CLINICAL RELEVANCE. College of Veterinary Medicine University of Missouri Columbia, MO 65211

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1 Veterinry Therpeutics Vol. 7, No. 1, Spring 26 Moultion of Leptin, Insulin, n Growth Hormone in Oese Pony Mres uner Chronic Nutritionl Restriction n Supplementtion with Rctopmine Hyrochlorie* Preston R. Buff, PhD Philip J. Johnson, BVSc, MS, MRCVS Chrles E. Wiemeyer, DVM, PhD c Venktseshu K. Gnjm, DVM, MS, PhD, Nt T. Messer IV, DVM Dune H. Keisler, PhD Deprtment of Veterinry Biomeicl Sciences Deprtment of Veterinry Meicine n Surgery c Deprtment of Veterinry Pthoiology Deprtment of Animl Sciences College of Veterinry Meicine University of Missouri Columi, MO 6211 CLINICAL RELEVANCE Horses tht re fe eyon their nutritionl requirement n re physiclly inctive will evelop oesity, which is often ccompnie y insulin resistnce n heightene risk of lminitis. The use of phrmcologic gents in comintion with nutritionl restriction my promote weight loss in oese horses tht re unle to exercise ecuse of lminitic pin. This stuy showe tht reucing fee intke of rome grss hy to 7% of liitum intke in oese pony mres reuce oy weight without inuce exercise. Aitionl supplementtion of rctopmine hyrochlorie for 6 weeks resulte in tenency for increse weight loss. Susequent moultion of the oesity-ssocite hormones leptin n insulin ws oserve s result of cloric restriction. INTRODUCTION Oesity is prolem in moern horses in lrge prt ecuse of the tenency of owners n triners to fee n exercise horses in mnner tht is not congruous with the wy the species evolve. 1 Oese horses hve excessive epots of ipose, which cn generlly e etermine y oy conition scoring system. Mny horses hve een ientifie s metoliclly efficient or esy keepers n re suscep- *Supporte in prt y grnt from the USDA Animl Helth Formul Funs, Committee on Reserch, College of Veterinry Meicine, University of Missouri Columi n the Animl Helth Fountion of St. Louis. 64

2 P. R. Buff, P. J. Johnson, C. E. Wiemeyer, V. K. Gnjm, N. T. Messer IV, n D. H. Keisler tile to eveloping oesity when fe se on the nutrient requirements of norml horses. 2 Oese horses re often preispose to insulin resistnce 3,4 n re potentilly susceptile to metolic synrome n lminitis. 6 In Jnury 2, the US FDA pprove the use of rctopmine hyrochlorie (Pylen, Elnco) (RAC) in swine finishing rtions to ugment weight gin, fee efficiency, n crcss lenness in finishing pigs. To te, ll in vivo experimenttion with RAC hs use growing-niml moel. Recently, RAC ws shown to e sore n metolize when fe to horses. 7 RAC n other β-renergic gonists shre similr structurl chrcteristics with the nturlly occurring ctecholmines opmine, norepinephrine, n epinephrine. 8 These ctecholmines regulte wie rnge of physiologic responses in mny ifferent tissues. We hypothesize tht RAC woul lter weight loss n peripherl concentrtions of leptin, insulin, n growth hormone in mture oese pony mres in negtive energy lnce. Our ojective ws to etermine the effects of negtive energy lnce, chieve y feeing oese pony mres t 7% liitum fee intke, n the effects of treting with RAC uring perio of 6 weeks. MATERIALS AND METHODS Animls n Proceures All proceures with live nimls were pprove y the University of Missouri Animl Cre n Use Committee. Fifteen oese pony mres with oy conition scores greter thn 7 were use in this experiment. 9 Ech mre ws ssigne to one of five sttisticl locks se on initil oy weight to ensure n even istriution of oy size (249.9 ±.1 kg) in ech tretment group. The three heviest mres were ssigne to lock 1, n the lloction continue until the three lightest weight mres were ssigne to lock. Ech lock ws represent- e eqully in ech of the three tretments, which consiste of control group n two tretment groups tht receive either.6 or 1. mg/kg/y of RAC. Dosge ws etermine y ose response stuy 1 to not increse oy temperture, respirtion, or hert rte. Mres were house iniviully in 16-m 2 ox stlls n fe free-choice choppe rome hy for ys to etermine ily liitum fee intke (ys 4 to ). Fresh hy ws offere twice ily 12 hours prt, with ech mre receiving 4, g/feeing. Refusls from ech previous feeing were weighe n iscre efore the next feeing. Once fee consumption h een etermine (4,34.8 ± 34.8 g/y), ech mre ws then fe t 7% of iniviul liitum intke y weight (strting on y 1). The proportionl mount fe ws 9% choppe rome hy (2,933.4 ± 2.8 g/y) n 1% ots cote with molsses (32.9 ± 22.9 g/y) (use s crrier for the RAC). This esign ensure tht ll nimls were nutritionlly restricte t the sme rte. The mres receive this rtion in two mels fe 12 hours prt (7 n 19) for the urtion of the stuy. Twice-ily ministrtion of RAC egn on y 3 n continue for 6 weeks. RAC ws premesure for ech niml n then mixe thoroughly with the ots efore ech feeing to ensure consumption. The RAC ots mixture or ots (control) were fe to the mres first, followe y hy fe no more thn 3 minutes fter consumption of ots. Mres h ccess to wter n slt throughout the stuy n receive turnout (y tretment group) in ry lot for 1 hour once every thir feeing. Feces ws remove from stlls n ry lot twice ily to prevent coprophgy. Bloo smples were collecte ily for the first 1 ys n then weekly for the remining weeks of the stuy; smples were collecte efore the evening feeing ws provie. Three loo smples were collecte from ech mre over 1-hour perio 6

3 Veterinry Therpeutics Vol. 7, No. 1, Spring 26 Boy Weight (kg) Initil Decreses in Boy Weight Tene to Be Greter in Horses Receiving RAC t 1. mg/kg Week Figure 1. Boy weight chnge of nutritionlly restricte pony mres (control nimls n those receiving ily tretment of.6 or 1. mg/kg RAC). Tretment egn on y 3. Vlues re men oy weight y tretment (lest squres mens ± SEM). Boy weight ecrese in ll mres throughout the stuy ( P <.1). Mres trete with 1. mg/kg RAC tene to hve greter ecrese in oy weight ( P =.91). to ccount for vritions cuse y hormone pulse or vriility. Bloo ws collecte vi jugulr venipuncture using Vcutiner tues contining EDTA (Becton, Dickinson n Compny, Frnklin Lkes, NJ). Bloo smples were immeitely centrifuge t 3, g for 2 minutes t 4 C, n plsm smples were store t 2 C until ssye. Boy weights were mesure weekly throughout the stuy using igitl scle. Hormonl Anlysis Plsm smples were nlyze for leptin vi oule-ntioy rioimmunossy (RIA) proceures previously vlite for equine plsm using n in-house rit nti ovine leptin primry ntiserum (numer 71). 11 Seril ilutions of pool of equine serum were prllel to the ovine leptin stnr curve. The intr- n interssy coefficients of vrition were less thn 1%. Plsm insulin concentrtions were mesure using single RIA kit vlite for Control 1. mg/kg.6 mg/kg equine plsm (Dignostic Proucts, Los Angeles, CA). 12 Seril ilutions of equine plsm were prllel to the humn insulin stnr curve. The interssy coefficient of vrition ws less thn 1%. Proceures of RIA were use to etermine equine growth hormone (GH) 13 using monkey nti porcine-gh ntiser (AFP1384, otine from A. F. Prlow, PhD, Hror- UCLA Meicl Center, Torrnce, CA). Seril ilutions of pool of equine serum were prllel to the equine GH stnr curve. The intr- n interssy coefficients of vrition were less thn 1%. Sttisticl Anlysis A repete mesures esign ws use to etermine the effect of RAC tretment on oy weight n concentrtions of plsm leptin, insulin, n GH. An nlysis of vrince (ANOVA) using the mixe proceures of SAS V8 (SAS Institute, Cry, NC) ws use to etermine sttisticl ifferences etween tretments. Effects within the moel inclue iniviul, lock, tretment, time, n the interctions of tretment n time. Initil vlues of hormones n oy weights were use s covrites. The repete sttement ws time n the error term ws iniviul within tretment y lock. Akike informtion criterion n Schwrz Byesin criterion were use to etermine the compoun symmetry heterogeneous moel s est fitting for nlyses of plsm concentrtions of leptin n oy weight, compoun symmetry moel s est fitting for nlysis of plsm concentrtions of insulin, n ntiepenent moel s the est fit for GH. 14 Lest squres mens n SE were generte in the oy weight moel for time 66

4 P. R. Buff, P. J. Johnson, C. E. Wiemeyer, V. K. Gnjm, N. T. Messer IV, n D. H. Keisler n tretment y time. Lest squres mens, SE, n lest squres ifferences were generte in the leptin, insulin, n GH moels for time, tretment, n tretment y time. RESULTS No significnt ifferences existe in ny vrile mesure efore the onset of tretment (P >.1). Boy weights significntly chnge throughout the urtion of the stuy following fee restriction (P <.1; Figure 1). This stey ecline in oy weights ws oserve in ll mres regrless of tretment, s expecte. Although the fining ws not significnt, mres trete with 1. mg/kg RAC exhiite tenency to hve greter ecrese in oy weight compre with the mres in the control n.6-mg/kg RAC groups in the first 2 weeks of the stuy (P =.91; Figure 1). After the immeite chnges in weight in the 1.-mg/kg RAC group, oy weight ppere to ecrese t similr rte in ll of the groups. Plsm concentrtions of leptin chnge over time in ll mres regrless of tretment (P <.1; Figure 2). These concentrtions ecrese y 16% immeitely following the restriction of fee. After the initil ecrese, the concentrtions of leptin increse Leptin (ng/ml) Leptin (ng/ml) Figure 2. Chnges in leptin concentrtion over time (lest squres mens ± SEM) in pony mres regrless of tretment. Fee restriction egn on y 1. A significnt chnge in leptin concentrtion occurre over time ( P <.1).,,c Differences of preplnne comprisons etween ifferent time points re inicte y ifferent letters ( P <.). c Plsm Concentrtions of Leptin Chnge over Time in All Mres Dy of Stuy No Significnt Differences Were Oserve in Concentrtions of Leptin etween Groups Dy of Stuy Control 1. mg/kg.6 mg/kg Figure 3. Chnges in leptin concentrtion over time (lest squres mens ± SEM) in nutritionlly restricte pony mres (control nimls n those receiving ily tretment of.6 or 1. mg/kg RAC). Tretment egn on y 3. No ifferences were oserve etween ny tretment group ( P =.33)., c over the next 2 ys followe y progressive ecrese for the next 4 ys. Oservtions throughout the remining weeks inicte tht concentrtions of leptin increse to prerestriction vlues within 3 weeks n then ecrese for the remining 3 weeks. No significnt ifferences were oserve in concentrtions of leptin etween tretments (P =.33; Figure 3). 67

5 Veterinry Therpeutics Vol. 7, No. 1, Spring 26 Insulin (µiu/ml) Insulin (µiu/ml) c Insulin Concentrtions Decline Significntly Following Fee Restriction c Dy of Stuy Figure 4. Chnges in insulin concentrtion over time (lest squres mens ± SEM) in pony mres regrless of tretment. Fee restriction egn on y 1. A significnt chnge in insulin concentrtion occurre over time ( P <.1).,,c, Differences of preplnne comprisons etween ifferent time points re inicte y ifferent letters ( P <.). No Significnt Differences in Insulin Concentrtions Were Oserve etween Tretments Dy of Stuy Figure. Chnges in insulin concentrtion over time (lest squres mens ± SEM) in nutritionlly restricte pony mres (control nimls n those receiving ily tretment of.6 or 1. mg/kg RAC). Tretment egn on y 3. No ifferences were oserve etween ny tretment group ( P =.1). Control 1. mg/kg.6 mg/kg We oserve significnt ecline in concentrtions of insulin in ll mres following the fee restriction (P <.1; Figure 4). Concentrtions of insulin ecline overll following the onset of restriction. During the first 1 ys, insulin increse on ys 4 to n 8 followe y susequent ecreses (Figure 4). No significnt ifferences in concentrtions of insulin were oserve etween tretments (P =.1; Figure ). GH concentrtions chnge over time in ll mres, regrless of tretment (P <.1; Figure 6). Concentrtions of GH ppere to ecrese uring the first week following the initition of fee restriction n increse uring the remining weeks of the stuy. No significnt ifferences were oserve in concentrtions of GH etween tretments (P =.1; Figure 7). DISCUSSION Mny reserchers elieve tht equine metolism evolve for survivl se on the sesonlly vrile vilility of forge. 1 The temporry evelopment of itionl oy ft t times when forge is plentiful provies survivl mechnism for times when conitions re hrsh n forge is scrce. One theory is the nturl selection of genotype for metolic efficiency, similr to the thrifty genotype propose y Neel in humns. 1 Through centuries of reeing for trits inconsistent with metolic efficiency, humns hve prouce horses tht re consiere to hve norml metolism. We theorize tht some horses referre to s metoliclly efficient or esy keepers my hve retine 68

6 P. R. Buff, P. J. Johnson, C. E. Wiemeyer, V. K. Gnjm, N. T. Messer IV, n D. H. Keisler these thrifty genes n thus hve lower nutrient requirements thn norml horses. Circin 16,17 n sesonl 17,18 vritions of leptin hve recently een reporte in 4 horses, which supports this theory 3 of oy ft ccumultion for survivl. During perios in which 2 forge is reltively unville, the oy ft stores, which were never 1 intene to ecome excessive, re eplete to provie energy for survivl. Uner mny moern horse- mngement systems, the comintion of feeing strch-rich rtions over mny yers n protrcte perios of stll confinement cn le to the cquisition n mintennce of sustntil iposity in omesticte horses. The β-renergic gonists re reporte to e metolic moifiers n re orlly ctive; the min effects on the crcss of the niml re incresing skeletl muscle n 4 reucing ipose tissue mss. 8 When growing nimls re trete 3 orlly with RAC, they respon y incresing muscle mss n ecresing ipose stores; this hs 2 een seen in swine, rts, 22 1 n steers. 23 Similr results re oserve when geneticlly oese n geneticlly len pigs re trete with RAC. 24 Skeletl muscle eposition is rmticlly enhnce y the β-renergic gonists clenuterol n cimterol, oth of which re specific for the β 2 receptor sutype. 2,26 Conversely, RAC is primrily β 1 gonist 27 n hs een shown to GH (ng/ml) GH (ng/ml) Figure 6. Chnges in GH concentrtion over time (men ± SEM) in nutritionlly restricte pony mres regrless of tretment. A significnt chnge occurre over time regrless of tretment ( P <.1). Tretment egn on y 3. No effect of tretment ws oserve ( P =.1)., Differences of preplnne comprisons etween ifferent points in time re inicte y ifferent letters ( P <.). GH Concentrtion Chnge in All Mres, But No Effect of Tretment Ws Oserve Dy of Stuy No Significnt Differences Were Oserve in GH Concentrtions etween Tretment Groups Dy of Stuy Control 1. mg/kg.6 mg/kg Figure 7. Chnges in GH concentrtions over time (lest squres mens ± SEM) in nutritionlly restricte pony mres (control nimls n those receiving ily tretment of.6 or 1. mg/kg RAC). Tretment egn on y 3. No ifferences were oserve etween ny tretment group ( P =.1)., hve high ffinity for mile sucutneous ipose tissue. 28 Clenuterol hs een investigte s possile therpeutic tretment for oesity in horses n hs resulte in reuction of ipose, 29,3 ut it hs een reporte to negtively ffect cric function. 31 Recently, RAC,, 69

7 Veterinry Therpeutics Vol. 7, No. 1, Spring 26 ws teste in horses n ws etermine to e metolize y the etection of metolites in urine. 7 Tht stuy showe tht when RAC is fe to horses in the sme mnner s in the present stuy, it is sore vi the gstrointestinl trct. The metolites recovere inicte tht RAC is highly metolize vi similr methos in other species. In the present stuy, RAC ws elivere in the sme metho n consistent osge s the previous stuy. In tht stuy, however, horses were ose once, wheres in the present stuy, nimls were ose twice ily for 6 weeks. Bse on the previous t, we elieve tht RAC ws sore n metolize y the nimls in the present stuy. However, no tests were performe in the present stuy to etermine if RAC ws sore or metolize. In the current stuy, we oserve tenency for the group receiving RAC t 1. mg/kg to hve greter ecrese in oy weight compre with the control n.6-mg/kg groups. The ifferences seen in this group pper to hve occurre very soon fter tretment egn. It cn e speculte tht the lck of continue response is result of tretment with RAC twice ily. This ose frequency my not llow the β 1 receptors sufficient time to recover; thus, greter refrctory perio my e neee to elicit greter response. It is possile tht feeing RAC twice ily my not llow time for the moleculr ctions following receptor ining to recover n, therefore, my not llow full response to ech tretment. 27 Reucing tretment to once ily or less frequently coul potentilly ffect receptor ctivity. Recently reporte t support our contention tht the ose n route of ministrtion tht we use were pproprite in terms of iovilility for horses. 7 Becuse of housing constrints, the mximum numer of nimls per group ws five, which limite sttisticl power. Chnges in ll hormones mesure were ttriute to restriction of fee n susequent weight loss. We i not oserve chnges in hormone concentrtions ttriutle to tretment with RAC. However, the chnges in hormone profiles s result of fee restriction o offer insight regring metolism in oese horses. Most notle ws the chnge in the leptin concentrtion, which ecrese uring the first y following the fee reuction n then grully increse over the next 2 ys. This increse ws followe y ily ecrese for the next 4 ys. The remining weekly smples revele nother rise n fll in the concentrtions of leptin. The oserve chnges in concentrtions of leptin my e expline y Kenney s liposttic theory. 32 Briefly, Kenney suggeste tht nimls regulte ppetite n energy expeniture ccoring to the mount of energy storge (oy ft) n foo ville. Leptin is elieve to e key regultor in this mechnism, cting to signl iposity n regulting energy lnce. 33 We conclue tht the chnges oserve in concentrtions of leptin were ttriutle to regultion (or resetting) of the lipostt s time psse n fee ws continully restricte. While the horses were mintine in negtive energy lnce, their oy weight ecline n, presumly, metolism n energy expeniture were juste. When fee is restricte t constnt rte, oy weight ecreses until equilirium is reche. 34 We ttriute the rise n fll of leptin to justments in metolism s the oy is reching equilirium. The concentrtions of leptin oserve in the current stuy gree with previous oservtions in which concentrtions of leptin ecrese following n cute fee restriction. 17 Concentrtions of insulin rpily ecline s result of fee restriction n continue to e suppresse for the reminer of the experiment. Within the first 1 ys of eclining insulin concentrtions, we oserve increses on ys 4 to n y 8 followe y susequent ecreses. These trnsient rises in concentrtions of insulin 7

8 P. R. Buff, P. J. Johnson, C. E. Wiemeyer, V. K. Gnjm, N. T. Messer IV, n D. H. Keisler coul e n pttion response to negtive energy lnce. Becuse insulin hs lipogenic n ntilipolytic properties, this suppression of insulin is inictive of fee restriction. Suppression of this hormone therefore llowe the oy to mintin homeostsis y using ipose stores. A moest ecrese followe y moest increse in GH ws oserve fter fee restriction. In previous experiment, we oserve twofol increse in concentrtions of GH when horses were fee eprive for 48 hours, 17 inicting tht moultion of GH likely epens on nutrient vilility. Thus, we conclue tht reucing fee intke to 7% of liitum intke is elow the threshol require to increse GH to mximum concentrtion. CONCLUSION One interprettion of these t tht tenency existe for RAC to increse weight loss t the 1.-mg/kg ose offers insight for the use of β 1 -renergic gonists for weight loss in oese horses. The frequency or quntity of tretment with RAC my hve limite our success to moerte weight loss. However, reuction in fee consumption to 7% of liitum intke with miniml exercise resulte in significnt weight loss without ny pprent verse effects. Assessing the liitum forge intke for n iniviul oese horse n restricting the intke se on this recommention my represent the est pproch for oesity reversl in horses t this time. REFERENCES 1. Epstein H: The Origin of the Domestic Animls of Afric, vol 2. New York, Africn, 1971, p Ntionl Reserch Council: Nutrient Requirements of Horses, e rev. Wshington, DC, Ntionl Acemy Press, Jeffcott LB, Fiel JR, McLen JG, et l: Glucose tolernce n insulin sensitivity in ponies n Stnrre horses. Equine Vet J 18:97 11, Hoffmn RM, Boston RC, Stefnovski D, et l: Oesity n iet ffect glucose ynmics n insulin sensitivity in Thoroughre gelings. J Anim Sci 81: , 23.. Johnson PJ: The equine metolic synrome: Peripherl Cushing s synrome. Vet Clin North Am Equine Prct 18: , Johnson PJ, Gnjm VK, Slight SH, et l: Tissue-specific ysregultion of cortisol metolism in equine lminitis. Equine Vet J 36:41 4, Lehner AF, Hughes CG, Hrkins JD, et l: Detection n confirmtion of rctopmine n its metolites in horse urine fter Pylen ministrtion. J Anl Toxicol 28: , Ntionl Reserch Council: Metolic Moifiers: Effects on the Nutrient Requirements of Foo-Proucing Animls. Wshington, DC, Ntionl Acemy Press, Henneke DR, Potter GD, Kreier JL, et l: Reltionship etween conition score, physicl mesurements n oy ft percentge in mres. Equine Vet J 1: , Buff PR, Johnson PJ: Unpulishe t, University of Missouri Columi, Columi, MO, Buff PR, Dos AC, Morrison CD, et l: Leptin in horses: Tissue locliztion n reltionship etween peripherl concentrtions of leptin n oy conition. J Anim Sci 8: , Freestone JF, Wolfsheimer KJ, Kmerling SG, et l: Exercise inuce hormonl n metolic chnges in Thoroughre horses: Effects of conitioning n cepromzine. Equine Vet J 23: , Thoms MG, Bennett-Wimush K, Keisler DH, et l: Plsm concentrtions of growth hormone n insulin-like growth fctor-i in prepuerl qurter horses n ponies. J Equine Vet Sci 18:2, Littell RC, Henry PR, Ammermn CB: Sttisticl nlysis of repete mesures t using SAS proceures. J Anim Sci 76: , Neel JV: Dietes mellitus: A thrifty genotype renere etrimentl y progress? Am J Hum Genet 14:33 362, Piccione G, Bertoluccci C, Foà A, et l: Influence of fsting n exercise on the ily rhythm of serum leptin in the horse. Chronoiol Int 21:4 417, Buff PR, Morrison CD, Gnjm VK, et l: Effects of short-term fee eprivtion n meltonin implnts on circin ptterns of leptin in the horse. J Anim Sci 83: , Gentry LR, Thompson DL Jr, Gentry GT Jr, et l: The reltionship etween oy conition, leptin, n reprouctive n hormonl chrcteristics of mres uring the sesonl novultory perio. J Anim Sci 8: , Mitchell AD, Solomon MB, Steele NC: Influence of lev- 71

9 Veterinry Therpeutics Vol. 7, No. 1, Spring 26 el of ietry protein or energy on effects of rctopmine in finishing swine. J Anim Sci 69: , Grnt AL, Skjerlun DM, Helferich WG, et l: Skeletl muscle growth n expression of skeletl muscle lph-ctin mrna n insulin-like growth fctor I mrna in pigs uring feeing n withrwl of rctopmine. J Anim Sci 71: , Willims NH, Cline TR, Schinckel AP, et l: The impct of rctopmine, energy intke, n ietry ft on finisher pig growth performnce n crcss merit. J Anim Sci 72: , Smith DJ, Pulson GD: Growth chrcteristics of rts receiving rctopmine hyrochlorie n the metolic isposition of rctopmine hyrochlorie fter orl or intrperitonel ministrtion. J Anim Sci 72:44 414, Smith DJ: The phrmcokinetics, metolism, n tissue resiues of β-renergic gonist in livestock. J Anim Sci 76: , Yen JT, Mersmnn HJ, Hill DA, et l: Effects of rctopmine on geneticlly oese n len pigs. J Anim Sci 68: , O Donnell SR: Selectivity of clenuterol (NAB 36) in guine-pig isolte tissue contining β-renoceptors. Arch Int Phrmocoyn 224:19 198, Kim YS, Sinz RD, Summers RJ, et l: Cimterol reuces et-renergic receptor ensity in rt skeletl muscles. J Anim Sci 7:11 122, Anerson DB, Veenhuizen EL, Jones DJ, et l: The use of phenethnolmines to reuce ft n increse crcss lenness in met nimls, in Herstroh C, Morris CE (es): Avnces of Applie Biotechnology Series. Ft n Cholesterol Reuce Foos: Technologies n Strtegies, vol 12. The Woolns, TX, Portfolio, 1987, pp Spurlock ME, Cusumno JC, Mills SE: The ffinity of rctopmine, clenuterol, n L-644,969 for the et-renergic receptor popultion in porcine ipose tissue n skeletl muscle memrne. J Anim Sci 71:261 26, Kerns CF, McKeever KH, Mlinowski K, et l: Chronic ministrtion of therpeutic levels of clenuterol cts s reprtitioning gent. J Appl Physiol 91:264 27, McMnus CJ, Fitzgerl BP: Effect of ily clenuterol n exogenous meltonin tretment on oy ft, serum leptin n the expression of sesonl nestrus in the mre. Anim Repro Sci 76:217 23, Sleeper MM, Kerns CF, McKeever KH: Chronic clenuterol ministrtion negtively lters cric function. Me Sci Sports Exerc 34:643 6, Kenney GC: The role of epot ft in the hypothlmic control of foo intke in rts. Proc Royl Soc Lonon Series B 14:78 92, Houseknecht KL, Bile CA, Mtteri RL, et l: The iology of leptin: A review. J Anim Sci 76:14 142, Broy S: Bioenergetics n Growth, with Specil Reference to the Efficiency Complex of Domestic Animls. New York, Reinhol, 194, pp