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to the expectations of the trainer at the time of the examination. All examinations were performed between 25 and 29 April 2016. Ambient temperature during exercise was 26–31°C and average humidity was 74%. Horses were ridden in an 80 9 50 m dirt arena by their usual equestrian team riders. Approval from the Texas A&M Institutional Animal Care and Use Committee was obtained (IACUC 2016-0077). The SET protocol included: historical questionnaire
(Supplementary Item 1), physical examination, subjective gait evaluation, objective gait analysis (Lameness Locator)1,resting and dynamic upper airway endoscopy, bronchoalveolar lavage fluid (BALF) cytology, echocardiogram, resting and exercising electrocardiography (ECG), measurement of blood lactate concentration (Lactate Scout)2,packed cell volume (PCV), heart rate (HR), respiratory rate (RR), sweat response during exercise, measurements of creatine kinase (CK; Vitros 4600)3 before and 4–6 h after exercise, resting plasma electrolytes (Nova Biomedical Stat Profile prime)4 and serum amyloid A (StableLab EQ-1)5. An exercise routine modified from a previously reported study (Haney 1998) was used and the timeline of the protocol is described below. The total duration of the test was approximately 30 min. • HR, RR, temperature and blood lactate were measured at rest.
○ Walk for 5 min and trot for 5 min.
○ Lope for 5 min to the left and gallop for 1 min to the left, lope for 5 min to the right and gallop for 1 min to the right.
• Blood lactate was measured immediately after horses stopped and horses were walked for 1 min.
○ Four sets of four spins with horses walked for 1 min between sets.
• Blood lactate was measured immediately after horses stopped and horses were walked for 1 min.
○ Two sets of two stops with 80 m gallop between stops and horses were walked for 1 min between sets.
• Blood lactate, PCV and RR measured immediately after horses stopped.
○ Horses were walked for 10 min.
• Temperature and sweating described and HR recorded. Subjective gait evaluation and objective gait analysis
were obtained prior to the exercise test by a single observer. Horses were evaluated in a straight line on synthetic pavers and in 10 m circles in both directions on firm packed dirt. Lameness in each affected limb was graded according to the AAEP lameness scale. Exercising upper airway endoscopy was performed with a
halter mounted dynamic endoscope (Tele-View model TV- 505)6. Recording began as each horse began to lope and was discontinued at the beginning of the final 10-min walk. Recordings were reviewed and abnormalities recorded by a single nonblinded observer. Scoring of tracheal mucus and determination of
presence of blood in the airway was performed 30–60 min after exercise with a 1.5 m endoscope. Tracheal mucus was evaluated as previously described (Cou€
etil et al. 2016) and a
score >1 was considered abnormal. Bronchoalveolar lavage was performed 30–60 min after finishing the gallop using a
3 m Bivona tube passed blindly into the airway until wedged. The balloon was inflated and 250 mL of 0.9% NaCl used to lavage the lower airways. Each horse was sedated with xylazine (approximately 0.4 mg/kg i.v., Rompun7) and butorphanol (approximately 0.01 mg/kg i.v., Dolorex8). Bronchoalveolar lavage fluid cytology was considered abnormal if >10% neutrophils, >5% mast cells or >5% eosinophils were detected (Cou€
etil et al. 2016). The cardiac rhythm was recorded throughout exercise
with a digital telemetry unit (Televet)9. Electrodes (Phillips M2202A)10 were placed in a modified base-apex fashion with two electrodes just in front of the saddle and two caudal to the girth near the ventral midline. Electrodes were secured with glue (Medical Adhesive)11. Each R-R interval was analysed manually and with electronic calipers by a single investigator. The investigator was nonblinded to the horse and analysis was performed before endoscopy and BALF results were obtained and with the investigator unaware of lameness examination results. Arrhythmias were defined as
previously reported (Buhl et al. 2010) and if a depolarisation fitting the description of a supraventricular premature complex (SVPC) occurred during prominent waxing and waning of the rate, it was instead classified as a sinus arrhythmia. Standard 2D, M-mode and colour Doppler echocardiographic images were obtained by one nonblinded investigator before exercise using portable ultrasound equipment (MyLab 30Gold)12 with a 2–3.5 mHz phased array probe using previously reported windows and measurements (Buhl et al. 2005). If valvular regurgitation was observed, it was graded as: clinically insignificant, mild, moderate or severe according to the size of the jet and the haemodynamic consequences. The sweating response was categorised as absent, mild (damp hair coat in the neck), moderate (abundant sweat in localised areas such as neck and between hind quarters) or full sweat (soaked or dripping hair coat in all or most body surfaces). Data were reported descriptively. When summary statistics were reported, means and s.d. were used to describe data following a normal distribution, and median and ranges are used to describe data that are not normally distributed.
Results
Sixteen horses completed the study protocol and one horse was withdrawn after the musculoskeletal examination due to severe (grade 4) lameness. There were 12 geldings, four
mares, and one stallion of age 10 3 years. Fifteen participants were Quarter Horses and two were Paints. Responses to historical questionnaires were heterogeneous and reflected that horses had competed four or five times during the spring season. Horses were performing up to expectations at the time of examination but in 13 horses, musculoskeletal problems had been diagnosed during the season. Twelve horses were ridden 5 days/week, two horses were ridden 4 days/week and two horses were ridden 2 days/week. In all horses, riding sessions were approximately one hour in duration. General physical examination at rest was normal in all horses with the exception of one horse that had a 2/6 holosystolic band shaped and coarse murmur with the point of maximal intensity over the mitral valve area. Resting plasma electrolytes and serum amyloid A (reference range 0– 20 mg/L) concentration were normal in all horses.
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