British Journal of Nutrition

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1 (1), 11, 8 87 q The Authors 1 doi:1.117/s711117x Chnges in plsm mino id profiles, growth performne nd intestinl ntioxidnt pity of piglets following inresed onsumption of methionine s its hydroxy nlogue Ho Li 1, Hifeng Wn 1, Yves Merier, Xioling Zhng 1, Cimei Wu 1, Xiuqun Wu 1, Li Tng 1, Linqing Che 1, Yn Lin 1, Shengyu Xu 1, Gng Tin 1,DeWu 1 nd Zhengfeng Fng 1 * 1 Key Lortory for Animl Disese Resistne Nutrition of the Ministry of Edution of Chin, Animl Nutrition Institute, Sihun Agriulturl University, Y n 1, People s Repuli of Chin Adisseo Frne S.A.S., CERN, Commentry, Frne (Sumitted July 1 Finl revision reeived Jnury 1 Aepted 1 Ferury 1 First pulished online 1 August 1) Astrt The im of the present study ws to determine whether erly wening-indued growth retrdtion ould e ttenuted y inresed onsumption of methionine s DL-methionine (DLM) or DL--hydroxy--methylthioutyrte (HMTBA) in oth ltting sows nd wened piglets. Therefore, diets ontining DLM nd HMTBA t % of the totl sulphur-ontining mino ids (AA) present in the ontrol (CON) diet were fed to ltting sows nd wened piglets nd their responses were evluted. Compred with the CON dietfed sows, the HMTBA diet-fed sows exhiited tendeny (P, 1) towrds higher plsm turine onentrtions nd the DLM dietfed sows hd higher (P, ) plsm turine onentrtions, ut lower (P, ) isoleuine onentrtions. Sukling piglets in the HMTBA tretment group hd higher (P, ) intestinl redued glutthione (GSH) ontent, lower (P, ) oxidised glutthione (GSSG):GSH rtio, nd higher (P, ) plsm ysteine nd glutthione peroxidse (GPx) tivity thn those in the CON nd DLM tretment groups. The feed intke (P, ) nd ody weight of piglets verged ross post-wening (PW) dys were higher (P, ) in the HMTBA tretment group thn in the DLM tretment group nd were higher (P, ) nd tended (P, 1) to e higher, respetively, in the HMTBA tretment group thn in the CON tretment group. Inresed (P, ) GSSG ontent nd GSSG:GSH rtio nd down-regulted (P, ) expression of nutrient trnsport genes were oserved in the jejunum of piglets on PW dy 7 thn on PW dy. On PW dy 1, the HMTBA diet-fed piglets hd higher (P, ) intestinl GSH ontent thn the CON diet-fed piglets nd higher (P, ) plsm GPx tivity, villus height nd golet ell numers thn the CON diet- nd DLM diet-fed piglets. In onlusion, erly wening-indued growth retrdtion ppers to e ttenuted through hnges in plsm AA profiles nd elevtion of growth performne nd intestinl ntioxidnt pity in piglets following inresed onsumption of methionine s HMTBA. Key words: Erly wening stress: Intestine: Methionine hydroxy nlogues: Antioxidnt pity Over the lst few yers, pigs in the swine industry hve lrgely een wened t 17 d of ge, with the verge ge eing 19 d (1). Wening-indued intestinl dysfuntion nd growth retrdtion (,) hve eome the mjor onstrints on the prtie of improving sow produtivity through erly wening. It hs een found tht gstrointestinl dysfuntion indued y erly wening ould e ttenuted y delyed wening in pigs (), whih indites the signifine of promoting timely growth of neontl intestine. Given tht milk is the min soure of nutrition in suking piglets, mternl nutrition my ffet their overll growth nd intestinl development vi the regultion of milk quntity nd qulity. Gun et l. () reported tht the dietry rtios of true digestile essentil mino ids (AA) inluding methionine:lysine required for milk synthesis in ltting sows should e higher thn the vlues reommended y the Ntionl Reserh Counil (NRC) (). Further studies (7) hve shown tht ompred with those reommended y the NRC (), the inlusion levels of methionine nd methionine þ ysteine should e inresed y 1 nd %, respetively, in n idel AA profile for Arevitions: AA, mino ids; CON, ontrol diet; DLM, DL-methionine; GPx, glutthione peroxidse; GSH, redued glutthione; GSSG, oxidised glutthione; HMTBA, DL--hydroxy--methylthioutyrte; NRC, Ntionl Reserh Counil; POGRG, perentge of oxidised glutthione to redued glutthione; PVHCD, perentge of villus height to rypt depth; PW, post-wening; SAA, sulphur-ontining mino ids. * Corresponding uthor: Professor Z. Fng, fx þ , emil fngzhengfeng@hotmil.om These uthors eqully ontriuted to the study.

2 8 H. Li et l. ltting sows. However, no physiologil explntion exists for the role of inresed methionine onsumption in ltting sows nd their offspring. In ddition to pre-wening intestinl development sttus, the low feed intke is usully onsidered to e nother mjor ftor responsile for intestinl dysfuntion nd trophy in wening piglets (8). Previous work hs shown gstrointestinl dysfuntion to e physiologilly ssoited with erly wening-indued oxidtive stress (). It hs een estlished tht the synthesis of oth the mjor ellulr redutnt, glutthione (9), nd the mjor extrellulr redutnt, ysteine (1), depends on the AA ysteine or its preursor, methionine (11). Moreover, sulphur-ontining mino ids (SAA) hve een shown to e essentil for norml intestinl muosl growth in neontl pigs (1), nd the suppression of intestinl epithelil growth y SAA defiieny hs lso een demonstrted in neontl pigs (1). These funtionl roles neessitte onstnt nutritionl intke of SAA. However, the low feed intke in wening pigs, espeilly during the first few post-wening (PW) dys, my derese the supply of nutritionl SAA to levels lower thn tht prtilly required. DL-Methionine (DLM) nd its hydroxy nlogue, DL--hydroxy--methylthioutyrte (HMTBA), re routinely used s methionine supplements in livestok industry. Our previous studies (1 17) in pigs hve shown the differenes in the sorption nd metolism of these two methionine soures. The ojetive of the present study ws to determine whether erly wening-indued growth retrdtion ould e ttenuted y inresed onsumption of methionine s DLM or HMTBA in oth ltting sows nd wened piglets. Plsm nd milk AA profiles nd intestinl morphology, ntioxidnt pity nd mrna undne of genes relted to nutrient trnsport were lso evluted, whih my provide iologil explntion for performne responses. Mterils nd methods Animls nd diets The study protool ws pproved y the Animl Cre nd Use Committee of Animl Nutrition Institute, Sihun Agriulturl University, nd the study ws rried out in ordne with the NRC Guide for the Cre nd Use of Lortory Animls. A totl of eighteen pregnnt rossed (Lndre Yorkshire) primiprous sows (with similr ody weight nd kft thikness) were used in the experiment. On dy 11 of gesttion, sows were moved into frrowing rtes ( 1 7 m) with n re ( 1 m) lloted to neworn piglets on eh side of the rtes in n environmentlly regulted frrowing house. Temperture ws mintined t ^ 18C in the frrowing house, nd het lmps provided supplementl het to the pigs. All sows were fed the ontrol (CON) diet until frrowing, fter whih six sows were ontinued to e fed the CON diet nd the other sows were fed either the DLM or the HMTBA diet (six sows per dietry tretment). The CON diet for ltting sows ws formulted sed on the NRC () ltting sow nutritionl requirement vlues (Tle 1). The DLM nd HMTBA diets were prepred y dding DLM nd HMTBA to the CON diet, respetively, t % of the totl SAA present in the CON diet, suh tht the rtio of methionine þ ysteine:lysine pprohed tht reommended y Dourmd et l. (7). Within 1 h of frrowing, ll litters were stndrdised to hve ten piglets per sow. On postntl dy 1, ll litters were stndrdised to hve nine piglets per sow, nd piglets were given free ess to the diets tht they ontinued to reeive fter wening. Piglets from the CON, DLM nd HMTBA groups of sows were fed the wened diets CON, DLM nd HMTBA, respetively. The CON diet for piglets ws formulted sed on the NRC () piglet nutritionl requirement vlues (Tle ). The DLM nd HMTBA diets were Tle 1. Ingredients nd omposition of the ontrol (CON) diet of sows* Ingredients Content (kg) Composition % Mize 8 CP 1 1 Whet rn Lysine 97 Soyen mel (CP %) Methionine Frutose gluose syrup Methionineþystine Gluose Threonine Soyen oil Tryptophn 19 Lysine-HCl 1 Vline 8 Threonine 8 Digestile Lysine 8 Vline (99 %) 9 Digestile methionine Dilium phosphte 1 1 Digestile methionineþystine Limestone 1 C 8 NCl Totl P Premix Aville P 8 Choline hloride ( %) Totl 1 CP, rude protein. * The DL-methionine (DLM) nd DL--hydroxy--methylthioutyrte (HMTBA) diets were prepred y dding 1 kg of DLM (99 %) nd 1 1 kg of HMTBA (88 %) to the CON diet t the expense of mize to supply % of the totl sulphur-ontining mino ids. Eh diet (per kg) ontined 1 kj digestile energy. Provided the following per kg of diet: retinol, mg; vitmin D, 7mg; vitmin E, 1 mg; mendione, mg; thimin, mg; rioflvin, 8 mg; niin, mg; D-pnthotheni id, 1 mg; vitmin B, mg; vitmin B 1,mg; D-iotin, mg; foli id, mg. Provided the following per kg of diet: Cu, 1 mg; Fe, 1 mg; Mn, mg; Zn, 8 mg; I, 1 mg; Se, mg; ntioxidnt, 1 mg; nti-mould dditive, mg.

3 Methionine nutrition nd erly wening pigs 87 Tle. Ingredients nd omposition of the ontrol (CON) diet of piglets* Ingredients Content (kg) Composition % Extruded mize CP 19 7 Extruded soyen 1 Lysine 1 Dried whey (CP %) 1 Methionine 9 Soyen mel (CP %) 11 Methionineþystine 7 Fishmel (CP %) Threonine 8 Frutose gluose syrup 1 Tryptophn Gluose Digestile lysine 1 Porine plsm Digestile methionine Lysine-HCl 1 Digestile methionineþystine 8 Methionine Digestile threonine 7 Threonine Digestile tryptophn Dilium phosphte 7 C 8 Limestone 7 8 Totl P NCl Aville P ZnO Premix Choline hloride ( %) 1 Totl 1 prepred y dding DLM nd HMTBA to the CON diet, respetively, t % of the totl SAA present in the CON diet. Piglets from three of the six sows in eh group were wened on postntl dys 1 nd 8, respetively. Colletion of lood, milk nd tissue smples CP, rude protein. * The DL-methionine (DLM) nd DL--hydroxy--methylthioutyrte (HMTBA) diets were prepred y dding 1 9 kg of DLM (99 %) nd 1 kg of HMTBA (88 %), respetively, to the CON diet t the expense of mize to supply % of the totl sulphur-ontining mino ids. Eh diet (per kg) ontined 1 kj digestile energy. Provided the following per kg of diet: retinol, mg; vitmin D, mg; vitmin E, mg; mendione, mg; thimin, mg; rioflvin, 7 mg; niin, mg; D-pnthotheni id, 18 mg; vitmin B, mg; vitmin B 1,mg; D-iotin, mg; foli id, 1 mg. Provided the following per kg of diet: Cu, mg; Fe, 1 mg; Mn, mg; Zn, 1 mg; I, mg; Se, mg; ntioxidnt, 1 mg; nti-mould dditive, 8 mg. At min efore the morning mel on postprtum dy 1, following n overnight period of feed withdrwl, lood smples (1 ml) were withdrwn from the superior ven v of eh sow into heprinised tues nd immeditely entrifuged for 1 min t g nd 8C. The superntnts were divided into four susmples nd stored t 8C until nlysis. Milk smples ( ml) were lso olleted from eh sow on dy 1 efore the morning mel s desried previously (18). Briefly, piglets were seprted from their dms for 9 min initilly, nd then 1 interntionl units (IU) of oxytoin ( nonpeptide, 1 IU of oxytoin is the equivlent of out mg of pure peptide) were injeted into the er vein of eh sow, nd the funtionl petorl nd inguinl glnds were milked mnully to ollet milk smples, whih were stored t 8C until nlysis. At min efore the morning mel on postntl dys 1, 8 nd, following n overnight period of feed withdrwl, lood smples were olleted from the jugulr vein of one pig in eh litter s desried previously (19). Immeditely fter the olletion of lood smples, pigs were nesthetised nd led y exsnguintion s desried previously (17). A totl of fifty-four piglets were slughtered ording to the tretment groups (Tle ). Immeditely fter slughter, the domen ws opened, nd the entire intestine ws rpidly removed, thoroughly fluxed with sterile sline to remove luminl digest nd then disseted free of mesenteri tthments nd pled on smooth, old-surfe try. A m Tle. Feeding tretments for piglets efore slughter* Slughter time Postntl dy 1 (n ) Postntl dy 8 (n ) Postntl dy (n ) CON-1 CON-8 CON-8þCON-7PW CON-1þCON-7PW CON-1þCON-1PW DLM-1 DLM-8 DLM-8þDLM-7PW DLM-1þDLM-7PW DLM-1þDLM-1PW HMTBA-1 HMTBA-8 HMTBA-8þHMTBA-7PW HMTBA-1þHMTBA-7PW HMTBA-1þHMTBA-1PW CON, ontrol; PW, post-wening; DLM, DL-methionine; HMTBA, DL--hydroxy--methylthioutyrte. * In CON-1 tretment, piglets were rered for 1 d y sows fed the CON diet; in CON-7PW tretment, piglets were fed the CON diet for seven PW dys; nd in CON-1þCON-7PW tretment, piglets were rered for 1 d y the CON diet-fed sows nd then wened to e fed the CON diet for seven PW dys. The rest of the tretments n e dedued y nlogy.

4 88 H. Li et l. Tle. Primers used in rel-time PCR nlysis of gene expression in pig jejunum Genes Aession numer Primers Length (p) 18S riosoml RNA NR_1.1 Forwrd: -GACTCAACACGGGAAACCTCAC- 11 Reverse: -ATCGCTCCACCAACTAAGAACG- Apo A-IV preursor NM_188.1 Forwrd: -GTGGCTACTGTGATGTGGGACTAC- 9 Reverse: -CCAAGTTTGTCCTGGAAGAGAGTG- FZHUI ftty id-inding protein intestinl NM_ Forwrd: -TACAGCCTCGCAGACGGAAC- 9 Reverse: -CCTCTTGGCTTCTACTCCTTCATAC- N- nd Cl-dependent retine trnsporter I NM_ Forwrd: -TCGACTACTACTCGGCCAGCG- 77 Reverse: -ACCAGCGGGGTGAAGAAAGAC- setion of tissue smples representing duodenum, jejunum nd ileum ws olleted s desried previously () nd stored t 88C for susequent RNA isoltion. Smples of intestinl segments were olleted for the ssessment of intestinl morphology (1). The intestinl segments were opened lengthwise nd pinned to piee of dentl wx with the serosl side fing the wx. Susequently, the smples were fixed in % prformldehyde solution with the muosl side fing downwrds to fix the villi vertilly (). Mesurements Growth performne determintion. The ody weight of sows ws determined efore the morning mel on postprtum dys, 1 nd 8. The kft thikness of sows ws mesured mm from the left side of the dorsl midline t the lst ri level (P) using ultrsound (Reno Len-Meter) on postprtum dys, 1 nd 8. The ody weight of piglets ws determined efore the morning mel on postntl dys, 1, 8 nd. The feed intke of eh sow nd eh litter of piglets ws reorded dily. Amino ids nlysis For free AA nlysis, the frozen plsm nd milk smples were thwed t 8C. The protein preipittion proedure ws rried out s desried previously (). Briefly, 1 ml of the smple nd ml of 7 % (w/v) TCA solution were mixed thoroughly nd entrifuged t 1 g nd 8C for 1 min. Then, the superntnt ws olleted nd nlysed for AA y ion-exhnge hromtogrphy using n L88 high-speed AA nlyser (Hithi) s desried y Yin et l. (). Histologil proedure Intestinl morphology ws exmined s desried in detil y Berkeveld et l. (). From eh segment, two trnsverse tissue setions were ut using stereo mirosope. These setions were dehydrted, emedded together in prffin wx nd setioned t mm. Lter, one setion ws trnsferred onto slide nd stined with hemtoxylin nd eosin. Hene, eh slide ontined two trnsverse tissue setions of gut segment. The height of twelve villi nd depth of twelve rypt were determined in eh slide using the Nikon Elipse 8I fluoresene mirosope (Nikon) equipped with n epifluoresene imge nlysis system. Villi nd rypts were only mesured when there ws omplete longitudinl setion of villus nd n ssoited rypt. The verge villus height nd rypt depth per slide were used s experimentl oservtion vlues. The numer of golet ells in the intestinl muos ws determined y periodi id Shiff hemtoxylin stining of intestinl setions (), nd it is expressed per 1 mm length of villus. Antioxidnt pity nlysis Frozen jejunl smples were rpidly thwed t 8C nd homogenised on ie in twenty volumes (w/v) of ie-old physiologil sline nd entrifuged t g for min t 8C, nd then the superntnts were olleted for redued glutthione (GSH) nd oxidised glutthione (GSSG) nlysis. GSH nd GSSG ontents in the smples were determined using method desried previously (7). Briefly, totl glutthione ontent ws determined y lulting the rte of redution of, -dithiois--nitroenzoi id y GSH t 1 nm nd ompring this with GSH stndrd urve. GSSG ontent in the smples ws determined using the sme method fter treting the smples with -vinylpyridine for min. GSH ontent ws lulted s follows: totl glutthione ontent GSSG ontent. Protein onentrtion in jejunl smples ws mesured using the Brdford method (8). The tivity of glutthione peroxidse (GPx) in plsm ws determined y quntifying the rte of H O -indued oxidtion of GSH to Tle. Bkft thikness, ody weight nd feed intke of sows on postprtum dys, 1 nd 8 (Men vlues with their pooled stndrd errors) CON DLM HMTBA Pooled SEM Bkft thikness (mm) Dy Dy Dy Body weight (kg) Dy Dy Dy Totl feed intke (kg) Dys Dys Energy intke (kj)* Dys Dys CON, ontrol; DLM, DL-methionine; HMTBA, DL--hydroxy--methylthioutyrte. * Clulted sed on dily digestile energy intke s perentge of metoli ody weight.

5 Methionine nutrition nd erly wening pigs 89 Tle. Comprison of the ody weight gin (kg) of piglets wened t 8 d of ge with tht of piglets wened t 1 d of ge t eh of the experimentl phses* (Men vlues with their pooled stndrd errors) Wening time Phses Diets 1 d 8 d Pooled SEM P Dys 1 8 CON , 1 DLM 1 7 1, 1 HMTBA , 1 Dys 8 CON DLM , HMTBA 1, Dys 1 CON DLM , 1 HMTBA 1 9 CON, ontrol; DLM, DL-methionine; HMTBA, DL--hydroxy--methylthioutyrte. * Within eh of the phses, there were no differenes (P. 1) mong the dietry tretment groups. GSSG s desried previously (9). One unit of GPx ws defined s the mount required to redue the level of GSH y 1 mmol/l in min per 1 ml of plsm. The tivity of GPx in plsm is expressed s units/1 ml. RNA extrtion nd rel-time PCR Jejunl tissue smples were used to determine the expression of genes relted to the intestinl trnsport of dietry nutrients. The nlysed genes inluded po A-IV preursor, FZHUI (ftty id inding protein humn intestine) ftty id-inding protein, nd N- nd Cl-dependent retine trnsporter I. Totl RNA ws extrted from the frozen smples using the RNAiso Plus regent (Tkr Bio, In.) ording to the mnufturer s speifitions. The onentrtion of RNA in the smples ws determined using DU-8 nulei nd protein detetor (Bekmn) t n optil density (OD) of nm; n OD :OD 8 rtio rnging etween 1 8 nd ws onsidered eptle. The integrity of RNA ws verified y eletrophoresis on 1 % grose gel stined with ethidium romide. Rel-time quntittive PCR nlysis ws rried out using the SYBR Green method nd the ABI 79HT Sequene Detetion System. Briefly, first-strnd omplementry DNA were synthesised from 1 mg of totl RNA s desried previously (1). The therml yling prmeters were s follows: 98C for s, followed y forty yles t 98C for 1 s nd 8C for s, followed y 98C for 1 s, 8C for 1 min nd 98C for 1 s. To onfirm speifi produt mplifition, melting urve nlysis ws rried out, nd the PCR produts were lso deteted y ethidium romide stining of the % grose gels fter eletrophoresis using Tris ette EDTA uffer. Primers for individul genes were designed using Primer Express. (Applied Biosystems). Primers used in rel-time PCR nlysis of gene expression in pig jejunum re given in Tle. The stndrd urve of eh gene ws run in duplite nd three times for otining relile mplifition effiieny vlues s desried previously (). The orreltion oeffiients (r) of ll the stndrd urves were. 99 nd the mplifition effiieny vlues were etween 9 nd 11 %. The reltive mrna undnes of the nlysed genes were lulted using the DDCT method (1), nd ll the dt were normlised to those of the 18S RNA () in the sme smples. Sttistil nlysis Dt were nlysed s desried y Littell et l. (). The MIXED proedures of SAS sttistil pkge (version 8.1; SAS Institute, In.) were used to nlyse repeted-mesures dt inluding ody weight, feed intke, intestinl morphology, ntioxidnt pity nd mrna undne dt. The generlised liner model proedures of SAS sttistil pkge (version 8.1; SAS Institute, In.) were used to nlyse dt suh s AA onentrtions, whih were mesured t single time point. Mens of the dietry tretment groups were ompred using the lest-signifint differene test when the F test in the ANOVA ws signifint. P vlues, were onsidered sttistilly signifint nd P vlues, 1 were onsidered tendeny towrds differene. Results Growth performne There ws no differene in the ody weight nd kft thikness of sows on postprtum dys, 1 nd 8 (P. 1) mong the dietry tretment groups (Tle ). There ws no differene in totl feed intke nd energy (A) 8 Body weight (kg) (B) 8 Body weight (kg) , Fig. 1. Effets of (A) dietry tretment (, ontrol (CON);, DL-methionine;, DL--hydroxy--methylthioutyrte) nd (B) ge (, 1d;, 8d; ; d) on the ody weight of piglets mesured over postntl dys. Vlues re mens, with their stndrd errors represented y vertil rs.,, Men vlues with unlike letters were signifintly different (P, ). * Men vlue tended to e different from tht of the CON group (P, 1). *

6 8 H. Li et l. (A) 1 1 Feed intke (g/d) (B) Feed intke (g/d) f 1 1 f e,,d d,e,d,e d,e 1 Fig.. Effets of (A) post-wening (PW) dy nd (B) dietry tretment (, ontrol;, DL-methionine;, DL--hydroxy--methylthioutyrte) on the feed intke of piglets wened t 1 d of ge. Vlues re mens, with their stndrd errors represented y vertil rs.,,,d,e Men vlues with unlike letters were signifintly different (P, ). intke per metoli ody weight (P. 1) mong the dietry tretment groups during eh of the experimentl phses. Piglets wened t 1 d of ge hd lower (P, 1) dy 1 8 weight gin, ut higher (P, ) dy 8 weight gin thn those wened t 8 d of ge (Tle ). There ws no differene in piglet weight gin (P. 1) mong the dietry tretment groups during these experimentl phses. However, it ws found tht the ody weight of piglets verged over postntl dys ws signifintly (P, ) higher in the HMTBA tretment group thn in the DLM tretment group nd tended (P, 1) to e higher in the HMTBA tretment group thn in the CON tretment group (Fig. 1(A)). With n inrese in ge, signifint (P, ) inrese ws oserved in the ody weight of piglets (Fig. 1(B)). A grdul inrese ws oserved in the feed intke of piglets from PW dy 1 to dy 1 (Fig. (A)). Speifilly, feed intke ws higher (P, ) on PW dy thn on PW dys 1 nd, nd further signifint (P, ) inrese ws oserved on PW dys 7 nd 1. Overll, feed intke verged ross PW dys ws higher (P, ) in the HMTBA tretment group thn in the CON nd DLM tretment groups (Fig. (B)). Plsm free mino id onentrtions of sows The effets of diets with supplementl DLM or HMTBA on the plsm free AA onentrtions of sows on postprtum dy 1 re summrised in Tle 7. Plsm methionine onentrtions, PW dys showed tendeny (P, 1) to e ffeted y the dietry tretments. Compred with those in the CON diet-fed sows, plsm turine onentrtions tended (P, 1) to e higher in the HMTBA diet-fed sows, nd higher (P, ) turine onentrtions ut lower (P, ) isoleuine onentrtions nd tendeny (P, 1) towrds lower lysine onentrtions were oserved in the DLM diet-fed sows. The DLM diet- nd HMTBA diet-fed sows hd higher (P, ) plsm free lnine onentrtions, ut lower (P, ) itrulline onentrtions thn the CON diet-fed sows. Milk free mino id onentrtions of sows The effets of diets with supplementl DLM or HMTBA on the milk free AA onentrtions of sows on postprtum dy 1 re summrised in Tle 8. Milk free leuine nd phenyllnine onentrtions were higher (P, ) in the DLM diet-fed sows thn in the CON diet- nd HMTBA diet-fed sows. Compred with the CON diet-fed sows, the HMTBA diet-fed sows hd higher (P, ) milk free turine onentrtions nd exhiited tendeny (P, 1) towrds higher vline onentrtions, wheres the DLM diet-fed sows hd higher (P, ) vline onentrtions nd exhiited tendeny (P, 1) towrds higher turine onentrtions. Milk free lnine onentrtions were higher (P, ) nd histidine onentrtions tended (P, 1) to e higher in the HMTBA diet-fed sows thn in the CON dietfed sows. The HMTBA diet-fed sows lso hd higher (P, ) milk free histidine onentrtions nd exhiited tendeny (P, 1) towrds higher lnine nd ornithine onentrtions ompred with the DLM diet-fed sows. Plsm free mino id onentrtions of piglets The effets of sow diets with supplementl DLM or HMTBA on the plsm free AA onentrtions of sukling piglets on Tle 7. Effets of diets with supplementl DL-methionine (DLM) or DL--hydroxy--methylthioutyrte (HMTBA) on the plsm free mino id onentrtions (mmol/l) of sows on postprtum dy 1 (Men vlues with their pooled stndrd errors) Amino ids CON DLM HMTBA Pooled SEM Met 7 7 Lys 19, 71 * 117 Leu Ile Phe Vl His Pro Arg Al Ser Glu Tyr Cit Orn 8 7 Tu 19, * CON, ontrol., Men vlues within row with unlike supersript letters were signifintly different (P, ). * Men vlue tended to e different from tht of the CON group (P, 1).

7 Methionine nutrition nd erly wening pigs 81 Tle 8. Effets of diets with supplementl DL-methionine (DLM) or DL--hydroxy--methylthioutyrte (HMTBA) on the milk free mino id onentrtions (mmol/l) of sows on postprtum dy 1 (Men vlues with their pooled stndrd errors) Amino ids CON DLM HMTBA Pooled SEM Met 1 Lys Thr Leu 9 8 Ile Phe Vl 7, * His, * 1 Pro 188 Arg Cys 18 8 Al 11 18, Ser Glu 71 Gly 19 Tyr 1 Orn Tu 1 179, * CON, ontrol., Men vlues within row with unlike supersript letters were signifintly different (P, ). * Men vlue tended to e different from tht of the CON group (P, 1). postntl dy 1 re summrised in Tle 9. Plsm free ysteine onentrtions were higher (P, ) in the HMTBA tretment group thn in the DLM nd CON tretment groups. Plsm free itrulline onentrtions were higher (P, ) in the CON tretment group thn in the DLM nd HMTBA tretment groups. Plsm free glutmte onentrtions showed tendeny (P, 1) to e ffeted y the dietry tretments. Intestinl morphology nd golet ell numers The effets of wening time, dietry tretment, wening time dietry tretment intertion, wening time intestinl site intertion nd dietry tretment intestinl site intertion on intestinl morphology nd golet ell numers of piglets re shown in Fig.. Representtive stining of golet ells in eh group of piglets is shown in Fig.. Overll, piglets wened t 1 d of ge hd higher (P, 1) villus height, lower (P, 1) rypt depth nd thus higher (P, 1) perentge of villus height to rypt depth (PVHCD) thn those wened t 8 d of ge (Fig. (A)). Dietry tretment lso hd signifint (P, 1) effet on intestinl morphology nd golet ell numers (Fig. (B)). Among the three dietry tretment groups, the HMTBA diet-fed piglets hd higher (P, ) villus height nd PVHCD thn the CON diet- nd DLM diet-fed piglets, wheres the CON diet-fed piglets hd lower (P, ) golet ell numers thn the DLM diet- nd HMTBA diet-fed piglets (Fig. (B)). In Fig. (C), the effet of wening time dietry tretment intertion on villus height (P, ) nd golet ell numers (P, 1) is shown. The villus height nd golet ell numers of piglets wened t 1 d of ge were higher (P, ) in the HMTBA tretment group thn in the CON nd DLM tretment groups, nd the golet ell numers of piglets wened t 8 d of ge were higher (P, ) in the DLM tretment group thn in the CON nd HMTBA tretment groups. In Fig. (D), the effet of wening time intestinl site intertion on villus height (P, 1), PVHCD (P, ) nd golet ell numers (P, 1) is shown. Compred with piglets wened t 1 d of ge, those wened t 8 d of ge hd lower (P, ) villus height nd PVHCD in oth the jejunum nd ileum, lower (P, ) golet ell numers in the duodenum, nd higher (P, ) golet ell numers in the jejunum. In Fig. (E), the effet of dietry tretment intestinl site intertion on golet ell numers is shown (P, 1). Both the HMTBA nd DLM tretment groups hd higher (P, ) golet ell numers in the duodenum nd ileum thn the CON tretment group. In ontrst, villus height, rypt depth nd PVHCD were not ffeted (P. 1) y dietry tretment intestinl site intertion. Antioxidnt pity In Fig. (A), the effet (P, 1) of dietry tretment on jejunl GSH ontent, GSSG ontent nd POGRG (perentge of GSSG to GSH) is shown. Compred with the CON tretment group, oth the DLM nd HMTBA tretment groups hd higher (P, ) intestinl GSH nd GSSG ontents. Moreover, mong the three dietry tretment groups, the HMTBA nd DLM tretment groups hd the highest GSH nd GSSG ontents, respetively. As result, POGRG ws lower (P, ) in the HMTBA tretment group thn in the CON nd DLM tretment groups. In Fig. (B), the effet of PW dy (P, 1) on plsm GPx tivity nd jejunl GSH ontent, GSSG ontent nd POGRG is shown. Plsm GPx tivity ws higher (P, ) on PW dy 7 thn on PW dys nd 1. Deresed (P, ) intestinl GSH ontent nd inresed (P, ) GSSG ontent were oserved on PW dy 7 thn on Tle 9. Effets of sow diets with supplementl DL-methionine (DLM) or DL--hydroxy--methylthioutyrte (HMTBA) on the plsm free mino id onentrtions (mmol/l) of sukling piglets on postntl dy 1 (Men vlues with their pooled stndrd errors) Amino ids CON DLM HMTBA Pooled SEM Met Lys Leu Ile Phe Vl His 7 11 Pro Arg Cys Al Ser Glu Gly Tyr Cit Orn 1 9 Tu CON, ontrol., Men vlues within row with unlike supersript letters were signifintly different (P, ).

8 H. Li et l. (A) Aordingly, POGRG ws higher (P, ) on PW dy 1 thn on PW dy, ut ws lower (P, ) thn tht oserved on PW dy 7. Plsm GPx tivity (Fig. (C)), intestinl GSH ontent (Fig. (D)), GSSG ontent (Fig. (E)) nd POGRG (Fig. (F)) were ffeted (P, 1) y dietry tretment PW dy intertion. As shown in Fig. (C), oth the CON nd DLM ** ** ** 1 1 Crypt depth (µm) Villus height (µm) (B) Golet ells (per 1 µm) PVHCD (%) (A), 1 1 (C) 1 1 Crypt depth (µm),, d (D) 1 d 8 d 1 d 1 1 Golet ells (per 1 µm) PVHCD (%) (B) 8 d Golet ells (per 1 µm) Villus height (µm),,d,, d, 1 d 8 d 1 d Villus height (µm) 8 d PVHCD (%) 1 d 8 d Golet ells (per 1 µm) (E) Golet ells (per 1 µm) Villus height (µm) 1, 1 (C) Duodenum Jejunum Ileum Fig.. Effets of (A) wening time (, 1 d;, 8 d), (B) dietry tretment, (C) wening time dietry tretment intertion, (D) wening time intestinl site intertion nd (E) dietry tretment intestinl site intertion (, ontrol;, DL -methionine;, DL --hydroxy--methylthioutyrte;, duodenum;, jejunum;, ileum) on the intestinl morphology nd golet ell numers of piglets on postntl dy. Vlues re mens, with their stndrd errors represented y vertil rs.,,,d Men vlues with unlike letters were signifintly different (P, ). ** Men vlues were signifintly different etween the dietry tretment groups (P, 1). PVHCD, perentge of villus height to rypt depth. PW dy. Inresed (P, ) GSH ontent nd deresed (P, ) GSSG ontent were oserved on PW dy 1 thn on PW dy 7, ut neither GSH ontent nor GSSG ontent ws restored to the level oserved on PW dy. Fig.. Representtive stining ( ) of ilel golet ells in the (A) ontrol, (B) DL -methionine nd (C) DL --hydroxy--methylthioutyrte diet-fed groups of pigs. 8

9 Methionine nutrition nd erly wening pigs 8 (A) (B) (C) GPx tivity (units/1 µl) (E) GSSG ontent (µmol/g proteinl) GPx (units/1 µl) e d,e GPx (units/1 µl),,,d e e, GSH (µmol/1 mg protein) GSH (µmol/1 mg protein) (D) 8 GSH ontent (µmol/1 mg proteinl) (F) 11 tretment groups hd higher (P, ) GPx tivity on PW dy 7 thn on PW dys nd 1. In ontrst, there ws little vrition in plsm GPx tivity in the HMTBA tretment group from PW dy to dy 1, nd it ws higher (P, ) thn tht in the CON nd DLM tretment groups on PW dys nd 1. On PW dy, intestinl GSH ontent ws highest in the HMTBA tretment group, followed y tht in the DLM tretment group, nd lowest in the CON tretment group (Fig. (D)), intestinl GSSG ontent ws higher (P, ) in the DLM tretment group thn in the CON nd HMTBA tretment groups (Fig. (E)), nd POGRG ws lower (P, ) in POGR (%) d d d GSSG (µmol/g protein) GSSG (µmol/g protein) d d d 1 PW dy PW dy 7 PW dy 1 PW dy PW dy 7 PW dy 1 d e 1 PW dy PW dy 7 PW dy 1 PW dy PW dy 7 PW dy 1 Fig.. Effets of (A) dietry tretment, (B) post-wening (PW) dy nd (C F) dietry tretment PW dy intertion (, ontrol;, DL-methionine;, DL--hydroxy--methylthioutyrte;,PWdy;,PWdy7;,PWdy1) on the ntioxidnt pity of piglets wened t 1 d of ge. Vlues re mens, with their stndrd errors represented y vertil rs.,,,d,e Men vlues with unlike letters were signifintly different (P, ). GPx, glutthione peroxidse; GSH, redued glutthione; GSSG, oxidised glutthione; POGRG, perentge of oxidised glutthione to redued glutthione POGRG (%) POGRG (%) the HMTBA tretment group thn in the CON nd DLM tretment groups (Fig. (F)). The three dietry tretment groups hd inresed (P, ) GSSG ontent (Fig. (E)) nd POGRG (Fig. (F)) on PW dy 7 thn on PW dy, wheres GSH ontent (Fig. (D)) in the DLM nd HMTBA tretment groups ws lower (P, ) on PW dy 7 thn on PW dy. On PW dy 1, oth intestinl GSH ontent (Fig. (D)) nd GSSG ontent (Fig. (E)) were higher (P, ) in the DLM nd HMTBA tretment groups thn in the CON tretment group, ut POGRG ws lower (P, ) in the HMTBA tretment group thn in the DLM tretment group (Fig. (F)). Gene expression in the jejunum of piglets In Fig., the effets of wening time, PW dy, dietry tretment nd wening time PW dy intertion on the reltive mrna undnes of genes relted to nutrient trnsport in the jejunum of piglets re shown. Overll, piglets wened t 1 d of ge hd higher mrna undnes of po A-IV preursor (P, 1) nd N- nd Cl-dependent retine trnsporter I (P, 1) thn those wened t 8 d of ge (Fig. (A)). The mrna undnes of po A-IV preursor (P, ), N- nd Cl-dependent retine trnsporter I (P, 1) nd FZHUI ftty id-inding protein (P, 1) were lower on PW dy 7 thn on PW dy (Fig. (B)). Dietry tretment lso hd signifint (P, ) effet on the mrna undne of FZHUI ftty id-inding protein, whih ws lower (P, ) in the HMTBA tretment group thn in the CON nd DLM tretment groups (Fig. (C)). In Fig. (D), the effet (P, ) of wening time PW dy intertion on the mrna undne of po A-IV preursor is shown; the mrna undne of po A-IV preursor ws lower on PW dy 7 thn on PW dy in piglets wened t 1 d, ut no differene ws oserved etween PW dys in piglets wened t 8 d of ge. Disussion The primry im of the present study ws to determine whether erly wening-indued growth retrdtion ould e ttenuted y inresed onsumption of methionine s DLM or its hydroxyl nlogue (HMTBA) in oth ltting sows nd wened piglets. Given tht the lttion performne of sows is, to gret extent, ffeted y feed intke (), whih is orrelted with prity, genotype nd ody sttus (), primiprous sows hving similr geneti kground, ody weight nd kft thikness were used in the present experiment. It hs lso een shown tht mternl tissue moilistion nd nutrient requirements during lttion period re ffeted y litter size (). Therefore, ll litters were stndrdised s desried previously (7) to hve the sme numer of piglets. Bsed on the ove ontrol, we found no sttistil differene in feed intke mong the dietry tretment groups during the experimentl phses. This my llow us to disuss the differene in lttion performne in ssoition with dietry tretments, speifilly methionine levels nd soures. In the present study, the ody weight of piglets verged over postntl dys ws found to e higher in the HMTBA

10 8 H. Li et l. (A) Reltive mrna undne (B) Reltive mrna undne (C) Reltive mrna undne (D) Reltive mrna undne of po A-IV preursor ** Apo A-IV preursor * Apo A-IV preursor Apo A-IV preursor N-nd Cl-dependent retine trnsporter I ** N-nd Cl-dependent retine trnsporter I N-nd Cl-dependent retine trnsporter I FZHUI ftty id-inding protein intestinl FZHUI ftty id-inding protein intestinl tretment group thn in the CON nd DLM tretment groups. To our knowledge, wening is lwys ssoited with redution in food intke in young mmmls inluding piglets (8,8). The higher feed intke verged over PW dys my ount for the fster growth of the HMTBA diet-fed piglets. Compred with ge-mthed piglets wened t 8 d of ge, piglets wened t 1 d of ge grew slower during postntl dys 1 8, ut grew fster during postntl dys 8, whih is in good greement with the results of previous studies (,9). These oservtions indite the negtive effet of wening on piglet growth, espeilly during the first few ** FZHUI ftty id-inding protein intestinl 1 d 8 d Wening time Fig.. Effets of (A) wening time (,1d;, 8 d), (B) post-wening (PW) dy, (C) dietry tretment (, ontrol;, DL-methionine;, DL--hydroxy-- methylthioutyrte nd (D) wening time PW dy intertion (, PW dy ;, PW dy 7) on the reltive mrna undnes of genes relted to nutrient trnsport in the jejunum of piglets. Vlues re mens, with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different (P, ). Men vlues were signifintly different: * P,, ** P, 1. Men vlues tended to e different etween the dietry tretment groups (P, 1). PW dys. In ddition, on postntl dy, piglets wened t 8 d of ge tended to weigh more thn those wened t 1 d of ge in the DLM tretment group; however, the ody weight of piglets on postntl dy in the HMTBA tretment group ws not different etween the wening ges. These results indite tht inresed onsumption of methionine s HMTBA might llevite stresses ssoited with erly wening. The seondry im of the present study ws to investigte the mehnisms underlying the responses of ltting sows nd their offspring to inresed onsumption of methionine soures. An importnt finding ws tht plsm nd milk AA profiles were responsive to the inresed onsumption of methionine s DLM or HMTBA. The DLM diet-fed sows hd lower plsm lysine nd isoleuine onentrtions thn the CON diet- nd HMTBA diet-fed sows. The deresed lysine onentrtions my e ssoited with the ft tht lysine nd methionine shre the B,þ nd,þ trnsport systems in mmmlin intestine (). In support of this view, Hgihir et l. (1) first demonstrted mutul inhiition etween rginine, ystine, lysine nd ornithine nd oserved some inhiition y methionine in the intestinl epithelium. A down-regultion effet of L-methionine on these trnsporters hs lso een reported for roiler hikens s mens to lower the risk of methionine intoxition (). Lysine is often onsidered to e the first limiting AA for ltting sows sustining milk synthesis, prtiulrly when diets re sed on mize nd soyen mel s the min protein soures (). Reent studies hve indited tht isoleuine plys n importnt role in the tivtion of mmmlin trget of rpmyin signlling, whih is ontrol point in milk protein synthesis (). It ppers tht the lower onentrtions of isoleuine nd lysine in the plsm of the DLM diet-fed sows my limit these two AA ville for delivery to the mmmry glnd nd thus limit milk synthesis. This my e further supported y the oservtion tht the higher free leuine, vline, phenyllnine nd tyrosine onentrtions in the milk of the DLM diet-fed sows did not use differene in the ody weight of 1-d-old piglets mong the dietry tretment groups. There is evidene tht intestinl muosl tolism of rnhed-hin AA my provide nitrogen for the synthesis of oth lnine nd glutmte (). It hs lso een oserved tht the extensive tolism of rnhedhin AA in extr-intestinl tissues stimultes the synthesis of glutmte nd glutmine (,). It ppers tht the higher onentrtions of leuine nd vline in the milk of the DLM diet-fed sows might explin the potentil hnge in piglet plsm glutmte onentrtions, whih exhiited tendeny to e ffeted y the dietry tretments. The onentrtions of non-essentil AA inluding turine, itrulline nd lnine in plsm nd milk were lso ffeted y inresed onsumption of methionine s DLM or HMTBA. Given tht turine is n end produt of methionine metolism, the higher turine onentrtions in the plsm nd milk of the DLM diet- nd HMTBA diet-fed sows thn in those of the CON diet-fed sows might e due to the inresed vilility of methionine, whih showed tendeny to e ffeted y the dietry tretments. Inresed lnine onentrtions nd

11 Methionine nutrition nd erly wening pigs 8 deresed itrulline onentrtions in plsm were lso oserved following DLM nd HMTBA supplementtion ompred with those in the CON diet-fed sows. In previous study, the uptke of glutmine in vivo nd the relese of itrulline y the smll intestine hve een oserved in pigs (7). In this regrd, the higher onentrtions of itrulline in plsm indite tht more mounts of glutmine were tolised in the intestine of the CON diet-fed sows. Brnhed-hin AA tolism hs een shown to e n importnt rte-limiting event in lnine prodution in vivo (8). The deresed plsm isoleuine onentrtions pper to e suggestive of more extensive tolism, whih my ount for the inresed lnine prodution in the DLM diet-fed sows. The intestine is onsidered to e most suseptile to wening stresses (8,8). Moreover, gstrointestinl dysfuntion hs een shown to e physiologilly strongly ssoited with erly wening-indued oxidtive stress (). Thus, the ntioxidnt pity ws evluted to determine the mehnisms tht ount for the differenes mong piglets onsuming different levels nd soures of methionine. Wening ws found to led to elevted plsm GPx tivity nd intestinl GSSG:GSH rtio in piglets in the CON nd DLM tretment groups, whih is in good greement with the results of reent studies (9).Iths een estlished tht GPx nd glutthione ply n importnt role in the mintenne of redox lne of ells through the elimintion of retive oxygen speies suh s H O (),whih n e onverted into oxygen through oupled retions with the onversion of GSH into GSSG, tlysed y GPx. The GSSG:GSH rtio hs een shown to e good mesure of oxidtive stress of n orgnism (1). The inresed GPx tivity ompnied y n inresed GSSG:GSH rtio, s hs een oserved in the present study nd previous studies (9), indites tht the vrition in GPx tivity is n dptive response to oxidtive stress ssoited with erly wening. Interestingly, inresed onsumption of methionine s HMTBA in ltting sows nd wened piglets resulted in reltively onstnt plsm GPx tivity in piglets from PW dy to dy 1. One possile explntion is tht the inresed plsm ysteine onentrtions my enhne the pity of retive oxygen speies elimintion. In support of this view, ysteine is prtiulrly sensitive to retive oxygen speies nd thus is quntittively importnt retive oxygen speies svenger ().Given tht the synthesis of oth the mjor ellulr redutnt, glutthione (9), nd the mjor extrellulr redutnt, ysteine (1), depends on the AA ysteine or its preursor, methionine (11), the higher intestinl GSH ontent nd plsm ysteine onentrtions t wening nd the lower GSSG:GSH rtio s oserved t wening nd PW dy 1 my reflet the high effiieny of methionine soure s HMTBA thn s DLM to enhne the ntioxidnt pity of piglets. In ddition, onsistent with previous studies (), it ws found tht the expression of intestinl trnsport genes inluding po A-IV preursor, FZHUI ftty id-inding protein nd N- nd Cl-dependent retine trnsporter I in the jejunum of piglets ws lower on PW dy 7 thn on PW dy, inditing the signifint role of wening in the redution of the expression of these trnsport genes. Interestingly, the expression of FZHUI ftty id-inding protein ws lso ffeted y the dietry tretments with down-regulted expression eing oserved in the HMTBA tretment group, the underlying mehnism of whih remins to e eluidted. Compred with the CON tretment group, higher villus height nd villus height:rypt depth rtio nd higher numer of golet ells were lso oserved in the intestine of piglets in the HMTBA tretment group, whih provides further evidene for the llevited wening stresses following HMTBA onsumption. In summry, inresed onsumption of methionine s HMTBA ws found to llevite erly wening-indued growth retrdtion in piglets, whih is physiologilly ssoited with elevted mounts of SAA ville for delivery to extr-intestinl tissues without ompromising lysine nd isoleuine vilility, nd promote growth performne nd ntioxidnt pity of the intestine. This novel finding my hve importnt implitions for the nutritionl mngement of ltting sows nd wening piglets. Aknowledgements The uthors thnk Professor Mei Yu t Huzhong Agriulturl University for her vlule ssistne in sttistil nlysis. The present study ws supported y the Rhodimet Reserh Grnt from Adisseo Frne S.A.S., Brind, Antony Cedex, Frne, the Ntionl Nturl Siene Foundtion of Chin (91), nd the Sihun Provine Siene Foundtion for Fostering Youths Tlents (11JQ1). The uthors ontriutions re s follows: Z. F. designed the study; H. L., H. W., X. Z., C. W., X. W., L. T., L. C., Y. L., S. X. nd G. T. rried out the study; Z. F., Y. M., D. W. nd H. 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