Horse Plasma Research Articles

The effect of foal or adult horse plasma on equine monocyte-derived dendritic cell phenotype and function

Immunological and endocrine immaturity in foals increases foal morbidity and mortality from bacterial sepsis. Dendritic cells (DC) are critical in activating the adaptive immune response, but foal DC are phenotypically and functionally different than those of adult horses. Age-related variations in availability of some soluble plasma factors, such as hormones, might govern some age-related differences in DC function. Effects of exposure to plasma factors on equine DC phenotype and function have not been described. We hypothesized that exposure to plasma from foals or adult horses would differentially impact monocyte-derived DC (MoDC) phenotype and function. Eight healthy adult horses and 8 healthy foals were divided into pairs of one adult horse and one foal. Blood was collected from each pair for MoDC generation when foals were 1 and 30 days of age. MoDC from horses and foals were then exposed to killed whole-cell bacteria in the presence of their own age-matched plasma, plasma from the opposite-aged animal in the pair, and serum-free medium alone (control). Expression of DC-relevant surface markers (MHC class-II, CD86, and CD14) and endocytosis capability were measured by flow cytometry. Supernatant cytokine concentrations (IL-4, IL-17, IFN-γ, and IL-10) were quantified with a validated bead-based immunoassay. Data were analyzed using linear mixed-effects and Tobit regression models (P < 0.05). The percentage of MoDC expressing surface markers MHC class-II and CD86 was reduced in MoDC derived from 1-day-old foals in comparison to adult horse MoDC when cultured in medium alone or with either source of plasma (P = 0.0001). Foal and adult horse MoDC cultured in either source of plasma expressed more CD86 and less CD14 than cells cultured in serum-free medium alone (P ≤ 0.02). Adult horse and foal MoDC exposed to bacterial antigen in the presence of 1-day-old foal plasma secreted less IL-10 (P ≤ 0.0008) compared to those cultured in adult horse plasma. Endogenous production of IL-17 by MoDC from foals at day 1 of age cultured in adult plasma was increased compared to foal MoDC cultured in serum-free medium (P = 0.004). Phagocytosis of killed, labeled Staphylococcus aureus was reduced when MoDC generated from foals or adult horses were exposed to plasma from foals at day 1 or 30 of age (P ≤ 0.03). Age-related variation in soluble plasma factors appear to regulate equine MoDC function, but specific plasma factors capable of regulating MoDC phenotype or function were not defined in this study.

An innovative derivatization-free IC-MS/MS method for the detection of bisphosphonates in horse plasma.

Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their use quite difficult. Current analytical strategies to detect such compounds often rely on a solid phase extraction (SPE) followed by detection by means of UHPLC-MS/MS after methylation with chemical reagents. To improve the analysis throughput and to eliminate the need for chemical derivatization, an innovative 96-well SPE followed by ion chromatography-mass spectrometry was developed. Analyses are conducted on an ICS-6000 HPIC system coupled to a TSQ Altis™ (Thermo Scientific™). The use of a 96-well SPE allowed 5-fold sample increase and a 6-fold throughput improvement. While preliminary results conducted on horse plasma exhibited similar performances to the method for the detection of non-nitrogen-containing bisphosphonates, the analytical performances of nitrogen-containing bisphosphonates were greatly improved.

Effects of endurance racing on horse plasma extracellular particle miRNA.

Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. To evaluate the effects of 160km endurance racing on horse plasma extracellular particles and their miRNA population. Cohort study. We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2hours after the race, which returned to baseline 15hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.

Determination of grapiprant plasma and urine concentrations in horses

ObjectiveNon-steroidal anti-inflammatory drugs are inhibitors of cyclooxygenase (COX) in tissues and used as therapeutic agents in different species. Grapiprant, a member of the piprant class of compounds, antagonizes prostaglandin receptors. It is a highly selective EP4 prostaglandin E2 receptor inhibitor, thereby limiting the potential for adverse effects caused by wider COX inhibition. The objectives of this study were to determine if the approved canine dose would result in measurable concentrations in horses, and to validate a chromatographic method of analysis for grapiprant in urine and plasma. Study designExperimental study. AnimalsA total of six healthy, adult mixed-breed mares weighing 502 ± 66 (397–600) kg and aged 14.8 ± 5.3 (6–21) years. MethodsMares were administered one dose of 2 mg kg–1 grapiprant via nasogastric tube. Blood and urine samples were collected prior to and up to 48 hours after drug administration. Drug concentrations were measured using high-performance liquid chromatography. ResultsGrapiprant plasma concentrations ranged from 71 to 149 ng mL–1 with the mean peak concentration (106 ng mL–1) occurring at 30 minutes. Concentrations were below the lower limit of quantification (50 ng mL–1) in four of six horses at 1 hour and in all six horses by 2 hours after drug administration. Grapiprant urine concentrations ranged from 40 to 4077 ng mL–1 and were still detectable at 48 hours after administration. Conclusions and clinical relevanceCurrently, there are no published studies looking at the pharmacodynamics of grapiprant in horses. The effective concentration needed to control pain in dogs ranges 114–164 ng mL–1. Oral administration of grapiprant (2 mg kg–1) in horses did not achieve those concentrations. The dose was well tolerated; therefore, studies with larger doses could be conducted.

Biotechnological potential of Phospholipase D for Loxosceles antivenom development.

Loxoscelism is one of the most important forms of araneism in South America. The Health Authorities from countries with the highest incidence and longer history in registering loxoscelism cases indicate that specific antivenom should be administered during the first hours after the accident, especially in the presence or at risk of the most severe clinical outcome. Current antivenoms are based on immunoglobulins or their fragments, obtained from plasma of hyperimmunized horses. Antivenom has been produced using the same traditional techniques for more than 120 years. Although the whole composition of the spider venom remains unknown, the discovery and biotechnological production of the phospholipase D enzymes represented a milestone for the knowledge of the physiopathology of envenomation and for the introduction of new innovative tools in antivenom production. The fact that this protein is a principal toxin of the venom opens the possibility of replacing the use of whole venom as an immunogen, an attractive alternative considering the laborious techniques and low yields associated with venom extraction. This challenge warrants technological innovation to facilitate production and obtain more effective antidotes. In this review, we compile the reported studies, examining the advances in the expression and application of phospholipase D as a new immunogen and how the new biotechnological tools have introduced some degree of innovation in this field.

Diagnostic value of plasma and peritoneal fluid procalcitonin concentrations in horses with strangulating intestinal lesions.

To assess the diagnostic value of plasma and peritoneal fluid procalcitonin concentrations for identification of horses with strangulating intestinal lesions. 65 horses with signs of colic of intestinal origin and 10 healthy (control) horses. For each horse, plasma and peritoneal fluid samples were obtained for a CBC and determination of total protein, procalcitonin, and lactate concentrations. Signalment and clinicopathologic findings were compared among control horses and horses with strangulating and nonstrangulating intestinal lesions. Mean ± SD plasma (274.9 ± 150.8 pg/mL) and peritoneal fluid (277 ± 50.6 pg/mL) procalcitonin concentrations for horses with colic were significantly greater than the mean ± SD plasma (175.5 ± 46.0 pg/mL) and peritoneal fluid (218.8 ± 48.7 pg/mL) procalcitonin concentrations for control horses. Mean procalcitonin concentration in peritoneal fluid, but not plasma, differed significantly between horses with strangulating lesions and those with nonstrangulating lesions. A peritoneal fluid procalcitonin concentration ≥ 281.7 pg/mL had a sensitivity of 81%, specificity of 69%, positive predictive value of 56.7%, and negative predictive value of 87.9% for detection of strangulating lesions. Results suggested that peritoneal fluid procalcitonin concentration, when evaluated in conjunction with other clinicopathologic results, might be a sensitive indicator of intestinal ischemia and facilitate early identification of horses that require surgery to address a strangulating lesion.

Association of plasma concentrations of oxytocin, vasopressin, and serotonin with docility and friendliness of horses

Oxytocin (OXT) and vasopressin (AVP) have been associated with social interaction and affiliative behavior in animals. Additionally, AVP is known to affect socially aggressive behavior. In addition, serotonin has an association with aggressive behaviors. The objectives of this study were (1) to evaluate OXT, AVP, and serotonin concentrations in the plasma of horses of different breeds, sexes, and ages and (2) to determine if the neurotransmitters are associated with horse docility and friendliness. This study was performed at Sangju International Equestrian Center. Blood samples were collected from 23 horses, including 6 Thoroughbreds (11 to 18 yr), 6 Warmbloods (15 to 26 yr), 6 ponies (8 to 17 yr), and 5 Quarter Horses (4 to 12 yr). The group of horses consisted of 13 mares and 10 geldings. The plasma concentrations of OXT and AVP were measured by enzyme-linked immunosorbent assay, and the serum concentration of serotonin was measured by high-performance liquid chromatography. The characteristics of each horse were surveyed by 3 horse trainers. The effects of breed, sex, and age on the concentration of each neurotransmitter were assessed by a 3-way ANOVA with LSD post-hoc analysis. Linear regression analysis was performed to determine if the concentration of neurotransmitters is related to the docility and friendliness of horses. As a result, the concentrations of OXT and AVP did not vary with the breed, sex, or age of horses. However, the serotonin concentration varied depending on the breed and age of horses. Interestingly, there was a trend toward the existence of a correlation between docility and OXT in Thoroughbred horses. However, AVP and serotonin concentrations had no correlation with the docility and friendliness of horses. In conclusion, the docility and friendliness of Thoroughbred might be related with the blood OXT concentration.

Administration study of recombinant human relaxin-2 in horse for doping control purpose.

The insulin-like peptide relaxin (RLX), an endogenous peptide hormone produced in human for pregnancy and reproduction, is also known to exert a range of physiological and pathological effects. Its use is banned in human sports, horseracing, and equestrian competitions due to its potential performance enhancing effect through vasodilation resulting in the increase of blood and oxygen supplies to muscles. Little is known about the biotransformation and elimination of RLX in horses. This paper describes an administration study of rhRLX-2 and its elimination in horses, and the development of sensitive methods for the detection and confirmation of rhRLX-2 in both horse plasma and urine by nano-liquid chromatography/high resolution mass spectrometry (nano-LC/HRMS) after immunoaffinity extraction with the objective of controlling the abuse of rhRLX-2 in horses. The limits of detection in plasma and urine are 2pg/mL and 5pg/mL, respectively. Two thoroughbred geldings were each administered one dose of 10mg rhRLX-2 subcutaneously daily for 3 consecutive days. The rhRLX-2 could be detected and confirmed in the plasma and urine samples collected 105h and 80h, respectively, after the last dose of administration. For doping control purposes, rhRLX-2 ELISA could be used as a screening test to identify potential positive samples for further investigation using the nano-LC/HRMS methods.

Comparison of plasma and intra-articular gentamicin levels after intravenous administration in healthy horses

Septic arthritis is considered one of the most severe disorders that affect horses’ joints. Therapy is intended to eliminate the microorganisms that cause this infection. The intravenous administration of antibiotics is an easily performed technique, but there is no guarantee that the antibiotics will reach the joint milieu at high concentrations and remain elevated for a certain period of time. This study aimed to determine and compare the levels of gentamicin in the plasma and synovial fluid of healthy horses after intravenous administration for seven days. Five horses received 6.6 mg/kg intravenous gentamicin every 24 h for seven consecutive days. Blood and synovial fluid samples were collected from the right metacarpal-phalangeal joint before the start of the treatment (T0) and after 6 h (T6), 12 h (T12), and every 24 h during antibiotic therapy for seven days (D1 to D7). The levels of gentamicin in serum and synovial fluid were quantified by Enzyme-Linked Immunosorbent Assay (ELISA). The intravenous administration of 6.6 mg/kg of gentamicin, in horses, after a period of 24 h, promotes synovial fluid concentrations below the MIC of the drug, in spite of reaching high plasma concentrations of gentamicin, daily mean serum concentration which was 29-fold higher than the minimum inhibitory concentration (MIC) of the drug. The intravenous administration every 24 h during seven consecutive days did not cause systemic side effects, such as hepatic or renal injury.

PSVII-17 The relationship of biochemistry of seminal plasma, ejaculate volume and sperm concentration in horses

Abstract The aim of the work was to study the relationship between biochemistry of seminal plasma with the ejaculate volume and the concentration of spermatozoa. Ejaculates from 35 stallions aged 5 to 15 were collected during the breeding season (February–May). The concentration of calcium (Ca), phosphate (P), potassium (K), magnesium (Mg), selenium (Se), zinc (Zn) and lithium (Li) in seminal plasma were determined by atomic emission and mass spectrometry (AES-ICP and MS-ICP) using an mass spectrometer Elan 9000 (Perkin Elmer, USA) and an atomic emission spectrometer Optima 2000V (Perkin Elmer,USA). The concentration of glucose, phospholipids, aspartate-amino-transferase (AST) and alkaline phosphatase (AIP) was determined by ChemWell 2902V analyzer (Awareness Technology, USA). The significance of differences between the indices was determined using Student’s t-test, the differences at P ≤ 0.05 were considered statistically significant. A significant negative correlation between the volume of ejaculate and concentration of Se (r = -0.42; P = 0.01), P (r =-0.39; P = 0.001), AIP (r = -0.48; P = 0.01), Zn (r = -0.40; P = 0.01), Li (r = -0.39; P = 0.02), phospholipids (= -0.52; P = 0.03) in seminal plasma was established. A correlation between the concentration of sperm and the concentration of P (r = 0.71; P = 0.01), Se (r = 0.39; P = 0.02), Li (r = 0.33; P = 0.05), AIP (r = 0.81; P = 0.001) in seminal plasma was established. Also a negative correlation between the concentration of sperm and glucose (r = -0.62; P = 0.01) was found. We assumed that selenium, zinc, phosphorus, lithium, phospholipids and AIP in seminal plasma have an epididymal origin and do not depend on the secretion of the accessory sex glands. Glucose enters the ejaculate with the secrets of the accessory sex glands. Authors acknowledge financial support from Russian Science Foundation, Grant No: 17-16-01109, development program of Bioresource collections “Cryobank of genetic recourses the All-Russian Research Institute for Horse Breeding.”

The Influence of Training and Simulated Race on Horse Plasma Serotonin Levels

Exercise represents a physical stress that challenges homeostasis affecting central and peripheral serotoninergic systems. The influence of the exercise on circulating serotonin (5-hydroxytryptamine [5-HT]) levels depends on training state as well as the exercise protocol. The purpose of the present research was to determine changes of plasma 5-HT in sport horses in response to training (T) and simulated race (SR) and in addition to assess the possible presence of significant differences on circulating 5-HT between two different sessions of exercise. In particular, the research was carried out on 18 trained horses performing daily training and race activity. Plasma 5-HT levels were analyzed in platelet poor plasma fraction by ELISA assay at T0 (before exercise), T1 (30 minutes after exercise) and T2 (2 hours after exercise). The hypothesis was that both exercise sessions could affect plasma 5-HT levels. Results showed a significant increase of plasma 5-HT levels at T1, compared with T0, both after simulated race and training activity. These effects are probably related to an increased 5-HT release from platelets and/or an increased peripheral 5-HT synthesis induced from exercise. At T2, plasma 5-HT concentrations showed a significant decrease to physiological levels in both sessions. Moreover, plasma 5-HT levels at T1 (SR) were significantly higher than those at T1 (T). Targeting peripheral 5-HT could be useful to assess the physiological adaptability of horses to the exercise, together with other selection techniques of sport horses.

Pharmacokinetics of furosemide in thoroughbred horses subjected to supramaximal treadmill exercise with and without controlled access to water

BackgroundThe primary objective of this study was to assess the disposition of furosemide in Thoroughbred horses treated intravenously with 1 mg/kg of furosemide 4 and 24 h before supramaximal treadmill exercise without and with controlled access to water, respectively. Another objective was to determine whether furosemide was detectable in the plasma of horses after exposure to supramaximal treadmill exercise. Thoroughbred horses (n = 4–6) were administered single intravenous doses of 1 mg/kg of furosemide at 4 and 24 h before supramaximal exercise on a high-speed treadmill, with controlled and free access to water, respectively. Plasma furosemide concentrations were determined using liquid chromatography.ResultsFurosemide was detected in all the horses, regardless of whether they were treated 24 h or 4 h before excersice. In both treatment sequence groups of 2 horses, the concentration time profiles of furosemide during the first 4 h after its administration were relatively similar. The average maximum observed concentrations, AUC0–1.5h, and AUC0–3h, of both groups of horses were not different (p > 0.05). There were no significant differences in systemic clearance based on the geometric mean (95% confidence interval) (409 (347–482) mL/h/kg) for 4 h and 320 (177–580) mL/h/kg) for 24 h) between horses that were exercised 4- and 24-h post-furosemide administration. The plasma concentration of furosemide in all the horses fell below the limit of quantification (25 ng/mL) within 12 h after drug administration. In the group treated 24 h before exercise, none of the horses had detectable furosemide at the time of supramaximal treadmill exercise. In the group treated 4 h before exercise, furosemide was detected 1 h before and 2 h after supramaximal treadmill exercise in 4/4 and 3/4 horses, respectively. The mean AUC3-last h of both groups of horses were not different (p > 0.05).ConclusionsWater restriction did not exert any apparent effect on the disposition of furosemide. It remains to be determined, however, whether the attained plasma concentration of furosemide in combination with other controlled water access protocols have any direct or indirect pharmacological effect that may affect the athletic performance of the horse.

Refinement strategy for antivenom preparation of high yield and quality.

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness.

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to Cviridis venom. We measured the effects of Cviridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation.

Differences in plasma and peritoneal fluid proteomes identifies potential biomarkers associated with survival following strangulating small intestinal disease.

Strangulating small intestinal disease (SSID) carries a poor prognosis for survival in comparison to other types of colic, particularly if resection is required. Identification of markers which aid early diagnosis may prevent the need for resection, assist with more accurate prognostication and/or support the decision on whether surgical intervention is likely to be successful, would be of significant welfare benefit. To apply an unbiased methodology to investigate the plasma and peritoneal fluid proteomes in horses diagnosed with SSID requiring resection, to identify novel biomarkers which may be of diagnostic or prognostic value. Prospective clinical study. Plasma and peritoneal fluid from horses presented with acute abdominal signs consistent with SSID was collected at initial clinical examination. Samples from eight horses diagnosed with SSID at surgery in which resection of affected bowel was performed and four control horses subjected to euthanasia for orthopaedic conditions were submitted for liquid chromatography tandem mass spectrometry. Protein expression profiles were determined using label-free quantification. Data were analysed using analysis of variance to identify differentially expressed proteins between control and all SSID horses and SSID horses which survived to hospital discharge and those which did not. Significance was assumed at P≤0.05. A greater number of proteins were identified in peritoneal fluid than plasma of both SSID cases and controls, with 123 peritoneal fluid and 13 plasma proteins significantly differentially expressed (DE) between cases and controls (P<0.05, ≥2 fold change). Twelve peritoneal fluid proteins (P<0.036) and four plasma proteins (P<0.05) were significantly DE between SSID horses which survived and those which did not. A low number of samples were analysed, there was variation in duration and severity of SSID and only short-term outcome was considered. Changes in peritoneal fluid proteome may provide a sensitive indicator of small intestinal strangulation and provide biomarkers relevant to prognosis.

Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control.

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.

Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

Alterations in red blood cell parameters, plasma amino acids, total cholesterol and fatty acids in Standardbred horses undergoing fitness training

Maintenance of metabolic homeostasis is vital to optimise the supply of nutrients to support exercise and facilitate recovery and repair processes in horses. This study assessed the effects of exercise on resting plasma homeostasis in horses upon initiation of a training program to attain fitness in preparation for competitive harness racing. Four Standardbred horses (three males and one female) that had not been in work for at least 6 months were assessed prior to the commencement of the training program and then progressively on a weekly basis for 8 weeks. Resting plasma samples were collected in the early morning prior to exercise training and feeding. Samples were analysed for amino acid composition, cholesterol, palmitic acid and stearic acid on a weekly basis, and red blood cell counts and haemoglobin were analysed at weeks 0, 4 and 8. The red cell counts and levels of haemoglobin increased progressively over the training period (P&lt;0.05). Specific amino acids in the plasma displayed temporal variations during the training period. Glycine was the most abundant amino acid in resting horse plasma and together with serine was reduced throughout the first half of the training period, eventually returning to initial levels at weeks 7 and 8 (P&lt;0.05). A number of amino acids were noted to increase in concentration throughout the 8 week training period including ornithine, histidine and hydroxyproline (P&lt;0.05). Cholesterol fell to substantially lower levels after 8 weeks of exercise (P&lt;0.05). Palmitic acid showed three ‘peaks’ of elevated concentrations in plasma (P&lt;0.05) following the initiation of exercise and then again at the transitions into harder fast work sessions. Stearic acid was relatively constant throughout the exercise period. Adjustments in the plasma composition of these key metabolites were consistent with supporting the increased metabolic demands associated with the higher levels of exercise training.

Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing.

ABSTRACTRecently, the illegal use of novel technologies, such as gene and cell therapies, has become a great concern for the horseracing industry. As a potential way to control this, metabolomics approaches that comprehensively analyze metabolites in biological samples have been gaining attention. However, it may be difficult to identify metabolic biomarkers for doping because physiological conditions generally differ between resting and exercise states in horses. To understand the metabolic differences in horse plasma between the resting state at training centres and the sample collection stage after racing for doping test (SAD), we took plasma samples from these two stages (n=30 for each stage) and compared the metabolites present in these samples by liquid chromatography-high resolution mass spectrometry. This analysis identified 5,010 peaks, of which 1,256 peaks (approximately 25%) were annotated using KEGG analysis. Principal component analysis showed that the resting state and SAD groups had entirely different metabolite compositions. In particular, the levels of inosine, xanthosine, uric acid, and allantoin, which are induced by extensive exercise, were significantly increased in the SAD group. In addition, many metabolites not affected by extensive exercise were also identified. These results will contribute to the discovery of biomarkers for detecting doping substances that cannot be detected by conventional methods.

Combined use of platelet rich plasma and micro-fat in sport and race horses with degenerative joint disease: preliminary clinical study in eight horses

Combined use of platelet rich plasma and micro-fat in sport and race horses with degenerative joint disease: preliminary clinical study in eight horses