The beneficial effect of astaxanthin in the horses has been suggested by in vitro studies³⁴ and in vivo observations³², but these data did not show the direct antioxidant effect of supplementation. Moreover, the recommended dosage of commercially available supplements is wide. Sato et al.³² administered to the horses supplement that contained 37.5 mg of astaxanthin, however, the manufacturer of AstaReal^® mentioned the studies with 75 mg and 30–100 mg doses³⁵ and other company recommended much higher dose: 937.5 mg twice daily³⁶. Astaxanthin has been proven to be safe and beneficial at very high doses: 50 mg/kg and even 200 mg/kg in rats^37,38. Beagle dogs and cats were safely dosed with up to 40 mg and 10 mg, respectively, which corresponds to 3–4 mg/kg³⁹. However, in the study lasting for 6 weeks the dose of 0.3 mg/kg was used⁴⁰. In the present study the dose of 250 mg per horse was chosen, which on one hand, seems to be high enough to clearly evaluate the effect of astaxanthin supplementation and on the other hand completely safe in the 3 months supplementation period according to currently available data.

The presented results have shown the effect of astaxanthin supplementation on general antioxidant status and the antioxidant defense measured as TAS, the activities of antioxidant enzymes and lipid peroxidation reflected by TBARS as well as the effect of the high-intensity training session on these measurements. The study design allowed also to investigate the exercise-related changes in the level of antioxidant status related to the beginning of race training in Arabian horses without supplementation. Previously, antioxidant status in Arabian horses has been described only in endurance effort (aerobic in nature), so this study is the first report on antioxidant status in racing Arabians (anaerobic effort).

Many methods have been proposed for measuring exercise-induced stress, including the markers of lipid peroxidation, reactive oxygen metabolites, antioxidant potential/status, the antioxidant enzyme activity, and the muscle enzyme activity being an indirect measurement of oxidative damage^5,9. The methods are limited by the study protocol, and the most common measurements are the activities of antioxidant enzymes and muscle enzymes in blood. However, the presence of reactive oxygen metabolites, expressed as reactive oxygen metabolites (dROMs) in blood, has also been shown as a result of exertion^5,10.

The indirect beneficial effect of astaxanthin supplementation in race horses has been reported as a reduction of muscle enzymes creatine phosphokinase (CK) and lactate dehydrogenase (LDH-5) leakage into circulation³². Continuous astaxanthin supplementation for 8 weeks led to the decreases in CK and LDH activities measured 4 h after an intense training session. In this study, we looked deeper in the mechanisms by measuring antioxidant parameters which give the insight into the level of oxidative stress that occurs as a direct result of the training as well as single bout of exercise. The activities of muscle enzymes were not measured, as marked CK increases occur at least 2 h after the training session^12,30,41. Instead we used the exercise test that allows to investigate the changes in post exercise LA concentrations and the decreases after 40-min. restitution. Compared to Sato et al.³² findings, in our study the antioxidant effect of astaxanthin supplementation was observed earlier, after 4 weeks of supplementation and then remained at the similar level till the end of the observation period.

Changes of hematological measurements and LA concentration confirmed that the training session examined in this study posed a significant effort for horses, required for the training progress, however, the effort did not exceed exercise capacity of the horses, as indicated by the decreases after 40-min. restitution. The measurements of oxidative stress changed after single training session and during whole training period and the effect of astaxanthin supplementation was significant.

Higher TAS values observed one month after astaxanthin supplementation accompanied by lower TBARS indicate the improvement in ROS removal, and diminished level of lipid peroxidation. The oxidant/antioxidant imbalance phenomenon has been described in race horses as occurring during a 3-month race period²⁷. The design of the present study covered this period and provided more details regarding the changes related to training and supplementation.

TAS or total antioxidant capacity (TAC) provide the information regarding overall antioxidant status, involving all the antioxidants in the body, including the ones not recognized yet or difficult to measure. It increased by 31.5% after one month of astaxanthin supplementation, together with the decrease in TBARS by 34.5%. The change was significant only in the 1st month, however, in both astaxanthin and control groups of horses a tendency to increase in TAS during 4 months of intensive training was seen with higher values in the supplemented group. Thus, long term changes related to the race training were proved, however, such changes were not detected in the horses that began moderate training⁴². It is expectable that supplementation with antioxidants should increase TAS and it was confirmed in our study. However, it has also been reported that 2 weeks of supplementation with vitamin E alone or in combination with coenzyme Q₁₀ did not affect either TAS at rest or after exercise in leisure horses²⁹.

Major antioxidant defense and so that antioxidant status is enzymatic. Antioxidant enzymes orchestrate in removing free radicals and in turn prevent lipid peroxidation. GR and GPx act as a system for reduction and peroxidation of glutathione disulfide, which allows to utilize hydrogen peroxide (H₂O₂), and other organic hydroperoxides with the electron from glutathione. SOD acts synergistically with GPx by catalyzing the dismutation of the superoxide radical (O⁻ ₂) into molecular oxygen (O₂) and H₂O₂, then converted by CAT into oxygen and water.

Results presented in this study show that GR activity significantly decreased by 45.4% one month after astaxanthin supplementation and remained lower than in control group by the end of the observation. GPx, SOD, and CAT activities also tended to be lower in astaxanthin-supplemented horses, but the differences were not significant. Studies report various patterns of the changes in antioxidant activities of enzymes in response to antioxidant supplementation, but the increases were interpreted as beneficial²⁷. However, astaxanthin’s mechanism of action is more complex than supplements tested in equine studies. Astaxanthin is a ROS scavenger acting by both donating electrons and by bounding free radicals and so that form a non-reactive product. Additionally, due to a unique chemical structure with a series of conjugated bounds in non-polar region, the molecule can remove free radicals by transporting them along its own carbon chain outside the cell where other antioxidant can neutralize them⁴³. It seems that astaxanthin supplementation posed an additional background for removing free radicals and increasing the redox capacity, leading to higher TAS values. Training itself does not seem to affect the activities of antioxidant enzymes, except SOD which increased in July (after 4 months of training) in control group but not the group supplemented with astaxanthin. The patterns of changes in the activities of antioxidant enzymes in response to training in horses vary across the literature. The decreases in SOD and GPx activities in Thoroughbred horses have been reported in 6th and 12th weeks of training but GPx increased in the horses supplemented with antioxidant vitamins and trace elements²⁷. In leisure horses subjected to training of moderate intensity SOD and GPx levels did not change²⁹ or varied, mostly decreased at the beginning of training and increased in 2nd (GPx) and 8th (SOD) weeks of exercise⁴². Human studies have shown that training, understood as repeating exposure to increased ROS levels leads to the upregulation of antioxidant defense including increasing SOD and GPx activities at rest^44,45. Similar phenomenon may be expected in horses, however, it is likely dependent on the time and intensity of training. It should be strongly indicated that the training loads recommended for horses are much lower than recommended for humans, which can also influence the type, level and time of changes in antioxidant enzymes’ activities.

The intensive training leads to ROS production which in turn may result in lipid peroxidation³. TBARS assay gives an insight into overall levels of oxidative stress due to the fact that it allows for the measurement of many products of lipid peroxidation. Thiobarbituric acid (TBA) used in the assay bounds malondialdehyde (MDA), the most commonly measured product of lipid peroxidation but also other molecules derived from this process⁴⁶. In the present study, TBARS was not affected by the training process, but significantly influenced by astaxanthin supplementation. It cannot be excluded that training related changes in lipid peroxidation occur earlier as it was shown in the 2nd week of moderate training in the horses that were not trained before⁴². The astaxanthin effect occurred after one month of administration when TBARS level was significantly lower by 34.5% in the horses supplemented with astaxanthin, then remained lower until the end of observation. Other studies regarding antioxidant supplementation have reported lower or no differences. After 30 days of vitamin E supplementation, MDA production did not change in Thoroughbred horses undergoing low-intensity exercise and interval training³⁰. Ergothioneine administration for 4 weeks in Arabian stallions resulted in the reduction of MDA concentrations by 5%³¹. Unfortunately, singularly assessed MDA production cannot be directly compared to TBARS measurements and even TBARS assay results should not be compared across different laboratories due to possible pre-analytical differences and assay conditions that do not allow even minor variations in the protocol⁴⁶.

Marked increase in TAS accompanied by marked decrease in TBARS (both by over 30%) proved the beneficial effect of astaxanthin on the antioxidant defense in the examined horses. Decrease in TBARS indicates also lower level of lipid peroxidation, which likely deals also with the oxidative damage of the muscles. Such high level of changes has not been reported before in the literature regarding dietary antioxidants for horses.

The training session produced significant increases in TAS immediately after exercise in all horses regardless of the supplementation, indicating that antioxidant defense was triggered by exercise. However, the values before and 40 min after exercise were higher in astaxanthin supplemented horses. Thus, the relative increase was lower in supplemented horses (1.6-fold vs. 2-fold in the control group). Similar TAS increases (1.6-fold and 1.7-fold) have been reported in Arabian horses after 1250- and 1400- meters races⁹. In contrast, moderate exercise in sedentary horses produced only a slight increase (by 7.5%) and only in the 1st days of training⁴². It confirms that TAS changes in response to exercise depend on the intensity of effort, but in the present study they were also influenced by astaxanthin supplementation.

Both GPx and GR activities tended to increase immediately after training session and decrease after 40-min. restitution. Still, only the increase in GPx activity in the control group was significant. The increases, visible although not significant, may show the compensation triggered by increased ROS production during an effort, while the depletion after restitution may be related to already reduced ROS amount. Lower activities of these enzymes in the supplemented group of horses seem to mirror lower pool of ROS that must be removed and a better antioxidant state, resulting from the presence of astaxanthin that poses an additional antioxidant.

The differences in SOD activities were slight and insignificant in the C group and the only significant difference was the decrease after 40-min. restitution in the horses supplemented with astaxanthin additionally suggesting its synergistic action with endogenous antioxidant system. Various effects of astaxanthin on the activities of antioxidant enzymes have been presented in the literature. The increases in SOD, CAT, and GPx activities have been reported in cell lines subjected to oxidative stress⁴⁷. However, in mice undergoing moderate intensity exercise the decreases have been observed⁴⁸. Similarly, various results have been reported regarding the activities of antioxidant enzymes in response to the effort.

Slight, insignificant changes in the activities of SOD and GPx have occurred after moderate exercise in leisure horses^29,42. Slight but significant decreases in GPx activities have been reported in Arabians after 1250- and 1400- meters races⁹. In contrast, significant post exercise increases dealing with all antioxidant enzymes (SOD, GPx, GR, CAT) activities have been reported in Arabians after 30 km training session and have been markedly amplified by ergothioneine supplementation³¹.

As it has been mentioned, a certain ROS concentration is beneficial for proper adaptation to the workload. Low concentrations of ROS increase Ca²⁺ release and force production, but further increase leading to high amounts of ROS result in the drop in force output⁴⁹. Moreover, adaptation occurs only when the stimulus (ROS concentrations) exceeds minimal threshold, overloading the system⁴⁵. Thus, the dose of antioxidant should be carefully chosen to allow proper adaptation resulting from the training sessions including the upregulation of the body’s antioxidant defense. The dose selected in the present study allowed for contribution to the antioxidant defense, thus it seems that the production of ROS necessary to induce adaptive changes was maintained.

TBARS values did not change significantly in response to the exercise in this study, however, the levels in the horses supplemented with astaxanthin were lower in all sampling points. There are reports showing increased or unchanged lipid peroxidation rate measured as MDA production in response to exercise. Pronounced increases have been found in endurance horses. In Arabian stallions after 30 km long training MDA production increased 1.8-fold, but at lower extent in the horses supplemented with ergothioneine³¹. An increase in TBARS has been reported also after 160 km endurance ride together with the decrease in TAS in the halfway⁶. Shorter exercise sessions generally produced less or no increases. Chiaradia et al.¹¹ have shown the slight but significant increase in MDA production after training session in Maremmana race stallions. Similarly, in untrained leisure horses subjected to moderate exercise MDA levels increased by 47%, but remained unchanged when vitamin E was supplemented²⁹. Other studies regarding single, short time training sessions have reported no changes in MDA concentrations after exercise. Such results have been obtained in Standardbreds conditioned for 8 weeks before the examined training session¹² and in Arabians after 1250- and 1400- meters races, regardless of the distance⁹.

The results presented in this study confirmed the beneficial effects of astaxanthin supplementation on the antioxidant status of Arabian horses that begin their race training. Astaxanthin seems to counterbalance the training-related oxidative stress partially. The selected dosing seems proper to shift the redox status towards more reducing environment, which is undoubtedly beneficial for the health and welfare of a horse. At the same time, the effects of training sessions which trigger ROS production and, in this manner, contribute to training progress are maintained.