ISSN X Jan - Mar 2017 / v.19 / n.1 / Author(s)

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1 Brzilin Journl of Poultry Siene Revist Brsileir e Ciêni Avíol ISSN X Jn - Mr 2017 / v.19 / n.1 / Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 Author(s) Tomszewsk E I Muszyński S II Dorowolski P III Kwieień M IV Winirsk-Miezn A IV Świetlik I II V I Deprtment of Animl Physiology, Fulty of Veterinry Meiine, University of Life Sienes in Lulin, Lulin, Poln; II Deprtment of Physis, Fulty of Proution Engineering, University of Life Sienes in Lulin, Lulin, Poln; III Deprtment of Comprtive Antomy n Anthropology, Mri Curie-Skłoowsk University, Lulin, Poln; IV Deprtment of Bromtology n Foo Physiology, University of Life Sienes in Lulin, Lulin, Poln; V Deprtment of Animl Antomy n Histology, University of Life Sienes in Lulin, Lulin, Poln. Mil Aress Corresponing uthor e-mil ress Ew Tomszewsk University of Life Sienes in Lulin, Akemik 13, Lulin, Poln Tel: Emil: ewrst@interi.pl Keywors Bone, helte, hiken, histomorphometry, zin. Sumitte: April/2016 Approve: August/2016 Astrt The im of this stuy ws to evlute the effet of ing zin (Zn) minerl supplements from orgni (ZnGly) n inorgni (ZnO) soures to growing mle Ross 308 hikens on the mehnil, geometri, n histomorphometril prmeters of long ones. A orn-soyen mel sl iet ws supplemente with Zn t 50 or 100 mg kg 1 of premix, exept the ontrol group (0 suppl). The serum onentrtion of insulin-like growth ftor 1 ws higher in the ZnGly50 group ompre with the ontrol group. Positive influene of Zn on the femur is highlighte when onsiering the signifint inrese in prmeters suh s men reltive wll thikness, n mximum elsti n ultimte strengths fter Zn ministrtion. Zin supplementtion i not ffet tiil prmeters. The histomorphometri nlysis showe positive impt of Zn supplementtion (irrespetive of soure n level) on femorl treulr thikness. Rpi loss in tul one volume in tiil metphysel treule ws oserve with ZnGly t 50 mg kg 1. In onlusion, this stuy showe tht ietry Zn supplementtion positively influenes one mehnil properties, onfirming its enefiil effet on the evelopment of the skeletl system n one tissue of roilers teste for nutritionl-osteoporoti ftor. Averse helth effets in treulr one s result of the use of Zn t 50 mg kg 1 of the premix show tht supplementing Zn t the reommene ose (100 mg kg 1 ) is essentil. Introution There hs een n inresing emn for poultry met, n therefore, rering fst-growing n well-musle roiler rees is more profitle. However, the geneti seletion of roilers for musle eposition n growth rte hve use growth n one minerliztion normlities. During the short life of roilers their skeletl system unergoes intensive growth (Cook, 2000). The proper funtion of the skeletl system plys n essentil role in poultry proution, euse it not only provies struturl support for the ir ut it is lso n importnt minerl soure for metoli nees (Shrei et l., 2012). Moreover, lim ones re essentil for proper loomotor funtion. Bone eformities or even frtures resulting from insuffiient pttion of the skeleton to high oy weight re oserve. There re severl usl ftors tht often operte in prllel or ylilly, exerting the effets of negtive impt on one evelopment. These ftors inlue unstle n unlne ietry energy, protein n minerls, poisoning, hormonl isorers, n geneti preisposition (Cook, 2000). Beuse mle hikens hve hevier oy weights thn femles, the overloing or norml loing onitions of pelvi lim ones, resulting in struturl mge n eforme lower legs, re more ommon in mles thn in femles (Śliw et l., 1996; Tomszewsk et l., 2017). Aoring to Pz 159

2 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 (2013), femorl egenertion ours in young irs, oes not hve efine etiology, n n ffet one or oth legs. Moreover, in ue to their fst growth rte, isorers suh s one trophy, tiil yshonroplsi or honroystrophy re ommon in roilers (Śliw et l., 1996; Pz et l., 2013). Zin (Zn) is n essentil mironutrient for nimls, n ply speifi physiologil funtions in ll living systems, suh s the mintenne of struturl n funtionl integrity of iologil memrnes n filittion of gene expression n protein synthesis (Co et l., 2002; Prk el l., 2004). Zn is ritil for ell prolifertion n ifferentition, n it is require for the mintenne of immune funtions, enzyme struture n funtion, n ppetite regultion in ll vin speies. Nutritionl Zn efiieny in experimentl nimls results in elye sexul mturity n low testosterone levels (Root et l., 1979). There is very little reserh ville on the reltionship etween zin n femle sex hormones. However, some sientists foun link etween zin n proltin (high loo levels of zin inhiite proltin seretion), the hormone tht regultes wie vriety of iologil proesses in the ovry (Brnão-Neto et l., 1995). Moreover, Zn is neessry for norml growth n one evelopment, espeilly in poultry for foot helth (Co et l., 2002; Prk el l., 2004). It plys ruil role s tlyst of mny enzymes tht ffet one evelopment, suggesting its role in one isorers (Ymguhi & Go, 1998). Zn funtions s metl omponent of lkline phosphtse, metlloenzyme tht plys key funtion in the formtion of new one (Strher et l., 1980). Moreover, Zn inreses the synthesis of growth ftors, suh s insulin-like growth ftor 1 (IGF-1), n influenes the tivity of lium-regulting hormones (Lowe et l., 2002). Thus, Zn ppers to hve multiple importnt funtions in one evelopment, formtion, n metolism (Seo et l., 2010). Bones ontin out 30% of totl oy Zn (Molokwu & Li, 2006). In poultry, Zn efiieny results in insuffiient one minerliztion, skeletl mlformtion, n reution of weight gin (Shrei et l., 2012). In ition, Zn-efiient iets reue egg proution n hthility in lyers n reeers (Kienholz et l., 1961). In ition, zin is essentil for neurogenesis, synptogenesis, neuronl growth, n neurotrnsmission; it is store in speifi synpti vesiles y lss of glutminergi neurons n relese s neuro-moultor in n tivity-epenent mnner (Mret & Snste, 2006). Poultry iets re ommonly supplemente with zin, euse mny nturl fee ingreients re mrginlly Zn-efiient (Fuge & Speer, 2001). Therefore, poultry 160 iets require supplementtion to ensure equte intke of tre minerls. In the fee inustry, orgni tre minerl supplements hve een inresingly use. Zin oxie is one of the inorgni fee-gre zin soures ommerilly use y the poultry fee inustry; however, it is less ioville thn other inorgni ompouns, euse of its possile intertions with other fee ompouns in the hime (inhiitory effets of phyti i) (Co et l., 2002; Prk et l., 2004). Moreover, Zn my intert with lium n iron (Hmrie et l., 1986). For this reson, inorgni zin is use t onentrtions higher thn those reommene, whih my result in environmentl pollution (Lesson, 2003; Avign, 2014; Prk et l., 2004). The reommene level of ietry Zn use to e mg kg 1 for ifferent ommeril roiler strins, exept for growing mle Ross 308 hikens (NRC, 1994). Currently, ietry Zn supplementtion level is se on the nutritionl reommentions for Ross 308 roilers (Avigen, 2014) s 100 mg kg 1 of Zn, irrespetive of its ontent in the feestuffs. Amino-i heltes re reporte to hve signifintly higher sorption rtes in the intestine ompre with solule inorgni metl slts (Ashme et l., 1985). Furthermore, ue to their unique hemil struture, these heltes o not pper to ionize s reily s inorgni tre minerl soures uner vriety of onitions, whih results in reution or elimintion of their retivity with vrious fee omponents, like phosphte or other nturl ftors (Vieir, 2008; Hung et l., 2009). However, onfliting results hve een reporte regring the iovilility of Zn heltes n tritionl inorgni forms (Co et l., 2002). The pplition of the vrious heltes is regulte y EU Diretive 1334/2003 (EC, 2003). Although severe zin efiieny is unommon in vin popultions, mrginl efiieny is likely to e muh more prevlent n ssoite with immune ysfuntion or restrite physil evelopment. Reltive to the role of Zn, it is hypothesize tht the supplementtion of more ville form of Zn ompre with n inorgni form my improve the evelopment of the skeletl system of roilers. The ojetive of this stuy ws to evlute the effet of the ition Zn supplements from n orgni (ZnGly) or n inorgni (ZnO) soure in growing mle Ross 308 roiler iets on the mehnil, geometri, n histomorphometril prmeters of the long ones. The effet of ifferent (reommene-100 mg kg 1 n limite to 50 mg kg 1 of the premix) levels of supplementtion ws lso evlute in this investigtion.

3 2. Mterils n methos The experimentl proeures use throughout this stuy were pprove y the Lol Ethis Committee on Animl Experimenttion of the University of Life Sienes in Lulin, Poln Bir mngement n experimentl esign A totl of 250 one-y-ol mle Ross 308 roilers were rnomly llote to five Zn tretments with five replites (ges) of 10 irs eh, totling 50 irs per tretment. All ges were lote in the sme room. Birs were kept uner stnr rering onitions, n ir temperture ws set t the optiml level oring to ge, s estlishe in the Europen Counil Diretive 2007/43/EC (EC, 2007). During the first week, room temperture ws mintine t 33 C, n grully reue in 2 C weekly until rehing 24 C. Birs h onstnt ess to fresh wter, n fee ws supplie liitum. The iets were formulte in orne with the stge of the proution yle (Tle 1): strter (rumles, 1-21 ys), grower (pellets, ys), n finisher (pellets, ys). At the en of the experiment, when irs were 42 ys ol, 10 irs (two irs per replite x five replites) per tretment group were rnomly selete, n srifie. Before srifie, irs were fee-fste, ut were llowe free ess to wter. Birs were weighe efore plement in the experimentl fility n efore slughter Bsl iet n fee nlyses The sl iet ws se on orn n soyen mel, n formulte to ontin equte levels of ll nutrients, s reommene (NRC, 1994), exept for Zn. The nutrient ontent of the iets ws lulte se on the hemil omposition n metolizle energy vlues of the feestuffs, oring to the equtions of the Europen Tles (Kwieień et l., 2014). Dry mtter, rue sh, rue protein, rue ft, n rue fier ontents of the experimentl iets were etermine y stnr AOAC (2000) methos. Dietry Cu, Fe, Zn n C ontents in fee smples were etermine, fter shing t 550 C, y the AAS flme tehnique in spetrometer (Unim 939 AA, Shimzu Corp., Tokyo, Jpn), oring to the methos of AOAC (2000). Totl P ontent in the fees ws olorimetrilly etermine (PN-ISO 6491:2000) in Helios Alph UV-VIS pprtus (Spetroni Unim, Lees, Unite Kingom) (Kwieień et l., 2014). Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 Tle 1 Composition n nutritive vlue of the experimentl iets Ingreient (%) Strter (1-21y) Grower (22-35 y) Finisher (36-42 y) Corn Whet Soyen mel * Soyen oil Phosphte 1-lium , Foer hlk Soium ironte Soium hlorie Premix vit-min (no Zn) Conentrte protein-ft ** DL-methionine 99% L-lysine HCl L-threonine 99% The nutritionl vlue of 1 kg iet: e ME, MJ kg Totl protein,% Crue fier,% Crue ft,% Lysine,% Methionine + Cystine,% Threonine,% Thr:Lys rtio Totl lium,% Totl phosphorus,% e Aville phosphorus,% e Totl ville lium/ phosphorus Zn from plnts in sl iet, mg Iron, mg Copper, mg ontent of vitmins n minerls per 1 kg of Strter iet: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg, vitmin A UI, vitmin D UI, vitmin E 75 mg, vitmin K 3 4 mg, vitmin B 1 3 mg, vitmin B 2 8 mg, vitmin B 6 5 mg, vitmin B 12 0,016 mg, iotin 0.2 mg, foli i 2 mg, niotini i 60 mg, pntotheni i 18 mg, holine mg; ontent of vitmins n minerls per 1 kg of Grower iet: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0,15 mg, vitmin A UI, vitmin D UI, vitmin E 50 mg, vitmin K 3 3 mg, vitmin B 1 2 mg, vitmin B 2 6 mg, vitmin B 6 4 mg, vitmin B μg, iotin 0.2 mg, foli i 1.75 mg, niotini i 60 mg, pntotheni i 18 mg, holine mg; ontent of vitmins n minerls per 1 kg of Finisher iet: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg, vitmin A UI, vitmin D UI, vitmin E 50 mg, vitmin K 3 2 mg, vitmin B 1 2 mg, vitmin B 2 5 mg, vitmin B 6 3 mg, vitmin B μg, iotin 0,05 mg, foli i 1.5 mg, niotini i 35 mg, pntotheni i 18 mg, holine mg; nlyse vlues; e lulte vlues; * 46% Totl protein in ry mtter; ** 1 kg onentrte protein-ft ontins: 2% rue ft, 39% rue protein, 10,8 MJ EM The mino i omposition in the iets ws etermine y ion-exhnge hromtogrphy using n INGOS AAA 400 mino i nlyzer with post-olumn erivtiztion of ninhyrin n spetrophotometri etetion, oring to the stnr mnufturer s 161

4 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 proeure, n MCMiAŻ/PB-03 test proeure, s previously esrie (Kwieień et l., 2014). Cysteine n methionine (sulfur mino is) were etermine in seprte nlysis, s previously esrie (Kwieień et l., 2014). Aville lysine ws etermine se on the ifferene etween totl lysine n the solle resiul lysine, whih i not ret with DNFB (initrofluoroenzene) (Kwieień et l., 2014). Following this retion, the teste smples were gin sujete to i hyrolysis (Žili et l., 2006). The irs lso were weighe t 10, 21, 35 ys of ge (t not shown) to etermine fee intke (FI) n fee onversion rtio (FCR) Supplementtion of Zn mino i helte n Zn oxie Birs were istriute into ontrol group (CONT=0 Suppl), whih iet ontine premix evoi of Zn itive, n groups supplemente with two ifferent Zn soures (Zn oxie s stnr Zn inorgni soure or Zn-glyine helte s Zn orgni form) n two Zn levels (100% or 50% of the reommene Zn level). Therefore, the following tretment groups were estlishe: ZnGly50 Zn-glyine t 50% of the reommentions (50 mg kg 1 of iet or 313 mg of helte/kg premix); ZnGly100 - Zn-glyine t 100% of the reommentions (100 mg kg 1 iet or 625 mg of helte/kg premix); ZnO50 Zn oxie t 50% of the reommentions (50 mg kg 1 iet or 64 mg of ZnO/ kg premix); n ZnO100 Zn oxie t 100% of the reommentions (100 mg kg 1 iet or 128 mg of ZnO/ kg premix). The Zn-glyine helte ontine 16% Zn, n zin oxie ontine 78% Zn, n were e to the premix whih i not ontin Zn (Arkop Sp. Z o.o., Bukowno, Poln). The nlyze Zn ontents of the sl iets (Tle 1) ws mg kg 1 of Zn on s-fe sis. The mount of Zn in the iet ws se on the nutritionl reommentions for Ross 308 roilers (Avigen, 2014), i.e., 100 mg kg 1 of Zn, irrespetive of its ontent in the sl iet. Aoring to those reommentions, iets shoul ontin the sme Zn levels uring the entire rering perio, whih ws tken into ount in this stuy Bone mesurements After slughter, the right n left tiie n femor were ollete, n their weight n length were mesure fter the removl of soft tissues. Eh lim one ws wrppe with guze soke in isotoni sline, n store t -25 ºC until further nlysis. Bone 162 weight/length inex ws lulte s weight (mg) to the length (mm) rtio. This is simple inex of one ensity introue for the first time y Seeor n oworkers. In generl, the higher the one weight/length inex, the enser is the one (Seeor et l., 1991; Ziie et l, 2011), n inites hnges in one minerliztion (Ziie et l. 2011; Ziie et l. 2011) Anlysis of one mehnil n geometri properties The mehnil properties of the ollete ones were etermine fter 3-hour thwing t room temperture y pplying the three-point ening test. A Zwik Z010 universl testing mhine (Zwik GmH & Compny KG, Ulm, Germny), equippe with mesuring he (Zwik GmH & Compny KG, Ulm, Germny) with n opertion rnge up to 10 kn, linke to omputer with test TestXpert II 3.1 softwre (Zwik GmH & Compny KG, Ulm, Germny) ws use, s previously esrie (Tomszewsk et l., 2013; Tomszewsk et l., 2015). Mximum elsti strength n ultimte strength were etermine s previously esrie (Ferretti et l., 1993; Tomszewsk et l., 2012). Bse on the mesurements of the horizontl n vertil imeters of the mi-iphysel ross-setion of the ones (t not shown), the ross-setionl re n men reltive wll thikness were erive (Ferretti et l., 1993). The ortil inex of the one ws estimte s previously esrie (Tomszewsk et l., 2015) Histomorphometry Cylinril smples (rtilge n one) with 20- mm thikness were tken from the sme ntomil position of the knee joint, i.e., from the mile of the lterl femorl n tiil onyle. Sgittl one setions were ut perpeniulr to the rtiulr surfe. The tissue smples were sujete to histology n mirosopy proeures, s previously esrie (Tomszewsk et l., 2013). Mirosopi right-fiel imges were ollete using onfol mirosope (Axiovert 200M, Crl Zeiss, Jen, Germny) equippe with mer (AxioCm HR, Crl Zeiss, Jen, Germny) n fluoresent lmp ( nm exittion wvelength). Bone volume n tissue volume (t not shown) were mesure in the photogrphs of the one tissue setions using pixel ount to etermine reltive one volume (BV/TV%), s previously esrie (Tomszewsk et l., 2015). Other treulr one

5 prmeters exmine were men n mximum treulr thikness (T.Th), n treulr seprtion (T.Sp), efine s the istne etween the eges of jent treule (iretly mesure) (Tomszewsk et l., 2012) Growth hormone n one turnover mrkers Eh hiken ws fste for 12 hours efore loo olletion y stnr venipunture from rhil vein. After lotting t room temperture, loo ws entrifuge n frozen t -80 C until further nlysis. All the smples were etermine in uplite. Serum onentrtions of hiken growth hormone, insulin-like growth ftor 1, osteolin, n leptin were etermine using n enzyme-linke immunosorent ssy kit (ELISA; Usn Life Siene In. Wuhn, Chin) with minimum etetle onentrtions of ng/ml, 7.4 pg/ml, 0.67 pg/ml, n 14.8 pg/ml, respetively Selete mro- n tre minerls in the serum The serum onentrtions of zin, lium, opper, iron, mngnese, n phosphorus were etermine y olorimetri metho using Metrol 2300 GL unit (Metrol SA, Argentin) n sets of iohemil regents proue y BioMxim (Lulin, Poln) Sttistil nlysis The results of the experiment re expresse s LSMEANS ± SEM (stnr error of the men). As our im ws to evlute the effet of n ition of orgni n inorgni zin minerl supplements, s well s ifferent levels of Zn supplementtion on the mehnil n histomorphometri prmeters n the geometry of long ones, the following sttistil moel ws pplie: x ij = µ + α i + ε ij Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 where: x ij = n oservtion (one prmeter vlue); i = onentrtion ftor (ZnO50, ZnO100, ZnGly50, ZnGly100, CONT); j = numer of oservtions; μ = onstnt; α i = the min effet of the i th level; ε ij = rnom error of the i th level of the j th oservtion. Beuse oth ftors were ontrolle, the onsiere moel is fixe-effet moel. One-wy nlysis of vrine ws pplie. Contrst nlysis ws use to ompre the results otine in Zn-supplemente groups vs. the ontrol group: (1) the ontrol iet (CONT- 0 Suppl) vs. the iets ontining inorgni Zn (ZnO50 n ZnO100); n (2) CONT vs. the iets ontining orgni Zn (ZnGly50 n ZnGly100), n to ompre Zn supplementtion level (50 or 100 mg) vs. no Zn supplementtion (ontrol group). Signifine ws elre t P<0.05. All sttistil nlyses were rrie out using Sttisti 10.0 softwre (SttSoft In., Tuls, OK, USA). 3. Results 3.1. Boy weight, fee intke (FI) n fee onversion rtio (FCR) Birs h pproximtely the sme initil oy weight n presente similr finl oy weight (Tle 2). Fee intke n FCR (fee intke per kg oy weight gin) t 10, 21, 35, n 42 ys of ge were not influene y Zn soure or level (t not shown) Bone morphology, geometry, n mehnil prmeters No ifferenes in femorl length, mss, n weight/ length inex were etete etween the ontrol n Zn-supplemente irs (Tle 3), irrespetive of Zn soure or level. However, the reltive one weight of irs fe 50 mg kg 1 ZnGly ws signifintly higher ompre with the ontrols. Higher femorl strength, men reltive wll thikness, n ortil inex were etermine in the ZnGly group ompre with the Tle 2 The initil oy weight fter hthing n the effet of inorgni or orgni zin ietry supplementtion on the finl oy weight of 42-y-ol Ross 308 roilers CONT= 0 Supp Diet SEM p-vlue ZnO100 ZnO50 ZnGly100 ZnGly ZnO ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Initil weight [g] Finish weight [g] Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. oth 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50

6 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 Tle 3 The effet of inorgni or orgni zin ietry supplementtion on the mehnil n geometri properties of the femur of 42-y-ol Ross 308 roilers. Diet SEM P-Vlue CONT ZnO100 ZnO50 ZnGly100 ZnGly50 ZnO ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Bone weight [g] Length [mm] Weight/length inex [mg/mm] Bone weight (g/100g of BW) Ultimte strength [N] Mx. elsti strength [N] Mishft Volume [m 3 ] <0.01 < < <0.01 Cross-setionl re [mm 2 ] Men reltive wll thikness Cortil inex [%] Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. Both 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50 BW oy weight ontrol group (0 Suppl), with higher vlues oserve for the ZnGly50 group (Tle 3). Signifintly higher ultimte strength n men reltive wll thikness vlues were otine in the ZnO50 group ompre with the ontrol group. Similrly, the ZnGly50 group presente lrger ross-setionl re ompre with the ontrol group. On the other hn, ZnO tretment influene signifintly the ross setion of re n lso with higher vlues note for the ZnO50 group. The iet ontining Zn, irrespetive of its form n onentrtion, i not influene tii evelopment (exept of reltive one mss) ompre with the ontrol, non-supplemente irs (Tle 4) Bone histomorphometry Histomorphometri nlysis revele tht the supplementtion of Zn s ZnO influene only femorl treulr thikness, with higher vlues for the ZnO100 group (Tle 5). On the other hn, tiil reltive one volume erese in the oth iets (ontining ZnO or ZnGly) ompre with the ontrol group (0 Suppl). Moreover, treulr thikness ws reue n Tle 4 The effet of inorgni or orgni zin ietry supplementtion on the mehnil n geometri properties of the tii of 42-y-ol Ross 308 roilers Diet SEM p-vlue CONT= 0 Supp ZnO100 ZnO50 ZnGly100 ZnGly50 ZnO 164 ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Bone weight [g] Length [mm] Weight/length inex [mg/mm] Bone weight (g/100g of BW) Ultimte strength [N] Mx. elsti strength [N] Mishft Volume [m 3 ] Cross-setionl re [mm 2 ] Men reltive wll thikness Cortil inex [%] Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. Both 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50 BW oy weight

7 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 Tle 5 The effet of inorgni or orgni zin ietry supplementtion on treulr one morphology of the metphysis of long ones of 42-y-ol Ross 308 roilers. CONT= 0 Supp Diet SEM P-Vlue ZnO100 ZnO50 ZnGly100 ZnGly50 ZnO ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Femur BV/TV [%] T.Th men [µm] T.Th mx [µm] T.Sp [µm] Tii BV/TV [%] <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 T.Th men [µm] <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 T.Th mx [µm] <0.01 < <0.01 <0.01 <0.01 T.Sp [µm] <0.01 < <0.01 <0.01 <0.01 Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. Both 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50 BV/TV% - reltive one volume; T.Th men the men treulr thikness; T.Th mx the mximl treulr thikness; T.Sp men - the men treulr seprtion; T.Sp mx - mximl treulr seprtion treulr spe ws enhne in the nellous one of tii, irrespetive of iets ompre with the ontrol group (0 Suppl) (Tle 5) Growth hormone, leptin, n one turnover mrkers The onentrtion of growth hormone, osteolin n leptin ws similr mong tretments. The onentrtion of insulin-like growth ftor 1 ws enhne only in the ZnGly50 group ompre to the 0 Suppl group (Tle 6) Selete miro- n mroelements in the serum The irs fe iets ontining Zn, irrespetive of its onentrtion n form, presente similr serum levels of selete mro n tre elements (exept for opper) to those reeiving the ontrol iet with no supplementtion (Tle 7), exept for opper, whih serum level ws higher in the ZnGly50 ompre with the ontrol group. 4. Disussion Tre minerls, suh s zin, re importnt for vriety of physiologil proesses tht re essentil for optiml ir growth n evelopment. The response of one evelopment to ifferent ietry Zn forms n onentrtions re not known, lthough physiologil mehnisms of Zn tion hs een propose. The results of presente stuy re novel n, to our knowlege, the effets of ietry Zn limite to hlf Tle 6 The effet of inorgni or orgni zin ietry supplementtion on the serum levels of growth hormone, insulinlike growth ftor 1, osteolin n leptin of 42-y-ol Ross 308 roilers Diet SEM p-vlue CONT ZnO100 ZnO50 ZnGly100 ZnGly ZnO ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Growth hormone [ng/ml] Insulin-like growth ftor 1 [pg/ml] < <0.01 Osteolin [pg/ml] Leptin [pg/ml] Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. Both 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50

8 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 Tle 7 The effet of inorgni or orgni zin ietry supplementtion on the serum levels of selete minerls of 42-yol Ross 308 roilers. Diet SEM p-vlue CONT ZnO100 ZnO50 ZnGly100 ZnGly50 ZnO ZnGly ZnO100 ZnO50 ZnGly100 ZnGly50 Zin [µmol/l] Copper [µmol/l] Clium [µmol/l] Mngnese [µmol/l] Iron [µmol/l] Phosphorus [µmol/l] , Dt given re LSMEANS ±SEM CONT = 0 Supp iet - without e Zn; 100 or 50 = 100 or 50 mg of Zn /kg of Premix e to sl iet; SEM = stnr error of the men (n = 10 irs for eh iet); ZnO = ontrol iet vs. iets ontining inorgni Zn (CONT vs. Both 100 n 50); ZnGly = ontrol iet vs. iets ontining orgni Zn (CONT vs. Both 100 n 50); ZnO100= ontrol iet vs. ZnO100; ZnO50=ontrol iet vs. ZnO50; ZnGly100= ontrol iet vs. ZnGly100; ZnGly50= ontrol iet vs. ZnGly50 of the reommene level, irrespetive of its soure, hve not een investigte yet in irs n ny other nimls on one evelopment, onsiering one tissue histomorphometry, geometry, n mehnis, s well s growth hormone n one turnover mrkers, hve not een investigte yet in irs n ny other nimls. Our stuy showe tht the Zn, irrespetive of soure n onentrtion, i not influene roiler fee intke or oy weight, neither the weight n length of leg ones. Rossi et l. (2007) i not oserve ny hnges in roiler oy weight or weight gin when Zn ws e in orgni form (0, 15, 30, 60 ppm). Our finings re lso in greement stuy showing tht the tiil weight of mle Ross roilers i not hnge when the sl iet ws supplemente with Zn t 100, 150, or 200 mg kg 1 s oxie or n orgni form (Shrei et l., 2012). However, in Shrei et l. (2012), the supplements were fe from the 8 th to 28 th y of the grower phse, wheres in our stuy, Zn ws supplemente uring the entire rering perio n t 100 mg kg 1. Moreover, our supplementtion perio is typil for the 42-y rering yle n ws onsistent with the stuy of Ziie et l. (2011), ut longer thn tht evlute y El-Husseiny et l. (2012), of ys of ge. The urtion of the urrent stuy might explin the lk of response to ietry Zn supplementtion s the experimentl perio ws not long enough to use epletion of Zn in the irs fe with Zn limite to the onentrtion of 50 mg kg 1. Furthermore, our results re in greement with the finings of Shrei et l. (2012) who i not oserve ny ifferenes in the iologil vlue etween the oxie n orgni zin supplementtion to roilers. On the other hn, some stuies report greter ioeffiy of orgni Zn soures ompre with Zn oxie (Świątkiewiz et l., 2001). Investigtion of one evelopment in reltion to tre minerls from inorgni n orgni soures in poultry is very importnt euse one wekness n lesions le to worse performne n poor rss qulity (Rth et l., 2000; Shrei et l., 2012). Wek legs re ssoite with reue fee intke, whih finlly ffets weight gin. Therefore, leg one qulity is ommonly use s n initor of minerl equy in poultry iets (Shrei et l., 2012). Although our iet i not influene the weight of oth evlute ones, Zn supplemente t 50 mg kg 1 enhne tiil reltive weight. This my inite tht the inrese of one weight reltive to rss weight my e ue to the reue weight gin of those irs. In ition, irrespetive of Zn form t the 50 or 100 mg kg 1 level i not influene the minerl ensity of oth ones, oring to the one weight/length inex. Although Zn one ontent ws not evlute, in n erlier stuy, the iet with 25 mg kg 1 of orgni Zn (25% of the reommene level) resulte in similr Zn one ontent s tht of the ontrol group (Tomszewsk et l., 2016). Moreover, the level of 50 mg kg 1, irrespetive of Zn soure, inrese femorl mehnil prmeter vlues, ross-setionl re, n wll thikness. It seems tht the geometry of the femur showe erlier mturtion, resulting in higher vlues of oth ultimte n mximum elsti strength, espeilly in the group supplemente with ZnGly. It is worth unerlining tht these positive effets were not influene y oy weight or sex, sine the irs were weight- n sex-mthe t the eginning of this experiment. Furthermore, the finl oy weight vlues etween the groups of hikens i not iffer. Consiering this positive effet of Zn supplemente t the ose of 50 mg kg 1 of premix, irrespetive of 166

9 Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 soure, on ortil one mehnil properties, it n e hypothesize tht, in ition to geometry, other minerls, suh s lium, were etter utilize, n therefore, improve one qulity; however, this nees to e further investigte. It is known tht Zn effets on one tissue metolism re ose-epenent. It stimultes DNA synthesis in the osteolsts, one mss inrese, n C ontent (M & Ymguhi, 2000). Zin exess or efiieny n use oy weight loss, inluing one mss n ensity, whih my result in one eformities, low one minerliztion, reue one n serum C levels (Rth et l., 2000). Although severe zin efiieny is unommon in vin popultions, mrginl efiieny is likely to e muh more prevlent, n ssoite with to immune ysfuntions or restrite physil evelopment (Srimgeour et l., 2007). The positive hnges in femorl mishft in our stuy were not oserve in the tii. This fining is ifferent from the results of El-Husseiny et l. (2012), who reporte higher tii strength in femle roilers supplemente with Zn t 50 mg/kg iet. The effet of Zn supplementtion on the one evelopment my e ue to its iret impt on protein synthesis, positively ffeting one formtion (Seo et l., 2010). Another ftor tht my ffet Zn requirements is sex. Mles require higher nutrient levels thn femles t similr ge; however, when expresse s perentge of the iet, there seems to e little ifferene in nutrient requirements etween sexes (Slim et l., 2012). In the present experiment, no ifferenes in the serum levels of Zn or other minerls were etete, irrespetive of ose n soure. It shoul e note tht high Zn intke result in opper, iron, or mngnese efiieny (Skřivn et l., 2005; Suttle, 2010). Currently, there re no sensitive n speifi iomrkers to etet Zn efiieny in nimls. Low plsm or serum Zn levels re typilly use s iomrkers of Zn sttus, lthough less thn 0.1% of oy Zn is present in the plsm n its onentrtion ppers to e uner strit homeostti ontrol (Towers et l., 1981). On the other hn, the tivities of Zn-epenent enzymes in the plsm my e useful s funtionl inies of Zn sttus in roilers or intervention stuies s their nlysis to fster n more essile ompre with one Zn ontent. The stuy of Weeking et l. (1990) shows tht the tiil Zn ontent of hikens fe ornsoyen mel iet ws mrkely inrese y ietry Zn supplementtion, ut i not provie n estimte of the requirements (Weekin & Bker, 1990). 167 As mentione ove, the three-point ening test on long ones emonstrte signifint ifferenes in the mehnil prmeters espeilly of the femur. The effet of Zn supplementtion on one evelopment my e ue to its iret impt on hormonl growth meitors. The noli effet of insulin-like growth ftor 1 on osteolsts is enhne y Zn (Wng et l., 2002). Moreover, the higher iovilility of ZnGly inites tht more Zn ws sore, n not only eposite in one tissue ompre with ZnO, lso influene ell funtion through its effets on hormones n growth ftors. It shoul e note tht the ZnGly50 group presente higher insulinlike growth ftor 1 (IGF-1) levels ompre with the ontrol group. It is suggeste tht IGF-1 lolly proue in the tissues plys more importnt role in growth thn tht irulting IGF-1 n the proue y the liver (Wng et l., 2002). Cellulr hnges, whih re inue y reue Zn ville from loo supply, re lolly oserve in res lote wy from loo vessels. For this reson, it is suggeste tht longituinl growth might e very sensitive to Zn sttus (Wng et l., 2002). Among the prmeters reommene y the Amerin Soiety for Bone n Minerl Reserh, we ssesse the volume of one treule n the men thikness of one treule (Moetti et l., 2000). The histomorphometri nlysis showe positive impt of Zn supplementtion, irrespetive of soure n level, on femorl treulr thikness n rpi loss in tul one volume in tiil metphysel treule in the irs fe with oth Zn soures, ut with lrgest eline oserve in those fe ZnGly t 50 mg kg 1 of premix. This result suggests preisposition to lesions of the joint, hinering movement. This influene of Zn on the skeletl system ws lso oserve in the Znsupplemente hikens reltive to the ontrol group. However, the mehnism responsile for these effets is iffiult to explin n nees to e further stuie. We hypothesize tht iet with no Zn supplementtion my hve supporte one evelopment ue to possile mximum intestinl Zn sorption n equte C utiliztion. In ition, the ompetition for nonspeifi multivlent tion hnnels etween low Zn levels n other ivlent tions, like lium, my hve een lne, resulting in suffiient minerliztion of the ortil one mishft. However, reue treulr one ws oserve. The positive influene of Zn on one tissue is highlighte when onsiering the signifint inrese in men reltive wll thikness, mximum elsti strength n ultimte strength in roilers fter Zn ministrtion.

10 The isrepny oserve etween femorl n tiil results in our stuy my e ue to ifferent iomehnil properties of these oth ones. An erlier stuy showe tht the mehnil strength of the femur n the tii turkeys with leg eformities were 20.6% n 22.6%, respetively, lower ompre with helthy ontrols (Ttr et l., 2004). 5. Conlusion In onlusion, this stuy emonstrte tht Zn ietry supplementtion up to 50 mg kg 1, irrespetive of its form, positively influenes mehnil properties of the femur, onfirming its enefiil effet on the evelopment of the skeletl system n one tissues of roilers teste for nutritionl-osteoporoti ftor. However, histomorphometry showe greter hnges in nellous one thn in ortil one. This osteoporoti effets on treulr one oserve when the Zn-efiient iet ws fe emphsizes the nee to supplement zin t the reommene ose (100 mg kg 1 ) to roilers. Conflit of interest There re no known onflits. Finnil support for this work oes no influene its outome. The mnusript hs een re n pprove y ll nme uthors n tht there re no other persons who stisfie the riteri for uthorship ut re not liste. The orer of uthors liste in the mnusript hs een pprove y ll uthors. Aknowlegment This work ws finnilly y the projet NN from the Ministry of Siene n Higher Eution, Poln. Referenes AOAC. Offiil methos of nlysis. 17th e. Githersurg: AOAC Interntionl; Ashme HD, Grff DJ, Ashme HH. Intestinl sorption of metl ions n heltes. Springfiel: Chrles C. Thoms; p Avigen. Ross 308 Broiler Nutrition speifition. Huntsville: Avigen Inorporte pulishing; Effet of Zin Level n Soure (Zin Oxie Vs. Zin Glyine) on Bone Mehnil n Geometri Prmeters, n Histomorphology in Mle Ross 308 metllothionein in hikens fe onventionl ietry zin onentrtions. Animl Fee Siene n Tehnology 2002;101: Aville from: Cook ME. Skeletl eformities n their use: introution. Poultry Siene 2000;79: Europen Community. Europen Communities Diretive No. 1334/2003 of The Europen Prliment n the Counil. Offiil Journl of the Europen Union 2007;L187/11-L187/15. Europen Community. Europen Communities Diretive No. 2007/43/EC of 28 June 2007 of The Europen Prliment n the Counil. Offiil Journl of the Europen Union 2003;L182/19-L182/28. El-Husseiny OM, Hshish SM, Ali RA, Arf SA, Lil D, El- Smee A, et l. Effets of feeing orgni zin, mngnese n opper on roiler growth, rss hrteristis, one qulity n minerl ontent in one, liver n exret. Interntionl Journl of Poultry Siene 2012;11: Ferretti JL, Cpozz RF, Monelo N, Znhett JR. Interreltionships etween ensitometri, geometri n mehnil properties of rt femor: inferenes onerning mehnil regultion of one moelling. Journl of Bone Minerl Reserh 1993;8: Fuge AM, Speer B. Selete ontroversil topis in vin ignosti testing. Seminrs in Avin n Exoti Pet Meiine 2001;10(2): Hmrie KM, Csey CE, Kres NF. Zin. In: Mertz W, eitor. Tre elements in humn n niml nutrition. 5th e. Orlno: Aemi Press; v.2, p Hung YL, Lu L, Li SF, Luo XG, Liu B. Reltive iovililities of orgni zin soures with ifferent heltion strengths for roilers fe onventionl orn-soyen mel iet. Journl of Animl Siene 2009;87: Kienholz EW, Turk DE, Sune ML, Hoekstr WG. Effets of zin efiieny in the iets of hens. Journl of Nutrition 1961;75: Kwieień M, Winirsk-Miezn A, Zwiślk K, Srok SZ. Effet of opper glyinte helte on iomehnil, morphometri n hemil properties of hiken femur. Annles of Animl Siene 2014;14: Lesson S. A new look t tre minerl nutrition of poultry: n we reue environmentl uren of poultry mnure? In: Lyons TP, Jques KA, eitors. Nutritionl iotehnology in the fee n foo inustries. Nottinghm: Nottinghm University Press; p Lowe NM, Lowe NM, Frser WD, Jkson MJ. Is there potentil therpeuti vlue of opper n zin for osteoporosis? Proeeings of the Nutrition Soiety 2002;61: M ZJ, Ymguhi M. Alterntion in one omponents with inresing ge of neworn rts: role of zin in one growth. Journl of Bone Minerl Metolism 2000;18: Mret W, Snste HH. Zin requirements n the risks n enefits of zin supplementtion. Journl of Tre Elements in Meiine n Biology 2006;20:3 18. Molokwu C, Li Y. Zin homeostsis n one minerl ensity. Ohio Reserh n Clinil Review 2006;15:1-12. Aville from: ohiou.eu/ms-li/pulition%20pdf/orr-li&moloku.pf. Brnão-Neto J, Mureir G, Menonç BB, Bloise W, Cstro AVB. Enorine intertion etween zin n proltin. Biologil Tre Element Reserh 1995;49: Co J, Henry PR, Dvis SR, Cousins RJ, Miles RD, Littell RC, et l. Reltive iovilility of orgni zin soures se on tissue zin n 168 Ntionl Reserh Counil. Nutrient requirements of poultry. 9th e. Wshington: Ntionl Aemi Press; Prk SY, Birkhol SG, Kuen LF, Niset DJ, Rike S C. Review on the role of ietry zin in poultry nutrition, immunity, n reproution. Biologil Tre Elements Reserh 2004;101:

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