Effect of Incubation Time and Level of Fungus Myceliated Grain Supplemented Diet on the Growth and Health of Broiler Chickens

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1 Interntionl Journl of Poultry Science 12 (4): , 2013 ISSN Asin Network for Scientific Informtion, 2013 Effect of Incubtion Time nd Level of Fungus Mycelited Grin Supplemented Diet on the Growth nd Helth of Broiler Chickens V. Hines, W.L. Willis, O.S. Isikhuemhen, S.L. Ibrhim, F. Anike, J. Jckson, S.L. Hurley nd E.I. Ohimn Deprtment of Animl Sciences, Deprtment of Nturl Resources nd Environmentl Design, 3 Deprtment of Fmily nd Consumer Sciences, North Crolin A&T Stte University, Greensboro, North Crolinm 27411, USA 4 Deprtment of Biologicl Sciences, Fculty of Science, Niger Delt University, Amssom, Wilberforce Islnd, Byels Stte, Nigeri 1 2 Abstrct: A study ws conducted to evlute the effects of different incubtion time nd level of inclusion of Fungus Mycelited Grin (FMG) in diets of broiler chickens. The nine different dietry tretments were s follows: (1) Control-No-FMG (2) 5% FMG-14 d (3) 10% FMG-14 d (4) 5% FMG-28 d (5) 10% FMG-28 d (6) 5% FMG-42 d (7) 10% FMG-42 d (8) 5% FMG-56 d nd (9) 10% FMG-56 d. Ech diet ws fed to 3 replicte pens of 10 chicks ech in floor pens for 49 dys. The 270 dy-of-htch stright-run chicks were fed FMG tht included Lentinul edodes, Cordyceps sp. Gnoderm sp. nd Pleurotus ostretus incubted in grin for 14, 28, 42, or 56 dys. FMG protein percentge, live weights, Eimeri oocyst, bifidobcteri counts, mortlity, feed consumption, blood differentil nd immunoglobulins (IgA, IgG) were evluted in this experiment. Percent protein between mushroom incubtion periods differed significntly. Body weight results showed tht ll experimentl tretments were significntly (P<0.05) different from tretment 1 (Control) for mle broilers but did not differ with the femles, while the overll feed intke ws significntly higher for the control broilers. Fecl oocyst count showed only one tretment (2) differed significntly (P>0.05) from tretment 1 (Control). Bifidobcteri counts were highest in tretment 4 (8.071og10) when compred to the tretment 1 control (7.691og10) t week 5. Some hemtologicl nlysis vlues showed some elevtion nd decresed protein levels s the incubtion time incresed long with the ntibody IgA nd IgG titers. The results from this study suggest tht the incubtion time, mixture nd level of FMG do not dversely ffect growth nd higher ntibody levels in broiler chickens could enhnce helth s percent protein decline with incresed bet-glucn nd incubtion time of the FMG. Key words: Broiler chickens, mushrooms, performnce, incubtion time, helth INTRODUCTION The removl of mny feed dditives/ntibiotics in poultry rering hs negtively impcted helth, production performnce nd product sfety (Montgne et l., 2003). Therefore, new strtegies nd lterntives feed dditives, including medicinl mushroom, re ttrcting heightened interest (Ohimin nd Ofongo, 2012). Reserch on medicinl mushrooms for nimls hs focused on compounds tht cn modulte the biologic response of immune cells. Those compounds tht pper to stimulte the niml s immune response re of interest so tht drugs/ntibiotics usge in poultry production cn be reduced or eliminted. Recent reserch hs pved the wy for utilizing nd furthering the potentil for mushroom pplictions in poultry production. For instnce, Guo et l. (2004) studied the effects of mushroom nd herb polyscchrides on cellulr nd humorl immune responses of Eimeri tenell-infected chickens nd concluded tht these supplementtions resulted in n enhncement of both cellulr nd humorl immune responses. In recent decdes, significnt gins hve been mde with utilizing mushrooms for promoting niml helth (Borchers et l., 2004). Dlloul et l. (2006) pioneered some studies on the immune-potentiting effect of lectin extrcted from Fomitell frxine in poultry during coccidiosis. Their results demonstrted tht the lectin extrction given to chickens ws effective in promoting growth nd enhncing immune-modultory ctivity during coccidiosis. More recently, Selegen et l. (2009) studied the effect of polyscchride extrct from the edible mushroom Pleurotus ostretus ginst infectious bursl disese virus nd found positive synergistic reltionship between the extrcts nd burs vccines in stimulting the production of ntibodies. In similr vein, wter extrct method hs been utilized to further Corresponding Author: W.L. Willis, Deprtment of Animl Sciences, North Crolin A&T Stte University Greensboro, North Crolin 27411, USA 206

2 study mushrooms in promoting poultry helth (Willis et l., 2007, 2009). Other usges of this product, pioneered by Willis et l. (2009), utilized the mushroom extrct to molt ging lying hens for dditionl eggs production cycles. As these positive findings hve evolved using mushroom extrcts nd their product in enhncing poultry helth, some other issues nd concerns hve surfced. These include: wht re the most effective type nd dosge nd wht is the cost of producing the mushrooms nd the time nd lbor needed to extrct the polyscchrides from them? These concerns nd others led to studies by Willis et l. (2009) tht utilized Fungus Mycelited Grin (FMG) insted of extrcts in reserch with broiler chickens tht cost less nd ws esy to dminister. These investigtions resulted in FMG product composes of cultures of mushroom spwn incubted in sorghum grin. The product derived from the methodology employed is more economicl to produce nd dminister to chickens vi the feed rther thn extrcts dded to wter nd other delivery methods. Their most recent studies hve utilized FMG inclusion in the feed to molt lying hens nd investigte coccidiosis infection protection, Slmonell popultion reduction bifidobcteri growth nd immune enhncements in broiler chickens (Willis et l., 2012, 2011, 2010). The present study reltes to issues surrounding methods, type of mushrooms, level of supplementtion, durtion of incubtion time, feeding regimes nd performnce. Accordingly, this specific experiment ws undertken to determine if specific ntibody levels, body weights nd other performnce trits of broiler chickens were positively or negtively ffected by FMG incubtion time nd inclusion level in their diet. MATERIALS AND METHODS Experimentl design nd husbndry: Two hundred seventy (n = 270) dy-old-mle nd femle broiler chickens (Ross x Ross) were obtined from commercil htchery. The chicks were weighed nd rndomly distributed into nine different tretment groups replicted three times with 10 chicks (5 mles + 5 femles) tht were fed control or 5 or 10 percent FMG mixture, ech mde from four different mushrooms nd with four different incubtion periods s follows: (1) Control-No FMG (2) 5% FMG-14 d (3) 10% FMG-14 d (4) 5% FMG-28 d (5) 10% FMG-28 d (6) 5% FMG-42 d (7) 10% FMG- 42 d (8) 5% FMG-56 d nd (9) FMG 10%-56 d. The mushroom mixture utilized in this study consisted of Lentinus edodes (Shiitke), Gnodermn lucidum (Reishi), Pleurotus ostretus (Oyster) nd Cordyceps sp. All FMG types were evenly mixed nd fed t the 5 nd 10 percent inclusion level of blnced bsl mel rtion (Tble 1). All chicks were vccinted ginst Mrek s disese, infectious bronchitis nd Newcstle disese prior to leving the htchery. Ech tretment group ws Tble 1: Composition of bsl diets Amount Ingredients Strter Grower Finisher Corn Soyben mel Corn micro-flush Limestone fine Diclcium phosphte (18.5%) Lysine (78.5%) Methionine (99%) Threonine Slt PX NCSU Br Minerl (TM90) Choline chloride (60) PX NSCU Br Vitmin (NCSU90) Selenium Premix NCSU (0.02%) Poultry ft (Miter) Totl btch weight housed nd mintined within 27 floor pen system 2 contining unused wood shving in ech (1.2 m ) pen with drinker nd one hnging feeder. Body weights of mles nd femles, mortlity, blood, fecl smples for oocyst nd bifidobcteri counts were collected nd evluted t 49 dys of ge. Mushroom cultivtion, protein determintion nd bsl diet: Shiitke, cordyceps, reishi nd oyster were cultivted t the Mushroom Biology nd Fungl Biotechnology Lbortory t North Crolin A&T Stte University. Sterile sorghum grin ws seprtely inoculted with selected fungi, t 25 C for 2, 4, 6 nd 8 weeks before use (Willis et l., 2010). The resulting mycelited grin ws processed by ir-drying t bout 25 C for pproximtely 6 hours nd ground into powder tht ws used for supplementing the bsl rtion in the experimentl trils. Smples were collected from the different mushrooms t different incubtion times nd sent to commercil lbortory for protein ssessment. The bsl diet composition is shown in Tble 1. The four different fungi were weighed nd put into the bsl diet t 5 nd 10 percent levels, mixed thoroughly nd plced into tube feeders in respective tretment pens. The bsl mel rtion ws free of ny mediction. The chickens were fed d libitum FMG which hd been inoculted with combintion of four species of mushroom spwn tht hve documented helth ttributes. Blood differentil cell count: For determintion of blood differentil cell counts, peripherl blood smples were collected vi the broilers jugulr veins in vcuum tubes contining EDTA to prevent clotting. Blood smered slides were prepred, llowed to dry nd then fixed nd stined with HEMA3 Stin. A totl of one hundred cells were counted nd the results were expressed s percentges of lymphocytes, heterophils, eosinophils nd bsophils. Whole blood smples for ELISA IgA nd IgG were obtined from broilers per tretment group t 207

3 d 49. The collected blood ws put on ice nd trnsported to lbortory for processing. Serum ws seprted from whole blood by centrifugtion, decnted nd stored o in minus 80 lbortory freezer until nlyzed. Chicken IgA nd IgG ELISA kits were used to quntify the mount of IgA nd IgG ntibody levels present within the collected serum smples. Both stndrds nd smples were nlyzed ccording to the Chicken IgA (Ct. No. E33-103) nd Chicken IgG (Ct. No. E33-104). Eimeri fecl nlysis: Fecl smples were collected t dy 49 from four birds in ech tretment tht hd been plced in cges prior to collection. The smples were trnsported to the lbortory where two grms of feces per tube were mesured out onto smll scle nd then plced into cler, sterile continer in which 30 ml sodium nitrte ws conjugted. The mixture of solution nd fecl mtter ws strined through cheese cloth nd the mixture ws pipetted nd trnsferred to the chmbers of McMster s slide. A totl of 5 minutes elpsed in order for the eggs to rech the surfce of the chmbers of the slide. A microscope using 10x lens nd 10x eye piece ws used to red the McMster s slide. The totl number of eggs in the two chmbers ws multiplied by 50, thus indicting the eggs-per-grm for ech smple (Hodgson, 1970). Bifidobcteri fecl nlysis: The popultion of Bifidobcteri in fecl smples ws determined using the stndrd lbortory method (Ibrhim nd Slmeh, 2001; Brown et l., 2005). Fecl smples were collected from floor droppings t four nd five weeks of the experimentl tril nd trnsported to the lbortory for nlysis. The smples (11 g ech) were diluted with 99 ml sterilized 0.1% peptone wter nd homogenized using stomcher 400 lb system 4 for 2 min nd 100 µl of pproprite dilution ws plted onto modified BIM 24 gr. Pltes were incubted t 35 C for t lest 3 d to llow for bifidobcteri cell growth. In ddition, the Grm Stin techniques were used to fcilitte microscopic exmintion of morphologicl chrcteristics of bifidobcteri. Fructose 6-phospnloketlse ctivity ws mesured to confirm the identity of the bifidobcteri. Sttisticl nlysis: Dt nlysis ws crried out using SPSS version 17.0 (SPSS Inc., Chicgo, IL). Anlysis of Vrince (ANOVA) ws performed to detect ny significnt differences between the tretment groups. The Duncn multiple rnge test ws subsequently used to detect the source of difference in the ANOVA output. Men vlues were considered significntly different t (P<0.05). Dt re expressed s men vlues±sem. The criticl level for the null hypothesis rejection in ll the sttisticl test ws 5% ( = 0.05). RESULTS AND DISCUSSION Protein percent of mushrooms: The effect of incubtion growth time on the percent protein is shown in Tble 2. There were significnt (P<0.05) differences in protein levels of tretments. The dt for the mushroom species showed decline or decrese s incubtion time incresed for most species, except for Reishi. As such, the feeding t vrious levels of inclusion ws expected to significntly decrese body weights. However, this ws not cler cut cse, s body weights were somewht constnt. The mixture of different types is believed to blnce the protein decline overll of the supplemented product into the bsl rtion t the levels of inclusion. Body weights: The effects of dietry supplementtion with FMG on body weights of mle nd femle broiler chickens re presented in Tble 3. Body weights of mle broilers differed significntly (P<0.05) in ll experimentl tretments when compred to the Control-No FMG; wheres the femles did not differ. The mixtures incubted for different periods of time did not show ny decline in body weights s ws shown in protein percent decline over extended periods of incubtion (Tble 2). Even t the 10% level of inclusion, no significnt differences (P>0.05) were observed with the four-wy mixture which is in contrst to previous reserch showing significnt decline in weight using single fungus t the 10% inclusion level. Moreover, s demonstrted in previous work by Willis et l. (2010), individul mushroom inclusion t the 5% level ws comprble to the control groups. The fct tht the femles in ech tretment did not differ from ech other Tble 2: Protein level of mushroom for different incubtion times Mushroom Incubtion dys Protein (%) Shiitke ±0.234de ±0.130de ±0.227b ± ±0.338 Cordyceps ±0.344de ±0.391de ±0.266e ±0.146b ±0.121b Oyster ±0.377de ±0.292de ±0.283cd ±0.551bc ±0.373bc Reishi ±0.372de ±0.363de ±0.188e ±0.757de ±0.367de Ech vlue is expressed s men±stndrd error (n = 3) Different letters in ech column indicte significnt differences t P<0.05 ccording to the Duncn Sttistics 208

4 Tble 3: Body weight, feed intke nd bifidobcteri popultion Body weight, kg 10 Bifidobcteri popultion, 1og cfu s Feed Trts Mle Femle intke (kg) Week 4 Week ±0.115b 2.552± ±0.139b 7.403±0.003c 7.690±0.083b ± ± ± ±0.066d 7.487± ± ± ±0.135b 6.723± ±0.032bc ± ± ± ±0.023e 8.070±0.015c ± ± ± ± ± ± ± ± ±0.137b 7.857±0.085bc ± ± ± ±0.024b 7.960±0.035c ± ± ±0.271b 6.430± ±0.114c ± ± ±0.132b 6.563±0.042b 7.560±0.055 Ech vlue is expressed s men ± stndrd error (n = 3). Different letters in ech column indicte significnt differences t P<0.05 ccording to the Duncn Sttistics presents chllenge s to why this occurred. At this time, no explntion is offered; however, Willis et l. (2007) demonstrted tht mle nd femle chicken dministered mushroom extrcts responded differently with regrds to body weight gin. Feed intke: The verge feed intke per bird is shown in Tble 3. There were some significnt (P<0.05) differences observed mongst tretments. The birds receiving feed from tretments 2, 4, 5, 6, 7 consumed less feed compred to the control (tretment 1). As mentioned previously, the verge protein for the control ws 7.42% which declined s the incubtion period incresed. Therefore, less feed intke ment less protein intke, thereby reducing weights. Bifidobcteri popultion: Bifidobcteri popultion results re presented in Tble 3. There were significnt (P<0.05) differences for weeks 4 nd 5 mongst tretments. Tretment 4 yielded the highest bifidobcteri popultion for both weeks. As noted in previous reports by Willis et l. (2009), bifidobcteri in chicken feces will not rise fr bove 8.00 cfu s t log10 nd indeed, the level pproched 8.00 cfu s in most tretment groups. This beneficil bcteri in the gut generlly supports good gut helth by competitive exclusion of hrmful bcteri from the gut of chickens (Ohimin nd Ofungo, 2012). Oocyst counts: The oocyst counts re shown in Tble 3. The egg count ws low nd consistent in ll tretments except 2 which ws significntly (P<0.05) higher thn the control nd ll others. It ws lso observed tht the lowest numericl IgA vlues (dt not shown) nd lymphocyte percent ws ssocited with tretment 2. The reson why this tretment hs the highest nd lowest vlues for oocyst count nd IgA level is not known t this time nd requires further investigtions. In recent experiment conducted by Willis et l. (2013), there ws very low level of nturl oocyst shed from broilers tht were fed different types nd levels of mushrooms which is in greement with the findings in this pper. Tble 4: Oocyst count, immunoglobulin (IgG) nd percent mortlity Trts Oocyst counts, IgG, ng/ml Mortlity 6 eggs/g 1og10 y (%) ± ± b b ± ± b ± ± bc 4 783± ± cd 5 933± ± d ± ± b ± ± bc ± ± ± ± bcd 3.33 Ech vlue is expressed s men±stndrd error (n = 3) Different letters in ech column indicte significnt differences t P<0.05 ccording to the Duncn Sttistics Blood differentil percentges: Blood differentil percents re shown in Tble 4. There were significnt differences in ll components of blood evlution. The most notbly ws seen with the heterophiles of tretment 6, 7, 8 nd 9 which were significntly higher thn the control. Differences were lso observed in the monocytes. Recently, reserch results from Willis et l. (2010) observed no dose response influence by feeding different levels of shiitke FMG on heterophils nd lymphocytes percentges. According to published work by Cmpbell (2007), helthy birds re expected to hve more lymphocytes thn heterophils in circultion which would influence the H:L rtio. However, Mxwell nd Robertson (1995) noted tht the numbers of heterophils increse during stressful conditions nd becuse of tht, the H:L rtio cn be used to detect the presence of physiologicl stress. Although, no experimentl chllenge ws involved here, birds ppered to be helthy bsed on performnce trits. So these results re inconclusive, becuse the vritions in hemtologicl vlues could be dependent on mny fctors tht influence the physiologicl stte of the broiler chickens. Mortlity nd serum IgA nd IgG levels: The percent mortlity for ech tretment is shown in Tble 3. There ws no significnt mortlity relted to tretments. The serum IgA levels were not significntly (P<0.05) influenced by the FMG supplementtion (dt not 209

5 Tble 5: Blood differentil percent of seven week old broilers Trts. # Heterophiles (%) Monocytes (%) Lymphocytes (%) Bsonophils (%) Eosinophils (%) ± ± ± ± ±0.376b ±0.607b ±1.151bcd ± ± ± ±0.140b ±0.543bcd ± ± ± ±2.014bc ±1.245bcd ± ± ± ±1.342b ±2.001bc ± ± ± ±2.939bc ±1.498b ± ± ± ±3.196bc ±2.490b ± ± ± ±4.367c ±2.754d ± ± ± ±2.589bc ±2.216cd ± ± ±1.819 Ech vlue is expressed s men ± stndrd error (n = 3) Different letters in ech column indicte significnt differences t P<0.05 ccording to the Duncn Sttistics shown). However, there ws trend towrd higher Cmpbell, T.W., Avin nd Exotic Animl elevtion trend compred to the control group. Therefore, rd Hemtology nd Cytology. 3 edition. Wiley, John & some helth nd performnce enhncements were Sons, Inc. relized with the higher immunoglobulin levels. There Dlloul, R.A., H.S. Lillehoj, J.S. Lee, S.H. Lee nd K.S. were significnt (P<0.05) IgG concentrtion differences Chung, Immunopotentiting effect of mongst tretments groups (Tble 4). Tretments 5 nd fomitell frxine-derived lectin on chicken immunity 6 produced higher IgG concentrtions thn ll other nd resistnce to coccidiosis. Poult. Sci., 85: 446- tretments with the highest. The recovery of oocyst 451. demonstrted low chllenge in ll tretments; thus, Guo, F.C., R.P. Kwkkel, B.A. Willims, H.K. Prmentier, the level of specific IgG in the ser of chickens my be W.K. Li, Z.Q. Yng nd M.W.A. Verstegen, ttributed to the increse in bet-glucns t the 42 d Effects of Musrhoom nd Herb Polyscchrides on incubtion period. Lee et l. (2009) demonstrted tht Cellulr nd Humorl Immune Responses of feeding chicks with hyper immune egg yolk, contining Eimeri tenell-infected Chickens. Poult. Sci., 83: IgY (IgG) ginst E. tenell nd E. mxim, provided them with significnt protection ginst coccidiosis. Ibrhim, S.A. nd M.M. Slmeh, Simple nd rpid Guo et l. (2004) sw higher production of IgG in method for screening ntimicrobil ctivities of infected nd non-infected E. tenell chicks fed Bifodbcterium species of humn isoltes. J. Rpid polyscchride extrcts. The effect of FMG on immune Methods Autom. Microbiol., 9: response does not pper to be relted to the inclusion Hodgson, J.N., Coccidiosis: Oocyst-counting levels of the FMG. Since body weights nd oocyst counts technique for coccidiostt evlution. Exp. did not differ gretly, further experiments re needed with Prsitol., 28: experimentl coccidil chllenged chicks to determine Lee, S., H. Lillehoj, D. Prk S. Jng A. Morles, D. n immune response to coccidiosis. Grci, E. Lucio, R. Lrious, G. Victori, D. Mrrufo nd E. Lillehoj, Protective effect of Conclusion: It is concluded tht mushroom incubtion hyperimmune egg yolk IgY ntibodies ginst time, with declining protein percent, does not imprt Eimeri tenell nd Eimeri mxim infections. Vet. declining body weights t the 5 nd 10% inclusion Prsitol., 163: levels. Additionlly, no impirment of helth nd well- Mxwell, M.H. nd G.W. Robertson, The vin being were cited in ssocition with level nd incubtion bsophilic leukocyte: A review. World s Poult. Sci. J., time during the preprtion time of the different FMG. 51: From this body of work nd the verge protein, 7.85% Montgne, L., J.R. Pluske nd D.J. Hmpson, A nd bet-glucn level of 202 mg/g t d 28; this tem of review of interctions between dietry fibre nd the scientists recommend the usge of the 28 d incubted intestinl mucos nd their consequences on FMG of the combined mixture t the 5% inclusion level. digestive helth in young nonruminnt nimls. Anim. Feed Sci. Technol., 103: REFERENCES Borchers, A.T., C.L. Kenn nd M.E. Gershwin, Mushrooms, tumors nd immunity: An updte. Exp. Biol. Med., 229: Brown, A., C. Shovic, S.H. Ibrhim, P. Holck nd A. Hung, A non diry probiotic influence on chnging the gstrointestinl trct s microflor environment. Alterin. Ther. Helth Med., 11: Ohimin, E.I. nd R.T.S. Ofongo, The effect of probiotic nd prebiotic feed supplementtion on chicken helth nd gut microflor: A review. Int. J. Anim. Vet. Adv., 4: Selegen, M., M.V. Putz nd T. Ruge, Effect of Polyscchride extrct from the edible mushroom Pleurotus ostretus ginst Infections Bursl Disese virus. Int. J. Mol. Sci., 10:

6 Willis, W.L., O.S. Isikhuemhen nd S.A. Ibrhim, Performnce ssessment of broiler chickens given mushroom extrct lone or combintion with probiotics. Poult. Sci., 86: Willis, W.L., K. King, O.S. Isikhuemhen nd S.A. Ibrhim, Administrtion of mushroom extrct to broiler chickens for bifidobcteri enhncement nd Slmonell reduction. J. Applied Poult. Res., 18: Willis, W.L., O.S. Isikhuemhen, S. Ibrhim, R.C. Minor, S. Hurley nd E.I Ohimn, 2010 Compring the Feeding of Fungus Myceilited Grin with other Anticoccidil Control Mesures on Oocyst Excretion of Eimeri Chllenged Broilers. Int. J. Poult. Sci., 9: Willis, W.L., O.S. Iskihuemhen, S. Hurley nd E.I. Ohimin, Effect of Phse Feeding Supplementl Fungus Mycelited Grin on Oocyst Excretion nd Performnce of Broiler Chickens. Int. J. Poult. Sci., 10: 1-3. Willis, W.L., D.C. Wll, O.S. Isikhuemhen, S. Ibrhim, R.C. Minor, J. Jckson, S.L. Hurley nd F. Anike, Effect of Different Mushrooms Fed to Eimeri- Chllenged Broilers on Rering Performnce. Int. J. Poult. Sci., 11: Willis, W.L., D.C. Wll, O.S. Isikhuemhen, J.N. Jckson, S. Ibrhim, S.L. Hurley nd F. Anike, Effect of Level nd Type of Mushroom on Performnce, Blood Prmeters nd Nturl Coccidiosis Infection in Floor-Rered Broilers. Open Mycol. J., 7: