Microstructural changes in cartilage and bone related to repetitive overloading in an equine athlete model.

The palmar aspect of the third metacarpal (MC3) condyle of equine athletes is known to be subjected to repetitive overloading that can lead to the accumulation of joint tissue damage, degeneration, and stress fractures, some of which result in catastrophic failure. However, there is still a need to understand at a detailed microstructural level how this damage progresses in the context of the wider joint tissue complex, i.e. the articular surface, the hyaline and calcified cartilage, and the subchondral bone. MC3 bones from non-fractured joints were obtained from the right forelimbs of 16 Thoroughbred racehorses varying in age between 3 and 8 years, with documented histories of active race training. Detailed microstructural analysis of two clinically important sites, the parasagittal grooves and the mid-condylar regions, identified extensive levels of microdamage in the calcified cartilage and subchondral bone concealed beneath outwardly intact hyaline cartilage. The study shows a progression in microdamage severity, commencing with mild hard-tissue microcracking in younger animals and escalating to severe subchondral bone collapse and lesion formation in the hyaline cartilage with increasing age and thus athletic activity. The presence of a clearly distinguishable fibrous tissue layer at the articular surface immediately above sites of severe subchondral collapse suggested a limited reparative response in the hyaline cartilage.

Evidence of purinergic neurotransmission in isolated, intact horse sweat glands.

BACKGROUND: Fluid secretion by sweat glands in response to heat and exercise is underpinned by increases in intracellular calcium. In horses, this is primarily via ß2-adrenoceptors, but studies in equine sweat gland cell lines have indicated a possible role for purinergic agonists. Knowledge of equine sweating stimulus-secretion mechanisms in intact glands from healthy animals would allow future comparison to determine whether these mechanisms are affected in equine anhidrosis. HYPOTHESIS/OBJECTIVES: To determine whether purinergic agonists can induce changes in intracellular calcium in intact, freshly isolated equine sweat glands. ANIMALS: Eleven healthy thoroughbred horses from the Hong Kong Jockey Club were used in this study. METHODS: Freshly isolated equine sweat glands were loaded with the calcium-sensitive fluorescent dye fura-2 AM, and changes in intracellular calcium were recorded before, during and after stimulation by purinergic agonists. RESULTS: Purinergic agonists ATP and UTP generated significant increases in intracellular calcium. CONCLUSIONS AND CLINICAL IMPORTANCE: The results show that it is possible to investigate stimulus-secretion coupling mechanisms by fluorescence imaging in equine sweat glands that have been isolated from fresh skin samples. Such isolated glands retain functional ß2-adrenoceptors and P2Y purinergic receptors that couple to a calcium-signalling pathway. Using isolated, intact sweat glands therefore offers a very useful model for the further study of secretory processes in equine sweat glands, and using this experimental approach could facilitate a better understanding of how these mechanisms are affected in equine anhidrosis.

Stress fracture of the palmar, distal cortex of the third metacarpal bone: A diagnostic challenge with a good prognosis.

BACKGROUND: Transverse stress fracture of the palmar cortex of the distal aspect of the third metacarpal bone (TSF PCD McIII) is poorly documented. OBJECTIVES: To describe the typical signalment of this injury, the common clinical and radiological signs and prognosis. STUDY DESIGN: A retrospective hospital-based case series with follow-up of racing records. METHODS: All cases of TSF PCD McIII diagnosed among racehorses in training at the Hong Kong Jockey Club between 2011 and 2019 were identified. Clinical records were reviewed and documented signs were recorded for each case. Findings from a predefined list of radiological features were listed. Descriptive statistics were used to describe the age and training profile of horses, the clinical and radiological findings and time to return to ridden exercise. RESULTS: Twenty-three cases (n = 23) were identified. The majority (57%) had recently started or returned to training from a break and 35% (n = 8) of cases had not yet undertaken timed gallops at the time of injury. Most cases (20/23; 87%) were lame; in nine (39%), lameness was severe. Localised swelling of superficial soft tissues was reported in 15/23 (65%) cases. Pain on palpation of the distal McIII was present in 15/23 (65%) cases and in response to fetlock flexion in 12/23 (52%). Diffuse, localised increase in radiopacity, disruption to the outline of the bone periosteal surface and outward displacement of the button of the splint bone were common radiological features in early cases (70%, 67% and 67% of all cases). Sixty-one per cent of cases (14/23) were initially misdiagnosed. Most horses (n = 18/23) resumed training and racing after a median of 83 and 246 days. MAIN LIMITATIONS: Clinical notes were not consistent between cases. Radiographs were taken at different times. CONCLUSIONS: Horses commencing training are at risk of TSF PCD McIII, which presents with confusing clinical signs and subtle radiological findings. The long-term outlook is favourable.

Design and preliminary experience of a transportable equine operating theatre.

BACKGROUND: Equine surgical facilities are not always available, even in situations where high-value horses are involved in relatively high-risk activities. OBJECTIVES: To develop a self-contained equine surgical unit that can be relocated and transported by road. STUDY DESIGN: Proof of concept. METHODS: A modular operating suite for horses, which included a theatre, scrub room, instrument preparation and storage room, and an anaesthetic induction/recovery box: Transportable Equine Operating Theatre (TEOT) was designed, constructed and tested. A second, larger unit was then built following the same design criteria. RESULTS: Both units were based on modifications of standard 6.06 × 2.35 m (20 × 8 ft), steel shipping containers that were fitted out to provide anaesthetic and surgical facilities in a functional and safe working environment. The two TEOTs consisted of either two or three containers that had selected sections of their sides removed so that they could be joined together to create a single large internal space. This space was subdivided to create separate working areas. A dedicated anaesthetic induction/recovery box was fabricated from a modular steel frame that could be disassembled for transport. The component parts could be loaded onto the bed of a standard lorry for road transport. The unit was designed to incorporate equipment that is found in any high-quality equine operating theatre. Surgical procedures on adult Thoroughbred horses have been successfully completed in both units. MAIN LIMITATIONS: The two units have had minimal use to date, and there is little data on their performance. CONCLUSIONS: The equipment described here is a way to provide equine surgical facilities that can be assembled and disassembled to allow their relocation to different sites.

The effect of stress fracture occurring within the first 12 months of training on subsequent race performance in Thoroughbreds in Hong Kong.

BACKGROUND: Racehorses are at an increased risk of stress fracture within the first 12 months of racing and when resuming training after a break. Research in these high-risk periods and on the effect of performance post-recovery is limited. OBJECTIVES: To describe the occurrence of stress fractures, diagnosed by nuclear scintigraphy (NS), in racehorses' first 12 months training in Hong Kong, and their impact on racing performance and career length. STUDY DESIGN: Retrospective 1:2 matched case-control study. METHODS: Clinical records of horses with NS-diagnosed stress fractures within 365 days of import between 2006 and 2018 were collated. Cases and controls were matched on import date. Univariable conditional logistic regression compared signallment, pre-fracture training and post-recovery racing performance between cases and matched controls. Shared Frailty Cox regression analysed time from import to fracture and total career length. RESULTS: Eighty-seven horses sustained a NS-diagnosed fracture within their first year in Hong Kong (incidence risk 1.7% [95% confidence interval (CI) 1.4%-2.1%; N = 5180]). The humerus (42.0%; 95% CI 31.8%-52.6%; n = 39) and tibia (28.0%; 95% CI 19.1%-38.2%; n = 26) were the most common stress fracture sites. Cases missed a median of 63 days (Interquartile range (IQR) 49-82) of training because of fracture. Within the 12 months following diagnosis, case horses had a median of four (IQR 2-4, P < .0001) fewer race starts and were down HK$ 206 188 (IQR HK$ 0-436 800, P = .007) in race earnings compared to controls. Career length did not significantly differ between cases and controls (median 2 years and 3 months; IQR 15.3-39.1 months; P = .2). MAIN LIMITATIONS: Only stress fractures diagnosed by NS were included, hence, the study is not representative of all stress fractures occurring in racehorses in Hong Kong. CONCLUSIONS: Racehorses sustaining a stress fracture within 1 year of entering Hong Kong lost significant time in training, earnings and race starts. However, overall career length was unaffected.

Using time-series analysis techniques to enhance the understanding of musculoskeletal injury in Thoroughbred racehorses.

BACKGROUND: Many racing jurisdictions monitor race-day musculoskeletal injury (MSI) but fail to evaluate injuries occurring during training. Additionally, previous risk factor analyses have failed to explore temporal trends in injury occurrence. OBJECTIVES: To use time-series analysis techniques to identify trends, cyclicity and peaks in MSI incidence, in racehorses training and racing at the Hong Kong Jockey Club (HKJC) from July 2010 to June 2018. STUDY DESIGN: Retrospective longitudinal study. METHODS: The monthly incidence of all MSI, superficial digital flexor tendon (SDFT) injury, suspensory ligament injury and appendicular skeletal fracture occurring in training and racing were collected from veterinary records. The number of horses in training was collated monthly from trainer records. Time-series analysis techniques were used to describe trends and cyclical patterns for injury types. For each injury, incidence risks above the 90th percentile were identified as peaks in incidence. RESULTS: A total of 1471 injuries were recorded over eight racing seasons; 605 fractures (41.1%), 550 SDFT injuries (37.4%), and 316 suspensory ligament injuries (21.5%). Evidence of seasonality was detected in fracture incidence risk; increasing from October (median 0.25 per 1000 horses) until May (median 0.71 per 1000 horses), coinciding with the racing season (ending mid-July). Elevated incidence of MSI occurred throughout 2012; however, the greatest incidence risks of SDFT (14.8 per 1000 horses) and fracture (1.3 per 1000 horses) occurred since 2017. MAIN LIMITATIONS: Monthly (opposed to daily) incidence risk of injury reduced the resolution of the data. Additionally, fracture was not described according to bone or fracture type, which may have confounded overall trends. CONCLUSIONS: Evidence for seasonal variation in the incidence of fracture occurrence has been demonstrated. Based on using time-series techniques, further epidemiological studies, retrospectively targeting periods of high peaks in injury incidence risk could be used to aid identification of risk factors for injury.

Evaluation of detailed training data to identify risk factors for retirement because of tendon injuries in Thoroughbred racehorses.

OBJECTIVE: To identify the risk factors for premature retirement because of tendon injury in a Thoroughbred racehorse population. ANIMALS: 175 Thoroughbred racehorses (cases) at the Hong Kong Jockey Club that were retired from racing because of tendon injury between 1997 and 2004 and for which the last preretirement exercise was at a fast pace were each matched with 3 control horses that were randomly selected from all uninjured horses that had galloped on the same date as that last exercise episode. PROCEDURES: Training data for all horses were examined. Conditional logistic regression analyses were performed to identify risk factors for retirement from racing attributable to tendon injury. Two multivariable conditional logistic regression models were created; each contained 8 explanatory variables. RESULTS: Compared with control horses, case horses were older at the time of import, accumulated more race distance soon after import, were more likely to have had previous official veterinary or ultrasonographic examinations, raced fewer times during their career, and were in training for a longer period and had exercised at a reduced intensity during the 180-day period preceding the last fast-paced work date. CONCLUSIONS AND CLINICAL RELEVANCE: In addition to identification of risk factors for tendon injury among racing Thoroughbreds, results have suggested that resources focused on obtaining accurate training data may be misdirected in the absence of internationally agreed criteria for incident tendon injury among racehorses. Nevertheless, changes in training intensity and findings of previous clinical examinations could be used to identify horses at risk of tendon injury-associated retirement.

Controlling the misuse of cobalt in horses.

Cobalt is a well-established inducer of hypoxia-like responses, which can cause gene modulation at the hypoxia inducible factor pathway to induce erythropoietin transcription. Cobalt salts are orally active, inexpensive, and easily accessible. It is an attractive blood doping agent for enhancing aerobic performance. Indeed, recent intelligence and investigations have confirmed cobalt was being abused in equine sports. In this paper, population surveys of total cobalt in raceday samples were conducted using inductively coupled plasma mass spectrometry (ICP-MS). Urinary threshold of 75 ng/mL and plasma threshold of 2 ng/mL could be proposed for the control of cobalt misuse in raceday or in-competition samples. Results from administration trials with cobalt-containing supplements showed that common supplements could elevate urinary and plasma cobalt levels above the proposed thresholds within 24 h of administration. It would therefore be necessary to ban the use of cobalt-containing supplements on raceday as well as on the day before racing in order to implement and enforce the proposed thresholds. Since the abuse with huge quantities of cobalt salts can be done during training while the use of legitimate cobalt-containing supplements are also allowed, different urinary and plasma cobalt thresholds would be required to control cobalt abuse in non-raceday or out-of-competition samples. This could be achieved by setting the thresholds above the maximum urinary and plasma cobalt concentrations observed or anticipated from the normal use of legitimate cobalt-containing supplements. Urinary threshold of 2000 ng/mL and plasma threshold of 10 ng/mL were thus proposed for the control of cobalt abuse in non-raceday or out-of-competition samples.

Control of the misuse of testosterone in castrated horses based on an international threshold in plasma.

Testosterone is an endogenous steroid produced primarily in the testes. Trace levels of testosterone are found in urine samples from geldings, as testosterone is also secreted by the adrenal. An international threshold of free and conjugated testosterone in urine (20 ng/mL) was adopted by the International Federation of Horseracing Authorities (IFHA) in 1996 for controlling testosterone misuse in geldings. In view of the recent popularity of using blood in doping control testing, it is necessary to establish a threshold for testosterone in gelding plasma. A liquid chromatography-mass spectrometry (LC/MS) method was developed for quantifying low levels of free testosterone in gelding plasma. Based on a population study of 152 post-race plasma samples, the mean ± SD concentration of plasma testosterone was determined to be 14.7 ± 6.8 pg/mL. Normal distribution could be obtained after square-root or cube-root transformation, resulting in respective tentative thresholds of 49 or 55 pg/mL (corresponding to a risk factor of less than 1 in 10 000). A rounded-up threshold of 100 pg/mL of free testosterone in plasma was proposed. Based on the administration of Testosterone Suspension 100 to six geldings, the same average detection time of 14 days was observed in either plasma or urine using the proposed plasma threshold and the existing international urine threshold. The maximum detection time was 18 days in plasma and 20 days in urine. The results demonstrated the proposed plasma threshold is effective in controlling the misuse of testosterone in geldings. Similar results were subsequently obtained in Europe, and this proposed threshold was adopted by IFHA in October 2013.

Circularly polarized light standards for investigations of collagen fiber orientation in bone.

Bone exhibits positive form birefringence dominated by and dependent upon the orientation of its collagen. The biomechanical efficacy of bone as a tissue is largely determined by collagen fibers of preferred orientation and distribution (and corresponding orientation of mineral crystallites), and evidence is accumulating to demonstrate that this efficacy extends to function at the organ level. This study has three aims. The first is to provide a Background to the study of circularly polarized light (CPL) investigations of collagen fiber orientation in bone. The significance of preferred collagen fiber orientation in bone, linearly polarized light and CPL imaging principles, and a short history of CPL studies of mammalian functional histology are reviewed. The second is to describe, in some detail, methodological considerations relating to specimen preparation and imaging appropriate for the quantitative analysis of preferentially oriented collagen. These include section transparency, section thickness, the uniformity of the illuminating system, and CPL paraphernalia. Finally, we describe a grey-level standard useful for quantitative CPL, based upon mineralized turkey tendon, which shall be provided to investigators upon request. When due consideration is paid to specimen preparation and imaging conditions, quantitative assessment of collagen fiber orientation provides insight into the effects of mechanical loading on the skeleton.

Comprehensive surface-based morphometry reveals the association of fracture risk and bone geometry.

Surface-based morphometry method is advantageous in its objectivity and increased capability in detecting focal morphological changes, but has not been applied in bone-related research. Orthopedics research in human has confirmed the association of the bone geometry in proximal femur and its fracture. In this study, surface-based morphometry is used to test the hypothesis that there is relationship between bone geometry and fracture risk of the proximal sesamoid bone (PSB) in forelimbs of Thoroughbred racehorses. The PSB surfaces were extracted from CT images of nonfractured forelegs (i.e., right foreleg in this study) of 6 racehorses with fractures in the contralateral (i.e., left) foreleg, and the right forelegs of 6 matched controls. Significant differences were detected at the abaxial margin of the medial PSB base which was found to be up to 3.5 mm more prominent in the fracture-group compared to the control-group. This study demonstrated a successful application of computational morphometry in bone. The detected anatomical differences may lead to a larger moment arm generated via the medial branch of the suspensory apparatus, increasing pressure on the sesamoid surface, and thus potentially predisposing to fracture. Findings from this pilot study not only increase the likelihood of accurate PSB fracture risk assessment, but also shed light on investigating the influence of sports and exercise on human athletes.

Brittle tail syndrome is an emerging infection in horses caused by a keratinolytic fungus Equicapillimyces hongkongensis gen. nov., sp. nov.

The newly described brittle tail syndrome causes weakening and breakage of the tail hair of horses. Extensive mycological and molecular studies showed that a novel fungus Equicapillimyces hongkongensis gen. nov., sp. nov. is the most likely cause of this syndrome. It is a septate branching hyaline mould which grows optimally at 30°C, requires nicotinic acid but is inhibited by cycloheximide, and specifically infects horse hair. Hyphae fill the core of infected hair shafts with short-necked structures resembling ascomata containing banana-shaped septate ascospore-like structures perforating the hair cortex from within. Compared to asymptomatic horses (n=31), horses with clinical signs of the syndrome (n=22) are significantly more likely to have positive E. hongkongensis gen. nov., sp. nov. smear (6.5% vs. 100%), culture (6.5% vs. 72.7%), and PCR (32.3% vs. 100%, P<0.001 for all). No other potential pathogens were found on bacteriological and mycological culture or PCR (for Trichophyton, Microsporum and Epidermophyton). Genotyping of pure E. hongkongensis gen. nov., sp. nov. isolates and their corresponding direct specimens by PCR and sequencing of the 18S rRNA, ITS1-5.8S-ITS2, 28S rRNA, beta-actin, beta-tubulin, and elongation factor 1 alpha showed that they are all identical but unique, and related distantly to fungi mostly in the class Sordariomycetes and the family Ophiostomataceae. Its geographical distribution, environmental or animal reservoirs are still unknown. Besides the ugly appearance of infected horse tails, this fungus may emerge as another equine pathogen if it affects the skin and hoof of horses.

Statistical analysis of bone mineral density using voxel-based morphometry-an application on proximal sesamoid bones in racehorses.

Voxel-based morphometry (VBM) is a computational technique that has been used to analyze statistical differences between groups of MR brain images. This study outlines a new VBM pipeline, designed for determining statistical variation in bone mineral density (BMD). CT images of proximal sesamoid bone (PSB) specimens from the right forelimb of six racehorses that had suffered PSB fractures were compared with six age-matched control specimens. Following segmentation, masked gray-scale images were co-aligned to a statistical template generated with all 12 CT datasets iteratively. Student t-tests were performed voxel-by-voxel on spatially aligned 3D images to reveal significant differences in the spatial variation of bone density between the fracture and control groups. Overall density and densities from the axial and abaxial areas of PSBs were compared between groups. The BMD in abaxial regions of the medial and lateral PSBs of the fracture group were 12.7% (p = 0.044) and 13.5% (p = 0.047) higher, respectively, than controls. The overall mean density of paired PSB and the medial and lateral PSBs separately were higher in the fracture group. The VBM pipeline facilitates detailed comparison of density variation between bone groups at the voxel level.