five gelding thoroughbred horses ( ages 57 year , 
 weight 468535 kg ) were used in the forelimb experiment , and four gelding thoroughbred horses ( ages 57 year , weight 468544 kg ) 
 were used in the hind limb experiment .
two 
 teflon - coated stainless steel electrodes ( a - m systems inc . , carlsborg , wa , u.s.a . ) with 1 cm of the tip bared were inserted into a 
 23 g needle of 6 cm in length , and 1 cm of the tip was bent back and twisted around the needle shaft .
the electrodes were inserted into the splenius ( sp ) at the level of the second cervical vertebra at 
 a depth of 20 mm , the middle point of muscle belly of the brachiocephalicus ( bc ) at a depth of 20 mm , the middle 
 point of the biceps brachii ( bb ) at a depth 50 mm , and the middle point of the pectoralis descendens 
 ( pd ) at a depth of 20 mm in the forelimb experiment ; in the hind limb experiment , they were inserted into the 
 longissimus dorsi ( ld ) at the level of the first lumbar vertebra at a depth of 50 mm , the biceps 
 femoris ( bf ) in the upper third portion of the muscle at a depth of 60 mm , the middle point of muscle belly of the 
 vastus lateralis ( vl ) at a depth of 80 mm , and the middle point of the gluteus medius ( gm ) at a 
 depth of 40 mm .
electromyography ( emg ) data were collected from right and left 
 side muscles ( type 271 nec san - ei sokki inc . ,
strain gauges 
 ( n22-fa-10 - 120 - 11-vs3 ; nec san - ei instruments , ltd . ,
tokyo , japan ) were attached with glue to the dorsal midline of the fore or hind 
 hooves in the applicable experiment , and they were connected to dynamic strain measuring instruments ( dp-612b , kyowa electronic 
 instruments , tokyo , japan ) .
emg and strain gauge data were recorded with pc using the windaq software ( dataq , akron , oh , u.s.a . ) . 

the horses performed a designated exercise protocol consisting of walking ( 1.6 m / sec ) and 
 trotting ( 3.5 m / sec ) on a treadmill ( sto ab , knivsta , sweden ) at five gradients ( 6% , 3% , 0% , + 3% , + 6% ) . because the inclination 
 of the slope for uphill training is 3 to 3.5% at the training center of the jra , the range of inclination was determined by doubling 
 the value in ordinary use .
the emg signal and strain gauge data were filtered with software ( bimutus , kissei comtec co. , ltd . , nagano , japan ) using a 
 high - pass butterworth filter with a 10 hz cutoff point and a low - pass butterworth filter with a 40 hz cutoff point , respectively . 

stride time and duty factor ( the percentage of stance to stride time ) were determined by the strain gauge data from randomly 
 selected right or left hooves .
the integrated electromyogram ( iemg ) values for each muscle in each stride were calculated from the 
 emg data .
one recording on the right or left side of the emg signal was selected depending on the signal condition .
comparisons between 
 conditions were made using repeated measures analysis of variance ( r 3.0.2 , r core team ( 2013 ) .
url http://www.r-project.org/ ) . where significant 
 differences ( p<0.05 ) were identified , a pair - wise ryan test was applied .
in the forelimb experiment , stride time and duty factor at a walk and a trot were not significantly different under any of the 
 inclinations of the treadmill ( fig .
1.mean (  sd ) stride time and duty factor at inclinations from 6% to + 6% at a walk ( 1.6 m / sec ) in the forelimb experiment ( a ; 
 n=5 ) and hind limb experiment ( b ; n=4 ) and at a trot ( 3.5 m / sec ) in the forelimb experiment ( c ; n=5 ) and hind limb experiment 
 ( d ; n=4 ) . ) . on the other hand ,
in the hind limb experiments , stride time on the 6% incline was significantly shorter than that on the 
 0% and + 3% inclines during walking ( fig .
stride time on the 6% incline was also 
 significantly shorter than that on the 0% , + 3% , and + 6% inclines during trotting ( fig .
duty factor at a walk and trot were not significantly different under any of the inclinations of the treadmill ( fig .
1 ) . mean (  sd ) stride time and duty factor at inclinations from 6% to + 6% at a walk ( 1.6 m / sec ) in the forelimb experiment ( a ; 
 n=5 ) and hind limb experiment ( b ; n=4 ) and at a trot ( 3.5 m / sec ) in the forelimb experiment ( c ; n=5 ) and hind limb experiment 
 ( d ; n=4 ) . at a walk ,
the iemg values for the muscles in the forelimb were not significantly different under any of the inclinations of the 
 treadmill ( fig .
iemg values for the forelimb ( left ; bc n=5 , bb n=3 , sp n=4 , pd n=5 ) and hind limb ( right ; ld n=3 , vl n=3 , gm n=4 , 
 bf n=4 ) during downhill ( 3 , 6% ) , level ( 0% ) , and uphill ( + 6 , + 3% ) walking .
significant differences 
 ( p<0.05 ) are indicated by different letters . ) . in the hind limb at a walk ,
the iemg values for the ld and vl were not significantly different under any of the 
 inclinations of the treadmill , while the iemg values for the gm at all inclinations other than + 6% were significantly less than that 
 at + 6% inclination ( fig .
the iemg of the bf significantly decreased as the inclination 
 of the treadmill decreased .
there was a significant difference between each different inclination with the exception of between 6% 
 and 3% ( fig .
2 ) . mean (  sd ) iemg values for the forelimb ( left ; bc n=5 , bb n=3 , sp n=4 , pd n=5 ) and hind limb ( right ; ld n=3 , vl n=3 , gm n=4 , 
 bf n=4 ) during downhill ( 3 , 6% ) , level ( 0% ) , and uphill ( + 6 , + 3% ) walking .
significant differences 
 ( p<0.05 ) are indicated by different letters . at a trot ,
the iemg values for the bc at the 6% , 3% , and 0% inclines were significantly less than that at the + 6% incline ( fig . 3fig .
3.mean (  sd ) iemg values for the forelimb ( left ; bc n=4 , bb n=2 , sp n=3 , pd n=5 ) and hind limb ( right ; ld n=3 , vl n=4 , gm n=4 , 
 bf n=4 ) during downhill ( 3 , 6% ) , level ( 0% ) , and uphill ( + 6 , + 3% ) trotting .
the iemg values for the other three muscles in the forelimb were not significantly different under any of the inclinations 
 of the treadmill at a trot ( fig .
the iemg values for the 
 vl were not significantly different under any of the inclinations of the treadmill , while the iemg values for the ld at the 6% and 
 3% inclines were significantly decreased compared to that at + 3% and + 6% inclination .
the iemg of the gm at 6% and 3% inclines 
 were also significantly decreased compared with that at + 6% inclination .
the iemg values for the bf at all inclinations other than 
 + 6% incline were significantly decreased compared with that at + 6% inclination ( fig . 
 3 ) .
mean (  sd ) iemg values for the forelimb ( left ; bc n=4 , bb n=2 , sp n=3 , pd n=5 ) and hind limb ( right ; ld n=3 , vl n=4 , gm n=4 , 
 bf n=4 ) during downhill ( 3 , 6% ) , level ( 0% ) , and uphill ( + 6 , + 3% ) trotting .
in the hind limb experiment , stride time at a walk and trot shortened as the inclination decreased , while stride time was not 
 different under any of the inclinations in the forelimb experiment . in theory , stride time does not change whether it is measured in 
 the forelimb or hind limb . in this study , the average stride time at a walk in the hind limb at a 6% incline
was reduced by 0.035 
 sec ( 3.3% ) compared with that at a 0% incline . at a trot , the average stride time in the hind limb at a 6% incline
was also reduced 
 by 0.024 sec ( 3.5% ) compared with that at a + 3% incline .
because the effect of inclination on stride time was very little , the 
 results might be different between the forelimb and hind limb experiments .
in previous reports , the effects of inclination on stride 
 time at a walk and trot were also inconsistent [ 2 , 5,6,7 , 11 , 13 ] .
the iemg values changes by horse and the position of the electrodes in each muscle .
this method evaluated the effect of incline within each horse . in the bb at a trot , a 
 statistical comparison could not be performed because emg data were only recorded for 2 horses . in some muscles at a walk and trot , 
 emg data could not be recorded for 1 or 2 horses .
the statistical powers of the test were low in these muscles . in the muscles of the forelimb
, the effects of the inclines were observed through the iemg values for the bc at a trot , which 
 decreased as the incline decreased .
the iemg values for the bc , which protracts the forelimb , decreased on the downward slope possibly because gravity assisted the protraction of the forelimb . on the other hand , 
 iemg values for
this result was consistent 
 with a previous report in which the iemg values for the bc increased on an upslope at a trot .
there was no influence at a walk and trot on the iemg values for the bb , which acts as an antigravity muscle , although it was reported that the vertical ground reaction force in the forelimb decreased on 
 an upslope . in the pd , which has a similar function as the bc
the iemg values for the sp , which supports the head , were not influenced 
 by the inclination because the movement of the head was not influenced by the inclination of the treadmill . in the hind limb muscles , the iemg values for the gm and bf decreased as the incline decreased at a walk and trot [ 2 , 7 ] .
the iemg values for these muscles decreased because the propulsive force decreased as the 
 incline decreased .
the inclination had no effect on the bending of 
 the back at a walk , while the iemg values for the ld increased significantly as the inclination increased .
it was reported that the vertical ground reaction force in the hind 
 limb increased on an upslope .
it was assumed from these studies that the hind limb force 
 decreases on a downslope .
however , the iemg values for the vl were not influenced by the inclination , as reported by crook 
 et al . , although this was different from the predicted result based on 
 the previous report about the vertical ground reaction force .
the inclination of + 6% might not be enough to influence the effect of the vertical ground reaction force on 
 the iemg values for the vl .
it was reported that heart rate and oxygen demand increased as the inclination increased [ 4 , 
 6 , 14 ] .
the iemg values for the bc , gm , and bf on an 
 upslope were higher than those on flat ground , while the iemg values for any muscles on a downslope were not different from those on 
 flat ground .
the rate of oxygen consumption was increased by about 250% on the 10% upslope compared with trotting on level ground 
 and was only reduced by about 50% on a 10% downslope [ 4 , 6 , 14 ] .
the present data suggested that the increased rate of oxygen consumption 
 reflected the increased muscle activities . in conclusion , in the forelimb , the iemg values for the bc increased on an upslope at a trot , while the iemg values for the muscles 
 that were measured did not change on a downslope at a walk and trot . in the hind limb ,
the iemg values for the gm and bf increased 
 on an upslope , while the iemg values for these muscles did not change or decreased on a downslope .
the effects of the inclination on 
 the muscle activities were larger on an upslope and in the hind limb muscles compared with the forelimb muscles .
 abstractit is important to know the effects of the inclination of a slope on the activity of each muscle , because training by running on 
 a sloped track is commonly used for thoroughbred racehorses . 
 the effects of incline ( from 6 to + 6% ) on the forelimbs and hind 
 limbs during walking and trotting on a treadmill were evaluated by an integrated electromyogram ( iemg ) . 
 the muscle activities in 
 the forelimbs ( 5 horses ) and hind limbs ( 4 horses ) were measured separately . 
 two stainless steel wires were inserted into each of 
 the brachiocephalicus ( bc ) , biceps brachii ( bb ) , splenius ( sp ) , and pectoralis descendens ( pd ) in the forelimb experiment and into 
 the longissimus dorsi ( ld ) , vastus lateralis ( vl ) , gluteus medius ( gm ) , and biceps femoris ( bf ) in the hind limb experiment . 
 the 
 emg recordings were taken at a sampling rate of 1,000 hz . at a walk , the iemg values for the forelimb were not significantly 
 different under any of the inclinations . in the hind limb , 
 the iemg values for the gm and bf significantly decreased as the 
 inclination decreased . at a trot 
 , the iemg values for the bc in the forelimb significantly decreased as the inclination of the 
 treadmill decreased . in the hind limb , 
 the iemg values for the ld , gm , and bf significantly decreased as the inclination 
 decreased . 
 uphill exercise increased the iemg values for the bc , ld , gm , and bf , while downhill exercise resulted in little 
 increase in the iemg values . 
 it was concluded that the effects of inclination on the muscle activities were larger for the uphill 
 exercises , and for the hind limb muscles compared with the forelimb muscles . 
Materials and Methods
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