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Vadadustat is a newly launched hypoxia-inducible factor stabilizer with anti-anemia and erythropoietic effects; however, its use in horses is expressly forbidden in both racing and equestrian competitions. Following our previous report on the pharmacokinetic study of vadadustat in horse plasma and urine, a long-term longitudinal analysis of vadadustat in horse hair after nasoesophageal administration (3 g/day for 3 days) to three thoroughbred mares is described in this study. Our main objective is to further extend the detection period of vadadustat for the purpose of doping control. Three bunches of mane hair from each horse were collected at 0 (pre), 1, 2, 3 and 6 month(s) post-administration. These hair samples were each cut into 2-cm segments and pulverized after decontamination of hair samples. The analyte in the powdered hair samples was extracted with liquid–liquid extraction followed by further purification by solid-phase extraction with strong anion exchange columns. The amount of vadadustat incorporated into the hair was quantified with a newly developed and validated method using liquid chromatography–high-resolution mass spectrometry. Our results show that vadadustat was confirmed in all post-administration hair samples, but its metabolites were not present. Thus, the detection window for vadadustat could be successfully extended up to 6 months post-administration. Interestingly, the 2-cm segmental analysis revealed that the tip of the drug band in the hair shifted along with the hair shafts in correspondence with the average hair growth rate (∼2.5 cm/month) but gradually diffused more widely from 2 cm at 1 month post-administration to up to 14 cm at 6 months post-administration. However, the loss in the total amount of vadadustat in hair over time was observed to most likely be due to the degradation of vadadustat. These findings will be useful for the control of abuse and/or misuse of vadadustat and the interpretation of positive doping cases. [ABSTRACT FROM AUTHOR]

Rationale: For the purpose of doping control, this is the first report of accurate quantification of four critical structural isomers of nicotine metabolites (trans‐3′‐hydroxycotinine, cis‐3′‐hydroxycotinine, 5′‐hydroxycotinine, and N′‐hydroxymethylnorcotinine) in equine plasma and urine for the establishment of their elimination profiles. Besides, the pharmacokinetic studies of trans‐3′‐hydroxycotinine and N′‐hydroxymethylnorcotinine in equine plasma and urine are also presented for the first time. Methods: The accurate quantification methods of the aforementioned four structural isomers in horse plasma and urine were successfully developed and validated using the solid‐phase extractions followed by liquid chromatography/tandem mass spectrometry analysis. Baseline chromatographic separation was achieved to completely differentiate these isomers, which shared the same selected reaction monitoring transition. Such methods were applied to post‐administration samples obtained from the nicotine and tobacco leaf administration studies for the establishment of pharmacokinetic profiles. Results: N′‐Hydroxymethylnorcotinine could be quantified for the longest period, ranging from 48 to 72 h in plasma and 96 h in urine after a single administration of 250 mg of nicotine and an equivalent amount of nicotine in tobacco leaves. In terms of detection, both N′‐hydroxymethylnorcotinine and trans‐3′‐hydroxycotinine could be detected up to the last sample collection time point (96 h), indicating that they are the most appropriate biomarkers for nicotine exposure. Conclusions: N′‐Hydroxymethylnorcotinine and trans‐3′‐hydroxycotinine were detected longest in plasma and urine samples after both nicotine and tobacco leaf administrations, and N′‐hydroxymethylnorcotinine was deemed most appropriate as a monitoring target due to its relatively higher abundance and slower elimination rate. These two biomarkers could also be used to differentiate sample contamination by tobacco products and genuine nicotine exposure to horse regardless of intentionality. [ABSTRACT FROM AUTHOR]

Summary: Background: For medication control in several jurisdictions, withdrawal time is the period of refrain from racing after drug administration. It is set by adding a safety period to an experimental detection time. However, there are no reports of statistical analyses of detection time for the determination of withdrawal time in flunixin meglumine‐treated horses. Objective: To analyse the population pharmacokinetics of flunixin in horses through the generation of a dataset for detection time statistical analysis and predictions via Monte Carlo simulation. Study design: Experimental study. Methods: Drug plasma and urine concentrations following single intravenous administration of flunixin 1.1 mg/kg bodyweight (BW) in 10 horses and multiple administration of q 24 hours for 5 days in 10 horses were measured using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Data were modelled using a nonlinear mixed effect model followed by Monte Carlo simulation. Irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were calculated using the Toutain approach. Detection times were obtained considering the time after the last administration for selected quantiles of 5000 hypothetical horses under the international screening limit (ISL) proposed by the International Federation of Horseracing Authorities (plasma: 1 ng/mL, urine; 100 ng/mL). Results: For a regimen of 1.1 mg/kg BW q 24 hours, the IPC and IUC values were 2.0 and 73.0 ng/mL respectively. Detection times in plasma above the ISL for 90% of simulated horses were estimated as 74 hours after a single 1.1 mg/kg dose administration, 149 and 199 hours after multiple doses over 5 days at either 24‐ or 12‐hour intervals respectively. Corresponding detection times in urine were 46, 68 and 104 hours respectively. Main limitation: Only female horses were investigated. Conclusions: Statistical detection times for different flunixin meglumine regimens indicated a delay of detection time in plasma after multiple administrations under ISL. [ABSTRACT FROM AUTHOR]

IOX4, a hypoxia‐inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long‐term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), 1, 2, 3, and 6 month(s) postadministration. Our results showed that the presence of IOX4 was identified in all postadministration horse hair samples, but no metabolite could be detected. The detection window for IOX4 could achieve up to 6‐month postadministration (last sampling point) by monitoring IOX4 in hair. In order to evaluate the longitudinal distribution of IOX4 over 6 months, a validated quantification method of IOX4 in hair was developed for the analysis of the postadministration samples. Segmental analysis of 2‐cm cut hair across the entire length of postadministration hair showed that IOX4 could be quantified up to the level of 1.84 pg/mg. In addition, it was found that the movement of the incorporated IOX4 band in the hair shaft over 6 months varied among the three horses due to individual variation and a significant diffusion of IOX4 band up to 10 cm width was also observed in the 6‐month postadministration hair samples. [ABSTRACT FROM AUTHOR]

The use of nicotine stimulants in horses is generally banned in horse racing and equestrian sports—accidental consumption of tobacco products is one of the possible causes of nicotine exposure in horses. The authors recently reported a comprehensive metabolic study of nicotine in equines, differentiating between nicotine exposure and sample contamination by means of a nicotine biomarker trans‐3′‐hydroxycotinine. To identify potential biomarkers for the differentiation of genuine nicotine administration and consumption of tobacco products, tobacco leaves (equivalent to 250 mg of nicotine) were nasoesophageally administered to three thoroughbred mares. Quantification methods of anatabine in plasma and urine were newly developed and validated and successfully applied to postadministration samples. Previously reported simultaneous quantification methods of eight target analytes including nicotine and its metabolites in plasma and urine were also applied to the samples. The results demonstrate that both trans‐3′‐hydroxycotinine and anatabine could be used as potential biomarkers in equine urine and plasma to indicate recent exposure to tobacco products in horses. As well, trans‐3′‐hydroxycotinine had the longest half‐life as a detectable metabolite in urine and plasma. To our knowledge, this is the first report of a comprehensive study of tobacco product detection in horses. [ABSTRACT FROM AUTHOR]

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron‐less genes from whole‐genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron‐less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron‐less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR‐based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR‐based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR‐based transgene detection in gene‐doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene‐doping detection as an additional confirmatory test to prevent false positives. [ABSTRACT FROM AUTHOR]

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real‐time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low‐copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low‐copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low‐copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene‐doping detection. [ABSTRACT FROM AUTHOR]

IOX4 is a hypoxia‐inducible factor prolyl hydroxylase (HIF‐PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF‐PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono‐hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post‐administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high‐resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O‐desbutyl IOX4, O‐desbutyl IOX4 glucuronide, four mono‐hydroxylated IOX4, N‐oxidized IOX4, and N‐oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O‐desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post‐administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h. [ABSTRACT FROM AUTHOR]

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix‐assisted laser desorption/ionisation‐time‐of‐flight mass spectrometry (MALDI‐TOF MS). As a model of therapeutic oligonucleotides, 22 bp‐long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short‐length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI‐TOF MS. In addition, a 17 bp sequence was determined using in‐source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control. [ABSTRACT FROM AUTHOR]

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)‐based transgene detection methods such as digital PCR and real‐time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real‐time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real‐time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real‐time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing. [ABSTRACT FROM AUTHOR]

Paracetamol is commonly used as an over‐the‐counter analgesic and antipyretic medication for humans, but not sold as a legitimate therapeutic medication for horses in Japan. However, paracetamol is commonly found in horses together with its two isomers, metacetamol and orthocetamol. We previously reported that paracetamol and orthocetamol were both present in selected feed consumed by Japanese racehorses. For the purpose of the doping control of paracetamol in local Japanese horses, we proposed establishing residue limits (Japanese residue limits, JRLs) to minimize the risk of reporting paracetamol from environmental contributions and to differentiate its presence from active administration. Recently, we proposed a preliminary JRL for paracetamol in equine plasma based on a population study of more than 300 Japanese racehorses. In this paper, we will present our studies on the urinary concentrations of paracetamol, metacetamol, and orthocetamol in postrace samples collected from 403 Japanese racehorses over a 1 year period, detected using liquid chromatography–electrospray ionization–tandem mass spectrometry. Our results revealed that the hydrolyzed urinary concentrations of paracetamol, metacetamol, and orthocetamol were in the range 15.7–2,360 ng/mL (median 363 ng/mL), 8.07–382 ng/mL (84.5 ng/mL), and 919–74,418 ng/mL (13,475 ng/mL), respectively. Based on our statistical model, the preliminary JRL of hydrolyzed paracetamol in equine urine was determined to be 7,400 ng/mL, at a risk factor of 1 in 10,000. Further investigations will be required to demonstrate the applicability and validity of our preliminary plasma and urine JRLs to local Japanese racehorses. [ABSTRACT FROM AUTHOR]

Objective: To clarify the detailed pharmacokinetics (PK) of orally administered voriconazole in tear fluid (TF) of horses for evaluating the efficacy of voriconazole secreted into TF against equine keratomycosis. Animals studied: Five healthy Thoroughbred horses. Procedures: Voriconazole was administrated through a nasogastric tube to each horse at a single dose of 4.0 mg/kg. TF and blood samples were collected before and periodically throughout the 24 hours after administration. Voriconazole concentrations in plasma and TF samples were analyzed using liquid chromatography‐electrospray tandem‐mass spectrometry. The predicted voriconazole concentration in both samples following multiple dosing every 24 hours was simulated by the superposition principle. Results: The mean maximum voriconazole concentrations in plasma and TF were 3.3 μg/mL at 1.5 h and 1.9 μg/mL at 1.6 h, respectively. Mean half‐life in both samples were 16.4 and 25.2 h, respectively. The ratio of predicted AUC0–24 at steady state in TF (51.3 μg∙h/mL) to previously published minimum inhibitory concentration (MIC) of Aspergillus and Fusarium species was >100 and 25.7, respectively. Conclusions: This study demonstrated the detailed single‐dose PK of voriconazole in TF after oral administration and simulated the predicted concentration curves in a multiple oral dosing. Based on the analyses of PK‐PD, the simulation results indicated that repeated oral administration of voriconazole at 4.0 mg/kg/d achieves the ratio of AUC to MIC associated with treatment efficacy against Aspergillus species. The detailed PK‐PD analyses against pathogenic fungi in TF can be used to provide evidence‐based medicine for equine keratomycosis. [ABSTRACT FROM AUTHOR]

Nonsteroidal anti-inflammatory drugs such as flunixin meglumine have been used to treat signs of systemic inflammatory conditions, but it is also known to have the side effect to small intestine mucosa. It may be considered to be due to inhibition of both cyclooxygenase (COX)-1 and COX-2. On the other hand, meloxicam is widely used in equine clinical practice and an effective nonsteroidal anti-inflammatory drug with the preferential inhibitory effect on COX-2. However, it has not yet been evaluated in equine systemic inflammation. The aim of this study was to evaluate the effect of meloxicam administered 60 minutes prior lipopolysaccharide (LPS)-induced inflammatory response in five Thoroughbred horses using a crossover test. Clinical parameters including body temperature, heart rate, respiratory rate, behavioral pain score, and hoof wall surface temperature were recorded, and plasma tumor necrosis factor-alpha, cortisol, and leukocyte counts were measured at various times before and after LPS infusion for 420 minutes. At time points 60, 90 (P <.01), 120, and 180 (P <.05) minutes, pain scores were significantly lower in meloxicam-treated horses. There was no significant difference in other parameters. In the present study, we revealed the analgesic effect of meloxicam using an equine low-dose endotoxin model. • The effect of meloxicam on lipopolysaccharide-induced inflammatory responses in horses was evaluated. • Pre-administration of meloxicam, a preferential cyclooxygenase-2 inhibitor, reduced pain scores in an equine low-dose endotoxin model. • Meloxicam might be a new therapeutic option for systemic inflammatory conditions. [ABSTRACT FROM AUTHOR]

Thoroughbred racehorses are produced by mating small numbers of Arabian stallions and native British mares, and have been improved by selection of horseracing performance for about 300 years. While these improvements led to good performance as racehorses, they exposed horses to numerous medical disorders, aggravated by extensive exercise. Fractures are frequent medical disorders in Thoroughbred racehorses. In this study, fracture heritability was estimated using 3,927 Japanese Thoroughbred racehorses to elucidate the risk of racehorse fractures. The heritability estimates of all examined fractures were low (h2 = 0.06), while those of fractures in carpal bone and carpus (carpal bone plus distal radius) were moderate (h2 = 0.37, 0.24, respectively). Fracture occurrence age for carpal bone and distal radius was both 3.3 years old and was younger than that for other fractures. These results indicated that a larger proportion of the variation in the studied population was due to genetic factors for carpal fractures than for other fractures, while the fractures at other bones were largely affected by environmental factors, correlated with the athlete period (number year in racing). These findings contribute to develop a management plan for suppressing racehorse fractures and improving horseracing safety. [ABSTRACT FROM AUTHOR]

• Effects of meloxicam (MX) and flunixin meglumine (FM) were compared. • The potential of MX for treatment of systemic inflammatory response syndrome (SIRS)/endotoxemia was investigated. • MX and FM had similar analgesic effects against endotoxin-induced inflammation. • Given the adverse effects of nonselective COX inhibitors such as FM, MX may be beneficial in thoroughbred horses with SIRS/endotoxemia. Flunixin meglumine (FM), a nonselective cyclooxygenase (COX) inhibitor, is most frequently selected for the treatment of equine systemic inflammatory response syndrome (SIRS)/endotoxemia. However, FM has considerable adverse effects on gastrointestinal function. The aims of this study were to compare the effect of meloxicam (MX), a COX-2 selective inhibitor commonly used in equine clinical practice, with FM, and to investigate the potential for clinical application in horses with SIRS/endotoxemia. Fifteen horses were divided into three groups of five and orally administered MX (0.6 mg/kg), FM (1.1 mg/kg), or saline as placebo at 30 minutes after LPS challenge. Clinical parameters, including behavioral pain scores, were recorded and blood for clinical pathological data was collected at various times from 60 minutes before to 420 minutes after LPS infusion. The pain score were significantly lower in both the MX and FM groups than in the placebo group, with no significant difference between them. Body temperature was significantly lower in the MX and FM groups than in the placebo group. Heart rates and respiratory rates, hoof wall surface temperature, and leukocyte counts changed similarly between the MX and FM groups. TNF-α and cortisol were lower in the FM group than in the MX group. The results suggest that MX suppresses the inflammatory response after LPS infusion and has an analgesic effect similar to that of FM. Given the adverse effects of nonselective COX inhibitors, clinical application of MX may be beneficial in horses with SIRS/endotoxemia. [ABSTRACT FROM AUTHOR]

• A total of 724 jockey falls occurred in 17,459 maiden-class race starts • The overall jockey fall rate was 41.5 of 1,000 (95%: CI 38.6–44.5) • Increased odds of a jockey fall with dual direction, mare, inexperienced horse and apprentice jockey • Decreased odds of a jockey fall with the summer season and years 2013 to 2017 • There risk factors can form the foundation of risk mitigation measures Jockey safety is an important subject from a welfare perspective and public perception. This is the first retrospective case-control study that aims to identify risk factors associated with jockey falls (JF) in Thoroughbred jump races held by the Japan Racing Association (JRA). JF in 17,459 maiden-class race starts at eight racecourses from 2003 to 2017 were retrospectively reviewed. Data were extracted from a database and official accident reports maintained by the JRA. Thirteen possible risk factors were evaluated using multivariable logistic regression to identify those that were significantly associated with JF. A total of 724 JF were recorded, with an incidence rate of 41.5 falls per 1,000 starts (95% CI: 38.6–44.5). Final model included stable, horse age, year, season, course, horse sex, horse experience, and jockey experience. No two-way interactions were observed. Six risk factors were significantly associated with JF: Year (2003–2007 or 2008–2012 > 2013–2017; P =.0011), season (spring, autumn, or winter > summer; P =.0006), course type (dual direction > single direction; P <.0001), horse sex (female > male or gelding; P =.0003), horse experience (inexperienced horse > experienced horse; P <.0001) and jockey experience (apprentice jockey > experienced jockey; P =.0332) significantly affected the odds of JF. In agreement with overseas reports, our results suggest that the occurrence of JF is multifactorial and associated with jockey- and horse-related factors as well as environmental factors. To safeguard the welfare of jockeys, implementation of measures according to identified risk factors is recommended. [ABSTRACT FROM AUTHOR]

• Jockey falls in 715,210 starts from 2003 to 2017 were retrospectively reviewed • Seventeen variables were evaluated using multivariable logistic regression • Catastrophic musculoskeletal injury, surface type, jockey experience, distance, track and field size were associated with jockey falls • Identification of six risk factors provides a basis to form targeted strategies Jockey safety is of paramount importance from welfare perspective and public perception. This retrospective case-control study aims to identify risk factors associated with jockey falls (JF) in flat races of Japan Racing Association (JRA). JF in 715,210 race starts by 74,328 horses at 10 racecourses from 2003 to 2017 were reviewed. Data were extracted from a database maintained by JRA and from official accident reports issued by race stewards. Seventeen possible risk factors were evaluated using multivariable logistic regression, to identify those significantly associated with JF. A total of 992 JF incidents were recorded, with an incidence rate of 1.39 falls per 1,000 starts (95% CI: 1.30–1.48). 6 risk factors were significantly associated with JF. Odds increased with horses that sustained catastrophic musculoskeletal injury (CMI) (OR: 203; CI: 169–241; P < 0.001). Increased odds were also associated with dirt track surfaces (OR: 1.99; CI: 1.74–2.29; P < 0.001), apprentice jockeys (OR: 1.43; CI: 1.21–1.68; P < 0.001), smaller track sizes (OR: 1.41; CI: 1.24–1.61; P < 0.001), larger fields (OR: 1.25; CI: 1.07–1.47; P = 0.005), and longer race distances (OR per 200 m: 1.05; CI: 1.01–1.09; P = 0.02). Since CMI was identified as a major contributing factor for JF, measures to minimize CMI may lead to improvement of jockey safety. The increased odds associated with apprentice jockeys may indicate the importance of jockey education and training. For jockey safety, proper staffing of medical professionals especially for races on dirt, smaller track, larger fields, and longer distances is recommended. [ABSTRACT FROM AUTHOR]

In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommendation from Clinical and Laboratory Standards Institute guidelines. Among 583 enterococcus isolates, E. faecium and E. faecalis were identified for 48.2% (281/583) and 7.4% (43/583), respectively. One isolate that was representing E. faecium (153 isolates) and E. faecalis (31 isolates) from each sample was selected for antimicrobial susceptibility test. The highest rate of resistance for E. faecium isolates was observed against enrofloxacin (57.5%; 88/153), followed by streptomycin (32.0%; 49/153), kanamycin (18.3%; 28/153), gentamycin (5.9%; 9/153), erythromycin (5.9%; 9/153), and oxytetracycline (4.6%; 7/153). For E. faecium isolates, the highest resistance was observed against streptomycin (90.3%; 28/31), followed by kanamycin (41.9%; 13/31), gentamycin (29.0%; 9/31), lincomycin (9.7%; 3/31), oxytetracycline (6.5%; 2/31), erythromycin (6.5%; 2/31), tylosin (6.5%; 2/31), enrofloxacin (6.5%; 2/31), and chloramphenicol (3.2%; 1/31). The results indicated that enrofloxacin and aminoglycosides were highly resistant among tested antimicrobials. Continuous monitoring studies are useful to increase the awareness of the potential for AMR bacteria to arise from imprudent use of antimicrobials in TB racehorses in Japan. • The first study identifying antimicrobial-resistant enterococci in Thoroughbred racehorses in Japan. • Enrofloxacin was highly resistant among tested antimicrobials. • Enterococcus (3.3%) isolates showed multidrug resistant. [ABSTRACT FROM AUTHOR]

Ever since 'One Health' concept was introduced in early 2000s, judicious use of antimicrobials by veterinarians has become an issue of great concern. Recently, findings of anti-inflammatory effects in certain types of antimicrobials have raised a subject for discussion among racing authorities. Regulatory framework of antimicrobials in racing should be based on best interest of horse welfare and doping control perspective, but basic data on prevalence of antimicrobials are lacking. Analysis of 100 postrace urinary samples collected from 10 Japanese racecourses by targeting 21 antimicrobials using ultra performance liquid chromatography–tandem mass spectrometry resulted in detection of ceftiofur, cefalotin, cefalotin metabolite, dihydrostreptomycin, gentamicin, kanamycin, and oxytetracycline. Detection of antimicrobials critically important for resistance in human medicine was limited to a single sample. Oxytetracycline, which is known to possess anti-inflammatory effects, was detected in three samples. This may suggest the need for establishing a regulatory framework from doping control perspective and further studies to clarify pharmacologically relevant concentration of antimicrobials with such properties. • One hundred postrace urinary samples were analyzed using UPLC-MS/MS. • Antimicrobials were detectable in 60% of the sample. • Six types of antimicrobials were detected of 21 antimicrobials that were targeted. • Detection of antimicrobials critically important for drug resistance was limited. • Presence of antimicrobials with anti-inflammatory effects warranted regulation. [ABSTRACT FROM AUTHOR]

ABSTRACT Salbutamol is a short‐acting and selective beta‐2 adrenergic agonist. Inhaled (IH) administration of salbutamol is widely used to control lower respiratory tract disease in horses. Here, we estimated the pharmacokinetic parameters of salbutamol after a single intravenous (IV) or IH administration in six horses, and we statistically analysed the detection times with various dosing regimens. Plasma and urine concentrations of salbutamol were measured by liquid chromatography–tandem mass spectrometry, and data were modelled by using a nonlinear mixed effect model followed by Monte Carlo simulation (MCS). With IH salbutamol, the maximum plasma concentration was 0.12 ± 0.06 ng/mL at 0.29 ± 0.17 h after administration. Typical values were, for clearance, 1.53 L/kg/h; distribution volume at steady state, 5.43 L/kg; terminal half‐life, 6.06 h; IH bioavailability, 19.0%; and urine to plasma ratio, 2057. Statistically estimated 95th percentile detection times in the urine at levels below the international screening limit (0.5 ng/mL) proposed by the International Federation of Horseracing Authorities, as simulated in 5000 horses by MCS, were 44 h after 1.6 μg/kg q 24 and 54 h after 1.6 μg/kg q 4 h over a 3‐day IH administration period. [ABSTRACT FROM AUTHOR]

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry. [ABSTRACT FROM AUTHOR]

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0–2 cm) at 0.375–0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z -scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair. • Segmental hair analysis of daprodustat is developed for doping control in horses. • Capability for long-term drug monitoring for at least 6 months is demonstrated. • Drug-band movement in mane hair is consistent with reported equine hair growth rate. • Drug-band chronological diffusion is likely due to changes in hair growth phases. • Results suggest passive diffusion for daprodustat incorporation into equine hair. [ABSTRACT FROM AUTHOR]

Background: Vadadustat, a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, is a substance which carries a lifetime ban in both horse racing and equestrian competition. A comprehensive metabolic study of vadadustat in horses has not been previously reported., Objective: Metabolism and elimination profiles of vadadustat in equine plasma and urine were studied for the purpose of doping control., Methods: A nasoesophageal administration of vadadustat (3 g/day for 3 days) was conducted on three thoroughbred mares. Potential metabolites were comprehensively detected by differential analysis of full-scan mass spectral data obtained from both in vitro studies with liver homogenates and post-administration samples using liquid chromatography high-resolution mass spectrometry. The identities of metabolites were further substantiated by product ion scans. Quantification methods were developed and validated for the establishment of the excretion profiles of the total vadadustat (free and conjugates) in plasma and urine., Results: A total of 23 in vivo and 14 in vitro metabolites (12 in common) were identified after comprehensive analysis. We found that vadadustat was mainly excreted into urine as the parent drug together with some minor conjugated metabolites. The elimination profiles of total vadadustat in post-administration plasma and urine were successfully established by using quantification methods equipped with alkaline hydrolysis for cleavage of conjugates such as methylated vadadustat, vadadustat glucuronide, and vadadustat glucoside., Conclusion: Based on our study, for effective control of the misuse or abuse of vadadustat in horses, total vadadustat could successfully be detected for up to two weeks after administration in plasma and urine., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR., (©2021 The Japanese Society of Equine Science.)

Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time PCR assays. Digital PCR yielded a similar copy number regardless of the probe (P-true: 1230.7, P-false: 1229.7), whereas real-time PCR revealed a decrease in sensitivity based on C q values (P-true: 23.5, P-false: 29.7). When substituted primers were used, the detected copy number decreased in the digital PCR assay, and the C q value in real-time PCR was much higher. Interestingly, digital PCR copy numbers improved by performing PCR at a low annealing temperature, even if a substituted probe was used. Thus, when primer and probe sequences did not completely match the template transgene, digital PCR was relatively robust, but real-time PCR was less sensitive. Although PCR specificity may be reduced, PCR sensitivity can be improved by lowering the annealing temperature. If the target sequence is substituted to escape doping detection, it may be desirable to set the annealing temperature lower and use a more robust method, such as digital PCR, to increase the detection of positive cases, which will also result in fewer false-negative results.

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

Jockey safety is of paramount importance from the standpoint of welfare and public perception. Thus, an understanding of the epidemiology and associated risk factors is necessary to implement measures to reduce the jockey falls (JFs) and jokey injuries (JIs). This descriptive epidemiological study investigated the occurrence of JFs and JIs in 715,210 and 25,183 rides in flat and jump races, respectively, from 2003 to 2017. In flat races, the incidence rates of JFs and JIs were 1.4 and 0.6 per 1,000 rides, respectively. In jump races, they were 44.4 and 18.1 per 1,000 rides, respectively. In flat races, 56.8% of JFs at corners resulted in JIs. In jump races, the major causes of JFs and JIs were lost balance and hampered by a fallen horse at an obstacle. Our findings provide a basis to design a future study analyzing risk factors for JFs., (©2020 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.)

Extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored bla CTX-M-2 , and both bla TEM-1 and bla CTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all β-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and bla CTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs., (©2019 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.

Objective: To investigate the pharmacokinetics and antihistaminic effects (pharmacodynamics) of olopatadine in a small population of healthy horses after administration via nasogastric tube., Animals: 4 healthy adult Thoroughbreds., Procedures: Olopatadine (0.1 mg/kg, once) was administered via nasogastric tube. Blood samples were collected at predetermined time points for pharmacokinetic analyses of the drug in plasma. Olopatadine effects were investigated by measurement of cutaneous wheals induced by ID histamine injection (0.1 mL [10 μg]/injection) at predetermined time points. Inhibition effect ratios were calculated on the basis of measured wheal size (area) after versus before olopatadine administration., Results: Mean ± SD maximum plasma olopatadine concentration was 48.8 ± 11.0 ng/mL approximately 1.5 hours after administration. Median terminal half-life was 6.11 hours. Mean ± SD maximal effect was 88.2 ± 4.9% inhibition approximately 3.5 hours after drug delivery, and the inhibition effect remained > 80% for 12.5 hours after treatment. No signs of adverse clinical effects were observed., Conclusions and Clinical Relevance: Results suggested olopatadine may have a strong, long-term inhibitory effect against histamine-induced wheals in the skin of horses. Clinical research with a larger number of horses is warranted.

This study compares clinical characteristics between induction with thiopental/guaifenesin and propofol/ketamine in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine. Clinical records of 214 horses that underwent arthroscopic surgery between 2015 and 2016 were retrospectively retrieved. Horses were premedicated with medetomidine and midazolam to sedate at the adequate level for smooth induction, and then induced with either thiopental (4.0 mg/kg) and guaifenesin (100 mg/kg) in Group TG (n=91) or propofol (1.0 mg/kg) and ketamine (1.0 mg/kg) in Group PK (n=123). Anesthesia was maintained using sevoflurane with constant rate infusion of medetomidine. Quality of induction/recovery, sevoflurane requirement, cardiovascular function and recovery characteristics were evaluated. Anesthetic induction scores (median, range) for Group TG (5, 2-5) and Group PK (5, 2-5) were not significantly different. There were no significant differences in end-tidal sevoflurane concentration (mean ± standard deviation) between Group TG and Group PK (both 2.4 ± 0.2%). Dobutamine infusion rate (µg/kg/min) required for keeping mean arterial blood pressure (MAP) above 70 mmHg in Group PK (0.43, 0.10-1.40) was significantly lower than in Group TG (0.67, 0.08-1.56). Recovery score in Group PK (5, 2-5) was significantly higher than in Group TG (4, 2-5). Both propofol/ketamine and thiopental/guaifenesin provided a smooth induction of anesthesia. Moreover, induction with propofol/ketamine resulted in lower dobutamine requirements for keeping MAP above 70 mmHg during maintenance, and better quality of recovery. Induction with propofol/ketamine would be preferable to thiopental/guaifenesin in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine during arthroscopic surgery.

Arthritis is thought to cause oxidative stress in synovial fluid in humans, but there have been few reports in horses. To evaluate oxidative stress in synovial fluid in horses, this study used 19 horses with unilateral fracture of the carpal joint bone. Synovial fluid was collected from the carpal joint on the fracture (arthritis group) and contralateral (control group) sides. Diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) were then measured, and the oxidative stress index (OSI) was calculated. d-ROMs and OSI of the arthritis group were significantly higher than the control group. BAP of the arthritis group was significantly lower than the control group. Thus, this study revealed that oxidative stress develops in the synovial fluid of horses during arthritis.

Synovium-derived mesenchymal stromal cells (SM-MSCs) from seven Thoroughbreds with naturally occurring intra-articular fracture proliferated to over ten million cells by the second passage. Using three experimental Thoroughbreds, columnar osteochondral defects were made arthroscopically at the bilateral distal radius. Five million allogenic SM-MSCs were implanted into the right defect, and another five million were injected into the right radio-carpal joint (implantation site). No SM-MSCs were implanted into the left defect or the same joint (control site). At 3 and 6 weeks after surgery, ten million autologous SM-MSCs were injected into the right joints. Radiolucent volumes of defects calculated by analysis of postmortem CT images 9 weeks after surgery were decreased in implanted sites compared with control sites in all horses. The average scores for ICRS gross and histopathological grading scales in implanted sites were equal to or higher than those of the controls. These results suggest that allogenic implantation and subsequent autologous injection of SM-MSCs might not obstruct subchondral bone formation in defects.

Equine scintigraphy has been legally permitted in Japan since 2009; however, it has not yet been a routine modality for horses. One reason is the legal regulations concerning the disposal of contaminated bedding. However, overseas, the bedding after scintigraphy can be disposed following radioactivity decay, but this is not allowed in Japan. Therefore, beddings are required to stored permanently in a controlled area, implying that large amounts of beddings such as straw would be kept untreated, which is quite unpractical. This may cause a hospital owner to hesitate to construct an equine scintigraphy facility. Therefore, it is proposed that water-dispersed paper bedding is disposed as aqueous waste after radioactivity decay. The purpose of this study was to check the availability of bedding, thus radioisotopes were not used in this study. Three horses were housed individually in stalls covered with water-dispersed paper bedding for 48 hr. Physical condition, including body weight, was monitored, and a complete blood cell count and biochemical analysis were conducted. The results revealed that physical conditions and results of blood analysis were all stable within the normal range, and the veterinarian did not find any specific abnormality in any of the three horses. No marked changes in the levels of blood cortisol were observed before and after stalling, suggesting almost no stress for the horses. Because the water-dispersed paper bedding did not negatively affect the horses, it can be used as a substitute for conventional straw bedding.

It is desirable to reduce surgery-induced oxidative stress (OS) because it can cause immune suppression and delayed wound healing. Propofol is known to have antioxidant potential and to reduce OS in humans, but there have been no studies of this issue in horses. This study was conducted to evaluate OS under three different propofol administration protocols in Thoroughbred racehorses undergoing arthroscopic surgery with sevoflurane anesthesia. Blood oxidative stress (bOS) and blood antioxidant power (bAP) were used as OS biomarkers. Both bOS and bAP significantly decreased after surgery in all groups, but no differences in these reductions were found among them. Different propofol administration protocols with sevoflurane anesthesia did not cause a difference in OS in Thoroughbred racehorses that underwent arthroscopic surgery.

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.

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.

The oxidant and antioxidant equilibrium is known to play an important role in equine medicine and equine exercise physiology. There are abundant findings in this field; however, not many studies have been conducted for reference ranges of oxidative stress biomarkers in horses. This study was conducted to determine the reference values of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) using blood samples from 372 (191 males, 181 females) Thoroughbred racehorse aged 2 to 5 (3.43 ± 1.10 (mean ± SD)) years old. There were obvious gender differences in oxidative biomarkers, and growth/age-related changes were observed especially in females. Gender and age must be considered when interpreting obtained oxidative stress biomarkers for diagnosis of disease or fitness alterations in Thoroughbred racehorses.