five thoroughbred horses ( two females and three geldings ) , aged 4 to 7 years old with 
 body weight of 493.8  26.6 ( mean  sd ) kg , belonging to the equine research institute , 
 japan racing association , were assigned to the experiment .
these horses had been routinely 
 grazed for 2 months and specially trained for treadmill exercise prior to this study .
hydrogen - rich water ( hw ) was made by special equipment ( high density hydrogen water , 
 melodian co. , tokyo , japan ) for the present study .
the hw consisted of filtrated water 
 ( ph=6.82 ) with a concentration of molecular hydrogen ( h2 ) higher than 1 ppm . 

fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum 
 bag ( 10 l in volume ) and tightly sealed in order to prevent hydrogen gas escape .
two 
 liters of hw were administered into the esophagus via a nasogastric catheter 30 min before 
 the treadmill exercise . for the placebo trial ,
the same volume of normal water without 
 hydrogen was administered by the same method .
horses 
 were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 
 and 13 m / s until they were exhausted .
the first treadmill exercise was performed 
 after placebo administration and the second treadmill exercise was performed after 
 hydrogen - rich water ( hw ) administration , after one - week interval .
two litters of 
 placebo water or hw were administered to the esophagus by a nasogastric catheter in 
 each horse at 30 min before the treadmill exercise .
blood samples ( serum ) were 
 collected 4 times : 30 min before the exercise , immediately before the exercise , 
 post - exercise , and at 30 min after the exercise .. treadmill exercise on a 6% incline was performed by each horse .
each horse was 
 exercised on the treadmill with stepwise increases of exercise intensity every 2 min , at 
 treadmill running speed of 1.6 , 3.6 , 7 , 10 , 12 , 13 and 14 m / sec , until the horses became 
 thoroughly exhausted .
by thoroughly exhausted , we mean that the horses were too exhausted 
 to maintain their position at the front of the treadmill with humane encouragement .
horses 
 were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 
 and 13 m / s until they were exhausted .
the first treadmill exercise was performed 
 after placebo administration and the second treadmill exercise was performed after 
 hydrogen - rich water ( hw ) administration , after one - week interval .
two litters of 
 placebo water or hw were administered to the esophagus by a nasogastric catheter in 
 each horse at 30 min before the treadmill exercise .
blood samples ( serum ) were 
 collected 4 times : 30 min before the exercise , immediately before the exercise , 
 post - exercise , and at 30 min after the exercise .
the first treadmill exercise was performed after the placebo administration , and the 
 second treadmill exercise was performed after the hw administration , after one week 
 interval . in order to measure serum d - roms and bap , venous blood samples of 10 ml
the blood samples were collected from the jugular vein immediately before the 
 administration of placebo water ( distilled water ) or hw , 30 min before the treadmill 
 exercise .
blood samples were also collected at pre - exercise , immediately before the onset 
 of treadmill exercise , at post - exercise , immediately after treadmill exercise , and at 30 
 min after the end of treadmill exercise .
in addition to the samples , blood was collected 
 at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine 
 their background levels of d - roms and bap .
the measurement of d - roms was performed using a colorimetric method of final derivatives , 
 i.e. , hydroperoxide produced by free radicals , in which hydroperoxide in the serum reacts 
 with n , n - diethyl - p - phenylenediamine to form [ a - nh2 ] 
 using a free radical analyzer ( free carpe diem , wismerll , tokyo , japan ) .
this d - roms test 
 was invented and developed by carratelli , m. and the validity of this method has been 
 demonstrated by comparisons with the results of the electron spin resonance ( esr ) method , 
 which serves as a direct measurement of unpaired electrons [ 2 , 39 , 40 ] .
bap was determined by color reaction of thiocyanate which reflects reduction potency from 
 fe to fe due to electrons ( e ) in the blood using 
 the same free radical analyzer .
the d - roms , bap and bap / d - roms values were statistically evaluated using two - way 
 repeated - measures analysis of variance ( two - way repeated - measures anova ) .
in addition , the 
 statistical significance of differences was tested by two - factor anova with only one 
 observation in each cell at pre - exercise , post - exercise and 30 min after exercise for each 
 of the placebo and hw groups , and the dunnet test was used to compare post - exercise and 30 
 min after exercise data with that at pre - exercise .
wilcoxon s signed - rank test was also 
 used to examine the data of the blood samples at 30 min before treadmill exercise , before 
 the placebo or hw administration , in order to examine if the basal conditions were the 
 same .
for all the data , differences were considered significant if the p value was less 
 than 0.05 .
five thoroughbred horses ( two females and three geldings ) , aged 4 to 7 years old with 
 body weight of 493.8  26.6 ( mean  sd ) kg , belonging to the equine research institute , 
 japan racing association , were assigned to the experiment .
these horses had been routinely 
 grazed for 2 months and specially trained for treadmill exercise prior to this study .
hydrogen - rich water ( hw ) was made by special equipment ( high density hydrogen water , 
 melodian co. , tokyo , japan ) for the present study .
the hw consisted of filtrated water 
 ( ph=6.82 ) with a concentration of molecular hydrogen ( h2 ) higher than 1 ppm . 

fresh hw that was made about 3 hr before the treadmill exercise was stored in an aluminum 
 bag ( 10 l in volume ) and tightly sealed in order to prevent hydrogen gas escape .
two 
 liters of hw were administered into the esophagus via a nasogastric catheter 30 min before 
 the treadmill exercise . for the placebo trial ,
the same volume of normal water without 
 hydrogen was administered by the same method .
horses 
 were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 
 and 13 m / s until they were exhausted .
the first treadmill exercise was performed 
 after placebo administration and the second treadmill exercise was performed after 
 hydrogen - rich water ( hw ) administration , after one - week interval .
two litters of 
 placebo water or hw were administered to the esophagus by a nasogastric catheter in 
 each horse at 30 min before the treadmill exercise .
blood samples ( serum ) were 
 collected 4 times : 30 min before the exercise , immediately before the exercise , 
 post - exercise , and at 30 min after the exercise .. treadmill exercise on a 6% incline was performed by each horse .
each horse was 
 exercised on the treadmill with stepwise increases of exercise intensity every 2 min , at 
 treadmill running speed of 1.6 , 3.6 , 7 , 10 , 12 , 13 and 14 m / sec , until the horses became 
 thoroughly exhausted .
by thoroughly exhausted , we mean that the horses were too exhausted 
 to maintain their position at the front of the treadmill with humane encouragement .
horses 
 were exercised with increment of load every 2 min at speeds of 1.6 , 3.6 , 7 , 10 , 12 
 and 13 m / s until they were exhausted .
the first treadmill exercise was performed 
 after placebo administration and the second treadmill exercise was performed after 
 hydrogen - rich water ( hw ) administration , after one - week interval .
two litters of 
 placebo water or hw were administered to the esophagus by a nasogastric catheter in 
 each horse at 30 min before the treadmill exercise .
blood samples ( serum ) were 
 collected 4 times : 30 min before the exercise , immediately before the exercise , 
 post - exercise , and at 30 min after the exercise .
the first treadmill exercise was performed after the placebo administration , and the 
 second treadmill exercise was performed after the hw administration , after one week 
 interval . in order to measure serum d - roms and bap , venous blood samples of 10 ml
the blood samples were collected from the jugular vein immediately before the 
 administration of placebo water ( distilled water ) or hw , 30 min before the treadmill 
 exercise .
blood samples were also collected at pre - exercise , immediately before the onset 
 of treadmill exercise , at post - exercise , immediately after treadmill exercise , and at 30 
 min after the end of treadmill exercise .
in addition to the samples , blood was collected 
 at 10:00 and 13:00 on a day without treadmill exercise from all five horses to determine 
 their background levels of d - roms and bap .
the measurement of d - roms was performed using a colorimetric method of final derivatives , 
 i.e. , hydroperoxide produced by free radicals , in which hydroperoxide in the serum reacts 
 with n , n - diethyl - p - phenylenediamine to form [ a - nh2 ] 
 using a free radical analyzer ( free carpe diem , wismerll , tokyo , japan ) .
this d - roms test 
 was invented and developed by carratelli , m. and the validity of this method has been 
 demonstrated by comparisons with the results of the electron spin resonance ( esr ) method , 
 which serves as a direct measurement of unpaired electrons [ 2 , 39 , 40 ] .
bap was determined by color reaction of thiocyanate which reflects reduction potency from 
 fe to fe due to electrons ( e ) in the blood using 
 the same free radical analyzer .
the d - roms , bap and bap / d - roms values were statistically evaluated using two - way 
 repeated - measures analysis of variance ( two - way repeated - measures anova ) .
in addition , the 
 statistical significance of differences was tested by two - factor anova with only one 
 observation in each cell at pre - exercise , post - exercise and 30 min after exercise for each 
 of the placebo and hw groups , and the dunnet test was used to compare post - exercise and 30 
 min after exercise data with that at pre - exercise .
wilcoxon s signed - rank test was also 
 used to examine the data of the blood samples at 30 min before treadmill exercise , before 
 the placebo or hw administration , in order to examine if the basal conditions were the 
 same .
for all the data , differences were considered significant if the p value was less 
 than 0.05 .
on the days of placebo and hw administration , the mean values of d - roms immediately before 
 administration of placebo or hw at 30 min preceding the treadmill exercise were 148.3    15.3 
 ( placebo ) and 152.8    9.4 ( hw ) , respectively .
likewise , the mean values of bap at 30 min 
 before treadmill exercise were 2,555.9    92.5 ( placebo ) and 2,774.1    32.7 ( hw ) , 
 respectively .
there were no significant d - roms or bap between the placebo and hw treatment 
 trials .
d - roms values of each horse at pre - exercise , post - exercise and 30 min after the treadmill 
 exercise are shown in fig . 2fig .
2.d - roms values at pre - exercise , post - exercise and 30 min after exercise of each horse 
 after administration of placebo and hw .. in all horses , d - roms tended to elevate at post - exercise , i.e. , immediately after 
 the treadmill exercise .
the average changes in d - roms induced by the treadmill exercise in 
 the placebo and hw trials are shown in fig . 3fig .
3.average changes of d - roms of all horses after administration of placebo and hw.30 min  : at 30 min after the treadmill exercise .
data are shown as means  se .. a significant difference in d - roms was found among the three measurement times of 
 pre - exercise , post - exercise and 30 min after exercise in the placebo 
 ( p<0.005 ) and hw ( p<0.005 ) trials .
the d - roms value significantly 
 increased at post - exercise , compared to pre - exercise , in the placebo ( p<0.005 ) and hw 
 ( p<0.001 ) trials .
no significant changes in d - roms were observed at 30 min after exercise 
 in both the placebo and hw trials .
the d - roms value at pre - exercise was lower for the hw 
 trial ( 148    14.3 u.carr ; mean    se ) than for the placebo trial ( 179    12.1 u.carr ; 
 mean    se ) , but the difference was not significant .
d - roms values at pre - exercise , post - exercise and 30 min after exercise of each horse 
 after administration of placebo and hw .
average changes of d - roms of all horses after administration of placebo and hw .
the bap values of each horse at pre - exercise , post - exercise and 30 min after the treadmill 
 exercise are shown in fig .
4.bap values at pre - exercise , post - exercise and 30 min after exercise of each horse 
 after administration of placebo and hw .. the bap value increased in all horses at post - exercise .
the average change in bap 
 values of the placebo and hw trials are shown in fig . 

5.average changes of bap of all horses after administration of placebo and hw.30 min  : at 30 min after the treadmill exercise .
data are shown as means  se .. there were significant differences among the three measurement times in the placebo 
 ( p<0.0001 ) and hw ( p<0.0001 ) trials ; the bap values at post - exercise increased to 150% 
 and 153% of the pre - exercise value in the placebo and hw trials , respectively .
significant 
 differences from pre - exercise were found at post - exercise ( p<0.0001 ) and 30 min after 
 exercise ( p<0.005 ) in the placebo trial , whereas a significant difference was present 
 only at post - exercise ( p<0.0001 ) in the hw trial .
bap values at pre - exercise , post - exercise and 30 min after exercise of each horse 
 after administration of placebo and hw .
the value of the bap / d - roms ratio is shown in fig . 

6.average changes of bap / d - roms of all horses after administration of placebo and 
 hw.30 min  : at 30 min after the treadmill exercise .
data are shown as means  se .. significant differences from pre - exercise were found in the placebo ( p<0.005 ) and 
 hw ( p<0.05 ) trials ; the differences were greatest at post - exercise in the placebo 
 ( p<0.001 ) and hw ( p<0.01 ) trials , and they were also significant at 30 min after the 
 exercise in the placebo ( p<0.001 ) and hw ( p<0.05 ) trials .
as for comparisons between 
 the placebo and hw trials on d - roms , bap and bap / d - roms ratio , a significant difference 
 ( p=0.04 ) was observed only for bap / d - roms ( two - way repeated - measures anova ) without 
 interaction between substance ( placebo or hw ) and measurement time points ( p=0.87 ) .
average changes of bap / d - roms of all horses after administration of placebo and 
 hw .
the excessive production of intramuscular or extra - muscular ros induced by intensive 
 exercise such as supra - maximum exercise might play an important role in enhancing 
 inflammation of the muscle .
however , concurrent action of protective factors , represented by 
 various antioxidative substances such as super oxide dismutase ( sod ) , catalase , peroxidase , 
 glutathione ( gsh ) , homocysteine , ascorbic acid ( vitamin c ) , -tocopherol ( vitamin e ) and 
 some minerals , might be enhanced or recruited against the oxidative stress .
therefore , the 
 balance of oxidative and antioxidative functions in tissues and blood are thought to be more 
 important than the production of oxidative substance alone . in the present study , 
 significant increases in d - roms and
bap were induced by the intensive treadmill exercise 
 ( figs . 3 and 
 5 ) , and were accompanied by elevation of the bap / d - roms ratio ( fig . 6 )
. there are several reports on the association of exercise with oxidative stress in equine . 

it has been suggested that biochemical parameters such as lipid hydroperoxides , which are 
 indicative of oxidative stress , are changed by exercise , and that the changes are 
 exacerbated during exercise at high temperature and humidity .
also , the exercise - induced increase in plasma lipid peroxide was 
 reduced by allopurinol - induced inhibition of xanthine oxidase , which resulted in the 
 formation of ros during exercise .
changes in 
 blood contents of malondialdehyde ( mda ) and gsh were evident in race horses ( ten 3 year - old 
 stallions ) subjected to physical exercise with a progressive strength of training , where the plasma mda and gsh significantly increased 
 after the ride and the increase in mda was still present at 18 hr after the exercise .
the present study demonstrated that the oxidative parameter ( d - roms ) and antioxidative 
 parameter ( bap ) in the blood were clearly and simultaneously elevated by intensive treadmill 
 exercise of thoroughbred horses .
the finding of increase in d - roms may be supportive of the 
 finding of increased lipid hydroperoxidase in horses after exercise [ 25 , 26 ] .
moreover , in the present 
 study the marked increase in bap observed at post - exercise and rapid return to the 
 pre - exercise level at 30 min after the exercise is worthy of note .
this evidence implies 
 that antagonistic action to strong oxidative stress is designed to onset in rapid 
 time - course and bap recovery to the pre - exercise level occurs within a short period after 
 the exercise .
this antioxidative function may be reinforced by exercise training of 
 thoroughbred horses because the antioxidative capacity has been suggested to be improved by 
 exercise training .
some evidence of the efficacy of dietary supplementation with antioxidants has previously 
 been presented .
the antioxidative influence of vitamin e and selenium supplementation in 
 3-year - old stallions has been reported .
the 
 antioxidant capacity , total antioxidant activities and thiobarbiturate reactive substances 
 in thoroughbred race horses were elevated after a race , and the intravenous administration 
 of ascorbate reduced the oxidative stress , although the creatine kinase activities were not 
 influenced by the administration . in recent years , possible therapeutic effects of hw , which contains molecular hydrogen 
 ( h2 ) , have been noticed in experimental studies with animals . in rats with 
 periodontitis , the intake of hw for 4 weeks lowered serum levels of ros and oxidised 
 low - density lipoprotein - cholesterol . in mice 
 subjected to physical restraint stress for 10 hr per day for 6 days each week ,
in addition , the inhalation of h2 gas protected the brain from 
 ischemia and reperfusion - induced damage in the rat .
brain slices derived from mice administered hydrogen - rich pure water for 33 
 days showed significantly less superoxide formation than the control . in a rat model of sepsis , the intraperitoneal administration of 
 hydrogen - rich saline inhibited the increase of oxidative responses , such as ros and 
 malondialdehyde , in the hippocampus in a dose - dependent manner . also , drinking hw significantly suppressed intimal hyperplasia of 
 the inferior vena cava in the rat .
it is of 
 interest to understand the biochemical mechanism of the antioxidative action of hw .
the 
 antioxidative action of molecular hydrogen ( h2 ) has been described in detail 
 .
h2 dissolved in culture medium of 
 pc12 cells significantly decreased the level of hydroxyl radicals ( oh ) without decreasing 
 the level of in super oxide anion radicals ( o2 ) or hydrogen peroxide 
 ( h2o2 ) derived from mitochondria .
the absorption of hw from the digestive organs and its time - course change in the body was 
 described in a rat study .
hydrogen in the blood 
 was detected at 3 min after the direct instillation of saturated hw into the stomach , and 
 the half - life of hydrogen in the muscle was estimated to be approximately 20 min after the 
 instillation .
therefore , it is assumed that the molecular hydrogen is rapidly absorbed by 
 the digestive organs and most of it can be metabolized within one hour in the living body . 
 in the present study ,
the d - roms value showed a tendency to decrease already at pre - exercise 
 after the intake of hw , compared to the placebo intake .
in addition , there were significant 
 increases in the bap / d - roms ration in the hw trial , compared with the placebo trial , at 
 pre - exercise , post - exercises and 30 min after exercise ( two - way repeated - measures anova ) , 
 while no significant difference was found in the same horses at 30 min before the treadmill 
 exercise .
this finding may indicate that the hw administered into the digestive tract of the 
 horse is rapidly distributed throughout the body after administration and acts to reduce a 
 part of free radicals in the blood . in addition , the earlier recovery of the bap / d - roms 
 ratio in the hw trial , compared to the placebo trial , may reflect lesser amounts of free 
 radicals ( d - roms ) during and immediately after the exercise , as compared to the placebo 
 trial . in conclusion , the present study demonstrated that oxidative and antioxidative changes in 
 the blood are significantly induced by treadmill exercise of thoroughbred horses , and that 
 the recent developed measurements , i.e. d - roms and bap tests , are useful and convenient 
 methods for determining exercise - related physiological changes in horses .
furthermore , we 
 suggested a possibility that the supplementation of hydrogen - rich water has efficacy in 
 lowering oxidative stress in horses .
 the present study aimed to clarify changes of oxidative stress and antioxidative 
 functions in treadmill - exercised thoroughbred horses ( n=5 , 3 to 7 years old ) , using 
 recently developed techniques for measurement of serum d - roms for oxidative stress , and 
 bap for antioxidative markers . also , the effect of nasogastric administration of 
 hydrogen - rich water ( hw ) or placebo water preceding the treadmill exercise on these 
 parameters was examined . 
 each horse was subjected to a maximum level of treadmill exercise 
 in which the horses were exhausted at an average speed of 13.2  0.84 m / sec . 
 blood samples 
 were taken 4 times , immediately before the intake of hw or placebo water at 30 min 
 preceding the treadmill exercise , immediately before the exercise ( pre - exercise ) , 
 immediately after the exercise ( post - exercise ) and at 30 min following the exercise . in 
 all horses , both d - roms and bap values 
 significantly increased at post - exercise . 
 the 
 increase in d - roms tended to be lower in the hw trial , as compared to the placebo trial at 
 pre - exercise . 
 the increase in bap was considerable at approximately 150% of the 
 pre - exercise values in both the hw and placebo treatment trials . 
 the bap / d - roms ratio was 
 significantly elevated at post - exercise in both treatment trials , while a significant 
 elevation was also observed at pre - exercise in the hw trial . 
 bap , d - rom , and the bap / d - rom 
 ratio tended to decline at 30 min after the exercise , except bap and bap / d - roms in the 
 placebo trial . 
 these results demonstrate that the marked elevation of oxidative stress and 
 anitioxidative functions occurred simultaneously in the intensively exercised horses , and 
 suggest a possibility that hw has some antioxidative efficacy . 
Materials and Methods
Horses
Supplementation of hydrogen water
Experimental protocol
Measurements of d-ROMs and BAP
Statistical analysis
Results
Discussion