Jie Luo, 1 Feiruo Huang, 1 Chenglin Xiao, 1 Zhengfeng Fang, 1 Jian Peng, 1 and Siwen Jiang Introduction

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1 BioMed Reserch Interntionl Volume 2013, Article ID , 9 pges Reserch Article Responses of Growth Performnce nd Proinflmmtory Cytokines Expression to Fish Oil Supplementtion in Lcttion Sows nd/or Wened Piglets Diets Jie Luo, 1 Feiruo Hung, 1 Chenglin Xio, 1 Zhengfeng Fng, 1 Jin Peng, 1 nd Siwen Jing 2 1 Deprtment of Animl Nutrition nd Feed Science, College of Animl Science nd Technology, Huzhong Agriculturl University, Wuhn , Chin 2 Key Lortory of Swine Breeding nd Genetics of Agriculturl Ministry, College of Animl Science nd Technology, Huzhong Agriculturl University, Wuhn , Chin Correspondence should e ddressed to Jin Peng; pengjin@mil.hzu.edu.cn nd Siwen Jing; jingsiwen@mil.hzu.edu.cn Received 15 My 2013; Revised 9 July 2013; Accepted 27 July 2013 Acdemic Editor: Krsten H. Weylndt Copyright 2013 Jie Luo et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. The study ws conducted to investigte whether dietry fish oil could influence growth of piglets vi regulting the expression of proinflmmtory cytokines. A split-plot experimentl design ws used with sow diet effect in the min plots nd differing piglet diet effect in the suplot. The results showed tht suckling piglets from fish oil fed dms grew rpidly (P < 0.05) thncontrol.it ws lso oserved tht these piglets hd higher ADG, feed intke, nd finl ody weight (P < 0.05) during postwening thn those piglets from lrd fed dms. Furthermore, there ws significnt decrese (P < 0.01) in the expression of interleukin 6 nd tumor necrosis fctor-α in longissimus dorsi muscle. In contrst, there ws tendency (P < 0.10) towrds lower ADG nd higher feed : gin in wened piglets receiving fish oil compred with those receiving lrd. Menwhile, splenic proinflmmtory cytokines expression ws incresed (P < 0.01) in piglets receiving fish oil during postwening period. The results suggested tht 7% fish oil ddition to sows diets llevited inflmmtory response vi decresing the proinflmmtory cytokines expression in skeletl muscle nd ccelerted piglet growth. However, 7% fish oil ddition to wened piglets diets might decrese piglet growth vi incresing splenic proinflmmtory cytokines expression. 1. Introduction The growth rte of piglet is most rpid during the erly postntl stge, nd it is very importnt to the susequent growth, finl ody weight, nd mrketing time of pigs. Mny reserches reveled tht fish oil could ffect young piglets growth [1 5]. It ws reported tht 3 5% of fish oil ddition to sows diets ws eneficil to suckling piglet growth [1, 5]. However, lcttion sow diet supplemented with 8 10% fish oil incresed piglets prewening moridity nd decresed sows milk production nd litter weight gin [2, 6]. Dietry supplementtion with 2 3% fish oil promoted wened piglets growth [3], wheres ody weight nd feed intke of wened piglets fed 5% or more fish oil were lower versus corn oil [4]. Previous studies hve demonstrted tht proinflmmtory cytokines could increse muscle protein degrdtion, reduce muscle protein synthesis, divert nutrients to the synthesis of components in the immune system, nd suppress niml growth [7 9]. Fish oil hs nti-inflmmtory nd immunomodultory effect. Studies in niml models nd in humn sujects generlly reported decresed production of proinflmmtory cytokines in immune cell in peripherl lood nd spleen fter fish oil supplementtion [10 12], nd the nti-inflmmtory effect hs lso een shown for suckling piglets of fish oil fed sows [13]. Further study reveled tht the immunomodultory effect ws cused y the (n-3) polyunsturted ftty cids ((n-3) PUFA), especilly, eicospentenoic cid (EPA, C20:5 (n-3)) nd docoshexenoic cid (DHA, C22:6 (n-3)) in the fish oil [14]. Remrkly, (n-3) PUFA could decrese the proinflmmtory cytokines (interleukin 1 (IL-1), IL-6, nd tumor necrosis fctor-α (TNF-α)) expression nd secretion in peripherl immune cells nd

2 2 BioMed Reserch Interntionl Tle 1: Ingredients nd composition of sow nd piglet diets (%). Ingredient Sow diet Piglet diet Sow diet Piglet diet Nutrients C T C T C T C T Corn DE (Mcl/kg) Whet rn Crude protein Dried whey Clcium Lrd oil Totl phosphorus Fish oil Aville phosphorus Fish mel Lys Soyen mel Met + Cys Sodium chloride Clcium cronte Diclcium phosphte L-Lysine HCl Methionine Vitmin minerl premix The control diet contins 70 g/kg lrd oil nd test group diet contins 70 g/kg fish oil; 500 mg/kg ethoxyquin ws dded to oil s nti-oxidtive. 2 Provided following per kg of diet. Cu: 5 mg; Fe: 80 mg; Zn: 50 mg; Mn: 20 mg; I: 0.14 mg; Se: 0.15 mg; V A : 2,000 IU; V D3 :200IU;V E :44IU;V k3 :0.5mg;V B1 : 1mg;V B2 :3.75mg;V B12 : mg; iotin: 0.2 mg; folic cid: 1.3 mg; nicin: 10 mg; pntothenic cid: 12 mg. 3 Providedfollowingperkgofdiet.Cu:6mg;Fe:100mg;Zn:100mg;Mn:4mg;I:0.14mg;Se:0.3mg;V A : 11,000 IU; V D3 :1,100IU;V E :44IU;V k3 :0.5mg; V B1 :1mg;V B2 :3.5mg;V B6 : 1.5 mg; iotin: 0.05 mg; folic cid: 0.3 mg; nicin: 15 mg; pntothenic cid: 10 mg. 4 By clcultion. suppress nimls inflmmtory response [5, 11, 15]. (n-3) PUFA could ttenute the growth inhiition effect y reducing the production of proinflmmtory cytokines in severl species [4, 16, 17]. Our previous study found tht intke of n-3 PUFA leds to significnt decreses in the expressions of proinflmmtory cytokine genes in loin muscle nd spleen, which my stimulte growth in growing-finishing rrows [18]. However, severl studies hve shown tht high level of fish oil (30% or more) supplementtion in mice diet incresed TNF-α production in splenocytes [19, 20]. In the present study, we hypothesized tht the inconsistent performnce of piglets tht ingested different levels of fish oil mentioned efore ws due to the different response of proinflmmtory cytokines expression to (n-3) PUFA supplementtion. Thus, piglets receiving lrd directly from the postwening diet or indirectly from the lcttion sows diets were used s the control. The im of the present study ws to investigte the effect of dietry supplementtion of 7% fish oil in lcttion sows nd/or wened piglets diets on growth performnce of piglets nd expression of proinflmmtory cytokines including IL- 1β,IL-6,nd TNF-α in skeletl muscle nd spleen. 2. Mterils nd Methods 2.1. Animls nd Diets. This tril ws crried out in ccordnce with Huzhong Agriculturl University Animl Cre nd Use Committee guidelines. A split-plot experimentl design ws used with sow diet effect (L) in the min plots nd differing piglet diet effect (PW) in the suplot. Lndrce lrge white sows (n = 20)t10deforeprturitionwere ssigned to 1 of 2 groups mtched for prity nd ody weight. The test group (T) received diet supplemented with 7% fish oil, while control group (C) received n isoenergetic, isonitrogenous, nd isolipidic diet with 7% lrd oil. Fish oil nd lrd were purchsed from Chin Ntionl Cerels, Oils & Foodstuffs Corp. (COFCO Limited). The oil nd ft were for food or feed-qulity grde. And 500 mg/kg ethoxyquin ws dded to fish oil nd lrd s ntioxidtive. The two diets were formulted to meet NRC requirements of nutrient stndrds for lcttion sow [21]. All diets were prepred weekly to keep them fresh. Two sows in the test group gve irth to less thn 3 piglets nd thus were dropped from the study. Upon frrowing, sows were fed their tretment diet twice dily (0800nd1600h).Sowswereinitillyfed2.0kg,ndthis ws incresed dily y 0.5 kg of feed until d 4 postprtum, depending on sows feed consumption nd recovery fter prturition. After d 4 postprtum, sows hd free ccess to their diets until wening. The composition of the sow diets is shown in Tle 1. Two sows of the fish oil fed group gve irth to less thn 3 piglets nd thus were rejected. At frrowing, litters were equlized within dietry tretments to the sme numer of piglets per litter ( 10). Prestrter feed ws freely ville to the nursing piglets from 7 d of ge until wening. At 28 d of ge, 56 piglets, 28 piglets (hlf femles nd hlf cstrted mles) per group of sows, were moved to cges nd rered in nursery room. All piglets remined in the sme tretment group defined y their dm; they were then sudivided into two groups of 14 piglets ech (one femle nd one cstrted mle s repliction) such tht totl of 4 experimentl groups otined: CC (control sows-control piglets), CT (control sows-treted piglets), TC (treted sows-control piglets), nd TT (treted sows-treted piglets). Piglets from CT nd TT groups were fed strter diet supplemented with 7% fish oil, nd n isoenergetic, isonitrogenous, nd isolipidic diet supplemented with 7% lrd ws used s the strter diet fed to CC nd TC piglets. The two diets were formulted to meet NRC requirements of nutrient stndrds for piglet [21]. The composition of the piglet diets is shown in Tle 1. Wened piglets were fed experimentl diets from 35 d to 70 d

3 BioMed Reserch Interntionl 3 Tle 2: Oligonucleotide polymerse chin rection primers. Gene 1 Accession no. Primer source Primer sequences (5 3 ) Orienttion Product size, p t 2 ( C) IL-1β M86725 Pig ATTCGAGTCTGCCCTGTA Forwrd TCTGGGTATGGCTTTCCT Reverse IL-6 M80258 Pig GCATTCCCTCCTCTGGTC Forwrd ATAGTGTCCTAACGCTCAT Reverse TNF-α AY Pig CTCCCTCTTTGTCTCCTCC Forwrd GCATTGGCATACCCACTCT Reverse β-ctin SSU07786 Pig GGACTTCGAGCAGGAGATGG Forwrd GCACCGTGTTGGCGTAGAGG Reverse IL-1β: interleukin 1β, IL-6: interleukin 6, TNF-α: tumor necrosis fctor-α. 2 t : optiml PCR nneling temperture. fter frrowing. The feed intke of piglets ws recorded dily. Theodyweightofpigletswsmesuredtd0,21,35,nd 70 postntl. The piglets verge dily gin (ADG) during 0 21 d nd d were clculted s ody weight gin/dys Collection of Milk nd Tissue Smples. At d 21 of lcttion, ml of milk ws collected from the functionl glnds of ech sow fter injection of 2 ml of oxytocin. The milk smples were immeditely frozen t 20 C for lter nlysis. The milk smples were nlyzed to determine ftty cid composition. Sixteen piglets (4 per tretment, hlf femles nd hlf cstrted mles) were slughtered t the end of the experiment. The pigs were deprived of feed for 12 h efore humnely slughter vi electriclly stun nd exsnguintion. The smples of the longissimus dorsi muscle were collected etween thetenthndlstris,ndspleensmpleswerecollectedt ccumen. All the collected smples were snp frozen in liquid nitrogenndstoredt 80 C for susequent RNA isoltion Ftty Acids Anlysis. Ftty cids composition of milk (10 ml), diets (1 g), nd diced muscle (2 g) were nlyzed y gs chromtogrphy. Lipids were extrcted y chloroform: methnol (1 : 1) s descried y Folch et l. [22]. Ftty cid methyl ester ws prepred for gs chromtogrphy determintion using KOH/methnol (0.4 mol/l) [23]. The CP-3800 gs chromtogrphy (Vrin, Inc., USA) equipped with 1177 injector, flme ioniztion detector, nd cpillry chromtogrphic column CPSil88 (Vrin, Inc., USA) (50 m 0.25 mm μm) for ftty cid methyl ester ws used in this experiment. The injector nd detector tempertures were kept t 250 Cnd270 C, respectively. Nitrogen ws used s crrier gs with flow rte of 1.0 ml/min, nd the split rtio ws 1 : 100. The column temperture ws progrmmed s follows: 100 C t first, incresed to 200 C(5 C/min), nd held constnt for 5 min; then, the temperture ws incresed to 225 C(2 C/min) nd kept constnt for 2 min. The totl nlysis time ws 39.5 min. The ftty cids were identified y compring the retention times of the peks with those of known stndrds (Sigm Chemicl Co., St. Louis, Mo). Response fctors for the ftty cidswereclcultedusingthesmestndrdmixturesplus n internl stndrd [24]. Ftty cid results re presented s g/100 g ftty cids. Sturted ftty cids re the sum of C14:0, C16:0, nd C18:0. The monounsturted ftty cids re the sum of C16:1 (n-7) nd C18:1 (n-9). The (n-3) PUFA re the sum of C18:3 (n-3), C20:5 (n-3), C22:5 (n-3), nd C22:6 (n-3). The (n-6) PUFA re the sum of C18:2 (n-6) nd C20:4 (n-6). The sum of the PUFA ws clculted s the sum of (n-3) PUFA nd (n-6) PUFA Reverse Trnscription Polymerse Chin Rection (RT- PCR). Totl RNA ws extrcted using the TRIzol regent (Invitrogen Corp., Crlsd, CA, USA) ccording to the mnufcturer s specifictions. The RNA smples were quntifiedspectrophotometricllyt260nd280nm.atwo-step semiquntittive RT-PCR method ws used to mesure gene expression [25]. Oligo-(dT) 20n (Toyoo, Osk, Jpn) ws used s the primer in the first step of cdna synthesis. Reverse trnscription rection solution (20 μl) consisted of 2 μg of totlrna,100uofmmlv(moloneymurineleukemi Virus) reverse trnscriptse (Toyoo, Osk, Jpn), 20 U of n RNAse inhiitor (Toyoo, Osk, Jpn), 0.5 mmol/l of deoxyrionucleotide triphosphtes (dntp) (Toyoo, Osk, Jpn), nd 0.5 μl oligo-dt primers. The cdna stock ws stored t 20 C. The yield of cdna ws mesured ccording to the PCR signl generted from the internl stndrd housekeeping gene, β-ctin, which ws mplified from 25 to 35 cycles strting with 0.5 μl ofthecdnasolution.the volume of ech cdna pool ws djusted to give the sme exponentil-phse PCR signl intensity for β-ctin fter 25 cycles [26]. Reltive RT-PCR [27] ws performed to mesure gene expression of IL-1β, IL-6, nd TNF-α of longissimus dorsi muscle nd spleen. Primer sequences nd optiml PCR nneling tempertures (t )relistedintle 2.PCRws performed on 2720 Therml Cycler (Applied Biosystems, USA). The PCR progrm egn with 94 C denturtion for 5 min, followed y cycles dentured t 94 Cfor 30 s, nneling for 30 s, nd extension for 30 s t 72 C, with finl extension t 72 C for 10 min. The liner mplifiction rnge for ech gene ws tested on the djusted cdna. The optiml cycle numer ws then considered to e two cycles lower thn the highest cycle of linerity. The PCR products were electrophoresed on 1.5% grose gel nd stined with ethidium romide (10 μg/ml). The gel imges were digitlly cptured with G:BOX (Syngene, Cmridge, UK) nd densitometry vlues were mesured using the Gene Tool softwre (Syngene, Cmridge, UK). RT-PCR vlues re

4 4 BioMed Reserch Interntionl Tle 3: Lipid concentrtion nd ftty cid composition of sow nd piglet diets 1. Sow diet Piglet diet C T C T Lipid, g/100 g diet Ftty cid composition, g/100 g totl ftty cid C14: C16: C18: SFA C16:1 (n-7) C18:1 (n-9) MUFA C18:2 (n-6) C20:4 (n-6) (n-6) PUFA C18:3 (n-3) C20:5 (n-3) C22:5 (n-3) C22:6 (n-3) (n-3) PUFA PUFA Ftty cids re designted y the numer of cron toms followed y the numer of doule onds. The position of the first doule ond reltive to the methyl (n) end of the molecule is lso indicted. 2 SFA: sturted ftty cids, MUFA: monounsturted ftty cids, PUFA: polyunsturted ftty cids; SFA: the sum of C14:0, C16:0, nd C18:0; MUFA: the sum of C16:1 (n-7) nd C18:1 (n-9); (n-6) PUFA: the sum of C18:2 (n-6) nd C20:4 (n-6); (n-3) PUFA: the sum of C18:3 (n-3), C20:5 (n-3), C22:5 (n-3), nd C22:6 (n-3); PUFA: the sum of (n-6) PUFA nd (n-3) PUFA. Tle 4: Lipid concentrtion nd ftty cid composition of milk of sows fed with nd without fish oil 1,2. C T SEM P-vlue Lipid, g/100 g milk solid Ftty cid composition, g/100 g totl ftty cid C14: C16: C18: SFA C16:1 (n-7) <1 C18:1 (n-9) <1 MUFA <1 C18:2 (n-6) <1 C20:4 (n-6) (n-6) PUFA <1 C18:3 (n-3) C20:5 (n-3) <1 C22:5 (n-3) <1 C22:6 (n-3) <1 (n-3) PUFA <1 PUFA <1 1 Vlues re mens ± pooled SEM, control n=9,tretmentn=6. 2 Ftty cids re designted y the numer of cron toms followed y the numer of doule onds. The position of the first doule ond reltive to the methyl (n) end of the molecule is lso indicted. 3 SFA: sturted ftty cids, MUFA: monounsturted ftty cids, PUFA: polyunsturted ftty cids; SFA: the sum of C14:0, C16:0, nd C18:0; MUFA: the sum of C16:1 (n-7) nd C18:1 (n-9); (n-6) PUFA: the sum of C18:2 (n-6) nd C20:4 (n-6); (n-3) PUFA: the sum of C18:3 (n-3), C20:5 (n-3), C22:5 (n-3), nd C22:6 (n-3); PUFA: the sum of (n-6) PUFA nd (n-3) PUFA. presented s rtio of the specified gene signl in the selected liner mplifiction cycle divided y the β-ctin signl. Dt for ech replicte represented the men of three individul RT-PCRs Sttistics. Experimentl nimls were ssigned to different tretments in completely rndomized design. Pen ws the experimentl unit. Sttisticl nlysis of the dt ws performed with the ANOVA procedure of SAS8.01 [28], nd the residul error ws used to test the min effect of dietry tretments. The multiple comprisons were preceded with DUNCAN procedure. Dt were presented s mens ± SEM. Growth performnce nd gene expression dt of postwening piglets were nlyzed s 2 2rndomizedANOVA with lcttion (L) nd postwening diet period (PW) s effects. The correltion etween ADG nd expressions of proinflmmtory cytokine genes ws performed with the CORR procedure of SAS8.01 [28]. Mens were considered sttisticlly different t P < Results 3.1. Ftty Acids Composition of Diets nd Milk. Ftty cid composition of sow nd piglet diets is shown in Tle 3.Ftty cid composition of sow milk t d 21 of lcttion is shown in Tle 4. Fish oil diets nd lrd diets hd the sme levels of lipids, while the (n-3) PUFA were nerly 8 9 times higher in fish oil diets s compred to lrd diets. Compred with lrd oil, fish oil dministrtion incresed the concentrtion of PEA, DHA nd totl (n-3) PUFA in sows milk (P < 0.01), wheres, the EPA, DHA, nd totl (n-3) PUFA contents in milkfromfishoilfedsowswerelowerthnthoseinfishoil dded diets which fed to piglets during postwening period (7.00 g/100 g totl ftty cid versus g/100 g totl ftty cid). As result, piglets from fish oil fed dms received fewer mount of (n-3) PUFA thn wened piglets fed 7% fish oil diet (0.41% versus 1.00%) Ftty Acids Composition in Longissimus Dorsi Muscle. Ftty cid composition of longissimus dorsi muscle is shown in Tle 5.The sturted ftty cids(c14:0,c16:0,c18:0,nd C20:0), C20:4n-6, nd n-6 PUFA contents in longissimus dorsi muscle of postwening piglets from fish oil fed dms were significntly lower (P < 0.05) thnthoseofpigletsfrom control sows. The C20:1n-9 (0.78% versus 0.62%) nd C22:5n- 3 (0.84% versus 0.2%) contents were significntly incresed (P < 0.05) in piglets from sows compred with those from control sows.

5 BioMed Reserch Interntionl 5 Tle 5: Ftty cids composition of longissimus dorsi muscle. Ftty cid Diet 1 P-vlue 2 SEM L PW L PW Ftty cid composition, g/100 g totl ftty cid C14: C16: C16:1n C18: C18:1n C18:1n C18:2n C18:3n C20: <0.01 < C20:1n <0.01 < C20:4n C20:5n < C22:5n < C22:6n < SFA <0.01 < MUFA n-6 PUFA n-3 PUFA < PUFA n-6/n <0.01 < P/S Vlues re mens ± pooled SEM, n=7. CC: ll period fed lrd diet; CT: post-wening period fed fish oil diet; TC: lcttion period fed fish oil diet; TT: ll period fed fish oil diet. 2 Effects of lcttion (L) nd the post-wening (PW) or the interction etween lcttion nd the strter diet period (L PW). 3 Ftty cids re designted y the numer of cron toms followed y the numer of doule onds. The position of the first doule ond reltive to the methyl (n) end of the molecule is lso indicted. SFA: sturted ftty cids, MUFA: monounsturted ftty cids, PUFA: polyunsturted ftty cids; SFA: the sum of C14:0, C16:0, nd C18:0; MUFA: the sum of C16:1 (n-7) nd C18:1 (n-9); (n-6) PUFA: the sum of C18:2 (n-6) nd C20:4 (n-6); (n-3) PUFA: the sum of C18:3 (n-3), C20:5 (n-3), C22:5 (n-3), nd C22:6 (n-3); PUFA: the sum of (n-6) PUFA nd (n-3) PUFA. Tle 6: Effect of fish oil supplementtion during lte gesttion nd lcttion on the performnce of suckling piglets 1. Item Diet C T SEM P-vlue Litter weight t delivery, kg Body weight t irth, kg Body weight t 21 d, kg Averge dily gin, g Vlues re mens ± pooled SEM, control n=10,tretmentn=8. The C16:0, C20:0, nd SFA contents in longissimus dorsi muscle of piglets receiving fish oil were significntly decresed (P < 0.05) compred with those receiving control diets during the postwening period. The C20:5n-3 (1.39% versus 0.13%), C22:5n-3 (0.56% versus 0.47%), C22:6n-3 (1.38% versus 0.34%), nd totl n-3 PUFA (4.38% versus 1.3%) contents in longissimus dorsi muscle of piglets receiving fish oilweresignificntlyincresed(p < 0.01) morethnthose receiving control diets during the postwening period Growth Performnce of Piglets. Growth performnce of suckling piglets is shown in Tle 6.Theodyweightstirth nd litter weights t delivery were not different (P > 0.05) etween groups. However, the verge dily gin (ADG) of suckling piglets ws significntly (P < 0.05) higher in the fish oil group thn in the control group. The growth performnce of wened piglets ws lso significntly (P < 0.05) ffected y sow diet effect (L) (Tle 7). The finl ody weights (21.29 kg versus kg) of postwening piglets from fish oil fed dms were significntly higher (P < 0.05) thn those of piglets from control sows. The ADG (325 g/d versus 265 g/d) nd verge dily feed intke (ADFI) (615 g/d versus 510 g/d) were lso significntly incresed (P < 0.05) in piglets from sows compred with those from control sows, wheres the feed conversion rte (1.91 versus 1.96) ws not different (P > 0.05) etween groups. The growth performnce of wened piglets ws not significntly (P > 0.05) ffected y piglet diet effect. However, there ws tendency (P < 0.10) towrds lower ADG (270 g/d versus 321 g/d) nd higher feed : gin (2.00 versus 1.87) in piglets receiving fish oil compred with those receiving control diets during the postwening period, ut the ADFI (534 g/d versus 591 g/d) nd finl ody weights (19.18 kg versus kg) were not significntly (P > 0.05) different etween groups. Additionlly, finl ody weight, ADG, nd ADFI were significntly higher in group TC thn those in group CT (P < 0.05).

6 6 BioMed Reserch Interntionl Tle 7: Growth performnce of wened piglets orn to fish oil-treted nd control sows nd fed diets with nd without fish oil supplementtion in the post-wening period. Item Diet 1 P-vlue 2 SEM L PW L PW Body weight (35 d), kg Body weight (70 d), kg Averge dily gin, g Averge dily feed intke, g Feed : gin Vlues re mens ± pooled SEM, n=7. CC: ll period fed lrd diet; CT: post-wening period fed fish oil diet; TC: lcttion period fed fish oil diet; TT: ll period fed fish oil diet. 2 Effects of lcttion (L) nd the post-wening (PW) or the interction etween lcttion nd the strter diet period (L PW)., Mens with different letters re significntly different (P < 0.05) mong groups. Tle 8: The correltion coefficients etween ADG nd expressions of proinflmmtory cytokine genes in longissimus dorsi muscle nd spleen. Item Longissimus dorsi muscle Spleen IL-1β 1 IL-6 1 TNF-α 1 IL-1β IL-6 TNF-α ADG Correltion coefficients (r) P-vlue IL-1β: interleukin 1β, IL-6: interleukin 6, TNF-α: tumor necrosis fctor-α Proinflmmtory Cytokines Gene Expression in Longissimus Dorsi Muscle nd Spleen. Figure 1 showstheresultsof cytokines expression in longissimus dorsi muscle nd spleen of wened piglets in C-C, C-T, T-C, nd T-T groups. IL-6 (Figure 1()) nd TNF-α (Figure 1(c)) expression in longissimus dorsi muscle ws significntly (P < 0.05) decresed in wened piglets from fish oil fed dms compred with those of piglets from lrd fed dms, while the IL-1β expression in longissimus dorsi muscle (Figure 1()) ws not different (P > 0.05). Additionlly, the IL-1β nd IL-6 (Figures 1(d) nd 1(e)) expression in spleen ws not different (P > 0.05). However, postwening piglets from fish oil fed dms hd higher (P < 0.05) TNF-α (Figure 1(f))expressioninspleen. Proinflmmtory cytokines expression of wened piglets in longissimus dorsi ws not ffected y postwening fish oil supplementtion (PW) or L PW interction (P > 0.05) (Figures 1(), 1(), nd1(c)). However, postwening fishoilsupplementtionresultedinsignificntlyincresed (P < 0.01) spleen IL-1β, IL-6, ndtnf-α mrna undnces (Figures 1(d), 1(e),nd1(f)). 3.5.TheCorreltionetweenADGndExpressionsofProinflmmtory Cytokine Genes. The mrna expressions for proinflmmtory cytokines in groups CC, CT, TC, nd TT were pooled nd compred with the ADG of the corresponding slughtered piglets in order to evlute possile correltion etween expressions of proinflmmtory cytokines nd growth performnce. The correltion coefficients etween ADG nd expressions of proinflmmtory cytokine genes re shown in Tle 8. Proinflmmtory cytokines expressions in longissimus dorsi muscleor in spleen were negtively correlted with piglets growth. There were sttisticlly significnt negtive correltions etween musculr IL-1β expression (r = , P < 0.01) or splenic IL-1β expression (r = , P < 0.01)levelsndADG. 4. Discussion Dily supplementry low level of cod liver oil (50 ml/d, pproximtely 1% 2%)to sows from dy 107 of gesttion until wening did not ffect weight gin nd overll moridity of pigletsinthestudyytugøletl.[29]. It ws suggested tht 1.75% (17.5 g/kg of diet) of fish oil to pregnnt diet nd 3.5% (35 g/kg of diet) of fish oil to lcttion diet for sows improved growth of their progeny [1]. The previous results showed tht the ody weight nd ADG of suckling piglets t 21 d postntl were incresed y 7% (70 g/kg of diet) fish oil supplementtion to the lte gesttion nd lcttion sows diets, which greed with other reserchers [1, 30]. We lso found tht wened piglets from fish oil fed dms hd higher growth rte thn those from lrd oil fed dms during the d35to70postntlperiod.theseresultsreveledtht7% of fish oil supplemented to sows diets could promote the growth performnce of their progeny, nd this effect even lsted during the postwening period. Interestingly, high level of fish oil supplementtion ws not eneficil for piglets growth. It ws found tht 8% (80 g/kg of diet) fish oil ddition to sow diet during lte gesttion nd lcttion period decresed litter weight gin of suckling piglets, compred with 8% niml ft (6). Similrly, 10% (100 g/kg of diet) fish oil ddition to lcttion sow diet resulted in incresed piglets prewening moridity nd decresedsowsmilkproduction(2).welsofoundthesme trend in the present study. The ADG nd feed conversion rte

7 BioMed Reserch Interntionl 7 Reltive gene expression nt nt nt nt Reltive gene expression Reltive gene expression () () (c) Reltive gene expression c c Reltive gene expression Reltive gene expression c, c (d) (e) (f) Figure 1: Reltive longissimus dorsi muscle nd spleen cytokines expression of wened piglets orn to fish oil-treted or control sows, nd piglets fed diets with nd without fish oil supplementtion during the postwening period. (), (), nd (c) were longissimus dorsi muscle cytokines expression; (d), (e), nd (f) were spleen cytokines expression. () nd (d): IL-1β expression; () nd (e): IL-6 expression; (c) nd (f): TNF-α expression. Gene signls were normlized to the expression of the housekeeping gene β-ctin opticl density signl. CC: ll period fed lrd diet; CT: postwening period fed fish oil diet; TC: lcttion period fed fish oil diet; TT: ll period fed fish oil diet. Dt shown re men vlues ± SEM, n=4., : mens lcttion decrese effect with P < 0.05, P < 0.01;, : mens postwening increse effect with P < 0.05, P < 0.01;, : mens with different letters re significntly different (P < 0.05)monggroups; nt : mens hve no significnt difference. (FCR) of postwening piglets were tended to decrese fter 7% of fish oil feeding. The results were in generl greement with nother study showing tht wened piglets receiving 5% (50 g/kg of diet) menhden fish oil + 1% (10 g/kg of diet) corn oilduringthed24to36postntlperiodhdlowerody weightginndadfithnthosefed6%cornoil[4]. The three proinflmmtory cytokines IL-1, IL-6, nd TNF-α could induce gret metolic chnges [31]. The cytokines pper to e primrily derived from mcrophgerich tissues, such s the liver nd spleen, nd vrious myelomonocytic cells s well. However, it ws reported tht cytokinesrelsoproducedycellsnottrditionllyconsidered to e prt of the immune system such s dipocytes nd myofiers [32, 33], which re effective sources nd trgets of cytokines [34, 35]. Proinflmmtory cytokines medite reprogrmming of metolism nd shift the prtitioning of dietry nutrients wy from skeletl muscle ccretion towrd metolic responses tht support the immune system [7, 31]. Skeletl muscle s the iggest ody tissue, mounting for 40 45% of the totl ody mss, is lso the most quickly growing tissues during erly postntl period except for one nd nervous system. Collectively, these dt suggest tht the utocrine/prcrine ctions of cytokines re of potentil importnce in muscle nd thus in postntl niml growth. There ws pucity of dt pertining to the cytokines expression fter (n-3) PUFA dministrtion in skeletl muscle. Previous study in our lortory showed tht during norml physiologicl processes feeding linseed diet (rich in α-linolenic cid) suppressed the expression of proinflmmtory cytokines in longissimus muscle of growing-finishing rrows nd decresed serum level of TNF-α from to ng/ml during norml physiologicl condition. These results showed tht the pproprite reduction of serum TNF- α levels tht rnged from to ng/ml might e eneficil to increse the longissimus muscle mss under norml physiologicl condition [18]. EPA nd DHA were considered to e more potent in regulting immune function [14]. Fish oil hs een reported to decrese production of proinflmmtory cytokines ecuse of the high content of EPA nd DHA[10 12]. The IL-6 nd TNF-α expression ws significntly suppressed in muscle of wened piglets from fish oil fed dms in the present study. We lso found negtive correltion etween ADG nd proinflmmtory cytokines expression. Thus, suppression of cytokines expression in muscle of piglets my positively reduce the inflmmtory response in skeletl muscle, ttenute the negtive effect of the potent proinflmmtory cytokines, nd promote piglet growth.

8 8 BioMed Reserch Interntionl Remrkly,itwsnotlwysconsistentwiththeresult which modertes fish oil supplementtion decresed production of proinflmmtory cytokines. The study in rodents suggested tht mice fed s high s 30% (300 g/kg of diet) of fish oil for 4 6 weeks incresed the TNF-α syntheses in splenocytes [19]. Brer et l. [20] lso reported tht TNF-α secretion ws incresed in splenocytes seprted from 4% of EPA (>30% fish oil ccording to our dt) fed mice fter stimultion with LPS. We lso found tht ll of the three proinflmmtory cytokines expressions were significntly incresed in spleen fter 7% of fish oil feeding during post-wening. High production of proinflmmtory cytokines in spleen, the iggest immune orgn, my increse proinflmmtory cytokines levels of the whole ody, suppress skeletl muscle protein ccretion, nd impct niml growth [7, 9, 36]. The results suggested tht dministrtion of 7% fish oil to lcttion sows significntly incresed the growth rte of their progeny during postwening period. However, the ddition of 7% fish oil to diets of postwening piglets ws likely to decresethegrowthrtendfcrofpiglets.thecontrry effects of 7% fish oil supplementtion to sows or wened piglets diets my e due to the different content of (n-3) PUFA tht the piglets received. The current results demonstrted tht the EPA, DHA, nd totl (n-3) PUFA contents of milk were lower thn those of piglets diets (2.71% versus 5.90%, 2.53% versus 2.94%, nd 7.00% versus 10.28%, resp.). As result of the lower level of lipid content in milk compred with tht in the diet, piglets only received 40% of (n-3) PUFA from milk reltive to tht ingested from fish oil diets directly. Our previous study found tht intke of n-3pufa couldincresethen-3pufacontentinlongissimus dorsi muscle of growing-finishing pigs [37]. In the current experiment, C22:5n-3 contents in longissimus dorsi muscle of postwening piglets from fish oil fed dms were incresed (0.84% versus 0.2%) more thn those of piglets from control sows. However, the C20:5n-3 (1.39% versus 0.13%), C22:5n-3 (0.56% versus 0.47%), C22:6n-3 (1.38% versus 0.34%), nd totl n-3 PUFA (4.38% versus 1.3%) contents in longissimus dorsi muscle of piglets receiving fish oil were incresed more thn those of piglets receiving control diets during the postwening period. These results reveled tht n-3pufa contents in the muscle were higher in piglets fed fish oil directly thn tht in wened piglets from fish oil fed dms. It ws further oserved tht the IL-6 nd TNF-α expression either in the muscle or in spleen ws lower in wened piglets from fish oil fed dms thn tht in piglets fed fish oil directly. It could e concluded tht moderte (n-3) PUFA intke ws eneficil to piglets growth y decresing proinflmmtory cytokines production nd suppressing inflmmtory response in skeletl muscle. However, high intke of (n-3) PUFA my promote splenic proinflmmtory cytokines production nd impct niml growth consequently. In conclusion, fish oil might regulte piglet growth through modulting proinflmmtory cytokines production in ody tissues. Approprite levels of fish oil supplementtion to sows diets my increse (n-3) PUFA content in milk nd (n-3) PUFA ingestion of their progenies, decrese the proinflmmtory cytokines expression nd their unfvorle effects on skeletl muscle, nd thus promote growth of piglets. However, high levels of fish oil supplementtion to postwening piglets diets my increse splenic proinflmmtory cytokines expression nd thus negtively impct the growth of wened piglets. Given tht the limited replicte numer of slughtered nimls in the present study my e not enough to generte definitive conclusion, further investigtion with more numer of smples is required to determine the pproprite fish oil supplementtion level, durtion, nd the precise mechnisms y which long chin (n-3) PUFA ffect piglets immunity nd growth. Conflict of Interests The uthors declre tht they hve no competing interests. Authors Contriution JieLuondFeiruoHungcontriutedequllytothiswork. Acknowledgments This reserch ws supported y the Ntionl High Technology R & D Progrm of Chin (no. 2006AA10Z140), the Ntionl Science Foundtion of Chin (no ), nd Mjor Science & Technology Industriliztion Projects in Wuhn City of Chin (no ). References [1] J. A. Rooke, M. Shnks, nd S. A. Edwrds, Effect of offering mize, linseed or tun oils throughout pregnncy nd lcttion on sow nd piglet tissue composition nd piglet performnce, Animl Science,vol.71,no.2,pp ,2000. [2] V. Dnielsen nd C. 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