Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica)

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1 University of Nebrsk - Lincoln DigitlCommons@University of Nebrsk - Lincoln Publictions from USDA-ARS / UNL Fculty U.S. Deprtment of Agriculture: Agriculturl Reserch Service, Lincoln, Nebrsk 2004 Betine improves growth, but does not induce whole body or heptic plmitte oxidtion in swine (Sus scrof domestic) Dine Wry-Chen USDA-ARS, dwc@cdrh.fd.gov Igncio Fernández-Fígres USDA-ARS Erkki Virtnen Finnfeeds Intl. Normn C. Steele USDA-ARS Thoms J. Cpern USDA-ARS Follow this nd dditionl works t: Prt of the Agriculturl Science Commons Wry-Chen, Dine; Fernández-Fígres, Igncio; Virtnen, Erkki; Steele, Normn C.; nd Cpern, Thoms J., "Betine improves growth, but does not induce whole body or heptic plmitte oxidtion in swine (Sus scrof domestic)" (2004). Publictions from USDA-ARS / UNL Fculty This Article is brought to you for free nd open ccess by the U.S. Deprtment of Agriculture: Agriculturl Reserch Service, Lincoln, Nebrsk t DigitlCommons@University of Nebrsk - Lincoln. It hs been ccepted for inclusion in Publictions from USDA-ARS / UNL Fculty by n uthorized dministrtor of DigitlCommons@University of Nebrsk - Lincoln.

2 Comprtive Biochemistry nd Physiology Prt A 7 (2004) Betine improves growth, but does not induce whole body or heptic plmitte oxidtion in swine (Sus scrof domestic), b Dine Wry-Chen *, Igncio Fernndez-Fıgres, Erkki Virtnen, Normn C. Steele, Thoms J. Cpern Growth Biology Lbortory, Animl nd Nturl Resources Insitute, USDA-ARS, Beltsville, MD, 20705, USA b Finnfeeds Intl., Wiltshire, UK Received My 2003; ccepted 7 September 2003 Abstrct Dietry betine my reduce crcss ft in growing pigs. We explored the effects of betine on short-term growth nd in vivo nd in vitro ftty cid oxidtion. Pigs were housed in metbolism crtes nd fed diets contining either 0% (control), 0.125% or 0.5% betine t 80% of d libitum energy intke. Ftty cid oxidtion ws mesured during intrvenous infusions of 1- C-plmitte nd in heptocytes incubted in the presence or bsence of betine nd crnitine. CO2 nd plmitte isotopic enrichments were determined by mss spectrometry. Pigs consuming 0.125% nd 0.5% betine for t lest 9 dys hd growth rtes tht were 38% nd 12% greter thn controls, respectively. Feed efficiency ws lso improved with betine. Fsting incresed plmitte oxidtion rtes 7 8-fold (P-0.01), but betine hd no effect in either the fed or fsted stte (P)0.1). For heptocytes, crnitine but not betine enhnced plmitte oxidtion. This response suggests tht previously observed reduction in dipose ccretion must be vi mechnism other thn oxidtion. Betine hd no effect on plsm non-esterified ftty cids or ure nitrogen. Under the confinement conditions in this study, dietry betine improved niml growth responses, but it hd no pprent effect on either whole body or heptic ftty cid oxidtion Elsevier Inc. All rights reserved. Keywords: Betine; Feed efficiency; Growth; Heptocyte; In vitro; In vivo; Oxidtion; Plmitte; Swine 1. Introduction Betine is nturlly occurring tertiry mine (trimethyl glycine), which is formed by the oxidtion of choline, nd it is present in most living Mention of trde nme, proprietry product or vendor does not constitute gurntee or wrrnty of the product by the US Deprtment of Agriculture or the US Food nd Drug Administrtion or imply its pprovl to the exclusion of other products or vendors tht lso my be suitble. *Corresponding uthor. Current ddress: FDAyCDRHyOST, LLAR (HFV-500), Lurel, MD 20708, USA. Tel.: q ; fx: q E-mil ddress: dwc@cdrh.fd.gov (D. Wry-Chen). orgnisms. It is highly concentrted in the sugr beet, from which it is extrcted s byproduct during sugr production, nd is redily vilble for use in niml feed (Virtnen, 1995). The use of betine s n ingredient in livestock diets is not new concept. It ws initilly introduced to the feed industry s replcement for methionine nd choline in poultry nd fish diets, where it is presumed to ct both s methyl donor nd s n osmoprotectnt (Kidd et l., 1997). In swine, it hs been suggested tht betine reduces crcss ft deposition (Cdogn et l., 1993; Fernndez- Fıgres et l., 2002) /04/$ - see front mtter 2003 Elsevier Inc. All rights reserved. doi: /j.cbpb

3 2 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) While not the only source of lbile methyl groups, betine plys n integrl role in 1-crbon metbolism. A methyl group from betine is trnsferred to S-denosylmethionine (SAM) vi methionine. S-Adenosylmethionine, or ctive - methionine, serves s the primry methyl donor for the biosynthesis of mny compounds including crnitine, cretine, nucleic cids, neurotrnsmitters, phospholipids nd hormones. Thus, betine my be integrlly involved in lipid metbolism vi its role on phosphtidylcholine synthesis nd in ftty cid oxidtion becuse crnitine is required for trnsport of long chin ftty cids into mitochondri. In ddition, Brk et l. hve shown tht betine increses heptic SAM nd tht betine cn both prevent nd reverse lcohol-induced heptotoxicity in lbortory rodents (Brk et l., 1994, 1997). A considerble portion of cellulr methionine is ctivted by ATP to form SAM. This process my lso be importnt in the coordinted regultion of prtitioning of homocysteine ( potentilly toxic intermediry metbolite), between de novo methionine synthesis (using methyl group from betine or folte), nd ctbolism through cystthionine (Selhub nd Miller, 1992). Addition of betine in swine diets hs incresed during the lst decde, but the experimentl results for growth nd crcss performnce hve not been consistent. Initil studies indicted tht dietry betine mrkedly decresed bckft thickness without influencing other growth prmeters (Cdogn et l., 1993). More recent studies in finishing pigs, using vrious nutritionl regimens nd experimentl conditions hve lso demonstrted decreses in some indictors of body ft (Lwrence et l., 2002) with potentil reduction in feed intke (Mtthews et l., 2001) under some conditions. In contrst, other studies in finishing pigs hve reported miniml or no effects of betine on growth, feed intke or body ft (Mtthews et l., 1998; Overlnd et l., 1999). In young, feedrestricted pigs, totl body composition nlysis indicted tht betine ws ssocited primrily with decresed crcss ft, nd n increse in protein deposition, prticulrly t higher levels of betine intke (Fernndez-Fıgres et l., 2002). Much of the evidence from growth trils, including from our lb (Fernndez-Fıgres et l., 2002), suggests tht betine my indeed depress overll ft deposition, nd this study focused on betine s potentil stimultory effects on lipid oxidtion s possible mechnism of ction for ft reduction in growing pigs. Informtion on the role of betine in ltering lipid metbolism in the pig is limited to growth studies. For this study, our specific ims were to determine the effect of dietry betine on long-chin ftty cid (plmitte) oxidtion both in the whole body nd in primry monolyer cultures of liver cells obtined from growing pigs. 2. Mterils nd methods 2.1. Diets nd nimls Crossbred (Yorkshire=Lndrce) femle pigs (gilts) were used for ll studies w(sus scrof domestic); from the Beltsville Agriculturl Reserch Center (BARC) swine herdx. All niml cre nd use procedures were pproved by the Beltsville Animl Cre nd Use Committee. From kg of body mss, pigs were restrictively fed corn soyben mel bsed diet (1.2% lysine,.5 MJykg) t 80% estimted d libitum consumption ccording to the Agriculturl Reserch Council (ARC, 1981) formul (dily digestible y0.0204=bw energy intke, MJs0.80=55 w1ye x). Pigs to be used for in vivo ftty cid oxidtion determintions were trined to brethe into breth collection msk t this time. At 40 kg of body mss pigs were moved to metbolism crtes nd plced on the control diet (Tble 1) for t lest 1 week before being ssigned to one of three tretment diets: control (0% betine dded to diet, wt.y wt.), 0.125% betine or 0.5% betine. Pigs were fed tretment diets for t lest 4 dys before inititing the whole body ftty cid oxidtion protocol in order to llow betine homocysteine methyltrnsferse levels to re-equilibrte (Finkelstein et l., 1983). Animls were weighed the dy before the initition of plmitte infusion studies in order to clculte the infuste dosge Growth prmeters Animls were weighed t lest weekly nd the mount of feed offered to ech pig ws djusted once week ccording to individul body mss bsed on the ARC eqution shown bove. All feed refusls were recorded. Averge dily gin (growth rte) nd feed intke were determined for ll nimls during the control diet period (8.4"0.4 dys) nd then fter initition of the tretment diets. Animls were fed the tretment diets for

4 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) Tble 1 Composition of bsl diet Ingredients Percentge of diet Corn Soyben mel (48% CP) Dried skim milk Animl ft (lrd) 1.00 Minerl vitmin mix b 2.50 Diclcium phosphte 1.08 L-Lysine 0.10 Clculted nutrient composition, % Crude protein (CP) 18.6 Ft 3.67 Metbolizble energy, MJykg diet.5 Clcium 0.97 Phosphorous 0.77 Lysine 1.20 Methionine 0.33 MethionineqCysteine 0.63 Tryptophn 0.22 Threonine 0.75 Arginine 1.12 Isoleucine 0.76 Leucine 1.65 Histidine 0.45 Vline 0.91 Phenyllnine 0.90 Glycine 0.65 Choline 0.11 Diets prepred by United Feeds Inc., Sheridn, IN. b Provided the following mounts per kilogrm of feed: Zn, mg; Fe, mg; Mn, 43.2 mg; Cu, 6.7 mg; I, 0.88 mg; Co, 0.34 mg; Se, 0.26mg; vitmin A, 4000 IU; vitmin D, 800 IU; vitmin E, 17 IU; vitmin B12, 0.02 mg; vitmin K ctivity, 1.9 mg; riboflvin, 8.3 mg; D-Pntotenic cid, 21.3 mg; nicin, 43.0 mg; choline, 1088 mg; thimine, 3.4 mg; pyridoxine, 6.9 mg; folic cid, 0.39 mg; biotin, 0.15 mg; N, 2.6 g. 11.6"0.5 dys for the collection of growth nd feed intke dt. Growth nd feed intke dt were cquired before the initition of infusion studies. The rtio of verge dily feed intke nd verge dily gin (feed to gin rtio; F:G) ws used s mesure of feed efficiency Ctheteriztion nd blood smpling For ll pigs, ctheters were non-surgiclly plced in the jugulr veins (vi both medil uriculr veins nd the sphenous rtery) under generl nesthesi w2 mg xylzine nd 2 mg telzol (Fort Dodge Lbortories, Inc., Fort Dodge, IA) per kgx to llow for simultneous smpling nd infusion in unnesthetized pigs (Wry-Chen et l., 1993). Blood smples were obtined from the ctheters during the control diet period (t lest 4 dys fter ctheteriztion) nd 1 week fter initition of tretment diet. Smples were collected in heprinized syringes nd plsm ws obtined fter centrifugtion (1800=g, 15 min 4 8C) nd frozen t y20 8C. Plsm non-esterified ftty cid (NEFA-C kit, Wko Dignostics, Richmond, VA) nd ure nitrogen (Sigm Dignostic Kit No. 640; Sigm, St. Louis, MO) concentrtions were determined for these smples nd on frozen liquots of plsm collected during the bsl period before ech isotope infusion. Kinetic mesurements were mde under stedy-stte fed or fsted conditions. Ftty cid oxidtion ws determined using method described by Wry-Chen et l. (2001) using C-plmitte-methyl-b-cyclodextrin complex s ftty cid trcer. The pigs receiving the control diet nd the high level of betine (0.5%) were studied under both fed nd fsted conditions, on two seprte, non-consecutive dys. Animls on the infusion study consumed the tretment diets for 14"3 dys. For the in vivo ftty cid oxidtion study, four pigs per tretment were infused during the fed stte nd five pigs per tretment were infused during the fsted stte. Kinetic mesurements were mde under stedy-stte fed or fsted conditions. Under fed conditions, pigs received one-tenth of their dily rtion, hourly, beginning 2 h before bsl smples were collected nd continuing throughout the collection period, so tht the entire rtion ws consumed over 9-h period. Under fsted conditions, collections begn following n 18-h fst. Feed refusls during the smpling period were recorded Infuste preprtion w1- Cx-Plmitte (potssium slt; 99AP) ws obtined from Mss Trce, Inc. (Woburn, MA). In the present investigtion, w1- Cx-plmitte ws bound to methyl-b-cyclodextrin (MCBD; Aldrich Chemicl Co., Inc., Milwukee, WI, USA; 33,261-5) t 3 g plmittey100 g methyl-b-cyclodextrin in 20% methyl-b-cyclodextrin solution, prepred in phosphte buffered sline (Wry-Chen et l., 2001). The mixture ws prepred t C nd filtered through 0.2 mm sterile filter into sterile bottle nd stored t 4 8C until infused. The finl infuste enrichments (99.22"0.06%) nd plmitte concentrtions were determined by gs chromtogrphy-mss spectrometry (Metbolic Solutions, Inc.; Merrimck, NH, USA). The finl

5 4 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) plmitte concentrtion fter filtering the methylb-cyclodextrin infuste ws 75.2"1.8% of the clculted vlues. Vlues used in the plmitte oxidtion clcultions were those determined by mss spectrometry nlysis Whole body ftty cid oxidtion: infusion protocol A 90 min primed-continuous (0.6or 1.2 y1 y1 mmoløkg Øh ; fed or fsted conditions, respec- tively) infusion of NH CO 3 (Mss Trce, Inc.) ws used to prime the CO2ybicrbonte pool (Wolfe, 1992) nd to determine bsl CO2 produc- tion. Immeditely following the NH CO3 infu- sion, w1- Cx-plmitte solutions were infused t constnt rte (0.068 mmol C-plmiy1 y1 tteømin Økg ) for 4 h. Breth nd blood sm- ples were collected during bsl, pre-infusion period, nd when isotopic enrichment plteu ws chieved (60 90 min for bicrbonte infusion nd min for plmitte infusion). One niml ws infused with the plmitte-methyl-b-cyclodextrin mixture for 360 min with smples tken every 30 min for further verifiction tht the plteu ws chieved before the min window. Additionl smples collected to ensure tht plteu hd been chieved (bicrbonte: t 30, 45, 60, 70, 80, 90 min; plmitte: t 30, 60, 90, 120, 5, 150, 165, 180 min) were not included in the clcultions. Smples (breth ndyor heprinized blood) were injected into evcuted vils for lter mesurement of CO2 enrichments. Blood smples were collected into heprinized syringes nd plsm ws hrvested fter centrifugtion for mesurement of plsm enrichments of w1- Cx-plmitte. Enrichments of breth nd plsm were determined by isotope rtio mss spectrometry (IRMS) nd gs chromtogrphy mss spectrometry (GCMS), respectively, (Metbolic Solutions, Inc., Merrimck, NH, USA) Heptocyte ftty cid oxidtion Isoltion of porcine heptocytes: Liver lobes were hrvested from growing pigs (30 60 kg) to estblish primry monolyer cultures of porcine liver cells (Cpern nd Gvelek, 1994). Crossbred cstrted mle pigs (brrows) were mintined on stndrd porcine diet nd fed pproximtely 85% of d libitum feed intke strting t 20 kg live mss. On the dy prior to killing, pigs were given unlimited ccess to feed. Pigs were stunned by cptive bolt device nd exsnguinted. Livers were immeditely excised, nd the left lterl lobe ws removed. Heptocytes were isolted by the twostep collgense perfusion technique essentilly s previously described for the entire neontl pig liver (Cpern et l., 1985) except only smll portion (pprox g) of the lobe ws used. A brnch of the portl vein ws cnnulted nd the cut surfces of the lobe were sewn with 00 silk suture mteril to crete bck pressure during the perfusion. Collgense buffer ws modified to contin 1:1 mixture of M199 nd 100 mm HEPES-buffered sline contining 67 mm NCl, 4.8 mm CCl2 nd 6.7 mm KCl. The buffer mixture ws mended with 0.1% BSA, 0.5% glucose, 0.075% collgense nd 5 mgyml bovine insulin. 6 Freshly isolted heptocytes (4=10 ) were seeded into T-25 flsks (Costr, Corning, NY) coted with pproximtely 150 mg pig til tendon collgen. Cells were initilly mintined in mixture of DMEM (low glucose)-m199 (1.25 gyl NHCO 3) medium contining 10% fetl bovine serum (FBS), nd mended with, b-mercpto- y4 y3 ethnol (1=10 M), glutmine (2=10 M), penicillin (100 unitsyml), streptomycin (0.1 mgy ml), gentmicin (50 mgyml) nd fungizone (mphotericin B, 1.25 mgyml) t 37 8C nd 5% CO. Three hours fter plting, monolyers were 2 wshed twice with HEPES-buffered sline nd medium ws replced with supplemented DMEM- M199 medium contining 5% FBS. On the following dy, monolyers were wshed with HEPES-buffered sline nd switched to serum-free M199 medium (M199 Hnks slts: M199 Erle s slts nd 1.25 gyl NHCO 3, 2:1 rtio) contining 25 mm HEPES, b-mercptoethnol, glutmine, penicillinystreptomycin, fungizone, gentmicin nd supplemented with 6.25 mgyml insulin, 6.25 mgyml trnsferrin, 6.25 ngyml selenium, 1.25 mgy ml lbumin nd 5.35 mgyml linoleic cid (ITSq, Collbortive Biomedicl Products, Bedford, MA, USA), 500 ngyml porcine glucgon (G-3157, Sigm), 1% bovine serum lipids (C-5555, Sigm), y6 1% DMSO (D-2650, Sigm) nd 1=10 M dexmethsone cette. Cells were mintined in n ir environment t 37 8C. Where noted, crnitine nd betine were dded to the culture medium t the strt of the serum-free culture period. Medium ws chnged dily. Except where noted, ll

6 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) medi components nd regents were purchsed from GIBCO BRL (Githersburg, MD, USA). In vitro plmitte oxidtion: On the fourth dy of culture, medi ws removed, cells were wshed with HEPES-sline nd fresh medi (6ml) without y5 lipids but contining 2=10 M 1- C-plmitte, prepred in 20% MBCD (finl concentrtion 0.2%), ws dded. Immeditely fter ddition of medium, the stndrd cp on ech flsk ws replced with cp contining rubber septum (Wheton, , Millville, NJ) nd ech flsk ws tightly seled nd incubted t 37 8C. After 6 h, cells were killed nd CO2 ws relesed into the flsk hedspce by ddition of 2.5 ml of 20% TCA, vi the septum using syringe nd 21 guge needle. Following incubtion t room temperture for 15 min, 10 ml gs smple ws removed nd injected into n evcuted tube (Exetiner, Lbco Ltd., Buckinghmshire, Englnd) for the determi- ntion of CO2 by mss spectrometry (IRMS, Metbolic Solutions, Nshu, NH). The C tom percent enrichment (APE) of controls (cultures incubted with unlbeled plmitte) ws subtrcted from the APE of experimentl cultures. The APE for controls in experiments reported ws " nd " for cultures without or with dded crnitine, respectively. Preliminry in vitro studies conducted under similr conditions indicted tht betine did not influence totl cell protein in heptocyte cultures Mss spectrometry nlysis w1- Cx-Plmitte enrichment in plsm, infuste nd cell culture medi ws determined by gs chromtogrphy-mss spectrometry (GCMS) on Hewlett Pckrd 5989A system (Plo Alto, CA) using Restek Rtx-1 cpillry column. Extrcted ftty cids were derivtized with ddition of MTBSTFA: cetonitrile (1:1 vyv) to produce t- BDMS derivtive. Quntifiction of the nturl nd w1- Cx-plmitte ftty cid derivtives ws crried out by GCMS in the electron impct mode. CO2 enrichment in breth nd heprinized whole blood smples ws determined by gs isotope rtio mss spectrometry (IRMS) on Europ Scientific (Crewe, UK) 20y20 Stble Isotope Anlyzer. The breth smples were loded directly on the IRMS vi Europ Scientific ABCA smple purifiction system. Blood smples were first cidified with sturted citric cid solution to liberte the crbon dioxide nd the relesed gs loded on the IRMS in the sme mnner s the breth smples Clcultions nd sttistics The plmitte entry rte wr (plmitte)x, the CO2 production rte wr (CO 2)x during the bicrbonte infusion, the plmitte oxidtion rte nd the percent of plmitte uptke oxidized were clculted s follows (Wolfe, 1992): R (plmitte)s w(e ye )y1 x=f ip plm plm R (CO )s w(e ye E )y1 x=f 2 ib pco2 bco2 bicrb Plmitte oxidtionswe =R (CO ) xye pco2 2 plm Plmitte uptke oxidized (%) swwe =R (CO ) xyf x=100 pco2 2 plm where Eip ws the isotope enrichment of the C- plmitte infuste, Eplm ws the plteu enrich- ment of plsm plmitte, Fplm ws the infusion rte of the C-plmitte infuste, Eib ws the isotope enrichment of the C-bicrbonte infuste, E bco2 ws the plteu enrichment of plsm or breth CO2 during the C-bicrbonte infusion, Fbicrb ws the infusion rte of the C-bicrbonte infuste, nd E pco2 ws the plteu enrichment of plsm or breth CO2 during the C-plmitte infusion. Averge plteu vlues were used in comprisons. The primry comprisons of interest between fed (both growth nd kinetic dt) pigs were: (i) control vs. betine (0.125 nd 0.5%); (ii) 0.125% vs. 0.5% betine nd for the fsted (kinetic dt) pigs; (iii) control vs. 0.5% betine. To compre dt from fed pigs, ANOVA ws used. Unpired t-tests were used to compre the fsted pigs. To determine the effect of betine on response of ftty cid oxidtion to fsting, the verge plteu vlues were compred using pired Student s t- tests for the control nd betine (0.5%) pigs studied under both fed nd fsted conditions. The level of significnce ws set t P Dt in tbles re expressed s men"stndrd error of the men (S.E.M.). For heptocytes studies control nd betine-treted cultures were compred by pired Student s t-test nlysis.

7 6 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) Tble 2 The effect of betine on growth performnce of pigs housed in metbolism crtes Control Betine 0.125% Betine 0.5% Men S.E.M. Men S.E.M. Men S.E.M. (ns19) (ns15) (ns15) Pre-tretment (control diet) ADG (kgyd) ADFI (kgyd) F:G Tretment diet ADG (kgyd) ADFI (kgyd) F:G Abbrevitions: ADGsAverge dily gin; ADFIsAverge dily feed intke; F:GsFeed to gin rtio (kilogrm of feed required per kg of body weight gin). Betine groups different from control (P-0.05). 3. Results 3.1. Growth performnce The growth performnce responses re presented in Tble 2. While housed in metbolism crtes, ll pigs grew t similr rtes during the pre-tretment period (consuming the control diet). Once switched to diets contining betine (0.125 nd 0.5%), pigs grew on verge more thn 20% fster thn did the nimls tht remined on the control diet (P-0.05), despite consuming similr mount of feed to the control nimls. The feed conversion efficiency (feed to gin rtio) of the pigs fed betine improved 25% or more s compred to controls (P-0.05) Blood prmeters Plsm concentrtions of non-esterified ftty cids (NEFA) nd plsm ure nitrogen (PUN) re presented in Tble 3. Dietry betine hd no effect on NEFA levels during either fed or fsting stte. Likewise, PUN levels were similr mong tretment groups In vivo whole-body ftty cid oxidtion Ftty cid oxidtion dt is presented in Tble 4. Fsting incresed plmitte oxidtion rtes for both tretment groups. On verge, fsting incresed the rte of plmitte oxidtion eight-fold (P-0.05) nd the percent of plmitte uptke oxidized four-fold (P-0.01). Dietry betine did not ffect this response of the pig to fsting. The CO2 entry rte during the fed stte tended to be lower in pigs consuming betine (P-0.10), but ll other prmeters were similr between tretments In vitro heptic ftty cid oxidtion The presence of dded crnitine hd mrked influence on C-plmitte oxidtion in monolyers Tble 3 The effect of dietry betine on plsm non-esterified ftty cids (NEFA) nd ure nitrogen (PUN) levels in pigs Control Betine 0.125% Betine 0.5% Men S.E.M. n Men S.E.M. n Men S.E.M. n NEFA (meqyl) Fed Fsted PUN (mgyml) Fed Fsted

8 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) Tble 4 Effect of dietry betine on whole body plmitte entry rte wr (plm)x nd plmitte oxidtion Control Betine (0.5%) Fed Fsted Fed Fsted Men S.E.M. Men S.E.M. Men S.E.M. Men S.E.M. (ns4) (ns5) (ns4) (ns5) Wt., kg y1 y1 R (plm), mmolømin Økg y1 y1b R (CO 2), mmolømin Økg Plmitte oxidtion, y1 y1c mmol min kg % Plmitte uptke oxidized b Betine group tended to be different from control (P-0.10). b Fsted group ws different from fed group (P-0.01). c Fsted group ws different from fed group (P-0.05). of porcine heptocytes (Fig. 1). Mximl stimultion of oxidtion ws observed t pproximtely 0.5 mm crnitine. Addition of 2 mm betine to monolyers of porcine heptocytes for 3 dys hd no influence (P)0.1) on C-plmitte oxidtion in the bsence or presence of 1 mm crnitine (Fig. 2). 4. Discussion In the present study, dietry betine enhnced the rte of body mss gin nd the feed efficiency of femle crossbred pigs in the kg mss rnge, compred to similrly treted controls. These findings were unexpected s erlier studies, including those from our lbortory, hve not Fig. 1. The influence of crnitine on the oxidtion of C plmitte in porcine heptocyte monolyers. Heptocytes were prepred nd mintined in culture s described in Section 2. Crnitine ws dded to flsks t vrious levels between 0.05 M nd 2 mm for the durtion of the culture period. Dt represent the men of duplicte flsks prepred from one pig. Fig. 2. The influence of crnitine nd betine on the oxidtion of C plmitte in porcine heptocyte monolyers. Heptocytes were prepred nd mintined in culture s described in Section 2. Where noted, 1 mm crnitine nd 2 mm betine were dded to the culture flsks for the durtion of the culture period. Dt represent the men"s.e.m. of duplicte cultures from five different pigs.

9 8 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) previously demonstrted growth enhncement in nimls fed vriety of experimentl diets contining similr levels of betine (Cdogn et l., 1993; Mtthews et l., 1998; Overlnd et l., 1999; Mtthews et l., 2001,b; Fernndez-Fıgres et l., 2002; Lwrence et l., 2002). A new pig growth tril from Finlnd hs recently been published reporting improved feed conversion rtios nd dily gins with lower level of dietry betine (Siljnder-Rsi et l., 2003). In tht study, the feed intke ws restricted by similr degree to tht in the current report (80 85% of d libitum). In the Finnish study, feed efficiency nd growth rtes improved with incresing level of dietry betine (for the two highest levels). The levels of betine used in the Finnish study were lower thn used in other published trils. It is possible tht the most effective dose is lower thn tht used in the current study nd in other studies. In the current study, the 0.5% betine diet showed no further improvement in growth performnce over the 0.125% betine diet. Perhps the 0.5% dose ws beyond the brek point of the response curve. However, this hypothesis is not supported by results from our erlier growth tril (Fernndez- Fıgres et l., 2002) where we observed liner reduction in crcss ft (per kg of dry mtter) from 0% to 0.5% dietry betine. Severl potentil fctors my hve contributed to the growth performnce findings in the current study. Most growth trils tke plce over mny weeks with d libitum feeding nd in conditions tht re potentilly less stressful thn found in production. The time frme of this study ws reltively short (under 2 weeks). The pigs were restrictively fed (80% d libitum) nd housed in very confined spce (metbolism crtes). In trils in which pigs re given d libitum ccess to feed, tendency towrds reduction in voluntry feed intke hs been reported, ccompnied by no chnge in overll growth (Mtthews et l., 1998, 2001). In the current experiment, where feed intke ws strictly controlled nd limited to 80% of expected d libitum intke, the improvement in feed:gin rtio resulted in the overll observed increse in growth rte. Gender my hve lso plyed role in the growth results of the current study. Only femles were used in the current growth study, while we used cstrted mles in our previous growth tril (Fernndez-Fıgres et l., 2002). When mle nd femle pigs hve been compred directly, conflicting results hve been previously obtined with respect to bility of betine to lter growth nd lipid metbolism (Lwrence et l., 2002). The previously noted decrese in voluntry feed intke hs been reported in betine growth trils with femles lone or with groups of mixed sexes (Mtthews et l., 1998, 2001). The Finnish study used mixed genders (femles nd cstrted mles), but they did not report their dt by gender (Siljnder-Rsi et l., 2003). We cnnot rule out tht the observed growth effect ssocited with betine ddition in this study is reltively trnsient nd my be msked or ttenuted in longer growth trils. Other reported studies with nutritionlly dequte diets nd similr levels of betine, hve been long-term evlutions. Even when seprted into more restrictive time periods, these hve still been considerbly longer thn the time frme of the current study (Mtthews et l., 1998; Lwrence et l., 2002). Finlly, the nimls in the current experiment were housed in metbolism cges. While the limited physicl movement might be ssocited with reduced energy expenditure, the pigs might lso be exposed to higher level of stress due to rtificil confinement nd feed restriction, compred to nimls mintined either in group pens or in lrger pens on d libitum feeding. It hs been previously suggested tht betine hs positive effects on growth nd metbolism in nimls tht re exposed to vrious physiologicl stresses (Virtnen 1995; Brk et l., 1997; Kidd et l., 1997; Junnil et l., 2000). The improvement in growth performnce my hve been ccompnied by better utiliztion of dietry protein, s suggested by the tendency for lower concentrtions of PUN with ddition of betine to the diet (P-0.11). Mtthews et l. (1998) observed decresed serum ure N levels under some conditions with dietry betine, but others hve reported no effect of betine on ure N levels in blood (Mtthews et l., 2001; Fernn- dez-fıgres et l., 2002). Although we did not detect chnges in PUN in our erlier growth tril, we did observe tendency for incresed efficiency for len gin (Fernndez-Fıgres et l., 2002). In the present study, we found betine to hve no significnt effect on plmitte oxidtion, either in vivo or in vitro. Although plmitte oxidtion ws significntly enhnced by fsting, dietry betine (0.5%) did not influence plmitte oxidtion or utiliztion in either the fsted or fed stte. In

10 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) the heptocyte model, it is cler tht vilble crnitine mrkedly limits the rte of plmitte oxidtion nd this my indicte tht t lest following 4 dys of culture, cellulr levels of crnitine re ttenuted. In other heptocyte studies where ftty cid utiliztion hs been investigted, crnitine hs been routinely dded to the extrcellulr environment presumbly to ensure mximl rtes of oxidtion (Pegorier et l., 1983; Odle et l., 1991). When betine ws dded to crnitine-free nd crnitine mended cultures, no enhncement of oxidtion ws observed. In ddition, since these cultures pper to be highly sensitive to levels of crnitine, the dt would suggest tht betine hd no influence on intrcellulr levels of crnitine. In the presence of high crnitine (1mM), vrying the levels of insulin nd glucgon hd only mrginl effects on plmitte oxidtion (dt not shown; see lso Pegorier et l., 1983) which suggests tht t lest in porcine heptocyte cultures mintined in complete serum-free medium, crnitine levels, nd, therefore, ftty cid entry into mitochondri re the key regultors of ftty cid oxidtion. Plsm levels of NEFA observed in this study were lso unffected by betine. It is possible tht lipid metbolism ws not ffected by betine in these nimls; however, given tht growth nd feed efficiency were ffected, we predict tht these pigs were ccruing less body ft. If this ws the cse, it my be more likely tht betine s effect on lipid metbolism in the pig is not due to its potentil effects on oxidtion; rther, decresed dipose ccretion my be result of reduced lipogenesis. Another possibility is tht different tissues ndyor dipose depots my respond differently to betine nd these loclized chnges would likely be undetectble on whole body bsis. In conclusion, betine ppers to enhnce growth performnce of pigs tht re housed in confined spce nd limit-fed. Our previously observed effects of betine on lipid ccretion (Fernndez-Fıgres et l., 2002) pper not to be due to n enhncement in ftty cid oxidtion, mesured either in vivo or in vitro. It is possible tht lipid synthesis ws ffected by betine, but this requires further study. Betine my be useful s prtitioning gent, especilly when limit feeding is employed ndyor housing conditions re stressful. However, further work will be required to estblish the reltionship of protein nd lipid turnover under these conditions. Acknowledgments The uthors wish to thnk Finnsugr Bioproducts Inc. (Schumburg, IL, USA) for prtil support of this study; S. Schneider-Firestone for ssistnce with smple collection nd for niml trining; D. Prsons nd J. Pitt for cre of the nimls nd technicl ssistnce; D. Gvlek for ssistnce with the in vitro study nd with niml trining; T. Chpmn t Metbolic Solutions, Inc., Merrimck, NH, USA, for dt nlysis nd technicl support. References ARC The Nutrient Requirements of Pigs. Agriculturl Reserch Council. Commonwelth Agriculturl Bureux, Slough, UK. Brk, A.J., Beckenhuer, H.C., Tum, D.J., S-denosylmethionine genertion nd prevention of lcoholic ftty liver by betine. Alcohol 11, Brk, A.J., Beckenhuer, H.C., Bdkhsh, S., Tum, D.J., The effect of betine in reversing lcoholic stetosis. Alcohol Clin. Exp. Res. 21, Cdogn, D.J., Cmpbell, R.G., Hrrison, D., Edwrds, A.C., The effects of betine on the growth performnce nd crcss chrcteristics of femle pigs. In: Btterhm, E.S. (Ed.), Mnipulting Pig Production IV. Austrlsin Pig Science Assoction, Attwood, Victori, Austrli, pp Cpern, T.J., Fill, M.L., Kornegy, E.T., Richrds, M.P., Steele, N.C., Isoltion nd culture of prenchyml nd non-prenchyml cells from neontl swine liver. J. Anim. Sci. 61, Cpern, T.J., Gvelek, D., Tissue ATPse ctivity in somtotropin-treted pigs. Thirteen symp. on Energy Metbolism of Frm Animls. Eur. Assoc. Anim. Prod. 76, Fernndez-Fıgres, I., Wry-Chen, D., Steele, N.C., Cmpbell, R.G., Hll, D.D., Virtnen, E., et l., Effect of dietry betine on nutrient utiliztion nd prtitioning in the young growing feed-restricted pig. J. Anim. Sci. 80, Finkelstein, J.D., Mrtin, J.J., Hrris, B.J., Kyle, W.E., Regultion of heptic betine-homocysteine methyltrnsferse by dietry betine. J. Nutr. 1, Junnil, M., Rhko, T., Sukur, A., Lindberg, L.A., Reduction of crbon tetrchloride-induced heptotoxic effects by orl dministrtion of betine in mle Hn-Wistr rts: morphometric histologicl study. Vet. Pthol. 37, Kidd, M.T., Ferket, P.R., Grlich, J.D., Nutritionl nd osmoregultion functions of betine. World s Poult. Sci. J. 53, Lwrence, B.V., Schinckel, A.P., Adeol, O., Cer, K., Impct of betine on pig finishing performnce nd crcss composition. J. Anim. Sci. 80,

11 140 D. Wry-Chen et l. / Comprtive Biochemistry nd Physiology Prt A 7 (2004) Mtthews, J.O., Southern, L.L., Pontif, J.E., Higbie, A.D., Bidner, T.D., Interctive effects of betine, crude protein nd net energy in finishing pigs. J. Anim. Sci. 76, Mtthews, J.O., Southern, L.L., Higbie, A.D., Persic, M.A., Bidner, T.D., Effects of betine on growth, crcss chrcteristics, pork qulity nd plsm metbolites of finishing pigs. J. Anim. Sci. 79, Mtthews, J.O., Southern, L.L., Bidner, T.D., Persic, M.A., 2001b. Effects of betine, pen spce nd slughter hndling method on growth performnce, crcss trits, nd pork qulity of finishing brrows. J. Anim. Sci. 79, Odle, J., Beneveng, N.J., Crenshw, T.D., Postntl ge nd the metbolism of medium- nd long-chin ftty cids by isolted heptocytes from smll-for-gesttionl-ge nd pproprite-for-gesttionl-ge piglets. J. Nutr. 121, Overlnd, M., Rorvik, K.A., Skrede, A., Effect of trimethylmine oxide nd betine in swine diets on growth performnce, crcss chrcteristics, nutrient digestibility nd sensory qulity of pork. J. Anim. Sci. 77, Pegorier, J.P., Duee, P.H., Girrd, J., Peret, J., Metbolic fte of non-esterified ftty cids in isolted heptocytes from newborn nd young pigs. Evidence for limited cpcity for oxidtion nd incresed cpcity for esterifiction. Biochem. J. 212, Selhub, J., Miller, J.W., The pthogenesis of homocysteinemi: interruption of the coordinte regultion by S- denosylmethionine of the remethyltion nd trnssulfurtion of homocysteine. Am. J. Clin. Nutr. 55, 1 8. Siljnder-Rsi, H., Peurnen, S., Tiihonen, K., Virtnen, E., Kettunen, H., Alviuhkol, T., et l., Effect of equimolr dietry betine nd choline ddition on performnce, crcss qulity nd physiologicl prmeters of pigs. Anim. Sci. 76, Virtnen, E., Piecing together the betine puzzle. Feed Mix 3, Wolfe, R.R., Rdioctive nd Stble Isotope Trcers in Biomedicine: Principles nd Prctice of Kinetic Anlysis. Wiley-Liss, New York, pp Wry-Chen, D., Boyd, R.D., Bumn, D.E., Ross, D.A., Effect of porcine somtotropin on the response of growing pigs to cute chllenges of glucose, insulin nd epinephrine nd during hyperinsulinemic-euglycemic clmp. Domest. Anim. Endocrinol. 10, Wry-Chen, D., Cpern, T.J., Steele, N.C., Methylbet-cyclodextrin: n lterntive crrier for intrvenous infusion of plmitte during trcer studies in swine (Sus scrof domestic). Comp. Biochem. Physiol. A 0,