Negative impacts dueto infectious diseasesare considerablein various types ofaquaculture, and. cost millions ofdollars in lost revenues due tomortality and reduced productionefficiency.

Therefore, interest continues indeveloping alternatives to traditionalmeans of combating diseases. Although theconcept of functional feeds is relatively newto the aquaculture industry, it representsan emerging new paradigm to developdiets that extend beyond satisfying basicnutritional requirements of the cultured

organism to improving health and resistanceto stress and disease-causing organisms(Gatlin 2002).

Disease prevention and treatmentstrategies, such as use of vaccines and drugs,ar.e generally limited in most aquaculturaloperations due to regulatory constraints orinconvenient administration options.

Therefore, our laboratory has

Cumulative Survival (%)

investigated the effects of various dietaryfactors on disease resistance as well asimmunostimulation of various fish species.

This research has focused on selectednutrients such as arginine, vitamin C, vitaminE and selenium, as well as various naturalfeedstuffs and synthetic compounds whichmay enhance immunity and fish health.

181 INTERNATIONAL AQUAFEED I November-December 08

PrebioticsPrebiotics are defined as non-digesti

food ingredients which beneficially affectthe host by selectively stimulating thegrowth of and/or activating the metabolisof one or a limited number of healpromoting bacteria in the intestinal tract,thus improving the host's intestinal balance(Gibson and Roberfroid 1995).

Some researchersalso have suggested thatthe interaction betweenintestinal microflora anenterocytes may triggerrelease of biologically-active substances aninfluence host immunity(reviewed by Pattersoand Burkholder 2003)_The health-promotingbacteria most commonlyaugmented by prebioticsinclude those of thegenus .Lactobacillus andBifidobacter which tendto limit the presence ofharmful bacteria.

Examples of prebiotics includemannanoligosaccharides (White et al2002), lactose (Szilagyi 2002), as well asoligofructose and inulin (Teitelbaum andWalker 2002). These natural compoundsare generally not altered by diet processingand require limited regulatory approsuch that their incorporation into diets is

much simpler than required for drugs orchemical therapeutic agents.

EvaluationTo explore potential use of a specialised

prebiotic preparation in aquaculture, ourlaboratory first evaluated the commercialproduct GroBiotic®-A in three separatefeeding trials with hybrid striped bass(Morone chrysops x M. saxatilis).

This product is a mixture of partiallyautolyzed brewers yeast, dairy ingredientcomponents and dried fermentationproducts containing 35.2 percent crudeprotein, 1.7 percent crude lipid and -53percent simple and complex carbohydratesincluding oligosaccharides. Hybrid stripedbass is an important aquacultured fish inthe United States and is negatively impactedby various bacterial pathogens, such asStreptoccocus iniae and Mycobacteriummarinum (Plumb 1997).

In each of the three trials with hybridstriped bass, a basal diet was formulatedto contain 40 percent crude protein,· 10percent lipid and 14.6kJ digestible energy/g.

Experimental diets were supplementedwith GroBiotic®-A at either onepercent or two percent of dry weightand maintained isonitrogenous andisoenergetic by adjusting the amount offishmeal and dextrin. After a seven-weekperiod in the first feeding trial, Significantlyenhanced weight gain (percent of initialweight) and feed efficiency (gram weight

gain/g feed) were observed in juvenile fish(initial weight of seven glfish) fed dietssupplemented with one or two percentGroBiotic®-A compared to those fed thebasal diet (Li and Gatlin 2004). Survivalduring the first feeding trial was high andno significant differences were observedamong treatments.

In the second feeding trial, survival ofjuvenile fish (initial weight of 19.7g/fish)fed diets containing GroBiotic®-A for fourweeks and then exposed by immersion to a

The shortness of the trial did not allowfor separation of juvenile hybrid stripedbass performance as influenced by dietaryGroBiotic®-A, thus no significant differenceswere observed in this regard.

Further studyTo further explore possible age/size-

related responses, a 2 I-week feeding trialwas conducted to evaluate GroBiotic®-Ain the diet of sub-adult hybrid striped bass(initial weight of 65-1 IBg/fish) exposed to.

o protein• organic mattero lipid8 energy

Figure 2: Digestibilitycoefficients of diets fedto the red drum. Basaland GroBiotic-A dietscontained 40 percentcrude protein fromapproximately halfdehulled soybean mealand half menhadenfishmeal while thecontrol diet had all ofits protein provided bymenhaden fishmeal (FMcontrol)

Digestibility coefficients of diets fed to red drum

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IBurr et a!. (2008\ Aquaculture Res. In press

standardized dose of Streptococcus iniae wassignificantly (P < 0.0 I) higher than fish fedthe basal diet (Li and Gatlin 2004).

Extra cellular superoxide anionproduction of head kidney macrophagesfrom fish fed GroBiotic®-A or brewersyeast also was Significantly higher than thatof fish fed the basal diet.

November-December 081 INTERNATIONAL AQUAFEED 119

1'=0.001

a chronic mycobacterial infection causedby Mycobacterium marinum (Li and Gadin2005).

Enhanced growth performancewas generally observed in fish fed dietssupplemented with GroBiotic®-A comparedto fish fed the basal diet throughout thefeeding trial with Significantly (P < 0.05)

Prebioticenhanced weight gain observed after 12weeks of feeding. The in situ mycobacterialchallenge employed in this experimentresulted in overall cumulative mortality ofapproximately 25 percent.

Fish fed two percent Grobiotic®-Ahad a significantly (P < 0.05) enhancedsurvival (80 percent) compared to theother treatments (72-73 percent) at theend of 21 weeks. It is known that ingestionof feed is a port of entry for mycobacteriain fish such as the snakehead Channa striatus(Chinabut et al. 1990). This could possiblybe a factor contributing to the positiveresponse associated with the GroBiotic®-Asupplement.

In vivo experimentsBased on the positive results obtained

with hybrid striped bass, we evaluatedGroBiotic®-A in a series of in vitro and invivo experiments with red drum (Sciaenopsocel/atus), a marine sciaenid cultured forfood and stock enhancement along the Gulfand Atlantic coasts of the United States.

An in vitro experiment revealed thatGroBiotic®-A altered the microbialcomposition of GI tract inoculum fromred drum, and the predominant microbialspecies was of the genus Lactococcus based

on DNA sequencing (Burr et al. 2008a).In a subsequent study, red drum

fed a diet containing 40 percent crudeprotein provided equally from soybeanmeal and fishmeal and supplementedwith GroBiotic®-A at one percent hadsignificantly elevated digestibility coefficientsfor crude protein, organic matter, lipid andenergy compared to a basal diet withoutprebiotic supplementation (see Figure 2)(Burr et al. 2008b). The prebiotic-inducedenhanced digestibility of the soybeanmeallfishmeal diet was comparable to thatobserved for the control diet in which all ofits protein originated from fishmeal.

Another feeding trial was then conductedwith red drum to evaluate their growthperformance, non-specific immune responsesand resistance to Amyloodinium ocel/atum,a parasitic dinoflagellate which may causesevere losses to various marine fishes.

In this feeding trial, red drum werefed soybean meal/fish meal-based dietsas previously described, and exposed toAmyloodinium ocel/atum after four weeksof feeding. Fish fed GroBiotic®-A at onepercent of diet had significantly enhancedweight gain, feed efficiency and survival(see Figure 3) after week six (Buentello etal. 2008).

Survival of red drum exposed toAmyloodinium ocellatus for 2 weeks

Basal GroBiotic-A

Diet

Cumulative Mortality of Rainbow TroutExposed to Infectious Hematopoietic

Necrosis Virus

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70

it 60~50

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Diet IP injection after 9 w'Ic feeding

seaiev et at, (2007) N. Am. J. Acuecutt, 69:400-406

20 I INTERNATIONAL AQUAFEED I November-December 08

In addition, red drum fed the dietsupplemented with GroBiotic®-A hadsignificantly elevated non-specific immumeresponses including serum lysozyme andintracellular superoxide anion production.The evaluation of GroBiotic®-A also hasbeen extended to other fish species such asrainbow trout Oncorhynchus mykiss (Sealeyet al. 2007) and golden shiner Notemigonuschrysoleucus, the predominant minnowspecies cultured in the United States for bait(Sink et al. 2007, Sink and Lochmann 2008).

In the study with rainbow trout,supplementation of GroBiotic®-A at twopercent of diet significantly increased feedefficiency at week three, although thedifference was not statistically distinct byweek nine.

However, fish fed the diet supplementedwith GroBiotic®-A had significantly greatersurvival after exposure to infectioushematopoietic necrosis virus (see Figure 4).

Two separate feeding trials have beenconducted with golden shiners in which theywere fed various practical diets includingthose supplemented with GroBiotic®-Aat two percent by weight. In one trial,golden shiners were fed diets containingfour or 10 percent lipid without prebioticsupplementation and one with 10 percentlipid and supplemented with GroBiotic®-Afor 16 weeks (Sink et al. 2007).

After the feeding trial, fish were subjectedto a Flavobacterium columnare challengeby immersion. Fish fed the prebiotic-supplemented diet had significantly lowermortality compared to fish fed the otherdiets (see Figure 5).

In another subsequent experiment(Sink and Lochmann (2008), goldenshiners were fed diets with or withoutsupplemental GroBiotic®-A for 10 weeks.Groups of fish fed each diet were eithersubjected to handling stress or directlyexposed to Flavobacterium columnareby immersion. Handling stress prior todisease exposure significantly increasedmortality of fish, but those fed the dietsupplemented with GroBiotic®-A hadreduced mortality compared to fish fedthe basal diet.

SummaryResearch to date has shown that

dietary supplementation of the prebioticGroBiotic®-A has consistently enhancedresistance to various pathogens byseveral fish species including hybrid

striped bass, red drum, rainbow trout

and golden shiners.

In addition, improvements in feed

efficiency, nutrient digestibility and

several different non-specific immune

responses also have been observed with

the supplementation of this prebiotic to

practical diets.

Thus, the combination of improved

growth performance and survival of fish

fed this prebiotic may result in considerably

augmented productivity in various

aquacultural enterprises resulting in a

beneficial return on investment.

References:Buentello, JA. Neill, W.H .• and D.M. Gatlin.III. (2008) Effects of dietary prebioticson growth. feed efficiency and non-specific immunity of juvenile red drumSciaenops ocellatus fed soybean-based diets.Aquaculture Research. In press.

Burr;G..M. Hurne, S.Rickeand D.M. GatlinIII (2008a) A preliminary in vitro assessmentof Grobiotic®-A. brewers yeast and fructo-oligosaccharide as prebiotics for the red drumSciaenops ocellat~s. J.Environ.Sci.Health. B 43:253-260.

Burr;G..M. Horne,W H. Neill. and D. M. GatlinIII (2008b) Effectsof prebiotics on nutrientdigestibility of a soybean-meal-baseddiet by reddrum Sciaenops ocellatusi. Aquaculture ResearchIn press.

Chinabut S..LimsuwanC. and Chanratchakool.P(1990) Mycobacteriosisin the snakehead,Channastriatus (Fowler).JoumalofFish Disease13.531-535.

Gatlin D. M. III.(2002) Nutrition and fish health. In:FishNutrition (ed. by J.E.Halver and R.W Hardy).pp. 67 1-702.Academic Press.SanDiego. CA. USA

Gibson G. R.and Roberfroid M. B. (1995) Dietarymodulation of the human colonic microbiota:introducing the concept of prebiotics. Journal ofNutrition 125. 1401-1412.

Li Pand Gatlin D. M. III (2004) Dietary brewersyeast and the prebiotic Grobiotic TMAE infiuencegrowth performance. Immune responsesandresistanceof hybrid striped bass(Marone chrysopsx M. saxadlis) to Streptococcus imae infection.Aquaculture 231.445-456.

Li Pand Gatlin D. M. III (2005). Evaluation of theprebiotic GroBiotic®-A and brewers yeast asdietary supplements for sub-adult hybrid stripedbass(Marone chrysops _ M. saxadlis) challengedin situ with Mycobacterium marinum. Aquaculture.248.197-205.

Patterson J.A and Burkholder K M. (2003)Application of prebiotics and probiotics in poultry

Sealey,W M..FT Barrows.K.A Johansen.K.OverturfS.E.LaPatraand R.WHardy (2007) Evaluationof the ability of partiallyautolyzed yeast and GroBiotic-A to improvediseaseresistance in rainbow trout. NorthAmerican Journal of Aquaculture 69. 400-406.

production. PoultryScience 82. 627-631.

Plumb J. A (1997)Infectious diseasesofstriped bass.In:StripedBassand Other MoroneCulture (ed. by R.M.Harrell). pp. 271-313.Elsevier.Amsterdam.Netherlands.

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4540

it 35-30~25~ 20o 15" to

5o

Cumulative Mortality of GoldenShiners Exposed to Flavobacterium

columnare

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GroBiotic-ABacterial challengeby immersion atter16 wk feedingDiet

Sink at al. (2007) N. Am. J. Aquaeult. 69:305·308

Sink,T D. and R.T Lochmann (2008) Preliminaryobservation of mortality reduction in stressed.Flavobacterium columnare - challengedgoldenshiners after treatment with a dairy-yeastprebiotic. North American Journal of Aquaculture70. 192-194.

SinkT D..R.T Lochmann.AE.Goodwn andE.Marecaux(2007)Mortality ratesin goldenshinersfedhigh-fatdietswith andwithouta dairy-yeastprebioticbeforechallengewithR(N()bocteriumcolumnae.North AmericanJoumalof Aquaculture69.305-308.

SzilagyiA (2002)Lactose- a potentialprebiotic. AlimentaryPharmacology andTherapeutics 16.1591-1602.

Teitelbaum J.E.andWalkerWA (2002)Nutrtional impact ofpre-and probioticsas protectivegastrointestinalorganisms.AnnualReview of Nutrition22. 107-138.

vvhite L.A. NewmanM. c.. Cromwell G.L.and Lindemann M.D. (2002) Brewersdried yeast as asource of mannanoligosaccharidesforweanling pigs.Journalof Animal Science80.2619-2628.

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November-December081 INTERNATIONALAQUAFEED 121