Use of δ-aminolevulinic Acid in Swine Diet: Effect on Growth Performance, Behavioral Characteristics and Hematological/Immune Status in Nursery Pigs*

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1 97 Use of δ-aminolevulinic Acid in Swine Diet: Effect on Growth Performnce, Behviorl Chrcteristics nd Hemtologicl/Immune Sttus in Nursery Pigs* R. D. Mteo 1, J. L. Morrow 2, J. W. Diley 2, F. Ji 1 nd S. W. Kim 1, ** 1 Deprtment of Animl nd Food Sciences, Texs Tech University, USA ABSTRACT : Certin mino cids re essentil precursors of vriety of importnt biomolecules in ddition to their mjor function s protein building blocks. δ-aminolevulinic cid (ALA) is synthesized from the condensed form of succinyl-coa with glycine fter decrboxyltion ctlyzed by ALA synthse. The objective of the study ws to determine the effects of ALA supplementtion on growth performnce, behviorl chrcteristics nd hemtologicl/immune sttus in nursery pigs. A totl of 144 pigs wened t 21 d of ge were llotted to three dietry tretments representing (-) control (w/o ntibiotics; NC), (+) control (w/crbdox t 50 mg/kg; PC), nd the tretment group with ALA supplementtion (0.05%; TA). Ech tretment hd 6 pens (replictes) with 8 pigs per pen. Pigs were fed phse 1 (21.9% CP, 1.40% Lys) nd 2 (20.6% CP, 1.15% Lys) experimentl diets for 3 nd 2 wks, respectively. Feed intke nd weight gin were mesured weekly during phse 1 nd t the end of phse 2. At the end of phse 2, blood smples were tken nd nlyzed using n utomted hemtology nlyzer. Skin color nd ctivity of pigs (48 h) from ll pens in ech tretment were mesured t the second week of phse 2. Growth performnce ws not ffected (p>0.05) by the dietry supplementtion of ALA during the 5 wk nursery period. Pigs in the TA (6.46) nd PC (6.68) hd higher (p<0.05) number of red blood cells (10 6 cell/µl) thn pigs in the NC (6.15). Pigs in PC (12.16) hd higher (p<0.05) hemoglobin level (g/dl) thn pigs in the NC group (11.29) nd the TA group (11.47). Pigs in the TA nd PC hd drker (p<0.05) nd less (p<0.05) yellow skin color thn pigs in the NC. Pigs in the PC tended (p = 0.081) to be less ctive thn pigs in the other groups. There were no differences in behviorl chrcteristics between the NC nd the TA. The dt suggest tht ALA supplementtion hs no dverse effects on growth performnce of nursery pigs. Moreover, ALA supplementtion incresed red blood cell counts which my be beneficil to pigs. (Asin-Aust. J. Anim. Sci Vol 19, No. 1 : ) Key Words : δ-aminolevulinic Acid, Antibiotics, Behvior, Hemtologicl Sttus, Nursery Pigs INTRODUCTION Certin mino cids re essentil precursors of vriety of importnt biomolecules such s heme in ddition to their mjor function s building blocks of protein. δ- Aminolevulinic cid (ALA) is chemicl produced from mino cids in the liver nd is ctlyzed by the pyridoxl phosphte (PLP) dependent enzyme, ALA synthse, from glycine nd succinyl CoA. This rection is known s the Shemin pthwy nd is rte limiting step for heme synthesis (Jover et l., 2000). The next series of steps in the synthesis of heme occurs in the cytosol wherein ALA dehydrtse converts two molecules of ALA to monopyrrol porphobilinogen (PBG). Two subsequent enzymtic steps convert four molecules of PBG into uroporphyrinogen III, which is then decrboxylted to form coproporphyrinogen III. The finl steps which include the insertion of ferrous iron into protoporhyrin IX by ferrocheltse occurs in the mitochondri (Ponk, 1997). Heme is regultory molecule tht controls metbolic * The uthors wish to cknowledge the finncil support from EsyBio Systems, Inc. Kore. ** Corresponding Author: Sung Woo Kim. 203 Animl nd Food Sciences Building, MS 2141, Lubbock Texs, USA. Tel: , Fx: , E-mil: sungwoo.kim@ttu.edu 2 USDA-ARS, Lubbock Texs, USA. Received September 26, 2004; Accepted September 8, 2005 pthwys nd the biosynthesis of vrious proteins (Jover et l., 2000) clled heme-proteins. These heme-proteins re involved in brod spectrum of crucil biologic functions including oxygen binding nd oxygen metbolism (Ponk, 1997). Compred with other cells in the orgnism, rpid heme biosynthesis occur in liver nd erythroid cells where lrge mounts of heme re needed not only for mitochondril cytochromes but lso s prosthetic groups for cytochrome P450 nd hemoglobin, respectively. Under physiologicl conditions, ALA is required for the synthesis of protoporphyrin IX, direct precursor of heme (Neumnn nd Brndsch, 2003) nd hs been used in vitro nd clinicl studies s n endogenous photosensitizer for photodynmic therpy in the tretment of vrious tumors in humns (Doring et l., 1998). However, the potentil beneficil effects of ALA supplementtion on growth nd immune sttus of pigs hve not been tested. Thus, it ws hypothesized tht the supplementtion of ALA to nursery pigs improves heme synthesis tht will in turn increse the synthesis of heme-proteins such s hemoglobin. These heme-proteins would then increse the number of red blood cells thereby improving the immune sttus of nursery pigs. MATERIALS AND METHOD A totl of 144 pigs (Cmborough 22 PIC bor, Pig Improvement Compny, Frnklin, KY) were used in the

2 98 MATEO ET AL. Tble 1. Experimentl diets Ingredients (%) Phse 1 (21 to 42 d of ge) Phse 2 (43 to 56 d of ge) Corn SBM (dehulled) Dried whey Slt Vitmin-minerl premix b Ft (vegetble oil) Diclcium phosphte Limestone Plsm protein (APC-920) Fish mel (Menhden) Zinc oxide Totl Chemicl composition Dry mtter (%) ME (Mcl/kg) Crude protein (%) Lysine (%) Cysteine+methionine (%) Tryptophn (%) Threonine (%) Clcium (%) Avilble phosphorus (%) Totl phosphorus (%) Either crbdox (0.1%) or δ-minolevulinic cid (0.05%) ws dded for PC or TA tretment, respectively by replcing the sme mount of corn. b Vitmin-minerl premix provided the following per kilogrm of complete phse 1:62.2 mg of mngnese s mngnous oxide, 100 mg of iron sulfte, mg of zinc s zinc oxide, 12.6 mg of copper s copper oxide, 0.96 mg of iodide s ethylenedimine dihydroiodide, 0.30 mg of selenium s sodium selenite, 10,074 IU of vitmin A s vitmin A cette, 1,100 IU of vitmin D 3, 82.6 IU of vitmin E, 3.6 IU of vitmin K s mendione sodium bisulfte, 73.2 µg of vitmin B 12, 18.4 mg of riboflvin, 58.6 mg of D-pntothenic cid s clcium pntothente, 73.2 mg of nicin, nd 2,210 mg of choline s choline chloride. Phse 2 diet contined 50% levels of vitmin nd minerl provided to the phse 1 diet. study. Pigs were wened t 21 d of ge nd llotted to one of three dietry tretments tht consisted of control group without ny ntibiotics (NC), control group with ntibiotics using crbdox t 50 mg/kg (PC), nd tretment group with the ALA supplementtion (0.05%) without ny ntibiotic included (TA). Ech tretment hd six replictes with eight pigs per replicte. The pigs were housed in rised-deck pens (2 2 m). Ventiltion ws provided by mechnicl system, nd lighting ws utomticlly regulted to pproximte the sesonl dy length. Ambient temperture within the room ws pproximtely 30 C immeditely fter wening nd ws djusted downwrd to pproximtely 22 C by the end of the experiment. The niml cre nd use protocol ws pproved by the Texs Tech University Animl Cre nd Use Committee. Pigs were fed the phse 1 experimentl diets for 3 wks nd the phse 2 experimentl diets for the following 2 wks (Tble 1). Tble 2. Growth performnce of pigs fed the experimentl diets during the 5 wk nursery period Tretment NC PC TA SEM Body weight (kg) Initil Week Week Week Week Averge dily gin (g) Week Week Week Week Overll Averge dily feed intke (g) Week Week Week Week 5 1, , , Overll Gin:feed rtio Week Week Week Week Overll NC: control group without ny ntibiotics, PC: control group with ntibiotics using crbdox t 50 mg/kg, TA: tretment group with the ALA supplementtion (0.05%) without ny ntibiotic included. The experimentl diets provided 21.9% CP nd 1.40% totl lysine during the phse 1 nd 20.6% CP nd 1.15% totl lysine during the phse 2. All the nutrients were provided to meet the nutrient requirement suggested by the NRC (1998). Pigs hd free ccess to feed nd wter. Feed intke nd weight gin were mesured weekly during the phse 1 nd t the end of phse 2. At the end of phse 2, blood smples (10 ml) were obtined s described by Kim et l. (2004) nd were then nlyzed for the number of white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, bsophils, red blood cells, hemoglobin, pltelets, etc using n utomted hemtology nlyzer (CELL-DYN 3200, Abbott Lb, Abbot Prk, IL). Eighteen cmcorders were plced t the ceiling of ech pen to monitor the movement of ll pigs in ech pen. Individul pigs hd their own unique number (one to eight) on their bcks written with blck permnent mrker mking it possible to identify the behvior of individul pigs. Pig behvior ws recorded for 48 h using video recorders with monitors tht were locted outside of the nursery room. Although, no one ws llowed to enter the nursery room during the 48 h period, we were still ble to observe the pigs through the monitors. After recording, tpes were plyed to mesure pig behvior dt. Number of pigs for ech behviorl ctegory ws counted for 48 h with 10 min intervl. Behviorl ctegory included stnding,

3 EFFECT OF δ-aminolevulinic ACID IN NURSERY PIGS 99 Tble 4. Color mesurement by Hunter Color System from the pigs skin fed experimentl diets during the 5 wk nursery period NC PC TA SEM L* b c c 0.23 * b* 9.39 b 8.46 c 8.29 c 0.20 NC: control group without ny ntibiotics, PC: control group with ntibiotics using crbdox t 50 mg/kg, TA: tretment group with the ALA supplementtion (0.05%) without ny ntibiotic included. b, c Mens in the sme row with different superscripts differ (p<0.05). lying, sitting, nd eting. Behviors including stnding nd eting were combined nd nmed ctive behvior wheres lying nd sitting were combined nd nmed inctive behvior. Averge percentges of ech behvior from ll pens from ech tretment were obtined nd recorded. After behviorl mesurement, the left side hm re of ll pigs from ech tretment group ws shved to remove ny hir. On the following morning (0900 h), pig skin color ws mesured from the shven spot using Minolt color recorder (MiniScn XE Plus, Hunter, Reston, VA). The L*, *, nd b* vlues were obtined. Colored pigs nd pigs with spots on their skin were excluded from the dt. The sttisticl nlysis ws performed with the Generl Liner Models procedure (PROC GLM) in SAS/STAT softwre (SAS Inst. Inc., Cry, NC). Tretment ws the min effect nd the initil pig weight ws used s covrite. Lest-squre mens, probbility of differences, nd stndrd errors were used to evlute the differences mong the different tretment groups. RESULTS Growth performnce of pigs fed the experimentl diets during the 5 wk nursery period is shown in Tble 2. Averge dily gin (g) of nursery pigs did not differ (p>0.05) mong the tretments during the 5 wk nursery period. Averge dily feed intke (g) of nursery pigs did not differ (p>0.05) mong the tretments s well s gin: feed rtio. Thus, growth performnce ws not ffected (p>0.05) by the dietry supplementtion of ALA during the 5 wk nursery period. Tble 5. Behviorl mesurements of pigs fed experimentl diets during the 5 wk nursery period NC PC TA SEM 48 h period Stnding Eting Sitting Lying 78.7 b b 1.0 Totl ctive c Totl inctive d h period ( h) Stnding Eting Sitting Lying Totl ctive c Totl inctive d , b Mens within row with different superscripts differ (p = 0.081). c Behvior mesurements including stnding nd eting. d Behvior mesurements including lying nd sitting. Hemtologicl evlution of pigs fed the experimentl diets during the 5 wk nursery period is presented in Tble 3. Pigs from both the TA nd PC groups hd higher (p<0.05) numbers of red blood cells thn pigs in the NC group. However, no differences (p>0.05) were noted between the TA nd PC. Pigs in the PC hd higher (p<0.05) hemoglobin levels compred to those pigs from both the TA nd the NC. Significnt differences (p<0.05) were noted in terms of bsophil counts between the NC nd the PC. Bsophil counts of the TA, however, did not differ (p>0.05) from tht of either the NC or the PC. No differences (p>0.05) were observed on ll other hemtologicl mesures in the study including white blood cells, neutrophils, lymphocytes, monocytes nd eosinophils. Pigs in the TA nd PC hd lower (p<0.05) L* vlues nd lower (p<0.05) b* vlues indicting tht they hd drker nd less yellow skin color thn pigs in the NC (Tble 4). There were no differences in behviorl chrcteristics between the NC nd the TA. However, the pigs in the PC tended (p = 0.081) to be less ctive thn pigs in other groups (Tble 5). Tble 3. Hemtologicl evlution of pigs fed the experimentl diets during the 5 wk nursery period NC PC TA SEM White blood cell (10 3 cell/µl) Red blood cell (10 6 cell/µl) 6.15 b 6.68 c 6.46 c 0.06 Hemoglobin (g/dl) b c b 0.12 Neutrophil (10 3 cell/µl) Lymphocyte (10 3 cell/µl) Monocyte (10 3 cell/µl) Eosinophil (10 3 cell/µl) Bsophil (10 3 cell/µl) 0.54 b 0.35 c 0.42 bc 0.04 NC: control group without ny ntibiotics, PC: control group with ntibiotics using crbdox t 50 mg/kg, TA: tretment group with the ALA supplementtion (0.05%) without ny ntibiotic included. b, c Mens in the sme row with different superscripts differ (p<0.05).

4 100 MATEO ET AL. DISCUSSION This study indictes tht the pigs fed diet supplemented with ALA or crbdox hd incresed number of red blood cells s compred to the pigs fed diet without both ALA nd crbdox supplementtion. The frction of whole niml fsting oxygen consumption used by the portl vein drined orgns (PVDO) ws found to be reduced (Yen nd Nienber, 1992), nd improved retention of dietry protein ccretion hve been reported in pigs fed crbdox supplemented diet (Roof nd Mhn, 1982). Yen nd Nienber (1992) suggested tht lthough crbdox reduces oxidtive demnd by PVDO, it does not decrese O 2 consumption of the whole niml, becuse crbdox probbly lso cuses n increse O 2 consumption s result of enhncement of muscle protein ccretion. This increse in oxygen consumption would hve required n increse in the production of red blood cells. However, in our experiment, pigs fed diets from ll three groups did not show ny significnt response in terms of growth performnce. Although pigs from both the PC nd TA obtined higher red blood cell counts compred to the NC, ll vlues were still within the norml rnge found in pigs (Merck Veterinry Mnul, 1991). Pigs in the TA nd the PC hd drker (p<0.05) nd less (p<0.05) yellow skin color thn pigs in the NC. This is probbly due to the higher numbers of red blood cells s shown in our experiment which my hve contributed lrgely to the drker skin color observed in pigs from both the PC nd TA. The lck of response of nursery pigs in both TA nd PC to growth performnce my hve been due to the previling reserch fcility conditions used in the study. It ws noted tht during the study period ll experimentl pigs used were rised under completely controlled environment wherein pthogenic nd other stress fctors were possibly mintined t very low levels. Yen nd Pond (1990) reported tht the min mode of ction of crbdox is tht it suppresses the microbil production of NH 3 in the gstrointestinl trct. This effect would probbly hve been more pprent in conditions where higher pthogen or stress lod is evident. Numerous studies hve indicted tht responses to ntibiotics under frm conditions my be twice s gret s those occurring in reserch sttion environment since the disese chllenge nd other stressors re generlly lesser (Cromwell, 2001) nd tht the level of growth performnce depends on the mngement nd housing conditions (Cromwell, 2000). This would suggest tht the nursery environment my hve been significnt in terms of the response of pigs towrds supplementl tretments. This my hve lso been the cse for the TA. In the study, both PC nd TA showed higher red blood cell counts compred to the NC. However, hemoglobin content did not show the sme trend s red blood cell counts did. Pigs from both the NC nd TA showed lower hemoglobin content compred to the PC. It hs been previously reported tht ALA synthse is the rte limiting enzyme in heme synthesis nd its expression is regulted by heme levels through negtive feedbck mechnism (Doring et l., 1998; Jover et l., 2000). Humn erythroid cells hve been reported to be subjected to end product feedbck regultion, which occurs t one or more rte limiting steps which led to the formtion of ALA (Grdner nd Cox, 1988). However, it hs lso been reported tht when ALA is pplied topiclly or systemiclly (Collud et l., 2004) or fter orl ppliction (Neumnn nd Brndsch, 2003), ALA bypsses the negtive feedbck control tht heme exerts on the enzyme ALA synthse, which ctlyses the nturl production of ALA. This would suggest tht in our experiment, the feed bck inhibition brought bout by heme would not be much of fctor since exogenous ALA ws provided in the diet. A probble explntion for the conflicting response between RBC number nd hemoglobin content in both the PC nd TA would involve the iron uptke by erythroid cells. According to Ponk (1997), heme-medited repression of ALA synthse-1 is responsible for rendering this enzyme the rte-limiting step in the non-erythroid heme biosynthetic pthwy, however in erythroid cells iron cquisition or the vilbility of iron for ferrocheltse, rther thn ALA production, is the rte-limiting step in heme synthesis. Grdner nd Cox (1988) reported tht negtive feedbck by heme mrkedly ffects iron utiliztion within humn cells nd control of its own synthesis must be exerted either t steps preceding the formtion of protoporphyrin IX or t the level of intrcellulr iron metbolism, including the insertion of ferrous iron into the protoporphyrin nucleus by ferrocheltse. Similrly, severl reports (Ponk nd Schulmn, 1985; Hrdilek nd Neuwrit, 1989) suggest tht heme blocks the uptke of iron from trnsferrin by erythroid cells. In vitro nd in vivo studies indicte tht trnsferrin is the only physiologic source of iron for erythroid cell heme synthesis (Ponk, 1997). In vitro studies with erythroid cells hve shown tht the only physiologiclly ctive chelte tht cn provide iron for their hemoglobin synthesis is trnsferrin (Ponk, 1997). Moreover, heme-induced block of heme synthesis in erythroid cells could not be restored by dded ALA (Ponk et l., 1973). A study by Gllo (1967) suggests tht even with phrmcologicl quntities of ALA, there is insignificnt entrnce into erythroid precursors or tht the site of hemin inhibition of heme synthesis is not ALA synthse. This mode of regultion of heme synthesis my be specific chrcteristic of the hemoglobin biosynthetic pthwy (Ponk nd Schulmn, 1985) since heme does not inhibit iron uptke in non-erythroid cells. This would suggest tht the exogenous supplementtion of ALA done in the present study my not hve prevented the

5 EFFECT OF δ-aminolevulinic ACID IN NURSERY PIGS 101 inhibition brought bout by heme in erythroid cells (red blood cells) since ALA synthse my not be the rte limiting step in erythroid cells. This would lso encourge further investigtion on the effect of ALA supplementtion on non-erythroid cells, ALA synthse being the rte limiting step in these cells. In terms of white blood cell differentil counts mesured in this experiment, only the bsophil counts responded to the different tretments. Pigs from the TA obtined vlues tht were not significntly different from either NC or PC. However, between the PC nd the NC, the former obtined lower bsophil counts. Although, differences were observed, the vlues were still within the norml rnge found in pigs. No significnt differences were observed in terms of lymphocyte counts mong tretments which my explin why there were lso no differences obtined in WBC count mong tretment groups. Other hemtologicl vlues mesured did not show ny significnt difference mong tretment groups. There were no differences noted in terms of behviorl chrcteristics between the NC nd the TA. However, it is interesting to note tht pigs in the PC group tended (p = 0.081) to be less ctive thn pigs from the other tretment groups. IMPLICATIONS Our results indicte tht t 0.05% inclusion, ALA hd no dverse effects on growth performnce of nursery pigs. Moreover, ALA supplementtion hd increse red blood cell counts which my be beneficil to pigs. In ddition, the results led us to investigte further regrding the potentil effects of ALA supplementtion especilly on non-erythroid cells. REFERENCES Collud, S., A. Juzeniene, J. Mon nd N. Lnge On the selectivity of 5-minolevulinic cid-induced protoporphyrin IX formtion. Curr. Med. Chem. Anti-Cnc. Agents. 3: Cromwell, G. L Why nd how ntibiotics re used in swine production, In: the proceedings of the pork industry conference on ddressing issues of ntibiotic use in livestock production (Ed. L. B. Shook). Univ. Illinois, Urbn, pp Cromwell, G. L Antimicrobil nd promicrobil gents In: Swine Nutrition 2 nd Ed edited by A. J. Lewis nd L. L. Southern CRC Press LLC, pp Doring, F., J. Wlter, J. Will, M. Focking, M. Boll, S. Amsheh, W. Cluss nd H. Dniel Delt-minolevulinic Acid Trnsport by Intestinl nd Renl Peptide Trnsporters nd Its Physiologicl nd Clinicl Implictions. J. Clin. Invest. 101: Gllo, R. C The Inhibitory Effect of Heme on Heme Formtion In vivo: Possible Mechnism for the Regultion of Hemoglobin Synthesis. J. Clin. Invest. 46: Grdner, L. C. nd T. M. Cox Biosynthesis of heme in immture erythroid cells: The regultory step for heme formtion in the humn erythron. J. Biol. Chem. 263: Hrdilek, A. nd J. Neuwirt Inhibition of cellulr iron uptke by hem in mouse erythroleukemi cells. Br. J. Hemtol. 73: Jover, R., F. Hoffmnn, V. Scheffler-Koch nd R. L. P. Lindberg Limited heme synthesis in porphobilinogen deminsedeficient mice impirs trnscriptionl ctivtion of specific cytochrome P450 genes by Phenobrbitl. Eur. J. Biochem. 267: Kim, S. W., L. E. Hulbert, H. A. Rchuonyo nd J. J. McGlone Reltive vilbility of iron in mined humic substnces for wenling pigs. Asin-Aust. J. Anim. Sci. 17: Merck Veterinry Mnul A hndbook of dignosis, therpy, nd disese prevention nd control for the veterinrin. 7 th ed. Merck nd Co., Inc.Rhwy, New Jersey, pp Neumnn, J. nd M. Brndsch δ-aminolevulinic Acid Trnsport in Cncer Cells of the Humn Extrheptic Biliry Duct. The Journl of Phrmcology nd Experimentl Therpeutics Fst Forwrd 305: NRC Nutrient Requirements of Swine, 9th Ed, Ntionl Acdemic Press, Wshington, DC. Ponk, P Tissue-Specific Regultion of Iron Metbolism nd Heme Synthesis: Distinct Control Mechnisms in Erythroid Cells. Blood 89:1-25. Ponk, P. nd H. M. Schulmn Regultion of heme synthesis in erythroid cells: hemin inhibits trnsferrin iron utiliztion but not protoporphyrin synthesis. Blood 65: Ponk, P., J. Neuwirt nd J. Borov The use of exogenous δ-minolevulinic cid for the studies of the regultion of hem synthesis in rbbit reticulocytes. Biochim. Biophys. Act. 304: Roof, M. D. nd D. C. Mhn Effect of Crbdox nd vrious dietry copper levels for wenling swine. J. Anim. Sci. 55: Yen, J. T. nd W. G. Pond Effect of crbdox on net bsorption of mmoni nd glucose into heptic portl vein of growing pigs. J. Anim. Sci. 68: Yen, J. T. nd J. A. Nienber Influence of Crbdox on fsting oxygen consumption by portl vein drined orgns nd by the whole niml in growing pigs. J. Anim. Sci. 70: