the animals were 24 one- to two - year - old thoroughbreds ( twelve males , twelve females ) clinically free of any 
 orthopaedic disorders and history . prior to a breaking and training period that begins in september and
ends in 
 april every year , twelve horses ( six males , six females ) were selected as subjects in the first study from 
 december 2013 to april 2014 .
the other twelve horses were used in the second study from december 2014 to april 
 2015 .
the running ( that is at a canter and a gallop ) distances for 1 month before the dates of the 
 ultrasonographic examination are presented in table 1table 1.changes of running distance for 1 month before the dates of the ultrasonographic examination in 
 december , february , and april2013 ( km / month)2014 ( km / month)december9.6  6.39.8  5.2february47.7  1.834.0  9.6april54.0  18.145.6  7.6running was performed at a canter and a gallop .. running was performed at a canter and a gallop .
the horses sdfts were ultrasonographically scanned on the same dates in december , february , and april . 
 before the examination ,
the horses had been kept in pasture for at least 3 hr following their daily exercise . 

the horses were retained in a treatment stall , and then sedated with medetomidine ( 2 mg / head intravenous 
 injection ) .
after completely clipping the palmar aspect from the proximal to the distal metacarpus , a linear 
 array transducer ( 50 mm of effective aperture length ) with a broadband frequency between 5 and 12 mhz was 
 directly placed onto the skin with abundant gel . using a mobile ultrasound system ( iu22 , philips medical 
 systems corporation , tokyo , japan ) , three longitudinal ( three equal lengths of the labelled 1 to 3 in order 
 from proximal to distal metacarpus ) and
six transversal ( separated by equal distance along the labelled 1a , 
 1b , 2a , 2b , 3a and 3b in order from proximal to distal , fig .
after completely clipping the palmar aspect of the metacarpus , a linear 
 array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly 
 outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in 
 the flattened sdft of the 2b , 3a and 3b images . )
images from the proximal end ( bottom of the accessory carpal bone ) to the distal end ( top of the 
 proximal sesamoid bone ) of the sdft were scanned .
the maximum depth of the image was 3.5 cm , and the depth of 
 focus was adjusted to the sdft .
both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz 
 and 6 to 11 hz , respectively .
after completely clipping the palmar aspect of the metacarpus , a linear 
 array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly 
 outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in 
 the flattened sdft of the 2b , 3a and 3b images .
blood flows in the tendon bundles 
 were visualized in the longitudinal and transversal cd images , in which the region of interest was set and the 
 gain setting was similar for all tendons , just below the noise level ( 7785% of maximal gain ) .
the positive signals of cd flow are grade 1 ( indicated by white 
 arrows ) , small color activities that were fixed and rhythmically blinking . )
was recorded and graded , as previously reported : grade 1 , tiny to small color activities that were 
 rhythmically blinking at a site , but for which flow rates were not measurable by pulsed doppler ; grade 2 , 
 unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed 
 doppler ; and grade 3 , unequivocal linear color activities appearing as blood streams in a regular direction 
 due to periodic changes in color .
the longitudinal cd scan was 
 inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2 , 
 therefore we used only the transversal images for the cd data analysis . a transversal cd image of the sdft
. the positive signals of cd flow are grade 1 ( indicated by white 
 arrows ) , small color activities that were fixed and rhythmically blinking .
the horses sdfts were ultrasonographically scanned on the same dates in december , february , and april . 
 before the examination ,
the horses had been kept in pasture for at least 3 hr following their daily exercise . 

the horses were retained in a treatment stall , and then sedated with medetomidine ( 2 mg / head intravenous 
 injection ) .
after completely clipping the palmar aspect from the proximal to the distal metacarpus , a linear 
 array transducer ( 50 mm of effective aperture length ) with a broadband frequency between 5 and 12 mhz was 
 directly placed onto the skin with abundant gel . using a mobile ultrasound system ( iu22 , philips medical 
 systems corporation , tokyo , japan ) , three longitudinal ( three equal lengths of the labelled 1 to 3 in order 
 from proximal to distal metacarpus ) and six transversal ( separated by equal distance along the labelled 1a , 
 1b , 2a , 2b , 3a and 3b in order from proximal to distal , fig .
after completely clipping the palmar aspect of the metacarpus , a linear 
 array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly 
 outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in 
 the flattened sdft of the 2b , 3a and 3b images . )
images from the proximal end ( bottom of the accessory carpal bone ) to the distal end ( top of the 
 proximal sesamoid bone ) of the sdft were scanned .
the maximum depth of the image was 3.5 cm , and the depth of 
 focus was adjusted to the sdft .
both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz 
 and 6 to 11 hz , respectively .
after completely clipping the palmar aspect of the metacarpus , a linear 
 array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly 
 outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in 
 the flattened sdft of the 2b , 3a and 3b images .
blood flows in the tendon bundles 
 were visualized in the longitudinal and transversal cd images , in which the region of interest was set and the 
 gain setting was similar for all tendons , just below the noise level ( 7785% of maximal gain ) .
the positive signals of cd flow are grade 1 ( indicated by white 
 arrows ) , small color activities that were fixed and rhythmically blinking . ) was recorded and graded , as previously reported : grade 1 , tiny to small color activities that were 
 rhythmically blinking at a site , but for which flow rates were not measurable by pulsed doppler ; grade 2 , 
 unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed 
 doppler ; and grade 3 , unequivocal linear color activities appearing as blood streams in a regular direction 
 due to periodic changes in color .
the longitudinal cd scan was 
 inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2 , 
 therefore we used only the transversal images for the cd data analysis . a transversal cd image of the sdft
. the positive signals of cd flow are grade 1 ( indicated by white 
 arrows ) , small color activities that were fixed and rhythmically blinking .
the running distance for 1 month before the date of the ultrasonographic examination was increased in the order 
 of december , february , and april in both of the two training periods . as the transversal gs images were obtained 
 using a linear array transducer that was directly placed onto the skin with abundant gels and not attached to a 
 coupler ,
the rounded sdft was clearly outlined in 1a , 1b , and 2a images , however the lateral and medial margins 
 could not be visualized in the flattened sdft of 2b , 3a and 3b ( fig . 
 1 ) .
there were no longitudinal or transversal gs images indicating injury in the sdfts in either of the 
 two training periods .
of 864 ( 6 images  2 forelimbs  24 horses  3 examinations ) transversal cd images , 56 images ( 6.4% ) showed the 
 cd flows in sdft bundles ( table 2table 2.among 864 transversal cd images , 56 images ( 6.4% ) showed positive cd flows in sdft bundles ) , which were categorized as rhythmically blinking ( grade 1 , 32 images , as presented in fig .
the cd flows 
 were unevenly distributed at 1a , 1b , 2a and 2b , and were more frequently detected in april than in either 
 december or february ( table 3table 3.the distribution of transversal cd images showing positive cd flows in sdft bundles1a1b2a2b3a3btotaldecember 4210007february104000014april1461230035total28121330056 ) .
there were 7 , 8 and 15 horses showing cd flows in december , february and april , 
 respectively .
a normal tendon is hypovascular , and angiogenesis or increased vascularity are associated with an injured 
 tendon for much of the healing process . within the first 24 hr after injury ( inflammatory phase ) , inflammatory 
 cells are recruited to the injured site , and vasoactive and chemotactic factors are associated with an increase 
 in vascular permeability and the initiation of angiogenesis [ 17 , 22 ] . within a few days ,
tenocytes gradually migrate to the injury site , 
 proliferate , and synthesize type iii collagen ( proliferative phase ) [ 19 , 
 22 ] . after approximately six weeks , the repair tissue changes from 
 cellular to fibrous ( remodelling phase ) .
the fibrous tissue gradually changes to scar - like tendon tissue over 
 the course of one year ( maturation stage ) [ 10 , 22 ] , and the vascularity finally declines in the latter half of this stage [ 1 , 22 ] .
therefore , we hypothesised that 
 the blood flows would also increase in equine sdft from a few days to six weeks after the injury , and that they 
 might be indicative of tendon injury and the subsequent healing process
. however , in this study of one- to 
 two - year - old horses , no macroscopic injury was found in the 56 transversal gs images in which positive signals 
 of blood flow were seen within sdft bundles .
some of the positive flow signals disappeared and/or then 
 reappeared during the study period .
therefore , we speculate that the positive signals of blood flow observed in 
 this study might be due to transient increases of blood flows in the inherent vessels in response to hypoxia 
  and/or hyperthermia [ 11 , 
 20 , 25 ] of tendon tissue which 
 were related to the increased exercise ( the increased mechanical behaviour of the tendon ) , rather than increased 
 vascularization associated with macroscopic injury of tendon bundles . based on the observance of increased 
 doppler flow in achilles tendons that was associated with weekly badminton hours and badminton years , it was 
 suggested that doppler flow may be a response to mechanical load on the tendon .
since doppler activity increased in human achilles tendons after running exercise performed by 
 both non - symptomatic subjects and symptomatic subjects in a rehabilitation program , it was proposed that all 
 tendons of both healthy and diseased subjects , have a certain amount of vascularization , which may be manifested 
 on demand up to a certain level . considering our result in reference 
 to these human studies ,
it is possible that the blood flows observed in the sdft of the one- to two - year - old 
 thoroughbreds were response to the increases in the intensity and distance of the running exercise during the 
 training period .
the main sources of blood supply are : the intrinsic systems at the myotendinous and osteotendinous junctions , 
 and the extrinsic system through the paratenon or the synovial sheath .
the ratio of the blood supply from the 
 intrinsic systems to that from the extrinsic system varies in the different parts of the tendon .
for example , 
 the central third , which could be a zone of hypovascularity , receives 35% of its blood supply from the extrinsic 
 system in the rabitt achilles tendon [ 14 , 18 ] .
an equine study reported that the blood supply of the normal sdft is primarily intratendinous 
 rather than paratendinous as previously thought . in this study ,
the 
 cd flows mainly increased in 1a ( 28 images ) , 1b ( 12 images ) , and 2a ( 13 images ) , which are proximal parts of the 
 sdft .
this result could be suggestive of increased blood supply from the intrinsic systems at the myotendinous 
 junction . at the myotendinous junction ,
perimysial vessels from the muscle continue between the fascicles of the 
 tendon , and extend to the proximal third of the human achilles 
 tendon [ 6 , 22 ] .
although we have no 
 data regarding the blood vessel distribution from the superficial digital flexor myotendinous junction to the 
 proximal third of metacarpal sdft in horses , this result might be explained as the perimysial blood supply to 
 the proximal third physiologically increasing in response to the increases in the running distance of the 
 clinically healthy horses . in conclusion , the increase of intratendinous blood flows in the proximal parts of the sdft could be responses 
 to the increase in exercise intensity as the training advanced . because no longitudinal or transversal gs image 
 indicating macroscopic injury of the sdft were found in either of the two training periods , increase of cd flows 
 in the proximal parts of the sdft are not necessarily predictive of tendon injury in the near future in the 
 training period of young thoroughbreds .
 abstractaim of this study was to evaluate the relationships of exercise and tendon injury with doppler flows 
 appearing in the superficial digital flexor tendon ( sdft ) of young thoroughbreds during training periods . 
 the 
 forelimb sdfts of 24 one- to two - year - old thoroughbreds clinically free of any orthopaedic disorders were 
 evaluated using grey - scale ( gs ) and color doppler ( cd ) images during two training periods between december 
 2013 to april 2015 . 
 twelve horses per year were examined in december , february , and april in training periods 
 that began in september and ended in april . 
 the sdft was evaluated in 3 longitudinal images of equal lengths 
 ( labelled 1 , 2 , 3 in order from proximal to distal ) , and 6 transversal images separated by equal lengths 
 ( labelled 1a , 1b , 2a , 2b , 3a and 3b in order from proximal to distal ) of the metacarpus using both gs and cd . 
 
 the running ( canter and gallop ) distance for 1 month before the date of the ultrasonographic examinations was 
 increased in december , february , and april in both of the two training periods . 
 cd flows defined as 
 rhythmically blinking or pulsatory colored signals were found in 56 of 864 ( 6.4% ) transversal cd images , in 
 28 , 12 , 13 , and 3 images of 1a , 1b , 2a and 2b , respectively , and in 7 , 14 , and 35 images captured in december , 
 february , and april , respectively . 
 there were no longitudinal or transversal gs images indicating injury in 
 the sdfts in either of the two training periods . 
 the increase of cd flows in the proximal regions of the sdft 
 are possibly related to the increase of the running distance during the training periods of the one- to 
 two - year - old thoroughbreds . because no injury was diagnosed in the sdfts by gs images during the training 
 periods , the increase of cd flows in the proximal parts of sdft is not necessarily predictive of tendon injury 
 in the near future during the training period of young thoroughbreds . 
Materials and Methods
Ultrasonographic examination
Results
Discussion