Culturing grass carp and grey mullet using food waste incorporated with traditional Chinese medicine, Baker's yeast and enzymes

Transcription

1 Hong Kong Bptist University HKBU Institutionl Repository Open Access Theses nd Disserttions Electronic Theses nd Disserttions 2013 Culturing grss crp nd grey mullet using food wste incorported with trditionl Chinese medicine, Bker's yest nd enzymes Wi Ming Choi Hong Kong Bptist University Follow this nd dditionl works t: Recommended Cittion Choi, Wi Ming, "Culturing grss crp nd grey mullet using food wste incorported with trditionl Chinese medicine, Bker's yest nd enzymes" (2013). Open Access Theses nd Disserttions This Thesis is brought to you for free nd open ccess by the Electronic Theses nd Disserttions t HKBU Institutionl Repository. It hs been ccepted for inclusion in Open Access Theses nd Disserttions by n uthorized dministrtor of HKBU Institutionl Repository. For more informtion, plese contct repository@hkbu.edu.hk.

2 CULTURING GRASS CARP AND GREY MULLET USING FOOD WASTE INCORPORATED WITH TRADITIONAL CHINESE MEDICINE, BAKER S YEAST AND ENZYMES CHOI WAI MING Ph.D. Thesis HONG KONG BAPTIST UNIVERSITY 2013

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4 Culturing Grss Crp nd Grey Mullet Using Food Wste Incorported with Trditionl Chinese Medicine, Bker's Yest nd Enzymes CHOI Wi Ming A thesis submitted in prtil fulfillment of the requirements for the degree of Doctor of Philosophy Principl Supervisor: Prof. WONG Ming Hung Hong Kong Bptist University September 2013

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6 Declrtion I hereby declre tht this thesis represents my own work which hs been done fter registrtion for the degree of PhD t Hong Kong Bptist University, nd hs not been previously included in thesis or disserttion submitted to this or ny other institution for degree, diplom or other qulifictions. Signture: Dte: Sept 2013 i

7 Abstrct The present study focused on using food wstes nd feed supplements, e.g. enzymes (bromelin nd ppin), bker s yest nd Trditionl Chinese Medicines (TCMs) for rering freshwter fish (grss crp nd grey mullet) in Hong Kong. Different types of food wstes, e.g. mets, bones, cerels, fruits nd vegetbles were collected from locl hotels, mixed in different rtios nd processed into feed pellets for feeding trils. The cerel dominnt food wste feed (FW A) ws more suitble for grss crp nd grey mullet, with the best growth performnce (e.g. feed conversion rtio (FCR), specific growth rte (SGR)) nd higher protein digestibility (in grss crp), compred to FW B nd FW C which contined higher proportions of met products. The NBT (Nitroblue Tetrzolium) ctivities in blood nd plsm protein levels were decresed in the grss crp, cultured with food wste feeds without ny supplements, compred to the commercil feed, Jinfeng, 613 formultion (Control). Upgrding FW A by the ddition of 1% nd 2% mixtures of bromelin nd ppin significntly incresed the feed protein solubility nd subsequent to growth (SGR nd reltive weight gin (RWG)) nd feed utiliztion (e.g. pprent net protein utiliztion (ANPU), protein efficiency rtio (PER)) in both fish species. The protein nd feed utiliztions by grss crp were lso promoted by the yest supplements with the optiml dose of 2.5% yest (S. cerevisie) dded to FW A upgrded by enzymes. This showed tht yest could further enhnce nutrient utiliztion contined in feeds fter ddition of bromelin nd ppin. The in vitro study on the grss crp s plsm treted with TCM extrcts lso showed tht TCM extrcts could stimulte plsm bctericidl ctivity (on Aeromons hydrophil), possibly through enhncing plsm complement ctivity. The ii

8 formultion with Rdix scutellri, Rhizom coptidis, Herb ndrogrphis nd Rdix sophore flvescentis in the rtio of 1:1:2:3 ws more effective in enhncing plsm bctericidl ctivity thn single TCM extrcts. Besides, R. coptidis nd R. scutellri possessed the strongest ntimicrobil ctivity (in vitro) on fish pthogens (such s A. hydrophil, Lctococcus grviee nd Vibrio cholere) mong the 17 tested TCMs. In ddition, TCMs were less likely for developing drug resistnt pthogens thn ntibiotics. Grss crp immunity (NBT ctivity in blood, plsm bctericidl ctivity nd totl immunoglobulin level) ws boosted by the ddition of TCM formultion nd bker s yest (S. cerevisie). The disese resistnce to pthogen (A. hydrophil) ws lso enhnced, with significntly lower mortlities observed in groups feeding with TCM (1 nd 2% for 21 to 28 dys) nd bker s yest (2.5 nd 5% for dys). The uses of yest nd TCMs led to positive effects on growth, immunity nd disese resistnce to pthogens in fish, but the effects (grss crp) were less effectul when both were supplemented in feed. The combined use of both supplements my impir the effects of TCM formultion or yest in the modultion of gut mircoflor, nd upset the blnce of beneficil microbil communities. The present study demonstrted the fesibility of using feed supplements (TCM nd bker s yest) to enhnce fish immunity nd enzymes upgrded food wste feeds for rering fish, for the development of more sustinble quculture in Hong Kong. iii

9 Acknowledgements I would like to express my deepest grtitude to my principl supervisor, Prof. Wong Ming-Hung, for providing me with professionl guidnce, continuous support nd encourgement throughout my reserch. I would lso like to extend my deepest grtitude to my co-supervisor, Prof. Bin Zho-xing, for providing me professionl knowledge on Trtitionl Chinese Medicine. Specil cknowledgement is given to Dr. Sheng-chun Wu for his vluble suggestions during my study. I would like to extend my deepest grtitude to ll stff nd students in the Croucher Institute for Environmentl Sciences (CIES), for their ssistnce nd friendship. Specil thnks re given to Mr. CL Lm nd Mr. WY Yin for providing technicl support nd ssistnce during my experiments nd field works. Lst but not lest. I would like to thnk my fmily, especilly my prents for their love nd morl support throughout the study period. iv

10 Tble of Contents Declrtion.. i Abstrct...ii Acknowledgements. iv Tble of Contents... v List of Tbles.. xii List of Figures xv Abbrevitions nd Acronyms xiii Chpter 1 The necessity of feed lterntives with supplements in current nd future quculture industry 1.1 Overview of world quculture sttus Hong Kong Inlnd Fisheries Sttus Emerging problems in fst growing quculture industry in worldwide nd Chin Disese prevlence nd buse of chemicls nd ntibiotics Rising feed mterils prices Using feed supplement s immuno-stimulnts Herbl supplement: Trditionl Chinese Medicine (TCMs) Probiotics: Bker s yest The necessity of lterntive protein sources in fish feed Utiliztion of food wste s fish feed in worldwide Using enzyme for enhncing feed conversion in fish feed Potentil uses of drug lterntives nd food wste s fish feed in Hong Kong quculture industry Aims nd objectives Contributions nd significnce of the present reserch. 32 v

11 Chpter 2 Using food wstes s fish feed ingredients for grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) cultivtion 2.1 Introduction Mterils nd Methods Food wste fish feed preprtion Feed digestibility of different food wstes formultions in grss crp Feed conversion of different food wstes formultions in grey mullet Fish growth performnce prmeters Chemicl nlysis on fish crcss nd feed Sttisticl nlysis Results Results of feed digestibility of different food wste formultions in grss crp Feed digestibility of different food wstes formultions nd growth performnce of grss crp Crcss composition of grss crp fed with different food wste formultions Results of feed conversion of different food wste formultions in grey mullet Growth performnce of grey mullet fed with different food wste formultions Crcss composition of grey mullet fed with different food wste formultions Discussion Growth of grss crp nd grey mullet fed with different food wste feeds Utiliztions of different food wste feeds by grss crp nd grey mullet Conclusion vi

12 Chpter 3 Upgrding food wstes by mens of bromelin nd ppin to enhnce growth nd immunity of grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) 3.1 Introduction Mterils nd Methods Effects of food wstes formultion upgrded by ppin nd bromelin on grss crp growth performnce Effects of food wstes upgrded by ppin nd bromelin on grey mullet growth performnce Fish growth performnce prmeters Fish immunologicl prmeters Sttisticl nlyses Results Results of grss crp feeding tril with different food wste feeds upgrded by ppin nd bromelin Grss crp growth performnce fed with different food wste feeds upgrded by ppin nd bromelin Grss crp crcss composition fed with different food wste feeds upgrded by ppin nd bromelin Grss crp immunologicl prmeters Results of grey mullet feeding tril with different food wste feeds upgrded by ppin nd bromelin Grey mullet growth performnce fed with different food wste feeds upgrded by ppin nd bromelin Grey mullet crcss composition fed with different food wste feeds upgrded by ppin nd bromelin Grey mullet immunologicl prmeters Discussion Growth of grss crp nd grey mullet fed with food wste supplemented with bromelin nd ppin mixture Utiliztions of food wste supplemented with bromelin nd ppin mixture Hemtologicl prmeters of fish fed with food wste supplemented with enzyme mixture..93 vii

13 3.5 Conclusion. 95 Chpter 4 Adding bker s yest to food wste to enhnce growth performnce nd immunity of grss crp (Ctenophryngodon idellus) 4.1 Introduction Mterils nd Methods Experimentl setup nd fish feed preprtion Feeding experiment design Fish immunologicl nd growth prmeters A. hydrophil injection to grss crp Sttisticl nlysis Results Feeding tril with yest supplement incorported in different feeds Growth performnce of grss crp Crcss composition of grss crp Hemtologicl prmeters of grss crp fed with different feeds Disese resistnce to Aeromons hydrophil of grss crp Discussion Growth nd feed utiliztions of grss crp fed with bker s yest supplemented feed Immunity of grss crp fed with bker s yest supplemented feed Fish disese resistnce to A. hydrophil Conclusion Chpter 5 The ntimicrobil ctivity of Trditionl Chinese Medicines (TCMs), potentil drug lterntive on deling with fish pthogens 5.1 Introduction Methods nd Mterils Antimicrobil ctivity of boiled queous extrcts of 17 TCMs Antimicrobil ctivity of selected queous nd orgnic TCMs extrcts Checkerbord method for the combined effect of C. phellodendri, R. scutellri, R. coptidis nd F. forsythie viii

14 5.2.4 Development of drug resistnt fish pthogens exposed to selected queous TCM extrcts Results Antimicrobil ctivity of boiled queous extrcts of 17 TCMs Antimicrobil ctivity of queous (boiled nd non-boiled) nd orgnic extrcts of 4 selected TCMs Antimicrobil ctivities of boiled queous mixtures of C. phellodendri, R. scutellri, R. coptidis nd F. forsythi Development of drug resistnt fish pthogens exposed to selected TCM extrcts Discussion Antimicrobil ctivities of boiled queous extrcts of 17 TCMs Antimicrobil ctivities of orgnic nd queous extrcts of R. coptidis nd R. scutellri, C. phellodendri nd F. forsythi Development of resistnt bcteri to single TCMs nd ntimicrobil ctivities of mixed TCM extrcts Conclusion Chpter 6 Effects of Trditionl Chinese Medicines (TCM) on the Immune Response of Grss Crp (Ctenophryngodon idellus) 6.1 Introduction Mterils nd Methods Experimentl fish feed preprtion Identifictions of TCMs Fish feeding experiment nd blood smpling A. hydrophil injection to grss crp Immunologicl prmeters nlysis Field tril in Yuen Long nd cost evlution on the ppliction of TCM feed Effects of TCM extrcts on plsm bctericidl ctivity of grss crp ginst A. hydrophil Sttisticl nlysis Results Immune prmeters in Grss crp blood feeding with TCM ix

15 formultion Fish growth nd disese resistnce to A. hydrophil in lbortory experiment Disese resistnce to A. hydrophil in field tril t Yuen Long nd cost evlution on TCM feed ppliction In vitro ctivtion on plsm bctericidl ctivity Discussion Hemtologicl prmeters of grss crp fed with TCM formultion Plsm bctericidl ctivity of grss crp in feeding tril nd in vitro study Disese resistnce to A. hydrophil Cost evlution of using TCM feed in quculture Conclusion Chpter 7 Upgrding food wste feed using TCMs, bker s yest nd enzyme on the immune responses of grss crp (Ctenophryngodon idellus) ginst Aeromons hydrophil 7.1 Introduction Mterils nd Methods Experimentl setup nd fish feed preprtion Feeding tril nd smple collections A. hydrophil injection to grss crp Sttisticl nlysis Results Growth performnce of grss crp fed with enzymes, bker s yest nd TCMs s supplements Immunity of grss crp fed with enzymes, bker s yest nd TCMs Disese resistnce to A. hydrophil of grss crp fed with enzymes, bker s yest nd TCMs Discussion Growth nd feed utiliztion of grss crp fed with enzyme upgrded food wste with bker s yest ndtcm x

16 7.4.2 Immunity prmeters of grss crp fed with enzyme upgrded food wste with bker s yest nd TCM Effects of bker s yest nd TCM on fish disese resistnce ginst A. hydrophil Conclusion 195 Chpter 8 Generl Discussion nd Mjor Conclusions 8.1 Introduction Generl Discussion The effects of dding enzymes in enhncing food wste utiliztion by fish The effects of dding of TCMs nd bker s yest to cope with fish infections Concerns on using feed supplements for ppliction in quculture Mjor conclusions Comments for future studies..215 References.218 Publiction & Conference Presenttions Curriculum Vite. 246 xi

17 List of Tbles Tble 1.1 Reduced fish mel inclusions in qu feed of different quculture species (FAO, 2012) Tble 2.1 Food wste ctegories collected from hotels Tble 2.2 Food wste feed formultions (contining 75% food wste).. 43 Tble 2.3 Composite nlysis of experimentl feeds Tble 2.4 Amino cid composition (% dry weight bsis) of the four experimentl feeds (Control, FW A, B nd C). 45 Tble 2.5 Growth performnce of grss crp fed with different food wste feeds...54 Tble 2.6 Proximte composition (wet weight bsis) of grss crp crcss fed withdifferent food wste feeds...55 Tble 2.7 Growth performnce of grey mullet fed with different food wste feeds Tble 2.8 Proximte composition (wet weight bsis) of grey mullet crcss fed with different food wste feeds 58 Tble 3.1 Food wste feed formultions (FW A & D, contining 75 & 60% food wste respectively)...73 Tble 3.2 Composite nlysis of experimentl feeds with 1% or 2% mixture of bromelin nd ppin in food wste A nd D (FW A & D) Tble 3.3 Growth performnce of grss crp fed with different food wste feeds upgrded by ppin nd bromelin. 80 Tble 3.4 Proximte compositions (%, wet weight bsis) of grss crp crcss fed with different food wste feeds upgrded by ppin nd bromelin Tble 3.5 Growth performnce of grey mullet fed with different food wste fish feeds upgrded by ppin nd bromelin 86 Tble 3.6 Proximte composition (%, wet weight bsis) of grey mullet crcss fed with different food wste fish feeds upgrded by ppin nd bromelin xii

18 Tble 4.1 Composite nlysis of three types of feeds: control (C) nd food wste formultion A (with (A-E) nd without (A) enzyme) feed supplement with or without bker s yest (Y)..101 Tble 4.2 Growth performnce of grss crp fed with control (C) nd food wste (with (A-E) nd without (A) enzyme) feeding groups with or without bker s yest (Y) Tble 4.3 Proximte composition of grss crp crcss (%, wet weight bsis) fed with control (C) nd food wste (with (A-E) nd without (A) enzyme) feeding groups with or without bker s yest (Y) Tble 5.1 Antimicrobil ctivity screening (mg/ml) using boiled queous extrcts of 17 Trditionl Chinese Medicines (TCMs) Tble 5.2 Antimicrobil ctivities (mg/ml) of different solvent extrcts of Rdix scutellri, Rhizom coptidis, Cortex phellodendri nd Fructus forsythi Tble 5.3 Rtio of MIC between non-boiled nd boiled queous extrcts Tble 5.4 The minimum inhibition concentrtion (MICs) frctionl inhibitory concentrtion (FIC) indices of boiled queous mixtures of four Trditionl Chinese Medicines (TCMs) 135 Tble 5.5 Development of drug resistnt fish pthogens fter seril pssges of Rdix scutellri, Rhizom coptidis, Cortex phellodendri nd Fructus forsythi Tble 6.1 The mjor chemicl components nd their medicl vlues of Rhizom coptidis, Rdix scutellri, Herb ndrogrphis nd Rdix sophore flvescentis identified by Thin-lyer Chromtogrphy (TLC) Tble 6.2 Weight gin rte (%) nd specific growth rte (%/dy) Tble 6.3 The product cost (in $USD) of 1% nd 2% Trditionl Chinese Medicine (TCM) feed Tble 7.1 Growth performnce of grss crp feeding with enzymes upgrded food wste (A-E) supplemented with bker s yest (Y) nd Trditionl Chinese Medicine (TCM). 185 Tble 8.1 Summry of feeding trils on growth nd feed utiliztion of food wstes supplemented with enzymes, TCM nd bker s yest by fish xiii

19 Tble 8.2 Summry of effects of TCM nd bker s yest on fish immunity Tble 8.3 In vitro studies of ntimicrobil ctivities nd immuno-stimulting properties of TCM xiv

20 List of Figures Fig. 1.1 World quculture production from 1950 to 2004 (showing Chin, rest of Asi nd the Pcific region) (FAO, 2006)... 3 Fig. 1.2 The pond nd mri-culture fish productions (tonnes) in Hong Kong quculture industry from 1984 to 2010 (Chn, 2005; AFCD, 2011)...6 Fig. 1.3 The number of unstisfctory qutic products in Hong Kong from (*Included dt from Jn to Oct only for 2012) (Source: CFS Food Surveillnce Progrmme)..10 Fig. 1.4 Frmework of reserch Fig. 2.1 The price index (2005=100) of ) food grins nd fish mel nd b) plnt nd fish oils, used for niml feeds production nd humn consumption (Rn et l., 2009) Fig. 3.1 ) Nitroblue tetrzolium (NBT) ctivity in blood, b) plsm totl protein (g/l) nd c) totl immunoglobulin (g/l) of grss crp in control nd food wste feeding groups, different superscripts (, b) within in sme smpling dy re significntly different (p<0.05) Fig. 3.2 ) Nitroblue tetrzolium (NBT) ctivity in blood, b) plsm totl protein (g/l) nd c) totl immunoglobulin (g/l) of grey mullet in control nd food wste feeding groups, different superscripts (, b, c) within in sme smpling dy re significntly different (p<0.05).. 88 Fig. 4.1 ) Totl plsm protein (g/l) nd b) totl immunoglobulin (g/l) of grss crp feeding with control (C) nd food wste (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05) Fig. 4.2 ) Nitroblue tetrzolium (NBT) ctivity nd b) plsm bctericidl ctivity (%) of grss crp feeding with control (C) nd food wste (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05) Fig. 4.3 Mortlity of grss crp ginst Aeromons hydrophil injection fter feeding with control (C) nd food wstes (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05) xv

21 Fig. 6.1 ) Totl protein (g/l) (Men ± SD) nd b) Totl immunoglobulin (IgI) (g/l) (Men ± SD) of grss crp plsm in control feed group nd feeding vrious doses of formulted TCM feed groups. Men in sme smpling dy with different superscripts re significntly different t p< Fig. 6.2 ) Bctericidl ctivity in plsm (%) (Men ± SD) nd b) Opticl density of NBT ssy in blood (Men ± SD) of grss crp in control feed group nd feeding vrious doses of formulted TCM groups. Men in sme smpling dy with different superscripts re significntly different t p< Fig. 6.3 Mortlity (%) (Men ± SD) of grss crp of different feeding groups fter intr-peritonel injection of A. hydrophil, ) in lbortory experiment (Control, 0.5%, 1% nd 2% formulted TCM) nd b) field tril (Control, 1% nd 2% formulted TCM).Men with different superscripts re significntly different t p< Fig 6.4 The in vitro effect of Rhizom coptidis (Rc), Rdix scutellri (Rs), Herb ndrogrphis (H) nd Rdix sophore flvescentis (Rsf) extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in ) het inctivted (HI) nd b) non-heted plsm, tretments mrked with sterisks showed significnt difference to control (*= p<0.05; ** = p<0.01) Fig 6.5 The in vitro effect of mixed TCM extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in het inctivted (HI), tretments mrked with sterisks showed significnt difference to control (* =p<0.05;**= p<0.01) Fig 6.6 The in vitro effect of mixed Trditionl Chinese Medicine extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in non-heted plsm, tretments mrked with sterisks showed significnt difference to control (*= p<0.05; **= p<0.01)..168 Fig. 7.1 ) NBT ctivity (bsorbnce t 540 nm) nd b) plsm bctericidl ctivity (%) of grss crp fed with commercil feed (control, C) nd enzymes upgrded food wste (A-E) supplemented with bker s yest (Y) nd Trditionl Chinese Medicine (TCM)..186 Fig. 7.2 ) Totl plsm protein nd b) totl immunoglobulin, IgI (g/l) grss crp fed with commercil feed (control, C) nd enzymes upgrded food wste (A-E) supplemented with bker s yest (Y) nd Trditionl Chinese Medicine (TCM) xvi

22 Fig. 7.3 Mortlity (%) of grss crp fed with commercil feed (control, C) nd enzymes upgrded food wste (A-E) supplemented with bker s yest (Y) nd Trditionl Chinese Medicine (TCM), fter 10 dys fter the intr-peritonel injection of A. hydrophil..189 xvii

23 Abbrevitions nd Acronyms AFCD AFFS Ah ANLU ANPU CFS CFU CHO: L CPI Cr 2 O 3 CSD Ec Ef EPD EU FAO FCR FDA FIC FIN FM FW A FW B FW C FW D GDPBS Agriculture, Fisheries nd Conservtion Deprtment Accredited Fish Frm Scheme Aeromons hydrophil Apprent net lipid utiliztion Apprent net protein utiliztion Center for Food Sfety Colony Forming Units Crbohydrtes to Lipid rtio Commodity Price Index Chromium Oxides Census nd Sttistics Deprtment ATCC Escherichi coli ATCC Enterococcus feclis Environmentl Protection Deprtment Europen Union Food nd Agriculture Orgniztion of the United Ntions Feed Conversion Rtio Food nd Drug Administrtion Frctionl Inhibitory Concentrtion Fishmel Informtion Network Fish Mel Food Wste Feed A Food Wste Feed B Food Wste Feed C Food Wste Feed D Gungdong Provincil Bureu of Sttistics xviii

24 GESAMP Joint Group of Experts on the Scientific Aspects of Mrine Environmentl Protection H IgI kg KOH Lg MDR mg MIC MPEDA NACA NOH NBT NCCLS NRC OIE P/E PER Rc Rs Rsf RWG S Sm SGR TCM Herb ndrogrphis Immunoglobulin kilogrm Potssium Hydroxide Lctococcus grviee Multi-drug Resistnce milligrm Minimum Inhibition Concentrtion Mrine Product Export Development Authority of Indi Network of Aquculture Centres in Asi Sodium Hydroxide Nitroblue Tetrzolium Ntionl Committee for Clinicl Lbortory Stndrds Ntionl Reserch Council World Orgnistion for Animl Helth Protein to Energy Rtio Protein Efficiency Rtio Rhizom coptidis Rdix scutellri Rdix sophore flvescentis Reltive Weight Gin ATCC Stphylococcus ureus ATCC Serrti mrcescens Specific Growth Rte Trditionl Chinese Medicine xix

25 Vc WWF WHO USD Vibrio cholere World Wide Fund for Nture World Helth Orgniztion US Dollr (United Sttes of Americ) xx

26 Chpter 1 The necessity of feed lterntives with supplements in current nd future quculture industry 1.1 Overview of world quculture sttus Aquculture is n importnt nd fst growing industry round the world nd the production expnded rpidly in recent 20 yers. Excluding qutic plnts nd nonfood products, the totl globl food fish production in quculture ws bout 60 million tonnes t 2010 which ws vlued US$119 billion (FAO, 2012). At the sme time, the nnul production from cpture fisheries ws only remined t bout 90 million tonnes in pst 20 yers (FAO, 2012); the fishery resource in nture is under tremendous pressure nd becoming questionble food source in future. To cope with this, quculture hs been gining importnce in the role of humn food source in the pst few decdes. The quculture industry in Asi is overwhelmingly importnt to globl quculture, contributing over 90% globl qutic products nd Chin lone contributed more thn 60% of totl world production (Fig. 1.1) (FAO, 2012). Chin dominted the globl quculture production (exclude qutic plnt nd non-food products) by 61.4% nd 49% in terms of quntity nd monetry vlue, respectively 1

27 (FAO, 2012). The freshwter finfish quculture production (33.9 million tonnes) dominted the world quculture industry, nd minly contributed by crps, 71.9% (~24.2 million tonnes) in 2010 (FAO, 2012). In the Gungdong Province of Chin, the economic vlue of quculture output reched 39.0 billion RMB in 2004 (GDPBS, 2005). The needs of rpid increse in quculture productions cn be predicted nd mintined in the ner future, but whether the growth rte cn be sustined in n environmentl friendly wy is in doubt. The rpid explosion in quculture production rised two mjor concerns round the world: the prevlence of fish diseses nd tremendous pressures on the demnd of feeding mterils, especilly fish mel re crucil problems in future quculture development. It is commonly known tht fish diseses re the results of interctions between hosts, pthogens nd environments. The fst growth of quculture industry during the pst 20 yers is ttributed to the high stocking density. This resulted in the fish more susceptible to infections, nd more nitrogenous sewge is generted due to intensive feeding nd excretions from fish. As consequence, fish diseses nd the deteriorted environment hve cused enormous economic loss. In 2010, Chin suffered 1.7 million tonnes of fish products owing to disese outbreks, nturl dissters nd pollutions, which worth $3.3 billion US dollrs (FAO, 2012). 2

28 Fig. 1.1 World quculture production from 1950 to 2004 (showing Chin, rest of Asi nd the Pcific region) (FAO, 2006) 3

29 According to the Food nd Agriculture Orgniztion (FAO), n dditionl 40 million tonnes of fish products which is 60% of the production in 2006, will be required to support the consumption in 2030 (FAO, 2006). Although the production incresed shrply, the fish mel usge for quculture feed ws stedy t bout 6 million tonnes in the pst 10 yers (FAO, 2012) nd the prediction of demnd ws even lower due to decresed supply nd higher prices (Tcon nd Metin, 2008). The development of quculture industry hs lso suffered from the prevlence of diseses, other concerns such s environmentl pollution nd fish fry sources lso endngered the industry. It is essentil to investigte strtegies for the sustinble development of fisheries, which is prticulrly true for Hong Kong fisheries s discussed below. 1.2 Hong Kong Inlnd Fisheries Sttus Inlnd fishery in Hong Kong ws vibrnt in the 1960s, with n expnsion of pond re during the 1960s to 1980s (Lu et l., 2003), but then it declined due to shift in industry sectors nd the development of new towns in the New Territories (Cheung, 1999; Lm, 1999). Pond re decresed by 40% from 1984 to 2004, while the number of inlnd fish frmers decresed from 1690 in 1991 to 637 in In 4

30 2010, 1,120 hectres of fish ponds re locted minly in the north-western New Territories of Hong Kong (HKSAR, 2012). Simultneously, the nnul production of pond fish decresed from 6,500 tonnes (HK$ 104 million) in 1984 to 2190 tonnes (HK$ 54 million) in 2010, reduced by 66 nd 48% of the quntity nd monetry vlue of totl fish production (Fig. 1.2), respectively (Chn, 2005; AFCD, 2011). The rpid development of quculture industry in the minlnd Chin nd the chnge of locl policy lso deeply impcted on the locl industry. Trditionlly, the fish-cum-duck prctice with ducks simultneously rered long the bunds of the crp polyculture ponds, utilized the duck mnure s orgnic fertilizer to enhnce pond productivity (Everitt nd Cook 1997). Due to the Livestock Wste Control Scheme introduced in 1994, the fish-cum-duck prctice cesed nd fish frmers used other orgnic mterils, minly penut cke, nd lso the infrequent use of pig mnure nd cttle mnure insted of duck mnure. The chnged prctice incresed the production cost nd reduced the income from selling edible ducks. Besides, the import of lowpriced freshwter fish from the minlnd further rendered the locl fishery becme less profitble nd drove some fishermen wy from the industry. 5

31 Fig. 1.2 The pond nd mri-culture fish productions (tonnes) in Hong Kong quculture industry from 1984 to 2010 (Chn, 2005; AFCD, 2011) 6

32 Currently, the mjority of fish frms use the polyculture mode which is similr to the pst, but grey mullet becme the mjor cultured species. The stocking composition included grey mullet (60-70%), grss crp (10%) nd bighed (15%) in combintion with smll number of silver crp, common crp nd tilpi (Lu et l., 2003). Grey mullet is the most populr species cultivted in the New Territories due to its reltively higher mrket vlue. In Hong Kong quculture industry, fish frmers (35%) usully strt the fish culture cycle (except grey mullet) from fingerlings (~3-4 in length) to mrketble size, but some frmers (~15%) strt from juvenile (> 8 ), this could reduce the grow-out period (Lu et l., 2003; Pritchrd, 2001). For exmple, the grss crp culture period is normlly 10 to 12 months from fingerlings to mrtketble size (1.5-2 kg), but only 3-4 months re needed if strted from juvenile (Lu et l., 2003). The decline in locl quculture industry should be revitlized in the future due to the emerging food sfety issues nd better public wreness on the environment. In recent yers, the Minlnd s fish products qulity hs been chllenged since number of food sfety issues hve been reported nd residents hve re-evluted the benefit of food produced loclly. Vrious undesirble chemicls re detected in imported fish, such s mlchite green which used for treting prsitic, fungl nd protozon diseses in fish nd is probbly crcinogenic to humns (Culp, 2004), ws detected in 7

33 turbots, eel, freshwter grouper, nd mud crp (CFS, 2006, b & c). Nitrofurns re ntimicrobil gents for veterinry use which my cuse cncer in experimentl nimls, were detected in silver crp nd freshwter grouper (CFS, 2006c). Endosulfn, n orgnochlorine pesticide which my cuse chronic kidney dmge, ws found in live eels (CFS, 2006d). Although lrge scle food sfety incidences on fish products were not found in recent few yers, the occurrence of unstisfctory qutic products cnnot be voided (Fig. 1.3) Due to the consecutive food sfety incidences, locl consumers hve become more nd more concerned over food sfety, nd some hve higher preference on consuming locl products. The consumers believed the locl fish products re good in qulity, lthough these products re usully sold t higher prices, especilly when the products re lbeled s ccredited or orgnic. The Agriculture, Fisheries nd Conservtion Deprtment (AFCD) implemented the voluntry Accredited Fish Frm Scheme (AFFS) to improve fish frms environmentl hygiene stndrds nd fish products qulity. The fish products tgged with specilly designed mrk were qulified from drug residues nd hevy metls nlyses before mrketing. Bsed on survey sponsored by WWF, there re potentilly 1.5 million households in Hong Kong who would be willing to consume eco-fish (fish lbeled s chemicl free). The eco-fish could be sold with 30-40% price premium (bout 8

34 HK$27.9/kg for grey mullet), nd the profits could ttrct fishermen, mking the industry economiclly sustinble (Boulnger et l., 2008). The mrket shre of locl fishery products from qulity frms re growing nd could be sustined in future. Moreover, edible mrine nd freshwter fishes for locl consumption re minly relied on the import from Chin nd other countries. The mrine fish culture production contributed bout 0.2% (re-clculted from HKSAR s sttistics) of totl sefood consumption in Hong Kong, while pond fish frmers produced only ~2190 tonnes, bout 4% of locl consumed freshwter fish (HKSAR, 2012). However, the demnd of locl quculture products is expected to be promoted once other sources of fishery product supplies re limited. A remrkble decrese on the supply of cpture fish products is foreseen in ner future, s the Hong Kong Specil Administrtive Region (HKSAR) Government (herefter the Government ) hs issued the legisltive mendments to bn trwling (including pir, stern, shrimp nd hng trwling) in Hong Kong wters nd the trwl bn becme effective since 31 December The locl fisheries should contribute more high qulity fish products nd reduce the demnd for cptured fish products. 9

35 Fig. 1.3 The number of unstisfctory qutic products in Hong Kong from (*Included dt from Jn to Oct only for 2012) (Source: CFS Food Surveillnce Progrmme) 10

36 1.3 Emerging problems in fst growing quculture industry in worldwide nd Chin The mss production nd the densely cultured systems re vulnerble to disese once the environmentl condition is deteriorted, nd severe economic loss is suffered in the resulted disese outbreks. In theoreticl bsis, fish disese is the closely relted to the interctions between host (fish), environment nd pthogen, the control of fish diseses should be bsed on multiple spects i.e. mintining good wter qulity nd voiding stressful condition for fishes. However, fish frmers re inclined culture fish t high stocking density due to economic fctors, nd pplied ntibiotics for combting fish diseses is commonly found in rel situtions. Aprt from ntibiotics, vitmins, hormones nd other chemicls (e.g. fungicide) re lso pplied in quculture s ntibcteril gent nd growth promoters (Jyprks nd Smbhu, 1996). All these production methods cused detrimentl impcts on the environment nd society. Another problem ccompnied with the rpid growth of quculture industry would be the fish feed supply. Fish mel is n importnt nd mjor ingredient nd its vilbility is expected to decrese in ner future becuse of the incresed prices nd rising concern on the sustinbility of the ocen sttus (Tcon et l., 2006). Currently, the supplies of other feeding ingredients e.g. soy ben, corn nd whet re tense s these mterils re lso consumed by niml husbndry sectors nd humns 11

37 (Rn et l., 2009). The development of quculture industry my not be environmentl nd economiclly sustinble nd chnges on feeding mterils nd drug pplictions re needed in the ner future Disese prevlence nd buse of chemicls nd ntibiotics With the rpid development of quculture, wide rnge of nturlly occurring nd synthetic ntibiotics hve been frequently pplied for curing the infections, the prctice of using veterinry ntibiotics nd chemicls hs been overwhelming nd the demnd on those drugs is on the rise. Chin rnked the first in terms of nnul production of penicillin, terrmycin nd doxycycline in the world, which ccount for 60% of the world s totl production (Grce, 2004). Vrious ntibiotic such s sulfonmide, terrmycin, penicillin, streptomycin, erythromycin, chlormphenicol nd oxolinic cid were commonly used through orl dministrtion, bth tretment nd injection in Chin (Southest Asin Fisheries Development Center, 2000). Most of these re used for quculture s well s livestock nd poultry frming. Antibiotics re very effective in combting bcteril diseses nd sved slmon frming in Norwy, which suffered from serious infections from Vibrio spp. during the 1980s (Grve et l., 1990). The ppliction of ntibiotics hs lso become the inevitble prctice in Chin 12

38 quculture (Xu et l., 2006), nd vrious types of ntibiotics re detected in the costl re in Chin, which is believed tht quculture dischrge is one of the mjor sources (Zou et l., 2011) Despite the impertive contributions of ntibiotics in controlling fish diseses, the dverse effects cused by the buse of ntibiotics nd their environmentl ftes hve stimulted public ttentions. Most of the veterinry drugs re only prtilly metbolized in the fish body before entering the wter environment vi urine nd excret (Hmscher et l., 2000), ccumultion of substntil mounts of ntibiotics in the river or fishpond sediment re resulted. They my lso enter into the soil together with the fishpond mud which is commonly used s fertilizer for crop production (Boxll et l., 2006; Zhu et l., 2007). Consequently, resistnce genes hve been developed ginst ntimicrobil gents nd re commonly found in qutic bcteri e.g. Vibrio spp. nd Aeromons spp. (Kim et l., 2004; Sørum, 2006). The buse of ntibiotics endngered the nturl environments, numerous studies showed ntibiotics resistnt bcteri re frequently detected round quculture sites nd closely relted to the quculture use of ntibiotics (Krunsgr et l., 1994; Chelossi et l., 2003; Shul Hmeed et l., 2003; Alcide et l., 2005). Not only therpeutic doses of veterinry ntibiotics, sub- 13

39 therpeutic doses in quculture which is lso generlly used in fish frms for prophylctic purposes nd both pplictions cused the development of ntibiotic resistnt bcteri (Smith et l., 2002). Even worse, terrestril veterinry pthogens nd humn pthogens my obtin the ntibiotic resistnce through horizontl trnsmission from bcteri in the qutic environment (Angulo nd Griffin, 2000). The recent ppernce of super-bcteri hs opened the Pndor s box for wide rry of upcoming problems. The sitution hs rised socil nd environmentl concern due to potentil risks for humn helth (Cbello, 2006). The ntibiotic residues my lso contminte the quculture products nd lso exert profound impcts on the economic development. The Europen Union (EU) bnned the shrimps nd prwns imported from Chin in 2002 due to their high residul ntibiotics (Hernández, 2005). The prohibition ws only cesed fter intensified monitoring nd testing for drug residues on qutic products (EU, 2002). In recent yers, the incidence of poison fish in the Hong Kong mrket is no longer rre occurrence, some qu-products contminted with ntibiotics (e.g. furzolidone) nd fungicide (e.g. mlchite green) re directly imported from the Minlnd s previous mentioned. Consumption of those contminted products my ssist the development of ntibiotic resistnce bcteri in humn intestine (Slyers et l., 2004), higher risk to humn in front of bcteril infections could be resulted (Sørum, 2006). 14

40 The sfegurding of food supply hs become serious public issue. There is noticeble demnd to seek n lterntive to ntibiotics vi environmentllysustinble nd ethiclly-cceptble pproches Rising feed mterils prices Another chllenging issue in sustining the rpid growing quculture industry would be the source of protein in fish feed. In the quculture industry, fish feeds re mde from different nturl plnt mterils nd relted by-products, such s soyben mel, cotton seed, corn gluten nd rice brn nd niml products e.g. fish mel nd fish oil. However, the prices of these mterils re rising due to competitions with niml husbndry sectors, nd humn consumption nd incresed fuel cost when trnsported from production countries (Chile, Brzil, Argentin nd U.S.) to mjor mrkets i.e. Asi (Rn et l., 2009). Fish mel (FM) nd fish oil re the importnt nd expensive ingredients s they re mjor protein nd lipid sources in quculture feeds. Fish mel is usully processed from by cught or non food fish cptured by fishery industry nd byproducts from fish processing industry i.e. fish offl or fish trimmings (FIN, 2006; FAO, 2012), bout 1/3 (30 million tonnes) of totl cpture fishery productions re 15

41 used to mnufcture fish oil nd FM (Anon, 2002). Approximtely 68% of FM in the world ws used for quculture feed productions (Tcon nd Metin, 2008). The FM production rised from 5 million tonnes in 1970s to 7.5 million tonnes in mid 1990s, then dropped to bout 6 million tonnes in 2009, while the demnd on FM ws froze in the pst 10 yers but the qutic resource cptured from environment hs been leveled off in the pst 20 yers (FAO, 2012). Sme trend ws lso observed in the supply of fish oil. The diminishing supplies nd incresing mrket price of FM nd fish oil lso forced decresing usge in quculture in future (Rn et l., 2009; Tcon nd Metin, 2008). The globl price for fishmel ws just US$500 to $700 per tonne in , but the price ws doubled to US$1,210 per tonne in 2008 (Rn et l., 2009). In fct, the relince on FM bsed feed ws lso disputed s inefficient nd uneconomicl, s the conversion from wild fish (for producing FM) to frmed fish (fish product) is bout 6 to 1 in rtio, especilly on crnivorous fish species, i.e. slmon, trout nd vrious groupers (Stniford, 2002; Milewski, 2002). Consequently, lterntive feeding mterils especilly for replcing FM re dvocted for production niml feeds. Besides, low FM consuming fish species (herbivores nd omnivores) e.g. tilpi nd crps re preferred over crnivorous species like trout nd slmon in quculture 16

42 industry nowdys. The nutritionl demnd of herbivores nd omnivores is lso esier to be fulfilled thn crnivores (Hrdy nd Tcon, 2002), nd those fish feed re less costly. 1.4 Using feed supplement s immuno-stimulnts As mentioned before, fish disese is the consequentil interctions between pthogen, host nd environment. A multiple pproch involving inhibiting nd eliminting pthogens, reducing host infections nd improving environmentl qulity is essentil for combting fish diseses. However, the pthogens especilly bcteri re becoming more resistnt to drugs nd the environment like quculture zones re getting more polluted due to humn ctivities. The enhncement on host immunity through feed supplement could be n dvnced progress in quculture reserch, while herbl medicine such s Trditionl Chinese Medicine (TCM) nd bker s yest re the novel quculture pplictions. Feed supplements hve been widely used in quculture industry nowdys, including probiotics e.g. yests, but the use of herbl medicine is reltively new topic in the industry. Herbl supplement s immunostimulnts, is preferred becuse it is nturl, biocomptible, biodegrdble, 17

43 cost effective, nd more importntly it is sfe for the environment (Ortuno et l., 2002) Herbl supplement: Trditionl Chinese Medicine (TCMs) Trditionl Chinese medicine (TCM) nd herbs hve been used s immunostimulnts in humn for thousnds of yers (Tn nd Vnith, 2004), but its use in quculture hve been only explored in recent yers, The herbl medicines cn serve s ntimicrobil gents or immunostimulnts to prevent fish disese nd could be potentil lterntive to replce vccines (Anderson, 1992; Secombes, 1994) nd ntibiotics (Glin et l., 2009). The principle of TCM is quite different from the Western phrmcologicl nd therpeutic principles which trgets on diseses or pthogens directly. The rtionle of TCM is minly bsed on few theories, such s the five elements theory nd the Yin- Yng blnce, which re considering the overll blnce of humn body (Cheng, 2000). TCM compound formultion contined vrious constituents which plyed different roles in disese tretments. The mjor constituent showed chief therpeutic effects e.g. nti-inflmmtion nd blnced the dishrmony in the body, while the djuvnt nd minister constituents (second component) ssisted the therpeutic ction 18

44 of mjor components. The messenger constituent (component mking the formul prescription trgeting pthologicl tissues) functioned s guider to modulte the formultion to trgeted orgn or to eliminte the dishrmony cused by other medicines in the formultion (Cheng 2000; Go nd Wu 2008). In other words, TCM is imed t mintining nd restoring the blnce of body or enhncing the immunity to defence diseses. Therefore, TCM formuls re not just n ddition of herbs but involve complex interctions between the herbs. Investigtion on the compound formultions of TCMs could be beneficil to quculture industry in combting fish diseses. In modern phrmcologiclogy, the therpeutic effects of TCM ctive ingredients hve been vlidted in some niml models such s mice, fish nd humn cells. Scutellri extrcts exhibited good ntibcteril nd ntivirl effects, nd boosted phgocytic ctivity in mice t low dose (Ci et l., 1994), nd with similr ctivity found in fish t low dose (Yin et l., 2006). Androgrphis pnicult extrcts were especilly importnt for treting inflmmtion by modulting mcrophge nd neutrophil ctivity in mouse (Chiou et l., 2000) nd humn blood cells (Shen et l., 2002). Rhizom coptidis exhibited superoxide nd hydroxyl rdicl scvenging properties in rt kidney nd brin tissues homogentes (Liu nd Ng, 2000). 19

45 The medicted vlues of TCMs in fish minly focused on the non-specific immune responses nd disese resistnces of fish to bcteril infections. Rohu, Lbeo rohit fed with Withni somnifer enhnced its phgocytic ctivity, lysozyme ctivity, totl immunoglobulin level nd Nitro blue tetrzolium (NBT) ctivity (Shrm et l., 2010). The disese resistnce to Aeromons hydrophil in rohu (L. rohit) ws lso enhnced when fed mngo kernel (Mgnifer indic) dded feed (Shu et l., 2007b). Common crp (Cyprinus crpio) fed with Astrglus root extrct showed better survivl rte fter chllenged with A. hydrophil (Yin et l., 2009). The use of compound formultion my hve better effect on immune-stimultion or improvement on disese resistnce of fish, compred with the use of single herb. Nile tilpi (Oreochromis niloticus) fed with mixed Astrglus nd Lonicer extrcts enhnced phgocytic nd respirtory burst ctivity of blood phgocytic cells nd its disese resistnce ginst A. hydrophil (Ardó et l., 2008). Mixed Scutellri rdix nd Herb euphorbie lso showed remrkble prevention nd tretment rte up to 85% ginst bcteril septicemi, the increse of phgocytic ctivity ws lso elevted ccompnied with higher herbl concentrtion nd longer feeding period to goldfish, Crssius urtus urtus (Zheng et l., 2006). All these bsic medicl vlues of herbs showed in fish dvocting the uses of TCMs in fish frming for replcing drugs like ntibiotics. 20

46 1.4.2 Probiotics: Bker s yest Probiotics is the feed supplement defined s live microbil feed supplement which beneficilly ffects the host niml by improving its intestinl blnce (Fuller, 1989). The probiotics is used in the form of feed dditives or directly introduced into the wter body, resulted enzymtic contribution in digestion, inhibition of pthogenic microorgnisms, ntimutgenic nd nti-crcinogenic ctivity, growth promoting fctors, nd incresed immune response (Ziei-Nejd et l., 2006; Wng et l., 2005; Wng nd Xu, 2004). The bcteri Bcillus sp. is reported to improve wter qulity, survivl nd growth rtes nd the helth conditions of juvenile shrimp (Peneus monodon) (Dlmin et l., 2001). Scchromyces cerevisie, the yest pplied in bking nd fermenting lcoholic beverges industries hs been used to improve feed utiliztion nd growth performnce in vrious crp species (Mohnty et l., 1996; Swin et l., 1996) nd Nile tilpi (O. niloticus) (Abdel-Twwb et l, 2008). These positive effects my be due to the dherence of yest cells to the fish gut nd secretions of mylse enzymes which incresed digestibility of diets (Scholz et l. 1999). Furthermore, the cell wll constituents of yest could stimulte the innte immune responses nd protect fish ginst infections (Estebn et l., 2001). 21

47 1.5 The necessity of lterntive protein sources in fish feed In modern quculture industry, fish feed is the most importnt nd the mjor cost for production nd it should contin blnced nutrients nd sufficient energy for fish growth. The feeds re minly mde from different ingredients from plnt nd niml derivtives, e.g. soy ben mel, rpeseed mel, rice brn, whet brn, corn gluten from plnts nd fish mel, blood mel, met mel, bone mel, nd fish oil from nimls, which provided different nutrients (e.g. mino cids, ftty cids) nd essentil elements (e.g. vitmins, trce metls) for fish growth. Different fish diets re formulted by djusting the mixture of ingredients s requirements on nutrients nd energy re vried between fish species nd different growth stges (lrve, fingerling, juvenile nd broodstock). In generl, fish mel is the mjor protein source used in quculture in the pst due to the low-cost, bundnce, good pltbility nd high protein qulity; but the preference of FM s feeding mterils no longer exists s described before. As result, lterntive sources of protein for fish feeding re essentil to support the industry in the coming future nd it is n importnt topic in quculture industry Utiliztion of food wste s fish feed in worldwide 22

48 In pst decdes, shifting from niml protein to plnt bsed protein ws mjor development in fish feed producing industry, the fish mel in quculture feed hs been grdully nd prtilly replced by other protein sources, such s soy ben mel nd rpeseed mel. The inclusion levels of fish mel were decresed in feeds for vrious species e.g. trout, Slmon nd crps (Tble 1.1) (Tcon et l., 2011). Vrious studies focused on the reduction of fish mel in feeds nd the less demnd of fish mel is predicted. It hs been shown tht slmon fed with diet contining 20% soyben protein chieved similr growth rte to those fed with high-qulity fish mel only (Olli et l, 1995). However, the previous motives of using low cost nd high vilbility of plnt bsed protein sources no longer exist due to the stress on food supply nd competition from livestock sector nd humn consumption (Fskin et l., 1999; Rn et l., 2009). When the feeding mterils re less vilble nd the production costs incresed, the industry tried to use food wstes nd industril by-products e.g. soy ben residue nd ppy wste for rering fish. Some ctegories of food wstes, such s met wste, fruit nd vegetble wste nd fish wste re rich in nutrients, they could be considered s substitutes for originl rw mterils for niml feed productions (Grcí et l., 2005). Vrious types of food nd industril wstes from poultry, soy suce producing, rice wine nd ppy processing industries, re 23

49 incorported into quculture feed production in recent yers s lterntive for fish mel re performed (Erturk nd Sevgili, 2003; Bke et l., 2009; Kng et l., 2010; Vechklng et l., 2011). In generl, met nd fish wste re good protein nd lipid sources for fish feed, met wste ws rich in ether extrct nd fish wste contined high level of crude protein, while fruit nd vegetble wste contined more nitrogen free extrct (digestible crbohydrtes) (Grcí et l., 2005). In Hwii, fermented ppy processing wstes by yest re implemented s fish mel substitute in shrimp feed, the result showed tht 50% of fish mel cn be replced by ppy wstes bsed on growth rte, feed conversion rtio (FCR) nd survivl rte fter 8 weeks of feeding (Kng et l., 2010). Similr results, comprble growth, survivl rte nd feed efficiency to control feed were lso found in soyben poultry by-product feed with egg supplemented in Pcific white shrimp (Smoch et l., 2004). Brewery wstes which contin bout 20% of crude protein nd possess very good mino cids profile such s lysine, rginine nd methionine (NRC, 1983). The brewery wste could replce the rice brn which is nother mjor ingredients in fish feed, but different growth performnce were observed in 3 crp spcies, Ctl ctl (Hm.), Lbeo rohit (Hm.) nd Cirrhin mrigl (Hm.). C. ctl nd L. rohit 24

50 Tble 1.1 Reduced fish mel inclusions in qu feed of different quculture species (FAO, 2012) Species/ Species group Fishmel inclusion in compound qufeed (%) * fed crp Tilpis Ctfishes Milkfish Miscellneous freshwter fishes Slmons Trouts Eels Mrine fishes Mrine shrimps Freshwter crustcens *Predicted vlue in Tcon et l. (2011) 25

51 (Hm.) lso showed best growth performnce nd FCR in 30% brewery wste incorported feed, C. mrigl (Hm.) showed retrded growth on the contrry (Kur nd Sxen, 2004). Poultry by products re other lterntive protein source which hs been studied in pst 30 yers (Higgs et l., 1979; Rwles et l., 2009), however, the results were vried in crnivorous nd omnivorous fishes. Rwles et l. (2006; 2009) showed the poultry by products could replce the fish mel by 40% or entirely in hybrid striped bss (crnivorous) diet, while poor growth in common crp, Cyprinus crpio fingerlings ws observed in ll tested feed with 33, 67 nd 100% replcement of fish mel by poultry by-product mel (Emre et l., 2003) Using enzyme for enhncing feed conversion in fish feed Enzyme supplementtion on niml feed mterils hs been studied to enhnce feed conversion nd utiliztion which cn be found in poultry nd swine industry (Cown et l., 1996; Kitchen, 1997). In fish processing industry, the wstes contined heds, viscer, skin, nd skeleton re dumped without further utiliztion in the pst, nd the quntity is believed to be more thn 20 million tonnes, or 25% of fish products (Rustd, 2003). However, these by-products re rich in protein nd cn be 26

52 utilized s lterntive protein source in the form of fish protein hydrolyste, which is soluble product of fish wstes treted with enzymes (Guerrd et l., 2002) nd it is potentil ingredient producing quculture feeds (Bhskr nd Mhendrkr, 2008; Nilsng et l., 2005). A commercil enzyme, Ronozyme TM VP enhnced growth performnce nd net protein utiliztion in tilpi fed with enzyme supplemented plm kernel mel bsed feed (Boonyrtplin et l. 2000). Other enzymes like bromelin nd ppin could be utilized in quculture, these proteses my enhnce the digestibility of protein through hydrolyzing connective tissue nd skin in rw mterils in food wstes. Bromelin is extrcted from the stem nd fruit of pinepple nd it could hydrolyse the feed proteins into smller protein peptides with higher digestiblity (Fennem, 1996). Previous study by Wong nd his collegues (1996) lso showed tht pretretment of soyben residue by ppin could enhnce the feed digestibility nd conversion of common crp (Cyprinus crpio). 1.6 Potentil uses of drug lterntives nd food wste s fish feed in Hong Kong quculture industry The rectivtion of locl inlnd fisheries is needed s incresing demnds on high qulity fish products nd preserving wild fisheries resources. Hence, more 27

53 technicl support is preferred for enhncing the quculturl productivity by environmentl friendly nd sustinble mens, idelly reducing production costs t the sme time. Locl pond fish frmers usully pplied whet brn, soyben nd penut cke s mjor fish feed, but the fish lso fed with corn mel, bred, noodles, flour nd biscuits (Lu et l., 2003). In those feed types, soyben, bred, noodles nd biscuits cn be regrded s industry by-products or food wstes. Only very smll mount of fish frmers dopted fish feed pellets s regulr feed. As cost is the dominnt considertion for frmers (Lu et l., 2003), the cost of food wste fish feed is therefore crucil point when ttrcting frmers ttentions s well s the fish grow rtes. The recycle of food wstes could lso be one of the wste mngement strtegies in Hong Kong. According to the Government, food wstes comprised of 38.4% of the domestic wste in 2007 (Leung et l., 2008). The Government s policy frmework ( ) minly focused on the municipl solid wste mngement through lndfill disposl bns, wste chrging nd producer responsibility schemes. More lterntives for food wste tretment would be importnt for esing lndfill disposl pressure. Recycling of food wstes cn utilize these vluble orgnic wstes nd 28

54 therefore llevite the loding of locl lndfills. A pilot plnt, the Wste Recycling Centre t Kowloon By, with 4 tonne/dy cpcity, ws commissioned in 2008 to hndle food wste from the Olympic equestrin events. A tril opertion for the recycling of source-seprted food wstes generted from selected commercil nd industril sectors including resturnts, hotels, wholesle mrkets nd genertors of the ctering, food production, bkery nd ben curd industries ws subsequently commenced (EPD, 2009). Bsed on the results obtined from these trils, lrger fcilities (Siu Ho Wn, Lntu Islnd nd Shling, North District operted in the lte 2010s) could hndle totl 400 tonnes (mostly food wste) for the production of biogs nd compost dily, but the cpcity is just bout 10% of dily produced food wstes (EPD nd ENB, 2008). The use of food wstes for rering fish could be sustinble strtegy to support fish feed nd quculture production ecologiclly nd economiclly. According to Kowloon Biotechnology Limited s website, the only food wste recycling fctory for niml feed production in Hong Kong, could hndle 50 tonnes of food wste dily (2012). The fctory sold their feed to locl fish frms, nonetheless the niml feed products were not endorsed by the Agriculturl, Fisheries nd Conservtion Deprtment (AFCD) (Asi City Network, 2012), which is the locl deprtment mnging locl cpture nd quculture fisheries industry. 29

55 As result, more scientific reserch to formulte qulity food wste feeds, nd the fesibility of ppliction in Hong Kong is needed. Incorporting of food wstes into fish feeds would be sound technology for esing food wste problem nd reducing economic costs of quculture industries. 1.7 Aims nd objectives This thesis focused on using food wstes nd feed supplements e.g. enzymes, bker s yest nd TCM for rering freshwter fish (grss crp nd grey mullet) (Fig. 1.4) with the following ims: 1) To investigte the pproprite food wste composition, minly met, cerel, vegetble nd fruit wstes s vible protein sources in the diets of grss crp nd grey mullet diets bsed on feeding trils (Chpter 2). The feed utiliztion nd fish growth of both species nd the dietry protein nd lipid digestibility by grss crp ws lso determined were evluted; 2) To investigte the effects of growth nd immunity of both species by food wste feed supplemented with bromelin nd ppin mixture bsed on feeding trils (Chpter 3). Different prmeters to indicte fish growth performnce e.g. specific 30

56 growth rte, feed utiliztion e.g. protein efficiency rtio nd some hemtologicl prmeters e.g. totl plsm immunoglobulin were mesured; 3) To investigte effects of growth nd immunity of grss crp with the dietry supplementtions of yest (Chpter 4), Trditionl Chinese Medicine (TCM) (Chpter 6) nd their combined form (Chpter 7). The hemtologicl prmeters e.g. Nitro- Blue tetrzolium (NBT) ctivity, totl plsm immunoglobulin nd plsm bctericidl ctivity nd the disese resistnce to pthogen through Aeromon hydrophil injection were determined; 4) To investigte the ntimicrobil ctivities of vrious TCM extrcts on severl field isolted fish pthogens, e.g. A. hydrophil, Vibrio cholere nd Lctococcus grviee, nd the development of drugs resistnt pthogens through broth microdilution method (Chpter 5); nd 5) To investigte the effects of single nd combined TCM extrcts on plsm bctericidl ctivity of grss crp by in vitro study (Chpter 6) It ws hypothesized tht the use of enzymes nd yest could improve feed conversion of food wstes feed, low feed conversion rtio (FCR) nd fster growth rte re observed in fish. It ws lso hypothesized tht the Trditionl Chinese 31

57 Medicine (TCM) could enhnce the fish immunity nd disese resistnce to pthogens. Finlly, using food wste s feed mteril, nd enzymes, bker s yest nd TCM s feed supplements for rering fish were generlly discussed, nd n overll conclusion ws drwn bsed on the results generted in this study. 1.8 Contributions nd significnce of the present reserch On the whole, this study is the first investigtion focused on food wste with feed supplements (e.g. enzymes, bker s yest nd TCM) for Hong Kong quculture industry. The results provided preliminry but importnt informtion on using feed supplements (TCM nd bker s yest) to enhnce fish immunity nd enzymes upgrded food wste feeds for rering fish. Food wstes contined notble protein levels nd other nutritionl contents which becme controversil issue for wste disposl, shortening the lifespn of locl lndfills. Utiliztion of food wstes could diminish the volume of dumped wste nd extend the lifespn of lndfills, while the protein rich food wste is vluble nd useful resource which could be incorported into fish feeds s rw mterils, nd should not be simply disposed s wste. 32

58 Chpter 1 Bckground The necessity of feed lterntives with supplements in current nd future quculture industry (Bckground) Chpter 2 Using food wstes s fish feed ingredients for grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) cultivtion Chpter 5 The ntimicrobil ctivity of Trditionl Chinese Medicines (TCMs), potentil drug lterntive on deling with fish pthogens Chpter 3 Upgrding food wstes by mens of bromelin nd ppin to enhnce growth nd immunity of grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) Chpter 6 Effects of Trditionl Chinese Medicines (TCM) on the Immune Response of Grss Crp (Ctenophryngodon idellus) Chpter 4 Adding bker s yest to food wste to enhnce growth performnce nd immunity of grss crp (Ctenophryngodon idellus) Chpter 7 Upgrding food wste feed using TCMs, bker s yest nd enzyme on the immune responses of grss crp (Ctenophryngodon idellus) ginst Aeromons hydrophil Chpter 8 Generl Discussion nd Conclusions Fig. 1.4 Frmework of reserch 33

59 The feed supplements (TCM nd bker s yest) could enhnce fish immunity nd reduce mortlity due to infections, in other words the yield could be incresed. These supplements my lso reduce the use of ntibiotics which hs been pplied in quculture s ntibcteril gents for mny yers (Jyprks nd Smbhu, 1996). The ultimte gol of this study is to ctivte locl inlnd fisheries through pplying low-cost nd relible food wste feeds supplemented with environmentl friendly feed supplements for rering fish. The sme notion could be pplied in mrine quculture, for the development of both sustinble mrine nd freshwter quculture in Hong Kong. Lst but not lest, the generl public could lso consume those high qulity nd green fish products, nd enjoy the better preserved fish ponds hbitts for leisure. The better mnged fish ponds could lso provide shelter nd foods for both locl nd migrtory birds. All these outcomes could contribute to the better development of the sustinble quculture industry, economiclly nd environmentl friendly nd these positive impcts on the environment, society nd the inlnd fisheries industry re precious nd intimtely interrelted. 34

60 Chpter 2 Using food wstes s fish feed ingredients for grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) cultivtion 2.1 Introduction Aquculture is growing in its importnce s one of the most importnt food supplies in the world. The industry contributed only 4% of globl fishery production in 1970 nd the expnded production ccounted for 36% of totl production in 2006 (Rn et l., 2009). The current production is only 60 million tonnes (FAO, 2012), but it hs been estimted tht 120 million tonnes will be needed by the yer 2020 (Delgdo et l., 2003). Fish feed ccounted for more thn 50% of the totl cost in the quculture industry (Rn et l., 2009). The mjor feed rw mterils re fish mel nd cerel grins e.g. soy ben, rice brn, whet, corn etc., which re the importnt sources of protein nd crbohydrte, respectively. The prices of these rw mterils re ffected by vrious fctors such s climte, wether conditions, globl economic growth nd fuel costs. From , the prices of fishmel, soyben mel, corn nd whet rose by 55, 67, 124, 130 nd 250 %, respectively (Rn et l., 2009) (Fig. 2.1). This ws due to the incresing energy cost (e.g. oils, gs) nd demnds, nd lso higher 35

61 feed cost from incresing mnufcturing nd trnsporttion cost. The supply nd cost of the rw mterils were lso ffected by wether, e.g. drought, flooding nd even globl wrming. Approximtely 68% of fish mel in the world ws used for quculture feed productions (Tcon nd Metin, 2008) nd the rest minly consumed by pig nd poultry sectors (FIN, 2007). Therefore, there is gret pressure on the production cost of fish mel due to competition between the two sectors. The tremendous demnd of quculture feed would lso provoke the price in the future. In 2020, the globl production of quculture feed is predicted rech 71 million tonnes, which is doubled the quntity in 2008 (29.2 million tonnes) nd lmost ten-fold of 1995 production (7.6 million tonnes) (FAO, 2012). It is estimted tht the quculture feed is minly consumed by crps, which ccounted for 31.3 % (9.1 million tonnes) of the totl feed production (FAO, 2012). As result, seeking lterntives for quculture feed re urgently needed; nd food wstes re one of the low-cost nd nutritious sources of feeding mterils which could be beneficil to the industry. Bsed on government sttistics, food wstes contributed 42.3% nd 34.9% of the totl domestic wste nd commercil nd industril (C&I) wste in 2011 respectively (EPD, 2012). More food wstes from C&I sectors hve been found in recent yers, with only 400 tonnes dily in 2001, but reched 1,056 tonnes in 2011 (EPD, 2012). 36

62 Fig. 2.1 The price index (2005=100) of ) food grins nd fish mel nd b) plnt nd fish oils, used for niml feeds production nd humn consumption (Rn et l., 2009) 37

63 The utiliztion of food wste could diminish the volume of dumped wste nd extend the lifespn of lndfills, while the protein rich food wste could be incorported into fish feed s rw mterils. Different types of food wstes from poultry, soy suce producing, rice wine nd ppy processing industries, hve been used s lterntive protein to replce fish mel in severl investigtions (Erturk nd Sevgili, 2003; Bke et l., 2009; Kng et l., 2010; Vechklng et l., 2011). It hs been shown tht slmon fed with diet contining 20% soyben protein chieved similr growth rte to those fed with highqulity fish mel (Olli et l, 1995). Rwles et l. (2009) showed the poultry by products could replce the fish mel completely in the diet of hybrid striped bss (Morone chrysops x M. sxtilis ). In the present study, vrious types of food wstes including fruit peel nd vegetbles, met, bone mel nd cerel wstes were collected from hotels nd food processing plnts nd trnsferred to Kowloon Biotechnology Limited in Lu Fu Shn for mnufcturing fish feed. Formulted feeds with different proportions of ech ctegory of wstes subsequently used for rering grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus). Herbivorous nd omnivorous fishes hve fewer requirements on dietry protein thn crnivorous species (NRC, 1993). Grss crp is herbivorous cyprinid, which consumes qutic mcroflor nd converts into met protein s nutritionl food source 38

64 (Gîlcă, 2010). Grey mullet is n omnivore nd feed on lge, detritus nd microscopic invertebrtes (Olukoljo, 2008). Polyculture is commonly dopted by locl fish frmers with the following stocking composition: grey mullet (60-70%), grss crp (10%) nd bighed (15%) in combintion with smll numbers of silver crp, common crp nd tilpi (Lu et l., 2003). Grss crp nd grey mullet re the mjor components, nd therefore, they re the idel species for the present investigtion to use food wste s fish feeds. The mjor objectives of the present study were 1) to determine the optimum formultion comprising vrious ctegories of food wstes for rering grss crp nd grey mullet 2) to evlute the effects of food wste on the growth performnce nd body composition of both species. It is hypothesized tht food wstes could be used s lterntive protein source for rering fish. It could be sustinble mens of quculture with both ecologicl nd economicl significnce. This pproch could lso promote recycling of food wstes to ese prt of the wste disposl pressure fced in Hong Kong. 2.2 Mterils nd Methods Food wste fish feed preprtion Food wstes minly consisted of food processing wste nd prtilly post 39

65 consumed wste, collected from locl hotels nd food processing plnts, grouped into 4 mjor ctegories: (1) fruit peel nd vegetbles, (2) met, (3) bone mel nd (4) cerel wstes. Fruit wste minly contined peels with some flesh of vrious fruits, bout 25% of pinepple, 25% wtermelon, 15% Cntloupe nd 35% other fruits, e.g. strwberry, bnn, pple. Met wstes included 60 to 70% of beef, pork nd chicken, 30 to 40% of fish such s slmon nd groupers. Vegetbles contined vrious types of lef vegetbles, such s lettuce nd spinch. Cerels usully included rice brn, soy ben mel, rice grin nd spghetti (Tble 2.1). All the food wstes were collected dily from the hotels nd food processing plnt nd trnsferred to the fctory of Kowloon Biotechnology Limited (t Lu Fu Shn) for further processing within 2 h. They were diced into smll pieces (~1cm dimeter), with excessive wter squeezed out, nd dried t 80ºC for 6 h. Ech type of food wstes ws grinded into powder to form different food wste products, under different rtio nd mixed with other rw mterils (non-food wste), such s fish mels, nd corn strch for pelletizing fish feed. Tble 2.2 shows the different types of food wstes prepred for lbortory experiments. They included Food wstes A (FWA- without met), B (FWB- with 25% met) nd C (FWC- control), A commercil feed: Jinfeng, 613 formulted feed (~30% protein), which minly contined whet middling, flour, ben pulp, rpeseed mel, fish mel, ben oil nd fish oil used s the control feed. The proximte 40

66 composition (sh, moisture, protein, fibre, lipid nd nitrogen free extrcts), mino cid profile, nd the feed protein solubility in KOH were determined bsed on the methods described in Section nd the results re shown in Tbles 2.3 nd 2.4. In generl, FWA minly comprised of cerel food wstes e.g. rice brn, soy ben mel, rice grin nd spghetti, which would be idel protein sources for herbivores nd omnivores, i.e. grss crp nd grey mullet, with some met products used to replce prts of cerels in FWB nd FWC. The control feed contined minly whet middling, flour, ben pulp, rpeseed mel, nd the formul ws similr to FWA Feed digestibility of different food wstes formultions in grss crp Three hundred fingerlings of grss crp Ctenophryngodon idell (herbivore) were used for testing different fish feeds, with 12 individuls stocked in ech tnk (~60 L wter) in triplictes. 0.5% by mss (dry weight) of the chromium oxides (Cr 2 O 3 ) (Sigm Aldrich) were extruded with the experimentl feed. Ech type of experimentl feed pellet (3mm) ws fed to the experimentl fish t feeding rte of 3% body weight wet weight diet per dy for 35 dys. Feeding with the control feed ws crried out t rte of 3% body weight dily in ech experimentl feeding group nd the fish were cclimted for 3 weeks before the strt of experiment. The wter temperture, ph nd dissolved oxygen were mesured three times week using portble Hnn 41

67 Tble 2.1 Food wste ctegories collected from hotels Ctegories Food Amount (%) Non-cooked or cooked fruit peel Non-cooked nd vegetbles - pinepple - wtermelon - Cntloupe - strwberry, bnn, pple - lef vegetbles, such s lettuce nd spinch Met - beef, pork nd chicken - slmon - groupers Cerels - rice brn - soy ben mel - rice grin - spghetti Bone - beef, pork nd chicken - slmon - groupers Cooked Non-cooked Cooked Non-cooked Cooked Cooked Non-cooked Cooked Non-cooked Cooked 42

68 Tble 2.2 Food wste feed formultions (contining 75% food wste) Formultion Food wste products Non-food wste products Totl (%) fruit/ vegetbles met products cerels bone mel other fish mel corn strch Food wste A Food wste B Food wste C

69 Tble 2.3 Composite nlysis of experimentl feeds Formultion Control FW A FW B FW C Dry mtter (%) 93.68± ± ± ±0.09 Ash (%) 8.24± ± ±0.03b 17.04±0.15b Protein (%) 30.16± ± ± ±1.64 Lipid (%) 5.17± ± ±1.81b 19.04±1.64c Fibre (%) 9.57± ± ±0.87 b 5.85±0.30 b Crbohydrtes (%) Energy (kj/g diet) CHO/L rtio P/E (mg/kj) Protein solubility (%) ± ±1.43b 38.74±1.35c 39.93±2.25c *Different superscripts (, b, c) mong feeding groups re significntly different (p<0.05) 1 Crbohydrtes (%) = 100 (crude protein % + crude lipid % + moisture % + sh %+ fibre %) (Cstell nd Tiews, 1980) 2 Energy (kj/g diet) = (% crude protein 23.6) + (% crude lipids 39.5) + (%crbohydrtes 17.3) (Chtzifotis et l., 2010) 3 Crbohydrtes to Lipid (CHO: L) rtio = % wt. in CHO/ % wt. in lipid 4 Protein to energy (P/E) (mg/kj) = crude protein (%) / energy 5 Protein solubility (%) = Protein in KOH / protein in smple x 100 % (Arb nd Dle, 1990) 44

70 Tble 2.4 Amino cid composition (% dry weight bsis) of the four experimentl feeds (Control, FW A, B nd C) Control FW A FW B FW C Essentil mino cid Threonine Vline Methionine Isoleucine Leucine Phenyllnine Lysine Histidine Arginine Sub-totl Non-essentil mino cid Asprtic Acid Serine Glutmic Acid Glycine Alnine Tyrosine Proline Sub-totl Totl

71 ph meter nd YSI digitl Dissolved Oxygen (DO) meter nd the vlues rnged from C, nd mg/ml respectively. Feces were collected 7 dys fter the experiment strted in order to llow for dpttion to the experimentl diet. Subsequently, feces were removed dily for 5 consecutive dys per week by siphoning out from the bottom of the quriums. The feces were recovered in the fternoon (3-4 h fter feeding) s soon s they were voided by the fish. The feces were filtered through fine nets, immeditely trnsferred to flsks, dried in n oven t 50 C for 48 h nd stored in desicctors until nlysis. The feces recovered on different dys, but coming from the sme qurium, were pooled. After 30 dys, the weight of fish ws recorded nd the fish were strved for 24 h before collecting crcss (3 fish per tnk) for chemicl nlyses. The fish were killed by MS-222, weighed, freeze dried, ground into homogeneous smple nd kept t - 20ºC. Five fish were collected t the beginning of the experiment for comprison. The sh, dry mtter, protein nd lipid content of fish crcss nd fish feces were nlysed, with detil procedures stted in Section Different prmeters: pprent digestibility on protein, specific growth rte, reltive weight gin, feed conversion rtio, protein efficiency rtio, pprent net protein utiliztion nd ft deposition rte were clculted for monitoring the growth performnce bsed on different tretments (Section 2.2.4) Feed conversion of different food wstes formultions in grey mullet Wild cught fries of grey mullet (omnivore) (4000, ~0.5 g ech) were imported 46

72 from Tiwn for testing different fish feeds (formultion A to C) with 400 individuls stocked in tnk (~1000 L wter ech, 10 tnks in totl) with continuous ertion nd wter flow-through, under 12 h-light:12 h-drk cycle. The experiment ws conducted in T Kwu Ling Opertion Centre of Agriculture, Fisheries nd Conservtion Deprtment (AFCD). Three food wste formultions ( FW A, B nd C) (Tble 2.3) nd commercil feed Jinfeng, 613 formulted feed (~30 % protein) were lso used to feed the grey mullet s control, ech with 2 replicte tnks ccordingly. Fish mortlity nd fish weight were recorded dily nd t the strt nd end of experiment, respectively. The wter temperture, ph nd dissolved oxygen were mesured three times week using portble Hnn ph meter nd YSI digitl Dissolved Oxygen (DO) meter (mintined t , ph , mg/L respectively). Thirty individuls (5 fish were grouped s smple nd 6 smples in totl representing the initil fish composition) were rndomly smpled, nd weighed t the beginning of the experiment. After 30 dys of the feeding experiment, the fish were strved for 24 h before collecting the crcss for chemicl nlyses, with 30 individuls (sme s the initil smpling) collected from ech tnk. They were killed by MS-222, weighed, freeze-dried, grounded into homogeneous smple by mechnicl blender nd kept t -20ºC until nlysis. Experimentl diets were dried to constnt weight t 50 C nd ground prior to nlysis. The sh, dry mtter, protein nd lipid content of fish crcss were nlysed, nd the detiled procedures were stted in Section Vrious prmeters: specific growth rte, reltive weight gin, feed conversion 47

73 rtio, protein efficiency rtio, pprent net protein utiliztion, ft deposition rte were clculted for monitoring the growth performnce bsed on different tretments (Section 2.2.4) Fish growth performnce prmeters Growth rtes were clculted for ech qurium s specific growth coefficient resulting from the following expression: ) Dily feeding rte (% body weight/dy) = Dily feed intke (g/fish) / initil verge body weight (g) b) Specific growth rte, SGR (%/dy) = 100 (Ln Wf- Ln Wi) / t c) Reltive Weight Gin, RWG (%) = (Wf Wi) x 100 / Wi where: Wf is the men finl body weight (g) for the fish in ech qurium, Wi is the men initil body weight of the fish in the sme qurium, nd t is time in dys. d) Feed conversion rtio, FCR = feed intke (g) / (Finl biomss Initil biomss (g)) e) Protein Efficiency Rtio, PER = weight gin (g) / protein intke (g). f) Apprent Net Protein Utiliztion, ANPU (%) = 100 (finl fish body protein (g) - initil fish body protein (g)) / totl crude protein intke (g) g) Apprent Net Lipid Utiliztion, ANLU (%) = 100 (finl fish body lipid (g) - initil fish body lipid (g)) / totl lipid intke (g) h) Apprent digestion coefficient (ADC) of protein nd lipid of diet (%) = 100 x [1-(dietry Cr 2 O 3 /fecl Cr 2 O 3 ) x (fecl nutrient / dietry nutrient)] 48

74 2.2.5 Chemicl nlysis on fish crcss nd feed Feed protein solubility in potssium hydroxide (KOH) The protein solubility ws determined ccording to the procedure described in Arb nd Dle (1990). Feed smple (~1.5g) ws dded to 0.2% KOH solution (75 ml) with mgnetic stirrer for 20 min (in duplicte). The smple ws then centrifuged t 3000 rpm for 15 min nd the superntnt ws filtered nd the totl nitrogen (15 ml of extrct ech, determined in triplicte) determined by the Kjeldhl method. The solubility of protein, expressed s percentge, ws clculted by dividing the protein content of the KOH extrcted solution by the protein content of the fish feed smple. Chromium nlysis Chromium (Cr) content in fish feed nd feces were nlysed by spectrometer (UV-1601, Shimdzu, Tokyo, Jpn) fter cid digestion of the smples ccording to Furukw nd Tsukhr (1966). Protein nlysis Smples of fish feed nd feces were nlyzed for nitrogen content with CHN nlyzer (Crlo Erb NA 1500, CE Instruments, Thermoquest Itli, Miln, Itly), the protein content (%) ws bsed on the nitrogen content of smple (%) multiplied by 6.25 (McKinney et l., 2004). Amino cid determintion 49

75 The mino cid profile in the fish feeds ws determined by the methods by GB/T of Stndrdiztion Administrtion of Chin (2001). In brief, the feeds were hydrolyzed with 6N HCl t 110 C for 24 h. The rectnt ws filtered through 0.45 μm filter membrne nd then the mino cid profile nlysed by n mino cid nlyzer (Hitchi, ). The chemicl nlysis ws performed in duplicte smples. Moisture nd sh content determintion The moisture nd sh contents of the experimentl diets nd fish crcss were determined ccording to the procedures of the Assocition of Officil Anlyticl Chemist (AOAC) (1990, b). For the moisture determintion (AOAC, 1990), the smple (fish feed or crcss) (~0.5g) ws weighed (W smple initil) nd dried t preweighed porcelin crucible with cover t 100 ± 2 C for 24 h. Then the crucible ws dried in desicctors for 2 h nd weighed to the nerest 0.1 mg immeditely (W smple finl). The crucible ws previously dried t 100 ± 2 C for 2 h nd weighed (W c ) s mentioned bove. The Percent Totl Dry Mtter (Totl DM, %) ws clculted s the following: % Totl DM = 100 x (W smple finl W c ) / (W smple initil W c ) W smple finl = smple nd crucible weight fter drying W smple initil = smple nd crucible weight before drying W c = dried crucible weight For the sh determintion (AOAC, 1990b), the smple (fish feed or crcss) (~0.5g) ws weighed (W sh initil) nd combusted t pre-weighed porcelin crucible 50

76 with cover t 600 ± 2 C for 2 h. Then the crucible ws dried in desicctor for 2 h nd weighed to the nerest 0.1 mg immeditely (W smple finl). The crucible ws previously dried t 100 ± 2 C for 2 h nd weighed (W c ) s mentioned. % Totl sh = 100 x (W sh finl W c ) / (W sh initil W c ) W sh finl = smple nd crucible weight fter combustion W sh initil = smple nd crucible weight before combustion W c = dried crucible weight Lipid determintion Ultrsound-ssisted extrction with modifictions ws dopted for lipid content determintion in fish feed nd fish crcss (Metherel et l., 2009). 0.1 g of smple ws extrcted with 3:2 v/v hexne: isopropnol mixture (12 ml) for 20 min with 40KHz soniction bth (Brnson Brnsonic 5510). The orgnic lyer ws collected fter centrifuged, the extrction ws then repeted for 2 times nd the orgnic lyer were pooled nd dried in pre-weighed glss tube under nitrogen nd the tube ws keep t 102 C for 30 min. The tubes were weighed gin fter keeping in desicctors for 3 h. Crude Fibre determintion Crude fibre ws determined by the loss on ignition (AOAC, 1984) of deftted feeds (2g) fter digestion with 200 ml of 0.255N H 2 SO 4 nd then NOH for 30 min ech. The residues were filtered nd wshed with hot distilled wter t lest twice. The residue ws wshed with 10ml of cetone twice fter ll digestions. The weight 51

77 of residue ws determined fter 105ºC (24 h) nd 550ºC (3 h). The percentge fibre ws determined s the following: % Fibre= Weight of residue fter 105ºC - Weight of sh fter 550ºC x 100 / Weight of smple Nitrogen-free extrct determintion Nitrogen-free extrct (non-fibrous crbohydrte) ws clculted by subtrcting the sum of (moisture % + crude protein % + crude ft % + crude fibre % + sh %) from 100 (Cstell nd Tiews, 1980) Sttisticl nlysis The ADC of experiment feed of grss crp nd effects of different feed types on the feed compositions, fish growth nd fish crcss compositions nd in two fish species were nlyzed by one-wy ANOVA, compring the men vlue with Duncn s multiple rnge tests (p<0.05) (SPSS Sttistics 17.0, Chicgo, Illinosis, USA). 2.3 Results Results of feed digestibility of different food wste formultions in grss crp Feed digestibility of different food wstes formultions nd growth performnce of grss crp The grss crp fed with FW A, the cerels dominnt (53%) feed showed the 52

78 most comprble growth performnce to the control, with no significnt difference ws found between the two groups (p>0.05) (Tble 2.5). SGR, RWG nd PER of the control group were significntly higher thn FW B nd FW C (p<0.05), while FCR of FW C ws significntly higher thn the control group (p<0.05). The protein nd lipid digestibility in the control group (97.05% nd 95.60% respectively) were significntly higher thn ll food wste feed groups (FW A, B nd C). The protein digestibility in FW A nd FW B were significntly higher thn FW C (p<0.05). The ANPU nd protein digestibility in FW A were significntly higher thn FW C (p<0.05), but there were no significnt differences on the ANLU nd lipid digestibility between two groups. In generl, grss crp fed with control diet showed the best growth which ws superior to tht of FW B nd C significntly in terms of APNU, protein digestibility, PER, SGR nd RWG (p<0.05), while FW C hd the lowest growth performnce Crcss composition of grss crp fed with different food wste formultions The crcss moisture ws significntly higher in FW A thn other groups (p<0.05). Significntly lower levels of protein (%) in grss crp crcss were found in FW C thn other groups (p<0.05). The lipid content in crcss were significntly lower in FW A thn ll other groups (p<0.05) (Tble 2.6). 53

79 Tble 2.5 Growth performnce of grss crp fed with different food wste feeds Mesurement Control FW A FW B FW C Initil body weight (g) 18.69± ± ± ±1.3 Finl body weight (g) 32.17± ±2.7b 25.97±1.2b 24.2±0.8b Feeding rte (% b.w./dy ) 3.03± ± ± ±0.1 SGR 1 (% b.w./dy) 1.94± ±0.24b 1.15±0.28b 1.05±0.23b Reltive weight gin (%) 72.34± ±10.3b 38.36±10.6b 34.38±8.5b Feed conversion rtio 1.34± ±0.13b 2.22±0.64b 3.03±1.47b Protein efficiency rtio 2.07± ±0.13b 1.16±0.25b 0.94±0.22b ANPU 2 (%) 48.39± ±1.7b 37.14±5.4bc 34.68±4.7c ANLU 3 (%) ± ±6.9b 54.75±12.2b 55.26±19.7b Protein Digestibility (%) 97.05± ±3.5b 78.64±4.0b 73.05±2.4c Lipid digestibility (%) 95.60± ±3.6b 75.50±3.3c 80.64±6.8bc *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 1 Specific growth rte, 2 Apprent net protein utiliztion, 3 Apprent net lipid utiliztion 54

80 Tble 2.6 Proximte composition (wet weight bsis) of grss crp crcss fed with different food wste feeds Mesurement Control FWA FWB FWC Moisture (%) 73.06± ±1.32b 73.81± ±1.32 Ash (%) 3.58± ± ± ±0.23 Protein (%) 15.18± ± ± ±0.64b Lipid (%) 5.56± ±0.78b 5.37± ±1.39 *Different superscripts (,b) mong feeding groups re significntly different (p<0.05) 55

81 2.3.2 Results of feed conversion of different food wste formultions in grey mullet Growth performnce of grey mullet fed with different food wste formultions The PER, RWG nd SGR were the highest in the control, followed by FW A nd FWC, while FW B ws the lowest (Tble 2.7). The sme pttern ws observed in terms of survivl rte of the control (89.63%) nd FW A (83.75%), compred with FW B (56.0%). The FCR ws the lowest in the control nd the highest in FW B. The ANPU nd ANLU were lso the highest in the control (20.11% nd 60.73% respectively) nd lowest in FW B (8.04% nd 20.43% respectively) Crcss composition of grey mullet fed with different food wste formultions No significnt difference ws found in crcss moisture nd protein levels between ll tretments (p>0.05) (Tble 2.8). A significntly lower sh content ws found in the control thn ll other groups (p<0.05), while lipid content in the control ws significntly lower thn FW A nd FW C (p<0.05). 56

82 Tble 2.7 Growth performnce of grey mullet fed with different food wste feeds (experiment in duplicte) Mesurement Control FW A FW B FW C Initil weight (g) Finl weight (g) Feeding rte (% b.w./dy) SGR 1 (% of b.w./dy) Survivl rte (%) Reltive weight gin (%) Feed conversion rtio Protein efficiency rtio ANPU 2 (%) ANLU 3 (%) Specific growth rte, 2 Apprent net protein utiliztion, 3 Apprent net lipid utiliztion 57

83 Tble 2.8 Proximte composition (wet weight bsis) of grey mullet crcss fed with different food wste feeds Mesurement Control FW A FW B FW C Moisture (%) 69.78± ± ± ±2.43 Ash (%) 4.55± ±0.83b 5.99±0.63b 5.48±0.52b Protein (%) 12.43± ± ± ±1.34 Lipid (%) 4.10± ±0.53bc 4.89±0.97b 6.50±1.31c *Different superscripts (,b,c) mong feeding groups re significntly different (p<0.05) 58

84 2.4 Discussion Growth of grss crp nd grey mullet fed with different food wste feeds The two fish species, grss crp nd grey mullet showed similr growth performnces (specific growth rte, reltive body weight gin nd feed conversion rtio) when fed with different feed formultions, in which the best growth performnce ws observed in the control feed. The cerels dominnt feed, i.e. FW A showed the best growth performnce mong ll food wste feeds, but it ws not stisfctory when compred to the control. The FW B nd FW C contining 25% nd 10% of met products showed significntly inferior growth (in terms of RWG, SGR, FCR nd PER) in grss crp, compred to the control group (p<0.05). The FCR in the control group ws the lowest mong ll feeding groups, while PER in the control group of grss crp ws significntly higher thn other food wste groups. It is known tht PER cn revel the reltionship between weight gin of fish nd protein consumed (Zeiotoun et l., 1973). The significntly lower PER lso indicted tht the proteins in food wstes were less effectively utilized by grss crp. FWA contined minly cerels (~53% in totl), which my be more suitble for both grey mullet (omnivore) nd grss crp (herbivore). Food wstes could provide nutrients nd energy for fish metbolism. In generl, met nd fish wste re good protein nd lipid sources for fish growth, while fruit nd vegetble wste contined high nitrogen free extrct (non-fibrous crbohydrte) (Grcí et l., 2005). Herbivorous fish possessed higher retention of plnt thn niml proteins (Jved nd Wtnbe, 2000), which my be relted to the enzyme system in the long gut of these 59

85 species such s grss crp (Smith, 1989). The digestion of cyprinids fish relied on the buccl cvity nd phrynx for replcing the bsented stomch function, nd higher trypsin ctivity nd lower pepsin ctivity were found in grss crp (Vsile nd Ciorne, 2009). Similr dpttion of intestine ws lso observed in grey mullet, nd the proximl intestine is highly folded, with enhnced surfce re nd bsorptive ctivity to omnivore diets (El-Bkry nd El-Gmml, 2010). As result, grey mullet nd grss crp digested nd utilized the FW A were more efficiently thn other food wste feeds, ccording to results of PER, FCR, RWG nd SGR. The differences in fish growth my be relted to the protein qulity e.g. mino cid profile nd digestibility of dietry protein. The mino cid profiles of the control, FW A nd FW B were similr (Tble 2.4), possessing similr levels of mino cid e.g. lysine, glycine, proline, phenyllnine nd tyrosine which re importnt for fish growth (Grg, 2007; Aksnes et l. 2008; Li et l., 2008). The vilbility nd digestibility of proteins re criticl for fish growth in this study. The differences of protein solubility in KOH in feed were closely relted to the growth performnce of fish. KOH protein solubility is convenient, chep nd fst method for monitoring the protein qulity of soy ben mel (Arb nd Dle, 1990). A study showed tht the growth of chicken ws correlted to the protein solubility nd protein dispersibility index of feeds (soyben mel with different het tretments) (Btl et l., 2000). However, KOH solubility is seldom use in evluting quculture feed nd growth performnce of fish, the present study ttempted to use this index to estimte the growth of grss crp nd grey mullet. The present results showed similr correltions, 60

86 with significntly higher growth rtes in fish in the control nd FW A, with the protein solubility 60.57% nd 51.91% respectively, which were significntly higher thn FW B nd FW C (p<0.05) (Tble 2.3). Both FW B nd FW C contined met product resulted in poor growth in both grss crp nd grey mullet, implying tht met product my not be suitble for these two species. Poultry by-products re good lterntive protein sources for mnufcturing fish feed (Higgs et l., 1979; Rwles et l., 2009), but it my be more suitble for crnivorous fish. Erturk nd Sevgili (2003), nd Rwles et l. (2006; 2009) showed tht the poultry by-products could replce the fish mel by 35% nd 40% or even entirely in hybrid striped bss (crnivore) diet. On the contrry, poor growth in common crp (Cyprinus crpio) fingerlings ws observed in ll tested feeds with 33, 67 nd 100% replcement of fish mel by poultry by-product mel (Emre et l., 2003) Utiliztions of different food wste feeds by grss crp nd grey mullet Grss crp fed with the control feed showed the most efficient use of protein nd lipid, reflected by ANLU, ANPU, nd pprent protein nd lipid digestibility. A similr trend ws lso observed in grey mullet, except tht FW B showed the poorest performnce ccording to different prmeters (no digestibility of nutrients ws investigted s different fish culture setup ws used nd fish feces were not collected due to the lnd-bsed setup of fish tnk in AFCD Center, siphoning out the feces ws not pplicble). High protein digestibility nd ANPU in grss crp demonstrted tht the control feed protein ws better utilized thn food wste feed proteins, implying 61

87 tht the sources of protein my be responsible for the efficiency on feed utiliztion. The mjor protein source in the control feed nd FW A ws derived from plnt bsed substnces, e.g. soy ben, rice brn, whet. These plnt bsed proteins re more efficiently digested by grss crp thn niml bsed proteins nd resulted in higher retention of protein in grss crp body (Jved nd Wtnbe, 2000). Besides, vried protein sources my result in the deficiency of some essentil mino cids contined in feed, which my led to poor utiliztion of dietry protein (Hlver nd Hrdy, 2002), s demonstrted by the lower protein digestibility of food wste groups (~73-83%). Therefore, the digestive systems of both grss crp nd grey mullet preferred plnt bsed thn niml bsed protein. As the experimentl feeds were isonitrogenous (sme protein level), only significnt differences in crbohydrtes nd lipid contents were noted between the 4 experimentl feeds. The protein sources contined in FW B nd FW C were prtilly derived from niml products, e.g. fish viscerl, pork, beef nd chicken which my possess higher mount of lipid leding to poor growth observed in both grey mullet nd grss crp. The non-protein energy sources i.e. lipids nd crbohydrtes could ffect the efficiency of protein utiliztion in fish (Wilson nd Hlver, 1986). However, fish re cpble to generte crbohydrte metbolites e.g. glucose nd glycogen from excessive lipid nd protein s energy, while crnivorous nd omnivorous fish re more cpble to utilize lipid s energy thn herbivorous fish (Du et l., 2009). Sufficient lipids in the diet promoted growth nd feed utiliztion in vrious fish species, s protein is spred in fish body nd converting crbohydrtes or 62

88 lipids into energy (De Silv et l., 2001; Sklli et l., 2004). As result, the growth performnce nd body composition re ffected by the lipid nd crbohydrte contents (crbohydrte to lipid (CHO: L) rtio) which served s mjor energy sources in feeds. The quntity of energy from crbohydrtes nd lipids re criticl to fish growth, but excessive mounts should be voided. The poor growth performnce of grey mullet nd grss crp fed with FW B nd FW C my be due to the high lipid nd low crbohydrtes contined in feed. FW C contined the highest mount of lipid (~19%), followed by FW B (~13%), while FW A nd the control feed showed similr lipid contents (5-6%). The mjority of energy contined in FW B nd FW C ws minly from lipids, rther thn from crbohydrtes, s shown by low CHO: L rtio (1.83 nd 1.04 respectively). Poor utiliztion of lipid (lower ANLUs) ws found in both grey mullet nd grss crp, but it ws surprised tht ANLU in grss crp fed with the low lipid content FW A ws the lowest. The lower vlue of ANLU lso indicted less retention of lipid in body, with more lipid converted to energy. Du et l. (2005) lso observed tht dietry lipid contents vried from 0 to 12%, which were djusted by fish nd corn oil, in their study involving grss crp. Go et l. (2010) lso reported tht grss crp showed the best growth t 7.5 of dietry CHO: L rtio, the tested rtios rnged from , with dietry energy nd protein levels mintined t 16.2 kj/g nd 39%, respectively. In generl, dietry lipids re minly converted to viscerl ft round different orgns e.g. livers nd kidneys, rther thn other prts of body (Sheridn, 1994). The 63

89 high dietry lipid (>10%) inhibited the growth of green grouper (Epinephelus coioides) with reduced feed intke quntity (Luo et l., 2004; Luo et l., 2005). A possible reson for lowered feed intke my be due to dequte energy supply from feed lipid nd finlly resulted in stunted growth with less protein intke (Luo et l., 2005). Bsed on the observtion fter 20 minutes of feeding, some feeds were not consumed in ll food wstes feed groups for grss crp nd grey mullet but the mount ws not significnt. This lowered feed intke my be due to the high lipid content in FW B nd FW C, or the lower pltbility of fish feeds due to the food wste components, which lso implied tht the food wste my reduce fish ppetite. High mortlity (10-46%) nd low utiliztion of lipid (~20-60% of ANLU) nd protein (~8-20% of ANPU) were found in the experiment deling with grey mullet fry (<1 g). The digestive system of fry is less developed nd much simpler thn tht in dult (Govoni et l., 1986); the proteolytic digestion in fry is dependent on exogenous sources e.g. live prey (Luff nd Hofer, 1984). The feed utiliztion in grey mullet fry ws the best when energy in feed ws minly from crbohydrte thn lipid (El-Dhhr, 2000). The present results lso showed tht control nd FW A (more crbohydrtes) hd better growth indices (SGR, FCR nd PER) nd survivl rte. Hence, the fry would be more susceptible to feed nutrients leding to high mortlity when feed protein nd lipid utiliztions were especilly low in FW B nd FW C. Aprt from growth performnce, the fish feed could lso ffect the body composition of fish. Both grss crp nd grey mullet fed with high lipid diets (FW C) showed the highest crcss lipid content nd stunted growth. Positive correltion 64

90 between crcss lipid content nd dietry lipid level ws observed in the present study, but the body lipid in FW A ws significntly lower thn the control (p<0.05). Similr correltion between crcss lipid content nd dietry lipid level ws lso found in grss crp (Go et l., 2010). Extensive studies indicted tht excessive ft deposition in fish viscerl nd tissues resulted from high lipid feeding (Lnri et l., 1999; López et l., 2006). Ali nd Al-Asgh (2001) showed tht dietry lipid content over 14% (CHO: L = 2.06) would hinder growth nd increse FCR in Nile tilpi, indicting poor utiliztion of feed, with higher crcss lipid contents. The lipid requirement vried mong different fish species. The present result illustrted lower lipid content (~6%) in feed fvored the growth of grss crp nd grey mullet thn high level of lipid (>13%). Köprücü (2012) lso noted tht protein utiliztion nd growth performnce of juvenile grss crp were the best in 6% lipid t 33% feed protein level, but not higher protein (37%) or lipid (8%). In generl, the growth of grss crp is more lible to lipid content in feed s poor utiliztion of excessive protein nd lipids ws found in grss crp, but crnivorous or omnivorous fish showed better utiliztion (Du et l., 2009). For the bove mentioned resons, the lipid content in food wste would impose dverse effects on fish growth, which should be removed prior mking fish feeds. In generl, the growth rtes of fish fed with food wste feeds were slower thn the control feed, further studies focusing on promoting feed utiliztion by upgrding FW A should be investigted. Supplementtion of exogenous enzymes in feed my enhnce digestion nd bsorption of nutrients by fish, leding to better weight gin 65

91 nd higher retention of protein in the body. It lso reduced wter pollution s fewer nutrients were excreted to the environment (Kolkovski et l., 1997). Enzymes e.g. ppin hve shown positive effects on growth rte, FCR, protein digestibility nd nitrogen retention efficiency, compred to diet without enzyme (Singh et l., 2011). Moreover, nutrients deficiency in feeds, especilly protein nd lipid would impir fish metbolic functions, e.g. proteins nd glycogen synthesis nd brekdown, nd hence reduce fish growth, immunity nd disese resistnce. It hs been noted tht the deficiency of essentil ftty cids dmged the ntibody production nd mcrophge function in rinbow trout (Kiron et l., 1995) nd serum complement ctivity in Nile tilpi (Lim et l., 2009). To our knowledge, there is severe lck of informtion on the effects of food wste treted with enzymes on fish immunity. Fish immunity is crucil fctor for fish frmers to dopt food wste for culturing fish. 2.5 Conclusion The hypothesis of this experiment ws rejected due to the poor growth performnce of fish fed with food wste feeds, further upgrding of food wstes should be investigted in order to utilize them s lterntive protein sources. In generl, both grss crp nd grey mullet performed the best growth (in terms of SGR, RWG, FCR nd PER) fed with cerel dominnt food wste fish feed (FW A), mong ll food wste feeds, but the growth ws lower thn the control. Met product contined feeds, FW B nd FW C showed inferior growth on both species, indicting grss crp nd grey mullet utilized plnt proteins better thn niml proteins. Being 66

92 herbivorous fish, grss crp would utilize crbohydrte s mjor energy source thn lipid. The high lipid content in feed ws lso possible reson for hindering growth, which could lso be ccumulted in body. Moreover, higher level of lipid ws observed in fish feeds contining met products. It is suggested tht lipid should be removed in the preprtion of food wste feed for grss crp. The growth performnce (reflected by RWG, SGR, FCR nd PER) of fish fed with food wste ws not stisfctory, nd further investigtions on upgrding FW A by dding enzymes re needed. 67

93 Chpter 3 Upgrding food wstes by mens of bromelin nd ppin to enhnce growth nd immunity of grss crp (Ctenophryngodon idellus) nd grey mullet (Mugil cephlus) 3.1 Introduction Protein is the most expensive component in feed which is mjorly from fish mel. Energy in feed is criticl to the utiliztion of dietry protein in fish (Wilson nd Hlver, 1986), energy is obtined from the ctbolism of proteins if the dietry energy is indequte. The protein is lso ctbolized into energy if dietry protein is excessive (Altise et l., 2006). Therefore, the feed is more cost effective if dietry protein is used for growth e.g. repiring nd building up tissue s much s possible nd with the lest mount broken down to energy (Guquelin et l., 2007). Protein utiliztion in fish could be enhnced by djusting feed protein to energy (P/E) rtio (McGoogn nd Gtlin, 2000) or pplying feed supplements such s medicinl herbs (Li et l., 2009) nd bker s yest (Osmn et l., 2010). Incresing the utiliztion of feed in quculture is importnt nd beneficil to quculture industry s well s the environment, s better feed conversion could reduce nutrient loss nd lower the feed cost in quculture production. In previous experiments (Chpter 2), the fish feeds (FWB & C) contined met product showed poor growth in both grss crp nd grey mullet while cerels dominnt feed (FW A) is most suitble to both species. This showed the met product my be more suitble for crnivores, but not omnivores nd herbivores. Although both grss crp nd grey 68

94 mullet fed with the cerel dominnt food wste feed (FW A) hd the best growth performnce (in terms of SGR, RWG nd PER) mong the food wste feeds, the growth performnces were poor when compred to the control, though not significntly. Supplementtion of enzymes ws dopted for the upgrding food wste feeds in this study. Nowdys, exogenous enzymes re extensively pplied s niml feed dditives to improve the nutritionl vlue nd reduce wter pollution (Kolkovski et l., 1997). Addition of enzymes in feed could enhnce the vilbility nd utiliztion of plnt protein by fish could be due to elimintion of nti-nutritionl fctors in plnt, e.g. phyttes (Liebert nd Portz, 2005; Singh et l., 2011). Enzyme mixtures (Frmzyme, including fungl xylnse, hemicellulse, pectinse nd cellulose) lso showed significnt improvement on the growth of Africn ctfish (Clris griepinus) (Yildirim nd Turn, 2010). Bromelin nd ppin could be considered s possible feed supplements used in quculture, with fruit wste is lso possible source for these two enzymes. Bromelin could be extrcted from the stem nd fruit of pinepple nd ppin obtined from ppy lef, unripe fruit nd ppy ltex. Bromelin incresed the digestibility of niml nd vegetble proteins by prtilly hydrolyzing the molecules into smller peptides (Fennem, 1996). Ppin cn hydrolyze proteins, lipids nd crbohydrtes nd function t wide rnge of ph nd temperture (Miymoto et l., 2004). Soy protein peptides were shortened by ppin tretment nd hence incresed its solubility (Wu et l., 1998). It hs been demonstrted tht pre-digestion of soyben 69

95 residues by ppin could enhnce the feed conversion in common crp (Cyprinus crpio) (Wong et l., 1996). A remrkble mortlity of grey mullet fries (20% more thn commercil feed) ws observed when fed with food wste feed (FW B nd C) in the previous experiment (Chpter 2). It rised concern on the effects of food wste on fish helth, probbly due to poor nutrient conversion from food wste feeds. Protein is constructed from vrious mino cids, which lso forms different biochemicl components e.g. hormone, enzyme nd immunoglobulin for metbolic functions. Hence, poor nutrition from diet could impir the fish immunity nd disese resistnce (Chndr, 1992). There is lso lck of informtion on the effects of food wste on the immunity of fish nd lso on the fesibility of upgrding food wste by dding enzymes. In the present study, more corn strch nd lipid (penut cooking oil) were dded into FW A to replce prt of the food wstes. The new food wste formultion (FW D), contined 60% food wstes, wheres corn strch nd lipid contents were incresed from 15% to 25% nd 6% to 12% respectively. The use of penut cooking oil ws to mimic the worst scenrio of high lipid content in food wstes, which ws lso observed in FW B nd FW C formultions. It hs been shown tht the growth of grey mullet fed with lower dietry protein could be enhnced by increse the energy content in the form of lipids (El-Dhhr, 2000b). Therefore, the present experiment ws intended to investigte whether lower protein of feed could support the growth of grss crp nd grey mullet by incresing the lipid content. 70

96 It is hypothesized tht the enzymes mixture of bromelin nd ppin could enhnce the utiliztion of food wste feeds by fish. As result, the objectives of present study were to investigte 1) the growth performnce of grss crp nd grey mullet; 2) the fish immunity when fed with food wste nd enzyme supplemented food wste; nd 3) the fesibility of improving the protein efficiency using lower protein nd higher lipid contents in feeds. 3.2 Mterils nd Methods Effects of food wstes formultion upgrded by ppin nd bromelin on grss crp growth performnce The procedures of food wste feed production were described in detils in Section In the present experiment, more corn strch nd lipid (penut cooking oil) were dded into FW A to replce prt of the food wstes s the new food wste formultion (FW D), contined 60% food wstes. Corn strch nd lipid contents contined in FW D were incresed from 15% to 25% nd 6% to 12 % respectively. Ppin nd bromelin were mixed with the food wste diets (FW A nd FW D contining 75% nd 60 % food wste respectively, Tble 3.1). The composition of FW D ws similr to FW A with dominnt cerel proportion, but higher portion of corn strch nd lipid ws replced prt of the food wste. Ppin (Sigm 76220, ppin from Cric ppy) nd bromelin (Sigm B-4882, EC , bromelin from pinepple stem) were mixed with the food wste diets (FW A nd FW D contining 75% nd 60% food wste respectively) with (50% by mss of fish diet) mol/l sodium 71

97 dihydrogen phosphte nd djusted to ph 8 with NOH, t 37ºC for 5 h. The commercil fish feed, Jinfeng, 613 formulted feed (~30 % protein) nd two food wste diets (FW A nd D) without enzyme tretment were used s the control. Then the substrtes were heted to 95 C for 5 min to inctivte the enzyme (Wu et l., 1998). The fish feed dough ws pelletized (3 mm dimeter) with met grinder nd dried t 50 C for 24 h. The proximte composition (sh, moisture, protein, fibre, lipid nd nitrogen free extrcts) nd the feed protein solubility in KOH were determined s mentioned in Section nd shown in Tble 3.2 Five hundred fingerlings of grss crp Ctenophryngodon idell (herbivore) were used for testing different fish feeds, with 18 individuls (~9-12 g) stocked in ech tnk (~60 L wter) in triplictes. The wter temperture, ph nd dissolved oxygen were mesured three times week using portble Hnn ph meter nd YSI digitl Dissolved Oxygen (DO) meter nd the vlues rnged from C, nd mg/ml respectively. Feeding with the control feed ws crried out t the rte of 2% body weight dily in ech experimentl feeding group nd the fish were cclimted for 3 weeks before the onset of experiment. The experiment ws conducted for period of 56 dys, with the fish blood smples collected t Dy 1, 14, 28 nd 56, by cudl venous puncture t vertebrl column of the fish (fish were nsthzied by MS-222). The oxidtive rdicl production (Nitroblue tetrzolium (NBT) ssy) in blood, totl protein nd totl immunoglobulin of plsm were determined (refer to Section for detils). 72

98 Tble 3.1 Food wste feed formultions (FW A & D, contining 75 & 60% food wste respectively) Formultion Food wste products Non-food wste products penut fruit/ vegetbles met products cerels bone mel other fish mel corn strch oil Food wste A Food wste D Totl (%) 73

99 Tble 3.2 Composite nlysis of experimentl feeds with 1% or 2% mixture of bromelin nd ppin in food wste A nd D (FW A & D) Formultion Control FW A 0% FW A 1% FW A 2% FW D 0% FW D 1% FW D 2% Dry mtter (%) 93.68± ± ± ± ± ± ±0.27 Ash (%) 8.24± ±0.46b 8.81±0.29b 8.68±0.25b 10.48±0.36c 10.41±0.33c 10.27±0.39c Protein (%) 30.16± ±0.31b 31.17±0.08b 32.07±1.68b ±0.35c 20.00±0.55 c 20.39±0.39 c Lipid (%) 5.17± ± ± ± ±1.66b 12.76±0.98b 12.71±1.05b Fibre (%) 9.57± ± ± ± ± ± ±0.85 Crbohydrtes (%) Energy (kj/g diet) CHO/L rtio P/E (mg/kj) Protein solubility (%) ± ±1.43b 72.89±4.18c 73.48±3.63c 56.21±2.51b 76.58±3.39cd 78.95±3.69d *Different superscripts (, b, c) mong feeding groups re significntly different (p<0.05) 1 Crbohydrtes (%) = 100 (crude protein % + crude lipid % + moisture % + sh % + fibre %) (Cstell nd Tiews, 1980) 2 Energy (kj/g diet) = (% crude protein 23.6) + (% crude lipids 39.5) + (% crbohydrtes 17.3) (Chtzifotis et l., 2010) 3 Crbohydrtes to Lipid (CHO: L) rtio = % wt. in CHO / % wt. in lipid 4 Protein to energy (P/E) (mg/kj) = crude protein (%) / Energy 5 Protein solubility (%) = Protein in KOH / Protein in Smple x 100% (Arb nd Dle, 1990) 74

100 The fish were strved for 24 h before collecting the crcss for chemicl nlyses, with 5 fish collected t the beginning of the experiment. After 56 dys of feeding experiment, 3 fish from ech tnk were collected nd killed by MS-222, weighed, freeze dried, ground into homogeneous smple nd kept t -20ºC until nlysis. The lipid, sh, moisture nd protein contents of the crcss were nlysed s described in Section Different prmeters: specific growth rte, reltive weight gin, feed conversion rtio, protein efficiency rtio, pprent net protein nd lipid utiliztion (Section for detils) were clculted for monitoring the growth performnce, bsed on different tretments Effects of food wstes upgrded by ppin nd bromelin on grey mullet growth performnce This experimentl setup ws lmost the sme s stted in Section 3.2.1, except the fish species ws grey mullet (~15-20g), insted of grss crp. The wter temperture, ph nd dissolved oxygen were mesured three times week using portble Hnn ph meter nd YSI digitl Dissolved Oxygen (DO) meter nd the vlues rnged from C, nd mg/ml respectively Fish growth performnce prmeters Growth rtes were clculted for ech qurium s specific growth coefficient resulting from the following expression: 75

101 ) Dily feeding rte (% of body weight/dy) = Dily feed intke (g/fish) / initil verge body weight (g) b) Specific growth rte, SGR (%/dy ) = 100 (Ln Wf- Ln Wi) / t c) Reltive Weight Gin, RWG (%) = (Wf Wi) x 100 / Wi where: Wf is the men finl body weight (g) for the fish in ech qurium, Wi is the men initil body weight of the fish in the sme qurium, nd t is time in dys. d) Feed conversion rtio, FCR = feed intke (g) / (Finl biomss Initil biomss (g)) e) Protein Efficiency Rtio, PER = weight gin (g) / protein intke (g). f) Apprent Net Protein Utiliztion, ANPU (%) = 100 (finl fish body protein (g) - initil fish body protein (g)) / totl crude protein intke (g) g) Apprent Net Lipid Utiliztion, ANLU (%) = 100 (finl fish body lipid (g) - initil fish body lipid (g)) / totl lipid intke (g) Fish immunologicl prmeters Plsm totl protein nd totl immunoglobulin ssys The totl immunoglobulin (IgI) in plsm ws determined using the method described in Siwicki et l. (1994) with slight modifictions. The protein concentrtion of the plsm ws determined ccording to the modified colorimetric method bsed on Brdford protein ssy (Brdford, 1976). Briefly, 5 μl of plsm nd 250 μl of Brdford solution were dded to 96-well microtiter pltes. After 20 min of incubtion t 22±1ºC, the bsorbnce ws mesured with microplte reder (Infinite 200, Tecn, 76

102 Austri) t 595 nm. The protein concentrtion (g/l) ws clculted from the stndrd curve. For totl immunoglobulin, 50 μl plsm nd 50 μl polyethylene glycol (10%) (PEG) were incubted t 22±1ºC for 2 h nd the mixtures were centrifuged t 1000 G for 15 min. The protein content of the superntnt ws determined by the ssy mentioned bove. The totl immunoglobulin concentrtion (Totl IgI) of the plsm (g/l) ws the difference between the totl protein (TP) level in plsm nd PEG treted plsm. Nitroblue tetrzolium (NBT) ssy The Nitroblue tetrzolium (NBT) ssy ws crried out bsed on the method described in Anderson nd Siwicki (1995). Heprinized fish blood (100 μl) ws dded to n equl volume of 0.2% NBT (Sigm-Aldrich, St. Louis, Missouri, USA) solution. The mixture ws then incubted t 22±1ºC for 30 min. The resultnt suspension (50 μl) ws dded into glss tube 1.0 ml N, N-dimethyl formmide (Sigm-Aldrich, St. Louis, Missouri, USA) nd centrifuged t 3000 G for 5 min. The opticl density (OD) of the superntnt ws mesured by spectrophotometer (UV- 1601, Shimdzu, Tokyo, Jpn) t 540 nm Sttisticl nlyses The effects of different feed types on the feed compositions, fish growth nd fish crcss compositions nd in two fish species were nlyzed by one-wy ANOVA, 77

103 compring the men vlue with Duncn s multiple rnge tests (p<0.05) (SPSS Sttistics 17.0, Chicgo, Illinosis, USA). The correltion between fish crcss lipid nd feed types ws tested by Spermn's test (p<0.01). 3.3 Results: Results of grss crp feeding tril with different food wste feeds upgrded by ppin nd bromelin Grss crp growth performnce fed with different food wste feeds upgrded by ppin nd bromelin For the growth performnce, the grss crp fed with FW A nd 1% mixed enzymes (FW A 1%) showed the highest specific growth rte (SGR),which ws significntly higher thn the control (p<0.05) (Tble 3.3). The fish fed with FW D 2% showed the lowest SGR which is significntly lower thn FW D 0% nd FW D 1% (p<0.05). The SGR in FW D with or without enzymes (ll FW D feed groups) were significntly lower thn the control (p<0.05). The reltive weight gin (RWG) in FW A 1% ws the highest, nd RWG in FW A 1% nd FW A 2% were significntly higher thn ll other groups (p<0.05), including the control. Besides, RWG of ll FW D feed groups were significntly lower thn the control (p<0.05), while FW D 2% showed the lowest RWG. The feed conversion rtio (FCR) of grss crp in ll FW D feed groups ws significntly higher thn the control nd ll FW A feed groups (p<0.05). The pprent net protein utiliztions (ANPUs) of FW A groups nd the control were comprble with FW D 1% hd the highest ANPU, but not significntly different 78

104 from the control (p>0.05), but significntly higher thn FW A 2% (p<0.05) which ws the lowest. The pprent net lipid utiliztions (ANLUs) ws the highest in FW A 1%, which ws significntly higher thn the control, while the ANLU in ll FW D feed groups were significntly lower thn the control (p<0.05). The ANLU of the FW A 2% ws the lowest mong FW A feed groups, with sme observtion ws found in FW D feed groups. The protein efficiency rtio (PER) of FW A 1% ws the highest mong ll feeding groups, which ws significntly higher thn the control group nd FW A 0% (p<0.05). It ws lso noted tht PER of FW D 2% ws significntly lower thn the control nd FW D 0% (p<0.05) Grss crp crcss composition fed with different food wste feeds upgrded by ppin nd bromelin The crcss moisture contents of grss crp fed with FW A 1% nd FW A 2% were significntly higher thn the control (Tble 3.4). On the contrry, crcss moisture contents in FW D 0% nd FW D 1% were significntly lower thn the control (p<0.05). No significnt difference in terms of sh contents were observed between ll feeding groups (p>0.05). The crcss protein contents in FW D 0% nd FW D 2% were significntly lower thn control (p<0.05). The crcss lipid contents of FW A 1% nd FW A 2% were significntly lower thn control (p<0.05) with FW D 0% hd higher crcss contents thn control (p<0.05). The decresing trends of lipid contents were significntly correlted with the increse of enzyme supplements (0%, 1%nd 2%) in FW A (r=0.630) nd FW D (r=0.669) groups (Spermn s test, p<0.01). 79

105 Tble 3.3 Growth performnce of grss crp fed with different food wste feeds upgrded by ppin nd bromelin Mesurement Control FW A 0% FW A 1% FW A 2% FW D 0% FW D 1% FW D 2% Initil weight (g) 10.76± ±0.14b 10.55±0.13d 10.89±0.05c 11.10±0.09b 10.96±0.08c 10.64±0.1 Finl weight (g) 15.02± ± ±0.41b 15.66±0.17b 14.23±0.06c 13.68±0.33d 12.63±0.16e Feeding rte (%b.w./dy) 2.08±0.05b 2.07±0.02b 2.08±0.05b 2.05± ±0.05b 2.11±0.05b 2.12±0.01b SGR (%b.w./dy) 0.60±0.02b 0.54± ±0.07c 0.65±0.02b 0.44±0.01d 0.40±0.04d 0.31±0.02e RWG (%) 39.56±1.66b 35.43± ±5.55c 43.80±1.40b 28.24±0.70d 24.86±2.48d 18.78±1.38e FCR 3.14±0.04b 3.46±0.29b 2.64±0.28b 2.85± ±0.14c 4.80±0.46c 6.19±0.46d ANPU (%) 30.66±3.64b 30.04±1.77b 33.66±3.56b 28.05± ±4.93b 37.54±6.55b 32.11±4.01b ANLU (%) 93.96± ± ±19.53b 47.11±6.50c 53.95±10.67c 41.71±5.88cd 25.31±8.39d PER 1.05±0.01b 0.92±0.08bc 1.22±0.14d 1.09±0.04d 1.19±0.04d 1.05±0.10b 0.80±0.06c *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 80

106 Tble 3.4 Proximte compositions (%, wet weight bsis) of grss crp crcss fed with different food wste feeds upgrded by ppin nd bromelin Control FW A 0% FW A 1% FW A 2% FW D 0% FW D 1% FW D 2% Moisture (%) 72.50± ± ±1.37cd 76.05±0.83d 69.64±2.60b 69.93±1.71d 73.33±2.33c Ash (%) 2.97± ± ± ± ± ± ±0.20 Protein (%) 10.04± ± ± ±0.86b 7.28±0.81c 8.92±1.74b 8.01±0.85bc Lipid (%) 7.96±0.81b 7.95±1.11b 6.08±0.68c 5.84±0.86d 10.47±1.47d 8.74±1.07b 6.85±1.03c *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 81

107 Grss crp immunologicl prmeters The NBT ctivities of FW D 0% nd FW D 1% were significntly lower thn the control t Dy 14 (p<0.05) (Fig. 3.1). At Dy 28, the ctivities of FW A 0% nd ll FW D feed groups were significntly lower thn the control, while FW A 1% showed significntly higher ctivity thn the control t Dy 56 (p<0.05). In generl, the plsm protein nd totl IgI in grey mullet fed with food wste without enzymes were lower thn the control (Fig. 3.1b & c). The plsm protein level of FW A 0% nd FW D 0% were significntly lower thn the control t Dy 14 nd 28 respectively (p<0.05). After 56 dys, FW A 0%, FW A 2% nd FW D 2% showed lower plsm protein thn the control (p<0.05). The totl IgI of FW A 0% ws significntly lower thn the control t Dy 14 nd 28 (p<0.05), but ll the tretments were not significntly different t Dy 56 (p>0.05) Results of grey mullet feeding tril with different food wste feeds upgrded by ppin nd bromelin Grey mullet growth performnce fed with different food wste feeds upgrded by ppin nd bromelin The SGR nd RWG of FW A 1% were the highest, which were significntly higher (p<0.05), while those prmeters of FW A 0%, FW D 0% nd FW D 2% were significntly lower thn the control (p<0.05) (Tble 3.5). The FCR of FW A 1% ws the lowest mong ll tretments but not significntly different from the control (p>0.05), while FCR of FW A 0%, FW D 0% nd FW D 2% were significntly higher 82

108 thn the control (p<0.05). The ANPU of FW A 0% nd FW D 1% ws significntly lower thn control (p<0.05), nd FW A 1% nd FW A 2% were significntly higher thn FW A 0% (p<0.05). The ANLU of FW A 2% ws the highest nd ws significntly higher thn ll other tretments (p<0.05). It ws lso noted tht ll FW D feed groups showed significntly lower ANLU thn the control (p<0.05). The PER of FW A 1% nd FW A 2% were significntly higher thn FW A 0% (p<0.05), but not significntly different from the control (p>0.05), while PER of FW D feed groups were significntly higher thn the control nd ll other groups (p<0.05). It ws lso noted tht FER of FW D 0% nd FW D 1% were significntly higher thn those of FW D 2% Grey mullet crcss composition fed with different food wste feeds upgrded by ppin nd bromelin No significnt difference in crcss moisture between ll tretments (p>0.05) ws observed. All tretments, except FW A 0% nd FW A 2% showed higher crcss sh contents thn the control (p<0.05) (Tble 3.6). The crcss protein contents in FW D 1% nd FW D 2% were significntly lower thn the control (p<0.05). The crcss lipid content of FW D 2% ws the highest which ws significntly higher thn the control (p<0.05) Grey mullet immunologicl prmeters There ws no significnt difference for the NBT ctivity between ll 83

109 tretments t Dy 14 nd 28 (p>0.05) (Fig. 3.2). At Dy 56, the NBT ctivity of FW D 2% ws significntly lower thn the control. The ctivity of FW A 1% ws the highest t Dy 56 which ws significntly higher thn FW A 0% nd ll FW D groups (p<0.05). The plsm protein level of FW A 2% ws significntly higher thn the control t Dy 14 (p<0.05) (Fig. 3.2b). The plsm protein levels of FW A 1%, FW A 2% nd FW D 2% were significntly higher thn the control t Dy 28 (p<0.05). However, the plsm protein levels of FW A 0% nd FW D 1% were significntly lower thn the control, while FW A 2% ws higher thn the control significntly t Dy 56 (p<0.05). The totl IgI level of FW A 2% ws significntly higher thn the control t Dy 14 (p<0.05) (Fig. 3.2c). At Dy 28 nd 56, the IgI levels of FW A 1% nd FW A 2% were not significntly different from the control (p>0.05) but significntly higher thn FW A 0% (p<0.05). FW A 2% showed the highest IgI level t Dy 56 which significntly higher thn the control (p<0.05). The sme trend of IgI level ws lso observed in FW D groups, with the totl IgI levels of FW D 1% nd FW D 2% significntly higher thn FW D 0% (p<0.05). 84

110 ) Absorbnce (540nm) Control FW A FW A 1% FW A 2% FW D FW D 1% FW D 2% b b b b b b b b b b b 0.1 b) Protein Concentrtion (g/l) Dy b bc b b b bc b b bc bc c bc bc bc c bc bc bc c) Dy IgI Concentrtion (g/l) b b b b b b c b bc b b bc Dy Fig. 3.1 ) Nitroblue tetrzolium (NBT) ctivity in blood, b) plsm totl protein (g/l) nd c) totl immunoglobulin (g/l) of grss crp in the control nd food wste feeding groups, different superscripts (, b) within in sme smpling dy re significntly different (p<0.05). 85

111 Tble 3.5 Growth performnce of grey mullet fed with different food wste fish feeds upgrded by ppin nd bromelin Mesurement Control FW A 0% FW A 1% FW A 2% FW D 0% FW D 1% FW D 2% Initil weight (g) 14.78±0.14b 14.76±0.25b 14.60±0.82b 15.44±1.30b 15.17±0.80b 13.86± ±0.62b Finl weight (g) 21.10±0.47bc 19.20±0.23d 21.53±1.42bc 21.90±1.60c 20.33±1.01bcd 19.50±0.78b 19.70±0.43bd Feeding rte (%b.w./dy) 2.10±0.07b 2.30±0.03b 2.15±0.09b 2.04± ±0.08b 2.24±0.08b 2.18±0.11b SGR (%b.w./dy) 1.27± ±0.03b 1.39±0.09c 1.25± ±0.02b 1.22± ±0.08b RWG (%) 42.73± ±1.04b 47.41±3.74c 41.89± ±0.80b 40.61± ±3.01b FCR 2.32±0.12b 3.43±0.08e 2.18± ± ±0.13cd 2.58±0.20b 3.00±0.15d ANPU (%) 21.15±1.88b 14.16±0.47c 20.60±3.40b 22.07± ±1.07b 14.10±4.15c 16.29±2.86bc ANLU (%) 71.65± ± ± ±7.38b 35.19±3.95c 32.38±2.66c 52.44±4.44d PER 1.43± ±0.02b 1.46± ± ±0.09d 1.94±0.15d 1.64±0.08c *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 86

112 Tble 3.6 Proximte composition (%, wet weight bsis) of grey mullet crcss fed with different food wste fish feeds upgrded by ppin nd bromelin Control FW A 0% FW A 1% FW A 2% FW D 0% FW D 1% FW D 2% Moisture (%) 65.79±1.79b 64.65±1.73b 67.03± ±2.26b 65.02±1.02b 63.72±1.31b 64.00±1.61b Ash (%) 5.21±0.18b 5.15± ±0.21c 5.51±0.08bc 5.78±0.37c 5.57±0.04c 5.80±0.24c Protein (%) 16.67± ± ± ± ±0.75b 14.87±0.98b 14.82±0.58b Lipid (%) 10.05± ±0.88b 11.14±3.19b 11.90±1.80b 11.04±1.46b 11.64±1.75b 13.29±1.95b *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 87

113 Absorbnce (540nm) ) Control FW A FW A 1% FW A 2% FW D FW D 1% FW D 2% bc bcd b cd bcd d Dy b) Protein Concentrtion (g/l) b b b c b c b b b b bc bc b b bc b b c) Dy IgI concentrtion (g/l) b b b b b b c bc b bc bc bc cd bc b d bcd bcd Dy Fig. 3.2 ) Nitroblue tetrzolium (NBT) ctivity in blood, b) plsm totl protein (g/l) nd c) totl immunoglobulin (g/l) of grey mullet in control nd food wste feeding groups, different superscripts (, b, c) within in sme smpling dy re significntly different (p<0.05) 88

114 3.4. Discussion Growth of grss crp nd grey mullet fed with food wste supplemented with bromelin nd ppin mixture In generl, the growth performnce of grss crp nd grey mullet ws the best when feeding with food wste A supplemented with 1% of mixed bromelin nd ppin (FW A 1%). The growth performnce ws evluted by SGR, RWG, FCR nd PER, nd grss crp fed with FW A 1% hd the best performnce, while grey mullet chieved the best SGR nd RWG in the sme tretment. The results were in line with the previous experiment (Chpter 2) on feeding with different food wste formultions, with FW A 0% showed comprble growth performnce in both grey mullet nd grss crp when compred with the control feed. The FW D formultion ws similr to FW A but contined lower protein level nd lower CHO: lipid rtio. The growth performnce of both fish species in most feeding groups of FW D were significntly lower thn the control (p<0.05). The poor growth of fish could be due to the difference in crbohydrte nd lipid (CHO/L) rtios in feed, which were bout 3.3 in FW D nd 6.3 in FW A. A study showed tht n inclusion of 10-20% of corn strch could promote growth nd PER in yellowfin sebrem (Sprus ltus), but dverse effect ws observed when percentge reched 26% (Wu et l., 2007). There is no specific informtion on the optimum crbohydrtes in feeds (NRC, 1993), lthough wrmwter fish like grss crp (Lin, 1991) re believed to hve better utiliztion of crbohydrtes thn coldwter fish like Atlntic slmon (Slmo slr) (Hellnd et l., 1991). In generl, 28-32% nd

115 42.5% dietry proteins nd crbohydrtes ccordingly re optiml for grss crp juvenile (Hsn et l., 2007), similr to the control feed nd FW A in the present study. Low P/E vlue in FW D indicted lrge portion of energy ws derived from non-protein source nd low CHO/L rtio indicted tht energy is minly from feed lipids. The non-protein energy sources could ffect the efficiency of protein utiliztion in fish (Wilson nd Hlver, 1986). However, it hs been noted tht crbohydrtes re more suitble energy source thn lipid for grey mullet (El-Dhhr, 2000), while high lipid content could hinder the growth in vrious fish, including green grouper (Epinephelus coioides), Nile tilpi (Oreochromis niloticus) nd grss crp (Ali nd Al-Asgh, 2001; Luo, 2004; Luo, 2005; Du et l., 2009). The high lipid content in feed my lso ffect the lipid content in crcss nd the feed utiliztion. The grss crp fed with FW D 0% retined higher lipid content in body nd showed poor growth. Ali nd Al-Asgh study (2001) lso showed tht Nile tilpi fed with high dietry lipid (>14%) resulted in poor growth nd higher crcss lipid. However, in the present study, both fish showed impired growth responses when fed with feeds contining high dietry lipid nd low protein contents, nd the lipid retentions in crcss were different. High lipid retention in the grey mullet ws not observed when fed with FW D without enzymes supplement (FW D 0%), while high retention ws observed in grss crp. Lower crcss protein retentions were observed in both fish fed with FW D nd protein spring effects were not shown. Protein spring effect occurred when fish re more cpble to metbolize feed protein 90

116 into body tissue from dietry energy of non-protein sources. This ws observed in mny fish species e.g. Jundi (Rhmdi quelen) nd hybrid clris ctfish (Clris mcrocephlus x Clris griepinus) (Jntrroti et l., 1998; Meyer nd Frclossi, 2004). The high dietry lipid did not compenste the effect of low dietry protein bsed on the retrded growth nd lower crcss protein contents, higher dietry energy level my be required in both fish to spre protein. On the contrry, higher PER nd similr RWG were observed in grey mullet of ll FW D groups compred to the control, but low retentions of protein nd lipids nd higher sh contents were found in crcss. This my lso indicte the indequte dietry energy in FW D nd protein ws prtilly converted into energy to support the growth of grey mullet (NRC, 1983). However, further study on the effects of higher level of dietry energy with low protein diet is needed to prove this. The present study lso suggested tht grey mullets possess different utiliztion efficiency of lipid in the body, nd re more cpble to utilize dietry lipid s energy source especilly with low protein nd high lipid diet. The present results showed tht grey mullet required less dietry protein thn grss crp, the PER in grey mullet ws improved when feeding with low protein diet (20%). Du et l. (2009) suggested tht dietry protein nd lipid t 35% nd 3% respectively re optiml for grss crp (body weight ws bout 3 g), while grey mullet fry (1.6 g) hd the best growth performnce t 28% feed protein (El-Dhhr, 2011). El-Dhhr (2000) lso reveled tht grey mullet utilized energy with low CHO/L rtio more efficiently. 91

117 3.4.2 Utiliztions of food wste supplemented with bromelin nd ppin mixture The ddition of bromelin nd ppin my enhnce the feed utiliztion or stimulte fish immunity. The present results showed the enhnced growth nd reduced lipid retention in grss crp when fed with enzyme supplemented feed. The decresed crcss lipid ws significntly correlted with the increse of bromelin nd ppin in FW A (r=0.630) nd FW D (r=0.669) (Spermn s test, p<0.01). Addition of 1% nd 2% of bromelin nd ppin mixture could enhnce ANLU in both grss crp nd grey mullet. The exogenous enzymes in feed my enhnce metbolism nd utiliztion of lipid in grss crp, with more energy converted from lipid, leding to more protein reserved in crcss s observed. However, there is no specific study on the effects of exogenous enzymes on the lipid utiliztion in fish. Exogenous enzyme supplementtion could ssist feed digestion nd utiliztion in fish. The ANPU of grss crp nd grey mullet ws lso improved when dding 1% of mixed enzymes in FW A. Bromelin nd ppin could hydrolyze proteins nd relese shorter peptides in feed, the key fctor to increse protein digestibility (Fennem, 1996; Singh et l. 2011). A few studies lso showed tht n ddition of exogenous ppin in feed could enhnce the growth of common crp nd improve wter qulity. Wong et l. (1996) showed common crp could digest the soyben residues treted with 1% ppin more efficiently, the turbidity of tnk wter nd hence fish mortlity were reduced in the ppin feed groups. A recent study lso showed the growth of common crp with 2% ppin ws promoted, lthough the wter prmeters were not 92

118 ffected (Singh et l., 2011). Another study on plm kernel mel lso indicted tht ddition of enzyme (Ronozyme VP) could enhnce the growth nd net protein utiliztion in tilpi (Boonyrtplin et l. 2000). It ws lso reveled tht exogenous enzymes (commercil products) could improve the protein digestibility nd feed conversion in rinbow trout (Oncorhynchus mykiss) (Frhngi nd Crter, 2007). The cost of enzyme supplemented fish feed could be further reduced if fruit processing wstes were utilized insted of pure enzymes. The use of pinepple wstes could lso promote the growth of Lbeo rohit fingerling, with the SGR, PER were significntly higher in feed contining 25% pinepple wstes, thn the control nd other fruit wste feeds such s ornge nd lime (Dek et l., 2003). In Hwii, the growth of Litopeneus vnnmei ws comprble to commercil feed when fed with 50% inclusion of fermented ppy wste in shrimp feed. The production cost could be further reduced under lrge scle production (Kng et l., 2010) Hemtologicl prmeters of fish fed with food wste supplemented with enzyme mixture The hemtologicl prmeters were ffected by the two food wste feeds nd supplemented enzymes. The totl protein, totl IgI nd NBT ctivity of fish feeding with food wste feeds were depressed to some extent during the experiment, e.g. significntly reduced NBT ctivities were found in grss crp fed with FW A 0% nd ll FW D groups t Dy 28 (p<0.05). The production of oxidtive rdicl by neutrophil ws quntified by the NBT ssy (Siwicki et l. 1994), nd noted tht the 93

119 oxidtive rdicl is n importnt defense system in fish (Anderson et l. 1992). The totl IgI nd totl serum protein levels of grss crp in FW A 0% were lso significntly lower thn the control during severl smplings (p<0.05). Serum protein level indicted blnce between nbolic nd ctbolic ctivity in fish. When nbolic processes exceeded ctbolic ones, the reserved protein is relesed into blood strem more to meet higher metbolic requirements (Helmy et l., 1974). Serum proteins re responsible for innte immune response of fish nd higher level of serum protein provided stronger response (Wiegertjes et l. 1996; Shu et l. 2007). The rise of plsm proteins could be resulted from the higher digestion of dietry protein (Lundstedt et l., 2002), while the lowered serum protein levels my show nutritionl deficiencies in fish (Siwicki et l., 1994). The nutrients contined in food wste in this study my not be utilized nd bsorbed by fish efficiently, nd therefore the growth nd immunity were lso ffected. Moreover, it is noted tht the impired immunity ws less obvious in grey mullet thn tht of grss crp. This further suggested different fish species possess different requirements on feed nutrients nd different feed utiliztion mechnisms for their growth. Although the food wste feeds hve negtive impcts on the tested hemtologicl prmeters, enzyme supplementtion could minimize the impcts nd even stimulte the NBT ctivity, totl protein nd totl IgI levels when fed with 1% or 2% mixed enzyme. Mixed exogenous enzymes of pepsin, ppin nd α-mylse could increse the totl serum protein nd globulin level nd enhnce the growth of Nile tilpi in terms of better SGR, PER nd FCR (God et l., 2012). A similr study dding lipse, 94

120 pepsin, trypsin in feed for common crp fry lso showed better growth performnce nd elevted totl serum protein, compred to feed without enzymes (Yousefin et l., 2013). However, the effects of ppin nd bromelin on fish immunity re not known, nd current study provided n induction on their effects on grey mullet nd grss crp. The use of enzyme s feed supplement my be n pplicble nd vible pproch in enhncing feed utiliztion nd fish immunity, but further investigtions on specific fish species re needed. To sum up, the present results indicted on growth performnce nd hemtologicl prmeters of grss crp nd grey mullet could be improved by dding enzymes to the food wstes. Other feed supplements such s bker s yest could be dded into food wste for further enhncement on feed utiliztions. Bker s yest is lso one of the populr supplements in quculture industry, serving s immunostimulnt nd growth promoters. 3.5 Conclusion The hypothesis of this experiment ws ccepted, the ddition of bromelin nd ppin could enhnce the utiliztion of food wste feeds by fish. In generl, the growth nd immunity of grss crp nd grey mullet fed with food wste feeds without enzymes were impired compred to the control, however, improvement superior to the control ws observed with the ddition of bromelin nd ppin supplement. Addition of 1% nd 2% mixture of bromelin nd ppin could significntly enhnce the ANLU in grss crp nd ANPU in grey mullet. The results lso showed tht grey 95

121 mullet ws more cpble to utilize protein nd lipid in the low protein diet thn grss crp, but the high dietry lipid did not compenste the effect from low dietry protein bsed on the retrded growth. Further experiments on the effects of bker s yest (Scchromyces cerevisie) on grss crp immunity nd disese resistnce were conducted in Chpter 4. 96

122 Chpter 4 Adding bker s yest to food wste to enhnce growth performnce nd immunity of grss crp (Ctenophryngodon idellus) 4.1 Introduction The use of probiotics in quculture industry hs been extensively investigted (Aly et l., 2008; Wng nd Xu, 2004; Wng et l., 2005). Probiotics is defined s live microbil feed supplement which beneficilly ffects the host niml by improving its intestinl blnce (Fuller, 1989). The probiotics cn be pplied in the form of feed supplements or directly introduced to the quculture system, for enhncing feed vlue, enzymtic digestion, pthogens inhibition, growth nd immune response of hosts (Ziei-Nejd et l., 2006; Wng nd Xu, 2004; Wng et l., 2005). Probiotics cn enhnce the estblishment of fvorble microbil communities e.g. Bcillus sp. nd other lctic cid bcteri in the gstrointestinl system. These beneficil communities could lter gut morphology nd generte enzymes for digestion nd bsorption of nutrients nd lso stimulte the host immunity (Verschuere et l., 2000). It ws observed tht the food wste feed ws not efficiently utilized by grss crp nd grey mullet, leding to impired growth (Chpter 2). Although supplementtion of bromelin nd ppin could enhnce the utiliztion of food wste, the immunity of grss crp ws not ffected significntly (Chpter 3). Therefore, ttempts will be mde to pply bker s yest s feed supplement to enhnce growth nd host immunity. 97

123 The yest, Scchromyces cerevisie hs been used in bking nd fermenting lcoholic beverges for thousnd yers. More recently, it hs been used to improve growth nd feed utiliztion in quculture, in vrious crp species (Mohnty et l., 1996; Swin et l., 1996), hybrid striped bss (Morone chrysops M. sxtilis) (Li nd Gtlin, 2005) nd Nile tilpi (Oreochromis niloticus) (Abdel-Twwb et l, 2008). S. cerevisie lso llevited the trnsporttion stress of tilpi nd strengthened their disese resistnce ginst Aeromons hydrophil (Zki, 2004). The positive effects of probiotics on fish growth my be due to the dhesion of yest cells to the intestinl wll, with their mylse enzymes incresing the digestibility of the diet (Scholz et l. 1999). S. cerevisie lso significntly enhnced mylse ctivity in Nile tilpi (Ess et l., 2010). Furthermore, the cell wll constituents of yest could stimulte innte immune responses nd protect fish ginst infections (Estebn et l., 2001). Bker s yest consisted of β-glucns, mnnn oligoscchrides nd nucleic cid which re regrded s immune-stimulnts (White et l., 2002). The β-glucn isolted from yest hs shown immune-stimulting properties on vrious fish species, such s Asin ctfish (Clris btrchus) nd common crp (Cyprinus crpio), enhncing their disese resistnces to A. hyrdophil (Kumri nd Shoo, 2006 & b; Selvrj et l., 2005). The ddition of whole yest in diet stimulted the immune response of chnnel ctfish (Icthrus puncttus), sebrem (Sprus urt) nd tilpi (O. niloticus) (Chen nd Anisworth, 1992; Rodriguez et l., 2003; He et l., 2009). Applying whole yest would be superior to extrcted nd purified constituents, s the cost nd technique re lessened. 98

124 In ddition, the whole bker s yest is potentil lterntive for fish mel protein (Oliv-Teles nd Gonclves, 2001). Yest is one of the mjor by-products in brewing industry followed by brewer spent grin nd could be collected esily nd economiclly. It is hypothesized tht the bker s yest could enhnce the growth nd immunity of grss crp through better feed utiliztion. In this Chpter, the bker s yest ws used s feed supplement (2.5% nd 5% w/w) by incorporting into 3 different types of feeds: commercil feed (Jinfeng, 613 formultion) (s the control) nd food wste feeds with (FW A-E) nd without (FW A) ddition of bromelin nd ppin mixtures. The objectives of this study were to investigte 1) the effects of dding bker s yest to food wste feed on growth nd feed utiliztion of grss crp; 2) chnges of fish immunity nd 3) disese resistnces of fish to Aeromons hydrophil. 4.2 Mterils nd Methods Experimentl setup nd fish feed preprtion The food wste feed formultion A (FW A) contining 75% food wstes with minly plnt mterils such s cerels, fruit nd vegetbles (Chpter 2, Tble 2.2), ws prepred ccording to the procedures mentioned in Section Ppin nd bromelin in the proportion of % (weight by weight) were dded into FW A by mixing with (50% by mss of fish diet) mol/l sodium dihydrogen phosphte nd djusted to ph 8 with sodium hydroxide (NOH), t 25ºC for 3 h. The commercil fish feed (Jinfeng, 613 formultion) nd FW A without enzyme tretment were used s the controls. The resulted substrtes were heted to 95 C for 5 99

125 min to inctivte the enzyme (Wu et l., 1998). The three types of feeds, i.e. the control feed (C-0%Y), enzyme treted FW A (A- 0%Y) nd non-enzyme treted FW A (A-E-0%Y) were supplemented with 2.5% nd 5% w/w of bker s yest, Scchromyces cerevisie (Type II, YSC2, Sigm-Aldrich) (nine groups in totl). Deionized wter ws dded nd the fish feed dough ws pelletized with met grinder nd dried t 50 C for 24 h. The proximte compositions (sh, moisture, protein, fibre, lipid nd nitrogen free extrcts) were determined in Section with the results shown in Tble Feeding experiment design Seven hundred fingerlings of grss crp Ctenophryngodon idell (herbivore) were purchsed from locl fish frm. Eighteen individuls (~20-25g) were plced into ech tnk (~60L). The wter temperture, ph nd dissolved oxygen were mesured three times week using portble Hnn ph meter nd YSI digitl Dissolved Oxygen (DO) meter nd the vlues rnged from C, nd mg/ml respectively. The control feed ws fed to fish t the rte of 2% body weight dily for 3 weeks before strting the experiment. The experiment ws conducted for 56 dys nd there were triplictes for ech tretment (9 in totl). 100

126 Tble 4.1 Composite nlysis of three types of feeds: control (C) nd food wste formultion A (with (A-E) nd without (A) enzyme) feed supplement with or without bker s yest (Y) Formultion C-0%Y C-2.5%Y C-5%Y A-0%Y A-2.5%Y A-5%Y A-E-0%Y A-E-2.5%Y A-E-5%Y Dry mtter (%) 93.68± ± ± ± ± ± ± ± ±0.16 Ash (%) 8.24± ± ± ±0.46b 8.68±0.34b 8.48±0.25b 8.81±0.25b 8.25±0.33b 8.10±0.20b Protein (%) 30.16± ± ±1.68bc 31.44±0.31b 30.14±0.35b 31.65±0.81bc 31.17±0.08b 30.74±0.35b 32.85±0.82c Lipid (%) 5.17± ± ± ± ± ± ± ± ±0.67 Fibre (%) 9.57± ± ± ± ± ± ± ± ±0.49 Crbohydrte (%) Energy (kj/g diet) CHO/L rtio P/E (mg/kj) *Different superscripts (, b, c) between feeding groups re significntly different, p< Crbohydrtes (%) = 100 (crude protein % + crude lipid % + moisture % + sh %+ fibre %) (Cstell nd Tiews, 1980) 2 Energy (kj/g diet) = (% crude protein 23.6) + (% crude lipids 39.5) + (% crbohydrtes 17.3) (Chtzifotis et l., 2010) 3 Crbohydrtes to Lipid (CHO: L) rtio = % wt. in CHO / % wt. in lipid 4 Protein to energy (P/E) (mg/kj) = crude protein (%) / energy 101

127 4.2.3 Fish immunologicl nd growth prmeters Fish blood smples were collected by cudl venous puncture t Dy 1, 14, 28 nd 56, nd the bctericidl ctivity (described s follows), neutrophil ctivity (Nitroblue tetrzolium (NBT) ssy) in blood, totl protein nd totl immunoglobulin of plsm were determined (refer to Section for detils). Vrious prmeters: specific growth rte, reltive weight gin, feed conversion rtio, protein efficiency rtio, pprent net protein utiliztion nd pprent net lipid utiliztion were monitored s prmeters to indicte fish growth performnce of different groups (Section 2.2.4). Bctericidl ctivity of plsm The bctericidl ctivity of blood plsm ws conducted bsed on the method of Abidov nd Mirismilov (1979). A. hydrophil (sme bcteri used for the infection, Section 4.2.4) ws inoculted in LB broth t 28ºC for 18 h. The cultures were centrifuged t 850 G for 15 min. The superntnt ws removed nd the bcteri pellet ws wshed by sterile 0.9% sline twice. The concentrtion of bcteri ws djusted to cfu/ml, bsed on the opticl density of suspension nd diluted to 10-4 by sterile sline. Equl volumes (80 µl) of diluted bcteril suspension nd plsm (0.9% sterile sline for growth control) were mixed nd incubted t 28ºC for 30 min. After incubtion, 50 µl of mixed solution ws poured onto the LB gr plte nd the dishes were incubted for 18 h t 28ºC. The bctericidl ctivity ws represented by the percentge decresed of colony counts in the smple compred to the control. 102

128 4.2.4 A. hydrophil injection to grss crp Aeromons hydrophil (isolted from disesed fish in the Perl River fishery Reserch Institute, Chin fishery Science Chin) ws inoculted in LB broth overnight t 28 ºC. The cultures were centrifuged t 850 G for 15 min. The superntnt ws removed nd the bcteri pellet ws wshed twice in sterile 0.9% sline. The suspension ws djusted to 1 x 10 8 cfu/ml, bsed on the opticl density of suspension with sterile sline (~0.13 bsorbnce t 625 nm). Suspended bcteri (0.1 ml) ws injected into the peritonel cvity of fish (12 fish for ech tnk) t Dy 56 of the feeding experiment. The mortlity rte ws recorded in the following 10 dys fter infection Sttisticl nlysis The results obtined t ech smpling dy (Dy 1, 14, 28, 56) were compred using one-wy ANOVA nd Duncn s multiple rnge tests (SPSS Sttistics 17.0, Chicgo, Illinosis, USA). Significnt differences between experimentl groups were expressed t the significnce level of p< Results Feeding tril with yest supplement incorported in different feeds Growth performnce of grss crp The highest vlues of SGR nd RWG were shown in A-E-5%Y nd SGR nd RWG of A-E-2.5%Y, A-E-5%Y nd C-5%Y were significntly higher thn those of C- 103

129 0%Y (p<0.05) (Tble 4.2). The lowest vlues of SGR nd RWG were observed in A- 0%Y which were significntly lower thn C-0%Y (p<0.05). The yest supplemented groups, C-5%Y, A-5%Y, A-E-2.5%Y nd A-E-5%Y lso showed significntly higher growth rte (in terms of SGR nd RWG) thn its bse feed without yest supplement i.e., C-0%Y, A-0%Y nd A-E-0%Y correspondingly. Similr observtions were lso found in FCR, with FCR of A-5%Y significntly lower thn A-0%Y, while FCR of A-E-2.5%Y nd A-E-5%Y significntly lower thn A-E-0%Y (p<0.05). However, FCR of A-0%Y nd A-2.5%Y were significntly higher thn C-0%Y (p<0.05). The PER of A-0%Y ws significntly lower thn C- 0%Y (p<0.05), while A-E-2.5%Y ws significntly higher thn C-0%Y (p<0.05). The ANPU of C-0%Y ws significntly lower thn ll other groups (p<0.05) nd ANPU of A-E-2.5%Y ws higher thn A-E-0%Y (p<0.05). A significntly lower ANLU ws only observed in A-2.5%Y (p<0.05) Crcss composition of grss crp The moisture content of A-0%Y ws significntly lower thn the control (p<0.05). The sh content of C-2.5%Y ws significntly higher thn C-0%Y (p<0.05) (Tble 4.3). Crcss protein contents of most of the groups (except C-5%Y, A-2.5%Y nd A-E-5%Y) were significntly higher thn C-0%Y (p<0.05). The yest supplemented groups C-2.5%Y nd A-E-2.5%Y showed significntly higher crcss protein contents thn C-0%Y nd A-E-0%Y (feed groups without yest) (p<0.05). A significntly higher crcss lipid content ws observed in A-0%Y thn C-0%Y, while A-2.5%Y ws significntly lower thn A-0%Y (p<0.05). 104

130 Tble 4.2 Growth performnce of grss crp fed with the control (C) nd food wste (with (A-E) nd without (A) enzyme) feeding groups with or without bker s yest (Y) C-0%Y C-2.5%Y C-5%Y A-0%Y A-2.5%Y A-5%Y A-E-0%Y A-E- A-E-5%Y 2.5%Y Initil weight (g) 14.10± ± ± ± ± ± ± ± ±0.31 Finl weight (g) 21.18±0.32bc 21.14±0.59bc 22.89±0.67cd 19.26± ±1.04b 22.40±1.20cd 20.23±1.06b 23.19±1.24d 22.87±0.97cd Feeding rte 2.01±0.04b 2.03±0.08b 2.04±0.07b 2.05±0.04b 2.17±0.12c 2.04±0.02b 2.05±0.06b 1.88± ±0.06b (%/dy) SGR 0.73±0.03bc 0.75±0.07bcd 0.89±0.08de 0.53± ±0.12b 0.85±0.08cde 0.66±0.08b 0.88±0.08de 0.90±0.04e (%/dy) RWG (%) 50.2±2.6bc 51.9±5.7bc 64.5±7.0d 35.0± ±9.4b 61.3±7.4cd 45.1±6.5b 63.6±6.8d 65.6±3.5d FCR 2.81±0.11b 2.78±0.35b 2.36±0.23b 3.94±0.77c 3.73±1.01c 2.45±0.27b 3.16±0.38bc 2.20± ±0.16 PER 1.18±0.05bcd 1.24±0.15cde 1.38±0.14de 0.83± ±0.23b 1.30±0.13de 1.03±0.12bc 1.49±0.16e 1.38±0.09de ANPU (%) 28.17± ±2.66b 36.30±3.06b 36.79±2.39b 35.97±3.91b 38.50±2.93b 37.87±2.05b 52.57±4.55c 38.96±1.21b ANLU (%) 102.6±41.4b 117.7±10.5b 117.7±4.2b 128.3±6.6b 97.6± ±1.8b 102.5±11.1b 105.4±1.3b 112.9±7.5b *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 105

131 Hemtologicl prmeters of grss crp fed with different feeds The plsm protein in ech group did not vry significntly during the experimentl period (Fig. 4.1). Significntly higher plsm protein levels were only observed in A-2.5%Y nd A-5%Y t Dy 14 nd 28, compred to A-0%Y (p<0.05), while the plsm protein level of A-0%Y ws significntly lower thn ll other groups t Dy 56 (p<0.05). There ws no significnt difference between ll feed groups nd the control in totl IgI levels t Dy 14 (p>0.05) (Fig. 4.1b). The IgI levels of A-5%Y nd A-E-5%Y were significntly higher thn A-0%Y t Dy 28 nd 56 respectively (p<0.05). There ws no significnt difference between ll feed groups nd C-0%Y (the control) in NBT ctivity t Dy 14 (p>0.05) (Fig. 4.2). The NBT ctivity of A-E- 5%Y ws significntly higher thn C-0%Y, A-E-0%Y nd A-E-2.5%Y t Dy 28 (p<0.05). The NBT ctivities of C-2.5%Y, C-5%Y, A-E-2.5%Y nd A-E-5%Y were significntly higher thn C-0%Y t dy 56 (p<0.05). It ws lso noted tht the ctivity of A-5%Y ws significntly higher thn A-0%Y (without yest) t Dy 56 (p<0.05). The serum bctericidl ctivities of A-5%Y, A-E-2.5%Y nd A-E-5%Y were significntly higher thn C-0%Y t Dy 14 (p<0.05) (Fig. 4.2b). The ctivities of A- E-2.5%Y nd A-E-5%Y were significntly higher thn A-E-0%Y (without yest) nd C-0%Y t Dy 14 nd 28 (p<0.05). The ctivities of C-5%Y nd A-5%Y were lso significntly higher thn C-0%Y t dy 28 (p<0.05). All groups except A-0%Y nd A- E-0%Y, i.e. ll yest supplemented groups showed significntly higher bctericidl ctivities thn C-0%Y (p<0.05). 106

132 Tble 4.3 Proximte composition of grss crp crcss (%, wet weight bsis) fed with the control (C) nd food wste (with (A-E) nd without (A) enzyme) feeding groups with or without bker s yest (Y) C-0%Y C-2.5%Y C-5%Y A-0%Y A-2.5%Y A-5%Y A-E-0%Y A-E-2.5%Y A-E-5%Y Moisture (%) 75.8± ±5.0b 75.2± ±2.0b 74.0±0.8b 73.0±2.1b 74.9±3.4b 74.8±1.2b 74.3±1.6b Ash (%) 2.59± ±0.62b 2.68±0.42b 3.15±0.42b 2.58± ± ±0.11b 2.77±0.49b 3.00±0.26b Protein (%) 9.48± ±2.55b 10.30±0.60b 11.53±0.60b 11.22±0.58b 11.38±1.06b 11.45±0.95b 13.72±1.06c 11.16±0.21b Lipid (%) 6.34±0.65b 6.00± ± ±0.21c 6.26±0.68b 6.99±0.20bc 5.99± ± ±0.73b *Different superscripts (, b) mong feeding groups re significntly different (p<0.05) 107

133 ) Plsm Protein (g/l) b) 12 Totl IgI (g/l) C-0%Y C-2.5%Y C-5%Y A-0%Y A-2.5%Y A-5%Y A-E-0%Y A-E-2.5%Y A-E-5%Y b b b b b b b bcbc bc bc bc b b b b b bc b b b b b b 1 14 Dy b b b b b b b b b b b b b b b b 1 14 Dy Fig. 4.1 ) Totl plsm protein (g/l) nd b) totl immunoglobulin (g/l) of grss crp feeding with the control (C) nd food wste (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05) 108

134 ) 0.6 C-0%Y C-2.5%Y C-5%Y A-0%Y A-2.5%Y A-5%Y A-E-0%Y A-E-2.5%Y A-E-5%Y c 0.5 bc bc b b b b bc c b b b b b b c b c bc c bc b c Absorbnce (540nm) b) Bctericidl ctivity (%) Dy c bc bc c bc b b b b 1 14 Dy b b c bc bc b c bc c Fig. 4.2 ) Nitroblue tetrzolium (NBT) ctivity nd b) plsm bctericidl ctivity (%) of grss crp feeding with the control (C) nd food wste (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05). 109

135 Disese resistnce to Aeromons hydrophil of grss crp The disesed grss crps showed lethrgy, expnded bdomen with red blotches nd redden nd swollen nus, with yellow mucus relesed from the nus when slight pressure ws pplied to the bdomen. They lost their ppetite fter infection. These signs mtched with enteritis infection observed in grss crp (NACA, 1989; Zheng et l., 2012). Mortlities of yest supplemented groups, C-5%Y, A-2.5%Y, A-E-2.5%Y nd A-E- 5%Y were significntly lower thn C-0%Y fter 10 dys of injections (p<0.05). The highest mortlity ws 69.4% noted in A-0%Y, while lowest ws 33.3% in A-E-2.5%Y (Fig. 4.3). 4.4 Discussion Growth nd feed utiliztions of grss crp fed with bker s yest supplemented feed In generl, the yest supplemented feed groups showed better growth rtes thn the groups without yest, in terms of FCR, PER, SGR nd RWG. The highest growth rte ws observed in the corresponding groups with 5% S. cerevisie (bker s yest) with different types of feeds, i.e. FW A with nd without bromelin nd ppin nd the control feed. This indicted the yest could fcilitte fish growth in different feeds. Lr-Flores et l. (2003) showed higher protein digestibility of feed in Nile tilpi by dding S. cerevisie, nd similr results were lso observed in se bss nd tilpi (Tovr et l., 2002; Ozório et l., 2012). Better feed utiliztion hence lower FCR suggested tht the feed quntity nd cost could be substntilly reduced. 110

136 C-0%Y A-0%Y Mortlity (%) b bc cd b b d cd 10 0 C-2.5%Y C-5%Y A-2.5%Y A-5%Y A-E-0%Y A-E-2.5%Y A-E-5%Y Fig. 4.3 Mortlity of grss crp ginst Aeromons hydrophil injection fter feeding with the control (C) nd food wstes (with (A-E) nd without (A) enzyme) feeds groups with or without bker s yest (Y), different superscripts (, b) within one smpling dy re significntly different (p<0.05). 111

137 Bker s yest itself is rich in protein nd is good feed lterntive for Cyprinus crpio fingerlings, replcing 30% of fish mel without impiring fish growth (Korkmz nd Ckirogullri, 2011). The bker s yest minly comprised of mnnn, glucns nd smll mounts of chitin nd nucleic cid (Anderson et l., 1995; Nguyen et l., 1998), which would be beneficil to fish (Li nd Gtlin, 2003). The dministrtion of β-glucn lso stimulted the immunity nd enhnced the survivl of L. rohit fingerlings, prt from promoting growth (Misr et l., 2006). Protein utiliztion s well s fish growth ws lso promoted by the yest supplements in the present study. All yest supplemented groups showed significntly higher ANPU thn the control feed without yest. The yest my lso help the estblishment of beneficil intestinl microbil community, which could lso enhnce enzyme ctivities for digestion (Tovr et l., 2002; Wché et l., 2006). For exmple, the mylse ctivity in tilpi ws incresed fter feeding with S. cerevisie (Ess et l., 2010). The gstrointestinl bcteri re lso involved in the brekdown of nutrients nd provided physiologiclly ctive nutrients to hosts (Birgi et l., 2004; Rmirez nd Dixon, 2003). As consequence of better nutrient digestion nd bsorption, higher protein ws retined in fish body fed with 2.5% or 5% yest supplemented feeds in the current study. The growth rte nd protein utiliztion were lso higher in both enzyme nd yest (2.5%) supplemented group thn the groups without enzymes. This suggested tht the yest could further enhnce the nutrient utiliztion in feeds, in ddition to the initil improvement fter dding bromelin nd ppin. 112

138 Lower lipid retentions in grss crp crcss could be due to more lipids being converted to energy nd hence protein ws reserved (Meyer nd Frclossi, 2004). Significnt reductions of lipid retentions were found in enzyme nd yest supplemented groups. On the contrry, Quentel et l. (2005) showed tht S. cerevisie incresed muscle lipids in rinbow trout muscle. The contrdictory results my be due to the ddition of ppin nd bromelin which reduced the lipid retention in grss crp (Chpter 3) Immunity of grss crp fed with bker s yest supplemented feed The immunity of grss crp could be stimulted by implementing yest in feeds, reflected by the higher oxidtive rdicl production ctivity in blood (NBT ctivity) nd bctericidl ctivity of plsm. The NBT ssy quntified the production of intrcellulr superoxide rdicls by leukocytes (Shu et l., 2007b; Ardó et l., 2008). In generl, higher NBT ctivities were found in the grss crp fed with yest supplemented feeds thn feeds without yest, e.g. C-2.5%Y nd C-5%Y showed significntly higher ctivities thn C-0%Y t Dy 56 (p<0.05) (Fig. 4.2). The NBT ctivity reched highest level t Dy 28 in A-E-5%Y which ws significntly higher thn ll other groups. Li nd Gtlin (2003) lso obtined similr results with 2% of brewer s yest improved the extrcellulr superoxide nion nd blood neutrophil oxidtive rdicl productions of hed kidney mcrophges in hybrid striped bss (M. chrysops M. sxtilis). The rective oxygen rdicls in fish immune system re hrmful for bcteril pthogens nd re importnt for killing bcteri in blood (Hrdie et l., 1996; Itou et l., 1996) 113

139 A more evident response on the immuno-stimulting effect of yest ws lso observed in the plsm bctericidl ctivity. All yest supplemented groups showed remrkble increses in bctericidl ctivities, compred to the corresponding types of feeds without yest (C-0%Y, A-0%Y nd A-E-0%Y). Compred to whole yest S. cerevisie, β-glucn (extrcted from S. cerevisie) nd lminrn (glucn from brown lge), β-glucn is more effective to stimulte serum bctericidl ctivity nd lysozyme ctivity in Nile tilpi (El-Boshy et l., 2010). Similr stimultions on lysozyme, serum bctericidl nd complement ctivities by β-glucn were found in Lbeo rohit (Misr et l., 2006). As result, the immune-stimulting effect of S. cerevisie should be minly due to the presence of β-glucn. On the other hnd, the yest did not ffect the totl plsm protein nd IgI levels to gret extent. Higher IgI levels in yest supplemented groups nd lower levels in nonyest supplemented groups were generlly observed, though not significntly different, when compred to the control feed without yest. Moreover, depressed totl protein level ws found in FW A without ny yest nd enzymes, this ws in line with the previous study (Chpter 3). This could be due to the poor utiliztion of feed, s nutritionl deficiencies my cuse lower plsm protein in fish (Siwicki et l., 1994) nd the level of immunoglobulin my lso be lowered Fish disese resistnce to A. hydrophil The disese resistnce to A. hydrophil of grss crp ws lso gretly enhnced 114

140 when supplemented with 2.5% yest in enzyme supplemented FW A. Similr result ws obtined in Nile tilpi ginst A. hydrophil with 0.1% w/w of live bker s yest (Abdel-Twwb et l., 2008). The mortlity of Nile tilpi ws closely relted to the doses dded in feed nd it ws significntly reduced s the yest supplementtion incresed from 0.1 to 0.6% (Osmn et l., 2010). The well-built resistnce to A. hydrophil in grss crp my be due to the enhnced NBT ctivity nd bctericidl ctivity. The present study bout grss crp fed with Trditionl Chinese Medicine (TCM) lso showed higher resistnce to A. hydrophil ccompnied with higher bctericidl ctivity (Chpter 6). In ddition, the modifictions of bcteri flor in guts by yest could lso ffect the disese resistnce to pthogens nd fish immunity. The dietry yest products contining S. cerevisie could lter the intestinl microbil community into more beneficil one, nd provide stronger protection for gibel crp (Crssius urtus) ginst Grm-negtive pthogens such s A. hydrophil (He et l., 2011) It is envisged tht combintion of bromelin, ppin nd bker s yest my further promote fish growth nd immunity. More prominent resistnce to A. hydrophil ws observed on the group combining bromelin (0.5%), ppin (0.5%) nd lso yest (2.5%). Only 33.3% mortlity ws recorded, compred with nerly 70% in the food wste groups without ny supplements. Positive results relted to growth nd bctericidl ctivity were pprent fter feeding fish with these supplements. The enzymes my further fcilitte the immuno-stimulting effects of yest, but no study hs been conducted on the 115

141 combined effects of enzymes nd bker s yest. Bker s yest is nturl substnce nd hs no dverse effects on nimls nd the environment, it is lso by-product redily vilble from industries such s brewery with low-cost (Tewry nd Ptr, 2011). The ppliction of bker s yest could be beneficil to both quculture industry nd the environment, serving s n immunostimulnt nd s n lterntive to ntibiotics. The present study indicted tht the whole yest feed supplement yielded positive results on fish growth nd immunity. The whole yest is more economicl in terms of the net profit of fish frmers s it promotes higher growth rtes nd better feed conversion. 4.5 Conclusion The hypothesis ws ccepted s the bker s yest enhnced the growth nd immunity of grss crp through better feed utiliztion. The yest supplemented feed groups generlly showed better growth rtes thn feeds without yest, in terms of FCR, PER, SGR nd RGW. The optiml dose ws 2.5% yest (S. cerevisie) with bromelin nd ppin (FW A) dded. Protein utiliztion ws enhnced by the yest supplements. Grss crp immunity ws stimulted by implementing yest in feeds, reflected by the higher NBT ctivity in blood nd bctericidl ctivity of plsm. This indicted tht bker s yest could fcilitte fish growth s supplements in different feeds. It ws concluded tht there my be dditive effects on growth nd immunity by mixing enzymes nd yest. This inexpensive industril product could reduce feed quntity nd enhnce 116

142 fish immunity in quculture, which subsequently reduces feed cost ccompnied with higher yields. 117

143 Chpter 5 The ntimicrobil ctivity of Trditionl Chinese Medicines (TCMs), potentil drug lterntive on deling with fish pthogens 5.1 Introduction Aquculture provides importnt food sources in the worldwide; its importnce is incresing s the cpture fishery production hs been sturted during the pst 20 yers (FAO, 2012). The quculture industry hs expnded rpidly in the pst few decdes, but the high stocking density in fish frms esily triggered off the disese outbrek such s prsitic nd bcteril diseses. Aeromons hydrophil, Streptococcus spp. nd Vibrio spp. re the common fish pthogens nd could cuse relentless economicl loss in fish frms. In fct, bcteril infection is one of the mjor fctors cusing mortlity in quculture (Grisez nd Ollevier, 1995). In generl, drugs nd ntibiotics such s Mlchite green, formlin, terrmycin, potssium permngnte, sulfonmides nd tetrcyclines re commonly used for controlling diseses (Rukyni, 1994; Rwn et l., 2009). The ppliction of drugs nd ntibiotics my not be beneficil nd sustinble pproches deling with diseses, especilly ntibiotics. The effectiveness of ntibiotics decresed s result of improper use nd buse. The potentil hzrd of ntibiotics to public helth hs lso been rised since the development nd spred of drug resistnt bcteri were found. The frequent occurrences of ntibiotic resistnt bcteri hve been 118

144 observed in recent yers nd re probbly due to the buse of veterinry ntibiotics nd sub-therpeutic doses used in quculture (Smith et l., 2002). As consequence, more nd more ntibiotics resistnt fish pthogens hve been detected round quculture sites (Furushit et l., 2005; Cbello, 2006; Sørum, 2006). Terrestril veterinry pthogens nd even humn pthogens my gin ntibiotic resistnce genes through horizontl gene trnsmissions from bcteri in the quculture environment (Angulo nd Griffin, 2000; Heuer et l., 2009). The presence of ntibiotics residues in quculture products nd the environment is lso ssocited with humn helth risks (Cbello, 2006). The drugs nd bcteri in the contminted products my be trnsferred with resistnt genes developed in the humn intestine (Slyers et l., 2004) leding to bcteril infections. Senderovich et l. (2010) reveled tht fish digestive systems re reservoirs of Vibrio cholere nd therefore improper mngement of quculture diseses nd drug pplictions would endnger humn helth. The Mrine Product Export Development Authority of Indi (MPEDA) hs recommended the htchery opertors nd fish frmers not to use vrious ntibiotics (Snndkumr, 2002), the Europen Union hs lso bnned the use of ntibiotics s feed dditives in food-producing niml unless uthorized fter Jnury The Food nd Drug Administrtion (FDA) lso restricted the use of vrious quculture drugs, e.g. oxytetrcycline nd chlormphenicol (FDA, 2011). World Helth Orgniztion (WHO), Food nd Agriculture Orgniztion of the United Ntions (FAO) nd the World Orgnistion for Animl Helth (OIE) jointly issued the guidelines for the responsible 119

145 use of ntimicrobil gents for veterinry uses (FAO/OIE/WHO, 2006). Unfortuntely, the uses of ntibiotics e.g. quinolones in fish frming in developing countries such s Chin nd Chile re still common (Cbello, 2004; Jcoby, 2005). Therefore, there is n urgent need to seek lterntives for ntibiotics to del with bcteril infections in quculture systems, otherwise more resistnt strin bcteri would be spred in the environment. Trditionl Chinese Medicines (TCMs) is one of the novel lterntives to ntibiotics, which cn be served s ntimicrobil gent nd immuno-stimulnt. TCMs hve been trditionlly used in Chin quculture industry but only being explored scientificlly in recent yers. Herbl medicines showed distinctive ntimicrobil ctivities on fish pthogens (Bkkli et l., 2008; Vn Vuuren nd Viljoen, 2008), which hve been suggested s n lterntive to ntibiotics (Glin et l., 2009). Using herbl medicines for treting humn pthogens with multi-drug resistnce (MDR) hs become nother populr topic, ntimicrobil ctivity on vrious pthogenic orgnisms, such s Stphylococcus ureus, Pseudomons eruginos, E. coli, Bcillus subtilis, Slmonell typhimurium nd Enterococcus feclis hve been studied extensively (Singh et l., 2010; Chn et l., 2011; Ibrhim et l., 2011). However, there is lck of informtion on drug resistnce development in TCMs on whether drug resistnt bcteri would be developed when exposed to TCM. Therefore, there is need to study the longer term effects of using TCMs in quculture. TCM hve been used for humn therpeutic purposes with thousnds yer of history, 120

146 detils of usge nd formultions of TCM hve been recorded in substntil ntionl litertures, e.g. Shen Nong s Mteri Medic (The Complier Group, 2009), Compendium of Mteri Medic (Li, 2009) nd Dictionry of Chinese Mteri Medic (Jingsu New Medicl College, 1977). Drug resistnce of bcteri to TCMs nd nturl herbs is seldomly recorded in literture, even for its long history in pplictions. To the best of our knowledge, there is limited informtion focusing on the development of drug resistnce in bcteri to TCM. The present study will focus on the ntimicrobil ctivities of selected TCMs nd drug resistnce development in fish pthogens to these TCMs. Seventeen types of TCMs used for treting disese in humn digestion systems were selected for the investigtions on their ntimicrobil ctivities relted to some common fish pthogens. Three common fish pthogens, V. cholere, A. hydrophil nd Lctococcus grviee were selected for this study. V. cholere is the custive gent of choler in humn nd vibrosis in fish. It is commonly found in nturl qutic environments nd the intestine of freshwter fish e.g. grss crp, grey mullet, common crp nd mrine fish e.g. soldierfish, nd fish serving s reservoirs of these pthogens (Senderovich et l., 2010). A. hydrophil could cuse the infections on skins, blood nd intestine in both humn nd fish (Cstro et l., 2008). L. grviee is grm-positive pthogen cusing lctococcosis in both mrine nd freshwter species such s yellowtil, rinbow trout nd grey mullet (Akhlghi nd Keshvrzi, 2002; Chen et l., 2002; Vendrell et l., 2006). It is lso infectious on humn urinry trct, blood nd skin (Elliot et l., 1991). 121

147 It hs been recognized tht totl contents of single herb could show more prominent effect thn single ctive ingredient or constituent in herb (Nhrstedt nd Butterweck, 2010). It ws hypothesized tht the TCM possessed good ntimicrobil ctivity which would defer the development of drug resistnce in fish pthogens, nd is therefore n idel lterntive to ntibiotics. The mjor objectives of this experiment were to 1) screen TCMs to identify which one(s) possess strong ntimicrobil ctivity; 2) investigte the ntimicrobil ctivities of TCM extrcts in different solvents; 3) revel the combined effects of selected queous TCM extrcts; nd 4) investigte the development of drug resistnt pthogens exposed to TCM extrcts, compred with tht of ntibiotics. 5.2 Methods nd Mterils Antimicrobil ctivity of boiled queous extrcts of 17 TCMs Seventeen Trditionl Chinese Medicines (TCMs) Rhizom coptidis, Rdix strgli, Herb ndrogrphis, Herb houttuynie, Rdix scutellrie, Rdicis ngelice sinensis, Astemisi cpillries, Cnidium monnieri, Rdix istidis, Folium istidis, Rdix glycyrrhize, Rhizom rhei, Cortex phellodendri, Semen sinpis, Fructus forsythie, Fructus grdenie jsminoidis nd Rdix sophore flvescentis were obtined from nd identified by the School of Chinese Medicine, Hong Kong Bptist University were tested for their ntimicrobil ctivity of TCM extrcts on vrious bcteri s stted below. The herbs were dried t 40ºC for 24 h nd then pulverized to powder using mechnicl blender. 122

148 In the present experiment, 3 species of grm-positive (Lctococcus grviee, Stphylococcus ureus nd Enterococcus feclis) nd 4 species of grm-negtive bcteri (Escherichi coli, Aeromons hydrophil, Vibrio cholere nd Serrti mrcescens) were involved. The minimum inhibitory concentrtions (MICs) of TCM extrcts on 7 species of bcteri, ATCC E. coli, ATCC S. ureus, ATCC E. feclis, L. grviee, V. choler, ATCC S. mrcescens nd A. hydrophil were determined by micro-dilution method (NCCLS, 2001). L. grviee nd V. cholere were isolted from liver of disesed grey mullet, from the fish frm in Yuen Long, Hong Kong nd A. hydrophil from the intestine of disesed grss crp in Institute of Hydrobiology, Jinn University, Gungzhou. The A. hydrophil nd L. grviee ws identified by API 20NE kit nd API 32 Strep kit, the V. cholere ws identified by 16SrDNA nlysis. Three lbortory strins of bcteri, ATCC S. ureus, ATCC E. feclisnd ATCC S. mrcescens re lso common pthogens for qutic diseses, while ATCC E. coli ws used s the control for the bcteri testings. The bcteri were cultured from -80 ºC bcteri stock in Mueller-Hinton Broth t 37ºC for 18h (except S. mrcescens nd A. hydrophil t 28 ºC). The overnight culture ws spred on Mueller-Hinton Agr for severl genertions nd then single colony ws re-trnsferred from gr plte into Mueller-Hinton broth nd cultured t optimum temperture (37 or 28 ºC) for 18 h. The cultures were centrifuged t 850G for 15 min nd the superntnt removed. The bcteri pellets were wshed with sterile sline with the turbidity of suspension djusted with sline to ~0.13 bsorbnce t 625nm 123

149 spectrophotometriclly. The bcteri were diluted by MHB nd the finl concentrtion of bcteri in the well ws bout 5 x 10 5 cfu/ml. The powdered TCM ws extrcted by boiling in deionized wter. The powder ws then soked for 30 min in deionized wter (1:10 w/v), nd boiled for 30 min. The extrct ws removed nd nother portion of deionized wter ws dded nd boiled for nother 30 min. The extrcts were pooled then filtered with Whtmn No. 1 filter pper by suction filtrtion nd were dried t 50 C. The dried extrcts were then kept t desicctors for 12 h before recording the weight. The extrcts yield (%) ws clculted s follows: (Mss of the dried extrct / mss of the ground plnt smple) x 100% The dried extrcts were then redissolved in 50% ethnol to finl concentrtion (328 mg/ml) nd were stored t -20 ºC. Two ntibiotics chlormphenicol ( to 64 μg/ml) nd streptomycin (0.125 to 256 μg/ml) were used s positive controls. The tested concentrtions of TCM extrcts were fixed t 0.04 to mg/ml, s MIC vlues within 1 to 8 mg/ml were generlly considered s possessing ntimicrobil ctivity (Fbry et l., 1998; Gibbons, 2004; Rios nd Recio, 2005). The powder of chlormphenicol (Sigm, Aldrich) nd streptomycin (Sigm, Aldrich) ws weighed nd dissolved in ethnol nd sterile deionized wter t the pproprite concentrtion (10 fold of highest tested concentrtion) respectively, nd kept t -20ºC s stock solution. 100 µl of ech TCM extrct (81.92 mg/ml) ws serilly diluted two-fold with 124

150 sterile deionized wter in the well. 100 µl of bcteri culture (~5 x 10 5 cfu/ml) in MHB ws dded. 2, 3, 5-Triphenyltetrzolium chloride ws used for indicting the bcteri growth (Lee et l., 2007), pink solution indicted the growth of bcteri nd the well t the lowest concentrtion with no sign of growth ws recorded s minimum inhibition concentrtion (MIC). 10% Ethnol (concentrtion of ethnol in diluted TCM extrcts) nd sterile deionsied were included s solvent control to test whether ny inhibitory effect on the growth of tested bcteri. Growth control without TCM extrcts or ntibiotics ws included to confirm tht the growth of bcteri. The negtive controls with TCM extrcts or ntibiotics with sterile broth were included to confirm the prepred solutions were not contminted Antimicrobil ctivity of selected queous nd orgnic TCMs extrcts TCMs were extrcted by soniction bth in vrious solvents: hexne, dichloromethne (DCM), 90% ethnol (EtOH) nd deionized wter (20ml/g) using the procedure described by Ncube et l., (2012) with modifictions. The extrction ws repeted 3 times nd ech soncition lsted for 45 min. The crude extrcts were pooled nd then filtered with Whtmn No. 1 filter pper by suction filtrtion. The orgnic extrcts were concentrted by rotry evportor below 50ºC nd then dried t room temperture (~23ºC) under strem of ir until compete dryness. Wter extrcts were freeze-dried nd kept in irtight continers. The TCM ws lso extrcted by boiling in deionized wter for compring ntimicrobil ctivity of wter extrcts. The extrction 125

151 procedures nd the clcultion of extrct yield of ech solvent (%) were the sme s stted in Section The dried extrcts were then redissolved in 50% ethnol to finl concentrtion (328 mg/ml) nd stored t -20ºC. The minimum inhibitory concentrtions (MICs) of ech extrction on 7 species of bcteri were determined by microdilution method s mentioned in Section The rtio of MICs between non-boiled nd boiled wter extrcts (results from this section nd Section 5.2.1) were clculted Checkerbord method for the combined effect of C. phellodendri, R. scutellri, R. coptidis nd F. forsythie The four herbs (C. phellodendri, R. scutellri, R. coptidis nd F. forsythie)tht showed strongest bcteril inhibition in Section were selected for investigting the combined effects of the boiled queous extrcts t equl rtio. MIC vlues were determined for ech of these combintions to estblish ny interction effects following Section The frctionl inhibitory concentrtion (FIC) indices were clculted to evlute the interction of two extrcts, but not the effects of mixtures with more thn two extrcts. The concentrtion of ech tested extrcts in combintion is expressed s frction of the concentrtion tht produced the sme effect when used independently (Berenbum, 1978). The FIC ws clculted s the MIC of the combintion divided by the MIC of ech individul component extrct. The FIC index ws the sum of ech extrct FIC in combintion. Synergistic ( 0.5), dditive (> ), indifferent (1 4.0) or ntgonistic ( 4.0) were interpreted bsed on the vlue of FIC index (Schelz et l., 126

152 2006) Development of drug resistnt fish pthogens exposed to selected queous TCM extrcts The queous extrct ws used to investigte the development of resistnt fish pthogens s it is the trditionl prctice nd the most common form prepred in homesteds. Three fish pthogens, L. grviee, V. cholere nd A. hydrophil were cultured in three of the most inhibiting TCM extrcts (i.e. with lowest MIC vlues) respectively t sub MIC (hlf of MIC), with E. coli included s control (Section 5.2.1). The boiled queous extrcts used were R. coptidis, R. Scutellrie, F. forsythie nd C. Phellodendri. The chnges of MIC in 21 consecutive dys of experiment were investigted by the modified method of Clrk et l. (2011). The development of resistnt fish pthogens on two ntibiotics, Chlormphenicol nd Streptomycin were lso investigted. In brief, the concentrtions of TCM extrcts pplied on the bcteri species were bsed on the results obtined in Section The bcteri were cultured in 200 ml of Mueller-Hinton broth with hlf of the MICs (Tble 5.1) s the initil dose. The MIC of TCM extrcts on the bcteri ws tested using the sme micro dilution method (Section 5.2.1) t Pssge 1, 4, 7, 14 nd 21. The subculture doses of ntibiotics or TCM extrcts were djusted until significnt increse of MIC (>4-fold) ws observed, compred to the initil or previous subculture doses. The MIC of TCM extrcts of ech bcteri species ws tested gin 127

153 fter 7 further pssges (Pssge 28), without ntibiotics or TCM extrcts. 5.3 Results Antimicrobil ctivity of boiled queous extrcts of 17 TCMs The extrct yields of TCMs in boiled extrcts rnged from 9.52 (S. sinpis) to 50.26% (R. scutellri). Fourteen out of seventeen wter boiled TCM extrcts showed inhibition on 7 species of bcteri (Tble 5.1), no MICs were determined in the tested concentrtions ( mg/ml) for the 3 speices: S. sinpis, F. istidis nd R. istidis. In this study, MICs vlues equl or below 1.28 mg/ml were considered s possessing strong ntimicrobil ctivity (highlighted in bolded in Tble 5.1) nd MICs vlues equl or below 1.28 mg/ml. C. phellodendri nd R. coptidis demonstrted the strongest ntimicrobil ctivities, with brod rnge of inhibition on both grm-positive nd - negtive bcteri. Both extrcts lso showed strong ntimicrobil ctivity on the two fish pthogens, L. grviee nd V. cholree. S. ureus ws the most susceptible pthogen to TCM extrcts, inhibited (MICs mg/ml) by 64.7%, 11 out of the 17 tested TCMs, while S. mrcescens ws the most tolernt bcteri species (0%), the only recorded MIC ws mg/ml of F. forsythie. Bsed on the results, four TCMs, C. phellodendri, R. scutellri, R. coptidis nd F. forsythie possessed strong ntimicrobil ctivities nd were selected for further studies on ntimicrobil ctivity. 128

154 Tble 5.1 Antimicrobil ctivity screening (mg/ml) using boiled queous extrcts of 17 Trditionl Chinese Medicines (TCMs) Herbs Extrct Bcteri strins yield (%) Vc Lg Ah S Ef Sm Ec R. scutellri * * F. forsythie F. grdenie jsminoidis * * * C. phellodendri * * R. coptidis * 1.28 H. ndrogrphis * * H. houttuynie * * 5.12 * * * F. lonicere jponice * * * * * R. strgli * * * * * * F. cnidii * * 5.12 * * * R. glycyrrhize * 2.56 * * * R. ngelice sinensis * * * * * S. sinpis 9.52 * * * * * * * R. sophore flvescentis * * 5.12 * * * F. istidis * * * * * * * H. stemisi cpillris * * * * * * R. istidis * * * * * * * Chlormphenicol (µg/ml) / Streptomycin (µg/ml) / Ah= Aeromons hydrophil, Vc= Vibrio cholere, Lg= Lctococcus grviee, Ec= ATCC Escherichi coli, S= ATCC Stphylococcus ureus, Ef= ATCC Enterococcus feclis, Sm= ATCC Serrti mrcescens * No minimum inhibition concentrtion (MIC) ws found in tested concentrtions ( mg/ml) 129

155 5.3.2 Antimicrobil ctivity of queous (boiled nd non-boiled) nd orgnic extrcts of 4 selected TCMs Higher extrct yields in queous extrcts ( %) thn orgnic solvents ( %) were generlly observed, especilly R. coptidis nd R. scutellri (Tble 5.2). The yields in non-boiled extrcts were lso higher thn boiled queous extrcts, except C. phellodendri which possessed more non-polr content nd strongly inhibited S. mrcescens (0.16 mg/ml). The queous, ethnol nd DCM extrcts of R. coptidis nd R. scutellri possessed similr ntimicrobil bilities to the three field isolted pthogens: V. cholere, L. grviee nd A. hydrophil, e.g. the queous, ethnol nd DCM extrcts of R. coptidis on L. grviee were 0.08 to 0.16 mg/ml. However, these ethnol nd DCM extrcts generlly possessed higher ntimicrobil ctivities to the other four bcteri species: S. mrcescens, E. feclis, S. ureus nd E. coli, compred to queous extrct. The DCM nd ethnol extrcts of R. coptidis on L. grviee were 0.16 nd 0.32 mg/ml respectively, compred to 2.56 mg/ml of queous extrcts. Among the four selected extrcts, F. forsythie nd R. coptidis possessed the wekest nd strongest ntimicrobil bility, respectively. 130

156 Tble 5.2 Antimicrobil ctivities (mg/ml) of different solvent extrcts of Rdix scutellri, Rhizom coptidis, Cortex phellodendri nd Fructus forsythie Herbs Solvent Yield MIC^ on different bcteri strins # (%) Vc Lg Ah S Ef Sm Ec R. scutellri H D E Wn Wb * * * F. forsythie H D E Wn Wb C. phellodendri H D E Wn Wb * * R. coptidis H D E Wn Wb * 2.56 Chlormphenicol Streptomycin Ah= Aeromons hydrophil, Vc= Vibrio cholere, Lg= Lctococcus grviee, Ec= ATCC Escherichi coli, S= ATCC Stphylococcus ureus, Ef= ATCC Enterococcus feclis, Sm= ATCC Serrti mrcescens ^: MIC unit of TCM extrcts (mg/ml) nd ntibiotics (µg/ml) #: Wn= non boiled wter extrct, Wb= boiled wter extrcts, D=dichloromethne, E=ethnol, H=hexne Solvent polrity index: Wter 9.0, ethnol 5.2, dichloromethne 3.1, hexne

157 In comprison, ntimicrobil bilities vried in non-boiled nd boiled queous extrcts mong 7 bcteri, only 3 out of 21 extrcts showed lower rtio (rtio of MIC between Non-boiled nd boiled queous extrcts below 0.5) nd 2 extrcts showed higher rtio (>2) (Tble 5.3). Lower rtios were observed in R. scutellri on V. cholere nd L. grviee nd R. coptidis on E. feclis while higher rtios were found in R. coptidis on V. cholere nd A. hydrophil Antimicrobil ctivities of boiled queous mixtures of C. phellodendri, R. scutellri, R. coptidis nd F. forsythie The FIC indices were generlly lger thn 1, indicted the mixed TCM extrcts possessed weker ntimicrobil ctivities thn tht involved only single herbs, nd therefore ntgonistic effects on ntimicrobil ctivities were found in the combintions of these four TCMs (Tble 5.4). For exmple, the MIC of R. scutellri nd R. coptidis mixture ws 0.64 mg/ml (FIC index=2.25) to A. hydrophil, compred to 2.56 (R. scutellri) nd 0.32 (R. coptidis) mg/ml. Additive interctions (FIC index > ) were found in combintions of R. scutellri nd F. forsythie on E. coli nd A. hydrophil (FIC index =0.63) nd R. scutellri nd C. phellodendri on A. hydrophil (FIC index =0.56). Combintion of C. phellodendri nd F. forsythie showed synergistic effect (FIC index 0.5) on the inhibition on A. hydrophil (FIC index =0.38), further 132

158 enhncement on inhibition observed when dded with R. scutellri (FIC index =0.34). Vrious ntgonistic effects on ntimicrobil ctivities (FIC index 4.0) were observed in the combintions which contined R. coptidis Development of drug resistnt fish pthogens exposed to selected TCM extrcts In generl, development of drug resistnt bcteri to TCM extrcts ws not found, with the exception of L. grviee exposed to C. phellodendri nd E. coli exposed to R. coptidis, with 4 folds nd 8-16 folds increses of MICs were found fter 21 pssges respectively, but ll of the MICs fell bck to originl vlues fter 7 extrcts free pssges (Tble 5.5). Remrkble increses of MICs were observed in bcteri species treted with streptomycin, rnged from folds, the increses were generlly obvious fter 14 pssges. The MIC of E. coli mintined t 128 µg/ml to streptomycin fter 7 pssges without ntibiotics. Less drug resistnce ws found in bcteri species exposed to chlormphenicol, only 2-8 folds increses were observed nd MICs of ech bcteri species returned ner to the initil vlues fter 7 pssges without chlormphenicol. 133

159 Tble 5.3 Rtio of MIC between non-boiled nd boiled queous extrcts Rtio of MIC on different bcteri strins* Herbs Vc Lg Ah S Ef Sm Ec R. scutellri / / / F. forsythie C. phellodendri 2 1 / / / R. coptidis / 1 Ah= Aeromons hydrophil, Vc= Vibrio cholere, Lg= Lctococcus grviee, Ec= ATCC Escherichi coli, S= ATCC Stphylococcus ureus, Ef= ATCC Enterococcus feclis, Sm= ATCC Serrti mrcescens *: The vlues below 1 indicted lower MIC for non-boiled wter extrcts, the vlues below 0.5 were highlighted in bolded. /: No numericl vlue ws shown if MIC greter mg/ml. 134

160 Tble 5.4 The minimum inhibition concentrtion (MICs) frctionl inhibitory concentrtion (FIC) indices of boiled queous mixtures of four Trditionl Chinese Medicines (TCMs) MIC (mg/ml) nd FIC indices of bcteri TCM combintions A. hydrophil L. grviee V. cholere E. coli Rc Rs 0.64 (2.25) 1.28 (16.13) 1.28 (16.25) 2.56 (2.06) Rc Cp 0.64 (2.03) 0.32 (4.50) 0.32 (4.25) 1.28 (2.00) Rc Ff 0.64 (2.06) 0.32 (4.03) 0.64 (8.13) 1.28 (1.13) Rs Cp 1.28 (0.56) 1.28 (2.13) 1.28 (1.25) 5.12 (4.13) Rs Ff 1.28 (0.63) 5.12 (1.00) 1.28 (0.50) 5.12 (0.63) Cp Ff 2.56 (0.38) 2.56 (4.25) Rc Rs Ff Rc Rs Cp Rc Cp Ff Rs Cp Ff Rc Rs Cp Ff Rs: Rdix scutellri, Rc: Rhizom coptidis, Cp: Cortex phellodendri, Ff: Fructus forsythie : Synergistic interctions ( 0.5) re printed in bold b: Additive interctions(> ) re printed in itlics --. :Not tested s the MIC vlue of mixture >40.96 mg/ml, no FIC index ws determined

161 Tble 5.5 Development of drug resistnt fish pthogens fter seril pssges of Rdix scutellri, Rhizom coptidis, Cortex phellodendri nd Fructus forsythie Herbs No. of pssge MIC on different bcteri strins Vc Lg Ah Ec R. scutellri Control b (1) d (1) (4) (1) 28 c 5.12 (1) (1) 2.56 (1) (1) F. forsythie Control (2) (2) (1) (1) C. phellodendri Control (1) 2.56 (4) (1) 0.64 (1) R. coptidis Control (1) 0.16 (1-2) 0.64 (2-4) (16) (1) 0.16 (1-2) 0.16 (1) 0.64 (1) Chlormphenicol Control (4) 16 (4-8) 4 (2-4) 64 (8) (1) 8 (2-4) 2 (2) 16 (4) Streptomycin Control (64-128) 512 (32) 64 (16-32) 128 (32-64) (4-8) 64 (4) 16 (4-8) 128 (31-64) MIC unit for TCM extrcts (mg/ml) nd ntibiotics (µg/ml) (-- Not Tested) b Control: pssges without ntibiotics or TCM extrcts c Pssges from No were without ntibiotics or TCM extrcts d Fold of increse of MIC t 21 nd 28 pssges compred to control re shown in brcket 136

162 5.4 Discussion Antimicrobil ctivities of boiled queous extrcts of 17 TCMs The present study results showed strong inhibiting effects on the growth of some fish pthogens which demonstrted potentil uses of TCMs s ntimicrobil gent for pthogens in quculture. MIC vlues below 8 mg/ml suggested tht the TCM extrcts possessed ntimicrobil ctivity (Fbry et l., 1998) nd MIC vlues below 1 mg/ml re considered s possessing remrkble ntimicrobil ctivity (Gibbons, 2004; Rios nd Recio, 2005). In this study, due to the difference in tested concentrtions, MICs equl to or below mg/ml were regrded s possessing ntimicrobil ctivities, nd equl to or below 1.28 mg/ml s possessing strong ctivities. Only 2 TCM extrcts (R. coptidis nd C. phellodendri) tested in this study showed strong inhibitions on the tested bcteri, R. coptidis exhibited strong ntimicrobil ctivity on two common fish pthogens V. cholere nd L. grviee (0.08 mg/ml) nd lso effective (MIC: mg/ml) on other bcteri e.g. A. hydrophil, S. ureus nd E. coli. Berberine hydrochloride is the ctive component of R. coptidis nd C. phellodendri, which showed strong ntimicrobil ctivity to vrious fish pthogens: A.hydrophil, Pseudomons fluorescens, Vibrio hrveyi, Edwrdsiell ictluri nd Streptococcus dysglctie, with MIC rnged from mg/ml (Zhng et l., 2010). Li et l. (2006) lso found similr results, with R. coptidis, R. scutellri nd C. phellodendri exhibited stronger ntimicrobil ctivity mong 10 tested TCMs. R. coptidis extrcts lso inhibited S. ureus (Yu et l., 2005) nd A. hydrophil (Zhng nd Yng 137

163 2006). However, Rttnchikunsopon nd Phumkhchorn (2009) found tht Androgrphis pnicult possessed strong ntimicrobil ctivity on Streptococcus glctie (31.25 µg/ml), while the present result did not showed wek ntimicrobil ctivities on H. ndrogrphis (lef of Androgrphitis) (MIC mg/ml). A study showed tht Androgrphitis extrcts hd n inhibitory effect on Bcillus cereus, E. coli nd Pseudomons eruginos (Singh et l., 2003), but no inhibition ws reveled on E. coli in the present study. The inhibitory effect of Androgrphitis extrct is possibly due to the presence of both rbinoglctn proteins nd ndrogrpholides (Singh et l., 2003). The difference in inhibitory effects on bcteri vried ccording to the types of solvent such s methnol, chloroform nd wter for extrctions nd bcteri strins. Similr vritions of bcteril inhibitions exposed to TCM extrcts were lso found in recent study (Philip et l., 2009), with different ctive compounds dissolved in different solvents bsed on different extrction methods Antimicrobil ctivities of orgnic nd queous extrcts of R. coptidis nd R. scutellri, C. phellodendri nd F. forsythie In generl, the present results showed tht the orgnic extrcts such s ethnol nd DCM possessed stronger ntimicrobil ctivities on bcteri thn queous extrcts, especilly in R. scutellri. Vorvuthikunchi et l. (2006) nd Muludzi et l. (2011) lso reported the orgnic extrcts (DCM, methnol nd ethnol) of medicinl plnts 138

164 possessed higher ntimicrobil ctivity thn queous extrcts. In terms of the solvent polrity, stronger ntimicrobil ctivities were found in DCM nd ethnol extrcts (polrity index =3.1 nd 5.2 respectively) of R. coptidis nd R. scutellri thn hexne extrcts, except prominent ntimicrobil ctivity ws found in hexne extrcts (polrity index =0) (0.16 mg/ml) of C. phellodendri on S. ureus. This result ws contrdictory to other findings tht non-polr extrcts (petroleum ether nd DCM) usully demonstrted stronger ctivity thn polr extrcts (e.g. ethnol nd wter) (McGw et l., 2001; Ncube et l., 2012). Grm-positive bcteri i.e. S. ureus, L. grivee nd E. feclis did not possess outer membrne s grm-negtive bcteri. The outer membrne could regulte the entry of hydrophobic substnce into the cell; therefore grm positive bcteri re more susceptible to ntimicrobil substnce especilly nonpolr contents, resulted in the distortions of the electron flow, proton motive force, cytoplsmic membrne, nd cogultion of cell contents (Burt, 2004). Most relevnt studies found tht grm-positive bcteri re more susceptible to TCM extrcts thn grm-negtive bcteri (Lopez et l., 2005, Shn et l., 2007), but the present experiment did not show such observtions. Although S. mrcescens (grm-negtive) nd S. ureus (grm-positive) were the most tolernt nd sensitive species which were inhibited by 0% nd 64.7% of 17 types of TCMs respectively, other grm-positive bcteri, L. grviee (29.4%) nd E. feclis (17.6%) showed similr susceptibilities s V. cholere (35.3%) nd A. hydrophil (23.5%). Further ntimicrobil studies of these 17 types of TCMs on more bcteri species should be investigted for concluding the susceptibility of grm- 139

165 positive nd -negtive bcteri. The heting during extrctions my ffect the ntimicrobil ctivities of TCMs, but the effects could be vried nd would be depended on the bcteri nd herb species. Nonboiled queous extrcts showed stronger inhibition compred to boiled ones, e.g., lower MICs were found in the non-boiled extrcts of R. coptidis on E. feclis nd R. scutellri on V. cholere nd L. grivee. This indicted heting during extrctions my impir the ntimicrobil ctions of TCMs. Herbl extrcts contined vrious groups of chemicls (such s tnnins, sponins, flvonoids, lkloids, phenols, glycosides) which cn be photo-degrded when exposed to light (Oyi et l., 2007). Heting could reduce the biovilbility nd hence ntimicrobil ctivity of ctive components such s sponins nd tnnins in queous extrcts (Bolji et l., 1997). On the contrry, stronger ntimicrobil ctivities were observed in heted queous extrcts found in R. coptidis on V. cholere nd A. hydrophil. The extrction methods could ffect the ntimicrobil bility of TCM extrcts, but the present results suggested the ntimicrobil ctivities of heted nd non-heted extrcts could be relted to the specific interctions between chemicls nd bcteri. Further studies should be performed on the mechnisms of inhibition of vrious pthogens by TCM extrcts Development of resistnt bcteri to single TCMs nd ntimicrobil ctivities of mixed TCM extrcts In the study on the development of resistnt bcteri to TCMs, MICs of V. cholere, 140

166 L. grivee nd A. hydrophil ginst TCM extrcts did not increse drmticlly (<4 folds). Although 8-16 folds increse of MICs were found in E. coli ginst R. coptidis, the MICs returned to the originl level (0.64 mg/ml) fter 7 pssges without TCM extrct. Similr trends were lso observed in the tretment of R. scutellri with A. hydrophil nd C. phellodendri with L. grviee, which ws completely different from pthogens ginst streptomycin. Pthogens treted with streptomycin developed tough drug resistnce nd high degree of resistnces ( 4 folds MIC of originl vlues) persisted fter drug free pssges. The results indicted drug resistnce ws less esily developed nd retined in bcteri treting with TCM extrcts thn ntibiotics. The results were in line with findings of Meng et l. (2003) who studied R. coptidis nd climed tht it ws probbly due to the multiple ctions of TCM extrcts to inhibit bcteri. Synergistic effects between selected TCMs were only found in 2 out of 22 combintions in 4 bcteri species in this study. F. forsythie mixed with C. phellodendri exerted stronger inhibition on A. hydrophil (MIC= 2.56mg/mL, FIC index = 0.38) thn single herbs. The combintion of F. forsythie nd R. scutellri lso showed stronger inhibition on V. cholere (MIC= 1.28mg/mL, FIC index = 0.50). It is surprising tht those three TCMs showed wek or no inhibition on A. hydrophil when on their own (MICs rnged from mg/ml). Synergistic ntimicrobil ction of TCM mixture my occur in the presence of some substnces with low ntimicrobil ctivity. Stermitz et l. (2000) showed tht ntimicrobil ctivity of berberine ws gretly enhnced in the presence of 5 -methoxyhydnocrpin (5 -MHC), compound isolted from chulmoogr 141

167 oil nd n inhibitor of multidrug resistnce (MDR) pumps of S. ureus. MDR pump is the common wy of drug resistnce in both grm-positive nd grm-negtive bcteri which could block the entry of ntimicrobil gent into the bcteri (Li nd Nikido, 2004; Poole, 2007). Similr effects were lso noted in berberine bounded with 5-nitro-2- phenylindole (INF-55), n inhibitor of MDR pump on inhibiting S. ureus nd E. feclis (Bll et l., 2006; Tomkiewicz et l., 2010). In South Afric, vn Vuuren nd Viljoen (2008) lso reported synergistic effects of the combined of trditionl herb extrcts on inhibiting Bcillus cereus, Cndid lbicns nd Cryptococcus neoformns. These studies indicted the presence of multiple ingredients in herbs which my be responsible for the defense ginst the drug resistnce development. However, the synergistic effects in combined TCM extrcts should not be lwys ssumed, combintions with ntgonistic effects re commonly found in different studies, including the present one (Ndhll et l., 2009; Ncube et l., 2012). Investigtions on ntimicrobil gents from mixed herbl extrcts could be ineffectul if only focused on the combintions of herbs with strong ntimicrobil ctivities. Some TCMs tht hve low ntimicrobil bility might enhnce the inhibition on bcteri when combined, e.g. combintions of C. phellodendri, F. forsythie nd R. scutellri. It is possible to investigte new ntimicrobil gents by combining different TCMs, nd not only focusing on herbs possessing strong ntimicrobil ctivities. However, it is time consuming to study in depth the synergistic effect between copious types of herbs nd the result is not gurnteed. A more efficcious step would be on 142

168 synergistic effects between different solvent extrcts from single herb which is beneficil to combintion chemotherpy to tret infectious diseses (Ncube et l., 2012). TCMs cn serve s ntimicrobil gents, it lso cn ct s immunostimulnts, s well s growth promoters nd potentil lterntive to vccines to fish (Anderson, 1992; Secombes, 1994). The herbl medicine cn be effective on enhncing fish disese resistnce through orl dministrtion (Shrm et l. 2010) or injection (Wu et l., 2010), nd this environmentl friendly drug should be further investigted for ctul ppliction in quculture, on lrge scle. 5.5 Conclusion The hypothesis ws ccepted tht the TCMs (R. coptidis R. scutellri nd C. phellodendri) generlly deffered the development of drug resistnce pthogens, compred to the tested ntibiotics. To conclude, the results of this study provided significnt informtion on using TCMs s lterntives to ntibiotics in quculture. R. coptidis seems to be superior with strong inhibitions to fish pthogens e.g. V. cholere nd L. grviee but with less drug resistnce developed. The ntimicrobil ctivities of TCM extrcts vried in different solvents with ethnol nd DCM extrcts (especilly R. scutellri) possessed stronger ntimicrobil ctivities on bcteri thn queous extrcts. Besides, R. coptidis did not show ny synergetic effect on ntimicrobil ctivity when combined with other TCMs, only two combintions of TCM extrcts: F. forsythie nd C. phellodendri on A. hydrophil, nd F. forsythie nd R. scutellri on V. cholere showed synergetic 143

169 effects (FIC index 0.5). Appliction of TCMs my ese the problem of the buse of ntibiotics nd other undesirble chemicls used in quculture. 144

170 Chpter 6 Effects of Trditionl Chinese Medicines (TCM) on the Immune Response of Grss Crp (Ctenophryngodon idellus) 6.1 Introduction Grss crp is the second lrgest production (fter silver crp) in freshwter quculture worldwide, which is more thn 3 million tonnes, nd Chin contributed 95.7% of the globl production in 2002 (FAO, 2009). Enteritis is one of the most common diseses in grss crp, especilly for young fish, with mortlity round 50% - 90% (Yng, 2008). Aeromons spp in the intestine proliferted rpidly nd cused dysfunction of cpillries on the intestine nd relesed endotoxin leding to septicemi, the mjor symptom of enteritis (Xu et l., 1988). Due to the high stocking density, fish diseses hve crucil effect on quculture yield. Antibiotics re commonly used to control nd tret diseses in quculture, with sulphgunidine nd furzolidone commonly used for treting enteritis in grss crp (FAO, 2009). The frequent occurrence of ntibiotic resistnt bcteri ner quculture site is probbly due to the buse of veterinry ntibiotics nd sub-therpeutic doses pplied in quculture (Smith et l., 2002). As consequence, more nd more ntibiotics resistnt fish pthogens hve been detected round quculture sites (Furushit et l., 2005; Cbello, 2006; Sørum, 2006). Terrestril veterinry pthogens nd even humn pthogens my gin ntibiotic resistnce determinnts through horizontl gene 145

171 trnsmission from bcteri in the quculture environment (Angulo nd Griffin, 2000; Heuer et l., 2009). Due to the vrious thretening effects of ntibiotics, the Europen Union hs bnned its use s feed dditives in food-producing nimls fter Jnury Other feed supplements which re environmentlly sfe nd cost-effective should be used, with herbl medicines one of the populr options. The possible use of Trditionl Chinese Medicine (TCM) for quculture hs been explored in recent yers, Direkbusrkom (2004) confirmed tht the usge plys n importnt role in the Asin quculture industry. Herbs re known to exert positive effects on growth, combt virl infection, stimulte ppetite nd relief stress (Frncis et l., 2005; Citrsu et l., 2006; Venketrmlingm et l., 2007). Therefore the use of TCMs my gretly reduce the disese outbrek in quculture production, nd replce the use of ntibiotics s n effective mesure for disese control. The principle of TCM is quite different from the western phrmcologicl nd therpeutic principles which trget on diseses or pthogens directly. The rtionle of TCM is minly bsed on the theories, such s the five elements theory nd the Yin-Yng blnce, which re considering the overll blnce in the humn body (Cheng, 2000). In other words, TCM is imed t mintining nd restoring the blnce or enhncing the host immunity to defend diseses. The medicinl vlues of TCMs in fish hve been studied, but they minly focused on the non-specific immune responses nd disese resistnces of fish to bcteril infections. Rohu (Lbeo rohit) fed with Achyrnthus showed enhnced superoxide 146

172 nion production level, lysozyme nd serum bctericidl ctivity (Ro et l., 2006). Mortlities in TCM fed fish due to bcteril infections e.g. Aeromons hydrophil in Nile tilpi (Oreochromis niloticus) nd common crp (Cyprinus crpio) (Ardó et l., 2008; Yin et l., 2009) nd Vibrio hrveyi in grouper (Epinephelus tuvin) (Punith et l., 2008) were significntly reduced. Better disese resistnce ws shown in different studies using herbl mixture rther thn single herb. Absli nd Mohmd (2010) showed the formultion contining Ocimum bsilicum, Cinnmomum zeylnicum, Jugpns regi nd Menth piperit enhnced the non-specific immunity nd disese resistnce to A. hydrophil in common crp. In generl, herbl medicine seems ble to exert ntimicrobil nd immuno-stimultory effects in the treted fish. Bsed on TCM theories, TCM compound formultions involved complex interctions between drugs nd my exert better effects on immune-stimultion or improvement on disese resistnce of fish. However, there is lck of informtion on the use of compound formultion contining more thn three types of TCM s feed supplements. Besides, there is limited reserch investigting the fesibility of its ppliction in rel quculture prctice, especilly on its cost effectiveness. It ws hypothesized tht the TCM formultion possess stimultory effects on grss crp immunity nd would be cost effective in preventing Aeromons infection in grss crp. The TCM extrct mixture ws lso expected to be more effective on ctivting plsm bctericidl ctivity thn single herb. 147

173 In this study, compound formultion of TCMs, in rtio of 1:1:2:3 with Rhizom coptidis: Rdix scutellri: Herb ndrogrphis: Rdix sophore flvescentis ws concocted. Two herbs (R. coptidis nd R. scutellri) which showed strong ntibcteril ctivities on A. hydriphil (Chpter 5) were combined with nother two herbs (Herb ndrogrphis: Rdix sophore flvescentis) which showed immunostimulting properties or enhnced disese resistnce (Li et l., 2005; Rttnchikunsopon nd Phumkhchorn, 2009). The effects of this formultion (in form of TCM supplemented fish feed) on immune prmeters of grss crp i.e. oxidtive rdicl production in blood (Nitroblue Tetrzolium (NBT) ssy), totl protein nd totl immunoglobulin nd bctericidl ctivity in plsm were investigted. Finlly, the cost effectiveness of pplying the TCM feed in quculture ws evluted. The mjor objectives of this study were to 1) investigte the effects of the TCM formultion on grss crp immunity nd disese resistnce; 2) evlute its effectiveness in quculture both biologiclly nd economiclly vi lbortory experiments nd field tril; nd 3) investigte the in vitro effects of single nd combined TCM extrcts on plsm bctericidl ctivity. 6.2 Mterils nd Methods Experimentl fish feed preprtion Trditionl Chinese Medicines Rhizom coptidis, Rdix scutellri, Herb ndrogrphis nd Rdix sophore flvescentis were obtined from the School of Chinese 148

174 Medicine, Hong Kong Bptist University for ll the experiments on TCMs. The herbs were dried t 30ºC for 24 h nd then pulverized to powder using mechnicl blender. The powdered herbl medicines were mixed ccording to the following rtio: Rhizom coptidis: Rdix scutellri: Herb ndrogrphis: Rdix Sophore flvescentis = 1:1:2:3. 0.5, 1 nd 2% w/w of TCM powder (1:10 w/v, TCM powder: wter) ws boiled for 30 min with 200 ml of deionized wter nd the queous extrcts were filtered through Whtmen No.1 filter pper. The TCM residues were boiled with nother 200 ml of deionized wter. The extrcts nd the TCM residues were pooled nd mixed with powdered commercil grss crp feed thoroughly. The commercil fish feed used ws Jinfeng, 601 Grss crp formulted feed, with 33.7 % protein, 4.2% crude ft nd 7.7% sh. Deionized wter ws dded nd the fish feed dough ws pelletized with met grinder nd dried t 50 C for 24 h. The fish feed contining no TCM ws used s the control Identifictions of TCMs The four selected TCMs were verified bsed on morphologicl, microscopic nd thin-lyer chromtogrphy (TLC) indentifictions by the School of Chinese Medicine of Hong Kong Bptist University. The morphologicl nd microscopic identifictions vlidted the plnts species of TCMs, ccording to the procedure listed in the Hong Kong Chinese Mteri Medic Stndrds (HKCMMS) Volume III (Deprtment of Helth, HKSAR). The TLC identifiction ws conducted using stndrd solutions of 149

175 corresponding herbs, the TLC pltes were observed under UV light (254nm) nd visible light (The Stte Prmcopoei Commission of PR Chin, 2005). The corresponding components in TCMs re identified nd lised in Tble Fish feeding experiment nd blood smpling Helthy grss crp fingerlings (27.1± 4.2 g) were bought from fish frm in Hong Kong, nd plced in 12 tnks (65 L) with 20 fish per tnk, nd the remining fish were cultured in 3 stock tnks (~200L). All the fish tnks were continuously erted with the wter temperture mintined t 22±2 o C. The wter temperture, vlues of ph nd dissolved oxygen were mesured three times week using portble Hnn ph meter nd YSI digitl Dissolved Oxygen (DO) meter nd the vlues were 21.9±1.7 C, 6.5±0.28 nd 6.7±0.48 mg/ml.the fish were fed with experimentl control feed, by 1% of body weight (g) per mel nd two mels per dy. The fish were cclimtized for 2 weeks nd dpted to the experimentl control feed, the experimentl fish were evluted by creful exmintion of physicl ppernce nd behvior (e.g. rpid responses to light, nd ctive feeding behvior) nd showing no sign of infection (body lesion, scrtching, lethrgy). After 2 weeks cclimtion in lbortory condition, the fish were redistributed into the 12 tnks (20 fish per tnk, 4 tretments in triplictes) rndomly. Ded fish were discrded nd replced by qulified fish from the stock tnks, which were cclimted for further 2 weeks (without ny mortlity during this period) before strt of experiment. 150

176 Tble 6.1 The mjor chemicl components nd their medicl vlues of Rhizom coptidis, Rdix scutellri, Herb ndrogrphis nd Rdix sophore flvescentis identified by Thin-lyer Chromtogrphy (TLC) Herbs Rhizom coptidis The chemicl components (Rf vlue)* Berberine chloride (0.41) nd Plmtine chloride (0.27) Medicl vlues of herbs Berberine reduced the mrna expression level of inflmmtion fctors (Choi et l., 2006); this herb showed effects on treting dirrhe, deintoxiction, nti-inflmmtory (Liu & Ng, 2000) Rdix scutellri Biclin (0.06), Biclein (0.30) nd Wogonin (0.48) Biclin enhnced the phgocytosis of mcrophges (Ci et l., 1994); this herb is responsible for the tretments of fever, ulcer, cncer, nd inflmmtion in humns (Horvth et l., 2005) Herb ndrogrphis Rdix sophore flvescentis Dehydrondrophilide (0.49) nd Andrpgrpholide (0.27) Oxymtrine (0.32) Andrpgrpholide enhnced rective oxygen species production by neutrophil (Shen et l., 2000) Oxymtrine mrkedly inhibit the growth of tumors (H22 murine heptom nd S180 murine srcom) (Li et l., 2006; Shen et l., 2005) *The Rf vlues of the TCMs were mtched with corresponding stndrd chemicls. 151

177 Three different dosges of the mentioned TCM formultion were used in the experiment: 0.5%, 1%, nd 2% (w/w). A tretment without TCM dded in fish feed ws used s the control. There were triplictes for ech tretment i.e., 12 tnks in totl. The fish blood smples (two fish per tnk) were collected by cudl venous puncture t Dy 1, 7, 14 nd 21 fter euthnized by 100 mg/l of Anesthetic Tricine, MS-222 (Sigm- Aldrich, St. Louis, Missouri, USA). The Nitroblue Tetrzolium (NBT) ssy, bctericidl ctivity nd protein nd immunoglobulin ssys were conducted. No mortlity ws observed during the 21 dys of experiment A. hydrophil injection to grss crp Aeromons hydrophil ws inoculted in LB broth overnight t 28ºC. The cultures were centrifuged t 850 G for 15 min. The superntnt ws removed nd the bcteri pellet ws wshed twice in sterile 0.9% sline. The suspension ws djusted to 1 x 10 8 cfu/ml, bsed on the opticl density of suspension with sterile sline (~0.13 bsorbnce t 625 nm). Suspended bcteri (0.1 ml) were injected into the peritonel cvity of fish (12 fish for ech tnk) t Dy 21 of the feeding experiment. The mortlity rte ws recorded in the following seven dys fter infection Immunologicl prmeters nlysis The totl immunoglobulin in plsm ws determined using the method described in Siwicki et l. (1994) with modifictions, the protein concentrtion of the plsm ws 152

178 determined ccording to the modified colorimetric method bsed on Brdford protein ssy (Brdford, 1976). The Nitroblue Tetrzolium (NBT) ssy ws crried out bsed on the method described in Anderson nd Siwicki (1995). The bove procedures were described in detils in Section The bctericidl ctivity of blood plsm ws conducted, bsed on the method of Abidov nd Mirismilov (1979) (Section for detils) Field tril in Yuen Long nd cost evlution on the ppliction of TCM feed The production cost of TCM feed for field tril experiment ws estimted, bsed on the cost of control feed sme s the lbortory experiment, nd compred with the cost of the TCM dded feed. Both the control fish feed nd the two types of TCM feeds (1% nd 2%) were processed by commercil fish feed mnufcturer in Lu Fu Shn, Hong Kong. The feeding costs using the 2% TCM feed in grss crp culture were estimted with the following ssumptions: the feeding period of 2% TCM feed ws 60 dys t 2% dily feeding rte on young grss crp (~0.028 kg, men weight with 1% dily weight gin) t two stocking densities (9000 nd fish/h) with the mrketble grss crp size of 1.5 kg (FAO, 2009). The wholesle sle price of grss crp ws converted from the verge wholesle prices ($15.62 HKD/kg) in Hong Kong during (CSD 2009), which ws $2.0 USD/kg (1 USD = 7.8 HKD). The profit of improved yield when using the TCM feed ws lso evluted bsed on the reduced mortlity observed in field tril. 153

179 Clcultion on pplying 2% TCM feed (i) Applying 2% TCM feed cost (USD/h) = DFR (% of b.w.) x MBW x SD (fish/h) x FD x TCM feed cost (USD/kg) (ii) Profit of improved yield when using the TCM feed (USD/h) = SD (fish/h) x RD (%) x HFW (kg/fish) x WP (USD/kg) where SD: Stocking density = 9000 or fish/h (FAO, 2009) HFW: Hrvested fish weight = 1.5 kg/fish (FAO, 2009) DFR: Dily feeding rte = 2% body weight per dy MBW: Men body weight in the 60 feeding dys= kg (clculted from 1% specific growth rte) FD: Feeding Dy = 60 dys 2% TCM feed cost = $ 1.02 USD/kg RD: Reduced mortlity due to feeding TCM feed = 20% (refer to field tril results) WP: Wholesle price = 2.0 USD/kg (CSD, 2009) A field tril on the effects of TCM formultion on the resistnce of grss crp to A. hydrophil ws conducted t the fish pond in Yuen Long, Au Tu Fisheries Centre of Agriculture, Fisheries nd Conservtion Deprtment (AFCD), Hong Kong SAR Government. Two types of TCM feeds (only 1% nd 2% were tested, bsed on lbortory 154

180 results) nd the feed without dding TCM serving s the control were used in this field tril. Fifty individuls of grss crp (~20 g) were cultured in fish cge (~2 m x 2 m x 1.5 m) plced in fish pond nd cclimtized for two weeks before experiment. The fish were fed bout 1% of body weight (g) per mel nd twice per dy (verge weight of fish ws bout 20g). The pond wter temperture, ph nd dissolved oxygen levels were recorded 5 times week by hndheld Multiprmeter Instrument YSI 556MPS which were 19.5±3.3ºC, 6.7±0.31 nd 6.2±0.35 mg/ml respectively. The sme screening procedures for fish followed those of the lbortory study nd the clinicl signs in disesed fish were lso exmined Effects of TCM extrcts on plsm bctericidl ctivity of grss crp ginst A. hydrophil The experiment ws performed bsed on Ji et l. (2012) with modifictions. Blood of grss crp ws collected from 5 grss crp with n verge weight of 500 g. The blood smples were tken from the cudl vein to serum tubes with Lithium heprin s nticogulnt. The plsm were seprted by centrifugtion t 4000 g for 15 min nd pooled nd stored t -80ºC. The complement ctivity in grss crp plsm ws inctivted t 52 ºC for 30 min (Ski, 1981). Single herbs nd combintions of 2 to 4 herbs of Rhizom coptidis (Rc), Rdix scutellri (Rs), Herb ndrogrphis (H) nd Rdix sophore flvescentis (Rsf) were tested. The queous herbl extrcts were prepred s described in Section The bctericidl ctivity of plsm ws tested bsed on the 155

181 modified methods of Abidov nd Mirismilov (1979) nd Ji et l. (2012). Sme strin of pthogens, A. hydrophil in the bcteril chllenge of Chpter 4 nd 6 (Section nd 6.2.6) ws inoculted in TSB t 28 ºC for 18 h. The cultures were centrifuged t 850 G for 15 min. The superntnt ws removed nd the bcteri pellets were wshed by sterile 0.9% sline twice. The concentrtion of bcteri ws djusted to bout cfu/ml, bsed on the opticl density of suspension nd diluted to 10-4 by sterile sline. A volume of 60 µl grss crp plsm or het-inctivted grss crp plsm ws mixed thoroughly with 30 µl of diluted A. hydrophil, nd then 30 µl of TCM extrcts (24, 80 or 240 mg/l) ws dded. The finl concentrtions of TCM extrcts were 6, 20 or 60 mg/l, respectively. After being incubted t 28 ºC for 30 mins, 50 µl of mixed solution ws spred onto the LB gr plte nd the dishes were incubted for 18 h t 28ºC. The plsm nd the TCM extrcts were replced by sterile phosphte buffered sline (PBS, ph 7) s growth control. The TCM extrcts were replced by PBS to demonstrte the bctericidl ctivity of grss crp without TCMs. The bctericidl ctivity ws represented by the percentge decresed of colony counts in the smple compred to growth control Sttisticl nlysis The results were compred t ech smpling dys (Dy 1, 7, 14, 21) using onewy ANOVA nd Duncn s multiple rnge tests (SPSS Sttistics 17.0, Chicgo, Illinosis, 156

182 USA). Significnt differences between experimentl groups were expressed t the significnce level of p< Results Immune prmeters in Grss crp blood feeding with TCM formultion No significnt difference in totl protein ws found mong ll TCM feed groups compred to the control feed group t ll smpling dys (p>0.05) (Fig. 6.1). A significntly (p<0.05) higher totl immunoglobulin level (IgI) ws observed in 1 nd 2% TCM feed groups thn the control feed group t Dy 21 (p<0.05), nd the totl IgI of 1% TCM group t Dy 14 ws lso significntly (p<0.05) higher thn the control group (Fig. 6.1b). All groups feeding the TCM formultion showed higher bctericidl ctivity of plsm t Dy 21 s compred to the control group. However, only significntly higher (p<0.05) bctericidl ctivity of plsm ws shown in 2 % TCM group, compred to the control feed group t Dy 21; while the bctericidl ctivity of 2% feed group ws grdully incresed since Dy 1, nd significntly improved t Dy 21 compred to the vlue t Dy 1 (Fig. 6.2). A higher NBT ctivity ws found in 2% TCM group t Dy 14 significntly, but no significnt difference ws found mong ll tretments for the neutrophil oxidtive ctivity in the NBT ssy (p>0.05) t Dy 21 (Fig. 6.2b). In generl, 2% TCM feed enhnced the totl immunoglobulin nd bctericidl ctivity of plsm Fish growth nd disese resistnce to A. hydrophil in lbortory experiment 157

183 The mortlity of fish ws monitored for seven dys fter bcteril infection. The disesed grss crps in lbortory nd field studies showed lethrgy, expnded bdomen with red blotches nd loss of ppetite fter infection; redden nd swollen nus ws observed, with yellow mucus relesed from the nus when slight pressure ws pplied to the bdomen. Hemorrhges in the intestinl wll nd liver enlrgement were observed in disesed fish. These signs mtched with enteritis infection observed in grss crp (NACA, 1989; Zheng et l., 2012). Generlly, ll groups fed with TCM showed lower mortlities fter feeding for 21 dys (Fig. 6.3). A significnt reduced mortlities (p<0.05) were observed in 1% nd 2% TCM groups, with mortlities of 43.3% nd 26.7% respectively, compred to the control group (60.0%). The TCM feeding groups i.e. 0.5, 1 nd 2% lso showed higher reltive weight gin nd specific growth rte (Tble 6.2) compred to the control group, but the difference ws not significnt (p>0.05). 158

184 ) Totl protein (g/l) control 0.5% TCM 1% TCM 2% TCM 5 b) Totl IgI (g/l) Dy b b b 1 7 Dy b Fig. 6.1) Totl protein (g/l) (Men ± SD) nd b) Totl immunoglobulin (IgI) (g/l) (Men ± SD) of grss crp plsm in the control feed group nd feeding vrious doses of formulted TCM feed groups. Men in sme smpling dy with different superscripts re significntly different t p<

185 ) Bctericidl Activity (%) Control 0.5% TCM 1% TCM 2% TCM b b) Absorbnce ( 540nm) Dy b b b Dy Fig. 6.2) Bctericidl ctivity in plsm (%) (Men ± SD) nd b) Opticl density of NBT ssy in blood (Men ± SD) of grss crp in the control feed group nd feeding vrious doses of formulted TCM groups. Men in sme smpling dy with different superscripts re significntly different t p<

186 ) Mortlity (%) b b b 0 Control 0.5% TCM 1% TCM 2% TCM b) b Mortlity (%) Control 1% TCM 2% TCM Fig. 6.3 Mortlity (%) (Men ± SD) of grss crp of different feeding groups fter intr-peritonel injection of A. hydrophil, ) in lbortory experiment (Control, 0.5%, 1% nd 2% formulted TCM) nd b) field tril (Control, 1% nd 2% formulted TCM). Men with different superscripts re significntly different t p<

187 Tble 6.2 Weight gin rte (%) nd specific growth rte (%/dy) Tretment Tnk experiment (n=20) Pond Tril (n=30) ^WGR, % #SGR, %/dy ^RWG, % #SGR, %/dy Control 12.66± ± ± ± % TCM 15.00± ±0.17 N.T* N.T* 1% TCM 13.42± ± ± ±0.11 2% TCM 13.58± ± ± ±0.12 Vlues in the sme column with different superscripts re significntly different (p<0.05) *N.T.: Not tested ^Reltive Weight Gin, RWG = [(Finl body weight - Initil body weight)/initil body weight ] X 100% #Specific Growth Rte, SGR= (Ln Weight finl-ln Weight Initil) x 100/dy 162

188 6.3.3 Disese resistnce to A. hydrophil in field tril t Yuen Long nd cost evlution on TCM feed ppliction The disesed grss crp showed sme clinicl signs of enteritis s observed in lbortory study. There were lower fish mortlities in both 1% nd 2% TCM feeding groups (77.8% nd 69.4%) respectively, compred to the control group (88.9%), significnt (p<0.05) mortlity reduction ws only observed in 2% TCM (Fig. 6.3b). The TCM feeding groups i.e. 1% nd 2% lso showed higher weight gin rte nd specific growth rte (Tble 6.2) compred to the control group, but the difference ws not significnt (p>0.05). The production cost of the TCM feed is listed in Tble 6.3. The TCM costs involved in the totl feed production cost were 7.3% nd 13.8% for 1% nd 2% TCM feeds respectively. The costs using the 2% TCM feed were nd USD/h, the profits of improved yields when using the TCM feed were 5400 nd USD/h, for low (9000 fish/h) nd high (18000 fish/h) stocking density respectively. The profits were bsed on 20% of the reduced mortlity in field tril when induced disese In vitro ctivtion on plsm bctericidl ctivity For the single herbs tretments, the bctericidl ctivity in het inctivted plsm ws significntly enhnced in the presence of Rsf nd H t 20 nd 60 mg/l (p<0.01; p<0.05 for Rsf 60 mg/l) (Fig. 6.4). The ctivity in norml plsm ws enhnced when 163

189 treted with Rc 60 mg/l, nd H 20 nd 60 mg/l (p<0.01), but inhibition ws observed when treted with Rsf t 6 mg/l (Fig. 6.4b). The highest bctericidl ctivity (50.92 ± 2.25%) ws observed in Rc 60 mg/l mong ll single TCM extrcts. In mixed TCM extrcts, the ctivity in het inctivted plsm ws generlly improved significntly (p<0.05 or <0.01), except Rc Rs t 20 mg/l, Rc Rs H (t rtio 1:1:2) t 6 mg/l, Rc Rs Rsf (1:1:2) t 60 mg/l, ll four TCM mixed t equl quntity t 6 mg/l nd ll tested doses in Rs H (Fig. 6.5). Generlly, extrcts with 2 types of TCM showed significntly higher bctericidl ctivity thn the norml plsm control (p<0.05 or <0.01), except Rc Rs nd Rsf H t ll doses, while Rc Rsf t 6 mg/l showed the highest bctericidl ctivity (52.10 ± 2.85%) in ll single nd mixed herbl extrcts which ws significntly higher thn control (p<0.01), but the ctivity decresed with incresing concentrtion (Fig. 6.6). Surprisingly, no ctivtion ws found in norml plsm treted with extrcts of 3 types of TCM nd suppression of bctericidl ctivity ws found in tretment Rc Rs H (1:1:2) t 6 mg/l (p<0.05) nd Rc Rs Rsf (1:1:3) t 60 mg/l (p<0.01). In the combintions of 4 types of TCM, significnt ctivtions were found in TCM mixed with equl rtio (t 6 mg/l) nd t the rtio 1:1:2:3 of Rc, Rs, H, Rsf t ll tested doses (p<0.01). 164

190 Tble 6.3 The product cost (in $USD) of 1% nd 2% Trditionl Chinese Medicine (TCM) feed Item Cost per kg 1% TCM feed 2% TCM feed ($USD/kg) Quntity Cost Quntity Cost (kg) $USD % (kg) $USD % Rw mteril nd processing cost (Control feed) Rhizom coptidis Rdix scutellri Herb ndrogrphis Rdix sophore flvescentis Totl / Control feed cost = $0.891 USD/kg 1% TCM feed cost = $0.961 USD/kg 2% TCM feed cost = $1.02 USD/kg 165

191 Fig 6.4 The in vitro effect of Rhizom coptidis (Rc), Rdix scutellri (Rs), Herb ndrogrphis (H) nd Rdix sophore flvescentis (Rsf) extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in ) het inctivted (HI) nd b) nonheted plsm, tretments mrked with sterisks showed significnt difference to control (*= p<0.05; ** = p<0.01) 166

192 Fig 6.5 The in vitro effect of mixed TCM extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in het inctivted (HI), tretments mrked with sterisks showed significnt difference to control (*= p<0.05; **= p<0.01) 167

193 Fig 6.6 The in vitro effect of mixed Trditionl Chinese Medicine extrcts (6, 20 nd 60 mg/l) on the bctericidl ctivity of grss crp in non-heted plsm, tretments mrked with sterisks showed significnt difference to control (*= p<0.05; **= p<0.01) 168