Differential expression of growth and immunity related genes influenced by in ovo supplementation of amino acids in broiler chickens

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1 Czeh J. Anim. Si., 59, 2014 (9): Originl Pper Differentil expression of growth nd immunity relted genes influened y in ovo supplementtion of mino ids in roiler hikens S.K. Bhnj, M. Sudhgr, A. Goel, N. Pndey, M. Mehr, S.K. Agrwl, A. Mndl Centrl Avin Reserh Institute, Iztngr, Indi ABSTRACT: The present study ws imed t investigting the role of in ovo dministered mino ids: lysine, rginine, threonine or methionine plus ysteine (Met+Cys) in 14-dy emryos on expression profile of growth (hiken growth hormone (GH), insulin like growth ftors (IGF) I nd II, nd muin) nd immunity relted genes (IL-2, IL-4, IL-6, IL-12, TNF-α, nd IFN-γ). On inution dy (ID) 18, higher (P < 0.01) GH nd muin gene expression ws oserved in lysine, threonine, rginine or Met+Cys injeted emryos, while IGF-II expression ws higher in threonine, rginine or Met+Cys injeted emryos on ID 20. Expression of growth genes ws down regulted (P < 0.01) on dy of hth in most of the mino ids injeted hiks. On dy 7 post-hth (PH), threonine or rginine exhiited higher expression of GH, IGF-I, nd IGF-II ut higher muin gene expression only on dy 14 PH. Threonine or Met+Cys injeted irds hd higher expression of IL-6 nd TNF-α, while rginine injeted irds hd higher TNF-α expression. Lysine, threonine or Met+Cys injeted irds hd higher IL-2, ut lower of IL-12 nd IFN-γ gene expression. It is onluded tht rginine nd threonine enhned the expression of growth relted genes, while threonine nd Met+Cys modulted expression of immune genes in roiler hikens. Keywords: in ovo injetion; ritil nutrients; growth genes; immunity genes; roilers INTRODUCTION Nutritionl nd hormonl ftors re the mjor determinnts of niml growth during fetl nd post-ntl development. The dietry intrinsi ftors nd relted metoli intertions hve diret s well s indiret influene on growth vi expression nd regultion of genes. Moreover, the genes present on the somtotropi xis respond to nutrition differently t the level of trnsription (Bevin et l. 2001). The growth hormone (GH) tht regultes the growth exerts its effets minly y insulin-like growth ftor-i (IGF-I), whih is synthesized in the liver under GH ontrol nd sereted into the irultion (M Murtry et l. 1997). Protein or energy defiieny leding to redued growth veloity ws ssoited with lowered levels of plsm IGF-I (Strus nd Tkemoto 1990, 1991). Wheres dietry supplementtion of high protein diet with rginine, methionine, nd ysteine enhned plsm IGF-I levels nd ody weight gin in young hiks (Kit et l. 2002). Thus the erly essiility to ritil dietry nutrients would e enefiil, ut it depends upon the initition of food onsumption. In ontrst, in the se of feeding the young irds with ritil nutrients y in ovo injetion, the initition of the feed intke does not depend on the ird. Previous studies lso suggested tht in ovo supplementtion of mino ids signifintly inresed growth in roiler Supported y the Deprtment of Biotehnology, Ministry of Siene nd Tehnology, Government of Indi (Projet No. BT/PR9519/AAQ/01/345/2007). 399

2 Originl Pper Czeh J. Anim. Si., 59, 2014 (9): hiks (Bhnj et l. 2004; Bhnj nd Mndl 2005; Kdm et l. 2008; Bkyrj et l. 2012). The development of muus sereting ells in smll intestine of hikens ours during lte emryoni ge nd immeditely fter post-hth. Muin is the min onstituent of the muus lyer whih n influene nutrient digestion nd sorption. Dietry omponents hve the potentil to indue hnges in muin dynmis. The presene of nutrient in gstrointestinl trt (GIT) is lso ruil for muosl development nd feed deprivtion for the first 48 h post hth (PH) resulted in delyed development of the muosl lyer nd muin synthesis in young hiks (Uni et l. 2003). Even the intr-mnioni nutrient dministrtion elerted smll intestine development nd hd n enhned effet on the funtion of enteroytes (Tko et l. 2004; Uni nd Fekret 2004). In reent work, it hd een reported tht Thr defiient diet redued MUC2 gene expression in duklings tht used hnges in muin seretion ffeting intestinl nutrition nd sorption (Horn et l. 2009). Besides growth, nutrients n lso ply n importnt role in funtioning of the immune system. Upon tivtion, the nive T ells (Th0) differentite into either T helper 1 (Th1) ells primrily responsile for ell medited immunity nd inflmmtion, or Th2 effetor ells, eliiting humorl immunity y sereting different ytokines whih medite very different kind of immune responses (Spellerg nd Edwrds 2001; Steheli et l. 2001). Few studies hve een onduted to evlute the effet of in ovo mino id dministrtion on the PH performne ut snty informtion is ville on the role of these nutrients on the expression profile of growth (hiken GH, IGF-I nd II, nd muin) nd immunity relted genes (IL-2, IL-4, IL-6, IL-12, IFN-γ, nd TNF-α) in roiler hikens. Emphsis is now lid on investigting the role of nutrients in growth proess t moleulr level. In ddition, it will e interesting to see whether the nutrients would e le to modulte the expression of ytokines responsile for humorl or ellulr immunity in hikens. Keeping in view the ove fts, n effort ws mde to ssess the effet of in ovo injetion of ritil mino ids (lysine: Lys, threonine: Thr, rginine: Arg, methionine nd ysteine: Met+Cys) on the expression profile of growth nd immunity relted genes during lte emryoni nd erly PH period. Result of the present study will help in preising the diet for etter growth nd immune responsiveness of neontl hiks. MATERIAL AND METHODS Experimentl mteril nd tretments. The study ws onduted using rystlline mino ids: Lys (55 mg), Thr (40 mg), Arg (62.5 mg), nd Met+Cys (45 mg t 1 : 1 rtio) s in ovo supplement. Fertile eggs (n = 500) were olleted from roiler reeder floks mintined on lned rtion s per Ntionl Reserh Counil reommendtions (NRC 1994) nd were distriuted into five groups (four mino ids nd one uninjeted ontrol) of 100 eggs eh. In our erlier study on in ovo feeding (Bkyrj et l. 2012) no sttistil vrition ws oserved in the response of uninjeted ontrol nd shm ontrol (only pleo with no nutrients), so to redue the smpling size we hd exluded shm ontrol from the present experimentl design. After olletion, the eggs were fumigted with formldehyde gs (t 1X onentrtion) nd inuted in fored drft inutor t temperture of 37.5 C with reltive humidity of 60%. In ovo mino id injetion. On inution dy (ID) 14, 0.5 ml of wrm (30 C) mino id solution ws injeted into the mnioti vity through pinhole mde t the rod end of the egg, using 24G hypodermi needle (25 mm long) following the tehnique of Bhnj et l. (2004). The whole injetion proedure ws rried out under lminr flow system nd the injetion site ws seled with sterile prffin wx. Immeditely fter injetion the eggs were returned to the inutor nd on ID 19, the eggs were trnsferred to the hther nd pled in pedigree-hthing oxes. The hthed hiks were rered in eletrilly heted ttery rooders nd provided stndrd diets nd mngement till 28 dys of ge. Tissue olletion for growth relted gene expression studies. Four emryos or irds from eh tretment group were killed through ervil dislotion to ollet 200 mg of liver (for hiken growth hormone (GH), IGF-I nd IGF-II gene expression) nd intestine tissue (jejunum, for muin gene expression) on ID 18 nd 20, on dy of hth (DOH), nd on dys 7 nd 14 PH. The tissue smples were then homogenized in n utomted Kinemti TM Polytron TM homogenizer PT 1200 E (Thermo Fisher Sientifi, In., Wlthm, USA) nd 50 mg of homogenized tissues were olleted for totl RNA isoltion. For ontingeny uses, out 150 mg of tissue were immersed in 500 µl of RNA stilizing solution in 1.5 ml entrifuge mirotues. These smples were kept t 4 C over- 400

3 Czeh J. Anim. Si., 59, 2014 (9): Originl Pper night nd then trnsferred to deep freezer nd kept t 80 C for further use. Colletion of smples for immune relted gene expression studies. Cndidte genes for humorl immunity: To quntify the ndidte genes of humorl immunity (IL-6, IL-10, nd TNF-α), four in ovo supplemented irds were hllenged with 1 ml of 1% (V/V) sheep red lood ells (SRBC) on dy 21 PH nd lood smples were olleted fter 5 dys post-injetion. Approximtely 1.5 ml of lood from eh ird ws mixed with 3 ml of PBS (1 : 2 dilutions). An mount of 3 ml of Histopque (Sigm Dignostis In., St. Louis, USA) ws tken in 15 m onil entrifuge tue nd the diluted lood ws refully lyered over the olumn nd entrifuged t 1800 g for 20 min t room temperture. After entrifugtion the opque interfe ontining peripherl lood mononuler ells (PBMC) ws refully trnsferred to len onil entrifuge tue. 10 ml of isotoni PBS solution ws dded, mixed y gentle spirtion, nd then entrifuged t 2500 g for 10 min. After entrifugtion, the pellet ws dissolved in denturing solution for totl RNA isoltion. Cndidte genes for ell-medited immunity: For quntifition of ellulr immune relted genes (IL-2, IL-12, nd IFN-γ), pproximtely 1.5 ml of lood ws olleted from the jugulr vein of nother four irds per group on dy 21 PH nd the PBMC ells were seprted (s detiled erlier). Viility of these ells were determined y Trypn lue (0.4%) stining methods. Cells were resuspended in the known volume of RPMI-1640 (Sigm Dignostis In.) to mke the finl onentrtion of 106 ells per ml of medium. PBMCs were plted in 6 well ulture pltes with RPMI-1640 (without phenol red) medium, supplemented with 10% FBS, 2mM l-glutmine, 2mM l-rginine under 5% CO 2 tension in humidified tmosphere. The PBMC ells were sensitized to 10 µg/ml Connvlin A for period of 4 h, then the PBMCs dhered to ulture pltes were hrvested into 1.5 ml Eppendorf tue (Eppendorf, Hmurg, Germny), nd entrifuged t 5000 g for 1 min. After entrifugtion, the pellet ws dissolved in denturing solution for totl RNA isoltion. RNA isoltion nd reverse trnsription. Totl ellulr RNA from liver nd jejunum of eh tretment group ws isolted using RNAgents Totl RNA Isoltion System (Promeg, Mdison, USA), purity nd quntity were ssessed y mesuring the optil density of eh smple t 260 versus 280 nm in nnodrop. Any possile tres of genomi DNA were removed y treting 5 mg of eh RNA smple with 5 U of RNse-Free DNse (Biogen Ide, In., Durhm, USA) t 37 C for 1 h. The DNse ws susequently intivted y inution t 65 C for 10 min. Eh DNse treted totl RNA smple (2 µg) ws reverse trnsried using the RevertAid First Strnd DNA Synthesis Kit (MBI Ferments, Hnover, USA) ording to the mnufturer s instrutions. The reverse trnsription retion ws rried out in finl volume of 20 µl. The resultnt DNA ws stored t 20 C for further use. Stndrdiztion of primers for PCR. The primers for this study were designed using DNASTAR Lsergene softwre (Version 5.0, 1997). The speifiity of primers ws heked y the NCBI lst progrm. The qpcr ssys were evluted y the genertion of stndrd urve. Clirtion urves for eh gene were done on n iq5 yler (Bio-Rd Lortories, Herules, USA) with five 10-fold seril dilutions (in triplites) nd were lulted y the Bio-Rd Optil System Softwre (Version 2.1, 2010) with the nlysis mode PCR se line sustrted. Amplifition effiieny (E) of qpcr retions ws lulted with the slope of the logliner portion of the lirtion urve ording to the eqution: E = 10 ( 1/slope) (Rsmussen 2001; Bustin et l. 2009). PCR produts for different genes were sequened nd the sequenes were sumitted to NCBI, USA or EMBL, UK for onfirmtion. The genes with the ssigned ession numers re reported in Tle 1. Expression of growth nd immune relted genes y rel time PCR. The mplifitions of growth nd immune relted genes were rried out using n iq5 yler (Bio-Rd Lortories) in 25 µl volume ontining 1X QuntiTet SYBR Green PCR Mster Mix (SYBR Green 1 dye, HotStrtTq DNA polymerse, nd dntps in optimized uffer omponents; Qigen GmH, Hilden, Germny), 0.2μM onentrtion of eh gene-speifi primer, nd 1 µl of DNA templte. PCR yling onditions inluded initil denturtion t 95 C for 10 min, followed y 40 yles of denturtion t 95 for 30 s, nneling for 30 s, nd extension t 72 for 45 s. For eh gene of interest, negtive nd positive ontrols were lso inluded. Negtive ontrols were smples in whih DNA ws not dded. A melting urve ws performed for eh smple fter ompletion of mplifition nd nlyzed in omprison to negtive nd positive ontrols to determine the speifiity of PCR retion. 401

4 Originl Pper Czeh J. Anim. Si., 59, 2014 (9): Tle 1. Oligonuleotide sequene of growth nd immune relted gene primers Gene 1 Sequene (5' 3') Mjor growth relted genes GH F-CACCACAGCTAGAGACCCACATC R-CCCACCGGCTCAAACTGC IGF-I F-GGTGCTGAGCTGGTTGATGC R-CGTACAGAGCGTGCAGATTTAGGT IGF-II F-GGCGGCAGGCACCATCA R-CCCGGCAGCAAAAAGTTCAAG Intestinl trt development relted gene Mu F-CTGGCTCCTTGTGGCTCCTC R-AGCTGCATGACTGGAGACAACTG Humorl immunity relted genes IL-4 F-AATGACATCCAGGGAGAGGTTTC R-GCTAGTTGGTGGAAGAAGGTACG IL-6 F-GAAATCCCTCCTCGCCAATCTGA R-TGAAACGGAACAACACTGCCATCT TNFα F-AGACCAGATGGGAAGGGAATGAA R-GAAGAGGCCACCACACGACAG Cell medited immunity relted genes IL-2 F-CCCGTGGCTAACTAATCTGCTG R-TGAGACACCAGTGGGAAACAGT IL-12 F-GCCGACTGAGATGTTCCTGG R-CCTTGCTTTTGTATTTCTTTGTGC IFNg F-AGCTGACGGTGGACCTATTATTGT R-CGGCTTTGCGCTGGATTC Referene gene 28S F-CAGGTGCAGATCTTGGTGGTAGTA R- GCTCCCGCTGGCTTCTCC Anneling temperture ( o C) Produt size (p) Aession numer E* (%) HE JN JN JN JN NM JN HE JN JN JN GH = hiken growth hormone, IGF = insulin-like growth ftor, Mu = muin, IL = interleukin, TNFα = tumor nerosis ftor lph, IFNg = interferon gmm, 28S = 28S rrna *effiieny (E) ws lulted y the slope of the stndrd urve y the eqution: E = 10 ( 1/slope) The reltive expression rtio (ER) of trget gene is omputed, sed on its rel-time PCR effiienies (E) or stti effiieny of 2, nd the yle threshold (Ct) differene ( ) of men ontrol versus eh unknown smple ( Ct ontrol tretment) s desried elow (Pfffl 2001) using 28S rrna s the referene housekeeping gene: Ct trget (ontrol tretment) (E trget) ER = Ct ref (ontrol tretment) (E ref) Sttistil nlysis. mrna expression levels (ER) of growth relted nd immune relted genes were nlyzed y One Wy ANOVA using the SPSS softwre pkge (Version 16.0, 2007). Differene in men vlues ws onsidered s signifint t the level of 95% (P < 0.05) nd 99% (P < 0.01). RESULTS Expression pttern of growth relted genes. During pre-hth (ID 18 or 20), the reltive expression of hepti GH gene ws inresed in ll the mino id (Lys, Thr, Arg, nd Met+Cys) injeted emryos ompred to uninjeted ontrol. While on DOH, hepti GH expression deresed (P < 0.01) in Lys or Thr treted emryos thn in the uninjeted ontrol. During PH on dy 7, Thr or 402

5 Czeh J. Anim. Si., 59, 2014 (9): Originl Pper Expression rtio () GH lysine threonine rginine metionine + ysteine uninjeted ontrol Expression rtio Expression rtio Expression rtio ID 18 ID 20 DOH 7D PH 14D PH ID 18 ID 20 DOH 7D PH 14D PH () IGF-I d () IGF-II (d) Muin ID 18 ID 20 DOH 7D PH1 4D PH d ID18 ID 20 DOH 7D PH 14D PH Figure 1. Reltive expression of hepti GH, IGF-I, IGF-II, nd intestinl muin genes during lte emryoni nd erly post-hth period. Expression rtio is the men of four oservtions ID = inution dy, DOH = dy of hth, D = dy, PH = post hth d signifint differenes (P < 0.05) Arg injeted emryos hd two fold higher hepti GH expression (P < 0.01) ut on dy 14 the expression ws similr in mino id (AA) injeted nd uninjeted ontrol (Figure 1). During pre-hth on ID 18 hepti IGF-I gene expression deresed (P < 0.05) in Lys, Thr or Arg injeted hiks, ut it inresed on ID 20. During PH, the expression inresed (P < 0.01) in ll the 403

6 Originl Pper Czeh J. Anim. Si., 59, 2014 (9): () Humorl immunity relted genes Expression rtio IL-4 IL-6 TNFα () Cell medited immunity relted genes lysine Expression rtio threonine rginine methionine + ysteine uninjeted ontrol 0 IL-4 IL-6 TNFα Figure 2. Expression rtio of humorl (IL-4, IL-6, TNFα) nd ell medited immunity (IL-2, IL-12, IFN-γ) relted genes. Vlues re men of four oservtions signifint differenes (P < 0.05) Arg injeted hiks nd in Lys or Thr injeted hiks on dy 7 PH when ompred to uninjeted ontrol (Figure 1). Expression of hepti IGF-II gene did not differ in AA injeted emryos on ID 18 ut on ID 20 higher (P < 0.01) expression ws oserved in Arg or Met+Cys treted emryos. During PH the expression ws higher in Thr or Arg injeted hiks on dy 7 PH (P < 0.01) nd in Arg injeted hiks on dy 14 PH (P < 0.05) thn in uninjeted ontrol hiks (Figure 1). On ID 18 nd dy 14 PH, higher (P < 0.01) intestinl muin gene expression ws oserved in Lys, Thr, Arg or Met+Cys injeted emryos or hiks, while no suh differenes were oserved on ID 20 or DOH. But on dy 7 PH, the expression deresed (P < 0.01) in Met+Cys hiks ompred to uninjeted ontrol (Figure 1d). Expression pttern of immune relted genes. No vrition ws oserved in the expression of IL-4 gene in AA injeted or ontrol hiks, however, the expression of IL-6 gene ws inresed (P < 0.01) in Thr, Arg or Met+Cys injeted hiks ompred to uninjeted ontrol group. The expression of TNF-α gene ws up regulted in Thr or Arg treted hiks ut down regulted in Lys treted hiks (Figure 2). Expression of IL-2 gene ws up regulted in Thr or Met+Cys treted hiks, while no vrition ws oserved in Lys or Arg injeted hiks in omprison to uninjeted ontrol group. The expression of IL-12 gene ws deresed (P < 0.05) in Lys or Met+Cys while no differene ws oserved in the expression of IFN-γ gene in AA injeted hiks when ompred to uninjeted ontrol group (Figure 2). DISCUSSION Growth hormone (GH) is neessry for differentition of musle, dipoytes, nd other ells to modulte development nd growth (Kim 2010). In the present study, inresed expression of hepti GH gene ws oserved in AA injeted emryos during pre-hth period, inditing n effetive wy to inrese the size of the hik without deresing hthility (Oht et l. 1999). The highest growth rte in domesti hiken ours during 404

7 Czeh J. Anim. Si., 59, 2014 (9): Originl Pper the lte emryoni to juvenile stges of development (Kuhn et l. 2002), nd injetion of the ritil AA (Lys, Thr, Arg or Met+Cys) might hve helped the developing emryo to overome the nutritionl stress, where yolk is the only soure. These results lso orrelted with the erlier studies where inrese in ody weight of roilers ws reported when GH ws dministered in ovo t speifi dys of emryogenesis (Komis et l. 1999). During PH, growth hormone n e very useful for the norml development of the emryo (King nd Snes 1986), whih ws quite evident from the present study s signifint inrese in the expression of GH gene ws oserved in Thr or Arg treted hiks. Erlier findings lso suggested tht in ovo dministrtion of ll 20 AA inresed hik weight y % (Oht et l. 2001; Bhnj et l. 2004). Even in ovo injetion of speifi AA (Lys+Arg, Lys+Met+Cys or Thr+Gly+Ser) indued y round 2.0% higher hik weight t hth nd y 10 13% higher ody weight t dy 21 PH (Bhnj nd Mndl 2005). In reent study lso higher PH ody weight ws oserved in in ovo Arg treted hiks (Bhnj et l. 2012). It is suggested tht IGF-I synthesis is GH-independent during emryogenesis (Tnk et l. 1996). This ws lso evident on ID 20, when oth IGF-I nd GH gene expression inresed in Thr, Arg, nd Met+Cys treted hiks tht might hve helped the emryos to hieve higher skeletl musle growth y reduing protein degrdtion nd/or y inresing protein synthesis (Toms et l. 1998; Conlon nd Kit 2002). Generlly, IGF-I nd -II re responsile for prolifertion of predipoytes, hondroytes, nd firolsts through mino id stimultion, gluose uptke, inresed DNA synthesis, tissue growth stimultion, nd overll emryogenesis regultion (MGuinness nd Cogurn 1990; Leh nd Rosselot 1992; Guerne et l. 2003). In the present study the expression of oth IGF-I nd IGF-II inresed in Arg treted groups, too. Vsnti (2009) reported tht reltive IGF-II expression ws higher in ontrol group thn in other tretments on DOH, ut the expression inresed with ge where ll PH supplemented groups showed higher reltive IGF-II levels thn ontrol. Previously, Uni et l. (2005) suggested tht in ovo injetion of Arg inresed roiler hthling weights, rest musle, nd improved hepti glyogen reserves over the ontrols. Dietry supplementtion of Arg enhned plsm IGF-I levels nd ody weight gin in young hiks (Kit et l. 2002). Similrly, there re reports tht GH mrna expression nd serum onentrtion were enhned y Arg, iotin, nd Lys supplementtion (Kim et l. 1991, 2004). Another AA whih lso enhned the GH nd IGF-I expression in our study ws Thr, tht hs speifi regultory role in the seretion of pnreti enzymes (mylse, trypsinogen, nd hymotrypsinogen) in hikens (Yng et l. 1989). Dietry Thr level hd lso signifint effet on the verge dily gin nd feed onversion rtio (Kdm et l. 2008). Moreover, Thr is preursor for synthesis of the mino id glyine required during prodution of uri id in hikens. Very few informtion is ville on the effets of dietry nutrients on muin synthesis in poultry speies. A deresed muin synthesis result of ompromised muus lyer ould redue nutrient utiliztion. It is lso reported tht in ovo rohydrte injeted emryos hd greter villus surfe re from dy 19 of inution until 3 dys PH, higher golet ell density of jejunum villi, nd higher muin gene expression thn the slineontrol irds (Smirnov et l. 2006). Certin mino ids suh s Thr, Ser, nd Cys re of prtiulr interest euse these re the key omponents in muin mino id kone (Gum Jr.1992). The hydroxyl group of Thr nd Ser is neessry for ester linkges to rohydrte groups tht mke up the mjority (50 80%) of the moleulr weight of muin (Montgne et l. 2004). When dietry Thr level inresed, there ws n inrese in intestinl golet ell density nd MUC2 expression (Horn et l. 2009). In the present study, signifintly higher expression of muin gene in Thr injeted hiks over ontrol during PH ws oserved. Further, higher expression of muin gene ws lso seen in Arg or Met+Cys injeted hiks, suggesting the presene of these mino ids might hve triggered the numer of golet ells nd prodution of idi muin in the GIT of hik emryo. The expression of muin gene is diretly relted with the development of smll intestine. In reent study, higher smll intestinl weight in Lys, Thr, nd Arg tretments ut lower weight in Met ws reported (Bhnj et l. 2012). These findings orroorted with our study wherein expression of muin gene ws inresed in Lys, Thr, nd Arg tretment while deresed signifintly in Met+Cys tretment. In the present study the expression of IL-4, IL-6, nd TNF-α ws higher in Met+Cys injeted irds thn in those of uninjeted ontrol nd lysine 405

8 Originl Pper Czeh J. Anim. Si., 59, 2014 (9): group, nd rehed signifint level only in IL-6 (for Met+Cys) nd TNF-α (for Lys). This is in line with the erlier report of Sirimongkolksem (2007), who suggested tht Met plys n importnt role in growth nd humorl immunity (ntiody prodution), nd Cys is in greter demnd thn lysine for humorl immunity. Cysteine lso dded to the glutthione nd essory protein produed y liver during the ute phse response (Grimle 2006). In nother study, oth Coidiosis nd Newstle Disese infetions were severe in hiks fed methionine defiient diets (Cook, 1991). Thr tretment ws found to inrese the expression of humorl immune genes (IL-4, IL-6, nd TNF-α), though IL-4 expression did not reh signifint level. Higher humorl immune response ould e ttriuted to higher Thr ontent in the immunogloulins (Tenenhouse nd Deutsh 1966). In the present study the expression of IL-2 gene inresed in Thr or Met+Cys tretment, possily relted to the ell-medited immunity (Kidd et l. 1996; Shym Sunder et l. 2008). In erlier studies signifint differene in ell medited immunity ws oserved in in ovo injetion of AA omintions ontining Lys, Thr or Met+Cys (Bhnj nd Mndl 2005; Bkyrj et l. 2012) nd they lso inresed the reltive weight of spleen (Bhnj et l. 2012). The expression of IL-12 gene deresed in ll the mino id injeted irds (Lys, Thr, Arg, nd Met+Cys) in omprison to uninjeted ontrol, ut it did not reh the signifint level in Thr nd Arg tretments. The expression of IFN-γ gene ws redued ut insignifintly in most of mino ids exept Arg in omprison to uninjeted ontrol hiks. There is lso ross regultion etween Th1 ells nd Th2 ells (Spellerg nd Edwrds 2001) nd it ws lso evident in the present study where expression of IL-4 suppressed the expression of IFN-γ nd it is lso reported tht IL-4 produed y Th2 ells lok the differentition of Th0 to Th1 (D Andre et l. 1993). CONCLUSION Bsed on the ove oservtion it ws onluded tht mino ids, prtiulrly rginine nd threonine, enhned the expression of growth relted genes (GH, IGF-I, IGF-II, nd muin gene) during pre- nd post-hth periods. Moreover, threonine nd sulphur ontining mino ids (methionine plus ysteine) plyed role in modulting immune gene expressions. There is need of further reserh on how these mino ids modulte t the protein level nd the result of this study ould e helpful in formulting the hik s diet for etter growth nd immunity. REFERENCES Bkyrj S., Bhnj S.K., Mjumdr S., Dsh B.B. (2012): Post-hth immunomodultion through in ovo supplemented nutrients in roiler hikens. Journl of Siene of Food nd Agriulture, 92, Bevin C., Chevlier B., Cogurn L.A., Simon J., Dulos M.J. (2001): Insulin-like growth ftors nd ody growth in hikens divergently seleted for high or low growth rte. Journl of Endorinology, 168, Bhnj S.K., Mndl A.B. (2005): Effet of in ovo injetion of ritil mino ids on pre- nd post-hth growth, immuno-ompetene nd development of digestive orgns in roiler hikens. Asin-Austrlsin Journl of Animl Siene, 18, Bhnj S.K., Mndl A.B., Johri T.S. (2004): Stndrdiztion of injetion site, needle length, emryoni ge nd onentrtion of mino ids for in ovo injetion in roiler reeder eggs. Indin Journl of Poultry Siene, 39, Bhnj S.K., Mndl A.B., Goswmi T.K. (2004): Effet of in ovo injetion of mino ids on growth, immune response, development of digestive orgns nd rss yields of roiler. Indin Journl of Poultry Siene, 39, Bhnj S.K., Mndl A.B., Agrwl S.K., Mjumdr S. (2012): Modultion of post-hth growth nd immunoompetene through in ovo injetion of limiting mino ids in roiler hikens. Indin Journl of Animl Siene, 92, Bustin S.A., Benes V., Grson J.A., Hellemns J., Huggett J., Kuist M., Mueller R., Noln T., Pfffl M.W., Shipley G.L., Vndesompele J., Wittwer C.T. (2009): The MIQE Guidelines: Minimum Informtion for Pulition of Quntittive Rel-Time PCR Experiments. Clinil Chemistry, 55, Conlon M.A., Kit K. (2002): Musle protein synthesis rte is ltered in response to single injetion of insulin-like growth ftor-i in seven-dy-old leghorn hiks. Poultry Siene, 81, Cook M.E. (1991): Nutrition nd immune response to the domesti fowl. Critil Review of Poultry Biology, 3, D Andre A., Aste-Amezg M., Vlinte N.M., M X., Kuin M., Trinhieri G. (1993): Interleukin 10 (IL-10) inhiits humn lymphoyte interferon-gmm prodution y suppressing nturl killer ell stimultory ftor/ 406

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