Interntionl Journl of Poultry Science 4 (6): 350-355, 005 ISSN 68-8356 sin Network for Scientific Informtion, 005 Effects of Dicetoxyscirpenol nd Fusric cid on Poults: Individul nd Comined Effects of Dietry Dicetoxyscirpenol nd Fusric cid on Turkey Poult Performnce.S. Firchild, J.L. Grimes, J.K. Porter, W.J. Croom, Jr., L.R. Dniel nd W.M. Hgler, Jr. Deprtment of Poultry Science, North Crolin Stte University, ox 7608, Rleigh, NC US R.. Russell griculturl Reserch Center, USD/RS, thens, G US E-mil: Jim_Croom@ncsu.edu strct: Turkey poults were rndomly plced in tteries nd fed one of four dietry tretments: control (C); control plus 4ppm dicetoxyscirpenol (DS); control plus 300ppm Fusric cid (F); nd control plus 4ppm DS nd 300ppm F (). There were 0 poults per pen with 6 replicte pens per tretment. Individul W, W gins (WG) nd feed consumption y pen were determined t d6, d, nd d8. Period nd cumultive feed to gin were clculted. Mouth lesions were scored for tretments t d8. On d8 poults were euthnized for determintion of orgn weights nd jejunl histomorphometrics. F hd no effect on W or WG t ny period compred to C. Poults fed hd reduced W nd WG compred to C, while poults fed DS hd lower W thn ll tretments t every period. Poults fed F or C hd etter feed to gin (P<0.05) thn poults fed DS or t d6. There were no differences mong the tretments t d or d8. Poults fed F hd significntly lower reltive intestine wt thn poults fed the other diets, nd significntly higher reltive urs wt t d8 when compred to poults fed DS or. DS, F nd ltered intestinl rchitecture. Poults fed DS or hd higher mouth lesion scores thn poults fed F or C, ut mouth lesion scores in DS nd poults were not different from ech other. Dietry DS resulted in decresed poult performnce, while dietry F hd little or no effect. Fusric cid fed in comintion with DS resulted in some protective effect towrds DS. Key words: Dicetoxyscirpenol, fusric cid, turkey, performnce, intestinl histomorphometrics Introduction Molds, present in corn nd other grins fed to poultry, produce mycotoxins tht re detrimentl to poultry production. fter consumption of contminted feed, the decresed weight nd weight gin tht results from poor helth nd performnce re two chrcteristics with finncil implictions. One such genus of molds commonly found nturlly occurring in feedstuffs is Fusrium, which produces numer of mycotoxins s secondry metolites. Two mycotoxins produced y Fusrium species re dicetoxyscirpenol (Ueno et l., 973) nd fusric cid (Hidk et l., 969). 4,5-dicetoxyscirpenol (DS) is type tricothecene tht hs een chrcterized y its effects of reduced ody weight gin nd mouth lesions in chickens nd turkeys when fed t rnge of to 4ppm DS in the feed for up to 3 weeks (demoyero nd Hmilton, 99; Leeson et l., 995; Kuen et l., 997). DS primrily inhiits protein synthesis t the riosoml level, followed y secondry disruption of DN nd RN synthesis. It ffects ctively dividing cells of the orl cvity nd gstrointestinl trct (Leeson et l., 995). The time from exposure needed to provoke the development of mouth lesions through contct with contminted feed hs een reported to e few dys, with the numer of mouth prts hving lesions tripling within wks(demoyero nd Hmilton, 99). Fusric (5-utylpicolinic) cid (F) is considered to hve low to moderte toxicity to poultry. The metolite is hypotensive gent due to its inhiition of dopmine-$hydroxylse, key enzyme in the regultion of the synthesis of the neurotrnsmitter norepinephrine, in drenl glnds (Hidk et l., 969). Chu et l. (993) descried potentition of the humorl immune response to ovine erythrocytes when F ws dded to the feed t 35 ppm. Chicks cell-medited cutneous response to phytohemgglutinin-p ws reduced fter F ws dded to the feed t 35, 75, nd 50 ppm. fter injection into the lterl ventricle, F produced dosedependent increse in feed intke (ungo et l., 999). However, F given in the feed up to 400 ppm yielded no chnge in W or ody weight gins (WG; Chu et l., 993; Oguno et l., 994). Under field conditions, feed my e contminted y more thn one mycotoxin, whether this occurs through mixing individul ingredients ech contminted y one mycotoxin, or y hving one feed ingredient contminted with mold cple of producing more thn one mycotoxin (Ueno et l., 973). Fusric cid hs een shown to e synthesized with other mycotoxins 350
Firchild et l.: Dicetoxyscirpenol, Fusric cid nd Poults Tle : Composition of the tretment rtions Ingredient Diet (%) ------------------------------------------------------------------------------------------------------------------------ Fusric cid Dicetoxyscirpenol Fusric cid + Dicetoxyscirpenol Corn 45. 45. 45. 45. Soymel 39.8 39.8 39.8 39.8 Limestone.4.4.4.4 Poultry ft.0.0.0.0 Dicl 8.5 Phos.... Poultry mel 8.0 8.0 8.0 8.0 DL Methionine 0. 0. 0. 0. Lysine 0. 0. 0. 0. Slt 0. 0. 0. 0. Choline Chloride 0. 0. 0. 0. Minerls 0. 0. 0. 0. Vitmins 0. 0. 0. 0. 3 Selenium 0. 0. 0. 0. 4 Fusric cid 0.03 0.03 5 Dicetoxyscirpenol 0.004 0.004 ----------------------------------------------- Clculted nlysis --------------------------------------------- Protein 7.9 7.9 7.9 7.9 ME (kcl/l),97.7,97.7,97.7,97.7 Ft 5. 5. 5. 5. Methionine 0.68 0.68 0.68 0.68 Methionine + Cystine.3.3.3.3 Lysine.7.7.7.7 Clcium.44.44.44.44 ville Phosphorus 0.73 0.73 0.73 0.73 Sodium 0.3 0.3 0.3 0.3 Content y nlysis 6 Fusric cid (mg/kg) 9 37 7 Dicetoxyscirpenol (mg/kg) 5 5 Minerls mix supplied the following per kilogrm of diet: 0 mg Zn s ZnSO 4 H O; 0 mg Mn s MnSO 4 H O; 80 mg Fe s FeSO4 H O; 0 mg Cu s CuSO 4 ;.5 mg I s Cu(IO 3) ;.0 mg Co s CoSO 4. Vitmin mix supplied the following per kilogrm of diet when dded t 0.%: vitmin, 6,600 IU; vitmin D 3, 000 ICU; vitmin E, 33 IU; vitmin, 9.8 mg; rioflvin, 6.6 mg; nicin, 55mg; d-pntothente, mg mendione, mg; folic cid,.mg; thimine, mg; pyridoxine, 4mg; d-iotin, 6mg; ethoxyquin, 50mg. 3 4 Selenium premix supplied.mg Se, s NSeO 3. dded to fusric cid nd fusric cid + dicetoxyscirpenol diets t rte of 5 300mg/kg of finished feed. Dicetoxyscirpenol dded to dicetoxyscirpenol nd fusric cid+ dicetoxyscirpenol diets t rte of 6 4mg/kg of finished feed. Fusric cid nlyzed y gs chromtogrphy for purity; verge of 43% recovery rte sed on clculted 7 mendment. Dicetoxyscirpenol nlyzed y gs chromtogrphy for purity; 00% recovery rte sed on clculted mendment. produced y Fusrium species (Porter et l., 995). Fusric cid hs een shown to exhiit synergism with nother Fusrium metolite, fumonisin, fter injection into fertile chicken eggs (con et l., 995). Synergism etween F nd DS on mortlity hs lredy een demonstrted in two species of insects tht yielded 4- to 7-fold increse in toxicity (Dowd, 988). Since DS nd fusric cid re common metolites of the fungi, Fusrium, shown to e present in poultry feedstuffs, study hypothesizing possile synergistic toxicity etween the two is wrrnted. The ojectives of this study were to determine the individul nd comined effects of dicetoxyscirpenol nd fusric cid on turkey poults d6 to d8. Mterils nd Methods Experimentl design nd diet preprtion: This study ws conducted under niml cre nd use guidelines estlished y North Crolin Stte University s niml Cre nd Use Committee. The experiment ws performed using 40, dy-of-htch Lrge White Hyrid turkey poults htched from fcilities locted t North Crolin Stte University. The turkeys were individully weighed, wing-nded nd rndomly plced in electriclly heted ttery pens. Dietry tretments; control (C), DS dded t 4 ppm, F dded t 300 ppm, nd comintion of 4 ppm DS plus 300 ppm F (), were ssigned y Ltin Squre rrngement. These dietry concentrtions of DS, F nd were used 35
Firchild et l.: Dicetoxyscirpenol, Fusric cid nd Poults Tle : Effect of dietry fusric cid (F), oservtions). dicetoxyscirpenol (DS) lone nd in There were 6 replicte pens of 0 turkey poults per comintion () on W (g) nd W gins dietry tretment, nd the poults were grown to 8 d of (WG, g) of poults ge. The poults were fed sl turkey strter diet W ------------------------------------------- Tretment d6 d d8 4. 47.8 45.7 Fusric cid 6.5 55.3 464.9 Dicetoxyscirpenol C 00.0 C 93. C 35.4 0.8 5.3 389.0 Men 5.7 8.9 46. SEM.. 3.8 ------------------ WG ------------------ 68.0 3. 03.6 Fusric cid 69.7 9.0 09.7 C C C Dicetoxyscirpenol 43.7 9. 57. 53.9 03.6 73.7 Men 59.3.5 86.8 SEM...0,, C Mens within column with no common superscript differ significntly (P < 0.0) Tle 3: Effect of dietry fusric cid (F), dicetoxyscirpenol (DS) lone nd in comintion () on feed to gin rtios of poults Tretment d6 d d8.04.3.4 Fusric cid.05.9.37 Dicetoxyscirpenol.9.5.49.5.37.5 Men.0.3.45 SEM 0.0 0.03 0.0, Mens within column with no common superscript differ, significntly (P < 0.05). Mens within column with no common superscript differ significntly (P < 0.0) Tle 4: Effect of dietry fusric cid (F), dicetoxyscirpenol (DS) lone nd in comintion () on reltive intestine nd urs weights of poults Tretment Reltive Reltive Intestine Wt urs Wt 5.97 0. Fusric cid 5.44 0.3 Dicetoxyscirpenol 6.3 0.9 5.84 0.0 Men 5.8 0. SEM 0.07 0.0, Mens within column with no common superscript differ significntly. ecuse previous studies in this lortory hd demonstrted tht they re levels tht cn e fed to poults without ffecting mortlity (Hgler, unpulished (Tle ) tht ws found to e elow detection limits for fltoxin, fumonisin, deoxynivlenol, zerlenone, nd T- toxin. The diet met or exceeded the Ntionl Reserch Council (994) requirements for essentil nutrients. The DS ws prepred from cultures of Fusrium smucinum NRRL 3495 using the method of Richrdson nd Hmilton (987), who confirmed the identity of the toxin y nucler mgnetic resonnce nd mss spectroscopy. The F ws purchsed from Sigm-ldrich (St. Louis, MO, US). Toxin purity for the F nd DS used in this experiment ws determined y gs chromtogrphy. The sl diet ws comined with DS nd F y dissolving ech pure crystlline toxin in cetone, pipetting ech over 0.45 kg of feed individully or in comintion, thoroughly mixing, nd letting the feed dry overnight in shllow pn. fter drying, these 0.45 kg quntities of feed were mixed with their respective dietry tretment ssignments to produce the three tretments contining dded toxins. Wter ws provided d liitum vi nipple drinkers in ech pen. Dt collection nd nlysis: Individul W, W gins (WG) nd feed consumption y pen were determined t d6, d, nd d8. Period nd cumultive feed to gin per pen were clculted through d nd d8. Mortlities were recorded dily. Mouth lesions were scored for ll tretments t d8 sed on five-point scle (0 = no lesions; 5 = severe lesions) y n individul with no knowledge of tretment ssignments. t d8, fter euthnsi y cervicl disloction nd dissection, liver, spleen, urs, intestinl, nd jejunl weights were mesured t d8. fter euthnsi, 0 poults per tretment were rndomly selected cross ll tretments nd pens nd portions of their mid-jejun prepred for histologicl nlysis s descried y ird et l. (994) in mice nd modified for turkey poults y Fn et l. (997). Jejunl segments were fixed in Crnoy s solution for 4 hours, sequentilly dehydrted nd nd stored in 70% ethnol (vol/vol). Smples were then emedded with prffin nd cut into 5µm sections, mounted on glss slide nd then stined with Feulgen regent nd counter-stined with 0.05% Fst Green. computerized microscropic imge TM nlyzer (Chemilmger 440, lph Innotech Corp., Sn Lendro, C, US) ws used to determine villus plnr perimeter length, villus width t mid-villus, villus height, height of enterocytes t mid-villus nd supporting muscle lyer thickness. Mesurements on individul histomorphometric prmeters were mde from rndomly selected slides exhiiting mid-section pln of villi. ecuse of orienttion of tissue smples, not ll prmeters could e mesured from ech section. No 35
Firchild et l.: Dicetoxyscirpenol, Fusric cid nd Poults Tle 5: Histomorphometric nlysis of jejunl tissue of poults fed fusric cid (F), dicetoxyscirpenol (DS) lone or in comintion () Villus Plnr Villus Villus Serosl Crypt Mid-Villus Surfce Height 3 Width 4 Thickness 5 Depth 6 Enterocyte re (µ) (µ) (µ) (µ) (µ) Height (µ) 08±75.6 567±4.69 05±7.8 69±9.8 57±3.7 0±9.66 F 77±3.3 585±7.83 30±3.0 c 9±6.78 56±3.30 4±3.07 DS 7±7.87 575±43.38 3±3. 38±6.99 49±3.6 4±5.8 6±96.395 509±6.35 45±4.05 c 4±8.0 46±4.9 MD* 3 4 5 6 Mens ± SEM n=07; n=0; n=06; n=09; n=44; n=09 c tretments in columns with different superscripts differ (p <0.05) * MD = dt not ville suitle sections for mesuring mid-villus enterocyte Tissue nd histomorphometric nlysis: Fusric cid height could e found for the tretment. significntly reduced reltive intestinl weight compred Sttisticl nlysis of performnce prmeters, mouth to ll other dietry tretments (Tle 4). The F lesions nd reltive orgn weights were performed tretment lso incresed reltive urs wt compred to using the Generl Liner Models procedure of the SS the DS nd tretments, ut no dietry tretment ws Institute, Inc. (989). The sttisticl design ws different from control (Tle 4). There were no fctoril rrngement of tretments which included the significnt differences in the reltive wts of the liver, min effects of tretment, level of ttery nd error. spleen, or jejunum (dt not shown). Reltive orgn weights were nlyzed using covrite Histomorphometric nlysis of jejunl tissue nlysis with individul ird weight s the fixed vrile. prmeters were within the rnge of vlues noted in Mens of performnce prmeters nd reltive orgn previous studies in this lortory with d4 poults (Fn weights were seprted using the Lest Squres et l., 997). Jejunl seros in F treted poults ws Mens procedure with significnce ccepted t p<0.05. 9% thinner (Tle 5) thn those of controls. DS ecuse of differences in smpling for histologicl tretment resulted in 8-33% greter serosl thickness nlysis compred to ll other prmeters, the (P<0.05) compred to controls nd F tretments. There sttisticl nlysis of histomorphometric dt were ws non-significnt trend (p = 0.07) for decresed conducted independently. Smples for crypt depth for ll tretments compred to controls (57µ histomorphometric nlysis were collected from the vs 56µ, 49µ nd 46µ for control, F, DS nd, jejun of 5-7 irds per tretment nd rndomly selected respectively). oth F nd DS tretment decresed cross ll pens. Slides for nlysis of morphometric enterocyte height t mid-villus y 59% (P = 0.05; Tle prmeters were rndomly selected sed on 5). pproprite sptil orienttion of the fixed tissue smples. Replicte prmeter mesurements were Mouth lesion scores: ll tretments were scored for mde on ech slide. Dt were nlyzed using one- mouth lesions in order to show no cross contmintion wy nlysis of vrince (Sttistix version 8, 003). with DS. ll scoring ws performed on sujective Pirwise comprison of mens ws conducted using scle of 0 for no visile lesions to 5 for the presence of Tukey s Method with significnce ccepted t p<0.05. severe lesions. Only tretments with DS dded were ssocited with mouth lesions. Since the poults fed the control nd F diets hd no mouth lesions, the DS nd tretments were sttisticlly compred to ech other for mouth lesion scores. The two tretments were not significntly different from ech other (DS = 4.48; = 4.64). Results Effects on poult performnce: There ws no effect of F on poult W (Tle ) or WG (Tle ) when fed t 300 ppm in the feed t d6, d, or d8. However, there ws significnt reduction in W nd WG for poults fed the DS diet for ech period performnce chrcteristics were mesured (P<0.0). The comintion diet, F plus DS () yielded significnt recovery in W nd WG t d6, d, nd d8 (P<0.0). t d6, the control diet nd F diet hd significntly etter feed to gin rtio thn the DS nd the diets (Tle 3). t d8, poults fed the F diet hd etter feed to gin rtio thn either the DS or fed irds (Tle 3), ut no tretment diet ws significntly different from control. There were no differences in cumultive feed to gin for d0-d or d0- d8 dys. Discussion Fusrium species produce vst rry of mycotoxins, mny of which re economiclly importnt in regrds to niml production. If mycotoxin is found in contminted feed source, there is high chnce of dditionl mycotoxins eing present, produced y severl or single species of mold (Prelusky et l., 994). Concentrtions of individul mycotoxins re usully low enough not to cuse prolems with niml performnce, ut there is potentil for interctions 353
Firchild et l.: Dicetoxyscirpenol, Fusric cid nd Poults etween metolites tht result in net effects of dditive or even synergistic reltionships. Fusric cid is common metolite of severl Fusrium species including F. moniliforme, F. suglutinns, nd F. oxysporum nd its simultneous synthesis with other Fusrium toxins including fumonisins nd trichothecenes hs een reported (Porter et l., 995). fter the ddition of F to the feed t 300 ppm in the current study, there ws no significnt effect on W or WG. These results concur with those reported y Chu et l. (993), who reported no effect of on W nd WG when chicks were fed up to 50 ppm, nd Oguno et l. (994) who reported similr results with chicks nd poults fed up to 400 ppm. The ddition of DS to the turkey strter diet hd expected negtive results of reduced weight gin nd severe mouth lesions. Similr results were reported y Kuen et l. (997). fter feeding 4 ppm DS, significnt reduction in WG ws seen t wk nd cumultive reductions of 3% in WG seen through d0. Only tretments with DS were ssocited with mouth lesions, in contrst to the lck of orl lesions with F. The two DS tretments were not significntly different from ech other (DS = 4.48; = 4.64). Kuen et l. (997) reported orl lesion scores for the DS tretment were 3.9 on to 4 scle. Skln et l. (003) reported modest increses in growth, ut not feed efficiency, in poults fed diets with 0.43 pp to 0.860 pp. In ddition they oserved significnt orl lesions fter 7 dys. In the present study, the effects seen fter the ddition of oth F nd DS to the diet were unexpected. The tretment resulted in significnt recovery in W nd WG t ech dy mesured, which included feeding t levels less thn the mximum used in previous pulictions. However, the comintion diet impired feed to gin t d6 compred to the control diet. Histomorphometric prmeters indicte tht DS, F nd diets lter physiologicl function in the intestinl trct (Tle 5). Hoerr et l. (98) reported tht cute dministrtion of lrge dosges of DS (.7 mg/kg ody weight) to d7 roilers, vi gvge, resulted in villus trophy nd decresed epithelil mitosis within 4 hours. In the present study, the serosl lyer ws decresed with F whilst it incresed with DS. The trend (P = 0.07) for decresed crypt depth long with sustntil decreses in (P<0.05) enterocyte height t mid-villus indicte tht oth DS nd F re ffecting the propgtion nd development of enterocytes. Enterocyte height t mid-villus is positively correlted with cell mturtion (ird et l., 994). The 59% decrese in enteroctye height ssocited with oth F nd DS is highly indictive tht these mycotoxins re ltering digestive nd sorptive function (Tle 5). This is supported y the work of Skln et l. (003) who reported incresed proliferting cells in jejunl crypts of d3-d34 poults fed DS t 0.860 ppm. Incresed production of enterocytes in the crypts of villi could result in fster migrtion of cells up the villus stlk t rte tht precludes norml development. Skln et l. (003), however, reported tht jejunl enterocyte migrtion rte ws not ffected in DS poults. lthough Skln et l. (003) dministered DS t concentrtions tht were only 0% tht used in this study, their oservtions indicte the decrese in enteroctye height t mid-villus (Tle 5), hd more to do with ltertions in cell metolism rther thn migrtion rte. Tricothecene mycotoxins re known to decrese protein synthesis, inhiit DN nd lipid synthesis nd inctivte some enzymes (Ueno, 977), which could ffect enterocyte size nd mturtion. con et l. (995) demonstrted synergistic toxic interction etween nd fusric cid fumonisin in the fertile chicken egg. Injected lone, fusric cid resulted in % mortlity. While keeping the fusric cid dose constnt, dose dependent mortlity curve ws oserved s fumonisin ws comined t incresing levels. Synergism ws lso demonstrted etween fusric cid nd DS in insects (Dowd, 988). Feeding oth fusric cid plus DS cused n increse in mortlity from 5% with DS lone to over 0% with fusric cid plus DS comined. The study of interctions etween mycotoxins often leds to etter understnding of how mycotoxicoses would present themselves in production situtions, where conditions re fvorle for the mixing of fungl metolites. It is difficult to predict the response from comined toxins sed on their individul responses. The comintion of mycotoxins used in this study did not produce n expected incresed toxicity, which could hve een descried s ddition, potentition or synergism (Klssen nd Eton, 99). Nor did the study demonstrte the ility of one toxin (DS) to msk ny effects of nother, such s fusric cid. It is hypothesized tht fusric cid ntgonized the toxic ctions of DS with regrds to W nd WG. Even though the poults hd the sme visul score for mouth lesions, they were le to significntly increse their feed intke nd wt gin throughout the length of the study compred to irds on the DS diet. We interpret the current dt s demonstrting tht for poult performnce prmeters, fusric cid plus dicetoxyscirpenol my e less toxic thn the individul toxins. For mny tissues, neither DS or F, lone or in comintion, hd ny effect on reltive orgn weights. However, dicetoxyscirpenol produced the sme severity of mouth lesions whether lone or with F in the diet of turkey poults. Further work is needed to fully evlute the mycotoxin interction reltionships of F with other Fusrium metolites nd their effects on poult performnce. 354
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