The

Veterinary Journal
The Veterinary Journal 174 (2007) 113–121
www.elsevier.com/locate/tvjl

Effects of exercise and oral antioxidant supplementation enriched
in (n  3) fatty acids on blood oxidant markers and
erythrocyte membrane fluidity in horses
B. De Moffarts a,1, K. Portier b,1, N. Kirschvink a,2, J. Coudert c, N. Fellmann c,
E. van Erck a, C. Letellier d, C. Motta e, J. Pincemail f, T. Art a, P. Lekeux a,*
a

Department for Functional Sciences B41, Faculty of Veterinary Medicine, University of Lie`ge, 4000 Lie`ge, Belgium
b
Equine Department, National Veterinary School of Lyon, France
c
Laboratory of Sport Physiology and Biology, Faculty of Medicine, University of Auvergne, France
d
CHU Hospital SUD, Laboratory of Biochemistry, Rennes, France
e
Faculty of Medicine, Laboratory of Biochemistry A, Rennes, France
f
Probiox S.A., CHU, University of Lie`ge, Belgium
Accepted 2 June 2006

Abstract
The aim of this study was to investigate in a placebo-controlled field study the effect of a (n  3)-vitamin supplementation on erythrocyte membrane fluidity (EMF), oxidant/antioxidant markers and plasmatic x3/x6 fatty acid ratio (FAR) in 12 eventing horses.
Venous blood was sampled at rest before (PRE) and after (POST) a three week treatment period with either the supplement (group
S, n = 6) or a placebo (group P, n = 6) as well as after 15 min (POST E15 0 ) and 24 h (POST E24h) after a standardised exercise test.
The following markers were analysed: EMF, plasma antioxidant capacity of water and lipid soluble components, ascorbic acid, uric acid
(UA), glutathione (reduced: GSH, oxidised: GSSG), vitamin E (Vit E), b-carotene, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, selenium, copper (Cu), zinc (Zn), oxidised proteins (Protox), lipid peroxides (Pool) and FAR.
EMF did not differ between group S and P after treatment, but GPx remained unchanged in group S whereas it decreased in group P
and plasma Cu/Zn ratio remained unchanged whereas it increased in group P. FAR were significantly increased in group S. Exercise
induced a significant decrease of EMF (POST vs. E24h) in both groups, but which was significantly lower at E15 0 in group S than in
group P. Exercise induced a significant increase of UA and ACW (POST vs. E15 0 ) and Protox (POST vs. E24h) in both groups. An exercise-related decrease in GSH and Pool (POST vs. E15 0 ) was found in group P, whereas Vit E and FAR (POST vs. E24h) significantly
decreased in both groups.
The study showed that exercise induced a decrease in EMF in horses associated with changes of blood oxidative balance. The (x-3)vitamin supplementation tested improved the oxidative balance poorly but delayed the exercise-induced decrease of EMF and increased
the FAR. 
2006 Elsevier Ltd. All rights reserved.
Keywords: Horses; Exercise; Antioxidant; Membrane fluidity; (x-3)PUFA

1. Introduction
*

Corresponding author. Tel.: +32 4 366 40 30; fax: +32 4 366 29 35.
E-mail address: Pierre.lekeux@ulg.ac.be (P. Lekeux).
1
B. de Moffarts and K. Portier contributed equally to this study.
2
Present address: Animal Physiology, Department for Veterinary
Medicine, University of Namur, Namur, Belgium.
1090-0233/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2006.06.001

Physical exercise is characterised by an increase in the
body’s oxygen consumption and is associated with a rise
in reactive oxygen species (ROS) generation (Cazzola
et al., 2003). The relationship between physical exercise
and oxidative tissue damage has been investigated in

. However.. copper (Cu). oxidised: GSSG).. 2000). 2004. similar age distribution within each group) were formed.b) as well as improved exercise variables (i. 1000 IU Vitamin E. the anti-inflammatory effects of (x-3) fatty acids seem to be optimal if an appropriate oxidant/antioxidant balance is maintained using antioxidants that prevent lipid peroxidation (D’Aquino et al. glutathione redox ratio: (GSSG)/(GSH + GSSG)..000 IU Vitamin A.. 2005). De Moffarts et al. (iii) plasma x3/x6 fatty acid ratio . 1996. 2004a. During a run-in period of 6 weeks. and a reduction in red blood cell membrane fluidity. proteins. 2002. The study was performed in healthy eventing horses. Marlin et al.1. antioxidant enzymes and vitamins. Therefore. Biochemical assessment of oxidative stress is usually performed using indirect markers such as antioxidant scavengers. 150 mg Zn and 2. blood was sampled at rest (POST treatment). The first blood sample was taken prior treatment when the horses were at rest and had not been exercised (PRE treatment). 2003). 2004) or a biosensor of oxidative stress (Benderitter et al. carbohydrates and (desoxy)ribonucleic acids (Clarkson and Thompson.. two homogenous groups of six horses (three geldings. Vitamin E. Study design After a 6 week run-in period. 2003. ROS alter the structural properties of erythrocyte cell membranes by changing the composition and distribution of their lipids. Materials and methods 2. 2002. zinc (Zn). 1996). lower heart rates were observed during exercise in a fish oil treated horse group) in horses (O’Connor et al. The effect of exercise on red blood cell fluidity has. In horses. The following marker analyses were performed: (i) assessment of EMF by determining the relaxation contraction time (Tc). Palozza et al.3 ± 3. / The Veterinary Journal 174 (2007) 113–121 numerous studies in men and animals (Sen and Packer. 2002. leading to exercise intolerance and poor performance in different animal species (Sen and Packer. 2000). Exercise has been shown to induce cellular and tissue damage by oxidation of cellular components. The mechanisms of action have yet to be elucidated... Horses Twelve clinically healthy and regularly trained eventing horses (six mares and six geldings. One group received over 3 weeks an oral x3/x6-vitamin supplement (S). three mares. selenium (Se).e. Accordingly.. oxidant/antioxidant markers and plasmatic x3/x6 fatty acid ratio. 2. cancer and autoimmune disease (Calder. to the best of the authors’ knowledge.. Recent researches has focused on the role of dietary fats. 1996. 2000)..114 B. 2000). (ii) assessment of oxidant/antioxidant markers: antioxidant capacity of water soluble components in plasma (ACW). de Moffarts et al. 1991. The horses were trained daily and were turned out to grass for 1 h per day. Regarding PUFA. 60 mg Cu. not yet been assessed in the equine species. 2000. 2005). but a role of (x-3) fatty acids in membranes’ lipid composition and thus in their function has been suggested (Ma et al. 15 min (E15 0 ) and 24 h (E24h) after a standardised exercise test (ET) performed on a race track. Cazzola et al. 2003). activity of glutathione peroxidase activity (GPx) and superoxide dismutase (SOD)... diabetes. 2002. 1996) in response to oxidants has been observed in humans (Borst et al. 2.25 mg Se per horse per day). 2004). b-carotene (b-car). Song et al.. whereas the other group received a placebo (P). providing 30. The aim of the present study was to investigate in a placebo-controlled field investigation the effect of a (x-3)-vitamin supplementation on EMF. 1996.. Several studies have suggested that (x-3) long chain poly-unsaturated fatty acids (PUFA) have beneficial effects on human health disorders. oxidised proteins (prot-ox). i. the quantification of ROS is difficult in practice because of their very short half-life and requires highly sophisticated techniques like spin trapping. 750 mg Vitamin C. such as coronary heart disease. ascorbic acid (AA). the potential anti-inflammatory properties of (x-3) fatty acid could be counterbalanced by their proinflammatory properties through their capacity to stimulate ROS production in neutrophils and macrophages (Costabile et al. 2004)... such as membrane lipids. Hall et al. all animals were stabled on straw and were fed twice daily with hay (4 kg/ day) and a commercial cereal blend (2–3 kg/day) enriched with vitamins and trace-elements (TWYDIL competition.2. a reduced ‘‘gliding capacity’’ of the phospholipid layers (Hollan. erythrocyte membrane fluidity (EMF) might be considered as an indirect marker (Keddad et al. Exercise-induced oxidative stress is believed to contribute to accelerated muscle fatigue and muscle fibre damage. Deaton et al. Cazzola et al.e. as well as to a decreased immune defence of the organism (Nieman. uric acid (UA). lipid peroxides (Pool). Following 3 weeks of x3/x6-vitamin or placebo treatment...5 years) were investigated in Spring 2004. Indeed. 2005). 1997). After the run-in period. some recent studies using orally administrated fish oil described in vitro and in vivo anti-inflammatory effects (O’Neill et al. the same feeding and training regimen was maintained for three further weeks but horses received additionally either an oral x3/x6-vitamin supplement or a placebo. which is inversely proportional to EMF (Hollan. which were investigated at rest and after exercise. glutathione (reduced: GSH. Hargreaves et al. numerous experimental and field studies have demonstrated that exercise-induced oxidative changes occur and support the rationale for using antioxidants in competition horses (Mills et al. antioxidant capacity of lipid soluble components in plasma (ACL). which play an important role in animal and human health by influencing physiopathological processes (Schmidt et al. mean ± standard deviation (SD): 10. 2004).

1000 m at 8. study of positioning and rotational movement of the probe. A final step of 1000 m was performed by each horse at its individual maximal velocity. 1000 m at 9. After incubation. At the end of each gallop step. respectively for Vit E and b-car.9 m/s. copper. b-car. Hb. Vit E. Supplement and placebo: composition and administration The x3/x6-vitamin supplement (100 mL/horse/day) provided 50 mL of fish oil extract (guaranteed composition: 40% of C20. 2. 2004. For determination of Protox and Pool.B.7. Glutathione.2. 2.1.5. Zn. xanthane gum. Belgium). 900g) and plasma was immediately frozen on dry ice and kept at 80 C. The ET was adapted to the eventing horses and comprised a warm-up walk (5 min) and trot (3000 m at low velocity) followed by three steps of gallop at determined velocity and length (1000 m at 6.7. C20.. Oxidant antioxidant markers Total antioxidant capacities. ESR experiments were performed with a Bruker ECJ 106 spectrometer equipped with a resonance cavity TMH 269 using similar protocol than Keddad et al. Plasma concentrations of Vit E and b-car were determined by high pressure liquid chromatography (HPLC) on reversed phase columns (C18 120. A groom systematically verified the ingestion of supplement or placebo. 900g) and the washing of red blood cells. C20. Plasma concentration of UA was analysed spectrophotometrically using an automatic analyser (Roche Hitachi). Boehringer Mannheim).7 m/s).5. (1996).6. C18.1.2. C18. 2. SOD. UA. Both treatments were distributed once daily with oats for three consecutive weeks. .. 2. 2. / The Veterinary Journal 174 (2007) 113–121 determination (based on the analysis of plasma C18. Cu. AA (AA). AA. ACL.5. Measurement and control of speed During the different exercise steps of horses. as well as technicians performing blood sample analyses. Blood sample collection and processing 2. 5000 mg of D-a-tocopherol acetate and 5000 mg of copper orotate. EDTA tubes were spun (15 min. Heart rate monitoring Heart rate (HR) was determined over 20 s at the end of each exercise step using a telemetric electro-cardiogram recorder (Life Scope 8. total protein) were processed and analysed according to previously detailed procedures (de Moffarts et al. Selenium. packed cell volume. GSH. Field exercise test A standardised ET with a final step at maximal velocity was performed on the race track of Ghlin (Mons. C22.3x6. Blood marker analysis 2.3x6. cholesterol. Whole blood was analysed spectrophotometrically using a RANSOD and RANSEL kit. 115 puncture for analysis of plasma lactate (LA) (Accusport. Erythrocyte membrane fluidity EMF was determined by electron spin resonance (ESR) in order to determine the Tc. Plasma concentrations of AA were spectrophotometrically determined by using the reduction of 2.5x3. Whole blood glutathione (GSH and GSSG) was determined spectrophotometrically using the Biooxytech kit GSH/GSSG 412 Assay (Oxis).5. Superoxide dismutase (SOD) and glutathione peroxidase (GPx). 2005).5x3 [eicosapentaenoic acid: EPA] and 20% of C22. Venous blood lactate determination The horses were shortly stopped after each gallop step and venous blood (1 mL) was sampled by jugular vene- Oxidant/antioxidant marker samples (ACW. 100 · 45) with an isocratic elution (methanol/ water 98:2) and ultraviolet (UV) detection at 280 and 450 nm. Plasma concentrations of traceelements were determined by coupled mass spectrometry (Agilent 7500a ICP-MS). arachnid oil. Uric acid.6x3). Both treatments were well tolerated by the horses and no adverse reactions were reported. tocopherol or copper orotate.3x3. After centrifugation of the heparin tube (5 min. C20.3. the horses were shortly stopped and venous blood was sampled for lactate determination. Supplement and placebo were provided by PAVESCO AG (Switzerland). Riders and trainer were blinded to the treatment allocation. zinc. 2. A cool-down walk of 5 min finished the ET.4x6.6x3 [docosahexanoic acid: DHA]). the rider had to reach reference points placed along the racetrack within a very tight time window.6dichloro-phenol-indophenol (Merck n 1. allowed to determine Tc (Keith et al. De Moffarts et al.7. a radical marked probe (16 nitroxide stearic acid [C18]) was added to the erythrocyte preparation.3. a-Tocopherol and b-carotene. Plasma ACW and ACL were determined by photochemiluminescence detection using the Photochem assay (Photochem Analytic). C20. Exercise monitoring 2.4. GPx.2x6. 1973).03028). sucran) but did not provide EPA nor DHA. Se.5x3 and C22. The placebo consisted of 100 mL of the supplement’s matrix (water. respectively (Randox Laboratories).2x6. Each horse was further equipped with the Equipilot GPS (Fidelak) system. 2. Nihon Kohden) that was placed along the race track. Fatty acid and EMF determination were performed on blood sampled in heparin tubes that were kept at 4 C until analysis (within 36 h of collection).3 m/s. which allowed a posteriori and continuous calculation of mean speed during each race step.

Pool. their concentrations were adjusted for variation in PCV.08 ± 0. placebo at rest At PRE treatment. E24h) concerning both groups. 3.41 ± 0. 0. 3. horses were treated during 3 weeks with an oral placebo (h. 0. Activity of GPx remained unchanged in group S whereas it decreased in group P (Table 1). .6x3 concentrations and x3/x6 fatty acid ratio were significantly increased in group S (Fig.1.35 ± 0.36 and POST: 2.05: (w) significantly different from POST and (d) significantly different from placebo at the same time.038 and POST: 1. 1.. Standardisation of blood markers’ concentration. 1. Se (PRE: 187 ± 10 vs. GSSG. 1.57 ± 0.24 vs.7.27 vs. E15 0 .12 vs. GSH. No supplement-related differences were detected for AA.56 ± 0. by gasliquid chromatography (Chrompack CP 9001) coupled with flame ionisation detectors. Data are shown as means ± SEM. (1996). Supplement vs. No significant differences between groups were found for Vit E (PRE: Placebo vs.33 and POST: 0.3. relative changes between PRE and POST (delta) were analysed by analysis of variance (ANOVA). Statistical analysis 5 Data are shown as means with their standard error of mean (SEM).028 vs.29 ± 0. 2a–c).99 ± 0. UA. whereas it increased in group P (1.94 ± 0.118 vs. POST. In order to test the treatment effect at rest.) was determined in jugular venous blood of 12 healthy trained event horses at rest before (PRE) and after a supplement period (POST) as well as 15 min (E15 0 ) and 24 h (E24h) after a exercise test.6 vs. De Moffarts et al. 2. As GSH and GSSG were determined in whole blood.97 ± 0. P < 0.56 ± 0. n = 6) or an antioxidant supplement enriched in (n  3) fatty acids (r.46 ± 0. after lipid extraction and preparation of methyl esters. The technique was adapted from Keddad et al. the following result description refers to statistical differences detected for either group (S or P) effects or time-exercise-related effects (PRE. (2002).05).44 vs. Markers determined at PRE.18 vs.9 ± 0.56 ± 0. Cu (PRE: 1.2 ± 0.).05 vs.05. with P < 0. 3. 194 ± 11 lg L1). Exercisetime effect is significant as well as interaction between exercise and supplement effect. At POST treatment. Correlation analyses were performed separately at T0 and at T1 by linear regression between x3/x6 ratios and oxidant markers and between Tc and oxidant markers. 209 ± 12. GPx and SOD by expressing their activities per gram of haemoglobin (Hb) and Protox by expressing per gram of protein.3. supplement and timesupplement interaction.046 and POST: 0. Results 3.49 mg/g chol).053 mg L1) and Zn (PRE: 0. n = 6). A significant correlation was observed between Tc and SOD. Tc did * 4 • 3 0 PRE POST E15’ E24h Fig.s. Protox.77 ± 0. / The Veterinary Journal 174 (2007) 113–121 Pool. Fatty acids Plasma fatty acid composition was determined. Plasma concentration of oxidised proteins was analysed spectrophotometrically allowing determination of carbonyl structures according to the method described by Reznick and Packer (1994). Indeed. Xobs is the measured marker concentration at different time points of sampling. Withingroup and between-group related differences at each time point are indicated in Table 1 by superscripts. the correlation indicated * Tc (ns) 6 * 2. POST.47 ± 0. n. plasma C20.116 B.71 ± 0.7 ± 0. Plasma concentration of lipid peroxides was analysed spectrophotometrically using OxyStat Kit (Biomedica) according to the method described by Hildebrandt et al. Adjustment was performed using the following equation (Hinchcliff et al.13. Concentration of hydrophilic plasma markers (AA and UA) and trace-elements was standardised by total protein concentration whereas lipophilic plasma markers (Vit E and b-car) were standardised by cholesterol.64 ± 0. 2.8 and POST: 179 ± 8. b-car (PRE: 0. Regarding oxidant/antioxidant markers shown in Table 1. E15 0 and E24h were analysed by a mixed linear model for repeated measures (SAS) in order to assess effects of sampling time. 0. with P < 0. the plasma Cu/Zn ratio remained unchanged (1.05 ± 0. 2000): X adj ¼ X obs þ ðX obs  ½ðM R  M obs Þ=M R Þ where Xadj is the adjusted marker concentration. 1). ACL.s. The relaxation–contraction time (Tc) (n. 0.046 mg L1). Differences were considered to be significant when P < 0. Vit E was standardised by calculating the Vit E/cholesterol ratio. GRR. At POST treatment. In group S. Supplement: 2. Protox and SOD (Table 1).05. 0.33 lg dL1). at PRE treatment. not differ between groups (Fig.5x3 and C22.41 vs.8. MR is the concentration of the standardising element determined at rest and Mobs is the concentration of the standardising element determined at the time point of sampling.056 vs. no significant differences between group S and P were found.

s.   Indicates that variation between PRE and POST (delta) value was significantly different between groups. * n.9 ± 3.7ad 21 ± 3.73 ± 0. but not in the supplemented group. * n.33ac 5. a positive and significant relation between the both markers (r = 0. E24h) in both groups.74 and P = 0.2 ± 23.s.9 ± 4.41 mg/g chol).s. At POST treatment.4 ± 0.s.01) (Fig.s.49ac 83 ± 21ac 45 ± 10ac 0. De Moffarts et al.s.036 ± 0.1ac 108 ± 12.9ac 9 ± 3.7 ± 8.056 ± 0.3 ± 0.7ac 9 ± 2.12ac 84 ± 19ac 58.7ac 67. Protox: oxidised protein.3 ± 1.086. 3. n.9ac 3.7 ± 1.8 ± 17ac 0.04 ± 0.53 ± 0.58 ± 1.05) if the second superscript is different (c.0ac 6. GSSG: oxidised glutathione. HR and lactate at each step allowed the determination of the speed at which plasma lactate of 4 mmol L1 (VLA4). n. E24h: 2. horses were treated over 3 weeks with an oral placebo (P. In group S.7 ± 2. An exercise-related decrease of GSH (POST vs.26ac 4.s.5 ± 2ac 9 ± 2. whereas no more significant regression was found in group S (r = 0. ACW: antioxidant capacity of water soluble components in plasma. P-values of group supplement or time effects are indicated in separate columns. 3. UA: uric acid.2ac 7.05) whereas it became non-significant in supplement-treated horses. E15 0 ) and Pool (POST vs.). E15 0 ) and Protox (POST vs. n = 6) Marker (unit) Part A ACW (gmol eq AA mL1) AA (lmol L1) UA (lg L1) GSH (lmol L1) GSSG (lmol L1) GRR (%) Part B ACL (gmol eq TROLOX mL1) Pool (lmol L1) Protox (lmol gprot1) SOD (IU gHb1) GPx (IU gHb1) Group Time 0 Group effect Exercise-time effect PRE (rest) POST (rest) E15 P S P S P S P S P S P S 50. n. ACW (POST vs.015).32ac 8.s.7 ± 2ac 61.42 ± 1.6ad 971 ± 106ae 997 ± 72ad 95 ± 16.B. Physiological variables during exercise test The determination of velocity. whereas only group P showed a significant increase at E15 0 (Fig.2 ± 3.4 ± 10ac 0.029).77ac 5. S: Post vs. ACL: antioxidant capacity of lipid soluble components in plasma.6 ± 2. placebo after exercise Exercise induced a significant increase of Tc in both groups at E24h.s.2 ± 19ac 32.9ad 24.8 ± 6.038 ± 0. SOD: superoxide dismutase and GPx: glutathione peroxidase.49vs.5x3.3 ± 12. the analysis was made separately within groups. At PRE. n. n. the linear regression indicated a positive and significant correlation between the x3/x6 ratio and Pool (r = 0.39 mg/g chol.8ac 1177 ± 71ad 1030 ± 69ad 108 ± 19.0ac 76 ± 18.1ac 9 ± 3.7ac 24. 3.18ac 66 ± 16ad 43 ± 11ac 0. Markers known to be unaffected in short term by a single exercise (Se.71ac 6.003ad 0. E24h) in the placebo group.7ad 86. GPx significantly increased (POST vs.87ac 15.5ad 16.7ac 63.005ac 1284 ± 101ac 1399 ± 89ac 259 ± 8ac 262 ± 16ac n.5ac 88 ± 25.72 ± 0.9ac 21.5ac 20. 2a– c).: non-significant.6 ± 32. whereas values for group S tended to decrease in a non-significant manner. C20.6ac 1113 ± 35ad 1137 ± 73ad 86 ± 19.71 ± 0.004ad 1377 ± 122ac 1289 ± 64ac 224 ± 13ad 253 ± 11ac  2.41 vs.2. Zn.s.3ac 9. At POST.s.002ac 0. b-carotene) were not assessed. 1). n. GSH: reduced glutathione. P S P S P S P S P S 3. Exercise induced a significant increase of UA (POST vs.68 and P < 0.86 and P = 0.64ac 54.71 ± 0. Pool: lipid peroxide.7 ± 7. 3).052 ± 0.45 ± 0. Differences between the placebo group and the supplement group at each sampling point are significant (P < 0. E24h.6x3 and x3/x6 ratio significantly decreased after exercise. E24h: 3.51 ± 1. E15 0 and E24h are shown in Table 1. E15 0 ) was observed in group P.051 ± 0.s.8 ± 6.s. whereas only the x3/ x6 ratio decreased in group P (POST vs.8bc 9 ± 3.69 ± 1.05) if the first superscript is different (a or b for with-in column comparisons).64 ± 1.s. A significant exercise-related decrease of Vit E occurred in both groups (P: Post vs. n. Mean value (±SEM) of blood markers assessed at rest (POST value). The regression remained positive and significant in placebo-treated horses (r = 0.6ac 67. Fig.003ac 0.36 ± 2.4 ± 2.3bc 9 ± 3. Cu. Linear regression remained positive and significant in placebo-treated horses (r = 0.17ac 61. 2.1ac 45.35ac 110 ± 35. C22. * E24h Data are shown as means ± SEM. n.3. n = 6) or an antioxidant supplement enriched in (x-3) fatty acids (S.8 ± 0.4ac 7.02ac n. n. * * n.29ac 6.99 ± 0.s.9ac 48 ± 23.049 ± 0.003ac 1360 ± 128ac 1275 ± 68ac 242 ± 13ac 244 ± 9.s.5 ± 13.s.5 ± 2.7 ± 0. For general comparison. SOD) was performed separately within each group.06ac 6. the same analysis (Tc vs.004ad 0. * n. a HR of 180 beats/ . whereas differences between sampling times are significant (P < 0.6ac 17. / The Veterinary Journal 174 (2007) 113–121 117 Table 1 Markers determined in jugular venous blood of 12 healthy trained event horses at rest before (PRE) and after a supplement period (POST) as well as 15 min (E15 0 ) and 24 h (E24h) after an exercise test.043 ± 0.8ac 8.2ac 27. n.7 ± 8. GRR: glutathione redox ratio (GSSG/[GSH + GSSG]). Supplement vs. AA: ascorbic acid. * n.004ad – – – – 4. d or e for within-line comparisons).2ac 839 ± 50ac 801 ± 45ac 92.76 and P < 0. E15 0 ).

53 ± 0. placebo at rest 5 * c 0 PRE POST E15’ E24h Fig. Discussion This study shows that intense exercise induced significant modifications of EMF and oxidant/antioxidant markers in healthy eventing horses.05. 0 b PRE POST E15’ E24h ω3/ω6 (% ) †• 20 †• 15 *†• and between x3/x6 fatty acid ratio and Pool were found in plasma.44 vs.29 m/s).42 vs.36 m/ s).6 ω3 (%) 2 SOD (IU/gHb) y = 472x .9 ± 0. Equation.1. No significant differences between group P and S were found for the following variables: VLA4 (P: 8.6x3 fatty acid relative fraction (%) were determined in jugular venous blood of 12 healthy trained event horses at rest before (PRE) and after a supplement period (POST) as well as 15 min (E15 0 ) and 24 h (E24h) after a exercise test. horses were treated during 3 weeks with an oral placebo (h. S: 10. Long chain x-3 fatty acid (EPA and DHA) plasma concentration increased in group S. 3..75 ± 0.5 ± 0. The (x-3)-vitamin supplement had a significant effect on the x3/x6 plasma ratio. Although other studies investigating the effect of fish oil supplements in equines also used a supplementation period of similar duration (3–4 weeks) (O’Connor et al.9 ± 2. Ochoa et al. / The Veterinary Journal 174 (2007) 113–121 C22. the fact that the horses were trained might further have reduced the treatment effect. 2.. 2004a. Significant and positive correlations between EMF and erythrocyte SOD activity A 3 week period of (x-3)-vitamin supplementation did not significantly affect EMF in resting horses.5x3 fatty acid and C22. S: 8.1 ± 1. Supplement vs.) at the beginning of the protocol (PRE) in 12 healthy eventing horses. 2004.59 vs. which was higher in group S at POST treatment.504 R = 0. In other species.. 2000. To our knowledge this is the first report using EMF as functional marker of oxidative stress in exercising horses.63 vs. maximal lactate concentration after the last step (P: 13. Data are shown as means ± SEM: ( ) significantly different from PRE.. Besides species. Similarly.5 0 a P < 0.32 vs.86 ± 0.and supplement-related differences contributing to this lack of effect. there is no scientific evidence that this supplementation period allowed reaching an optimal effect. regular exercise has been reported to increase EMF in man (Cazzola et al. 2005) and human requirements that were adapted to the metabolic weight of a medium sized warm blood horse as used in this study.4 ± 0. Exercise-induced changes of EMF could partially be modulated by oral administration of a (x-3)-vitamin supplement. (w) significantly different from POST. the dose regimen of fish oil and Vit E chosen for this study was based on earlier field investigations (de Moffarts et al. De Moffarts et al. n = 6). 1988) has been described. Hall et al. Correlation between superoxide dismutase (SOD) (IU/gHb) determined in venous jugular blood and corresponding relaxation contraction time (Tc) (n. 2003).39 m/s). V200 (P: 10. C20. S: 13. (a–c) The plasmatic(x-3)/(x-6) fatty acid ratio (x3/x6) (%). S: 8. 2003) or x-3-fatty acid administration (Mueller and Talbert.5 *†• 1 0. . maximal velocity during the last step (P: 11. In fact. S: 11 ± 0.3 ± 0. an increase in EMF after antioxidant complementation (Drieu et al.. min (V180) or of 200 beats/min (V200) was reached.118 B.28 m/s).b). V180 (P: 8.5 ω3 (%) 800 0 3 †• 6 4 5 Tc (ns) †• 4 *†• 2 Fig. 10 4. 4.81 mmol L1). Given that the training regimen was unchanged throughout the runin period as well as during the supplementation period. 68 †• †• 1.01 2000 1600 1200 PRE POST E24h E15’ C20. a training-related increase of EMF might have occurred before the study was started. coefficient of correlation and P-value are indicated. n = 6) or an antioxidant supplement enriched in (n  3) fatty acids (r. with P < 0. and (d) significantly different from placebo at the same time..s.

As several oxidant/antioxidant markers were significantly affected at E15 0 and E24h in our study. Belgium. Among the blood oxidant/antioxidant markers. de Moffarts was supported by a grant from the ‘‘Fonds pour la Recherche en Industrie et en Agriculture’’ (FRIA). The activity of GPx remained unchanged in group S after treatment.. The main reason for this decrease was probably the fact that supplement and placebo administration were stopped the day of the exercise test and that plasma PUFA concentrations decreased within 24 h. but did not reduce their phagocytic capacity and tumor necrosis factor-a excretion (Hall et al.b). This study was partially supported by TWYDIL-PROBIOX and the ‘‘Hippodrome de Wallonie’’. 2005). which mainly contained arachnid oil. 4.. Despite the provision of Vit E in the (x-3)-vitamin supplement. Cazzola et al. In our study. The positive and significant correlation that was found between Tc and SOD further suggests that oxidative processes negatively affect EMF. Recent studies realised in horses indicate that PUFA might have anti-inflammatory properties.. It was observed that fatty acid (c20. This decrease was observed as soon as 15 min after the test and lasted for at least 24 h. but it should be remembered that the horses were tested under field conditions that are less standardised than during treadmill tests.B.6x3) and x3/x6 plasma ratio decreased significantly at E24h in both groups. remained unchanged.5x3 and c22. no treatment-related effects on exercise variables were found. no significant changes of plasma Vit E was observed in group S. / The Veterinary Journal 174 (2007) 113–121 These results indicate that feeding EPA and DHA-containing oil is sufficient to change plasma lipid content to high amounts of (x-3) fatty acids. Given that (x-3) supplementation increases tissue membranes’ susceptibility to peroxidation. Vit E consumption for membrane protection might have been increased (Song et al. a decrease also occurred in group P. placebo after exercise The horses were subjected to a standardised ET whose monitoring did not show any difference between groups. 2000). In comparison to the placebo group. Although no significant effect on plasma Cu and Zn was observed in group S. Acknowledgements We wish to thank the family Caulier for providing horses and collaboration during the study and M. a significant decrease was observed in both groups between E15 0 and E24h. 2002) and this observation is therefore interesting. Crosby et al. Leblond for preparing the manuscript. In group S. . possibly because exercise-related decrease or redistribution of PUFA occurs (O’Connor et al. the administration of a (x-3)-vitamin supplement did not affect the oxidant/ antioxidant balance but delayed the exercise-induced decrease of EMF and increased the plasma x3/x6 ratio. B. this decrease was mostly due to the fact that the treatment was administrated for the last time prior ET. The slight and non-significant decrease of the x3/x6 ratio in group P at POST was probably due to the supplement itself.. This finding might further explain why fish oil treated horses were found to have lower heart rates during a standardised treadmill exercise (O’Connor et al. whereas a significant decrease was noted in group P. An increase of the Cu/Zn ratio might have pro-inflammatory effects (Ojuawo and Keith.6x3 were likely to be decreased 24 h after the last administration. whereas an increase was observed in group P. Exercise significantly decreased EMF in group P and group S. the results obtained in group P cannot support or reject this hypothesis. 2002). DHA and/or Vit E. Indeed.. Lemaıˆtre et al.. which plays an important role for SOD activity. Regarding x3/x6 plasma ratio. 1990). 2004. which was associated with changes of the blood oxidant/antioxidant balance. Conclusion This study has shown that exercise induced a decrease in EMF in healthy eventing horses.. GPx activity and/or expression have been found to be increased after DHA supplementation. suggesting that the onset of red blood cell rigidity was 119 delayed. Interestingly. there was no significant change at E15 0 ... As plasma PUFA concentration is directly depending on PUFA ingestion. An exercise-induced alteration of rheological properties of red blood cells has been associated with oxidative processes in human athletes (Senturk et al. 1994. possibly because the implication of GPx in PUFA-peroxides (Venkatraman et al. The comparison of two feeding regimens enriched in fish or corn oil has shown that the fish oil regimen inhibited the LPS-induced increase of PGE2 in bronchoalveolar macrophages. (2003) consider EMF as a ‘‘functional’’ marker of oxidative stress.5x3 and c22. which might be affected negatively by a single and intense exercise but could be positively modulated by training. Blood viscosity and vascular resistance might be modulated by (x-3) fatty acids and could therefore account for lower heart rate during exercise.. the Cu/Zn ratio.. De Moffarts et al.2. flaxseed supplementation has been associated with a reduction of skin test reactivity in horses with Culicoides hypersensitivity (O’Neill et al. 2005) but did not seem to be affected by the (x-3)vitamin treatment. a relation between oxidative stress and decreased EMF was likely. 5. 2004). exercise-related changes were similar to those reported in earlier studies performed by our group (de Moffarts et al. 1996. Supplement vs. 2004). Although the EMF was decreased to a similar extent in group P at E24h. Increased PUFA intake has been associated with decreased Vit E concentration in rat tissue (Leibovitz et al. 1997). c20. 2004a. As the placebo treatment did not provide EPA.

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