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Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations of wild and domestic animals. In horse racing industries, they are extremely important for identifying and registering individuals and doping control to ensure fair competition. In this study, we constructed an individual identification panel for horses by using insertion and deletion (INDEL) markers. The panel included 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89 Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180-0.489 in all markers. The total probability of exclusion for paternity testing, power of discrimination, and probability of identity were 0.9994271269, >0.9999999999, and 0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals), and no contradictions were observed in genetic inheritance among the trios. When this panel was applied to the trios (52 trios) containing false fathers, an average of 7.3 markers excluded parentage relationships. In addition, genomic DNA extracted from the urine of six horses was partially genotyped for 39 markers, and 6-28 markers were successfully genotyped. The newly constructed panel has two advantages: a low marker mutation rate compared with short tandem repeats and a genotyping procedure that is as simple as short tandem repeat typing compared with single nucleotide variant typing. This panel can be applied for individual identification, paternity determination, and urine-sample identification in Thoroughbred horses., Competing Interests: The authors declare no competing interests concerning this study, including patents, products in development, or marketed products., (©2023 Catalyst Unit.)

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples., (©2023 The Japanese Society of Equine Science.)

The secretion of prolactin and growth hormone in response to exercise and emotional stresses was investigated in Thoroughbreds. Two experiments were performed: one with loading of only exercise stress and one with simultaneous loading of exercise and emotional stresses. Exercise stress was loaded in 4 steps using a treadmill for horses: pre-exercise period (5 min), walking period (6.5 min), galloping period (3 min), and cooling down period (10 min). Emotional stress was loaded by showing a loud video of an audience at a racetrack during the walking period. The results clearly demonstrated that exercise stress rapidly increased the secretion of prolactin and growth hormone and that secretion of them persisted for a specific period after the exercise. In addition, emotional stress promoted prolactin secretion., (©2021 The Japanese Society of Equine Science.)

Many studies have reported that advancing age in broodmares has a negative impact on the reproductive performance of horses. However, although the ages at first and last mating vary among broodmares, it is unknown how this variation affects the correlation between age and reproductive performance in mares. Here, in order to examine the effects of the ages at first and last mating, we analyzed all recorded mating events for Thoroughbreds in Japan from 1997 to 2017. We found that the live foal birth rate of mares with a younger age at first mating indeed declined at an earlier age than those with an older age at first mating and that the number of years since the first mating also contributes to the decline in the birth rate. We also found that the live foal birth rate and mean earnings of the produced foals are much higher for mares with an older age at last mating compared with mares with a younger age at last mating. Our results should aid breeders in assessing the value of broodmares and designing breeding strategies., (©2021 The Japanese Society of Equine Science.)

In the past decade, mass spectrometry has become an important technology for protein identification. Recent developments in mass spectrometry allow a large number of identifications in samples; therefore, mass-spectrometry-based techniques have been applied to the discovery of biomarkers. Here, we conducted a proteomic study to compare the proteomes in sera between healthy Thoroughbreds and Thoroughbreds with respiratory disease associated with transport (RDT). We found that four proteins, apolipoprotein F, lipopolysaccharide binding protein, lysozyme and protein S100-A8, were upregulated, while keratin 1 was downregulated in the RDT group. It is assumed that inflammation and immune response are involved in the changes of these proteins. The findings suggested that these proteins are potentially useful for elucidating the mechanism of development of RDT., (©2021 The Japanese Society of Equine Science.)

Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term., (©2020 The Japanese Society of Equine Science.)

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses., (©2020 The Japanese Society of Equine Science.)

Despite growing recognition of post-race exertional heat illness (EHI) in the horse racing industry, reports on its prevalence are limited. The purpose of this study was to investigate the prevalence of post-race EHI and climate conditions at racecourses in Japan. The overall prevalence of EHI from 1999 to 2018 was 0.04% (387 cases for 975,247 starters) in races operated by the Japan Racing Association (JRA). The yearly prevalence has been increasing, exceeding 0.07% in the last four years of the studied period. The overall prevalence in summer (May-September) was 0.086% (352 cases for 409,908 starters). The monthly prevalence varied among the 10 JRA racecourses, which are distributed from latitude 34 to 43°N, ranging from no cases to 0.459%. During summer, prevalence of post-race EHI was high when the mean monthly ambient temperature was high at a racecourse. To evaluate climate conditions, we investigated the wet-bulb globe temperature (WBGT, °C) from 9 AM to 5 PM on sunny race days in July and August of 2017 and 2018 at three racecourses with a high prevalence of EHI among the 10 racecourses. The durations of time during which WBGT was between 28 and 33°C at these three courses were 95, 94, and 65% of the minutes measured, respectively. This result indicated that most races on the sunny summer days were held when WBGT was between 28 and 33°C at the three racecourses. These findings could be useful in developing the appropriate countermeasures to be taken during hot weather at each of the studied racecourses.

Twelve Thoroughbred racehorses were diagnosed as epiglottic entrapment at the Korea Racing Authority equine hospital. Four different surgical correction techniques were used to treat epiglottic entrapment: the transnasal hook bistoury, transoral unshielded hook bistoury, transnasal shielded hook bistoury, and transendoscopic laser techniques. Eleven cases were surgically resolved eventually, with one case of recurrence. Five complications related to surgical correction occurred: a severe nasal passage laceration and bleeding (n=1), epiglottic laceration (n=1), epiglottis tip burns (n=2), and moderate nasal passage laceration (n=1). Intraoperative complications occurred in approximately 41.7% of cases. Thus, the possibility of surgical complication should be considered thoroughly when choosing a surgical technique for correction of epiglottic entrapment.

There have been few reports about temporohyoid osteoarthropathy in Japanese horses. The aim of this study was to describe the symptoms and management of temporohyoid osteoarthropathy and to investigate its association with crib-biting behavior, which is commonly observed in Japanese horses. The data concerning case details, signs, diagnosis procedures, treatment, and outcomes were collected retrospectively from the medical records of 11 Thoroughbreds with temporohyoid osteoarthropathy. The trainers and owners were asked whether the horses had displayed crib-biting behavior. Nine of the horses were diagnosed by guttural pouch endoscopy, and two were diagnosed by skull radiography. Eight horses were treated medically, of which three (37%) showed improvement; the other three horses underwent ceratohyoidectomy, with two (67%) showing improvement. Crib-biting behavior was observed in eight (73%) of the horses. A comparison of data regarding crib-biting behavior among 437 Thoroughbreds from six local riding schools indicated a significant association between temporohyoid osteoarthropathy and crib-biting behavior: the odds ratio for a horse with temporohyoid osteoarthropathy to exhibit crib-biting behavior compared with the general population was 12 (95% confidence interval, 3-45), and horses exhibiting crib-biting behavior were significantly more likely to have temporohyoid osteoarthropathy (P<0.001, Fisher's exact test). This suggested that crib-biting behavior may be a risk factor for temporohyoid osteoarthropathy., (©2019 The Japanese Society of Equine Science.)

Thoroughbred racehorses are commonly affected with superficial digital flexor (SDF) tendinopathy. This study aimed to identify risk factors for SDF tendinopathy in racing horses. The authors selected racehorses (n=292) with SDF tendinopathy from the medical records of a racetrack. As a risk factor associated with track-related variables, the SDF tendinopathy odds ratio (OR) was significantly high for a sloppy track surface compared with a standard track surface. Regarding risk factors associated with race-related variables, the SDF tendinopathy OR was significantly high in the following cases: when the order of arrival was worse than or equal to the 10th place; when the racehorses started to run a short race and when the racehorses' favourites were worse than or equal to the 8th place. Regarding risk factors associated with racehorse-related variables, the body weight of racehorses with SDF tendinopathy was significantly heavier than that of control horses. When there was a decrease in body weight since previous racing, the SDF tendinopathy OR was significantly high. Regarding risk factors associated with race career-related variables, when the charge in the race distance was short, the SDF tendinopathy OR was significantly high. As a countermeasure to prevent SDF tendinopathy, a sloppy track surface should be avoided during the race by guiding the horse toward to more solid track surface. Selecting long-distance races with slow speed, if possible, could reduce the risk of SDF tendinopathy., (©2019 The Japanese Society of Equine Science.)

The lying behavior of Thoroughbred foals on breeding farms was continuously measured using triaxial accelerometers. Accelerometers were attached on the side of the hind limb cannon and under the halter of six foals to record triaxial angle data every 10 sec for a period of 24 hr. Lying behavior was divided into sternal lying and lateral lying based on head angle. Sampling was performed for two consecutive days each week until weaning. Sampling periods were divided into two periods on the basis of pasturing time: daytime pasturing (period A: 7-hr pasturing period, 2-60 days of age) and overnight pasturing (period B: 19-hr pasturing period, 32-152 days of age). Lying duration and frequency were longer and higher, respectively, in period A (44.6% of the time and 26.8 ± 7.4 times per day) than those in period B (27.7% of the time and 15.3 ± 4.2 times per day). In addition, foals spent more time laterally in period A (48.1% of total lying time) than in period B (38.9% of total lying time). Foals lie down longer in stalls than in pastures (Period A, 56.3 vs. 16.0%; Period B, 52.5 vs. 21.1%). Lateral lying was also longer in the stall than in the pasture. Lying duration and frequency changed with age in period A. Wet weather prevented lying behavior in the pasture. These results suggested that age, weather, and pasturing time affected the lying behavior of Thoroughbred foals under management at a breeding farm. Triaxial accelerometers may be useful for monitoring equine lying behavior.

Although severe blood coagulopathy in horses with acute colitis causes multiple organ failure, which may be fatal, few studies have focused on the correlation between the fluctuations of coagulation parameters and severity of colitis. In this study, we evaluated the fluctuations of coagulation parameters in 14 Thoroughbred racehorses with acute colitis for 5 days from the day of hospitalization and compared them between 5 survivors and 9 non-survivors. Noteworthy features in the non-survivors were that antithrombin activity and fibrin degradation products continuously decreased and increased, respectively, for 4 days or more and that thrombin-antithrombin complexes increased in the last 2 days before death. Thus, these parameters should be continuously monitored to observe these fluctuations in assessing the severity of acute colitis.

Although hyperglycemia at admission with colic has been reported to have a poor prognosis, there is no report specifically about acute colitis with systemic inflammatory response syndrome (SIRS) in horses. In this study, we measured blood glucose (Glu), insulin (Ins), and cortisol (Cor) levels in 17 Thoroughbred racehorses diagnosed as having acute colitis with SIRS, and examined the relationship between time-dependent changes in Glu, Ins, and Cor and prognosis. Glu levels were high in 3 horses at admission, but thereafter no horses had persistently high Glu levels. There was no significant difference in Glu, Ins, and Cor levels within 72 hr between surviving and non-surviving horses. In conclusion, the Glu level is unlikely to be a useful prognostic biomarker in acute colitis with SIRS.

The purpose of this study was to evaluate foaling rates of mares that were rebred after the pregnancy loss in same reproductive season and to examine factors influencing them in Hidaka, Japan. The study included 82 Thoroughbred mares that had experienced pregnancy loss. The foaling rate of the mares that were rebred after pregnancy loss was 57.3%. The foaling rate decreased as the period until detection of pregnancy loss increased. Aging and lower body condition score of mares decreased the foaling rate.

Global positioning system (GPS) units are now lightweight and compact. They have proven useful for analyzing the behavioral characteristics of horses in pastures. Because the GPS records data in latitude and longitude, it may be feasible to calculate the distance between GPS units. The present study aimed to confirm the applicability of GPS units in behavioral studies on horses. For this, we analyzed the accuracy of the distances calculated from GPS units using Hubeny's distance formula and of the monthly changes in interindividual distances obtained from GPS units worn by Thoroughbred dams and their foals in a pasture until weaning. The calculated inter-GPS distances were highly accurate. The regression line was linear, and the squared correlation coefficient (r 2 ) was 0.9998. During the first month of age, the interindividual dam-dam and foal-foal distances were significantly greater than the dam-foal distance. During the second month of age, the dam-foal distance increased once and gradually decreased up to the fifth month of age. During the sixth month of age, the dam-foal distance was significantly greater than the foal-foal distance. The GPS distances calculated using Hubeny's distance formula were useful for analyzing changes in interindividual distances in a herd of Thoroughbred dams and their foals. Most likely, calculation of the distance between GPS units worn on equine head collars is likely to become a very useful tool as an objective index for quantifying equine behavioral observations.

Body weight is an important trait to confirm growth and development in humans and animals. In Thoroughbred racehorses, it is measured in the postnatal, training, and racing periods to evaluate growth and training degrees. The body weight of mature Thoroughbred racehorses generally ranges from 400 to 600 kg, and this broad range is likely influenced by environmental and genetic factors. Therefore, a genome-wide association study (GWAS) using the Equine SNP70 BeadChip was performed to identify the genomic regions associated with body weight in Japanese Thoroughbred racehorses using 851 individuals. The average body weight of these horses was 473.9 kg (standard deviation: 28.0) at the age of 3, and GWAS identified statistically significant SNPs on chromosomes 3 (BIEC2&#95;808466, P=2.32E-14), 9 (BIEC2&#95;1105503, P=1.03E-7), 15 (BIEC2&#95;322669, P=9.50E-6), and 18 (BIEC2&#95;417274, P=1.44E-14), which were associated with body weight as a quantitative trait. The genomic regions on chromosomes 3, 9, 15, and 18 included ligand-dependent nuclear receptor compressor-like protein (LCORL), zinc finger and AT hook domain containing (ZFAT), tribbles pseudokinase 2 (TRIB2), and myostatin (MSTN), respectively, as candidate genes. LCORL and ZFAT are associated with withers height in horses, whereas MSTN affects muscle mass. Thus, the genomic regions identified in this study seem to affect the body weight of Thoroughbred racehorses. Although this information is useful for breeding and growth management of the horses, the production of genetically modified animals and gene doping (abuse/misuse of gene therapy) should be prohibited to maintain horse racing integrity.

Transcriptome analyses based on DNA microarray technology have been used to investigate gene expression profiles in horses. In this study, we aimed to identify exercise-induced changes in the expression profiles of genes in the peripheral blood of Thoroughbred horses using DNA microarray technology (15,429 genes on 43,603 probes). Blood samples from the jugular vein were collected from six horses before and 1 min, 4 hr, and 24 hr after all-out running on a treadmill. After the normalization of microarray data, a total of 26,830 probes were clustered into four groups and 11 subgroups showing similar expression changes based on k-mean clustering. The expression level of inflammation-related genes, including interleukin-1 receptor type II (IL-1R2), matrix metallopeptidase 8 (MMP8), protein S100-A8 (S100-A8), and serum amyloid A (SAA), increased at 4 hr after exercise, whereas that of c-Fos (FOS) increased at 1 min after exercise. These results indicated that the inflammatory response increased in the peripheral blood cells after exercise. Our study also revealed the presence of genes that may not be affected by all-out exercise. In conclusion, transcriptome analysis of peripheral blood cells could be used to monitor physiological changes induced by various external stress factors, including exercise, in Thoroughbred racehorses.

Five Thoroughbred foals (age, 8-33 weeks; median age, 31 weeks; weight, 122-270 kg; median weight, 249 kg) exhibiting ataxia with suspected cervical myelopathy (n=4) and limb malformation (n=1) were subjected to computed tomographic (CT) myelography. The areas of the subarachnoid space and cervical cord were measured on transverse CT images. The area of the cervical cord was divided by the area of subarachnoid space, and stenosis ratios were quantitatively evaluated and compared on the basis of histopathological examination. The sites with a ratio above 52.8% could have been primary lesion sites in the histopathological examination, although one site with a ratio of 54.1% was not a primary lesion site. Therefore, in this study, a ratio between 52.8-54.1% was suggested to be borderline for physical compression that damages the cervical cord. All the cervical vertebrae could not be scanned in three of the five cases. Therefore, CT myelography is not a suitable method for locating the site of compression, but it should be used for quantitative evaluation of cervical stenosis diagnosed by conventional myelography. In conclusion, the stenosis ratios determined using CT myelography could be applicable for detecting primary lesion sites in the cervical cord.

Ligand-dependent nuclear receptor compressor-like (LCORL) encodes a transcription factor, and its polymorphisms are associated with measures of skeletal frame size and adult height in several species. Recently, the single nucleotide polymorphism (SNP) BIEC2-808543 located upstream of LCORL was identified as a genetic diagnostic marker associated with withers height in Thoroughbreds. In this study, 322 Thoroughbreds-in-training were genotyped for BIEC2-808543 to evaluate the association between genotype and body composition traits, including body weight, withers height, the ratio of body weight to withers height, chest circumference, and cannon circumference. Of these, withers height and cannon circumference were significantly associated with LCORL genotypes throughout almost the entire training period in males and females. Animals with a C/T genotype had higher withers height (maximum differences of 1.8 cm and 2.1 cm in males and females, respectively) and cannon circumstance (maximum differences of 0.65 cm and 0.48 cm in males and females, respectively) compared with animals with a T/T genotype. These results suggested that the regulation of LCORL expression influences the skeletal frame size in Thoroughbreds and thus, indirectly affects the body weight. Although LCORL and BIEC2-808543 would be useful for selective breeding in Thoroughbreds, the production of genetically modified animals and gene doping based on genetic information should be prohibited in order to maintain racing integrity.

Oxidative stress has been reported to occur during surgery. It is important to reduce intraoperative oxidative stress to improve the postoperative prognosis. However, there are no reports regarding oxidative stress related to surgery in horses. In the present study, we measured pre and postsurgical diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP); the oxidative stress index (OSI) was then calculated (OSI=d-ROMs/BAP × 100). d-ROMs were not significantly different between the pre and postsurgical periods. However, BAP significantly decreased after surgery (P=0.02), and OSI significantly increased after surgery (P=0.02). Based on these results, it suggested that castration surgery under inhalation anesthesia decreases the antioxidant potential and causes oxidative stress in horses.

This study aimed to evaluate the influence of radiographic abnormalities of 2-year-old Thoroughbred horses that were listed at in-training sales in Japan, on whether they started to race or not at 2-3 years of age. Radiographs of 850 2-year-old Thoroughbreds in the in-training sales repository from 2007 to 2010 were reviewed, and 26 categories of radiographic abnormalities were found. Forty-three horses (5.1%, 43/850) did not start a race at 2-3 years of age. In accordance with the racing results for this age category, as determined by Fisher's exact test and multiple logistic regression analysis, none of the radiographic abnormalities were significantly related to failure to start a race. At 2 years of age, 198 horses (23.3%, 198/850) did not start a race. Horses with enlargement of the proximal sesamoid bones in the fore (9 of 19 horses) and hind limbs (5 of 9 horses) did not start a race at the age of 2 years, and fewer of these horses (fore, P=0.021; hind, P=0.030) started a race at the age of 2 years compared with the population of horses without these radiographic abnormalities. These results suggest that identification of radiographic enlargement of the proximal sesamoid bones during training sales could derail the racing debut of horses at the age of 2 years. However, this might not necessarily indicate a poor prognosis and resulting in retirement from racing at 2-3 years of age.

This study evaluated induction of anesthesia and cardiorespiratory and anesthetic effects during maintained anesthesia with the combination of alfaxalone, medetomidine, and butorphanol. Alfaxalone (1.0 mg/kg) was administered to induce anesthesia after premedication with medetomidine (7.0 µg/kg), butorphanol (25 µg/kg), and midazolam (50 µg/kg) in six Thoroughbred horses. Intravenous general anesthesia was maintained with alfaxalone (2.0 mg/(kg∙hr)), medetomidine (5.0 µg/(kg∙hr)), and butorphanol (30 µg/(kg∙hr)) for 60 min. Electrical stimulation of the upper oral mucosa was used to assess anesthetic depth at 10 min intervals during anesthesia. Heart rate (HR), respiratory rate (RR), and mean arterial pressure (MAP) were measured. All horses became recumbent within 1 min after alfaxalone administration. Induction scores were 5 (best) in five horses and 4 in one horse. During the 60-min anesthesia, average HR, RR, and MAP were 35.8 ± 2.6 beat/min, 4.7 ± 0.6 breath/min, and 129 ± 3 mmHg, respectively. No horse moved with electrical stimulation; however, two horses experienced apnea (no respiration for 1 to 3 min). Recovery scores were 5 (best) in two horses and 3 in four horses. These results suggest that alfaxalone is effective for induction and maintenance of anesthesia and analgesia when combined with butorphanol and medetomidine for 60 min in Thoroughbreds. However, respiratory depression might require support.

Autopsy imaging (Ai), postmortem imaging before necropsy, is used in human forensic medicine. Ai was performed using computed tomography (CT) for a 1-month-old Thoroughbred foal cadaver found in a pasture. CT revealed pericardial effusion, collapse of the aorta, bleeding in the lung lobe, gas in the ventricles and liver parenchyma, and distension of the digestive tract. Rupture in the left auricle was confirmed by necropsy; however, it was not depicted on CT. Therefore, Ai and conventional necropsy are considered to complement each other. The cause of death was determined to be traumatic cardiac tamponade. In conclusion, Ai is an additional option for determining cause of death.

The running performance of Thoroughbred racehorses has been reported to peak when they are between 4 and 5 years old. However, changes in their racing speed by month or season have not been reported. The purposes of this study were to reveal the average racing speed of Thoroughbreds, and observe changes in their average speed with age. The surveyed races were flat races on turf and dirt tracks with firm or standard track conditions held by the Japan Racing Association from January 1st, 2002 to December 31st, 2010. The racing speed of each horse was calculated by dividing the race distance (m) by the horse's final time (sec). Average speeds per month for each age and distance condition were calculated for each gender group when there were 30 or more starters per month for each age and distance condition for each gender group. The common characteristic change for all conditions was an average speed increase up until the first half of the age of 4 years old. The effect of increased carry weight on average speed was small, and average speed increased with the growth of the horse. After the latter half of the age of 4 years old, the horses' average speed remained almost constant, with little variation. It is speculated that decreases in the weight carried; and the retirement of less well performing horses; are responsible for the maintenance of average speed.

Bone mineral density (BMD) is one of the indications of the strength and health. BMD measured by quantitative computed tomography (QCT) was compared with that measured by dual energy X-ray absorptiometry (DXA) and radiographic bone aluminum equivalence (RBAE). Limbs were removed from horses that had been euthanized for reasons not associated with this study. Sixteen limbs (left and right metacarpals and metatarsals) from 4 horses were used to compare BMD as measured by QCT with those measured by DXA and RBAE. There was a strong correlation between BMD values measured by QCT and those measured by DXA (R(2)=0.85); correlation was also observed between values obtained by QCT and those obtained by RBAE (R(2)=0.61). To investigate changes in BMD with age, 37 right metacarpal bones, including 7 from horses euthanized because of fracture were examined by QCT. The BMD value of samples from horses dramatically increased until 2 years of age and then plateaued, a pattern similar to the growth curve. The BMD values of bone samples from horses euthanized because of fracture were within the population range, and samples of morbid fracture were not included. The relationship between BMD and age provides a reference for further quantitative studies of bone development and remodeling. Quantitative measurement of BMD using QCT may have great potential for the evaluation of bone biology for breeding and rearing management.

Aim 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.

Rupture of the gastrocnemius muscle is occasionally found in neonatal foals associated with dystocia and assisted delivery. In this report, 3 cases of gastrocnemius muscle disruption in newborn Thoroughbred foals (6, 5 and 2 days old) are reported. In all cases, the foals were presented with inability to rise unassisted postpartum, a dropped tarsus and swelling in the caudal aspect of the thigh accompanied by a hematoma. Ultrasonography, radiography, computerized tomography (CT) and subsequent autopsy were performed to confirm the clinical and pathological features of these cases.

We propose an approach of estimating individual growth curves based on the birthday information of Japanese Thoroughbred horses, with considerations of the seasonal compensatory growth that is a typical characteristic of seasonal breeding animals. The compensatory growth patterns appear during only the winter and spring seasons in the life of growing horses, and the meeting point between winter and spring depends on the birthday of each horse. We previously developed new growth curve equations for Japanese Thoroughbreds adjusting for compensatory growth. Based on the equations, a parameter denoting the birthday information was added for the modeling of the individual growth curves for each horse by shifting the meeting points in the compensatory growth periods. A total of 5,594 and 5,680 body weight and age measurements of Thoroughbred colts and fillies, respectively, and 3,770 withers height and age measurements of both sexes were used in the analyses. The results of predicted error difference and Akaike Information Criterion showed that the individual growth curves using birthday information better fit to the body weight and withers height data than not using them. The individual growth curve for each horse would be a useful tool for the feeding managements of young Japanese Thoroughbreds in compensatory growth periods.

The purpose of this study was to create a lower forelimb model of the Thoroughbred horse for measuring the force in the superficial and deep digital flexor tendons (SDFT and DDFT), and the suspensory ligament (SL) during a trot. The mass, centers of gravity, and inertial moments in the metacarpus, pastern, and hoof segments were measured in 4 Thoroughbred horses. The moment arms of the SDFT, DDFT, and SL in the metacarpophalangeal (fetlock) and distal interphalangeal (coffin) joints were measured in 7 Thoroughbred horses. The relationship between the fetlock joint angle and the force in the SL was assessed in 3 limbs of 2 Thoroughbred horses. The forces in the SDFT, DDFT, and SL during a trot were also measured in 7 Thoroughbred horses. The mass of the 3 segments, and the moment arms of the SDFT and DDFT in the fetlock joint of the Thoroughbred horses were smaller than those of the Warmblood horses, whereas the other values were almost the same in the 2 types. The calculated force in the SDFT with this Thoroughbred model reached a peak (4,615 N) at 39.3% of the stance phase, whereas that in the DDFT reached a peak (5,076 N) at 51.2% of the stance phase. The force in the SL reached a peak (11,957 N) at 49.4% of the stance phase. This lower forelimb model of the Thoroughbred can be applied to studying the effects of different shoe types and change of hoof angle for the flexor tendon and SL forces.

We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared the horse probiotics LacFi(TM), consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21, and Bifidobacterium boum HU. Here, we found that the five LacFi(TM) constituent strains remarkably suppressed pro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-β. The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumor necrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protective activity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFi(TM) constituent strains were detected in neonatal LacFi(TM)-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gel electrophoresis and culture methods. These five strains were found to be the predominant lactobacilli and bifidobacteria in the intestinal microbiota of LacFi(TM)-administered Thoroughbreds. Administration of LacFi(TM) to neonatal Thoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFi(TM)-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth. LacFi(TM) treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration of diarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). These results indicate that the LacFi(TM) probiotics regulates intestinal function and contributes to diarrhea prevention.

It 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.

The aim of this study was to clarify the relationship between the placental retention time (PRT) and the reproductive performance following mating at the foal heat in Thoroughbreds. For this purpose, we interviewed 292 farmers over a period of 3 years with questionnaires evaluating foaling, expulsion of placenta and reproductive performance at the foal heat in 1,432 mares. The obtained data were later compared with a previous study of heavy draft mares. The average of the PRT of the 1,432 Thoroughbred mares was 58 ± 88 min (mean ± SD). The mean PRT of Thoroughbreds was significantly shorter than that of the148 min of heavy draft mares. The incidences of retained placenta (RP) occurring in the Thoroughbred mares were 5.2 and 4.0%, for over 3 and 4 hr after foaling, respectively. The incidence of RP over 4 hr was significantly lower than that of 25% in heavy draft mares. The pregnancy rate at foal heat of the mares in which PRT was less than 3 hr was 37%, and it significantly decreased to 11% for those with PRT of more than 3 hr. In the comparison of the reproductive performance between Thoroughbred and heavy draft mares, the pregnancy rate of Thoroughbreds dropped drastically to 10% when PRT exceeded 40, and in consequence, the pregnancy rate of Thoroughbreds was significantly lower than the 30% of heavy draft mares, which had a PRT of over 4 hr. In conclusion, the Thoroughbred mares had a low incidence of RP, however, a PRT exceeding 3 hr severely affected the reproductive performance at the foal heat.

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.

Although high oxygen consumption in skeletal muscle may result in severe oxidative stress, there are no direct studies that have documented free radical production in horse muscles after intensive exercise. To find a new parameter indicating the muscle adaptation state for the training of Thoroughbred horses, we examined free radical formation in the muscle by using electron paramagnetic resonance (EPR). Ten male Thoroughbred horses received conventional training for 18 weeks. Before and after the training period, all horses performed an exhaustive incremental load exercise on a 6% incline treadmill. Muscle samples of the middle gluteal muscle were taken pre-exercise and 1 min, 1 hr, and 1 day after exercise. Muscle fiber type composition was also determined in the pre-exercise samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. We measured the free radical in the muscle homogenate using EPR at room temperature, and the amount was expressed as relative EPR signal intensity. There was a significant increase in Type IIA muscle fiber composition and a decrease in Type IIX fiber composition after the training period. Before the training period, the mean value of the relative EPR signal intensity showed a significant increase over the pre-exercise value at 1 min after the exercise and an incomplete recovery at 24 hr after the exercise. While no significant changes were found in the relative EPR signal intensity after the training period. There was a significant relationship between percentages of Type IIA fiber and change rates in EPR signal intensity at 1 min after exercise. The measurement of free radicals may be useful for determining the muscle adaptation state in the training of Thoroughbred horses.

To investigate the breeding structure in the Japanese Thoroughbred population, we applied a demographic analysis to the populations of foals produced from 1978 to 2005. The migration rate estimated from the proportion of foals produced by imported breeding horses was around 40% over the investigated period. After early 1990s, the migration rate through stallions imported from USA sharply increased. The average generation interval was within range of 10.5-11.5 years. The longer generation interval of Thoroughbred was considered to be a reflection of the fact that Thoroughbred horses begin breeding only after completing their performance in races. After the peak of 729 in 1993, the number of sires of foals progressively declined to 358 in 2005. Although the coefficient of variation of the progeny number of sires was within range of 1.0-1.2 until early 1990s, it gradually increased and reached the value of 1.6-1.7 in recent years. The effective number of sires consistently decreased after the peak of 302.6 in 1992, and reached 120-130 in recent years, which is 25-30% of the actual number of sires. In parallel, the demographic estimate of the effective population size declined after early 1990s. The main cause of the observed change in the breeding structure was inferred to be the intensive use of a limited number of stallions for breeding.

Using a dataset of 274 male Thoroughbred racehorses in the United States, we study the effect of age on racing performance. Beyer speed figures, which are uniform measures of racing performance across distance and racing surface, are utilized in this study. A system of equations is estimated to determine quadratic improvement and decline in racing performance. We find that a typical horse's peak racing age is 4.45 years. The rate of improvement from age 2 to 4 1/2 is greater than the rate of decline after age 4 1/2. A typical horse will improve by 10 (horse) lengths in sprints (less than 1 mile) and 15 lengths in routes (one mile or greater) from age 2 to 4 1/2. Over the next five years the typical decline is 6 lengths for sprints and 9 1/2 lengths for routes.

We evaluated differences in muscle fiber recruitment patterns between continuous and interval training to develop an optimal training program for Thoroughbred horses. Five well trained female thoroughbred horses (3-4 years old) were used. The horses performed two different exercises on a 10% inclined treadmill: 90%VO2 max for 4 min (continuous) and 90% VO2 max for 2 min × 2 times with 10-min interval (interval). Muscle samples were obtained from the middle gluteal muscle before and immediately after the exercises. Four muscle fiber types (type I, IIA, IIA/X, and IIX) were immunohistochemically identified, and the optical density of periodic acid Schiff staining (OD-PAS) in each fiber type and glycogen content of the muscle sample were determined by quantitative histochemical and biochemical procedures, respectively. No significant differences were found in the OD-PASs and glycogen contents between the continuous and interval exercises, but the decreases in OD-PAS of fast-twitch muscle fibers were obvious after interval as compared to continuous exercise. Interval exercise may be a more effective training stimulus for the glycolytic capacity of fast-twitch muscle fiber. The data about muscle fiber recruitment can provide significant insights into the optimal training program not only for thoroughbred horses, but also for human athletes.

At 114 ranches in the Hidaka District of Hokkaido, a total of 1,118 Thoroughbred foals that were born from January to June 2003 were examined at different times from May to September of the same year to ascertain the occurrence of clubfoot. Clubfoot was seen in a total of 179 foals (16%) at 89 ranches (78%). Also, 124 of the 179 foals (69.3%) had clubfoot of grade II or higher, and it was found that grade I was likely to be overlooked or rapidly advance. In the present study, the occurrence of clubfoot was higher than expected in the investigated region. Hence, it is necessary to establish accurate diagnostic techniques and clubfoot guidelines to minimize the impact.

To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO2max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. SR Ca(2+)-ATPase activity of the muscle and glycogen content of each muscle fiber type were determined with biochemical analysis and quantitative histochemical staining, respectively. As compared to the pre value, the glycogen content of each muscle fiber type was reduced by 15-27% at 1 min, 20 min, and 60 min after the exercise and recovered to the pre value at 24 hr after exercise test. These results indicate that 24 hr is enough time to recover glycogen content after short-term intensive exercise. The mean value of the SR Ca(2+)-ATPase activity showed a slight decrease (not significant) immediately after exercise, and complete recovery at 60 min after exercise. There were no significant relationship between the changes in glycogen content of each muscle fiber type and SR Ca(2+)-ATPase. Although further studies are needed, SR Ca(2+)-ATPase is not a useful parameter to detect muscle fitness, at least in Thoroughbred horses.