Carey A. Williams, PhD
Ever tried looking up 'oxidative stress' on the Internet? Google brings up just over 2.5 million results; Google Scholar brings up about 716,000. When adding 'horse' to this search you still get about 470,000 results from Google and just under 14,000 from Google Scholar. Try 'antioxidant' and you get over 13 million. So either way you look at it, oxidative stress and antioxidants are a hot topic. Several reviews have recently been published on the topic that detail oxidative stress and antioxidant supplementation specifically in horses1,2. My laboratory has been focusing on antioxidants and oxidative stress in many aspects of the exercising horse for about eight years. Even within these studies alone there are conflicting results. In the following few pages and in the presentation I will try to highlight studies from my laboratory along with other pertinent studies of antioxidant supplementation in exercising horses.
Oxidative Stress Background
The welfare of competing sport horses has attracted public attention following deaths at the Olympics and other championships. Welfare may be assessed partially by objective indicators of stress (heart rate, and various blood metabolites). Evidence of oxidative stress in horses has been described in reports dealing with intense3,4 and endurance exercise5-7.
Oxidation provides energy for maintenance of cellular integrity and functions. Most of the consumed oxygen forms carbon dioxide and water, however, 1 to 2% of the oxygen is not completely reduced and forms reactive oxygen species (ROS). When antioxidant systems are insufficient, oxidative processes may damage DNA, lipids, and contribute to degenerative changes, including aging and cancer. Lipids are protected directly by α-tocopherol (TOC) in the membranes and by other antioxidants, including ascorbic acid (ASC) in the cytisol or external spaces around cells.
Antioxidants are inter-related and may prevent oxidant damage in several ways: scavenging of ROS; decreasing the conversion of less reactive ROS to more reactive ROS; facilitating repair of damage caused by ROS; and providing an environment favorable for activity of other antioxidants8. Lipid peroxidation occurs in tissues with a high concentration of poly-unsaturated fatty acids, such as cell and organelle membranes, lipoproteins, adipose tissue, and brain. Vitamin E is the most efficient in preventing lipid oxidation in lipoproteins. Membrane concentration of TOC is approximately one TOC molecule to 1000 lipid molecules. The phytyl tail of the tocopherol molecule allows the positioning of the molecule within the membrane bilayer so that the active chroman ring lies close to the surface of the membrane.
Antioxidant Supplementation Trials
Vitamin E is the most commonly supplemented antioxidant in horses. In one study, vitamin E was supplemented above and below current recommendations and plasma thiobarbituric acid reactive substances (TBARS; an indicator of oxidative stress) increased with exercise, especially in horses with low plasma TOC9. Another study found that a single bout of submaximal exercise does not affect plasma TOC concentration, but horses conditioned for several weeks, may require higher levels of vitamin E supplementation than recommended10. It has been found in various species that vitamin C potentiates the effects of vitamin E by reducing the tocopheroxyl radicals and restoring its activity11. Under maintenance conditions horses have the ability to synthesize sufficient ascorbate, but the demand increases as stress on the body is increased.
Oxidative stress has been confirmed as a result of endurance exercise in the horse. One study looking at vitamin E and C interaction used 40 endurance horses competing in an 80-km race for the purpose of research12. Three weeks prior to the race the horses were divided into two groups; vitamin E alone (5,000 IU/d alpha-tocopheryl acetate) or vitamin E plus vitamin C (same vitamin E dose, plus 7 g ascorbic acid/d). New findings concerning LPO and WBC antioxidants demonstrate possible oxidative stress. Differences between present results and other studies relate to ASC correlations with muscle enzymes and improvements in antioxidant status with ASC supplementation. The 27% increase in RBC glutathione peroxidase (GPx) observed in the last two stages of this race in both treatment groups likely reflects a response to utilize reduced glutathione during the radical scavenging process (reduced glutathione donates an electron to reduce a wide variety of hydroperoxides using GPx as a catalyst). It also reflects the consumption of pro-oxidants generated during exercise. In contrast to the RBC changes, novel findings here were the changes in the WBC glutathione system. Fluctuations of WBC GPx during exercise and the sharp 41% increase during recovery may reflect replenishment of reduced glutathione, however, the reduced:oxidized ratio was not measured for technical reasons during this field study. Compared to RBC, the higher concentration of WBC GPx and lower WBC total glutathione (GSH-T) may affect phagocyte oxidative burst and other immune functions during prolonged exercise. Although moderate endurance training in athletes may enhance the immune system, some reports indicate that exhaustive endurance exercise might be detrimental13.
Plasma ASC concentrations were lower in the E group than the EC group at rest. This difference progressively diminished during the race as ASC increased in the E group but remained unchanged in the EC group. This could be due to an increased mobilization of intracellular ASC stores in the E group, whereas the EC group was able to maintain ASC levels using the exogenous source for its antioxidant capacity. These findings contrast with a previous result from our laboratory, where a decrease in plasma ASC during a highly competitive and difficult 80 km race was found14. Plasma ASC also decreased during a race and through the racing season in sled dogs15,16; however, this season decline was prevented by vitamin C supplementation, 1 g/Mcal ME15, compared to about 0.3 g/Mcal ME in the present study.
The plasma TOC range was wide in the present study (about 2 to 13 µg/mL). Vitamin E intakes of horses in this study were about 5-times the NRC17 recommended minimum when on the treatments. The previous plasma TOC ranges were similar to the present results, which suggest comparably high vitamin E intakes of endurance horses in Spain6 and England7,as in the United States14. A beneficial effect of high intakes of vitamin E on oxidative stress may partially account for the universal acceptance of this supplementation.
A study of polo ponies used similar E and EC groups18. Throughout the polo season plasma TOC and ASC were higher in the EC group than the E group in hard-working ponies, but not those in light work. These observations may reconcile the endurance findings where changes were observed in the highly competitive, mid-season race14, but not in this lightly competitive, early season race. In a survey taken after the race, riders ranked the exertion level of the endurance ride easier than most of the rides later in the competition season. Also, ambient temperature was cooler in this race than in the summer when the majority of endurance competitions are held.
Vitamin E intake was calculated in competitive endurance horses via a pre-ride survey detailing intake two weeks prior to the 80-km endurance race19. Pasture intake was estimated using 2.5% body weight eaten per day and subtracting amount of grain, hay, bran and/or other supplements obtained from the surveys. Horses were consuming 1150 to 4700 IU/d of vitamin E in their total diet during this time period. This level is 1.2 to 5-times higher than the recommended levels given by the NRC17; which, at this intake, averages 1000 IU/d. The horses with the lower vitamin E intake generally were the horses receiving mostly pasture and minimal grain to supplement their diet. The higher levels of E in the diets consisted of around 40 to 50% grain and included supplements with at least 1000 IU vitamin E. A negative correlation was found between the vitamin E intake and CK, and AST, and a positive correlation was found with intake and plasma TOC adjusted for albumin at all sample times. Enzyme activity in plasma is used as an indicator of muscle leakage during exercise. They can fluctuate for a number of reasons, including alteration of the membrane permeability, cell necrosis, impaired enzyme clearance, and increased enzyme synthesis. As apparent in the correlations found in the present study dietary intake of vitamin E is also a contributing factor in muscle enzyme plasma concentrations during exercise.
A negative correlation was found between finishing time and vitamin E intake for the 24 horses that finished the race. One hypothesis for this finding could be that the higher placed horses were working at a greater intensity and/or being trained harder, thus having more sweet feed or supplements in the diet. Their higher level of conditioning may also have allowed these horses to work harder with lower muscle enzyme activities. Careful consideration needs to be taken when reporting these results because of the possibility of confounding the data due to competitiveness of the rider. Many riders be training harder and adding more supplements to their horses' feed to ensure they have a competitive advantage. It is also important to note that this study failed to determine an optimal level of supplementation. Therefore feeding even higher amounts than given in the present study is not recommended. Previous research has shown possible detrimental effects in various species fed with high doses of vitamin E including a decreased feed and protein efficiency20, increased liver weights, decreased hematocrit and hemoglobin21, decreased prothrombin and blood coagulation time22, and an increased demand for vitamins D and K23.
However, other studies in my laboratory have investigated pharmaceutical levels of vitamin E on its impact of oxidative stress, muscle enzymes and antioxidant status. Horses supplemented with vitamin E at nearly 10-times the NRC17 recommended level did not experience lower oxidative stress compared to control horses24. Additionally, there was found to be lower plasma β-carotene (BC) levels observed in this group compared to control or a moderately supplemented group, which may indicate that vitamin E, has an inhibitory effect on BC metabolism. This study failed to show that supplementation above control levels is more beneficial to oxidative stress and antioxidant status in intensely exercising horses. However, this research has proven that supplementing with levels in 10-times excess may be detrimental to BC and should be avoided.
Arabians trained to run on an equine treadmill were supplemented with vitamin E (E), lipoic acid (LA), or nothing (CON) before they underwent an endurance exercise test (EET). The EET consisted of 3 exercise bouts totaling 55 km, with 20 min vet checks in-between to simulate an endurance race. These results showed that apoptosis (programmed cell death) occurs in WBC during exercise and it can be moderated by supplementation with vitamin E or LA. The E group had 50% lower and the LA group had 40% lower apoptosis compared to the CON group. The increase in antioxidant status in the E and LA groups aided the WBC in scavenging the ROS triggering the apoptosis in these cells.
Antioxidants are linked together in various ways; this explains the increase in antioxidant status with supplementation of E and LA. In the present study LA increased the GSH-T concentrations in whole blood compared to CON. The LA group also had increased levels of ASC and TOC in the plasma throughout the study. Both the E and LA groups had about 40% more GSH-T, 30% more TOC, and 15% more ASC than the CON group. This illustrates recycling and scavenging of antioxidant radicals using the exogenous sources of the vitamin E and LA.
Beta-carotene supplementation in horses has not been proven effective in reducing exercised-induced oxidative stress, or increasing antioxidant status in healthy horses25,26. Studies of BC alone or when in combination with other antioxidants has not increased the level of plasma BC in supplemented horses, however, when exercised did significantly decrease plasma CK and AST concentrations in supplemented horses after exhaustive exercise.
Other Exercise Studies
Older horses are another group that might require antioxidant supplementation, especially if in combination with exercise. In my laboratory we have found that evidence of a disequilibrium oxidant balance during exercise and aging showed varying results27. In old horses (22 ± 2 years), the amount of lipid peroxidation and blood antioxidant concentrations are similar to those found in mature but younger (12 ± 2 years) horses. Neither group had lipid peroxidation changes with either acute exercise or 8-weeks of training, but there was a higher concentration of GSH-T in the pre- vs. post-training tests in both age groups. The observation that more GSH-T was needed during the pre-training graded exercise test (GXT) for both old and younger groups of horses suggests that training helped the horses prime their systems for the intense post-training exercise tests. Our study also found that WBC apoptosis was significantly lower in the younger than in the older horses, signifying that age might have more of an impact on the immune system than on the oxidant/antioxidant system.
Elite 3-day event horses competing internationally at a CCI** or CCI*** were found to have no differences between divisions for cortisol, TOC, retinol, BC, AST, and GPx28. Total glutathione, however, had an effect of division, with horses in the CCI** showing higher concentrations than horses in the CCI***. Total glutathione also had a main effect of sample, with the peak immediately after the cross-country phase. Other measures including CK, AST, GPx, BC, retinol, cortisol and lactate also peaked immediately after the cross-country phase and were typically lower before the competition started compared to 24 h after the cross-country. Overall these results provided the first report of antioxidant status of horses competing in either a CCI** and CCI*** 3-day event28. Many other studies have looked at horses competing at different levels during 3-day events and found varying results in terms of heart rate, temperature, plasma lactate, CK and AST responses. These studies indicated that environmental factors at various events along with nutritional status could play an important role in the response of individual horses. Pre-event nutritional surveys were also undertaken in the previous study28 to determine the intake level of antioxidants and other nutrients that would affect the level of stress during competition. These correlations have not yet been determined but they may explain, at least in part, the possible differences or lack of differences between divisions.
Even though there have been many studies examining the levels of lipid peroxidation, antioxidant status and other related metabolites or markers in the horse during exercise, we still have a long way to go before we fully understand the large variation in results both with and without antioxidant supplementation.
On-going studies investigating the effects of superoxide dismutase (SOD) on oxidative stress and inflammation in exercising horses systemically and locally (synovial fluid) are currently being performed. Previous studies in rats and humans have shown beneficial results. Current research is seeking to determine if supplemental SOD has any effect on endogenous SOD activity and oxidative stress. Studies have tested a SOD derivative on exhaustively exercising rats and found that it provided effective protection against oxidative stress in the liver and kidney along with skeletal muscle in exercising rats29. Recently, in humans, the effects of an oral SOD supplement on preseason collegiate soccer players determined that performance (measure by VO2max, VLT, and time-to-exhaustion) improved by a greater magnitude in the group supplemented with SOD30. Another study by the same team tested the effects of a nutraceutical supplement mix containing SOD on college football players after an anaerobic exercise test. They found that the supplemented subjects had greater improvements in peak power and lowered muscle breakdown, measured with CK, along with lower levels of 8-isoprostane31. Both of these studies indicate that SOD can cross over into the bloodstream and elicit protective effects on an exercising individual.
Overall these recent exercise studies have shown that oxidative stress was observed during endurance, intense, and treadmill exercise. The extent of the oxidative stress and muscle enzyme leakage was dependent on the ambient temperature, conditioning level of the horse, and the intensity of work. Supplementing antioxidants like vitamin E, vitamin C, lipoic acid, and potentially superoxide dismutase is beneficial to horses by decreasing the oxidative stress and muscle enzyme leakage, and increasing antioxidant status. Thus, we can provide better health and welfare to our equine athletes by supplementing with antioxidants before they are asked to perform under intense conditions.
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