Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma.

OBJECTIVE: To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). ANIMALS: 6 adult university-owned horses. METHODS: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-ß1, IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays. RESULTS: There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP. CLINICAL RELEVANCE: These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Pharmacokinetics of grapiprant and effects on TNF-alpha concentrations following oral administration to horses.

Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses received a single oral administration of 15 mg/kg. Plasma concentrations were determined for 96 h using liquid chromatography-tandem mass spectrometry. Non-compartmental analysis was used to determine pharmacokinetic parameters. Pharmacodynamic effects were assessed ex vivo by stimulating blood samples with PGE2 and determining TNF-ɑ concentrations. Maximum concentration, time to maximum concentration and area under the curve were 327.5 (188.4-663.0) ng/ml, 1 (0.75-2.0) hour and 831.8 (512.6-1421.6) h*ng/ml, respectively. The terminal half-life was 11.1 (8.27-21.2) hr. Significant stimulation of TNF alpha was noted for 2-4 h post-drug administration. Results of this study suggest a short duration of EP4 receptor engagement when administered at a dose of 15 mg/kg.

The effect of stress and exercise on the learning performance of horses.

Domestic horses are widely used for physically demanding activities but the effect of exercise on their learning abilities has not been explored. Horses are also frequently exposed to stressors that may affect their learning. Stress and exercise result in the release of glucocorticoids, noradrenaline and other neurotransmitters that can influence learning. It is not currently possible to directly measure concentrations of neurotransmitters in the brains of behaving horses, however the inference of neurobiological processes from peripheral markers have been widely used in studies of human cognition. We assigned 41 horses to either ridden exercise, uncontrollable stress or inactivity and evaluated their acquisition of an industry-style aversive instrumental learning task. Exercised horses achieved the learning criterion in the fewest number of trials compared to the stressed and inactive horses whose performance did not differ. The exercised horses' salivary cortisol concentrations decreased during learning whereas the concentrations of the other groups increased. Spearman's correlations revealed that horses with the highest cortisol concentrations required the most trials to reach the criterion. We present novel data that exercise prior to learning may enhance the acquisition of learning in horses. Conversely, activities that expose horses to uncontrollable stressors causing strong cortisol release may impair learning. It is proposed that these effects may be due to the influence of neurotransmitters such as cortisol and noradrenaline on brain regions responsible for learning.

Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis.

Trypanozoon infections in equids are caused by three parasite species in the Trypanozoon subgenus: Trypanosoma equiperdum, T. brucei and T. evansi. They are respectively responsible for infectious diseases dourine, nagana and surra. Due to the threat that Trypanozoon infection represents for international horse trading, accurate diagnostic tests are crucial. Current tests suffer from poor sensitivity and specificity, due in the first case to the transient presence of parasites in the blood and in the second, to antigenic cross-reactivity among Trypanozoon subspecies. This study was designed to develop a microsphere-based immunoassay for diagnosing equine trypanosomosis. We tested beads coated with eight Trypanosoma spp. recombinant antigens: enolase, GM6, PFR1, PFR2, ISG65, VSGat, RoTat1.2 and JN2118HU. Of these, GM6 was identified as the best candidate for the serological diagnosis of Trypanozoon infections in equids. Using a receiver operating characteristic (ROC) analysis on 349 equine sera, anti-GM6 antibodies were detected with an AUC value of 0.994 offering a sensitivity of 97.9% and a specificity of 96.0%. Our findings show that the GM6 antigen is a good target for diagnosing equine trypanosomosis using a microsphere-based immunoassay. This promising assay could be a useful alternative to the official diagnostic tool for equine trypanosomosis.

Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

Data on multiple regression analysis between boron, nickel, arsenic, antimony, and biological substrates in horses: The role of hematological biomarkers.

The objective of this study was to evaluate the biomarkers of exposure to boron, nickel, arsenic, and antimony in an industrial region, evaluating the bioaccumulation in biological substrates and the correlation with biomarkers such as hematological parameters. Through indication of the accumulation of some minerals in the horse's biological substrates reflects environmental pollution. Moreover, an additional aim of the study was to show whether these contaminants have an influence on the hematological parameters in horses. Blood, serum, mane, and tail samples from 20 horses from an industrial area were analyzed to determine boron (B), nickel (Ni), arsenic (As), antimony (Sb) concentration. Hematological parameters (red blood cell [RBC], white blood cells [WBC], hemoglobin [Hb], hematocrit [Hct], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], platelet [PLT]) as a biomarker of blood in relation to the bioaccumulation of these elements were analyzed also. Descriptive statistical analysis was performed and single regression analysis (Pearson) and multiple regression analysis (p < 0.05) between blood factors, As, B, Ni, and Sb concentrations, and for each mineral in different substrate, respectively. Results showed a significant correlation between tail and mane concentrations with serum and blood for boron concentration (r = -1 p < 0.05). No significant correlation between sample (feed, hay, mane, tail, and water) concentrations and As, Ni, and Sb were found. A significantly negative correlation with blood parameters (r = -1 p < 0.05) was observed in Boron concentration for mane and tail. This suggests that the mane and tail may be a potential means to investigate suspected exposure to excessive levels of trace minerals.

The pharmacokinetics of a fentanyl matrix patch applied at three different anatomical locations in horses.

BACKGROUND: Matrix fentanyl patches have not been investigated in horses and may represent an effective means of providing analgesia over an extended time period without venous catheterisation. OBJECTIVES: To describe the pharmacokinetics of a matrix transdermal fentanyl patch in horses. STUDY DESIGN: Randomised experiment, Latin-square design. METHODS: Six adult horses were given each of three treatments with a 96-hour washout. For each treatment, two 100 µg/h matrix fentanyl patches were applied to the inguinal region (TXA), metacarpus (TXM) or ventral tail base (TXT) for 72 hours. Blood samples for fentanyl analysis were obtained and heart rate (HR), respiratory rate (RR) and rectal temperature (RT) were measured at various time points for 96 hours. Fentanyl plasma concentrations were measured with LC-MS/MS for pharmacokinetic analysis. A mixed-effects model was used to analyse pharmacodynamic variables. RESULTS: The time to maximum plasma concentration, maximum plasma concentration and area under the curve extrapolated to infinity were 10 ± 3.79, 14.3 ± 5.13 and 10.3 ± 4.8 hours; 2.07 ± 0.74, 1.55 ± 0.53 and 2.07 ± 0.72 ng/mL; and 46.6 ± 9.3, 44.6 ± 6.0 and 46.2 ± 7.68 ng hours/mL for TXA, TXM and TXT respectively. There was no significant difference among groups. There was no significant change from baseline or among treatment groups with regard to HR, RR or RT (P > .1 for all). MAIN LIMITATIONS: There was no intravenous treatment group for determination of bioavailability. CONCLUSIONS: Fentanyl was rapidly absorbed and persisted in the plasma for up to 96 hours. No adverse effects of treatment on HR, RR or RT were observed. Further controlled prospective studies are needed to determine what plasma concentration, if any, of fentanyl achieves an analgesic effect in horses when administered via a transdermal patch system.

Relationship between maximum eye temperature and plasma cortisol concentration in racehorses during intensive training.

The aim of the study was to determine the utility of maximum eye temperature measured by infrared thermography (IRT) as a stress indicator compared with plasma cortisol concentration in Thoroughbred and Arabian racehorses. The study included thirty racehorses undergoing standard training for racing. Measurements of maximum eye temperature and blood collection for plasma cortisol concentration were carried out before training (BT), and within 5 (5AT) and 120 minutes (120AT) after the end of the each training session in three repetitions, with a monthly interval. Both parameters were elevated at 5AT compared to BT (p⟨0.001). Compared to BT, at 120AT the maximum eye temperature remained elevated (p⟨0.001) and plasma cortisol concentration decreased (p⟨0.001). The study indicated significant weak correlations (r=0.220; p⟨0.001) between both measurements at all time points. The results support the use of IRT technique to monitor the response of horses to stress, potentially improving animal management and welfare.

Ivermectin treatment in lactating mares results in suboptimal ivermectin exposure in their suckling foals.

The management of equine strongyles has become problematic over the last decade because of an increased prevalence of drug-resistant isolates worldwide. Therapeutic options are therefore limited, leaving macrocyclic lactones as the most often effective drug class. However, their lipophilic properties result in a long-lasting elimination that could favour drug resistance selection. As a result, ivermectin treatment in lactating mares could promote suboptimal exposure of their foal parasites to ivermectin, thereby selecting for more resistant worms. To test for this putative transfer, we selected two groups of six foal-mare pairs, one group of mares receiving ivermectin and the other being left untreated. We compared faecal egg count trajectories in foals from the two groups and quantified plasma ivermectin concentrations in ivermectin treated mares and their foals during seven days. Our results showed limited but sustained plasmatic exposure of foals associated with non-significant faecal egg count reduction (P = 0.69). This suggests that ivermectin treatment in lactating mares results in suboptimal exposure to the drug in their foal.

Pre-analytical stability of sorbitol dehydrogenase in equine heparinized plasma.

Sorbitol dehydrogenase (SDH) activity is one of the most sensitive and specific markers for hepatocellular injury in horses, but its reported lability makes it impractical for use in many clinical settings. To date, stability of SDH in equine samples has only been evaluated in a limited number of studies in serum samples of horses with activities within reference intervals. The objective of the study was to determine pre-analytical stability of equine SDH activity in heparinized plasma stored at different temperatures for up to 72 h. Twenty client-owned horses admitted to a veterinary teaching hospital for any reason were included in the study. Blood samples collected in lithium-heparin tubes were immediately centrifuged and SDH activity was analyzed within 1 h of collection (T0). Aliquots of plasma were stored at room temperature, 4 °C and -20 °C and SDH activity was re-analyzed after 4 h (T4), 24 h (T24) and 72 h (T72). A significant difference from values measured at T0 was found for samples stored at room temperature (P = 0.022) and -20 °C (P < 0.001), but not at 4 °C. The activity of SDH was within ±20% of that measured at T0 for all samples under all temperature conditions stored for 4 h, and for all samples stored at 4 °C for 24 h. Bland-Altman plots revealed narrow limits of agreement at T4 for all storage temperatures and at T24 for samples stored at 4 °C. The mean absolute percentage error and 95th percentile of the absolute percentage error were lower for samples stored at 4 °C than those stored at room temperature or -20 °C. The activity of SDH has adequate stability for 4 h regardless of storage temperature and 24 h if stored at 4 °C across a wide range of values. Knowledge of the pre-analytical stability of SDH may permit its broader use in assessing hepatic disorders in horses.

Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy.

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young's modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.

Evaluation of the i-STAT Alinity v in a veterinary clinical setting.

Many point-of-care (POC) analyzers are available for the measurement of electrolytes and acid-base status in animals. We assessed the precision of the i-STAT Alinity v, a recently introduced POC analyzer, and compared it to 2 commonly used and previously validated POC analyzers (i-STAT 1, Stat Profile pHOx Ultra). Precision was evaluated by performing multiple analyses of whole blood samples from healthy dogs, cats, and horses on multiple i-STAT Alinity v analyzers. For comparison between analyzers, whole blood samples from dogs and cats presented to the emergency room were run concurrently on all 3 POC instruments. Reported values were compared by species (dogs and cats only) using Pearson correlation, and all values from all species were analyzed together for the Bland-Altman analysis. Results suggested that the i-STAT Alinity v precision was very good, with median coefficients of variability <2.5% for all measured parameters (except the anion gap), with variable ranges of coefficients of variation. In addition, good-to-excellent correlation was observed between the i-STAT Alinity v and i-STAT 1, and between the i-STAT Alinity v and Stat Profile pHOx Ultra for all parameters in both cats and dogs, respectively. In this cohort, the i-STAT Alinity v had clinically acceptable bias compared to the currently marketed analyzers and can be used for monitoring measured analytes in cats and dogs, although serial measurements in a single animal should be performed on the same analyzer whenever possible.

A Quest for a Universal Plasma-Derived Antivenom Against All Elapid Neurotoxic Snake Venoms.

This review describes the research aimed at the development of universal antivenom against elapid neurotoxic snake venoms. The antivenoms produced in Thailand in the 1980s were of low potency, especially against the elapid venoms. This was thought to be due to the low immunogenicity of the α-neurotoxins, which are the most lethal toxins in these venoms. Comparisons of various α-neurotoxin conjugates and polymers, and also different immunological adjuvants, showed that the adjuvant used is the major determinant in the antibody response in horses. The potent Freund's adjuvant was not used due to its severe local side-effect in horses. Therefore, a novel immunization protocol termed 'low dose, low volume multi-site' was developed for use in horses. This immunization protocol has led to the production of highly potent monospecific antivenoms against several elapid and viperid venoms, and two potent polyspecific antivenoms, one against 4 neurotoxic and another against 3 hematotoxic venoms. The immunization protocol has also led to other improvements in antivenom production including: several fold increases in antiserum potency, a reduction in the time required to reach therapeutically useful antibody titers, a 90% reduction in the amount of venom used, and 100% of the horses responding to the immunization program. This development is partly responsible for significant decrease in the Thailand's annual snakebite death toll from a few dozens to mostly nil in recent years. Finally, a simple and novel immunization strategy, using a 'diverse toxin repertoire' composed of numerous elapid toxin fractions as immunogen, was proposed and tested. This immunization procedure has resulted in the successful production of a widely paraspecific antiserum against at least 36 neurotoxic venoms of 28 species encompassing 10 genera and from 20 countries on four continents, and possibly against all elapid venoms with α-neurotoxins as the lethal toxins. These results indicate that, with optimizations of the composition of the 'diverse toxin repertoire', the immunization scheme and antibody fractionation to increase the antivenom neutralizing potency, an effective universal antivenom against the neurotoxic elapid snakes of the world can be produced.

Long-term detection of clodronate in equine plasma by liquid chromatography-tandem mass spectrometry.

Clodronate is a non-nitrogen-containing bisphosphonate drug approved in equine veterinary medicine. Clodronate is prohibited for use in competition horses; therefore, to set up an appropriate control, detection times and screening limits are required. The quantitative method in plasma consisted of addition of chloromethylene diphosphonic acid as internal standard. Automated sample preparation comprised a solid phase extraction with weak anion exchange properties on microplate. After methylation of the residue with trimethyl orthoacetate, analysis was conducted by high-performance liquid chromatography-tandem mass spectrometry. Using a weighting factor of 1/(concentration)2 , good linearity was observed in the range of 1 to 500 ng/ml, with low limits of detection and quantification of 0.5 and 1 ng/ml, respectively. Precision and accuracy determined at four concentrations were satisfactory, with an error percentage less than 15%. Absence of carry-over and good stability of clodronic acid in plasma after a long-term storage at -20°C were verified. The method was successfully applied to the quantification of clodronic acid in plasma samples from horses administered with a single intramuscular administration of Osphos® at a mean dose of 1.43 ± 0.07 mg/kg. The observed detection time will be verified in a clinical population study conducted in diseased horses.

Clock gene per 2 daily rhythm: Correlation with the serum level of uncoupling protein 1 (UCP1) in goat and horse.

Recent studies evidenced that the circadian rhythm of Per2 is involved in adaptive thermogenesis by the modulating transcription of uncoupling protein 1 (UCP1). For this purpose, we investigated the linkage between the daily rhythm of Per2 and UCP1 in ruminant and non-ruminant mammalian species. Five clinically healthy, not pregnant, and not lactating Maltese female goats and five clinically healthy, not pregnant, and not lactating Italian Saddle horses were enrolled in the study. All animals were housed under natural photoperiod (sunrise 05:05, sunset 20:55) and environmental temperature and humidity. Goats were kept individually in 3.0 × 2.0 m box, horses were housed individually in 3.5 × 3.5 m box; all boxes were equipped with an opening window. On each subject, blood samples were collected every 4 h for a 48-h period. The Per2 gene expression was determined on blood samples collected in PAX gene Blood RNA Tube, whereas UCP1 concentration was assessed on serum. Per2 and UCP1 levels were statistically influenced by the species (p < 0.0001) and the time of data collection (p < 0.0001), but not by the day of monitoring. Per2 showed daily rhythmicity, statistically different in mesor and amplitude between the two species, diurnal in goats, nocturnal in horses; with the same robustness. UCP1 did not show daily rhythmicity. During the experimental period the two parameters showed a negative correlation in horses. According to the findings herein obtained, we can claim that the role of Per2 in the thermogenesis induced by the beige adipocytes throughout UCP1 activation did not reflect what found in other mammal species, but further studies are required to establish their correlation in equids.

The Effect of the Clenbuterol-ß2-Adrenergic Receptor Agonist on the Peripheral Blood Mononuclear Cells Proliferation, Phenotype, Functions, and Reactive Oxygen Species Production in Race Horses In Vitro.

Clenbuterol, the ß2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reactive oxygen species (ROS) production, as well as cell proliferation, were evaluated by flow cytometry. In addition, IL-1ß, IL-4, IL-6, IL-10, IL-17, INF-γ and TNF-α concentrations were evaluated by ELISA. ß2-adrenoceptor stimulation leads to enhanced anti-inflammatory properties in well-trained horses, as do low doses in untrained animals. In contrast, higher clenbuterol doses create a pro-inflammatory environment in inexperienced horses. In conclusion, ß2-adrenoceptor stimulation leads to a biphasic response. In addition, the immune cells are more sensitive to drug abuse in inexperienced individuals under physical training.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton.

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and ß- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Topical ophthalmic atropine in horses, pharmacokinetics and effect on intestinal motility.

BACKGROUND: Topical ophthalmic atropine sulfate is an important part of the treatment protocol in equine uveitis. Frequent administration of topical atropine may cause decreased intestinal motility and colic in horses due to systemic exposure. Atropine pharmacokinetics are unknown in horses and this knowledge gap could impede the use of atropine because of the presumed risk of unwanted effects. Additional information could therefore increase safety in atropine treatment. RESULTS: Atropine sulfate (1 mg) was administered in two experiments: In part I, atropine sulfate was administered intravenously and topically (manually as eye drops and through a subpalpebral lavage system) to six horses to document atropine disposition. Blood-samples were collected regularly and plasma was analyzed for atropine using UHPLC-MS/MS. Atropine plasma concentration was below lower limit of quantification (0.05 µg/L) within five hours, after both topical and IV administration. Atropine data were analyzed by means of population compartmental modeling and pharmacokinetic parameters estimated. The typical value was 1.7 L/kg for the steady-state volume of distribution. Total plasma clearance was 1.9 L/h‧kg. The bioavailability after administration of an ophthalmic preparation as an eye drop or topical infusion were 69 and 68%, respectively. The terminal half-life was short (0.8 h). In part II, topical ophthalmic atropine sulfate and control treatment was administered to four horses in two dosing regimens to assess the effect on gastro-intestinal motility. Borborygmi-frequency monitored by auscultation was used for estimation of gut motility. A statistically significant decrease in intestinal motility was observed after administration of 1 mg topical ophthalmic atropine sulfate every three hours compared to control, but not after administration every six hours. Clinical signs of colic were not observed under any of the treatment protocols. CONCLUSIONS: Taking the plasma exposure after topical administration into consideration, data and simulations indicate that eye drops administrated at a one and three hour interval will lead to atropine accumulation in plasma over 24 h but that a six hour interval allows total washout of atropine between two topical administrations. If constant corneal and conjunctival atropine exposure is required, a topical constant rate infusion at 5 µg/kg/24 h offers a safe alternative.

A highly prevalent SINE mutation in the myostatin (MSTN) gene promoter is associated with low circulating myostatin concentration in Thoroughbred racehorses.

Horse racing is a popular and financially important industry worldwide and researchers and horse owners are interested in genetic and training influences that maximise athletic performance. An association has been found between the presence of a short interspersed nuclear element (SINE) mutation in the myostatin (MSTN) gene promoter and optimal race distance in Thoroughbred horses. There is previous laboratory evidence that this mutation reduces MSTN expression in a cell culture model and influences skeletal muscle fibre type proportions in horses. Manipulating MSTN expression has been proposed for illicit gene doping in human and equine athletes and already, researchers have generated homozygous and heterozygous MSTN-null horse embryos following CRISPR/Cas9 editing at the equine MSTN locus and nuclear transfer, aiming artificially to enhance performance. To date however, the role of the naturally-occurring equine MSTN SINE mutation in vivo has remained unclear; here we hypothesised that it reduces, but does not ablate circulating myostatin expression. Following validation of an ELISA for detection of myostatin in equine serum and using residual whole blood and serum samples from 176 Thoroughbred racehorses under identical management, horses were genotyped for the SINE mutation by PCR and their serum myostatin concentrations measured. In our population, the proportions of SINE homozygotes, heterozygotes and normal horses were 27%, 46% and 27% respectively. Results indicated that horses that are homozygous for the SINE mutation have detectable, but significantly lower (p < 0.0001) serum myostatin concentrations (226.8 pg/ml; 69.3-895.4 pg/ml; median; minimum-maximum) than heterozygous (766 pg/ml; 64.6-1182 pg/ml) and normal horses (1099 pg/ml; 187.8-1743 pg/ml). Heterozygotes have significantly lower (p < 0.0001) myostatin concentrations than normal horses. Variation in serum myostatin concentrations across horses was not influenced by age or sex. This is the first study to reveal the direct functional effect of a highly prevalent mutation in the equine MSTN gene associated with exercise performance. Determining the reason for variation in expression of myostatin within SINE-genotyped groups might identify additional performance-associated environmental or genetic influences in Thoroughbreds. Understanding the mechanism by which altered myostatin expression influences skeletal muscle fibre type remains to be determined.

Effect of uterine ozone therapy and anticoagulant sampling on oxidative stress parameters in mares.

With the purpose of assessing the effects of uterine ozone therapy and anticoagulant sampling on oxidative stress (OS) parameters in mares, ten mares underwent three consecutive days of uterine ozone therapy by flushing the uterus with ozonated lactated Ringer's solution followed by insufflation with ozone­oxygen gas. Serum samples were obtained at baseline and days 3, 6, 10 and 17 to determine the effect of ozone therapy on OS markers. Plasma obtained with anticoagulants citrate, ethylenediaminetetraacetic acid (EDTA) and heparin were at baseline and 6 days following therapy to determine the effect of anticoagulant on OS parameters. Antioxidants albumin and uric acid, total antioxidant capacity (TAC) using four different methods, total oxidant capacity (TOC) and lipid peroxidation were determined through photocolorimetry. Statistical analyses comprised repeated measures ANOVA followed by Dunnett's test or Friedman followed by Dunn's post-hoc test. Differences were considered significant when p < 0.05. Uterine ozone therapy significantly decreased uric acid, TAC in all four different methods, concomitantly with an increase on TOC at days 3 and 6 following therapy. No changes were observed on albumin and lipid peroxidation levels. Anticoagulants prevented the detection of oxidative stress induced by uterine ozone therapy depending on the method of analysis. In conclusion, uterine ozone therapy causes systemic oxidative stress in mares and the choice of anticoagulant sampling interferes with laboratory tests.