of DM)Protein 41.44 39.78 41.30 40.56 39.66Lipid 15.41 16.80 13.99 14.19 14.41Carbohydrate including fiber 27.73 28.03 29.08 29.41 29.83Ash 15.42 15.39 15.63 15.84 16.10Arginine 1.67 1.64 2.27 2.20 2.11Histidine 0.87 0.73 1.07 1.07 1.06Isoleucine 1.21 1.26 1.61 1.58 1.53Leucine 2.57 2.30 3.29 3.25 3.20Lysine 2.14 1.93 2.85 2.79 2.72Methionine 0.62 0.62 0.84 0.82 0.79Phenylalanine 1.42 1.29 1.82 1.81 1.78Threonine 1.11 1.05 1.53 1.49 1.44Tryptophan 0.38 0.37 0.49 0.49 0.48Valine 1.79 1.61 2.27 2.26 2.23Fatty acidsC14:0 0.02 0.02 0.05 0.05 0.04C16:0 2.07 2.40 2.52 2.55 2.59C18:0 0.76 0.85 0.91 0.93 0.95Ct8:bv9 3.65 4.25 4.19 4.29 4.40C18:2w6 2.94 3.06 3.35 3.44 3.53C18:3w3 0.17 0.19 0.20 0.21 0.21C20:4 0.21 0.21 0.25 0.26 0.26C20:5w3 0.02 0.02 0.11 0.10 0.08C22:5w3 0.01 0.01 0.02 0.02 0.02C22:6w3 0.01 0.01 0.22 0.19 0.15Results and discussionFrozen Artemia were incorporated into the test diets with four expectedadvantages: (1) to supply unidentified growth factors which are reportedly present inArtemia (Fluchter, 1982); (2) to eliminate the drawback that Artemia may compete forfood with larvae (Leger et al., 1986); and (3) to use excess product from the harvestingseason.Table 5.5 Percent survivals and overall diet acceptance score responses for common carplarvae fed microparticulate diets in experiment 2.3DietsPercentSurvivalRelativesurvival(%)Overall dietacceptancescore2.3.1 53.7 75.4 2.02.3.2 55.6 78.1 2.02.3.3 65.9 92.6 3.02.3.4 68.2 95.8 3.52.3.5 65.2 91.6 3.0Artemia nauplii 71.2 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent.Artemia nauplii proved to be the most acceptable and best diet for supporting thesurvival of common carp larvae. The results are shown in Table 5.5. Larvae fed diets2.3.3 to 2.3.5 had percent survivals in the range 91.6-95.8 and diet acceptance scores of 3or better. By contrast, values for the same parameters for larvae ingesting diets 2.3.1 and2.3.2 were 75.4 and 78.1 and 2, respectively. The presence or absence of raw squid as adietary component influenced the performance of the larvae fed the foregoing diets. Itsdietary inclusion resulted in better performance of larvae. These findings appear to be65contrary to the results of experiment 2.1 where raw squid as a single dietary source didnot result in good performance. The optimum inclusion level of raw squid in the test dietsappeared to range from 15-20%. Raw squid used in combination with others ingredientsincluding the supplemental vitamins and minerals can be considered to be an excellentdietary component for growing larvae. Raw squid has been reported to have a balance ofessential amino acids that enhances the performance of fish (Asgard, 1987). It has arelative advantage over squid meal in that it can be sieved conveniently through a 53 tirnsize mesh. In diets 2.3.1 and 2.3.2, raw squid was replaced with squid meal. The latterdiets supported relatively poor performance. The main reasons for the poor larvalperformance appeared to be poor diet acceptance.Experiment 2.4Moist diets are softer and more palatable than microparticulate diets (Pigott andTucker, 1989). Lemm and Hendrix (1981) observed that fish feeding responses dependednot only on chemical composition but also on the physical properties of the diet. In termsof preparation, moist diets are more advantageous than the other types of larval diets (Choet al., 1985). Fresh products can be used as ingredients to prepare moist diets. Also, noheating and drying are required, and consequently nutrient losses with the exception ofascorbic acid are negligible (Lovell, 1989). The purpose of this experiment was to evaluatethe efficacy of several moist diets for rearing common carp larvae.Materials and methodsFour moist diets (Table 5.6) were prepared as described in chapter 3 and they werechopped by hand to a cube size of approximately 5x5x5 mm3. Raw squid was included in66all diets on the basis of its acceptability as a dietary component in experiment 2.3. Theexperiment was conducted for a duration of 15 days, from 3-18 days of age, betweenAugust 18 and September 2, 1989. Artemia nauplii were used as the control diet. Bodylength, and percent survival were recorded at the termination of the experiment. Theperformance of larvae fed the formulated diets was assessed in relation to larvaeconsuming the Artemia nauplii. Overall diet acceptance scores were also noted.Results and discussionThe results of this experiment are given in Table 5.7. As was noted in theprevious experiments, there existed a general direct relationship between feed acceptanceand larval performance. At the termination of the experiment, the larvae fed on Artemianauplii had grown larger than those fed the experimental diets.67Table 5.6 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.4Ingredients (% as fed)Diets2.4.1 2.4.2 2.4.3 2.4.4Wet mixRaw pork liver 16.0 19.7 18.5 22.8Cooked chicken blood 8.0 8.8 7.1 5.7Whole chicken egg 18.0 19.7 18.5 22.7Raw Squid 16.0 17.5 18.5 25.6Frozen Artemia 25.6 16.4 18.5Corn oil 1.5 1.6 1.7 2.1Dry mixSkim milk 4.4 4.8 5.1 6.3Rice flour 4.8 5.3 5.6 6.9Water-soluble vitaminsbl 0.4 0.4 0.5 0.6Fat-soluble vitaminsb2 0.04 0.04 0.04 0.04cn Mineralsb3 3.2 3.5 3.7 4.6co K-carrageenan 2.0 2.2 2.3 2.8Total 100.0 100.0 100.0 100.0Dry matter % 32.9 35.4 35.8 41.5hi, b2, b3sa as shown in Table 5.4Table 5.6 (continued)Diets2.4.1 2.4.2 2.4.3 2.4.4Calculated nutrient levels of diets (% of DM)Protein 40.69 40.66 39.25 37.84Lipid 15.11 14.92 14.70 14.53Carbohydrate including fiber 28.57 29.08 30.70 32.67Ash 15.63 15.34 15.35 14.96Arginine 2.26 2.25 2.18 2.10Histidine 1.01 1.03 0.95 0.89Isoleucine 1.61 1.64 1.58 1.58Leucine 3.23 3.23 3.05 2.85Lysine 2.81 2.79 2.65 2.47Methionine 0.81 0.83 0.80 0.81Phenylalanine 1.79 1.80 1.69 1.59mco ThreonineTryptophan1.390.501.470.491.380.471.450.45Valine 2.20 2.23 2.09 2.00Fatty acidsC14:0 0.06 0.05 0.05 0.04C16:0 2.79 1.73 2.66 2.61C18:0 0.96 0.99 0.94 0.96Ci8:tw9 4.68 4.59 4.46 4.39C18:2w6 3.39 3.42 3.52 3.64C18:3w3 0.33 0.25 0.26 0.12C20:4 0.26 0.27 0.26 0.25C20:5w3 0.12 0.10 0.11 0.09C22:5w3 0.02 0.02 0.02 0.02C22:6w3 0.18 0.18 0.19 0.22Table 5.7 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed different diets in experiment 2.4Body Relative Percent Relative Overall dietDiets length (mm) body length Survival survival acceptancemean+ SD (%) (%) score*2.4.1 7.9+0.9 91.0 73.6 97.5 3.52.4.2 7.2+1.3 82.8 65.7 87.0 3.02.4.3 7.0+1.7 79.8 50.0 66.2 2.02.4.4 6.5+1.7 73.9 47.5 62.9 2.0A rtemia nauplii 8.7+1.4 100.0 75.5 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent.Common carp are characteristically bottom feeders (Jhingran and Pullin, 1988).The preparation of formulated diets in accordance with the nutritional requirements of thefish is clearly of benefit for the success of intensive aquaculture (Hasen and Macintosh,1992). To accommodate the feeding habits of this species, the moist diets, designated as2.4.1 to 2.4.4 which sank rapidly to the bottom of the container were used in thisexperiment. The observations on feeding behavior revealed that the larvae usuallypreferred to pick food from the bottom of the aquarium. However, occasionally they tooksinking food particles from the lower water column or they came to the surface to feed onfloating particles. Hasen and Macintosh (1992) also found that common carp fry preferredto feed from the bottom. Alternatively, Hecht and Viljoen (1982) reported that the larvaefed mostly in the water column and to a lesser degree from the bottom of the tank. Thelatter discrepancy in feeding behavior may have been due to differences in rearingconditions such as type of aquarium or water depth.At the beginning of the experiment, the average larval body length was recorded tobe 4.9±0.2 mm. After 15 days, maximum mean body length and percent survival of thelarvae fed diet 2.4.1 were 7.9 mm and 73.6%, respectively. Poorest percent survival was70noted in the group fed diet 2.4.4 (47.5%). The estimated protein content of 40.7% in diet2.4.1 was very close to the recommendation of NRC (1977). The reduced level of proteinin diet 2.4.4 (37.8%) may have contributed to poor larval performance. Diet 2.4.1,therefore, was selected to be the control diet in the experiment described below.The amino acid compositions of organisms used as natural food by fish larvae havebeen considered to be helpful in approximating their dietary requirements. Theserequirements have not been established and remain controversial (Nose and Murai, 1990).The essential amino acid content, expressed as percent of protein, in the test diets (Table5.6) was found to be comparable to that of the Artemia nauplii amino acid profile reportedby Watanabe et al. (1983).Freshwater fish require C18:2w6 and/or C18:3w3 while marine fish require w3HUFA such as C20:5w3 and C22:6w3 (Watanabe, 1988). The fatty acid requirements ofsome fish larvae have been investigated. Red sea bream, Pagrus major (Fujii and Yone,1976) and turbot, Scophthalmus maximus (Owen et al., 1975) require relatively highdietary percentages of eicosapentaenoic acid (EPA, C20:5w3) and docosahexaenoic acid(DHA, C22:6w3). Webster and Lovell (1990b) have reported that striped bass larvae,Morone saxatilis require a dietary source of C20:5w3. Unlike those larvae, carp fryreportedly can grow on a diet with no fat for a fairly long period without any appreciableproblem, although requirements for methyl linoleic (C 18:2w6) and methyl linolenic(C18:3w3) acids for proper growth and development of fry weighing 0.65 g have beendemonstrated (Watanabe et al., 1975). All diets in this experiment were formulated toinclude sufficient quantities of these essential fatty acids (Table 5.6).71Experiment 2.5Materials and methodsFive diets were formulated with slight changes in the proportions of ingredientsfrom the diets used in previous experiments, especially those tested in experiment 2.4.Diet 2.4.1 and Artemia nauplii were used as controls. Raw squid and frozen Artemia wereincluded in all the experimental diets. Frozen Artemia was added in comparatively greaterquantities in diet 2.5.3. The dietary compositions are provided in Table 5.8. Body length,larval survival and overall diet acceptance scores were recorded at the termination of theexperiment.72Table 5.8 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.5DietsIngredients (% as fed) 2.4.1 2.5.1 2.5.2 2.5.3 2.5.4(Control)Wet mixRaw pork liver 16.0 16.7 16.4 15.3 15.7Cooked chicken blood 8.0 9.9 9.7 7.7 9.8Whole chicken egg 18.0 15.9 17.5 15.3 15.7Raw squid 16.0 15.9 15.6 15.3 17.6Frozen Anemia 25.6 25.4 24.9 30.7 25.2Corn oil 1.5 1.5 1.5 1.4 1.5Dry mixSkim milk 4.4 4.4 4.3 4.2 4.3Rice flour 4.8 4.7 4.6 4.6 4.7Water-soluble vitaminsbl 0.4 0.4 0.4 0.4 0.4Fat-soluble vitaminsb2 0.04 0.04 0.04 0.03 0.04Mineralsb3 3.2 3.2 3.1 3.1 3.1K-carrageenan 2.0 2.0 2.0 2.0 2.0Total 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.7 32.6 31.6 32.6bl, b2, b3same as shown in Table 5.4Table 5.8 (continued)Diets2.4.1(Control)2.5.1 2.5.2 2.5.3 2.5.4Calculated nutrient levels of diets (% of DM)Protein 40.69 41.45 41.57 40.93 41.63Lipid 15.11 14.44 14.90 14.74 14.35Carbohydrate including fiber 28.57 28.47 28.07 28.60 28.42Ash 15.63 15.64 15.46 15.73 15.60Arginine 2.26 2.28 2.29 2.29 2.30Histidine 1.01 1.07 1.07 1.01 1.07Isoleucine 1.61 1.60 1.62 1.60 1.61Leucine 3.23 3.34 3.35 3.25 3.34Lysine 2.81 2.90 2.90 2.84 2.91Methionine 0.81 0.81 0.82 0.80 0.82--.1.p• Phenylalanine 1.79 1.84 1.86 1.79 1.84Threonine 1.39 1.42 1.43 1.35 1.43Tryptophan 0.50 0.50 0.51 0.50 0.50Valine 2.20 2.27 2.29 2.19 2.27Fatty acidsC14:0 0.06 0.06 0.06 0.07 0.06C16:0 2.79 2.61 2.74 2.67 2.60C18:0 0.96 0.92 0.96 0.92 0.90C18:1w9 4.68 4.36 4.60 4.47 4.33C18:2w6 3.39 3.34 3.33 3.36 3.31C18:3w3 0.33 0.33 0.33 0.38 0.32C20:2 0.01 0.01 0.01 0.01 0.01C20:4 0.26 0.27 0.27 0.27 0.26C20:5w3 0.12 0.12 0.12 0.14 0.13C22:5w3 0.02 0.02 0.02 0.02 0.02C22:6w3 0.18 0.18 0.18 0.18 0.20Results and discussionSimilar to the findings of experiment 2.4, larvae fed on diet 2.4.1 exhibited almostthe same growth and percent survival as those fed freshly hatched nauplii (Table 5.9).Thus, diet 2.4.1 was judged to be very suitable for culturing common carp larvae on thebasis of body length gain, percent survival and overall diet acceptance score. Diet 2.4.1and Artemia nauplii were continued as control diets in experiment 2.6.Table 5.9 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to moist diets in experiment 2.5DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*2.4.1 (control) 9.2+0.7 98.4 85.3 98.2 3.52.5.1 7.8+2.1 83.9 75.0 86.3 3.02.5.2 7.5+1.3 79.7 73.1 84.1 3.02.5.3 8.4+0.8 89.3 80.9 93.1 3.02.5.4 8.1+1.2 86.1 78.4 90.2 3.0Artemia nauplii 9.4+1.5 100.0 86.9 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent75Experiment 2.6Materials and methodsFive diets were formulated with diet 2.4.1 and Artemia nauplii serving as referencecontrols. Proportional changes were made in the same ingredients that were used in theprevious experiment. Two out of four experimental diets (2.6.3 and 2.6.4) were preparedwith lower contents of frozen Artemia and higher contents of whole chicken egg with thecomposition of the other two (2.6.1 and 2.6.2) in inverse proportion. The experiment wasconducted for a duration of 15 days, from 3-18 days of age, between October 4 and 19,1989 (Table 5.10). Body lengths, percent survivals and overall diet acceptance scoreswere recorded.76Table 5.10 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.6Ingredients (% as fed)Diets2.4.1(Control)2.6.1 2.6.2 2.6.3 2.6.4Wet mixRaw pork liver 16.0 15.7 15.9 16.3 16.6Cooked chicken blood 8.0 8.6 8.7 8.1 8.3Whole chicken egg 18.0 16.9 17.1 18.3 18.7Raw Squid 16.0 17.7 16.7 16.3 16.6Frozen Anemia 25.6 25.1 25.4 24.4 22.8Corn oil 1.5 1.5 1.5 1.5 1.5Dry mixSkim milk 4.4 4.3 4.4 4.5 4.6Rice flour 4.8 4.7 4.7 4.9 5.0Water-soluble vitaminsbl 0.4 0.4 0.4 0.4 0.4--1-.1Fat-soluble vitaminsb2Mineralsb30.043.20.043.10.043.20.043.30.043.3K-carrageenan 2.0 2.0 2.0 2.0 2.1Total 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.6 32.7 33.2 33.7bl, b2, b3same as shown in Table 5.4Table 5.10 (continued)Diets2.4.1(Control)2.6.1 2.6.2 2.6.3 2.6.4Calculated nutrient levels of diets (% of DM)Protein 40.69 41.31 41.07 40.60 40.48Lipid 15.11 14.74 14.8 15.09 15.06Carbohydrate including fiber 28.57 28.40 28.51 28.66 28.78Ash 15.63 15.55 15.62 15.65 15.68Arginine 2.26 2.30 2.28 2.26 2.24Histidine 1.01 1.04 1.03 1.01 1.01Isoleucine 1.61 1.62 1.61 1.61 1.61Leucine 3.23 3.29 3.27 3.22 3.21Lysine 2.81 2.87 2.85 2.80 2.78Methionine 0.81 0.82 0.81 0.81 0.81-4co PhenylalanineTlueonine1.791.391.821.421.811.401.791.401.781.40Tryptophan 0.50 0.50 0.50 0.49 0.49Valine 2.20 2.24 2.23 2.20 2.20Fatty acidsC14:0 0.06 0.06 0.06 0.06 0.06C16:0 2.79 2.7 2.71 2.78 2.77C18:0 0.96 0.93 0.94 0.96 0.96C18:1 4.68 4.51 4.54 4.68 4.67C18:2w6 3.39 3.33 3.36 3.40 3.42C18:3w3 0.33 0.33 0.33 0.32 0.31C20:2 0.01 0.01 0.01 0.01 0.01C20:4 0.26 0.26 0.26 0.26 0.26C20:5w3 0.12 0.13 0.13 0.12 0.12C22:5w3 0.02 0.02 0.02 0.02 0.02C22:6w3 0.18 0.20 0.19 0.18 0.18Results and discussionDiets 2.6.1 and 2.6.2 with higher levels of frozen Artemia and lower amounts ofwhole chicken egg relative to diets 2.6.3 and 2.6.4 promoted greater final body lengths andpercent survivals. The finding is further evidence that frozen Artemia have a highnutritive value for common carp larvae. The control diets, 2.4.1 and Artemia naupliisupported best larval survival and growth in terms of body length. The results arepresented in Table 5.11.Table 5.11 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.6DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*2.4.1 (control) 8.5±1.1 91.9 80.9 94.4 3.52.6.1 7.9±1.3 83.5 75.8 88.5 3.02.6.2 7.9±1.7 84.2 76.0 88.7 3.02.6.3 7.7±1.8 85.9 74.7 87.2 3.02.6.4 7.8+0.3 85.5 73.3 85.5 3.0Artemia nauplii 9.2+1.6 100.0 85.7 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent79Experiment 2.7Materials and methodsThe findings of experiments 2.4 to 2.6 clearly showed that diet 2.4.1, (raw porkliver 16%, cooked chicken blood 8%, whole chicken egg 18%, raw squid 16%, frozenArtemia 25.6%, corn oil 1.5%, skim milk 4.4% and rice flour 4.8%) provided a dietarycomposition that was most suitable for larval performance in terms of food acceptance,survival and growth.Common carp is an omnivorous fish species whose diet can contain large amountsof complex carbohydrates (Jauncey, 1982). The purpose of this experiment was toevaluate the effects of level of dietary carbohydrate on the performance of common carplarvae. Accordingly, three diets were formulated (Table 5.12) to contain varying levels ofrice flour. Artemia nauplii and diet 2.4.1 were included as controls. The experiment wasconducted from October 25, to November 9, 1989. Final body lengths, percent survivaland overall diet acceptance scores were recorded and compared to the Artemia naupliicontrol.80Table 5.12 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.7Ingredients (% as fed)Diets2.4.1(Control)2.7.1 2.7.2 2.7.3Wet mixRaw pork liver 16.0 15.8 16.0 16.2Cooked chicken blood 8.0 7.9 8.0 8.1Whole chicken egg 18.0 17.8 17.9 18.2Raw Squid 16.0 15.8 16.0 16.2Frozen Anemia 25.6 25.3 25.5 25.9Corn oil 1.5 1.5 1.5 1.5Dry mixSkim milk 4.4 4.4 4.4 4.5Rice flour 4.8 5.9 5.2 3.7Water-soluble vitaminsbl 0.4 0.4 0.4 0.4a) Fat-soluble vitaminsb2 0.04 0.04 0.04 0.041- Minerals1'3 3.2 3.2 3.2 3.2K-carrageenan 2.0 2.0 2.0 2.0Total 100.0 100.0 100.0 100.0Dry matter % 32.9 33.5 33.1 32.2bl, b2, b3sa as shown in Table 5.4Table 5.12 (continued)Diets2.4.1(Control)2.7.1 2.7.2 2.7.3Calculated nutrient levels of diets (% of DM)Protein 40.69 39.63 40.33 41.82Lipid 15.11 14.69 14.97 15.56Carbohydrate including fiber 28.57 30.52 29.23 26.49Ash 15.63 15.16 15.47 16.13Arginine 2.26 2.21 2.25 2.32Histidine 1.01 0.99 1.00 1.04Isoleucine 1.61 1.62 1.57 1.60Leucine 3.23 3.24 3.15 3.20Lysine 2.81 2.73 2.78 2.89Methionine 0.81 0.79 0.81 0.83ooND PhenylalanineThreonine1.791.391.751.351.781.381.841.43Tryptophan 0.50 0.48 0.49 0.51Valine 2.20 2.15 2.18 2.26Fatty acidsC14:0 0.06 0.06 0.06 0.07C16:0 2.79 2.71 2.76 2.87C18:0 0.96 0.93 0.95 0.99C18:1w9 4.68 4.55 4.64 4.82C18:2w6 3.39 3.30 3.36 3.50C18:3w3 0.33 0.33 0.33 0.34C20:2w6 0.01 0.01 0.01 0.01C20:4 0.26 0.25 0.26 0.27C20:5w3 0.12 0.12 0.12 0.13C22:5w3 0.02 0.02 0.02 0.02C22:6w3 0.18 0.18 0.18 0.18Results and discussionOnce again Artemia nauplii and diet 2.4.1 supported the best larval performance(Table 5.13). Larval performance was noted to be negatively correlated with increasingamounts of rice flour in diets 2.7.1 and 2.7.2. The diet with the least amount of rice flouri.e. 2.7.3 gave better results than diets 2.7.1 and 2.7.2. The control diet 2.4.1, appearedto contain the best level of rice flour for larval survival and growth in terms of bodylength.Table 5.13 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.7Body Relative Percent Relative Overall dietDiets length (mm) body length Survival survival acceptancemean + SD (%) (%) score*2.4.1 (control) 8.3+0.8 90.2 82.0 96.5 3.52.7.1 7.1+1.5 77.2 73.2 86.1 2.02.7.2 7.4+1.2 80.4 75.6 88.9 2.02.7.3 7.8+1.8 84.7 77.5 91.2 3.0Artemia nauplii 9.2+1.4 100.0 85.0 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent83Experiment 2.8Materials and methodsArtemia nauplii and diet 2.4.1 were used as the control diets. Diets 2.8.1 to 2.8.3were prepared with reduced concentrations of whole chicken egg, corn oil, skim milk andincreased amounts of frozen Artemia (Table 5.14). Raw pork spleen replaced raw porkliver in diet 2.8.4 since spleen had been found to be beneficial for better food acceptance(Degani and Levanon, 1986). Diet 2.8.4 also controlled a lower level of corn oil relative todiet 2.4.1. The experiment was conducted between April 5-20, 1990. Final body length,percent survivals and overall diet acceptance scores were recorded and compared to theArtemia nauplii control.84Table 5.14 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.8Ingredients (% as fed)Diets2.4.1(Control)2.8.1 2.8.2 2.8.3 2.8.4Wet mixRaw pork liver 16.0 16.0 16.1 16.0Raw pork spleen 16.1Cooked chicken blood 8.0 8.0 8.0 8.0 8.1Whole chicken egg 18.0 17.6 17.2 16.8 18.1Raw squid 16.0 16.0 16.0 16.0 16.1Frozen Anemia 25.6 26.9 27.3 27.9 25.8Com oil 1.5 0.9 0.9 0.9 1.0Dry mixSkim milk 4.4 4.0 4.0 4.0 4.4Rice flour 4.8 4.8 4.8 4.8 4.8oocy,Water-soluble vitaminsblFat-soluble vitgminsb20.40.040.40.040.40.040.40.040.40.04Mineralsb3 3.2 3.2 3.2 3.2 3.2K-carrageenan 2.0 2.0 2.0 2.0 2.0Total 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.0 31.9 31.8 32.8bl, b2, b3same as shown in Table 5.4Table 5.14 (continued)Diets2.4.1(Control)2.8.1 2.8.2 2.8.3 2.8.4Calculated nutrient levels of diets (% of DM)Protein 40.69 41.46 41.55 41.48 37.38Lipid 15.11 13.73 13.45 13.56 13.40Carbohydrate including fiber 28.57 29.18 28.92 28.91 33.50Ash 15.63 15.63 16.08 16.05 15.72Arginine 2.26 2.33 2.34 2.33 2.37Histidine 1.01 1.04 1.04 1.04 1.07Isoleucine 1.61 1.65 1.65 1.65 1.73Leucine 3.23 3.32 3.33 3.32 3.03Lysine 2.81 2.89 2.90 2.90 2.93Methionine 0.81 0.83 0.83 0.83 0.92oocm Phenylalanine 1.79 1.84 1.84 1.84 1.66Threonine 1.39 1.42 1.42 1.41 1.36Tryptophan 0.50 0.51 0.51 0.51 0.35Valine 2.20 2.26 2.26 2.25 2.11Fatty acidsC14:0 0.06 0.07 0.07 0.07 0.05C16:0 2.79 2.65 2.61 2.60 2.41C18:0 0.96 0.95 0.94 0.93 0.59C18:1w9 4.68 4.36 4.26 4.26 4.08C18:2w6 3.39 2.48 2.35 2.47 2.26C18:3w3 0.33 0.34 0.34 0.35 0.31C20:2w6 0.01 0.01 0.01 0.01 0.01C20:4w6 0.26 0.27 0.27 0.27 0.05C20:5w3 0.12 0.13 0.13 0.13 0.13C22:5w3 0.02 0.02 0.02 0.02 0.02C22:6w3 0.18 0.19 0.19 0.19 0.17Results and discussionSimilar to experiments 2.4 to 2.7 larvae fed on diet 2.4.1 performed better interms of growth in body length and percent survival than larvae fed any of the otherexperimental diets (Table 5.15). The reductions in the amounts of corn oil and skim milkand progressive increases in the amounts of frozen Artemia in diets 2.8.1 to 2.8.2 were notbeneficial for growth. Replacement of pork liver by pork spleen in diet 2.8.4 reducedperformance. Likewise, Dabrowski et al. (1983) reported that common carp larvae grewbetter on a diet supplemented with freeze-dried liver than with freeze-dried spleen.Table 5.15 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed moist diets in experiment 2.8DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*2.4.1 (control) 8.0+0.8 95.7 56.6 97.1 3.52.8.1 7.5+1.4 89.3 53.0 90.9 3.02.8.2 7.2+1.5 86.5 52.7 90.0 3.02.8.3 7.1+1.3 85.6 51.0 87.5 3.02.8.4 7.3+1.4 87.1 54.2 93.0 3.0A rtemia nauplii 8.3+1.1 100.0 58.3 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent87Experiment 2.9Materials and methodsSix moist diets were prepared according to the formulations presented in Table5.16. Artemia nauplii and diet 2.4.1 were used as the control diets. The experiment wasconducted from July 25 to August 9, 1990. In this experiment, the sources of most of thevitamins and minerals were changed from laboratory grade to technical grade to reducethe cost as recommended by Kanazawa et al. (1977) and Sujaritvongsanon (1984). Thecompositions of the vitamin and mineral supplements are shown in Appendix 4. Diets2.4.1 and 2.9.2 were compared with 2.9.1 and 2.9.3, respectively. Altered amounts ofskim milk and rice flour were tested in diets 2.9.2 to 2.9.5. Peanut protein is deficient inthe essential amino acids lysine, threonine, methionine and tryptophan (NutritionMonitoring Division, 1984). Peanuts in various forms, however, have been found to bevery desirable from a sensory quality point of view for the human diet (Singh and Singh,1991). Diets 2.9.2 to 2.9.5, therefore, contained steamed ground peanuts to study larvalresponses to this product.88Table 5.16 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.9Ingredients (% as fed)Diets2.4.1(Control)2.9.1 2.9.2 2.9.3 2.9.4 2.9.5Wet mixRaw pork liver 16.0 16.0 16.3 16.3 16.4 16.1Cooked chicken blood 8.0 8.0 8.1 8.1 8.2 8.2Whole chicken egg 18.0 18.0 16.3 16.3 16.4 16.1Raw squid 16.0 16.0 16.3 16.3 16.4 16.1Frozen Artania 25.6 25.6 26.0 26.0 26.3 25.8Steamed peanut 0.8 0.8 0.8 0.8Corn oil 1.5 1.5 1.5 1.5 1.5 1.5Dry mixSkim milk 4.4 4.4 4.1 4.1 4.1 5.7Rice flour 4.8 4.8 4.9 4.9 4.1 4.1oo Water-soluble vitaminsbl 0.4 0.4co Fat-soluble vita1jinsb2 0.04 0.04Mineralsb3 3.2 3.3Vitamins and mineralsc 3.6 3.7 3.7 3.6K-carrageenan 2.0 2.0 2.0 2.0 2.1 2.0Total 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.8 32.9 32.9 32.4 33.5bl, b2, b3, C same as shown in Table 5.4C The vitamin supplement supplied the following levels of nutrients/kg of do, diet: p-amino benzoic acid, 100 mg; biotin, 4 mg; inositol, 4000 mg; nicotinic acid, 400 mg; Ca-pantothenate, 600 mg; pyridoxine, 120 mg; riboflavin, 80 mg; thiamine-HCl, 40 mg; cyanocobalamine, 0.8 mg; Na-ascorbate, 2000 mg; folic acid, 8 mg; cholla. chloride, 6000 mg;menadione, 40 mg; beta-carotene, 96 mg; alpha-tocopherol, 200 mg; cholecalciferol, 12 mg. The mineral supplement supplied the following nutrients/kg dry diet: MgSO4.7H20,30410 mg; NaH2PO4.2H20, 7900 mg; K2HPO4, 20000 mg; C1130'002, 27200 mg.Table 5.16 (continued)Diets2.4.1(Control)2.9.1 2.9.2 2.9.3 2.9.4 2.9.5Calculated nutrient levels of diets (% of DM)Protein 40.69 40.74 40.38 40.42 41.18 41.09Lipid 15.11 15.13 15.27 15.29 15.59 14.88Carbohydrate including fiber 28.57 28.59 28.70 28.64 27.24 28.70Ash 15.63 15.54 15.65 15.65 15.99 15.33Arginine 2.26 2.27 2.29 2.29 2.33 2.23Histidine 1.01 1.01 1.01 1.01 1.03 1.00Isoleucine 1.61 1.62 1.60 1.60 1.63 1.57Leucine 3.23 3.23 3.22 3.22 3.28 3.17Lysine 2.81 2.81 2.80 2.81 2.86 2.76Methionine 0.81 0.81 0.80 0.80 0.82 0.79coo Phenylalanine 1.79 1.80 1.79 1.79 1.82 1.75Threonine 1.39 1.39 1.38 1.38 1.41 1.36Tryptophan 0.50 0.50 0.49 0.49 0.50 0.48Valine 2.20 2.21 2.19 2.20 2.24 2.16Fatty acidsC14:0 0.06 0.42 0.06 0.06 0.07 0.06C16:0 2.79 2.79 2.71 2.71 2.76 2.64C18:0 0.96 0.96 0.95 0.95 0.97 0.92C18:1 4.68 4.69 4.62 4.63 4.72 4.50C18:2w6 3.39 3.40 3.55 2.55 5.63 3.46C18:3w3 0.33 0.33 0.34 0.34 0.35 0.33C20:2w6 0.01 0.01 0.01 0.01 0.01 0.01C20:46 0.26 0.26 0.26 0.26 0.27 0.26C20:5w3 0.12 0.13 0.13 0.13 0.13 0.12C22:5w3 0.02 0.02 0.02 0.02 0.02 0.02C22:6w3 0.18 0.18 0.18 0.18 0.19 0.18Results and discussionIn this experiment diet 2.9.3, which was modified from diet 2.4.1 by changing thesource of the vitamin and mineral premix and through addition of steamed peanuts, gavethe best results (Table 5.17). When peanuts are heated, the sugars and free amino acidsreact to produce the pleasant aroma (Woodroof, 1983). Thus, adding 0.8% steamedpeanuts in the diets may affect the sensory quality for larvae since the olfactory organ ofthis species is functional at an early stage (Appelbaum, 1980).Table 5.17 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to moist diets in experiment 2.9DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*2.4.1 (control) 8.2±0.7 89.1 83.4 94.3 3.02.9.1 8.2±1.4 89.5 84.0 95.0 3.02.9.2 8.2±1.6 89.7 85.1 96.3 3.02.9.3 8.4±1.3 91.3 86.8 98.2 3.52.9.4 8.1± 1.2 87.8 82.2 93.0 3.02.9.5 8.2±1.7 88.8 83.3 94.2 3.0A rtemia nauplii 9.2+1.6 100.0 88.4 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent91Experiment 2.10Materials and methodsSix moist diets, including diet 2.9.3 as a control, were formulated. Diets 2.10.1,2.10.4 and 2.10.5 contained half the amount of steamed peanuts. Corn oil was added todiets 2.10.1 to 2.10.4 in reduced amounts compared to the control diet because of the highoil content of the steamed peanuts. The experiment was conducted for 15 days (larvae 3-18 days of age) from August 27 to September 11, 1990 (Table 5.18). Final body lengthsand percent survivals were recorded at the termination of the experiment. Overall dietacceptance scores were also noted.92Table 5.18 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 2.10Ingredients (% as fed)Diets2.9.3(control)2.10.1 2.10.2 2.10.3 2.10.4 2.10.5Wet mixRaw pork liver 16.3 16.3 16.4 16.3 16.2 16.2Cooked chicken blood 8.1 8.1 8.2 8.2 8.1 8.1Whole chicken egg 16.3 17.5 16.4 16.3 17.4 17.4Raw squid 16.3 16.3 16.4 16.3 16.2 16.2Frozen Artemia 26.0 26.1 26.2 26.1 25.9 25.8Steamed peanut 0.8 0.4 0.8 0.8 0.4 0.4Corn oil 1.5 0.6 0.7 1.1 1.1 1.5Dry mixSkim milk 4.1 4.1 4.1 4.1 4.1 4.0Rice flour 4.9 4.9 4.9 5.0 4.9 4.8co Vitamins and mineralse 3.7 3.7 3.7 3.7 3.7 3.60., K-carrageenan 2.0 2.0 2.0 2.0 2.0 2.0Total 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.1 32.4 32.6 32.2 32.3'same as shown in Table 5.16coTable 5.18 (continued)Diets2.9.3(control)2.10.1 2.10.2 2.10.3 2.10.4 2.10.5Calculated nutrient levels of diets (% of DM)Protein 40.42 41.80 41.38 40.89 41.77 41.31Lipid 15.29 12.85 13.29 14.31 12.47 13.86Carbohydrate including fiber 28.64 29.27 29.31 28.97 29.58 28.94Ash 15.65 16.08 16.02 15.83 16.18 15.89Arginine 2.29 2.36 2.34 2.32 2.37 2.33Histidine 1.01 1.05 1.04 1.03 1.05 1.04Isoleucine 1.60 1.66 1.64 1.62 1.65 1.64Leucine 3.22 3.34 3.30 3.26 3.28 3.30Lysine 2.81 2.91 2.87 2.84 2.90 2.87Methionine 0.80 0.84 0.82 0.81 0.83 0.83Phenylalanine 1.79 1.85 1.83 1.81 1.85 1.83Threonine 1.38 1.43 1.41 1.40 1.43 1.42Tryptophan 0.49 0.51 0.50 0.50 0.51 0.51Valine 2.20 2.28 2.25 2.22 2.27 2.25Fatty acidsC14:0 0.06 0.07 0.07 0.07 0.07 0.07C16:0 2.71 2.54 2.52 2.62 2.44 2.64C18:0 0.95 0.94 0.93 0.94 0.92 0.95C18:1vv9 4.63 4.14 4.17 4.4 3.98 4.37C18:2w6 2.55 1.89 2.27 2.93 1.74 2.54C18:3w3 0.34 0.33 0.33 0.34 0.34 0.33C20:26 0.01 0.01 0.01 0.01 0.01 0.01C20:4w6 0.26 0.27 0.27 0.26 0.27 0.27C20:5w3 0.13 0.13 0.13 0.13 0.13 0.13C22:5w3 0.02 0.02 0.02 0.02 0.02 0.02C22:6w3 0.18 0.19 0.19 0.19 0.19 0.19Results and discussionLarvae fed on Artemia nauplii and diet 2.9.3 still showed the best performance(Table 5.19). Diets containing reduced amounts of corn oil or steamed peanut supportedpoorer growth. In terms of percent survival and growth, common carp larvae can bereared successfully by using diet 2.9.3. The data suggest that the formulated diet 2.9.3has a nutritive value similar to that of live Artemia nauplii for culturing of common carp.This finding is in direct contrast to the results of other studies in which common carplarvae reluctantly accepted formulated diets and hence their growth was compromised(Grudniewski et al., 1979; Dabrowski and Poczyczynski, 1988).Table 5.19 Final body lengths, percent survivals and overall diet acceptance scoreresponses of common carp larvae to moist diets in experiment 2.10DietsBodylength (mm)mean+ SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*2.9.3 (control) 9.2+1.2 99.2 85.8 96.7 4.02.10.1 8.5+1.4 92.5 75.0 84.6 3.02.10.2 8.7+1.6 94.3 80.8 91.1 3.02.10.3 8.9+1.8 96.0 76.1 85.8 3.02.10.4 8.7+1.3 93.9 72.6 81.9 3.02.10.5 8.9+1.9 96.9 84.4 95.2 3.0Artemia nauplii 9.2+1.6 100.0 88.7 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentIn summary, the use of formulated diets as a first feed for fish larvae is possible.To be successful, a formulated diet must have the following attributes: suitable particlesize, palatability, and the balance of nutrients required by the larvae for growth andhealth.95II. EXPERIMENT 3: DRY DIETS FOR COMMON CARP LARVAEEXPERIMENTS 3.1 TO 3.5: RESPONSES OF COMMON CARP LARVAE TODIFFERENT DRY DIET FORMULATIONSDry diets contain 12% or less moisture in contrast to 18-45% in moist diets (New,1987). The advantages of dry versus moist diets have been reviewed extensively (Cho etal., 1985; Hardy, 1989b). Dry diets are easier to manufacture, transport and store. Also,they are less expensive than moist diets, when the costs of moist diets are expressed on adry weight basis. Further, the supply of dry ingredients can be more regular and theirquality can be defined more consistently.A series of experiments directed to assessment of the response of common carplarvae to dry diets of different formulations was conducted from the time of yolkabsorption to 10 to 15 days of age. The objective of these experiments was to determinesuitable proportions of dietary ingredients for promotion of the best performance of larvae.Fish meal, soybean meal, peanut meal, egg meal, corn gluten meal, shrimp meal, skimmilk and yeast were used as dietary protein sources. Beef tallow, cod liver oil, sesame oil,corn oil, soybean oil and rice bran oil were used as dietary lipid sources for the provision ofenergy and essential fatty acids. Corn flour, rice flour and wheat flour were used asdietary carbohydrate sources.Fish meal is a comparatively expensive feed ingredient and a considerable savingsin feed cost may be realized if the dietary concentration of fish meal can be loweredwithout compromising fish performance. Partial replacement of fish meal with inexpensiveplant protein products such as peanut meal, soybean meal and rice bran may be possible.Peanut meal has better binding capacity than fish meal but it is deficient in lysine and96methionine when compared with soybean meal. Soybean meal is a good source of essentialamino acids and is high in lysine. However, it has a relatively low concentration ofmethionine which can be corrected by addition of synthetic forms. Protease inhibitorswhich are present in raw soybeans are usually destroyed by heat during the oil extractionprocess and by toasting. Rice bran is often available at reasonable cost in the developingcountries (FAO/UNDP, 1983) and it contains 13% protein, 21% fat and 50% totalcarbohydrate (Nutrition Monitoring Division, 1989).Common carp require both w3 and w6 fatty acids (Watanabe et al., 1975). Marinefish oils such as cod liver oil have significant levels of highly unsaturated fatty acids (w3HUFA) (Singh and Chandra, 1988) which are not present in plant oils. Plant oils such ascorn oil or soybean oil are generally rich in C18:2w6 (March, 1990). Corn oil contains 58%linoleic acid, 24.2% oleic acid, 10.9% palmitic and 0.7% linolenic acid (Consumer and FoodEconomics Institute, 1979a). The fatty acid composition of soybean oil varies dependingupon its source, but palmitic, stearic, oleic, linoleic and linolenic are the predominant acidsfound (Yang and Peng, 1990). Linoleic acid accounts for the major part of the unsaturatedfatty acid content. Soybean oil has a higher content of linolenic acid (6.8%) than marinefish oil (1-2%). Sesame oil is inexpensive in Thailand (FAO/UNDP, 1983). Sesame oilcontains about 80% unsaturated fatty acids of which oleic and linoleic acids are the majorfatty acids present in approximately equal amounts. Linolenic acid is present in verysmall quantities (0.3%), whereas palmitic and stearic acids are the major saturated fattyacids. Rice bran oil is typically an oleic-linoleic acid type oil having 39.1% oleic and 33.4%linoleic and 16.9% palmitic acid (Consumer and Food Economics Institute, 1979a).Carbohydrate digestibility in fish depends on many factors including the structure,source, and method of processing of the carbohydrate, and fish species. A review by97Steffens (1989) shows that cereal flour is better digested than potato starch in trout. Thedigestibility of cereal flour at a dietary level of 37% was 55% while potato starch at 31%was 39% digestible in trout. Gelatinization of starch improves it digestibility. Bergot andBreque (1983) reported 90% digestibility of gelatinized corn starch at a dietary level of30% in rainbow trout. Carbohydrate digestibility varies greatly among different species offish. Carp, for instance, show higher digestibility of carbohydrates than salmonids(Steffens, 1989).98Experiment 3.1Materials and methodsCommon carp larvae were obtained from Burirum Freshwater Fisheries Station.The larvae were transferred in lots of 10 to 12 glass containers as explained in experiment2.1. The experiment was conducted over a 10-day feeding period (larvae 3-13 days of age)from July 20 to 30, 1989. Different diet formulations were studied with respect to theireffect on the performance of common carp larvae. Twelve dry diets (Table 5.20) wereformulated based on the recommendations of NRC (1977), Sitasit et al. (1982) and New(1987). Dry ingredients were ground to small particles and sieved through screens with125 and 250 [Jrn openings. The ingredients were blended thoroughly before adding the oil.Diets 3.1.1 to 3.1.6 were modified according to New (1987). Diet 3.1.7 was identical incomposition to NIFI 1 for common carp larvae. Diets 3.1.8 to 3.1.12 varied only in thekind of edible oil used. They were similar to diet 3.1.7 except that 3% rice bran wasreplaced with edible oil (Table 5.20). Larval survival was recorded. Based on continuedobservations of feeding behavior during the course of the experiment, overall dietacceptance scores were noted at the end of the experiment as described in chapter 3.99Table 5.20 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 3.1DietsIngredients (% as fed) 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8 3.1.9 3.1.10 3.1.11 3.1.12Fish meal 4.0 10.0 10.0 10.0 14.0 15.0 30.0 30.0 30.0 30.0 30.0 30.0Soybean meal 2.0 5.0 - 19.0 35.0Peanut meal 2.0 5.0 35.0 35.0 35.0 24.0 24.0 24.0 24.0 24.0 24.0Egg meal - - 20.0 - -Corn gluten meal - 10.0 20.0Shrimp meal 10.0 25.0Skim milk 15.0Yeast 5.0 - - 14.0Corn flour - 28.5 -Rice flour 39.0 25.0 19.0Wheat flour - 47.0 - - - - -1.-■ Rice bran 39.0 25.0 - - 45.0 42.0 42.0 42.0 42.0 42.0oo Beef tallow oil - 2.5 - - - -Cod liver oil 4.0 - - 3.0 -Sesame oil 4.0 4.0 3.0Corn oil 3.0Soybean oil - - 3.0Rice bran oil 3.0Dicakium phosphate - 3.0 - - - -Vitamins & mineralsa 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 88.1 86.4 87.5 90.2 88.7 90.6 89.4 89.7 89.7 89.7 89.7 89.7asame as shown in Table 5.2Table 5.20 (continued)Diets3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8 3.1.9 3.1.10 3.1.11 3.1.12Calculated nutrient levels of diets (% of DM)Protein 16.56 27.36 41.45 31.14 23.63 46.58 32.63 32.11 32.11 32.11 32.11 32.11Lipid 13.29 11.80 8.79 5.97 2.08 2.86 10.53 13.28 13.28 13.28 13.28 13.28Carbohydrate including fiber 59.64 44.67 36.25 49.12 66.55 36.33 35.64 33.88 33.88 33.88 33.88 33.88Ash 10.51 16.17 13.51 13.77 7.74 14.23 21.21 20.73 20.73 20.73 20.73 20.73Arginine 1.05 1.36 3.22 2.70 1.51 3.86 3.18 3.13 3.13 3.13 3.13 3.13Histidine 0.36 0.49 1.06 0.90 0.64 1.34 1.11 1.10 1.10 1.10 1.10 1.10Isoleucine 0.96 1.77 1.91 1.62 1.10 2.23 1.79 1.77 1.77 1.77 1.77 1.77Leucine 1.63 2.89 3.66 3.62 1.78 3.79 2.96 2.92 2.92 2.92 2.92 2.92Lysine 1.16 2.24 1.89 1.31 1.33 2.75 2.16 2.13 2.13 2.13 2.13 2.13Methionine 0.48 0.86 0.73 0.61 0.47 0.78 0.81 0.79 0.79 0.79 0.79 0.79Phenylalanine 1.02 1.80 2.27 2.06 1.17 2.56 1.94 1.91 1.91 1.91 1.91 1.911-■ Threonine 0.85 1.52 1.51 1.22 0.90 1.94 1.51 1.49 1.49 1.49 1.49 1.49)--, Tryptophan 0.21 0.39 0.44 0.31 0.28 0.60 0.41 0.40 0.40 0.40 0.40 0.40Valine 0.79 0.98 2.33 1.96 1.17 2.59 2.21 2.17 2.17 2.17 2.17 2.17Fatty acidsC14:0 0.04 0.04 0.01 0.11 0.02 0.03 0.06 0.06 0.06 0.06 0.07 0.08C16:0 2.14 1.66 1.04 1.09 0.15 0.31 2.02 2.12 2.19 2.26 2.24 2.45C18:0 0.41 0.39 0.18 0.60 0.03 0.07 0.26 0.25 0.40 0.31 0.37 0.30C18:1w9 5.31 4.38 2.60 2.41 0.29 1.24 4.64 4.80 5.66 5.17 5.12 5.65C18:2w6 5.12 4.14 0.98 1.29 0.16 0.79 3.98 3.79 5.07 5.61 5.39 4.82C18:3w3 0.19 0.14 0.07 0.05 0.04 0.03 0.20 0.21 0.20 0.21 0.41 0.24C20:4w6 0.01 0.02 0.09 0.02 0.03 0.03 0.06 0.10 0.06 0.06 0.06 0.06C20:5w3 0.01 0.03 0.49 0.03 0.04 0.04 0.08 0.37 0.08 0.08 0.08 0.08C22:5w3 0.01 0.01 0.10 0.01 0.02 0.02 0.04 0.09 0.04 0.04 0.04 0.04C22:6w3 0.03 0.08 0.57 0.08 0.12 0.12 0.24 0.54 0.24 0.24 0.24 0.24Results and discussionArtemia nauplii were accepted readily among the diets. Visual observationsrevealed that the larvae continued to ingest Artemia nauplii until their gut was filledcompletely. Then, they became inactive. There was an initial period when the larvaeattempted to eat dry diets, but usually they rejected them. A point was reached when foodwas swallowed after which time the fish appeared to recognize the dry diet particles asfood. Thereafter, they soon began feeding vigorously. Some larvae, however, neverlearned to accept their prescribed dry diet, and in the absence of live food, they died ofstarvation. An overall diet acceptance of 4 was shown by larvae ingesting Artemia naupliiwhereas larvae consuming other diets had diet acceptances of between 2 to 3 (Table 5.21).Table 5.21 Survivals and overall diet acceptance score responses for common carp larvaefed different dry diets in experiment 3.1DietsLarvalsurvivalOverall dietacceptancescorenumber of fish(out of ten)3.1.1 5 23.1.2 6 23.1.3 6 23.1.4 6 23.1.5 7 23.1.6 8 33.1.7 7 23.1.8 7 23.1.9 6 23.1.10 9 33.1.11 8 33.1.12 6 2Artemia nauplii 10 4* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent102The best results in this experiment were obtained in response to diet 3.1.10 whichcontained 3% corn oil. Corn oil has a high C18:2w6 and C18:1w9 content. Murata andHigashi (1980) reported that C18:1w9 and C18:2w6 were utilized preferentially as energysources for carp while C22:6w3 was used only to a small extent. The findings arecontrary to those of Lee et al. (1967) who reported that rainbow trout which received 10%corn oil as the sole dietary source of fat exhibited poor growth, inferior feed utilization andhigh mortality. Diet 3.1.11 with 3% soybean oil was also well accepted. Theseobservations indicate that common carp larvae can efficiently utilize corn and soybean oilsand they suggest that these oils have beneficial effects on the performance of common carplarvae. Dietary inclusion of cod liver, sesame and rice bran oils did not result in similarperformance effects.Diet 3.1.6, despite its much higher protein content, was ranked second best interms of larval performance. These beneficial effects may have been due to the inclusionof 14% yeast in diet 3.1.6. They also show that properly complemented plant proteinsources can be a good replacement for fish meal in larval diets. Poorest performance wasshown by larvae fed diet 3.1.1 which contained higher carbohydrate and lipid levels but aconsiderably lower level of protein. Diets 3.1.1, 3.1.2, 3.1.4 and 3.1.5, which containedlow protein and high carbohydrate levels, had showed low acceptance scores and the larvaeshowed varying degrees of survival.103Experiment 3.2Materials and methodsFour dry diets were prepared (Table 5.22) to evaluate the growth performance ofcommon carp larvae. Artemia nauplii were used as the control diet. Either corn orsoybean oil was included as the supplemental lipid source. Comparisons were made of thenutritional benefits of rice bran and rice flour, corn oil and soybean oil, inclusion or absenceof soybean meal and lower or higher levels of fish meal in the diets. The test dietcompositions are provided in Table 5.22. Rice flour was used as the carbohydrate sourcein diets 3.2.1, 3.2.2 and 3.2.4 whereas rice bran was employed in diet 3.2.3. Theadvantages of using rice bran instead of rice flour relate to its low cost and wideavailability especially in Thailand. Because of its high lipid and carbohydrate content(Nutrition Monitoring Division, 1989) rice bran has been used successfully in fish feeds forboth warm and cold water species (FAO/UNDP, 1983). The experiment duration wasextended from the 10-day period that was used in experiment 3.1 to 15 days in thisexperiment to observe the effects of feeding the diets over a prolonged period. Theexperiment was conducted when the larvae were 3-18 days of age, between October 4 to19, 1989. A total of 1,000 larvae were kept in each aquarium. A twice-a-day feedingschedule was maintained until the larvae were 18 days of age when the experiment wascompleted. One-half to two-thirds of the water in each aquarium was replaced daily. Asample of 40 larvae was taken for measurement of individual body lengths at the end ofthe experiment. The percent survivals of the larvae were recorded (Table 5.23) andexpressed relative to Artemia nauplii as control.104Table 5.22 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 3.2DietsIngredients (% as fed) 3.2.1 3.2.2 3.2.3 3.2.4Fish meal 12.5 30.1 30.1 30.1Soybean meal 18.5Peanut meal 18.5 24.1 24.1 24.1Rice bran 42.0Rice flour 45.7 42.0 42.0Com oil 2.8 2.8 2.8Soybean oil 2.8Vitamins & mineralse 2.0 1.0 1.0 1.0Total 100.0 100.0 100.0 100.0Dry matter % 89.6 89.2 89.7 89.2'same as shown in Table 5.16Table 5.22 (continued)Diets3.2.1 3.2.2 3.2.3 3.2.4Calculated nutrient levels of diets (% of DM)Protein 27.00 29.61 32.19 29.61Lipid 5.45 5.97 13.08 5.98Carbohydrate including fiber 56.90 48.91 33.95 48.91Ash 10.65 15.51 20.78 15.50Arginine 2.36 2.90 3.14 2.90Histidine 0.78 1.01 1.10 1.01Isoleucine 1.29 1.63 1.77 1.63Leucine 2.24 2.69 2.93 2.69Lysine 1.52 1.94 2.14 1.94Methionine 0.49 0.72 0.80 0.72Phenylalanine 1.52 1.78 1.92 1.781-,o Threonine 1.07 1.34 1.49 1.34cm Tryptophan 0.33 0.39 0.41 0.39Valine 1.53 1.94 2.18 1.94Fatty acidsC14:0 0.03 0.03 0.06 0.03C16:0 0.69 0.79 2.31 0.81C18:0 0.11 0.21 0.31 0.15Ci8qw9 1.66 1.96 5.30 2.01C18:2w6 2.35 2.27 5.68 2.49C18:3w3 0.08 0.29 0.22 0.10C20:4w6 0.06 0.06 0.06 0.06C20:5w3 0.08 0.08 0.08 0.08C22:5w3 0.02 0.04 0.04 0.04C22:6w3 0.25 0.25 0.24 0.25Results and discussionDietary acceptance scores, percent larval survivals and gains in body length weredirectly related (Table 5.2.3). Diet 3.2.1 supported similar performance to that noted forlarvae fed diets 3.2.2 and 3.2.3 in terms of growth survival and diet acceptance. Hence, itmay be possible to partially replace fish meal with soybean meal and peanut meal,although it is noteworthy that diet 3.2.1 had extra supplementation with vitamins andminerals. The control diet, Artemia nauplii, supported best larval performance which isconsistent with the results of previous experiments. The second best body length, percentsurvival and overall diet acceptance score were shown by larvae fed on diet 3.2.4. Thisdiet contained rice flour and corn oil. Artemia nauplii and diet 3.2.4 were, therefore,selected as control diets for experiment 3.3.Table 5.23 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.2Body Relative Percent Relative Overall dietDiets length (mm) body length Survival survival acceptancemean+ SD (%) (%) score*3.2.1 8.0+2.4 92.0 66.3 82.5 3.03.2.2 8.1± 2.1 93.1 67.5 84.0 3.03.2.3 8.0+2.2 92.0 64.2 79.9 3.03.2.4 8.3±1.8 95.4 72.1 89.7 3.5Artemia nauplii 8.7±1.4 100.0 80.4 100.0 4.0Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentCorn oil and soybean oil are both rich in C18:2w6, which is considered to beimportant for good performance of warmwater fish larvae (Watanabe, 1988). Thereplacement of rice bran (diet 3.2.3) by rice flour (diet 3.2.4) improved growth. Rice bran107is a relatively high in dietary fiber which can, by physical action, accelerate passage offood in the gut.Experiment 3.3Materials and methodsFour diet compositions were tested relative to Artemia nauplii and diet 3.2.4. Thesame ingredients were used in diets 3.3.1 and 3.3.4 as those included in control diet 3.2.4.However, slight changes in the proportions of fish meal, peanut meal and rice flour weremade. Diet 3.3.2 was dissimilar in composition because peanut meal was partly replacedwith soybean meal and the vitamin and mineral supplements were deleted. Diets 3.3.3and 3.3.4, were similar in composition except rice flour completely replaced rice bran indiet 3.3.4. Final body lengths, percent survivals and overall diet acceptance scores wererecorded as in previous experiments. The experiment was conducted for a duration of 15days (larvae 3-18 days of age) between October 25 to November 9, 1989.108Table 5.24 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 3.3DietsIngredients (% as fed) 3.2.4 3.3.1 3.3.2 3.3.3 3.3.4(control)Fish meal 30.1 30.1 30.4 32.1 32.1Soybean meal 6.1Peanut meal 24.1 26.1 18.2 24.1 24.1Rice bran 40.0Rice flour 42.0 40.0 42.5 40.0Corn oil 2.8 2.8 2.8 2.8 2.8Vitamins & mineralsc 1.0 1.0 1.0 1.0Total 100.0 100.0 100.0 100.0 100.0Dry matter % 89.2 89.2 89.2 89.7 89.21-,c.0^csame as shown in Table 5.16Table 5.24 (continued)Diets3.2.4(control)3.3.1 3.3.2 3.3.3 3.3.4Calculated nutrient levels of diets (% of DM)Protein 29.61 30.26 30.61 33.07 29.61Lipid 5.98 6.02 6.06 12.82 5.98Carbohydrate including fiber 48.91 47.97 49.24 32.88 48.91Ash 15.50 15.75 14.09 21.23 15.50Arginine 2.90 3.00 2.79 3.21 2.90Histidine 1.01 1.03 1.01 1.13 1.01Isoleucine 1.63 1.66 1.65 1.83 1.63Leucine 2.69 2.76 2.68 3.00 2.69Lysine 1.94 1.97 2.02 2.22 1.94Methionine 0.72 0.73 0.74 0.83 0.72i--, Phenylalanine 1.78 1.83 1.76 1.96 1.78o Threonine 1.34 1.37 1.36 1.54 1.34Tryptophan 0.39 0.40 0.40 0.42 0.39Valine 1.94 1.99 1.91 2.23 1.94Fatty acidsC14:0 0.03 0.03 0.03 0.06 0.03C16:0 0.81 0.82 0.78 2.24 0.81C18:0 0.15 0.15 0.15 0.31 0.15C18:1w9 2.01 2.06 1.87 5.16 2.01C18:2w6 2.49 2.53 2.43 5.53 2.49C18:3w3 0.10 0.10 0.10 0.21 0.10C20:4w6 0.06 0.06 0.06 0.07 0.06C20:5w3 0.08 0.08 0.08 0.09 0.08C22:5w3 0.04 0.02 0.02 0.02C22:6w3 0.25 0.25 0.25 0.26 0.25Results and discussionThe control diets i.e. Artemia nauplii and diet 3.2.4, supported the bestperformance (Table 5.25).Table 5.25 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.3DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*3.2.4 (control) 8.5±1.2 93.4 72.2 80.8 3.53.3.1 8.2±2.1 90.1 71.3 79.8 3.03.3.2 8.2±1.6 90.1 70.3 78.6 3.03.3.3 8.1+1.4 89.0 73.7 82.4 3.03.3.4 8.3+1.8 91.2 67.4 75.4 3.0A rtemia nauplii 9.1+1.7 100.0 89.4 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentThe partial replacement of peanut meal with soybean meal (diet 3.3.2) did notimprove performance. Lovell (1989) suggested that soybean protein has one of the bestessential amino acid profiles of the protein rich plant feedstuffs which included peanut andcottonseed meals for meeting the essential amino acid requirements of fish. Hence, it isinteresting that replacement of 6% peanut meal with soybean meal did not have abeneficial effect on larval performance. The deletion of the vitamin and mineral andsupplements also did not adversely influence larval performance. Thus, the data suggestthat the amounts of vitamins and minerals supplied by the dietary ingredients aresufficient to meet the demands of the larvae for rapid growth. Replacement of rice branwith rice flour (diet 3.3.3 versus 3.3.4) also did not improve performance as might havebeen expected because of the high fiber content in rice bran. The higher levels of dietary111lipid and a marginal elevation in protein content caused by the inclusion of rice bran in diet3.3.3 might have been responsible for maintaining similar larval performance byovercoming any possible negative effects of high dietary fiber contents.Experiment 3.4Materials and methodsThis experiment was designed to evaluate further the effects of soybean meal inthe larval diet. In this case, graded levels of soybean meal were included in the diet byreplacement of fish meal. Six diets, 3.4.1 to 3.4.6 were formulated with progressivelymore soybean meal (2% increments). The dietary soybean meal levels ranged from 0-12%(Table 5.26). All diets were modified from the control diet 3.2.4 and they had the sameingredient compositions except for dissimilar amounts of fish meal and soybean meal.Larvae were fed the test diets for 15 days. The experiment was conducted from March 16to 31, 1990. Final body lengths, percent survivals and overall diet acceptance scores wererecorded. Artemia nauplii also served as a reference control diet.112Table 5.26 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 3.4DietsIngredients (% as fed) 3.2.4 .^3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6(control)Fish meal 30.1 28.1 26.0 24.0 22.0 20.0 18.0Soybean meal 2.0 4.0 6.0 8.0 10.0 12.0Peanut meal 24.1 24.0 24.1 24.1 24.1 24.1 24.1Rice flour 42.1 42.1 42.1 42.1 42.1 42.1 42.1Corn oil 2.8 2.8 2.8 2.8 2.8 2.8 2.8Vitamins & mineralsc 1.0 1.0 1.0 1.0 1.0 1.0 1.0Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 89.2 89.2 89.3 89.3 89.4 89.4 89.51-L^csame as shown in Table 5.16Table 5.26 (continued)Diets3.2.4(control)3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6Calculated nutrient levels of diets (% of DM)Protein 29.61 29.47 29.33 29.20 29.06 28.93 28.79Lipid 5.98 5.95 5.91 5.87 5.82 5.78 5.74Carbohydrate including fiber 48.91 49.69 50.47 51.25 52.04 52.82 53.60Ash 15.50 14.89 14.29 13.68 13.08 12.47 11.87Arginine 2.90 2.87 2.83 2.80 2.76 2.73 2.69Histidine 1.01 0.99 0.97 0.95 0.93 0.91 0.89Isoleucine 1.63 1.60 1.57 1.54 1.51 1.48 1.45Leucine 2.69 2.66 2.63 2.59 2.56 2.53 2.50Lysine 1.94 1.90 1.86 1.82 1.77 1.73 1.69I-.I-,MethioninePhenylalanine0.721.780.701.760.671.750.651.730.621.720.601.700.571.6841- Threonine 1.34 1.32 1.29 1.27 1.24 1.22 1.20Tryptophan 0.39 0.39 0.38 0.38 0.37 0.36 0.36Valine 1.94 1.91 1.88 1.84 1.81 1.78 1.75Fatty acidsC14:0 0.03 0.03 0.03 0.03 0.03 0.03 0.03C16:0 0.81 0.80 0.79 0.78 0.77 0.76 0.75C18:0 0.15 0.15 0.14 0.14 0.14 0.13 0.13C18:1w9 2.01 1.99 1.97 1.95 1.93 1.90 1.88C18:2w6 2.49 2.50 2.50 2.50 2.50 2.50 2.50C18:3w3 0.10 0.10 0.10 0.09 0.09 0.09 0.08C20:4w6 0.06 0.06 0.06 0.05 0.05 0.04 0.04C20:5w3 0.08 0.08 0.07 0.06 0.06 0.05 0.05C22:5w3 0.04 0.04 0.03 0.03 0.03 0.03 0.02C22:6w3 0.25 0.23 0.21 0.20 0.18 0.16 0.15Results and discussionBest performance was noted for larvae ingesting Artemia nauplii and control diet3.2.4 which contained the maximum level of fish meal and no soybean meal. Larvalperformance was not improved by increasing the dietary soybean meal content. Gains inbody length relative to those of larvae fed Artemia ranged from 82-95% (diet 3.2.4)whereas relative percent survivals ranged from 66-99%. The findings suggest that fishmeal may be of slightly higher nutritive value for larvae than soybean meal and/or bemore palatable.Table 5.27 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae fed dry diets in experiment 3.4DietsBodylength (mm)mean+ SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*3.2.4 (control) 8.5+2.1 94.7 77.1 98.9 3.53.4.1 7.4+2.6 82.2 55.4 71.0 3.03.4.2 7.2+2.1 79.9 62.7 80.4 3.03.4.3 8.0+1.9 88.9 54.3 69.6 3.03.4.4 7.6+1.7 84.4 51.3 65.8 3.03.4.5 7.8±1.5 86.7 53.4 68.5 3.03.4.6 8.4+1.7 93.3 72.0 92.3 3.5Artemia nauplii 9.0+1.6 100.0 78.0 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent115Experiment 3.5Materials and methodsThis is the last of this series of experiments. The inclusion level of fish meal waskept constant at 18% in all diets except 3.2.4 (control). The level of fish meal was adaptedfrom diet 3.4.6 of experiment 3.4. Three dietary levels of peanut meal and two levels ofsoybean meal were used. Rice flour was included at a constant level in all diets except diet3.5.2 where it was replaced with rice bran. The purpose of this experiment was to observethe performance of the larvae given diets prepared with various proportions of only thoseingredients which consistently resulted in better performance in previous experiments.The ingredients included fish meal, soybean meal, peanut meal, rice bran, rice flour, cornand soybean oils. The experiment was conducted from June 7 to 22, 1990. Artemianauplii also served as a control diet. Final body lengths, percent survivals and overall dietacceptance scores were recorded at the end of the experiment.116Table 5.28 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 3.5Ingredients (% as fed)Diets3.2.4(control)3.5.1 3.5.2 3.5.3 3.5.4% % %Fish meal 30.1 17.7 18.0 18.0 18.0Soybean meal 11.8 12.0 12.0Peanut meal 24.1 25.5 24.1 24.1 36.1Rice bran 42.1Rice flour 42.1 41.3 42.1 42.1Corn oil 2.8 2.3 2.3 2.3 2.8Soybean oil 0.5 0.5 0.5Vitamins & mineralsc 1.0 1.0 1.0 1.0 1.0Total 100.0 100.0 100.0 100.0 100.01-, Dry matter % 89.2 89.5 90.0 89.5 89.4-4°same as shown in Table 5.16Table 5.28 (continued)Diets3.2.4(control)3.5.1 3.5.2 3.5.3 3.5.4Calculated nutrient levels of diets (% of DM)Protein 29.61 29.00 31.37 30.10 27.48Lipid 5.98 5.68 12.81 5.73 5.74Carbohydrate including fiber 48.91 53.42 38.66 53.17 54.03Ash 15.50 11.90 17.16 11.00 12.75Arginine 2.90 2.74 2.93 2.41 2.97Histidine 1.01 0.89 0.98 0.88 0.90Isoleucine 1.63 1.46 1.60 1.45 1.46Leucine 2.69 2.53 2.73 2.42 2.57Lysine 1.94 1.69 1.89 1.80 1.58Methionine 0.72 0.57 0.65 0.59 0.56)-,1-, Phenylalanine 1.78 1.71 1.82 1.62 1.75oo Threonine 1.34 1.20 1.35 1.21 1.18Tryptophan 0.39 0.36 0.37 0.36 0.36Valine 1.94 1.77 1.99 1.64 1.86Fatty acidsC14:0 0.03 0.03 0.06 0.03 0.02C16:0 0.81 0.74 2.24 0.67 0.82C18:0 0.15 0.14 0.30 0.13 0.15C18:1w9 2.01 1.90 5.16 1.55 2.21C18:2w6 2.50 2.45 5.64 2.27 2.69C18:3w3 0.10 0.11 0.23 0.12 0.08C20:4w6 0.06 0.04 0.04 0.04 0.04C20:5w3 0.08 0.05 0.05 0.05 0.05C22:5w3 0.04 0.04 0.04 0.04 0.04C22:6w3 0.25 0.14 0.15 0.15 0.15Results and discussionControl diet, 3.2.4 was the true reference diet for this experiment since all otherdiets were simple modifications of this diet except for the fact that all experimental dietshad 18% instead of 30% fish meal. Diet 3.2.4 supported similar larval performance tothat observed in previous experiments. However, when the diet was modified to thecomposition used in diet 3.5.2 by replacing rice flour with rice bran and including 12%soybean meal by replacement of fish meal, there was improved performance both inpercent survival and growth in body length. Diet 3.5.1 was formulated by partiallyreplacing about 12% fish meal in diet 3.2.4 with soybean meal. This modification resultedin some loss of performance which is consistent with the findings of previous experimentsi.e. responses of larvae to diet 3.4.2. In diet 3.5.4, the peanut meal content was increasedby 1.5 times relative to that in diet 3.2.4 by replacement of fish meal. A small loss ofperformance resulted, as a consequence of this formulation change which possibly can beattributed to the lower protein content of this diet. Diets 3.5.1 and 3.5.4 were similar incomposition except that the peanut meal content in diet 3.5.1 was about 70% of that indiet 3.5.4 and the former diet contained 12% soybean meal whereas the latter did not.There was no difference in the performance of larvae fed these two diets which suggeststhat soybean meal and peanut meal had similar nutritional value under the test conditionsemployed.The findings of this experiment strongly suggest that products like rice bran, whichis commonly designated as a roughage, can be included in larval diets at fairly high levels.This will reduce the cost of larval feed many fold compared to the situation where dietarynutrient levels are maintained using concentrate sources. It is also noteworthy thatpeanut meal, which is much cheaper than soybean meal, had similar nutritive value119relative to soybean meal. Further, the data suggest that corn oil can be replaced partlywith soybean oil without any loss in larval performance.Diet 3.5.2 resulted in almost the same performance as was obtained with Artemianauplii (Table 5.29). This diet was, therefore, selected for common carp larvae culture.Diet 3.2.4 contained a high amount of fish meal which is highly nutritious increases thecost of the diet. Most of the protein in diet 3.5.2 was derived from peanut meal andsoybean meal, which are not only generally available in world markets, but also less costlythan fish meal. Since larvae fed diet 3.5.2 exhibited the same performance as Artemianauplii, it is recommended that this diet be used for rearing common carp larvae.Table 5.29 Final body lengths, percent survivals and overall diet acceptance scoreresponses for common carp larvae to dry diets in experiment 3.5DietsBodylength (mm)mean + SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*3.2.4 (control) 8.4± 1.7 94.4 70.4 90.3 3.03.5.1 8.2+2.1 92.1 70.0 89.7 3.03.5.2 8.6+1.4 96.6 75.3 96.5 3.53.5.3 8.2+1.9 92.1 68.7 88.1 3.03.5.4 8.2+1.4 92.1 65.1 83.5 3.0Artemia nauplii 8.9+2.0 100.0 78.0 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentDry diet 3.5.2 can be used solely in the rearing of common carp larvae from thetime of first feeding to 15 days of age. Once again, this diet resulted in similar growthwhen compared to groups fed on Artemia nauplii which is generally accepted as the bestdiet for common carp larvae. The mass rearing of larvae with live foods is generallylimited by food availability and sometimes by problems of food quality as mentioned120previously. The technology of the rearing system such as waste management and rearingconditions must be emphasized for better results. Daily replacement of water and thecleaning of the tanks are strongly recommended to prevent mold growth in the tanks. Atechnical bulletin describing the important characteristics of an intensive rearing systemfor common carp larvae is needed. Appropriately formulated diets promote good larvalgrowth without any signs of nutrient deficiency and enable high larval survival. Inconclusion, it has been demonstrated that the feeding of common carp larvae exclusivelywith a formulated, high quality dry feed is clearly feasible when special attention is givento the techniques of feeding i.e. constant availability of food and water quality in therearing tanks.121III. EXPERIMENT 4: RESPONSES OF COMMON CARP LARVAE FEDSELECTED FORMULATED DIETS, THE NIFI 1 DIET AND ARTEMIA NAUPLIIMethods and materialsThis was the final experiment that was conducted on common carp larvae at theBurirum Freshwater Fisheries Station. The experimental diets (Table 5.30) consisted ofselected formulated diets from experiments 2 (moist diet 2.9.3), experiment 3 (dry diet3.5.2), the NIFI 1 diet which is commonly used for rearing common carp larvae, andArtemia nauplii. The diets were prepared as described in experiment 3. The proximatecompositions of the diets were determined as explained in chapter 3 at the National InlandFisheries Institute (NIFI), Bangkok. The experiment was carried out with duplicate lots oflarvae on each of the experimental diets. Larvae were obtained from induced spawning ofadults as described in chapter 3. At 3 days of age, the larvae were placed into the aquaria(45x90x45 cm3) at a density of 1,000 larvae per aquarium. Larvae were fed theirprescribed diets twice a day from 3-18 days of age for 15 days between October 1 and 16,1990. The aquaria were cleaned once a day by siphoning out debris and fish faeces andany dead larval fish. The water quality was recorded. Dissolved oxygen, alkalinity andhardness were measured by Standard Winkler Method (Swingle, 1969). Watertemperature was determined by placing a thermometer in each of the aquaria and pH wasmeasured by a pH meter.At the end of the experiment, a random sample of 40 fish was taken from eachreplicate aquarium for measurement of individual body length. The larvae remaining ineach aquarium were counted to determine the percentage survival. Analysis of variancewas used to detect significant differences in body length. Tukey 's honestly significant122difference (HSD) test was used to detect significance differences among the means for bodylength in relation to dietary treatments. Treatment means were considered significant atthe 0.05 level of probability. Percent survival data were subjected to arcsinetransformation to achieve homogeneity of variance followed by ANOVA and the TukeyHSD test.123L.)Table 5.30 Ingredient and nutrient compositions of diets fed to common carp larvae for 15 days from 3-18 days of age in experiment 4Diets*Ingredients (% as fed) NIF1 1 Moist diet Dry diet Anemia2.9.3 3.5.2 naupliiRaw pork liver 16.3Cooked chicken blood 8.1Whole chicken egg 16.3Raw squid 16.3Frozen Anemia 26.0Steamed peanut 0.8Com oil 1.5 2.3Soybean oil 0.5Skim milk 4.1Rice flour 4.9K-carrageenan 2.0Fish meal 30.0 18.0Soybean meal 12.0Peanut meal 24.0 24.1Rice bran 45.0 42.1Vitamins & minerals' 1.0Vitamins & minerals' 3.7 1.0Total 100.0 100.0 100.0Dry matter % 89.4 32.87 89.4Same as shown in Tables 5.2 and 5.16NIP! 1 commonly used diet for common carp larvae, moist diet 2.9.3 from experiment 2.9 or 2.10 and dry diet 3.5.2 from experiment 3.5.Table 5.30 (continued)DietsNIFI 1 Moist diet2.9.3Dry diet3.5.2AnemianaupliiCalculated nutrient levels of diets (% of DM)Protein 32.63 40.42 31.37 61.10Lipid 10.53 15.29 12.81 19.42Carbohydrate including fiber 35.63 28.64 38.66 9.38Ash 21.21 15.65 17.16 10.10Arginine 3.18 2.29 2.93 4.00Histidine 1.11 1.01 0.98 1.06Isoleucine 1.79 1.60 1.60 2.10Leucine 2.96 3.22 2.73 5.00Lysine 2.16 2.81 1.89 5.00Methionine 0.81 0.80 0.65 0.701--,l\Dcr 1PhenylalanineThreonine1.941.511.791.381.821.352.600.00Tryptophan 0.41 0.49 0.37 0.81Valine 2.21 2.20 1.99 2.60Fatty acidsC14:0 0.07 0.06 0.06 0.42C16:0 2.09 2.71 2.24 3.75C18:0 0.27 0.95 0.30 0.77C18:1w9 4.80 4.63 5.16 5.88C18:2w6 4.12 2.55 5.64 1.35C18:3w3 0.21 0.34 0.23 2.74C20:4w6 0.06 0.26 0.04 0.52C20:5w6 0.08 0.13 0.05 0.67C22:5w3 0.04 0.02 0.04 0.01C20:66 0.24 0.18 0.15 0.05Results and discussionThe proximate compositions of the three formulated diets and of some ingredientsare presented in Tables 5.31 and 5.32, respectively. The protein concentrations in thethree test diets were 31% (NIFI 1), 32.5% (dry diet 3.5.2) and 40.4% (moist diet 2.9.3)and the lipid concentrations were 8.8, 11.8 and 15.6%, respectively.Table 5.31 Proximate compositions of diets fed to common carp larvae in experiment 4(All values in parentheses are expressed as a percentage of dry matter.)Diets DM" CP2/ EV/ NFE4/including fiberAsh% % % % %NIFI 1 90.79 28.11 5.90 46.41 10.37(100.00) (30.96) (8.77) (48.85) (11.42)Moist diet 2.9.3 82.75 33.39 12.91 27.62 8.83(100.00) (40.35) (15.61) (33.37) (10.67)Dry diet 3.5.2 90.60 29.41 10.74 37.48 12.97(100.00) (32.46) (11.85) (41.37) (14.32)Note 11DM = dry matter; 2/CP = crude protein; 31EE = ether extract (crude lipid); 4/NFE = nitrogen freeextract which was estimated by difference.The body lengths of the common carp larvae in relation to diet and treatment areshown in Table 5.33, Fig. 5.1 and Appendix 2. The linear growth of the larvae fed themoist diet 2.9.3, dry diet 3.5.2, NIFI 1 and Artemia nauplii was significantly different atday 15 after yolk sac absorption.126Table 5.32 Proximate compositions of some ingredients used in diets for common carpand walking catfish larvae(Refer to Table 5.31 for additional information)Feed name DM" CP2/ EE 3/^NFE4/^Ashincluding fiber% % % % %Chicken blood 92.31 78.23 3.39 5.25 5.44(100.00) (84.75) (3.67) (5.69) (5.89)Chicken liver 94.31 70.31 15.47 2.86 5.67(100.00) (74.55) (16.40) (3.03) (6.01)Corn meal 86.07 15.17 0.87 66.52 3.51(100.00) (17.63) (1.01) (77.29) (4.08)Egg meal 93.55 51.17 28.09 8.99 5.3(100.00) (54.70) (30.03) (9.61) (5.66)Fish meal 88.37 50.93 4.38 0.57 32.49(100.00) (57.63) (4.96) (0.64) (36.77)Krill 83.08 59.01 3.82 2.42 17.83(100.00) (71.03) (4.60) (2.91) (21.46)Peanut meal 89.53 35.69 2.60 38.99 12.25(100.00) (39.36) (2.90) (43.55) (13.68)Peanut, steamed 93.34 33.51 49.19 14.95 2.35(100.00) (35.90) (52.70) (16.02) (2.52)Pork liver 95.14 74.98 2.37 11.79 6.00(100.00) (78.81) (2.49) (12.39) (6.31)Rice bran 90.68 11.84 16.62 54.74 7.48(100.00) (13.06) (15.33) (60.37) (8.25)Skim milk 93.45 25.65 2.96 57.63 7.21(100.00) (27.45) (3.17) (61.66) (7.72)Soybean meal 91.56 45.54 2.60 21.80 21.62(100.00) (49.74) (2.84) (23.81) (23.61)Spirulina, dried 91.72 53.72 0.27 23.27 14.46(100.00) (58.57) (0.29) (25.37) (15.77)Squid 83.64 66.85 3.39 0.96 12.44(100.00) (79.93) (4.05) (1.15) (14.87)Note 1/DM = dry matter; 2/CP = crude protein; 3/EE = ether extract (crude lipid); 4/NFE = nitrogen freeextract which was estimated by difference.Larvae fed on Artemia nauplii exhibited the highest growth which was significantlydifferent from larvae fed the other diets except diet 2.9.3. The growth of larvae fed onmoist diet 2.9.3 was higher than that of larvae fed on NIFI 1. In contrast to the results127obtained by Grudniewski et al. (1979) and Dabrowski et al. (1979) who used dry diets,there was no evidence that dry food improved survival or growth of common carp larvae.Larvae fed on the NIFI diet had the poorest linear growth, but their growth in length wasnot significantly different from that of larvae fed on dry diet 3.5.2. Moreover, diettreatment did not significantly influence the percent survival of the larvae (Table 5.33).Table 5.33 Mean values for final body lengths (mm) and percent survivals of commoncarp larvae in experiment 4Type of food* NIFI 1^Moist diet^Dry diet Artemia2.9.3 3.5.2^naupliiMean** body length (± SD)^8.21±0.39a 8.92±0.65bc 8.43±0.51ab 9.07+0.50cMean percent survival (%)^64.00^68.00^69.75^69.00Note: *NIFI 1 diet is a commonly used diet for rearing common carp larvae, moist diet 2.9.3 fromexperiment 2.9 or 2.10 and dry diet 3.5.2 from experiment 3.5.** Mean values from duplicate tanks based upon 40 fish per tank.Numbers with the same superscript are not significantly different at the 5% level of significance.The present results indicate that it is possible to rear common carp larvae on aformulated diet as early as the time of first feeding. As mentioned in chapter 2, commoncarp have a worldwide distribution. There are numerous varieties and subvarieties orstrains of common carp (Jhingran and Pullin, 1988). The larvae of the big belly strain ofcommon carp were used in these experiments and they proved to have satisfactory growthwhen fed on formulated moist diet 2.9.3. This is contrary to previous studies in whichlarvae fed on formulated diets demonstrated reduced growth rate and increased mortality(von Lukowicz, 1979; Bryant and Matty, 1980; Dabrowski et al., 1983; Opuszynski et al.,1281989). Previously, only larvae that were fed on natural food for 2-3 days and laterweaned to formulated diets (Bryant and Matty, 1981; Dabrowski, 1984b) or a combinationof live food with formulated diets (Mires, 1976; Prolubnikov and Kokova, 1984;Szlaminska and Przybyl, 1986) or only on live foods (Lubzens et al., 1984; Lubzens et al.,1987) showed satisfactory growth rates.Dissolved oxygen, alkalinity and water hardness were 8 mg/I, 620 ppm and 880ppm. The water temperature in the rearing tanks ranged from 25-29°C and water pHwas 7.65. It was not possible to measure ammonium concentration in water because oflack of proper instruments and facilities, the larval vigor did not reflect any signs of anyammonia excess. According to Potipitak (1981), the water quality used during theexperiments was suitable with respect to dissolved oxygen content and temperature andthese variables should not have exerted any adverse effects on growth or survival of thelarvae.SummaryIn conclusion, a formulated diet, developed over a 2-year period for rearingcommon carp larvae (Cyprinus earpio) was tested against Artemia nauplii in a 15-dayfeeding trial. The results showed that first feeding common carp larvae can be fed on aformulated diet without any live foods under the above conditions. These resultsconsequently achieved the initial objective of the study in that they demonstrated that it ispossible to rear larvae entirely on a formulated diet. The uncertainties and labourrequirements associated with the capture of live food can, thus, be avoided. The mortalitycharacteristically associated with failure of fish to convert from live to formulated diet doesnot apply or is thereby avoided.129109.57.5bcaNIFI 1^Moist Diet^Dry Diet^Allemla2.9.3 3.5.2 naupillDietsFigure 5.1 Body lengths of common carp larvae fed with different diets.Each value represents the mean ± SD. Body lengths which were similar as determinedby Tukey HSD Test (P > 0.05) are identified by the same superscript i.e. a, b and c.NIFI 1 is a commonly used diet for culturing common carp larvae, dry diet 3.5.2 is fromexperiment 3.5 and moist diet 2.9.3 is from experiment 2.9 or 2.10.130CHAPTER 6DEVELOPMENT OF DIET FORMULATIONS FOR WALKING CATFISH LARVAE(EXPERIMENTS 5 TO 7)IntroductionThe farming of walking catfish (Clarias spp.) in Thailand has become one of themost important aquacultural activities in this region. There are considerable fluctuationsin the success of production due to various factors such as genetics (Jarimopas et al.,1988), diseases and parasites (Areerat, 1987), environment control, nutrition and feeding(Chuapoehuk and Boonyaratpalin, 1983). One of the main constraints for the expansion ofcatfish culture is the insufficient number of fingerlings available to stock the outdoor ponds(Hepher, 1981).Under natural conditions, larvae feed on protozoa, phytoplankton, and smallcrustaceans. Fry can feed on worms, crustaceans, insects and organic matter at thebottom of the pond. Marine trash fish mixed with rice bran and boiled broken rice,manufactured diets, intestines of chickens, and chicken waste from a slaughter house areused commonly to feed adult catfish (Areerat, 1987).Catfish larval rearing has been mainly based on live food, especially Moina, whichare collected from natural waters or from culture. During the rainy season (May-October),the high cost and poor availability of live food reduces the success of larval rearing.Therefore, it is important to develop a formulated diet which can completely, or at leastpartially, replace live food for walking catfish larvae. The formulation of catfish larvaldiets requires certain considerations. The catfish is carnivorous throughout its life historyand prefers live foods (Carreon et al., 1976). To formulate diets, it is important to create a131diet that is similar to live foods in nutritional value, palatability, and acceptance. To date,the rearing of walking catfish larvae under laboratory conditions using formulated dietsalone has been generally unsuccessful (Chuapoehuk and Boonyaratpalin, 1983). Thischapter includes the results of investigations which were undertaken to assess the meritsof a series of moist and dry diets for culturing walking catfish larvae. Experiments 5, and6 evaluated respectively, the potential benefits of using moist and dry diets. Theperformance of walking catfish larvae fed Artemia, the best formulated diets fromexperiment 5 and experiment 6, and the NIFI 2 diet is outlined in experiment 7.132I. EXPERIMENT 5: MOIST DIETS FOR WALKING CATFISH LARVAEEXPERIMENT 5.1: SCREENING OF ACCEPTABLE DIETARY SOURCES FORWALKING CATFISH LARVAEMaterials and methodsMost of the feed ingredients that were used in the diet formulations for commoncarp larvae were tested for their nutritive value for walking catfish larvae. Twelve non-living food items (5.1.1 to 5.1.12) and two live foods, Moina and Artemia nauplii, were fedindividually to the larvae for an experimental period of 10 days (larvae 3 to 13 days ofage). The choice of food items depended mainly on their availability and inexpensive cost.Squid meal and dried Spirulina are commercial products and were not processed furtherbefore they were fed. Diets 5.1.3 to 5.1.12 were prepared as described in experiment 2.1of chapter 5. In short, these ingredients were ground raw and then seived through ascreen with 125 pan mesh openings. Live Moina and Artemia nauplii were prepared asexplained in chapter 3. The test food items were as follows:Food items^ Food items5.1.1 Squid meal 5.1.8 Raw sea mussel (Mytilus sp.)5.1.2 Dried Spirulina^ 5.1.9 Raw ark shell (Arca sp.)5.1.3 Raw chicken liver 5.1.10 Frozen Moina5.1.4 Cooked chicken blood 5.1.11 Frozen Artemia5.1.5 Raw pork liver^ 5.1.12 Frozen bloodworm5.1.6 Cooked pork blood 5.1.13 Live Moina5.1.7 Raw squid 5.1.14 Artemia naupliiWalking catfish larvae in these experiments were obtained from induced spawning,as described in chapter 3 at Burirum Fisheries Station, Thailand. During the first two133days of age, ten larvae were transferred from the incubation tanks to each of theexperimental glass jars of 200 ml capacity. These jars were aerated continuouslythroughout the course of the experimental period. Larvae were fed twice a day during theexperimental period and unused food and fecal matter were siphoned routinely from thebottom of the aquaria on a daily basis. Larval behavior during the experimental feedingperiod was observed twice a day. Percent survival was recorded after 10 days of feeding.The food items that were found to support growth of the walking catfish larvae wereselected for use in the formulation of moist diets in the following experiments.Results and discussionYolk absorption was completed nearly 10-12 hours after the start of feeding. Mostof the larvae preferred feeding at the bottom of the glass jars and they fed less frequentlyin the water column. They were considered satiated when they stopped searching for feedand assembled in the corners of the aquaria.Larvae fed on the live foods Artemia nauplii or Moina, showed 10 and 9 survivinglarvae out of 10, respectively and they had an overall diet acceptance score of 4 as givenin Table 6.1. Larvae showed preference for live foods by snapping at them more readilythan the non-living foods. The result is consistent with the findings of many previousexperiments especially of Hogendoorn (1980) who reported that live Artemia nauplii andlive zooplankton feeding to African catfish larvae gave good results. Artemia nauplii havebeen used as food for larval culture with considerable success (Leger et al., 1986). Theproximate composition (%+SD) of Artemia nauplii as calculated from 26 references andexpressed on a dry matter basis is: 52.2±8.8% protein, 18.9 ± 4.5% lipid, 14.8±4.8%carbohydrate and 9.7+4.6% ash (Leger et al., 1987). High dietary protein content is134probably the first requirement of most carnivorous predator fish. The bright color ofArtemia nauplii and their continuous movement make them an easy prey for sole larvae(Dendrinos et al., 1984). Some studies, however, suggest that the use of Artemia does notabsolutely guarantee success (Sorgeloos, 1980). For example, starved nauplii haverelatively lower free amino acid content (Dabrowski and Rusiecki, 1983), which reducestheir digestibility. The availability of different geographical strains of Artemia has resultedin the appearance of various symptoms in fish and crustacean larvae such as abnormaldevelopment, and metamorphosis, and high mortality (Leger et al., 1987).Table 6.1 Survivals and overall diet acceptance score responses for walking catfish larvaefed different food items in experiment 5.1FooditemsLarvalsurvivalOverall dietacceptancescorenumber of fish(out of ten)5.1.1^Squid meal 8 35.1.2^Dried Spirulina 6 25.1.3^Raw chicken liver 8 35.1.4^Cooked chicken blood 8 35.1.5^Raw pork liver 7 35.1.6^Cooked pork blood 7 35.1.7 Raw squid 8 35.1.8 Raw sea mussel (Mytilus sp.) 4 25.1.9^Raw ark shell (Arca sp.) 3 25.1.10 Frozen Moina 6 25.1.11 Frozen Artemia 7 35.1.12 Frozen bloodworm 5 25.1.13 Live Moina 9 45.1.14 Live Artemia 10 4* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent135A total of 8 out of 10 larvae survived when they were fed squid meal, raw chickenliver, cooked chicken blood or raw squid. Moreover, the overall diet acceptance score forthese food items was 3. The ingestion of each of these foods offered no difficulties which issimilar to the observations reported by Hogendoorn (1980) for African catfish larvae.Squid meal and raw squid have been reported to improve growth performance in manyshrimps (Cruz-Suarez et al., 1992). The reasons for these effects remain unknown butthey can be due to high available protein content and/or the presence of one or moregrowth factors (Cruz-Suarez et al., 1992). Raw chicken liver seems to be an excellent foodfor walking catfish larvae. Lemm and Hendix (1981) found that diets supplemented withchicken liver improved the growth of Atlantic salmon (Salmo solar). This beneficialresponse to liver could be explained by its high nutrient content and enzyme levels.Chicken blood may induce fish to start feeding earlier due to the presence of an attractivesubstance. The latter was suggested by the study of Degani and Levanon (1986) on eels(Anguilla anguilla L.).Larvae fed frozen Artemia, raw pork liver or cooked pork blood showed 70%survival and an overall diet acceptance score of 3. Frozen Artemia were accepted better bythe larvae after thawing. Performance on frozen Moina was similarly poor. Larvalsurvival on dried Spirulina was 6/10. Thus, the physical form and texture of food may beimportant factors determining larval food intake. Lowest percent survivals and overalldiet acceptance scores were recorded for larvae consuming raw sea mussels, raw ark shellsor frozen bloodworms. These foods were poorly recognized and ingested, and they havebeen reported to be inadequate in nutrient composition (Van der Wind, 1979).136EXPERIMENTS 5.2 AND 5.3: RESPONSES OF WALKING CATFISH LARVAE TODIFFERENT MOIST DIET FORMULATIONSOn the basis of the larval performance noted in experiment 5.1, raw pork liver,cooked chicken blood, whole chicken egg, raw squid, frozen Artemia, skim milk, rice flourand corn oil were selected as ingredients for moist diet development. These ingredientswere used to formulate the moist diets tested in experiments 5.2 and 5.3.Experiment 5.2Materials and methodsRaw chicken liver, cooked chicken blood, whole chicken egg, frozen Artemia, andraw squid were each included in the six experimental diets at constant levels. Thesupplemental lipid source was a mixture of sesame oil, corn oil and lard each at aninclusion rate of 0.2% in the diet. In three of the diets, krill meal was either included at2.8%, or was totally replaced with either squid liver meal or steamed peanut meal. In theremaining three diets, the krill meal content of 2.4% was replaced either by 1.4% each ofkrill meal and squid liver meal, squid liver and peanut meals, or krill and peanut meal.Krill meal is known to contain attractant properties for larval fish and the objective of thisexperiment was to determine whether differences in overall diet acceptance wouldinfluence larval performance. Fish larvae were obtained from induced spawning asdescribed in chapter 3. Soon after yolk sac absorption was nearly completed, larvae weretransferred into the indoor aquaria at a density of 1000 larvae per aquarium (45x90t45cm3). Water in the aquaria was aerated continuously and the larvae were fed twice aday.137Six moist diets were fed over a 15-day experimental period when the larvae were3-18 days in age (Table 6.2). Details of the preparation of the moist diets are described inchapter 3. Ingredients were selected on the basis of larval performance (experiment 5.1).Dried Spirulina, which has a fishy smell, was also added to the diets to improve theacceptance of the non-living food as discussed by Kay (1991). Raw squid was added to thediets to enhance palatability as shown in studies on salmonids (Asgard, 1987) and manypenaeids (Fenucci and Zein-Eldin, 1979). Artemia nauplii were used as the control diet. Asample of 40 larvae was taken from each treatment for measurement of individual bodylengths at the end of the experiment. Percent survivals of the larvae were recorded andare shown in Table 6.3.138Table 6.2 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 5.2Ingredients (% as fed)Diets5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6Wet mixRaw chicken liver 18.2 18.2 18.2 18.2 18.2 18.2Cooked chicken blood 4.5 4.5 4.5 4.5 4.5 4.5Whole chicken egg 18.2 18.2 18.2 18.2 18.2 18.2Frozen Artemia 26.8 26.8 26.8 26.8 26.8 26.8Raw squid 13.4 13.4 13.4 13.4 13.4 13.4Sesame oil 0.2 0.2 0.2 0.2 0.2 0.2Corn oil 0.2 0.2 0.2 0.2 0.2 0.2Lard 0.2 0.2 0.2 0.2 0.2 0.2Dry mixDried Spirulina 1.3 1.3 1.3 1.3 1.3 1.3Skim milk 4.0 4.0 4.0 4.0 4.0 4.01-,COtoKrill mealSquid liver meal2.8 -2.81.41.4 1.41.4Steamed peanut meal - 2.8 1.4 1.4Corn flour 3.5 3.5 3.5 3.5 3.5 3.5Vitamins & minerxdsc 4.5 4.5 4.5 4.5 4.5 4.5K-carrageenan 2.2 2.2 2.2 2.2 2.2 2.2Total 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 32.9 32.9 32.9 32.9 32.9 32.9C The vitamin supplement supplied the following levels of nutrients/kg of dry diet: p-amino benzoic acid, 100 mg; biotin, 4 mg; inositol, 4000 mg; nicothic acid, 400 mg; Ca-pantothenate, 600 mg; pyridoxine, 120 mg; riboflavin, 80 mg; thiamine-HC1, 40 mg; cyanocobalamine, 0.8 mg; Na-ascorbate, 2000 mg; folic acid, 8 mg; choline chloride, 6000 mg;menadione, 40 mg; beta-carotene, 96 mg; alpha-tocopherol, 200 mg; cholecalciferol, 12 mg. The mineral supplement supplied the following autriersdka dry diet: MgSO4.71120,30410 mg; NaH2PO4.2H20, 7900 mg; K2HPO4, 20000 mg; Ca3(PO4)2, 27200 mg.Table 6.2 (continued)Diets5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6Calculated nutrient levels of diets (% of DM)Protein 41.08 40.78 40.06 40.93 40.44 40.59Lipid 14.69 13.43 13.46 14.06 13.44 14.10Carbohydrate including fiber 18.11 19.37 19.56 18.74 19.46 18.81Ash 26.12 26.42 26.92 26.27 26.66 26.50Arginine 2.19 2.14 2.11 2.17 2.13 2.15Histidine 0.91 0.89 0.89 0.90 0.89 0.90Isoleucine 1.81 1.81 1.73 1.81 1.77 1.77Leucine 3.25 3.25 3.20 3.25 3.22 3.22Lysine 2.88 2.65 2.68 2.77 2.66 2.78Methionine 0.83 0.86 0.79 0.85 0.83 0.81Phenylalanine 1.82 1.81 1.78 1.82 1.80 1.801-,4:. Threonine 1.47 1.46 1.40 1.47 1.43 1.44o Tryptophan 0.51 0.49 0.50 0.50 0.49 0.50Valine 2.21 2.19 2.18 2.20 2.19 2.19Fatty addsC14:0 0.48 0.06 0.07 0.27 0.06 0.28C16:0 3.18 2.64 2.85 2.91 2.74 3.02C18:0 0.95 0.93 1.01 0.94 0.97 0.98C18:1 4.82 4.36 4.71 4.59 4.53 4.77C18:2w6 1.61 1.56 1.69 1.59 1.62 1.65C18:3w3 0.30 0.30 0.32 0.30 0.31 0.31C20:4 0.11 0.11 0.12 0.11 0.11 0.11C20:5w3 0.12 0.12 0.13 0.12 0.13 0.13C22:5w3 0.30 0.01 0.01 0.15 0.01 0.16C22:6w3 0.30 0.16 0.18 0.23 0.17 0.24Results and discussionThe dietary inclusion of krill meal did not improve food intake as measured by theoverall diet acceptance score (Table 6.3). Maximum food acceptance was shown by larvaefed diet 5.2.3 in which krill meal was completely replaced by peanut meal. The improvedperformance may have been a consequence of peanut meal having a higher nutritive valuethan krill meal. In diet 5.2.5, krill meal was replaced with 1.4% each of squid liver mealand peanut meal. This diet did not give an acceptance score as high as that given by diet5.2.3, (3 vs. 3.5). Relative body length, however, a measure of larval growth, differedonly from that of larvae fed diet 5.2.3 (96.7% vs. 97.6%). The other diet compositions didnot differ markedly from each other and they appeared to result in similar responses tothat noted for larvae ingesting diet 5.2.3. It can be concluded that replacement of krillmeal by peanut meal or by a blend of squid liver meal and peanut meal slightly improvedlarval performance. The control diet, Artemia nauplii, had the best overall diet acceptancescore and larvae consuming this diet had the highest final body length.Table 6.3 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed moist diets in experiment 5.2DietsBodylength (mm)mean+ SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*5.2.1 11.6+1.45 94.3 56.3 85.3 3.05.2.2 11.4+1.36 92.9 54.2 82.1 3.05.2.3 12.0±1.81 97.6 58.4 88.5 3.55.2.4 11.5±1.46 93.5 56.9 86.2 3.05.2.5 11.9±1.94 96.7 57.6 87.3 3.05.2.6 11.7±1.67 95.12 58.3 88.3 3.0Artemia nauplii 12.3 + 1.77 100.0 66.0 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent141Experiment 5.3Materials and methodsThree experimental diets were made (Table 6.4). Their compositions wereessentially similar to that of the best diet 5.2.3, which was used in experiment 5.2 i.e. thediets did not contain krill meal but peanut meal instead. The supplemental dietary lipidsources were the same as used in diet 5.2.3 namely sesame oil, corn oil, and lard.However, in this experiment equal proportions of each oil were used with overall lipidinclusion levels of 0.6%, 0.9%, 1.2% or 1.8%. Thus, this experiment examined the effectsof increasing concentration of dietary lipids on larval performance. Diet 5.2.3 was used asa control in addition to the live food, Artemia nauplii. At the end of the experiment, asample of 40 larvae was taken from each group for measurement of individual bodylength. Larval survival and overall diet acceptance scores were recorded (Table 6.5).142Table 6.4 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 5.3Ingredients (% as fed)Diets5.2.3(Control)5.3.1 5.3.2 5.3.3Wet mixRaw chicken liver 18.2 18.0 17.9 17.7Cooked chicken blood 4.5 4.5 4.5 4.5Whole chicken egg 18.2 18.0 17.9 17.7Frozen Anemia 26.8 26.8 26.8 26.6Raw squid 13.4 13.5 13.4 13.4Sesame oil 0.2 0.3 0.4 0.6Corn oil 0.2 0.3 0.4 0.6Lard 0.2 0.3 0.4 0.6Dry mixDried Spirulina 1.3 1.3 1.3 1.341.caSkim milkSteamed peanut mealI.- 4.02.74.02.74.02.74.02.7Corn flour 3.6 3.6 3.6 3.6Vitamins & mineralsc 4.5 4.5 4.5 4.5K-carrageenan 2.2 2.2 2.2 2.2Total 100.0 100.0 100.0 100.0Dry matter % 35.2 33.0 33.2 33.5'same as shown in Table 6.2Table 6.4 (continued)Diets5.2.3(Control)5.3.1 5.3.2 5.3.3Calculated nutrient levels of diets (% of DM)Protein 40.06 40.76 40.26 39.76Lipid 13.46 14.29 15.36 16.40Carbohydrate including fiber 19.56 18.43 18.20 17.97Ash 26.92 26.52 26.18 25.87Arginine 2.11 2.03 2.01 1.98Histidine 0.89 1.13 1.12 1.10Isoleucine 1.73 1.42 1.40 1.38Leucine 3.20 3.45 3.40 3.36Lysine 2.68 2.81 2.78 2.74Methionine 0.79 0.70 0.69 0.68--,.p. Phenylalanine 1.78 1.82 1.89 1.874 Threonine 1.40 1.39 1.38 1.36Tryptophan 0.50 0.48 0.47 0.47Valine 2.18 2.37 2.35 2.32Fatty acidsC14:0 0.07 0.06 0.07 0.07C16:0 2.85 2.72 2.87 3.01C18:0 1.01 0.85 0.93 1.0C18:1w9 4.71 5.02 5.39 5.75C18:2w6 1.69 2.23 2.66 3.08C18:3w3 0.32 0.34 0.34 0.34C20:4 0.12 0.06 0.06 0.06C20:5w3 0.13 0.12 0.12 0.12C22:5w3 0.01C22:6w3 0.18 0.15 0.15 0.15Results and discussionThe diets did not differ markedly in terms of the larval performance which theysupported or with respect to their acceptability (Table 6.5). Larvae fed diet 5.2.3 hadsimilar performance to those fed this diet in experiment 5.2. Dietary inclusion of 0.4% ofeach of the supplemental lipid sources i.e. sesame oil, corn oil and lard gave the bestperformance in this experiment. Hence, the data suggest that a total of 1.2% lipid fromthese sources is optimum for larval performance. Increasing the dietary concentration ofthe supplemental lipid to 1.8% resulted in poorest larval growth. It should be pointed outthat the dietary lipid requirements of walking catfish have not been established for thisstage of the life history. Thus, the results of this experiment may useful as a guideline forfuture formulations.Table 6.5 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed moist diets in experiment 5.3Body Relative Percent Relative Overall dietDiets length (mm) body length Survival survival acceptancemean ± SD (%) (%) score*5.2.3 (control) 12.1± 1.20 96.8 58.2 90.9 3.05.3.1 12.2±1.44 97.6 59.9 93.6 3.05.3.2 12.3±1.11 98.4 61.3 95.8 3.55.3.3 11.8±1.45 94.4 60.4 94.4 3.0Artemia nauplii 12.5± 1.34 100.0 64.0 100.0 4.0* Overall diet acceptance score described as 1 poor, 2 fair, 3 good and 4 = excellent145II. EXPERIMENT 6: DRY DIETS FOR WALKING CATFISH LARVAEEXPERIMENTS 6.1 TO 6.4: RESPONSE OF WALKING CATFISH LARVAE TODRY DIETS WITH DIFFERENT FORMULATIONSExperiment 6.1Materials and methodsThe overall objective of this study was to develop a dry diet which would supportexcellent growth of walking catfish larvae. To compare the effects of different types ofsupplemental lipid on larval performance ten experimental diets were prepared bymodification of NIFI 2 (diet 6.1.1); a diet prescribed for carnivorous larvae and fry (Sitasitet al., 1982). Changes in the source and content of lipid were made, and the cod liver oil inthe NIFI 2 diet was either replaced with a different oil or a combination of different oils.The compositions of the test diets are shown in Table 6.6. The NIFI 2 diet is composed of56% fish meal, 12% peanut meal, 12% rice bran, 14% alpha-starch, 0.4% wheat gluten,4% fish oil and 1.6% vitamins and minerals. Fish meal constitutes the main dietaryprotein source. Some of the oils that replaced cod liver oil have characteristic fatty acidprofiles. Soybean oil, corn oil and sesame oil contain high concentrations ofpolyunsaturated (w6) fatty acids (Yermanos et al., 1972; Yang and Peng, 1990).Cottonseed oil is rich in linoleic, palmitic and oleic acids and lard is a source of linoleic acidand oleic acid (Austic and Nesheim, 1990) and is easily obtained in the local market.146Table 6.6 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 10 days from 3-13 days of age in experiment 6.1DietsIngredients (% as fed) 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 6.1.7 6.1.8 6.1.9 6.1.10 6.1.11 6.1.12Fish meal 56.0 56.0 56.0 56.0 56.0 56.0 56.0 56.0 56.0 56.0 56.0 56.0Peanut meal 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0Rice bran 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0Starch 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0 14.0Wheat gluten 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4Sesame oil 4.0 3.2 2.6 2.0 2.0 1.3 1.3 1.3 1.3Soybean oil 2.0 0.7 1.0 1.0 1.3 1.3Corn oil 4.0 0.8 1.3 1.3Cod liver oil 4.0 2.0 1.3 1.3Cottonseed oil 0.7 1.0Lard 1.0 1.3 1.3Vitamins & minerals' 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0Dry matter % 92.0 92.0 92.0 92.0 92.0 92.0 92.0 92.0 92.0 92.0 92.0 92.0a The vitamin supplement supplied the following levels of nutrients/kg of dry diet: niacin, 100 mg; d-pantothenate, 24 mg; pyridoxine HC1, 20 mg; riboflavin, 8 mg; thiamine, 5 mg;Na-ascorbate, 500 mg; folic acid, 1 mg; choline chloride, 1400 mg; vitamin IC, 4 mg; vitamin A, 12,000 IU; vitamin E, 100 mg; vitamin D3, 4,000 1U; BHT, SO mg. The mineralsupplement supplied the following nutrients/kg dry diet: NaC1, 3000 mg; MgSO4.7H20, 1400 mg; C6H5Fe07.H20, 200 mg; MnSO4.4H20, 250 mg; K1, 10 mg;Ca(H2PO4)2.21120, 6000 mg; CuSO4, 10 mg; KC1, 1000 mg; ZnCO3, 130 mg.Table 6.6 (continued)Diets6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 6.1.7 6.1.8 6.1.9 6.1.10 6.1.116.1.12Calculated nutrient levels of diets (% of DM)Protein 45.38 45.38 45.38 45.38 45.38 45.38 45.38 45.38 45.38 45.38 45.38 45.38Lipid 10.24 10.24 10.24 10.24 10.23 10.23 10.23 10.23 10.24 10.23 10.23 10.23Carbohydrate including fiber 26.86 26.86 26.86 26.86 26.86 26.86 26.86 26.86 26.86 26.86 26.86 26.86Ash 17.52 17.52 17.52 17.52 17.52 17.52 17.52 17.52 17.52 17.52 17.52 17.52Arginine 1.36 1.36 1.36 1.36 1.36 1.36 1.36 1.36 1.36 1.36 1.36 1.36Histidine 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29Isoleucine 2.19 2.19 2.19 2.19 2.19 2.19 2.19 2.19 2.19 2.19 2.19 2.19Leucine 3.34 3.34 3.34 3.34 3.34 3.34 3.34 3.34 3.34 3.34 3.34 3.34Lysine 2.89 2.89 2.89 2.89 2.89 2.89 2.89 2.89 2.89 2.89 2.89 2.89Methionine 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09Phenylalanine 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.131-, Threonine 1.84 1.84 1.84 1.84 1.84 1.84 1.84 1.84 1.84 1.84 1.84 1.84oo Tryptophan 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50Valine 2.43 2.43 2.43 2.43 2.43 2.43 2.43 2.43 2.43 2.43 2.43 2.43Fatty acidsC14:0 0.06 0.06 0.06 0.06 0.08 0.06 0.07 0.06 0.06 0.06 0.08 0.08C16:0 1.11 1.20 1.29 1.22 1.91 1.23 1.38 1.24 1.20 1.19 1.45 1.44C18:0 0.16 0.36 0.23 0.34 0.25 0.34 0.45 0.32 0.25 0.28 0.45 0.48C18:1w9 2.50 3.65 3.00 3.52 3.02 3.43 3.49 3.31 3.05 3.03 3.46 3.44C18:2w6 1.36 3.08 3.80 3.22 1.52 3.26 2.84 3.36 2.74 2.64 2.86 2.76C18:3w3 0.16 0.14 0.16 0.14 0.15 0.19 0.22 0.21 0.15 0.24 0.16 0.24C20:4w6 0.17 0.12 0.12 0.12 0.14 0.12 0.12 0.12 0.13 0.13 0.12 0.12C20:5w3 0.53 0.15 0.15 0.15 0.34 0.15 0.15 0.15 0.28 0.28 0.15 0.15C22:5w3 0.07 0.10 0.07 0.11 0.11 0.07 0.07 0.07 0.10 0.10 0.07 0.07C22:6w3 0.85 0.44 0.44 0.44 0.65 0.44 0.44 0.44 0.58 0.58 0.44 0.44Results and discussionThe results showed that larvae responded well to diets 6.1.6, 6.1.7 and 6.1.11 aspresented in Table 6.7.Table 6.7 Final body lengths and overall diet acceptance score responses of walkingcatfish larvae fed dry diets in experiment 6.1DietsBodylength (mm)(mean + SD)Relativebody length(%)Overall dietacceptancescore6.1.1 8.2+1.9 88.2 3.06.1.2 7.7+2.1 82.8 2.06.1.3 7.3+2.5 78.5 2.06.1.4 8.2+2.7 88.2 3.06.1.5 7.3+2.6 78.5 2.06.1.6 8.9+1.3 95.7 3.56.1.7 8.8+1.8 94.6 3.06.1.8 8.4+1.7 90.3 3.06.1.9 7.6+ 2.1 81.7 2.06.1.10 7.1+ 2.7 76.4 2.06.1.11 8.6+1.4 92.5 3.56.1.12 8.5+2.6 91.4 3.0Artemia nauplii 9.3+1.1 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent.It has been shown that fatty acid requirements vary among freshwater fish. Forexample, Tilapia zilli require w6 fatty acids only (Kanazawa et al., 1980), while both w3and w6 fatty acids are required by Cyprinus carpio (Watanabe et al., 1975; Takeuchi andWatanabe, 1977). In this experiment, appropriate amounts of soybean oil, sesame oil,cottonseed oil and lard were shown to be suitable for inclusion in practical feeds forwalking catfish larvae. Previously, it has been shown that replacement of fish oil withsoybean oil had no effect on growth of rainbow trout (Suzuki et al., 1986) and Atlantic149salmon (Hardy at al., 1987). Moreover, lard can be used as a dietary energy source fortrout (Salmo gairdneri; Yu et al., 1977) and milkfish fingerlings (Chanos chanos; Alava,1986).Experiment 6.2Materials and methodsFive diets were prepared using constant levels of fish meal, peanut meal, corn mealand rice bran (Table 6.8). The effects of altering the dietary lipid composition were studiedfurther in this experiment. The level of supplemental dietary lipid was 4.6%. This lipidwas supplied by either 4.6% corn oil, 2.3% each of soybean oil and cottonseed oil, or 2.1%each of soybean oil and cottonseed oil with addition of 0.4% sesame oil, corn oil, or lard.Fish meal was included at a level of 57% in all diets. Artemia nauplii were used as thecontrol diet. Alterations in the dietary lipid were accomplished without alteration in theconcentrations of any of the other ingredients. Final body lengths of the larvae, percentsurvivals and overall diet acceptance scores were recorded as in previous experiments.150Table 6.8 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 6.2Ingredients (% as fed)Diets6.2.1 6.2.2 6.2.3 6.2.4 6.2.5Fish meal 57.0 57.0 57.0 57.0 57.0Peanut meal 10.8 10.8 10.8 10.8 10.8Corn meal 8.9 8.9 8.9 8.9 8.9Rice bran 8.9 8.9 8.9 8.9 8.9Sesame oil 0.4Soybean oil 2.1 2.1 2.1 2.3Cottonseed oil 2.1 2.1 2.1 2.3Corn oil 0.4 4.6Lard 0.4Vitamins & minerals' 9.8 9.8 9.8 9.8 9.8i-, Total 100.0 100.0 100.0 100.0 100.001-, Dry matter % 90.1 90.1 90.1 90.1 90.1csame as shown in Table 6.2Table 6.8 (continued)Diets6.2.1 6.2.2 6.2.3 6.2.4 6.2.5Calculated nutrient levels of diets (% of DM)Protein 39.17 39.17 39.17 39.17 39.17Lipid 9.87 9.87 9.87 9.88 9.88Carbohydrate including fiber 16.70 16.70 16.70 16.70 16.70Ash 34.26 34.26 34.26 34.25 34.25Arginine 3.33 3.33 3.33 3.33 3.33Histidine 1.40 1.40 1.40 1.40 1.40Isoleucine 2.25 2.25 2.25 2.25 2.25Leucine 3.44 3.44 3.44 3.44 3.44Lysine 2.96 2.96 2.96 2.96 2.96Methionine 1.12 1.12 1.12 1.12 1.12Phenylalanine 2.16 2.16 2.16 2.16 2.161-,CI Threonine 1.88 1.88 1.88 1.88 1.88t■D Tryptophan 0.51 0.51 0.51 0.51 0.51Valine 2.47 2.47 2.47 2.47 2.47Fatty acidsC14:0 0.08 0.08 0.09 0.06 0.08C16:0 1.55 1.56 1.63 1.31 1.59C18:0 0.32 0.30 0.36 0.24 0.31C18:1w9 2.91 2.83 2.92 3.03 2.81C18:2w6 3.75 3.84 3.59 4.15 3.81C18:3w3 0.29 0.29 0.29 0.16 0.30C20:4x6 0.12 0.12 0.12 0.12 0.12C20:5w3 0.15 0.15 0.15 0.15 0.15C22:5w3 0.07 0.07 0.07 0.07 0.07C22:6w3 0.46 0.46 0.46 0.46 0.46Results and discussionBest survival and food acceptance were noted for larvae fed Artemia nauplii (Table6.9). The results show that the diets supplemented exclusively with corn oil or an equalcombination of soybean oil and cottonseed oil gave the best performance of all theexperimental diets. Poorest larval performance was seen when the supplemental lipidpartially originated from lard or sesame oil. The diet acceptance scores were generallypositively correlated with final body lengths.Table 6.9 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed dry diets in experiment 6.2DietsBodylength (mm)mean+ SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*6.2.1 11.0+1.4 89.4 58.4 92.1 3.06.2.2 11.5+1.8 93.5 57.8 91.2 3.06.2.3 11.1+1.6 90.2 57.6 90.9 3.06.2.4 11.8+0.9 95.9 60.1 94.8 3.56.2.5 11.8+1.5 95.9 61.7 97.3 3.5Artemia nauplii 12.3+1.2 100.0 63.4 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent153Experiment 6.3Materials and methodsThe experiment assessed the effects of varying the fish meal content in diets forwalking catfish larvae (Table 6.10). The dietary concentrations of fish meal were 57%,58%, 59% and 60%. The increments in fish meal concentration were made by reducing theamounts of rice bran included in the respective diets. Soybean oil and cottonseed oils wereeach added to the diets at a level of 2.3%. Thus, the total supplemental lipid content in thediets was 4.6%. Final body lengths, percent survivals and overall diet acceptance scoreswere recorded.154Table 6.10 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 6.3DietsIngredients (% as fed) 6.3.1 6.3.2 6.3.3 6.3.4Fish meal 57.0 58.0 59.0 60.0Peanut meal 9.8 9.8 9.8 9.8Corn meal 7.9 7.9 7.9 7.9Rice bran 10.9 9.9 8.9 7.9Soybean oil 2.3 2.3 2.3 2.3Cottonseed oil 2.3 2.3 2.3 2.3Vitamins & minerals° 9.8 9.8 9.8 9.8Total 100.0 100.0 100.0 100.0Dry matter % 90.1 90.1 90.1 90.11--,^csame as shown in Table 6.2alCriTable 6.10 (continued)Diets6.3.1 6.3.2 6.3.3 6.3.4Calculated nutrient levels of diets (% of DM)Protein 39.17 39.35 39.73 40.16Lipid 9.88 9.90 9.94 9.80Carbohydrate including fiber 16.70 16.27 15.55 15.03Ash 34.25 34.48 34.78 35.01Arginine 3.33 3.32 3.36 3.40Histidine 1.40 1.41 1.43 1.44Isoleucine 2.25 2.26 2.29 2.32Leucine 3.44 3.45 3.48 3.52Lysine 2.96 2.99 3.03 3.07Methionine 1.12 1.14 1.15 1.17Phenylalanine 2.16 2.16 2.19 2.21I-cn Threonine 1.88 1.89 1.92 1.94cn Tryptophan 0.51 0.51 0.52 0.52Valine 2.47 2.47 2.50 2.53Fatty acidsC14:0 0.08 0.08 0.08 0.08C16:0 1.59 1.59 1.59 1.56C18:0 0.31 0.31 0.31 0.31C18:1w9 2.81 2.80 2.80 2.73C18:2w6 3.81 3.79 3.78 3.70C18:3w3 0.30 0.30 0.31 0.30C20:4,.6 0.12 0.12 0.13 0.13C205w3 0.15 0.15 0.16 0.16C22:5w3 0.07 0.08 0.08 0.08C22:6w3 0.46 0.47 0.48 0.49Results and discussionThe results showed that larvae fed on Artemia nauplii had the greatest final bodylengths (Table 6.11). Performance was increased almost linearly as the dietary fish mealcontent was raised at the expense of rice bran meal. Mean body lengths relative to that ofcontrol fish fed Artemia nauplii ranged from 95.1-97.6 % whereas the relative percentsurvivals ranged from 92.1-96.4 % for larvae fed the experimental diets.Table 6.11 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed dry diets in experiment 6.3Body Relative Percent Relative Overall dietDiets length (mm) body length Survival survival acceptancemean + SD (%) (%) score*6.3.1 11.7+1.8 95.1 52.6 94.9 3.06.3.2 11.7±1.7 95.1 51.0 92.1 3.06.3.3 11.8±1.3 95.9 52.6 94.9 3.06.3.4 12.0±1.2 97.6 53.4 96.4 3.5Artemia nauplii 12.3±1.4 100.0 55.4 100.0 4.0* Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellent157Experiment 6.4Materials and methodsExperiment 6.4 was the last experiment of this series. The test diet formulationsare provided in Table 6.12. Further manipulations of the dietary ingredient levels weremade and larval performance was recorded. The level of fish meal inclusion was increasedto 60% in all diets. This concentration of fish meal had resulted in improved performanceof larvae in the previous experiment (diet 6.3.4). Diet 6.3.4 was therefore included as areference diet in this experiment. Lard and sesame oil were not used in the diets andsupplemental lipid was provided only from corn, soybean, or cottonseed oils. Corn oil wasincluded in diets at levels of 1%, 1.8%, 2.8%, 3.6% and 4.6%. Cottonseed and soybean oils,in variable proportions to each other, were used to replace the corn oil content. The levelof supplemental lipid in all diets was 4.6%. Final body lengths, percent survivals andoverall diet acceptance scores of larvae fed the diets were recorded.158Table 6.12 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 6.4DietsIngredients (% as fed) 6.3.4 6.4.1 6.4.2 6.4.3 6.4.4(control)Fish meal 60.0 60.0 60.0 60.0 60.0Peanut meal 9.8 9.8 9.8 9.8 9.8Corn meal 7.9 7.9 7.9 7.9 7.9Rice bran 7.9 7.9 7.9 7.9 7.9Soybean oil 0.5 0.9 1.4 1.8Cottonseed oil 0.5 0.9 1.4 1.8Corn oil 3.6 4.6 2.8 1.8 1.0Vitamins & mineralsc 9.8 9.8 9.8 9.8 9.8Total 100.0 100.0 100.0 100.0 100.0Dry matter % 90.1 90.1 90.1 90.1 90.1csame as shown in Table 6.2Table 6.12 (continued)Diets6.3.4(control)6.4.1 6.4.2 6.4.3 6.4.4Calculated nutrient levels of diets (% of DM)Protein 40.16 40.16 40.16 40.16 40.16Lipid 9.80 9.80 9.81 9.81 9.80Carbohydrate including fiber 15.03 15.03 15.03 15.03 15.03Ash 35.01 35.01 35.00 35.00 35.01Arginine 3.40 3.40 3.40 3.40 3.40Histidine 1.44 1.44 1.44 1.44 1.44Isoleucine 2.32 2.32 2.32 2.32 2.32Leucine 3.52 3.52 3.52 3.52 3.52Lysine 3.07 3.07 3.07 3.07 3.07Methionine 1.17 1.17 1.17 1.17 1.171-,cs4 Phenylalanine 2.21 2.21 2.21 2.21 2.210 Threonine 1.94 1.94 1.94 1.94 1.94Tryptophan 0.52 0.52 0.52 0.52 0.52Valine 2.53 2.53 2.53 2.53 2.53Fatty acidsC14:0 0.07 0.06 0.07 0.07 0.08C16:0 1.33 1.27 1.39 1.44 1.50C18:0 0.26 0.24 0.27 0.28 0.29C18:1w9 2.90 2.94 2.86 2.81 2.77C18:2w6 3.98 4.04 3.91 3.84 3.77C18:3w3 0.19 0.16 0.22 0.25 0.27C20:46 0.13 0.13 0.13 0.13 0.13C20:5w3 0.16 0.16 0.16 0.16 0.16C22:5w3 0.07 0.07 0.07 0.07 0.07C22:6w3 0.49 0.49 0.49 0.49 0.49Results and discussionThe larvae fed diet 6.3.4 exhibited similar performance to that noted for the larvaefed this diet in experiment 6.2. The results show that 60% inclusion of fish meal in drydiets improves larval performance (Table 6.13). Further, the performance of the larvaegenerally declined as the level of supplemental corn oil was decreased (from 4.6% in diet6.4.1 to 1% in diet 6.4.4). It therefore appears that the fatty acid composition of thesupplemental corn oil was best suited to satisfy the fatty acid requirement of walkingcatfish larvae. Similarly, the amino acid profile of fish meal also had an enhancing effecton the growth and survival of these larvae. Therefore, corn oil and fish meal should beincluded in proper amounts when formulating diets for carnivorous walking catfish larvae(Table 6.13).Table 6.13 Final body lengths, percent survivals and overall diet acceptance scoreresponses for walking catfish larvae fed diets in experiment 6.4DietsBodylength (mm)mean+ SDRelativebody length(%)PercentSurvivalRelativesurvival(%)Overall dietacceptancescore*6.3.4 (control) 11.6±1.3 93.5 63.4 97.2 3.06.4.1 12.2±1.4 98.4 64.2 98.5 3.56.4.2 11.8±1.9 95.2 62.6 96.0 3.06.4.3 11.7±1.7 94.4 62.7 96.2 3.06.4.4 11.6±1.8 93.5 63.3 97.1 3.0Artemia nauplii 12.4+1.5 100.0 65.2 100.0 4.0Overall diet acceptance score described as 1 = poor, 2 = fair, 3 = good and 4 = excellentIn contrast, it has been shown in channel catfish (Ictalurus punctatus) thatsupplemental corn oil initially resulted in a positive growth response and protein sparing.Thereafter, however, growth inhibition was observed (Dupree and Sneed, 1966). It161appears that channel catfish may have a different requirement for essential fatty acids(Stickney and Andrews, 1971 and 1972). Tuncer and Harrell (1992) reported that larvalstriped bass (Morone saxatilis) and palmetto bass (M. saxatilis x M. chrysops) that were feddiets with corn oil showed signs of essential fatty acid deficiency.It should be mentioned that the most important management factor to considerduring the experimental period was aquarium hygiene. From my observations, theaccumulation of uningested food on the bottom of the aquaria always promoted fungaldevelopment. Accordingly, the tanks should be cleaned everyday in order to eliminate theundigested food and feces. In the next experiment, the performance of larvae fed diet6.4.1 was compared to that of larvae fed the moist diet 5.3.2 and Artemia nauplii162III. EXPERIMENT 7: RESPONSES OF WALKING CATFISH LARVAE FEDSELECTED FORMULATED DIETS, THE NIFI 2 DIET AND ARTEMIA NAUPLIIMaterials and methodsThis final experiment on walking catfish larvae was conducted at the BurirumFreshwater Fisheries Station to devise formulated diets that would promote satisfactiongrowth and survival. The best diets from experiment 5 (moist diet 5.5.3) and experiment6 (dry diet 6.4.1) were compared against NIFI 2, the conventional dry diet for culturingwalking catfish larvae, and Artemia nauplii (control diet). All diets were analyzed forproximate composition. The procedures used for proximate analysis were discussedpreviously in chapter 3.Four diets were each fed to duplicate groups of walking catfish larvae. Thecompositions of the experimental diets are given in Table 6.14. Larvae were obtainedfrom induced spawning using methods described in chapter 3. The experiments were 15days starting from the third day after hatching. The experiment was conducted fromSeptember 5 to 20, 1990. At 2 days of age, the larvae were placed into the aquaria(45x90x45 cm3) at a density of 1,000 larvae per aquarium. The aquaria were aeratedcontinuously and larvae were fed twice-a-day. Every morning before the first feeding,dead larvae were removed and the tanks were cleaned. About two-thirds of the water wasreplaced after each cleaning. At the end of the experiment, all larvae were counted todetermine the percentage survival and individual body lengths were measured on 40larvae that were picked randomly from each replicate aquarium. Statistical analyses werecarried out according to Zar (1984) for body length and percent survival data weresubjected to arcsine transformation before analysis. Tukey's honestly significant163difference test (P = 0 . 0 5) was used to detect significant differences among the means forbody length in relation to diet treatment.1641-,Table 6.14 Ingredient and nutrient compositions of diets fed to walking catfish larvae for 15 days from 3-18 days of age in experiment 7Diets*Ingredients (% as fed) NWI 2 Moist diet Dry diet Artenda5.3.2 6.4.1 naupliiRaw chicken liver 17.9 ■••■Cooked chicken blood 4.5Whole chicken egg 17.9Frozen Artemia 26.8Steamed peanut 2.7Raw Squid 13.4Corn oil 0.4 4.6Lard 0.4Sesame oil 0.4Cod liver oil 4.0Skim milk 4.0Dried Spirulina 1.3Corn flour 3.6K-carrageenan 2.2Fish meal 56.0 60.0Corn meal 7.9Peanut meal 12.0 9.8Rice bran 12.0 7.9Wheat gluten 0.4Starch 14.0Vitamins & minerals' 1.6Vitamins & mineralsc 4.5 9.8Total 100.0 100.0 100.0Dry matter % 92.6 33.2 92.4a, c same as shown in Tables 6.6 and 6.2* NIFI 2 commonly used diet (for walking catfish larvae); moist diet 5.3.2 from experiment 5.3 and dry diet 6.4.1 from experiment 6.4.Table 6.14 (continued)DietsNIFI 2 Moist diet5.3.2Dry diet6.4.1AnemianaupliiCalculated nutrient levels of diets (% of DM)Protein 45.38 40.26 40.16 61.10Lipid 10.24 15.36 9.80 19.42Carbohydrate including fiber 26.86 18.20 15.03 9.38Ash 17.52 26.36 35.01 10.10Arginine 3.29 2.01 3.40 4.00Histidine 1.36 1.12 1.44 1.06Isoleucine 2.19 1.4 2.32 2.10Leucine 3.34 3.4 3.52 5.00Lysine 2.89 2.78 3.07 5.00Methionine 1.09 0.69 1.17 0.701-,cs) Phenylalanine 2.13 1.89 2.21 2.60o.) Threonine 1.84 1.38 1.94 0.00Tryptophan 0.50 0.47 0.52 0.81Valine 2.43 2.35 2.53 2.60Fatty acidsC14:0 0.06 0.07 0.06 0.42C16:0 1.11 2.87 1.27 3.75C18:0 0.16 0.93 0.24 0.77C18:1 2.50 5.39 2.94 5.88C18:2w6 1.36 2.66 4.04 1.35C18:3w3 0.16 0.34 0.16 2.74C20:4 0.17 0.06 0.13 0.52C20:5w3 0.53 0.12 0.16 0.67C22:5w3 0.10 0.07C22:6w3 0.85 0.15 0.49 0.05Results and discussionThe proximate compositions of the three formulated diets are presented in Table6.15. The analyzed protein concentrations in the three diets expressed on a dry weightbasis were 44.5% (NIFI 2), 41.8% (moist diet 5.3.2) and 39.9% (dry diet 6.4.1) and thelipid concentrations were 11.8, 15.2, 9.4%, respectively.Table 6.15 Proximate composition of diets fed to walking catfish larvae in experiment 7(All values in parentheses are expressed as a percentage of dry matter.)Diets DM" CP21 EE3/ NFE4/including fiberAshNIFI 2 91.22 40.63 10.77 24.42 15.40(100.00) (44.54) (11.81) (26.77) (16.88)Moist diet 5.3.2 82.57 34.51 12.59 21.52 13.95(100.00) (41.80) (15.25) (26.06) (16.89)Dry diet 6.4.1 77.13 30.75 7.22 11.90 27.25(100.00) (39.87) (9.37) (15.43) (35.33)Note 11DM = dry matter; 2/CP = crude protein; 31EE = ether extract (crude lipid); 4/NFE = nitrogen freeextract which was estimated by difference.Results pertaining to the influence of diet treatment on larval performance arepresented in Table 6.16, Fig. 6.1 and Appendix 3. The experiment demonstrated thatlarvae can be reared on formulated diets provided that adequate attention is given to therearing methods and diet composition. Growth of larvae fed the formulated diets wassatisfactory. Indeed, the linear growth of larvae fed Artemia nauplii and dry diet 6.4.1were not found to be significantly different. Larvae fed on Artemia nauplii had the highestaverage body length (12.52 mm), followed those fed dry diet 6.4.1 (12.07 mm), moist diet5.3.2 (12.02 mm) and NIFI 2 (10.81 mm). The survival of larvae fed on Artemia nauplii167and dry diet 6.4.1 likewise did not differ significantly and was higher than larvae fed theother two diets.Table 6.16 Mean values for final body lengths (mm) and percent survivals of walkingcatfish larvae in experiment 7Type of food* NIFI 2^Moist diet^Dry diet Artemia5.3.2 6.4.1^naupliiMean** body length (±SD)^10.81+0.70a 12.02+1.12b 12.07+1.07bc12.52+1.12cMean percent survival (%)^56.25a^48.45a^62.75ab^65.55bNote: *NIFI 2 is a common diet for walking catfish larvae; moist diet 5.3.2 from experiment 5.3; dry diet6.4.1 from experiment 6.4.** Mean values from duplicate tanks with each based on 40 fish.Numbers with the same superscript are not significantly different at the 5% level of significance.The foregoing results agree with these of a study using formulated dry feed forrearing Clarias gariepinus larvae and fry (Appelbaum and Van Damme, 1988). Severalstudies have suggested that rearing catfish (Clarias lazera) larvae by using live or frozenArtemia nauplii in combination with a formulated diet gives good results (Hogendoorn,1980; Verreth et al., 1987). Likewise, a formulated dry diet can be used successfully forthe rearing of Clarias gariepinus when it is supplemented with live Artemia nauplii (Uysand Hecht, 1985; Hecht and Appelbaum, 1987) or after 2 and 4 days of feeding Artemia(Verreth and Van Tongeren, 1989). Decapsulated and dried Artemia cysts have beenshown to be an effective fish feed (Verreth et al., 1987).16815141 0aNIFI 2^Moist Diet^Dry Diet^Artemis5.3.2 6.4.1 nauplIIDietsFigure 6.1 Body lengths of walking catfish larvae fed with different diets.Each value represents the mean ± SD. Body lengths which were similar as determinedby the Tukey HSD Test (P > 0.05) are identified by the same superscript i.e. a. b and c.NTFI 2 is a commonly used diet for walking catfish larvae, moist diet 5.3.2 is fromexperiment 5.3 and dry diet 6.4.1 is from experiment 6.4.169CHAPTER 7OVERALL CONCLUSIONSThe present study has demonstrated that it is possible to formulate adequatepractical diets for rearing common carp and walking catfish larvae. The results drawnfrom the experiments are as follows:1) Artemia nauplii were the most readily consumed food by both common carp andwalking catfish larvae (experiment 1, chapter 4). Growth of both common carp andwalking catfish larvae fed on Artemia nauplii for 15 days was significantly better than thatof larvae fed on the NIFI diet. Artemia nauplii were, therefore, selected as a reference dietfor further experiments. However, since the NIFI diet is the commonly used practical feedfor freshwater larval culture in Thailand, it was therefore included in the study. Mouthsizes of larvae at the time of feeding commencement ranged from 425-450 pm, and thisrange in mouth size value was used to determine the optimal particle size of the testformulated diets.2) Moist and dry diets were assessed in experiments 2 to 4 for common carp(chapter 5), and in experiments 5 to 7 (chapter 6) for walking catfish larvae. The bestresponse in terms of linear growth and percent survival, in common carp larvae, wasobtained with moist diet 2.9.3. This diet will henceforth be designated Burirum 1 diet andit is composed of 16.3% raw pork liver, 8.1% coagulated cooked chicken blood, 16.3% wholechicken egg, 16.3% raw squid, 26% frozen Artemia, 0.8% cooked peanut, 1.5% corn oil,4.1% skim milk, a vitamin-mineral supplement, and 2% K-carrageenan. 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Icthyol, 13: 791-793.Zar, J. H., 1984. Biostatistical Analysis (2nd edition). Prentice-Hall, Englewood Cliffs,N.J., 718 pp.191ANOVA of mean body length of common carp larvaeTreatment^0.603^2^0.301Error 0.084 3 0.028Total 0.687^5^0.32910.811*^0.043APPENDICESAppendix 1. Analysis of variance of the data from experiment 1 (Table 4.3)Source^Sum of squares df^Mean square^F^ProbabilityANOVA of meanTreatmentErrorTotalbody length of walking catfish larvae^17.72^2^8.86^0.59 3 0.2018.31^5^9.0645.12**^0.006*significant difference (P < 0.05)** highly significant difference (P < 0.01)Appendix 2. Analysis of variance of the data from experiment 4 (Table 5.33)Source^Sum of squares df^Mean square^F^ProbabilityANOVA of mean body length of common carp larvaeTreatment^0.993^3^0.331^22.371** 0.006Error 0.059 4 0.15Total 1.052^7^0.481ANOVA of mean survival of common carp larvaeTreatment^0.009^3^0.003^0.866ns 0.528Error 0.013 4 0.003Total 0.022^7^0.006** highly significant difference (P < 0.01)ns non significant difference192Appendix 3. Analysis of variance of the data from experiment 7 (Table 6.16)Source^Sum of squares^df^Mean square^F^ProbabilityANOVA of mean body length of walking catfish larvaeTreatment^3.22^3^1.07^83.53**^0.000Error 0.05 4 0.01Total 3.27^7^1.08ANOVA of mean survival of walking catfish larvaeTreatment^0.052^3^0.017^21.149** 0.006Error 0.003 4 0.001Total 0.055^7^0.018** highly significant difference (P < 0.01)193Appendix 4. Composition of vitamin and mineral premixVitamins a b cWater-soluble vitamins (mg / kg of dry diet)p-amino benzoic acid 225.7 100Biotin 3.4 4Inositol 2266.7 4000Niacin 100 453.3 400Ca-pantothenate 24 158.7 600Pyridoxine HC1 (136) 20 27 120Riboflavin (B2) 8 113.3 80Thiamine HC1 (131) 5 34 40Cyanocobalamine (B12) 0.05 0.8Na-ascorbate 500 1133.3 2000Folic acid 1 8.5 8Choline-chloride 1400 4633.3 6000Fat-soluble vitaminsMenadione 4 27 40Vitamin A 120001U0-carotene 56.7 96of.-Tocopherol 100 226.7 200Vitamin D3 4000 IUCholecalciferol 5.7 12Source: a Sitasit et al. (1982); b Halver (1957) and Teshima et al. (1982); c Kanazawaet al. (1977); Sujaritvongsanon, (1984)194Appendix 4. (continued)Minerals a b c(mg / kg of dry diet)NaC1 3000 4350MgSO4.7H20 1400 13700 30410C6H5Fe07•H20 200 2970MnSO4•4H20 250 80KI 10 15Ca(H2PO4)2.2H20 6000 13580NaH2PO4.2H20 8720 7900K112PO4 23980C3H6Ca04 32700AlC13.6H20 15ZnSO4.7H20 30CuC12 10CoC12 100K2HPO4 20000Ca3(PO4)2 27200CuSO4 10KC1 1000ZnCO3 130Total 12000 100250 85510Source: a Sitasit et al. (1982); b Halver (1957) and Teshima et al. (1982); c Kanazawaet al. (1977); Sujaritvongsanon, (1984)195

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