Research Article Antioxidant Status in Elite Three-Day Event Horses during Competition

Oidtive Medicine nd Cellulr Longevity Volume 2012, Article ID 572090, 8 pges doi:10.1155/2012/572090 Reserch Article Antioidnt Sttus in Elite Three-Dy Event Horses during Competition Crey A. Willims 1 nd Amy O. Burk 2 1 Deprtment of Animl Sciences, Rutgers, The Stte University of New Jersey, New Brunswick, NJ 08901, USA 2 Animl nd Avin Sciences Deprtment, University of Mrylnd, College Prk, MD 20742, USA Correspondence should be ddressed to Crey A. Willims, cwillims@esop.rutgers.edu Received 14 Februry 2012; Accepted 22 April 2012 Acdemic Editor: Mnfred Lmprecht Copyright 2012 C. A. Willims nd A. O. Burk. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Theobjective of this studyws to determine if competition intensity would hve n effect on ntioidnt sttus in horses before nd during three-dy event. Body weight, body condition score, nd blood ws smpled from (n = 19) nd (n = 23) horses before the strt of dressge, 20 to 30 min following cross-country, nd 18 24 h fter cross-county. Dt were nlyzed using PROC MIXED in SAS. There were no differences between nd horses during competition for plsm cortisol, lctte, α-tocopherol, retinol, or erythrocyte glutthione peroidse. After cross-country, horses hd higher serum cretine kinse (P = 0.003) nd sprtte minotrnsferse (P <0.0001) thn the horses. Plsm β-crotene ws higher in the horses compred to the horses (P = 0.0001). Totl erythrocyte glutthione ws lso higher in the horses versus horses (P <0.0001). These results re the first report of ntioidnt sttus of horses competing in this level of three-dy event. The chnges in ntioidnt nd muscle enzymes observed between divisions re likely due to the incresed nerobic nd musculoskeletl demnd on the upper level horses nd the fitness required to compete t tht level. 1. Introduction Equine thletes competing t the top levels of their disciplines eperience physiologicl stress tht my compromise helth nd performnce. Oidtive stress results in oidtive dmge to ll cell components. Oidtive stress is cused by n imblnce between the rective oygen species (ROS) produced during cellulr respirtion nd the body s ntioidnt system used to scvenge these ROS [1]. Oidtive stress hs been observed in horses eposed to intense bouts of eercise [2, 3] nd endurnce eercise [4, 5]. Antioidnts re vitmins, minerls, nd enzymes tht must be synthesized in the body or obtined from the diet. Therefore, eercise level nd diet re both fctors tht ply role in influencing the oidtive stress nd ntioidnt sttus of the equine thlete. Eventing is rigorous eercise chllenge, similr to the humn trithlon tht tests the physicl bility nd skill of both horse nd rider during three seprte phses. In dressge, stndrd set of movements is performed nd wrded subjective score. Cross-country jumping requires horses to jump over 35 to 40 obstcles covering 5 to 7 km of terrin within limited time. For stdium, jumping horses rejumpedover10to12obstclesrrngedincourse set in n enclosed ren. The Interntionl Federtion for Equestrin Sports (FEI) governs the sport nd hs divided it into four levels of difficulty. The highest or Olympic level is denoted s Concours Complet Interntionle Four-Str Event (CCI4 ) followed by the,, nd CCI1 divisions. Mny studies previously hve reserched electrolyte loss nd fluid shifts, lctte response, nd other biochemicl mesures of eercise intensity of horses performing in 3- dy event competitions. However, none hve mesured the ntioidnt sttus long with their dietry profile. Therefore, the min objective of this study ws to determine the effects of rigorous, high-level three-dy event on the ntioidnt sttus of horses. Recognizing tht diet cn influence ntioidnt sttus, second objective ws to chrcterize the diet nd feeding mngement prctices of three-dy event horses prior to nd during the eercise chllenge. It is hypothesized

2 Oidtive Medicine nd Cellulr Longevity Tble 1: Specifictions of the cross-country jumping phse in the nd divisions of the 2006 Jersey fresh three-dy event. Distnce 4.4 5 km 5.7 6.8 km Speed 9.2 m/s 9.5 m/s No. of jumps 36 40 M. height 1.15 m 1.2 m M.drop 1.8m 2m tht the oidtive stress mrkers would be higher fter crosscountry jumping in both divisions, but the would be higher thn the due to the higher level of eercise intensity. 2. Mterils nd Methods 2.1. Competition. Horses nd riders prticipting in this study were competing in either the or division of the Jersey fresh three-dy event in Allentown, NJ from My 31 to June 4, 2006. The event consisted of postrrivl veterinry horse inspection on dy 1, dressge on d 2 or 3, cross-country jumping followed by the second veterinry horse inspection on d 4, nd then third veterinry horse inspection followed by stdium jumping on d 5. Competitors rode the 2005 FEI or event dressge test B for their respective divisions. The distnce, optimum speed nd jumping efforts for the cross-country jumping phse re presented in Tble 1. Men mbient temperture during the fivedysofcompetitionws22.9 C (rnge: 16.1 to 33.3 C) with 5.4, 3.3, nd 0.36 cm of precipittion occurring on dys 3, 4, nd 5 (Wether Dt Services, Inc, Wichit, KS). 2.2. Subjects. Prior to the first veterinry horse inspection, horse owners or riders were sked to complete relese sttement tht voluntrily enrolled their horses s prticipnts in the study. Nineteen of 33 (58%) horses nd 23 out of 41 (56%) horses were enrolled. Horses in the division completed dressge from 0900 to 1200 on dys 2 nd 3 of the competition nd cross-country jumping between 0900 nd 1100 on dys 4. Horses in the division completed dressge from 1300 to 1800 on d 2 nd 3 nd cross-country jumping between 1300 nd 1600 on dys 4. All stdium jumping strted t 1200 on dys 5 with horses competing first, immeditely followed by horses. 2.3. Smple Collection nd Processing. Hert rte, rectl temperture, nd respirtory rte were recorded by licensed veterinrin immeditely following completion of the crosscountry jumping phse. Whole blood ws collected on d 1 immeditely fter the first veterinry horse inspection (PRE), on d 4 20 30 min following the cross-country jumping phse s prt of the second veterinry horse inspection (XC), nd on d 5 immeditely fter the third veterinry horse inspection but prior to the commencement of stdium jumping (POST). The POST smple ws therefore tken 18 to 24 h fter XC. Body weight (BW) ws determined using n electronic scle, nd body condition score (BCS) [6] ws nlyzed t the time of blood collection. Whole blood (40 ml) ws collected by venipuncture into sodium heprin, EDTA, nd serum seprtor Vcutiner tubes (Becton Dickinson nd Compny, Frnklin Lkes, NJ), immeditely plced on ice, nd then processed into red blood cell, plsm, or serum liquots t n on-site lbortory within 30 min of collection using previously described methods [7]. All processed smples were stored on dry ice until trnsferred to 80 C, where they remined until nlysis (within 24 h of smple collection). 2.4. Smple Anlysis. Whole blood ws nlyzed for hemtocrit (Hct) using microhemtocrit technique (Spircrit, Oford Lbwre-Division of Sherwood Medicl, St. Louis, MO). Plsm ws nlyzed for totl protein (TP) using refrctometry. Plsm cortisol (CORT) ws nlyzed by rdioimmunossy (Dignostic Products Corportion, Los Angeles, CA). Plsm lctte (LAC) ws nlyzed using YSI model no. 1500 lctte nlyzer (YSI Inc., Yellowsprings, OH). Serum ws nlyzed for cretine kinse (CK) nd sprtte minotrnsferse (AST) using VetTest 8008 nlyzer (Model VT8008, IDEXX Lbortories Inc., Westbrook, ME). The ntioidnts α-tocopherol (TOC), retinol (RET), nd β-crotene (BC) were nlyzed by HPLC (Michign Stte Dignostic Lbortories, Est Lnsing, MI) by first running plsm smples through precolumn followed by reverse phse C-18 HPLC column eluted isocrticlly t 1.2 ml/min with n injection volume of 50 ul. Absorbnce ws mesured for TOC (292 nm), RET (325 nm), nd BC (450 nm). Red blood cells were nlyzed for totl glutthione (GSH-T) nd glutthione peroidse (GP). Methods for nlysis of GSH- T (Biotech GSH-420, kit #21023; Ois Helth Products Inc., Portlnd, OR; interssy CV 7.0%, intr-ssy CV 5.6%) nd cellulr GP (Biotech GP-340, kit #21017; Ois Helth Products Inc., Portlnd, OR; interssy CV 4.2%, intr-ssy CV 5.0%) were previously described [7]. 2.5. Sttisticl Anlysis. Dt re summrized s men ± SE unless otherwise noted. Effects of division, smple time, nd their interction were evluted on ntioidnt nd oidtive stress mrkers, long with performnce prmeters, using repeted model ANOVA with smple s our repeted effect (PROC MIXED, SAS Inst Inc., version 9.1, Cry, NC). To further test for differences between competition divisions nd smple times, Tukey-Krmer djustment ws used with significnce determined using P < 0.05. Associtions between eercise nd ntioidnt vribles were tested using Person s product-moment correltion. Horse within division ws included in the model to test for significnce; if insignificnt, then it ws removed from the model. Significnce ws determined using P<0.05. 3. Results 3.1. Horse Dt. Demogrphic informtion, initil BW, nd initil BCS of horses prticipting in the study re shown

Oidtive Medicine nd Cellulr Longevity 3 Tble 2: Subject informtion for horses nd riders competing in the nd divisions of the 2006 Jersey fresh three-dy event. Item Divisions Subject prticiption 1, % 57.5 56.1 Age, yr 11.3 ± 0.7 11.3 ± 0.5 Se of horse, % Mle 78.9 91.3 Femle 21.1 8.7 Breed of horse, % Thoroughbred 78.9 59.1 Thoroughbred cross 10.5 22.7 Wrmblood 5.3 9.1 Other 5.3 9.1 Initil BW, kg 529.2 ± 7.7 529.7 ± 7.1 Initil BCS 5.2 ± 0.1 5.1 ± 0.1 1, n = 19 out of 33 entered;, n = 23 out 41 entered. in Tble 2. There were no differences in ge, initil BW, nd initil BCS between nd horses. For both divisions, the mjority of horses were of the mle gender nd Thoroughbred breed. Men ge ws 11.3 ± 0.7 nd 11.3 ± 0.5 yrs for nd horses, respectively. Horses competing in the hd been competing t tht level for 0 to 1 yr (50.0%), 1 to 2 yr (30.0%), or 3 to 5 yr (20.0%), wheres horses in the hd been competing t tht level for 0 to 1 yr (52.2%), 1 to 2 yr (26.1%), or 3 to 5 yr (21.7%). Twelve subjects completed the (63.2%) with seven plcing in the top 10 (1st, 2nd, 3rd, 6th, 8th, 9th, nd 10th). Of the nonfinishers, three withdrew before cross-country jumping, three withdrew or were eliminted during crosscountry jumping, nd one did not pss the finl veterinry horse inspection. In the, 13 subjects completed the event (56.5%) with four plcing in the top 10 (3rd, 6th, 9th, nd 10th). Nonfinishers included one tht ws withdrwn before cross-county jumping, si tht were withdrwn or were eliminted during cross-county jumping, nd three tht were either withdrwn fter cross-country jumping for unknown resons or did not pss the finl veterinry horse inspection. Men hert rte, rectl temperture, nd respirtory rte immeditely following the completion of cross-country jumping were not different between divisions (dt not shown). For ll horses completing the cross-country jumping phse, men hert rte ws 102 ± 2.8 bets per min, rectl temperture ws 40.4 ± 0.09 C, nd respirtory rte ws 100 ± 3.2 breths per min. There were no correltions observed between these vribles nd blood vribles ssessed t XC (dt not shown). 3.2. Diet Dt. Diets of horses could not be controlled in this study, nd s result, horses were fed ccording to the rider or owner s preference. A nutritionl mngement survey ws conducted prior to nd during the competition to gther informtion on ech horse s feeding times; type of psture nd hours spent turned out on psture prior to competition; type nd mount of hy, concentrte, nd supplements offered t competition; feeding ssocited with trnsport to the event nd cross-country jumping. All feeding mngement dt collected during the study hve been previously reported [8]. 3.3. Blood Dt 3.3.1. Effect of Division. There ws no min effect of the competition division on plsm TP, CORT, LAC, AST, TOC, RET, BC, nd erythrocyte GP; therefore, dt were verged cross division (Tble 3). Figure 1 shows blood vribles tht differed between horses competing in the nd divisions. There ws min effect of division on Hct (P = 0.002), nd erythrocyte GSH-T (P < 0.0001) with the horses hving higher blood concentrtions thn the horses. For serum CK (P = 0.04), however, serum concentrtions were lower in the horses compred to the horses. For Hct, horses were higher t XC thn horses (P = 0.001; 55.1 ± 1.7versus50.1 ± 1.0%, resp.; Figure 1()).SerumCKwslowerin horses t XC thn horses (P = 0.003; 235 ± 16 versus 576 ± 123 mmol/l; Figure 1(b)). Red blood cell GSH-T ws higher in horses t XC (P <0.0001; 237 ± 12 versus 163 ± 12 um, resp.) compred to horses (Figure 1(c)), nd time by division interction ws observed (P = 0.006). 3.3.2. Effect of Time. The Hct ws highest t the XC smple for ll horses (P <0.0001, Figure 1()), but ws not different between PRE nd POST. Plsm TPws different for ll three smples (P <0.0003, Tble 3) with the highest occurring t XC. Plsm CORT ws highest fter XC nd remined higher thn PRE levels t POST (P <0.005, Tble 3). Serum CK, AST, nd plsm LAC were ll lowest t PRE (P < 0.01, Figure 1(b),ndTble 3, resp.). Plsm TOC did not chnge in horses throughout the competition (Tble 3). Plsm BC ws highest in horses t PRE (P = 0.003; Tble 3)nd decresed to XC nd POST. Plsm RET incresed in ll horses from PRE to XC, but it then decresed to its lowest vlue t POST (P < 0.03, Tble 3). Red blood cell GSH-T ws similr from PRE to XC, but it then decresed below bseline t POST (P <0.0001, Figure 1(c)). Red blood cell GPpekedtXCcompredtoPREndPOST(P = 0.004, Tble 3). 3.3.3. Correltions. Significnt correltions between the eercise nd ntioidnt vribles re shown in Tble 4.Someof these include positive correltion between LAC nd GSH- T, GP, CORT nd RET (P <0.05). Plsm CORT, ws lso positively correlted with CK (P = 0.007), RET (P = 0.003), nd GSH-T (P = 0.04) nd negtively correlted with TOC (P = 0.04). Plsm AST, however, ws not correlted with CORT s CK ws, but ws positively correlted with TOC (P = 0.002) nd negtively correlted with GP (P = 0.04).

4 Oidtive Medicine nd Cellulr Longevity 60 55 A b 800 700 600 B y Hct (%) 50 45 40 35 y CK (U/L) 500 400 300 200 100 30 PRE XC smple POST 0 PRE XC smple POST () (b) 300 250 E b GSH-T (µm) 200 150 100 c 50 y 0 PRE XC smple POST (c) Figure 1: Hemtocrit (Hct; ()), serum cretine kinse (CK; (b)), nd red blood cell totl glutthione (GSH-T; (c)) in nd horses before competition (PRE), 20 30 min fter cross-country (XC), nd 18 24 hr fter XC but before stdium jumping (POST). denotes difference between nd horses within smpling time (P <0.001). Letters, b, nd c denote differences cross smpling time for horses (P <0.05). Letters, y,ndz denote differences cross smpling time for horses (P <0.05). Tble 3: Blood vribles ssessed before the competition (PRE), 20 30 min fter cross-country jumping (XC), nd 18 24 h fter crosscountry jumping (POST) for horses competing in the nd divisions of the 2006 Jersey fresh three-dy event. Vrible b PRE XC POST Plsm TP, g/dl 7.01 ± 0.06 7.31 ± 0.07 y 6.76 ± 0.07 z Plsm CORT, ug/dl 5.47 ± 0.43 11.1 ± 0.70 y 7.55 ± 0.36 z Plsm AST, U/L 353.2 ± 33.8 413.4 ± 34.0 y 424.1 ± 36.2 y Plsm LAC, mmol/l 0.82 ± 0.04 11.2 ± 1.60 y 0.62 ± 0.03 Plsm TOC, ug/ml 5.50 ± 0.28 5.50 ± 0.26 5.75 ± 0.33 Plsm RET, ng/ml 163.4 ± 4.6 177.0 ± 5.7 y 152.9 ± 4.4 z Plsm BC, ng/ml 0.61 ± 0.07 0.47 ± 0.07 y 0.47 ± 0.07 y RBC GP, mu 260.6 ± 7.7 308.9 ± 12.8 y 254.5 ± 16.2 For ech vrible, no difference between divisions ws observed; therefore, dt ws verged cross divisions. b TP: plsm totl protein; LAC: plsm lctte; CORT: cortisol; AST: plsm sprtte minotrnsferse; TOC: plsm lph-tocopherol; RET: plsm retinol; BC: plsm bet-crotene; GP: erythrocyte glutthione peroidse.,y,z Within row, mens with unlike superscripts differ (P < 0.05).

Oidtive Medicine nd Cellulr Longevity 5 Tble 4: Significnt correltions between performnce nd ntioidnt blood mesures,b. Y X R P Y X R P Y X R P BW CORT 0.21 0.02 TP GP 0.23 0.02 CORT GSH-T 0.19 0.04 BW TP 0.22 0.03 TP CORT 0.25 0.002 CORT CK 0.25 0.007 Hct RET 0.22 0.03 LAC GSH-T 0.52 <0.0001 RET GSH-T 0.40 0.01 Hct GSH-T 0.66 <0.0001 LAC GP 0.19 0.04 TOC BC 0.40 <0.0001 Hct GP 0.27 0.007 LAC CORT 0.28 0.002 TOC AST 0.30 0.002 Hct CORT 0.44 <0.0001 LAC RET 0.27 0.004 GSH-T GP 0.28 0.003 TP CK 0.25 0.01 CORT RET 0.28 0.003 GP AST 0.19 0.04 TP GSH-T 0.33 0.0007 CORT TOC 0.19 0.04 CK AST 0.50 <0.0001 Horse ws included in the model to test for significnce, if insignificnt, then it ws removed from the model. b Initil BW (n = 112), hemtocrit (Hct; n = 104 totl points), plsm totl protein (TP; n = 104), lctte (LAC; n = 114), cortisol (CORT; n = 114), lphtocopherol (TOC; n = 114), bet-crotene (BC; n = 114), retinol (RET; n = 114), serum cretine kinse (CK; n = 113), sprte minotrnsferse (AST; n = 113), erythrocyte totl glutthione (GSH-T; n= 114), nd glutthione peroidse (GP; n = 111). 4. Discussion The min objective of this study ws to determine the effects of rigorous eercise ctivity, like three-dy event, on the ntioidnt sttus of horses. To our knowledge, this is the first pper to revel the ntioidnt levels of horses competing in upper level divisions of three-dy event. A compnion study studying inflmmtory prmeters nd diet on similr popultion of competitive horses ws recently published [9]. Overll, our findings suggest tht the cross-country jumping phse of three-dy event ws rigorous cute eercise bout tht chllenged the ntioidnt systems of the horse, with more of chllenge being plced on the horses. Collectively, the competing horses hd higher blood levels of RET, GP, nd GSH-T, but lower BC t XC compred to precompetition (PRE) indicting these ntioidnts were mobilized to be used in response to chllenge to the ntioidnt system s result of the crosscountry jumping phse. Specificlly within divisions, the horses hd 2.5 times greter serum concentrtions of CK, 1.5 times greter serum concentrtions of AST, pproimtely 50% lower plsm BC, nd 70% lower erythrocyte GSH-T, s compred to the horses t XC. This might pper to contrdict the bove sttement; however, it might lso led us to believe tht the higher level horses were more ble to hndle tht level of competition nd therefore did not need s much GSH mobilized. 4.1. Time Effects. We found tht GSH-T incresed t XC in both divisions but decresed below bseline fter 18 to 24 hr of recovery. One recent study did look t ntioidnt sttus in horses during moderte level jumping competition [10]. They found tht nonprotein sulfhydryl groups (this group included glutthione) did not increse until 24 h fter competition, but did theorize tht the response ws due to compenstory rection to the increse in pro-oidnts produced during eercise. Another study observed decline in GSH (both totl nd reduced) within 15 min fter stepwise field eercise test of 7000 m in wrmblood eventing horses [11]. The uthors theorize the reson why the GSH- T in the current study did not follow the sme pttern s in previous studies could be due to the dded stress of competition nd jumping efforts during competition in combintion with glloping t pproimtely 9.2 to 9.4 m/s. The dded stress cretes higher demnd for the mobiliztion of GSH, hereby cusing the increse fter crosscountry followed by compenstory decline during the recovery period of bout 24 hrs. A study involving endurnce horses during competitive rce designed for reserch purposes evluted ntioidnt sttus with supplementtion of vitmin E lone or in combintion with vitmin C [7]. The 27% increse in RBC GP observed in the lst two stges of the 80 km rce in both tretment groups likely reflected response to utilize reduced glutthione during the rdicl scvenging process (reduced glutthione dontes n electron to reduce wide vriety of hydroperoides using GP s ctlyst). It lso reflected the consumption of pro-oidnts generted during eercise. This sme trend ws found in the current study with GP in horses competing in both divisions incresing bout 18% over bseline. A similr increse ws found with GSH- T in both studies, with horses in our study incresing t XC ( = 60%; = 27%) nd with endurnce horses in the previous study peking fter the first 21 km nd then slowly decresing to below bseline by the recovery smple [7]. In generl, blood nd plsm GSH-T reflects recent fluctutions in these concentrtions in muscle cells [12, 13]. The typicl pttern observed is n increse in concentrtion fter the onset of eercise tht continues for period of time during recovery then drops below bseline by bout 18 to 24 hr. This pttern ws observed here nd in previous studies of horses engged in intense nd endurnce eercise [2, 5, 14]. Previous studies on the eventing horse hve focused more on performnce mesures, including plsm LAC nd CORT, serum CK, AST, nd ion nd wter loss, rther thn on ntioidnt sttus [15 19]. This eercise chllenge did elicit mny other biochemicl chnges similr to tht observed in other studies on the event horse. These mesures re indictors of the rigor or intensity of the eercise being performed. They lso help the resercher determine the level of fitness of the horse. Linden et l. [15] found similr blood concentrtions s found in the current study of Hct, TP, nd LAC in horses before nd fter the cross-country

6 Oidtive Medicine nd Cellulr Longevity phse of low level event (similr to CCI1 ). The CORT levels found in the current study were bout 40 to 70% higher thn wht ws observed in the previous study [15]. The similr LAC concentrtions indicte the horses re probbly more fit nd ble to hndle the higher intensity of eercise thn the lower level of eercise in the previous study. However, the higher CORT concentrtions observed here my be due to the fct tht our study ws conducted on horses eposed to more rigorous cute eercise nd higher level of competition thn in the prior study. This higher concentrtion of CORT my be indictive of more physiologicl stress plced on the horses during this high level of competition. This cn lso be seen with recently published study using horses subjected to lower level of jumping competition, where the posteercise smple hd two to three times less LAC, CK, nd AST concentrtions thn the horses in the current study [10]. 4.2. Division Effects. Severlstudieshveinvestigtedhorses competing t different levels during three-dy events nd found vrying results in terms of hert rte, temperture, LAC, CK, nd AST responses [17 20]. One study investigted CCI1 nd horses competing in the long-formt version of three-dy eventing including rods, trcks nd steeplechse phses during the sme dy of competition s the cross-country jumping phse [19]. The LAC, CK, nd AST concentrtions were similr between divisions, which the uthors stted ws not epected due to the considerble differences in speeds nd distnces of the two divisions. The LAC results fter cross-country jumping were higher in the current study, despite the smples before eercise being similr in both studies. This could be due to the dditionl phses needed to complete before cross-country jumping. The current study did not find differences in LAC or AST concentrtions between the nd divisions, similr to wht ws found previously, probbly due to the degree of fitness nd thletic bility of the higher level horses. As for the CK in the previous study [19], there were no differences found between divisions, which does not gree with the results found in the current study; the horses hd higher levels of the enzyme fter crosscountry jumping (2.5 times higher CK thn PRE). Also AST ws 1.5 times higher numericlly with only trend for division difference (P = 0.07). However, the numericl vlues were similr between studies. No recovery smples were tken in the previous study [19], so no comprisons cn be mde; however, it is importnt to point out tht the AST concentrtions remined higher in the horses 18 to 24 hr fter cross-country jumping. Another study looking t horses competing either with or without rods, trcks nd steeplechse lso found no differences between these divisions for LAC, CK, AST, nd other electrolyte mesures [20]. Overll, these physiologicl mesures re mrkers of eercise intensity nd musculr effort during the eercise; however, they vry with horses physicl fitness, ge, gender, seson of the yer, nd so forth. The current study shows tht these subjects hve fllen within wide vrition of norml rnges for eercise t this level of intensity despite the differences between divisions. Division effects were lso found with GSH-T. One theory to eplin the higher concentrtion in the horses fter cross-country jumping is tht they were less conditioned or physiclly fit in order to hndle tht level of eercise stress compred to horses therefore required higher concentrtions of ntioidnts. However, the CK response ws lower for the horses, indicting tht course plced lower demnd on the muscle cells of those horses compred to horses. In study looking t endurnce rces of vrying intensity, there ws higher GSH-T in the horses competing in the more intense endurnce rce, which is the opposite of wht ws found in the current study [21]. However, the endurnce rces did tke plce during different yers nd hd drsticlly different environmentl conditions; the more intense rce hd hotter nd more humid mbient conditions could lso hve contributed to the higher GSH-T concentrtions. The horses competing in the more intense endurnce rce lso hd higher CK concentrtions, which reflect wht we observed in the present study. Another theory for the higher concentrtions of GSH-T in the horses s compred to the horses could lso be due to dietry effects. Diet plys n importnt role in ntioidnt sttus in humns s well s horses. As mentioned below in more detil, the horses were found to be receiving more ntioidnt supplements nd hd longer ccess to fresh green psture (number one source for ntioidnts in horses) thn the horses. Whether these mounts re enough to definitely increse ntioidnt sttus is unknown, but it is possibility given the current results. 4.3. Correltions. A positive correltion between serum AST nd plsm TOC, like tht observed in this study, supports the hypothesis tht free rdicls produced during eercise chnge membrne permebility of muscle cells [1]. This sme correltion hs been observed in previous studies looking t the effects of endurnce competition on horses [21, 22]; however, the current study only found TOC to be correlted (positively) with AST. Even though most of the correltions presented could be used to infer tht mesures of oidtive stress nd ntioidnt sttus re poor predictor of muscle enzyme lekge becuse it only eplins smll mount of the vrition, the uthors feel tht physiologiclly there ws enough relevnce to wrrnt the discussion. Plsm LAC correlted positively with GSH-T, GP, RET nd CORT, which cn be eplined by the fct tht ll these mesures peked t XC nd returned or ws lower t POST. As for CORT, we found positive correltion with RET, GSH-T, nd CK, which gin ll increse with eercise, but negtive correltion with TOC. This negtive correltion is little hrder to eplin physiologiclly. Due to structurl cellulr chnges during eercise like the increse in hemtocrit due to splenic contrction nd epulsion of erythrocytes in horses nd n increse in musculr mitochondri tht uptke dditionl vitmin E, ny chnge in vitmin E plsm concentrtions remins constnt becuse the production nddegrdtion re overshdowed by the redistribution. The current study did not hve n effect of eercise on plsm TOC, which is further evidence ginst the belief tht TOC is relesed during lipolysis ssocite with

Oidtive Medicine nd Cellulr Longevity 7 eercise. Other studies in humn subjects hve never been shown to be true nd leve vitmin E biovilbility fctors not welldefined [23]. When tking into ccount the direction of responses, it mkes more sense due to the fct tht TOC does not increse t the XC smple nd return to bseline or lower by POST s with the other mesures s mentioned bove. Correltions with GP nd AST in the current study were negtive, which is similr to one of the endurnce competitions compred in previous study [21]. However, the previous study in endurnce horses found positive correltion with second endurnce competition compred. These differences in the previous study were thought to be due to the difference in mbient conditions, terrin of the competition nd level of difficulty. The present study showed correltion similr to tht of the more intense endurnce competition in the previous study [21]. Thtendurnce rce ws lso n erly seson competition, with more horses tht my not hve been fully conditioned for the eercise required by the rce. This could hve been similr to the competition in the present study, which took plce in the spring. Eplntion of ech correltion is difficult due to the trnsient nture of ech mrker nd the mount of vribility in ech though worth reporting to shre the comprtive spect of the results. 4.4. Diet Effects. Becuse feeding mngement of horses could not be controlled prior to competition, the effects of diet on ntioidnt sttus could not be evluted. However, nutritionl survey ws conducted with the results summrized nd reported elsewhere [8]. As mentioned erlier, the two most notble findings tht could hve possibly influenced ntioidnt sttus of horses were the differences in hours of psture turnout prior to rriving t the competition venue nd ntioidnt supplement use between nd horses. In the current study, horses were turned out for more hours ech dy prior to the competition compred to horses (12.3 ± 1.3 versus 9.1 ± 1.3 hr, resp.). Since psture is good source of bet-crotene, the precursor to vitmin A tht my be n eplntion for why the horses hd higher BC levels before nd fter cross-country jumping. Another notble finding ws tht horses in both divisions received n verge of 4 supplements per dy nd tht more horses received supplements specificlly formulted s ntioidnt supplements s compred to horses (42.1% versus 8.7%, resp.). Antioidnt supplements fed typiclly contined vitmins E, C, nd selenium, which, due to the ntioidnt interction nd recycling, could prtilly eplin the higher concentrtions GSH-T in the horses s compred to the horses. Despite the difference in ntioidnt supplements, we did not observe difference in the number of or horses fed multi-vitmin nd minerl supplements (21.0% versus 21.7%, resp.) or ll-in-one supplements (5.3% versus 8.7%, resp.), which often contin vitmin E, C, nd selenium s well. Given the horses good BCS, sound feeding mngement progrms, nd supplement dministrtion, it is likely tht mny of the horses were consuming ntioidnts in dequte mounts to combt the oidtive stress eperienced s result of the three-dy event. However, if specific recommendtions for ntioidnt supplementtion for three-dy event horses were to be developed, the ntioidnts fed to the horses should be investigted further. 5. Conclusions In conclusion, the cross-country jumping phse of threedy event is chllenge to the horse s ntioidnt systems, with the competitions being more rigorous eercise bout thn the. The increse or upregultion of mrkers of ntioidnt sttus is potentilly due to incresed scvenging of ROS creted by oidtive stress eperienced by horses during intense eercise. The ROS could lso cuse incresed muscle membrne permebility s observed by the increse in muscle enzyme concentrtions in the blood. Horses tht re more fit my be better ble to hndle the stress of three-dy competition, but the incresed intensity lso plces gret del of stress on the muscle cells more thn in lower levels of competition. It is lso likely tht levels of ntioidnts in the diet could enhnce the horse s bility to cope with the physicl demnds of the crosscountry jumping phse of three-dy event. The question now becomes how much oidtive stress is too much nd how much supplementtion is relly necessry to combt this stress or if by combting the stress we re disturbing the nturl dpttion mechnisms necessry to llow the body to cope with physicl nd psychologicl stresses. Acknowledgments The uthors thnk the Jersey Fresh event orgnizers, J. Cory nd L. Mthews, nd Technicl Delegte, R. Muller, for mking this study possible. They lso thnk the FEI hed veterinrin, D. Willimson, DVM., the competitors who llowed us to collect dt on their horses during the competition, nd the volunteers who helped with dt collection. Mny thnks re due to Lur Gldney, Progrm Associte, Rutgers University for her edits. References [1] J.M.McBridendW.J.Kremer, Freerdicls,eercise,nd ntioidnts, Journl of Strength nd Conditioning Reserch, vol. 13, no. 2, pp. 175 183, 1999. [2] E. Chirdi, L. Avellini, F. Ruec et l., Physicl eercise, oidtive stress nd muscle dmge in rcehorses, Comprtive Biochemistry nd Physiology B, vol. 119, no. 4, pp. 833 836, 1998. [3] A. White, M. Estrd, K. Wlker et l., Role of eercise nd scorbte on plsm ntioidnt cpcity in thoroughbred rce horses, Comprtive Biochemistry nd Physiology A, vol. 128, no. 1, pp. 99 104, 2001. [4] A. Frnkiewicz-Jóźko nd E. Szrsk, Anti-oidnt level to eercise in the blood of endurnce horses, Biology of Sport, vol. 17, no. 3, pp. 217 227, 2000.

8 Oidtive Medicine nd Cellulr Longevity [5] D. J. Mrlin, K. Fenn, N. Smith et l., Chnges in circultory ntioidnt sttus in horses during prolonged eercise, Journl of Nutrition, vol. 132, no. 6, pp. 1622S 1627S, 2002. [6] D. R. Henneke, G. D. Potter, J. L. Kreider, nd B. F. Yetes, Reltionship between condition score, physicl mesurements nd body ft percentge in mres, Equine Veterinry Journl, vol. 15, no. 4, pp. 371 372, 1983. [7] C. A. Willims, D. S. Kronfeld, T. M. Hess et l., Antioidnt supplementtion nd subsequent oidtive stress of horses during n 80-km endurnce rce, Journl of Animl Science, vol. 82, no. 2, pp. 588 594, 2004. [8] A. O. Burk nd C. A. Willims, Feeding mngement prctices nd supplement use in top level event horses, Comprtive Eercise Physiology, vol. 5, pp. 85 93, 2008. [9] C. A. Willims nd A. O. Burk, Nutrient intke during n elite level three-dy event competition is correlted to inflmmtory mrkers nd ntioidnt sttus, Equine Veterinry Journl, vol. 42, no. 38, pp. 116 122, 2010. [10] J. C. M. Sores, R. Znell, C. Bondn et l., Biochemicl nd ntioidnt chnges in plsm, serum, nd erythrocytes of horses before nd fter jumping competition, Journl of Equine Veterinry Science, vol. 31, no. 7, pp. 357 360, 2011. [11] B. de Moffrts,N.Kirschvink,E.vnErck,T.Art,J.Pincemil, nd P. Lekeu, Assessment of the oidnt-ntioidnt blood blnce in field eercise test in Stndrdbred nd eventing horses, Equine nd Comprtive Eercise Physiology, vol. 2, pp. 253 261, 2006. [12] T. D. Nokes, Effect of eercise on serum enzyme ctivities in humns, Sports Medicine, vol. 4, no. 4, pp. 245 267, 1987. [13] P. A. Hrris, Musculoskeletl disese, in Equine Internl Medicine, S. M. Reed nd W. M. Bylye, Eds., pp. 375 397, WB Sunders, Phildelphi, P, USA, 1998. [14] P. C. Mills, Effects of eercise intensity nd environmentl stress on indices of oidtive stress nd iron homeostsis during eercise in the horse, EuropenJournlofApplied Physiology nd Occuptionl Physiology, vol.74,no.1-2,pp. 60 66, 1996. [15] A. Linden, T. Art, H. Amory, A. M. Mssrt, C. Burvenich, nd P. Lekeu, Quntittive buffy cot nlysis relted to drenocorticl function in horses during three-dy event competition, Zentrlbltt fur Veterinrmedizin. Reihe A, vol. 38, no. 5, pp. 376 382, 1991. [16] G. L. Ecker nd M. I. Lindinger, Wter nd ion losses during the cross-country phse of eventing, Equine veterinry journl. Supplement, no. 20, pp. 111 119, 1995. [17] S. L. White, L. H. Willimson, P. L. Mykuth, S. P. Cole, F. M. Andrews, nd D. R. Geiser, Hert rte response nd plsm lctte concentrtions of horses competing in the cross-country phse of combined trining events, Equine Veterinry Journl. Supplement, no. 20, pp. 47 51, 1995. [18] S. L. White, L. H. Willimson, P. L. Mykuth, S. P. Cole, F. M. Andrews, nd D. R. Geiser, Hert rte response nd plsm lctte concentrtions of horses competing in the cross-country phse of combined trining events, Equine Veterinry Journl. Supplement, no. 18, pp. 463 467, 1995. [19] L. H. Willimson, F. M. Andrews, P. L. Mykuth, S. L. White, nd E. M. Green, Biochemicl chnges in three-dy-event horses t the beginning, middle nd end of Phse C nd fter Phse D, Equine veterinry journl. Supplement, no. 22, pp. 92 98, 1996. [20] J. K. Murry, J. M. Senior, nd E. R. Singer, A comprison of cross-country recovery rtes t CCI 2 with nd without steeplechse competitions, Equine veterinry journl. Supplement, no. 36, pp. 133 138, 2006. [21] C. A. Willims, D. S. Kronfeld, T. M. Hess et l., Comprison of oidtive stress nd ntioidnt sttus in endurnce horses in three 80 km rces, Equine nd Comprtive Eercise Physiology, vol. 2, pp. 153 157, 2005. [22] C. A. Willims, D. S. Kronfeld, T. M. Hess, K. E. Sker, nd P. A. Hrris, Lipoic cid nd vitmin E supplementtion to horses diminishes endurnce eercise induced oidtive stress, muscle enzyme lekge, nd poptosis, in The Elite Rce nd Endurnce Horse, A. Lindner, Ed., pp. 105 119, CESMAS, Oslo, Norwy, 2004. [23] M. G. Trber, Vitmin E, in Modern Nutrition in Helth nd Disese, M. Shils, J. Olson, M. Shike, nd A. C. Ross, Eds., pp. 347 362, Willims & Wilkins, Bltimore, Md, USA, 9th edition, 1999.

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