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Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Inflammatory bowel disease (IBD) is a chronic disease characterized by different cell infiltrates in the intestine. The aims of this study were to report the clinical and clinicopathological findings in horses with histological changes compatible with IBD in the duodenum. Further, the clinical progression of IBD and survival were investigated. Patient records were reviewed for horses in which histological evidence of IBD was found in duodenal biopsies collected during endoscopy. The histological changes were classified as mild, moderate or severe and the predominant infiltrating cell type was recorded. Clinical improvement was assessed by the owner via a questionnaire at 6 weeks after biopsy, along with survival after one year. In total, 149 horses were included, and the most common clinical signs were weight loss, reduced performance and pain during abdominal palpation. Most horses showed partial malabsorption during an oral glucose absorption test, and the horses with severe IBD had lower serum protein concentrations. Lymphoplasmacytic enteritis was the most common type of IBD (78.5% of cases), while in six horses neutrophilic infiltration of the duodenum was present. Overall, 71% of the cases had improved clinically after six weeks, mostly following treatment with corticosteroids. The results of a second biopsy were a poor predictor of improvement, and the horses that improved after 6 weeks were more likely to be alive after one year.

Despite its proven research applications, it remains unknown whether surface electromyography (sEMG) can be used clinically to discriminate non-lame from lame conditions in horses. This study compared the classification performance of sEMG absolute value (sEMGabs) and asymmetry (sEMGasym) parameters, alongside validated kinematic upper-body asymmetry parameters, for distinguishing non-lame from induced fore- (iFL) and hindlimb (iHL) lameness. Bilateral sEMG and 3D-kinematic data were collected from clinically non-lame horses ( n = 8) during in-hand trot. iFL and iHL (2-3/5 AAEP) were induced on separate days using a modified horseshoe, with baseline data initially collected each day. sEMG signals were DC-offset removed, high-pass filtered (40 Hz), and full-wave rectified. Normalized, average rectified value (ARV) was calculated for each muscle and stride (sEMGabs), with the difference between right and left-side ARV representing sEMGasym. Asymmetry parameters (MinDiff, MaxDiff, Hip Hike) were calculated from poll, withers, and pelvis vertical displacement. Receiver-operating-characteristic (ROC) and area under the curve (AUC) analysis determined the accuracy of each parameter for distinguishing baseline from iFL or iHL. Both sEMG parameters performed better for detecting iHL (0.97 ≥ AUC ≥ 0.48) compared to iFL (0.77 ≥ AUC ≥ 0.49). sEMGabs performed better (0.97 ≥ AUC ≥ 0.49) than sEMGasym (0.76 ≥ AUC ≥ 0.48) for detecting both iFL and iHL. Like previous studies, MinDiff Poll and Pelvis asymmetry parameters (MinDiff, MaxDiff, Hip Hike) demonstrated excellent discrimination for iFL and iHL, respectively (AUC > 0.95). Findings support future development of multivariate lameness-detection approaches that combine kinematics and sEMG. This may provide a more comprehensive approach to diagnosis, treatment, and monitoring of equine lameness, by measuring the underlying functional cause(s) at a neuromuscular level.

Speed alterations affect many gait analysis parameters. How horses adapt to speed is relevant in many equestrian disciplines and may differ between breeds. This study described changes in gait parameters in 38 Warmblood (WB) and 24 Franches-Montagnes (FM) horses subjected to an incremental speed test at walk (1.35–2.05 m/s) and trot (3.25–5.5 m/s). Time, force and spatial parameters of each limb were measured with an instrumented treadmill and analysed with regression analysis using speed as the independent variable. With higher speeds, stride rate, length, over-tracking distance and vertical ground reaction forces increased while the impulses decreased. The parameters followed the same linear or polynomial regression curves independent of breed, while the slope (linear) or incurvation (polynomial) often differed significantly between breeds. Some differences between the breeds were associated with height and speed (e.g. stride length at walk), and would disappear when scaling the data. The main differences between the breeds seem to stem from the movement of the hind limbs, with the FM obtaining long over-tracking distances despite the shorter height at withers. Some parameters relevant to gait quality could be improved in the FM to resemble WB movement by strict selection using objective measurements systems.

To optimise the performance and welfare of horses during equestrian competitions in hot climates, it is advised to acclimate them to the heat. The effects of training in a heated indoor arena were studied. Four Olympic horses (13.3 ± 2.2 years; three eventers, one para-dressage horse) were trained for 14 consecutive days in a heated indoor arena (32 ± 1 °C; 50-60% humidity) following their normal training schedule in preparation for the Tokyo Olympic games. Standardised exercise tests (SETs) were performed on Day 1 and Day 14, measuring heart rate (HR; bpm), plasma lactate concentration (LA; mmol/L), deep rectal temperature (T rec; °C), sweat loss (SL; L), and sweat composition (K +, Cl - and Na + concentration). The data were analysed using linear mixed models. The T rec and HR were significantly decreased after acclimation (estimate: -0.106, 95% CI -0.134, -0.078; estimate: -4.067, 95% CI -7.535, -0.598, respectively). Furthermore, for all the horses, the time taken to reach their peak T rec and heat storage increased, while their LA concentrations decreased. The SL, Cl -, and Na + concentrations decreased in three out of the four horses. Conclusions: Fourteen days of normal training in a heated indoor arena resulted in a reduction in cardiovascular and thermal strain. This is advantageous because it shows that elite sport horses can be acclimated while training as usual for a championship.

The use of surface electromyography in the field of animal locomotion has increased considerably over the past decade. However, no consensus exists on the methodology for data collection in horses. This study aimed to start the development of recommendations for bipolar electrode locations to collect surface electromyographic data from horses during dynamic tasks. Data were collected from 21 superficial muscles of three horses during trot on a treadmill using linear electrode arrays. The data were assessed both quantitatively (signal-to-noise ratio (SNR) and coefficient of variation (CoV)) and qualitatively (presence of crosstalk and activation patterns) to compare and select electrode locations for each muscle. For most muscles and horses, the highest SNR values were detected near or cranial/proximal to the central region of the muscle. Concerning the CoV, there were larger differences between muscles and horses than within muscles. Qualitatively, crosstalk was suspected to be present in the signals of twelve muscles but not in all locations in the arrays. With this study, a first attempt is made to develop recommendations for bipolar electrode locations for muscle activity measurements during dynamic contractions in horses. The results may help to improve the reliability and reproducibility of study results in equine biomechanics.

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death. REVIEW QUESTION: Does pharmacological therapy decrease either the incidence or severity of disease or infection caused by EHV-1 in domesticated horses? METHODS: A systematic review was preformed searching AGRICOLA, CAB Abstracts, Cochrane, PubMed, Web of Science, and WHO Global Health Index Medicus Regional Databases to identify articles published before February 15, 2021. Selection criteria were original research reports published in peer reviewed journals, and studies investigating in vivo use of therapeutic agents for prevention or treatment of EHV-1 in horses. Outcomes assessed included measures related to clinical outcomes that reflect symptomatic EHV-1 infection or virus infection. We evaluated risk of bias and performed a GRADE evaluation of the quality of evidence for interventions. RESULTS: A total of 7009 unique studies were identified, of which 9 met the inclusion criteria. Two studies evaluated valacyclovir or small interfering RNAs, and single studies evaluated the use of a Parapoxvirus ovis-based immunomodulator, human alpha interferon, an herbal supplement, a cytosine analog, and heparin. The level of evidence ranged between randomized controlled studies and observational trials. The risk of bias was moderate to high and sample sizes were small. Most studies reported either no benefit or minimal efficacy of the intervention tested. CONCLUSIONS AND CLINICAL IMPORTANCE: Our review indicates minimal or limited benefit either as a prophylactic or post-exposure treatment for any of the studied interventions in the mitigation of EHV-1-associated disease outcome.

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with respiratory and neurologic disease, abortion, and neonatal death. HYPOTHESIS: Vaccines decrease the occurrence of clinical disease in EHV-1-infected horses. METHODS: A systematic review was performed searching multiple databases to identify relevant studies. Selection criteria were original peer-reviewed research reports that investigated the in vivo use of vaccines for the prevention of disease caused by EHV-1 in domesticated horses. Main outcomes of interest included pyrexia, abortion, neurologic disease, viremia, and nasal shedding. We evaluated risk of bias, conducted exploratory meta-analyses of incidence data for the main outcomes, and performed a GRADE evaluation of the quality of evidence for each vaccine subtype. RESULTS: A total of 1018 unique studies were identified, of which 35 met the inclusion criteria. Experimental studies accounted for 31/35 studies, with the remainder being observational studies. Eight vaccine subclasses were identified including commercial (modified-live, inactivated, mixed) and experimental (modified-live, inactivated, deletion mutant, DNA, recombinant). Risk of bias was generally moderate, often because of underreporting of research methods, and sample sizes were small leading to imprecision in the estimate of the effect size. Several studies reported either no benefit or minimal vaccine efficacy for the primary outcomes of interest. Meta-analyses revealed significant heterogeneity was present, and our confidence in the quality of evidence for most outcomes was low to moderate. CONCLUSIONS AND CLINICAL IMPORTANCE: Our review indicates that commercial and experimental vaccines minimally reduce the incidence of clinical disease associated with EHV-1 infection.

Equine herpesvirus-1 (EHV-1) is a highly prevalent and frequently pathogenic infection of equids. The most serious clinical consequences of infection are abortion and equine herpesvirus myeloencephalopathy (EHM). The previous consensus statement was published in 2009 and considered pathogenesis, strain variation, epidemiology, diagnostic testing, vaccination, outbreak prevention and control, and treatment. A recent survey of American College of Veterinary Internal Medicine large animal diplomates identified the need for a revision to this original consensus statement. This updated consensus statement is underpinned by 4 systematic reviews that addressed key questions concerning vaccination, pharmaceutical treatment, pathogenesis, and diagnostic testing. Evidence for successful vaccination against, or effective treatment of EHV-1 infection was limited, and improvements in experimental design and reporting of results are needed in future studies of this important disease. This consensus statement also updates the topics considered previously in 2009.

Electrical disturbances in the heart, known as cardiac arrhythmias, are common in the horse buttheir clinical signficance remains a topic of debate. Due to their electric nature, arrhythmias have a tendency to go unnoticed, as there are often a lack of clincal signs associated with them. While arrhythmias have been associated with sudden cardiac death, in other instances the presence of a cardiac arrhythmia is not indicative of disease. This discrepency inspires the need to determine when cardiac arrhythmias are clinically relevant and what factors may predispose a horse to develop them. Perhaps more iminently, there is a need to scratch the surface on what a veterinarian may consider “normal”.I begin this thesis by exploring, in Chapter 1, the prevalence and risk factors for cardiac arrhythmias in a sedentary mixed breed equine population over the course of 24-hour electrocardiogram recordings. This chapter served as an exploration of what is to be expected in a sedentary herd of horses and aims to provide baseline data on prevalence in an average herd of North American horses. Our data shows that arrhythmias occur at a high prevalence despite a lack of clinical signs, and that increased body condition may put a horse at risk for the development of arrhythmias.Cardiac remodeling, due to stress from disease or exercise, alters the structure of the heart, which in turn effects electrical conduction in the heart. The relationship between arrhythmogenesis and cardiac remodeling remains unclear. In Chapter 2, the investigation of arrhythmogenesis continues by introducing and investigating the relationship between structure and electric functionality of the heart. Here, we demonstrate that myocardial staining for fibrosis and the matricellular protein tenascin C may serve as valuable indicators of myocardial remodeling following damage from exercise or disease.The continued investigation of equine arrhythmogenesis may provide the basis for the development of screening pr

The infectious agents Streptococcus equi subspecies equi (S. equi, the cause of strangles) and EHV-1 (a cause of mild cold-like symptoms, but also of abortion and neurological disease) occur frequently in horses in the Netherlands, as in most of the rest of the world. Research into the best control strategies for these diseases does not currently include (mathematical) models of transmission. This thesis contains several building blocks which will help apply such models to the control effort of these diseases. The basic reproduction number (R0) for S. equi was estimated from real-world outbreaks, and for EHV-1, we attempted to demonstrate an effect of herd vaccination on transmission potential of the virus, but were unable to show a significant effect. By comparing model outputs to real-world epidemiological data on S. equi, we were able to demonstrate that a long-held belief that one in four horses do not mount lasting immunity after strangles may be incorrect. We surveyed apparently healthy horses and ponies from throughout the Netherlands and found that around 1 in 26 of these were strangles carriers. Combining this information with 2022 competition records and disease surveillance reports, simulations showed that carriers and susceptible horses very frequently meet at competitions (hundreds of thousands of times per year), but that very few of these contacts lead to infection and disease in the susceptible horses. A questionnaire survey revealed that very few equine premises in the Netherlands implement effective bio-security measures, which is similar to reports from other countries. Approximately 4/5 of surveyed premises reported horses temporarily leaving the premises to participate in competitions or for training. Network analysis using sports and racing competition records revealed that the “social network” of sport horses and of racehorses are each highly connected. The Dutch sport horse network has five degrees of separation, meaning there is a connection

Horses have been used and bred for centuries for their movements. However, specific breeds are expected to have different movement capabilities. We have measured 425 horses from four different breeds at walk and trot on a straight line using an inertial measurement unit (IMU) system (EquiMoves®). This article describes how the data was collected, filtered and analysed to provide a useable dataset of 28 movement variables. It provides a full protocol for field measurements and requirements for adequate trials for analysis. Intra-class correlation coefficient estimates are also provided to assess repeatability of the measurements.

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.

Facial expressions are important in pain recognition in horses, but current observation-based pain scales remain subjective. A promising technique to quantitatively measure subtle changes in expression patterns, including changes invisible to the human eye, is surface electromyography (sEMG). To achieve high-quality and reliable sEMG signals, unilateral placement of bipolar electrodes is required in relation to the motor endplates (MEP). We aimed to localize the nerve entry points (NEPs; where the nerve branch first pierced the muscle belly) and the direction of the terminal nerve endings to estimate MEP locations of the innervating nerves in five equine facial muscles involved in pain expression. Three cadaveric Dutch Warmblood horse heads were dissected to identify the NEPs in the musculi caninus, levator anguli oculi medialis, nasolabialis, masseter and zygomaticus. These points were marked with pins and measured in relation to a reference line between two anatomical landmarks near the origin and insertion of the respective muscle. Relative distances were calculated from the most caudally situated landmark. NEPs were located at 33%–38% (caninus), 69%–86% (levator anguli oculi medialis) and 0%–18% (zygomaticus) from the caudal landmark. The nasolabialis showed two innervations zones. Its NEPs were located at 47%–72% (dorsal muscle branch) and 52%–91% (ventral branch). All terminal nerve endings were found to run in rostral direction. The masseter showed numerous NEPs diffusely spread within the muscle belly. Therefore, calculation of relative positions was not performed. These results could form the basis for feasibility studies and standardization of bipolar electrode positioning in vivo to measure facial muscle activity patterns in horses.

In order to improve the ability of clinical diagnosis to differentiate articular cartilage (AC) injury of different origins, this study explores the sensitivity of mid-infrared (MIR) spectroscopy for detecting structural, compositional, and functional changes in AC resulting from two injury types. Three grooves (two in parallel in the palmar-dorsal direction and one in the mediolateral direction) were made via arthrotomy in the AC of the radial facet of the third carpal bone (middle carpal joint) and of the intermediate carpal bone (the radiocarpal joint) of nine healthy adult female Shetland ponies (age = 6.8 ± 2.6 years; range 4-13 years) using blunt and sharp tools. The defects were randomly assigned to each of the two joints. Ponies underwent a 3-week box rest followed by 8 weeks of treadmill training and 26 weeks of free pasture exercise before being euthanized for osteochondral sample collection. The osteochondral samples underwent biomechanical indentation testing, followed by MIR spectroscopic assessment. Digital densitometry was conducted afterward to estimate the tissue's proteoglycan (PG) content. Subsequently, machine learning models were developed to classify the samples to estimate their biomechanical properties and PG content based on the MIR spectra according to injury type. Results show that MIR is able to discriminate healthy from injured AC (91%) and between injury types (88%). The method can also estimate AC properties with relatively low error (thickness = 12.7% mm, equilibrium modulus = 10.7% MPa, instantaneous modulus = 11.8% MPa). These findings demonstrate the potential of MIR spectroscopy as a tool for assessment of AC integrity changes that result from injury.

This paper explored the treatment options available for insect bite hypersensitivity (IBH) in horses through a literature study. The conclusion was that drugs used today to treat IBH are primarily systemic glucocorticosteroids and antihistamines. However, antihistamines have not been proven effective enough by themselves, even when high plasma concentrations are achieved. Systemic glucocorticoids have a good effect on IBH but carry many severe adverse effects and other treatment alternatives are therefore warranted. Traditional treatment methods such as rugging and stalling horses inside, alongside the use of fans and insect repellants are often used. New drugs such as vaccines against IL-5 and IL-31 have shown great potential as possible treatments of IBH. These types of vaccines have shown good effect on pruritus in dogs. At the moment these vaccines are too expensive to produce for treatment of equines and therefore not commercially viable. To summarize, improved therapies that are cost efficient are needed to treat IBH in horses., Detta arbete har genom en litteraturstudie utforskat de olika behandlingsalternativen som finns tillgängliga för sommareksem hos häst. Slutsatsen som drogs var att de läkemedel som primärt används idag för att behandla sommareksem är glukokortikoider samt antihistaminer. Dock så har antihistaminer inte funnits vara tillräckligt effektiva även när höga plasmakoncentrationer har uppnåtts. Systemiska glukokortikoider har en god effekt på sommareksem men har risk för flera allvarliga bieffekter, det är således motiverat att ta fram andra behandlingsalternativ. Traditionell, konservativ behandling såsom att täcka och använda insektsmedel på hästarna, samt hålla hästar inomhus tillsammans med andra åtgärder för att hålla området insektsfritt används ofta för behandling. Nya läkemedel såsom vacciner mot IL-5 och IL-31 har visat stor potential som möjliga behandlingar för sommareksem. Dessa typer av vacciner används framgångsrikt för att behandla klåda hos hund, de är dock i dagsläget för dyra för att kunna producera för häst och är därmed inte kommersiellt gångbara. Sammanfattningsvis så krävs det nya förbättrade behandlingar som är kostnadseffektiva för att behandla sommareksem hos häst.

National trotting races held in Spain between 1990–2022 were used to estimate genetic para meters for 5 precocity criteria: age at first ranked race (AFR), age when the animal reached 50% of ranked races (A50%), age when the animal reached the first 10 ranked races (A10), total num ber of races in which the animal has competed before the first ranked race (NRC) and percent age of first placings/year of participation (PFP), in order to evaluate their usefulness for genetic evaluation and their possible inclusion in the Spanish Trotter Horse official genetic program. A total of 176,137 records belonging to 4947 animals were used. A Bayesian multivariate BLUP ani mal model showed heritability values between 0.08 (NRC) and 0.44 (A10). Medium-high genetic co-rrelations were obtained between precocity traits, ranging between −0.78 (A10-PFP) and 0.96 (A50%-A10). NRC and PFP exhibited negative correlations, with the lowest range for NRC. Additionally, a Random Regression Model (RRM) was applied to PFP to evaluate changes in per formance during the animals’ sporting life. Heritability values decreased slightly up to 4 years of age (0.26–0.17), with a slight increase after the age of 5 (0.22). Genetic and permanent environ mental correlations between age groups were in the high range between the nearest age groups. Variations of the expected breeding values (EBV) over the trajectory of all the age groups allowed us to differentiate robust and plastic animals (animals capable of maintaining sporting potential throughout their lives or not). Correlations between the EBV estimated with both methodologies evidenced the benefits of PFP for the early selection of performance traits in STH. Therefore, the application of an RRM for the PFP trait is recommended as it has shown acceptable heritability and adequate correlations with the rest of the precocity criteria studied. The incorporation of this trait in the breed improvement program will allow breeders to have early genetic infor

The low rate of reproductive efficiency in horses may be linked to the equine industry’s practice of maintaining breeding stock that stands out for its athletic or morphological performance but exhibits poor reproductive efficiency. In this study, the age at first foaling, age at last foaling, interval between first and second foaling, average interval between foaling, foaling number and reproductive efficiency, and their relationship with the morphological records in 19,758 Pura Raza Española (PRE) mares were analyzed. After a partial least squares analysis height at withers, lateral hock angle, dorsal-sternal diameter, perimeter of anterior cannon bone, angle of shoulder, thoracic perimeter, hip-stifle distance, and angle of croup were the most important traits related with reproductive traits in PRE mares. A multivariate animal model with both morphological and reproductive traits was applied, including age, geographical area, coat color, and average stud size in the decade of the mare’s first foaling. The results indicate that reproductive selection is feasible, and is enhanced by the use of morphological traits, given the moderate to high magnitudes of heritability values in both types of traits, which oscillated between 0.1 (interval between first and second foaling) and 0.95 (height at withers). The resulting genetic parameters were used to develop a series of selection indexes based on morphological or morphological and reproductive combination traits to improve reproductive efficiency traits in PRE mares and thus compute the expected genetic response (EGR) for the different strategies. The increase in EGR, when comparing EGR using only reproductive traits as selection criteria vs. using a combined index of both morphological and reproductive traits, oscillated between 4.0% for the age at first foaling to 46.8% for the interval between first and second foaling. In addition, two precocity indexes showed positive EGR when the age at last foaling and the interv

African horse sickness (AHS) is a highly severe disease caused by a viral etiological agent, African horse sickness virus (AHSV). It is endemic in sub-Saharan Africa, while sporadic outbreaks have occurred in North Africa, Asia, and Europe, with the most recent cases in Thailand. AHSV transmission between equines occurs primarily by biting midges of the genus Culicoides, especially C. imicola, with a wide distribution globally. As research in horses is highly restricted due to a variety of factors, small laboratory animal models that reproduce clinical signs and pathology observed in natural infection of AHSV are highly needed. Here, we investigated the expression profile of several pro-inflammatory cytokines in target organs and serum of IFNAR (-/-) mice, to continue characterizing this established animal model and to go deep into the innate immune responses that are still needed.

A 6-year-old female Friesian horse was admitted with pyrexia, dullness, weight loss, watery faeces and mild bilateral mucoid nasal discharge. Radiographic and ultrasonographic examination of the thorax indicated a large mid-thoracic soft tissue mass, and the horse was euthanased because of a poor prognosis. At postmortem examination, it was found that the lung tissue was partly replaced by multiple to coalescing soft tissue lesions with a yellow gelatinous aspect, varying in size up to 15 ? 15 ? 20 cm. Histologically, the mass showed mild pyogranulomatous inflammation associated with large numbers of globular encapsulated 10?20 ?m yeasts with or without narrow-based budding, consistent with multiple coalescing cryptococcomas. Quantitative PCR testing of formalin-fixed paraffin-embedded tissue led to the identification of Cryptococcus gattii species complex. To the authors? knowledge, this is the first case reported in the Netherlands of pulmonary cryptococcomas due to C. gattii sensu lato in a horse that had never travelled abroad.

Speed alterations affect many gait analysis parameters. How horses adapt to speed is relevant in many equestrian disciplines and may differ between breeds. This study described changes in gait parameters in 38 Warmblood (WB) and 24 Franches-Montagnes (FM) horses subjected to an incremental speed test at walk (1.35-2.05 m/s) and trot (3.25-5.5 m/s). Time, force and spatial parameters of each limb were measured with an instrumented treadmill and analysed with regression analysis using speed as the independent variable. With higher speeds, stride rate, length, over-tracking distance and vertical ground reaction forces increased while the impulses decreased. The parameters followed the same linear or polynomial regression curves independent of breed, while the slope (linear) or incurvation (polynomial) often differed significantly between breeds. Some differences between the breeds were associated with height and speed (e.g. stride length at walk), and would disappear when scaling the data. The main differences between the breeds seem to stem from the movement of the hind limbs, with the FM obtaining long over-tracking distances despite the shorter height at withers. Some parameters relevant to gait quality could be improved in the FM to resemble WB movement by strict selection using objective measurements systems.

Background: Over-the-wire (OTW) catheter placement is performed frequently in horses. Intravascular loss of a guidewire has been anecdotally reported, but there is limited information regarding the treatment and outcome of horses that have experienced this complication of OTW catheter placement. Objectives: Describe the clinical and diagnostic features, treatment, and outcome of horses experiencing IV guidewire loss at the time of OTW catheter placement. Animals: Thirteen horses. Methods: Multicenter retrospective study to identify horses with IV guidewire loss. Horses of all ages were considered for inclusion. Horses were excluded from the study if complete medical records of signalment, indication, and outcome were not available. Intravenous guidewire loss was defined as the guidewire being lost IV at the time of OTW catheter placement. Results: No horses in this study experienced adverse clinical signs associated with the loss of a guidewire. Eight horses had the guidewire removed and the guidewire was left in situ in 5 horses. None of the horses with the guidewire in situ had experienced long-term effects. Conclusions and Clinical Importance: Intravenous guidewire loss seems to have a good long-term prognosis even in horses in which removal of the guidewire was not possible. Thus, in horses where guidewire removal is not feasible, guidewires that remain in situ may have limited to no adverse effects.

Background: Over-the-wire (OTW) catheter placement is performed frequently in horses. Intravascular loss of a guidewire has been anecdotally reported, but there is limited information regarding the treatment and outcome of horses that have experienced this complication of OTW catheter placement. Objectives: Describe the clinical and diagnostic features, treatment, and outcome of horses experiencing IV guidewire loss at the time of OTW catheter placement. Animals: Thirteen horses. Methods: Multicenter retrospective study to identify horses with IV guidewire loss. Horses of all ages were considered for inclusion. Horses were excluded from the study if complete medical records of signalment, indication, and outcome were not available. Intravenous guidewire loss was defined as the guidewire being lost IV at the time of OTW catheter placement. Results: No horses in this study experienced adverse clinical signs associated with the loss of a guidewire. Eight horses had the guidewire removed and the guidewire was left in situ in 5 horses. None of the horses with the guidewire in situ had experienced long-term effects. Conclusions and Clinical Importance: Intravenous guidewire loss seems to have a good long-term prognosis even in horses in which removal of the guidewire was not possible. Thus, in horses where guidewire removal is not feasible, guidewires that remain in situ may have limited to no adverse effects.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Objective: To provide a comprehensive anatomical description of the normal equine nasolacrimal duct using magnetic resonance imaging, and to develop a practical protocol for performing magnetic resonance dacryocystography (MRD) in horses. Animal studied: The heads of ten fresh horse cadavers with no history of sinonasal or ophthalmologic disorders were used for this study. Procedure: An MRD technique was developed in this study, which consisted of two parts. In the first part, olive oil and gadolinium were compared as contrast medium, using five horses. Two concentrations of gadolinium were used: 0.25 mmol/mL and 0.5 mmol/mL. In the second part, the influence of different head recumbencies using olive oil as the sole contrast medium was studied in the remaining five horses. Results: Injection with olive oil resulted in better distention and a complete filling of the nasolacrimal duct (ND) in all horses. Injection of 0.25 mmol/mL of gadolinium resulted in incomplete filling of the ND, whereas injection of 0.5 mmol/mL of gadolinium resulted in slightly better filling of the duct. The nondependent duct showed better distention compared to the dependent duct when scanning in lateral recumbency. No differences were observed between ducts in a dorsal recumbency. The degree of distention in dorsal recumbency was slightly less than the degree of distention observed in the nondependent ND in lateral recumbency. Conclusions: Magnetic resonance dacryocystography is a suitable technique for evaluating the nasolacrimal duct and can be performed during a normal magnetic resonance examination., Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Background: Ultrasonographic visualisation of the mesenteric vasculature of the large colon (LC) from the right side of the abdomen in cases of displacement and volvulus has been described. However, the LC can move freely within the abdomen and its mesentery can potentially contact both sides of the abdominal wall. Methods: Thirty-four horses presented with LC-related colic that had visible LC mesenteric vasculature visible on abdominal ultrasound were included. A control group was made including horses with confirmed small intestinal-related colic. The objective of this study was to evaluate the visibility of LC mesenteric vasculature with transabdominal ultrasonography in horses with LC-related colic and to determine its diagnostic value. Results: The LC mesenteric vasculature was identified on the right side of the abdomen in 16/34 horses with right dorsal displacement of the LC (RDDLC), 180° LC volvulus (LCV), 540° LCV or LC impaction. On the left side of the abdomen, LC mesenteric vessels were identified in 17/34 horses with left dorsal displacement of the LC (LDDLC), 180° LCV or RDDLC. Vessels were visualised on both sides in one horse with a 180° LCV. Presence of LC mesenteric vasculature in the dorsal aspect on the left side of the abdomen was significantly associated with LDDLC. Conclusion: LC mesenteric vasculature can be visualised on transabdominal ultrasound from either side of the abdomen in horses with different forms of LC-related colic., Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Objective: To provide a comprehensive anatomical description of the normal equine nasolacrimal duct using magnetic resonance imaging, and to develop a practical protocol for performing magnetic resonance dacryocystography (MRD) in horses. Animal studied: The heads of ten fresh horse cadavers with no history of sinonasal or ophthalmologic disorders were used for this study. Procedure: An MRD technique was developed in this study, which consisted of two parts. In the first part, olive oil and gadolinium were compared as contrast medium, using five horses. Two concentrations of gadolinium were used: 0.25 mmol/mL and 0.5 mmol/mL. In the second part, the influence of different head recumbencies using olive oil as the sole contrast medium was studied in the remaining five horses. Results: Injection with olive oil resulted in better distention and a complete filling of the nasolacrimal duct (ND) in all horses. Injection of 0.25 mmol/mL of gadolinium resulted in incomplete filling of the ND, whereas injection of 0.5 mmol/mL of gadolinium resulted in slightly better filling of the duct. The nondependent duct showed better distention compared to the dependent duct when scanning in lateral recumbency. No differences were observed between ducts in a dorsal recumbency. The degree of distention in dorsal recumbency was slightly less than the degree of distention observed in the nondependent ND in lateral recumbency. Conclusions: Magnetic resonance dacryocystography is a suitable technique for evaluating the nasolacrimal duct and can be performed during a normal magnetic resonance examination., Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality eja-culates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three dif-ferent procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared be-tween treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6 h of in-cubation. The remaining sperm parameters assessed were similar among treatments. In conclu-sion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in parti-cular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples., Dr JMM provided the Androcoll-E and Halotech DNA SL the Sperm-Halomax kits used in the experiments, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

CRediT authorship contribution statement E. Da Silva-Alvarez: performed experiments and, Methodology. J.M. Ortiz-Rodríguez: performed experiments and, Methodology. G. Gaitskell-Phillips: English review (native speaker), Writing e review & editing, Software. C. Serres: Formal analysis, performed the analysis of data, Validation, and, Writing e review & editing, Writing e original draft. B. García-Rodríguez: Formal analysis, performed the analysis of data, Validation, and, Writing e review & editing. L. Gutierrez-Cepeda: performed experiments and English, Writing e review & editing. F.E. Martín- Cano: Methodology, and, Data curation. A. Echegaray: Formal analysis, performed the analysis of data with Ecotext, Validation, and . N. Escartin-Casas: Formal analysis, performed the analysis of data with Ecotext, Validation, and, Writing e review & editing, Writing e original draft. F. Requena: Formal analysis, statisticalstadistical analysis, Data curation. M.C. Gil: performed experiments and, Methodology. F.J. Pena: ~ Conceptualization, Validation, and, Writing e review & editing. C. Ortega-Ferrusola: Conceptualization, conceived the study, Supervision, Formal analysis, and roles, Writing e original draft., This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as “control stallions” (n:21, acceptable semen quality), and “stallions with TD” (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 106 spz), that were subdivided into “induced TD group” (immunized, anti-GnRH vaccine) and “acquired TD group”. The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cu, Ministerio de Ciencias, Innovación y Universidades, Junta de Extremadura, FEDER, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Equine bronchoalveolar lavage (BAL) is usually performed with 250–500 mL of isotonic saline at pH 5.5. The acidic pH of saline may cause an increase in airway neutrophil count 48 h after BAL. Other isotonic solutions such as Ringer’s solution, phosphate-buffered saline (PBS) or Plasma-Lyte 148® have a neutral pH of 7.4 and might be a better choice for BAL by not provoking inflammation and the influx of neutrophils into airways. BAL was performed in four healthy horses in four different lung lobes using four different solutions in a randomized crossover design. In each lobe, BAL was performed twice with a 48 h interval using 250 mL of solution. Automated total nucleated cell counts (TNCs) were recorded, and differential cell counts in lavage fluid were determined by two investigators blinded to treatments. The mean volume of BAL fluid retrieved was 51 ± 14%. The mean neutrophil percentage (%N) increased from 1.5 ± 0.9% to 14.7 ± 9.6% at 48 h (p < 0.001) but was not significantly affected by the solution used or the lung lobe sampled. In conclusion, in this study, the influx of neutrophils into airways after BAL was independent of the type of isotonic solution used and the lung lobe sampled. Saline remains an appropriate solution for BAL in horses.

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals ( n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses.

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts ( n = 3) and blastocysts ( n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro ( n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in v

Equine squamous gastric disease (ESGD) is common in horses and poses a serious welfare problem. Several risk factors have been identified and ESGD is routinely treated with omeprazole. Fourteen mares, previously used as embryo recipients and diagnosed with ESGD, were selected. Horses were confined to individual stalls, exercised once daily, and fed ad libitum hay, 1 kg of a low starch compound complementary feed and a mineral supplement. Half of the horses received a compound containing hydrolysed collagen (supplement) and the other half did not (control). At the start of the study, ESGD scores were 3.57 and 3.36 for the supplement and control group, respectively. After 4 weeks, the ESGD grades were significantly reduced in both groups (1.89 and 1.43, respectively, p < 0.01), and healing (ESGD < 2) occurred in 7 out of 14 horses. No treatment effect was observed ( p = 0.75), and it was concluded that the change in husbandry overshadowed any potential effect of the compound. Severe ESGD can improve, and even heal, with the provision of a diet of ad libitum forage and a small amount of a compound complementary feed, without the use of omeprazole. A predictable daily routine, with a limited number of dedicated caretakers, may have contributed to the improvement of gastric health.

Musculoskeletal injuries are a major cause of injury and death for racehorses. Underrun heel conformation in racehorses has been associated with catastrophic fetlock breakdowns during racing and training. While the cause of the development of underrun heels is not fully understood, two factors that could contribute to their development are the method that horseshoes are attached to the hoof and length of the shoe branches. As racehorses are often shod with shoes with shorter branches and with nails placed palmar to the widest portion of the hoof, the research presented here aimed to investigate the effect of nail placement location and shoe branch length, to determine whether either factor could play a role in the development of an underrun heel. Two in vitro experiments examined the effects of nail placement (increasing number dorsal to palmar) and 3 different shoe branch lengths on hoof wall surface strains and deformations during limb loading that simulated the middle of stance through gallop loads. Rosette strain gauges placed on the lateral side of the equine hoof in the heel and quarter regions of the lateral side of the hoof wall measured strains. Kinematic markers placed on the hoof tracked hoof expansion and distortion. A third in vivo 30 week long longitudinal crossover study examined the effect of shoes with either Full or Short branch lengths on forelimb hoof shape and growth. The most notable findings are related to nail placement configuration. In vitro, differences were observed in hoof expansion and hoof wall surface strains and deformations among hooves with nails placed in three configurations to attach the shoe to the hoof wall: toe nails (T), toe and quarter nails (TQ), and toe, quarter, and heel nails (TQH). Heel expansion decreased (P<0.001) when nails were placed under the TQH configuration. The largest changes in hoof wall principal strain directions occurred distally with the TQH configuration. With the hoof fixed to the ground, the direc

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death. OBJECTIVE: To determine if there is an association between the level and duration of EHV-1 viremia and either abortion or equine herpesvirus myeloencephalopathy (EHM) in domesticated horses? METHODS: A systematic review was performed searching numerous databases to identify peer reviewed reports that evaluated viremia and EHM, or viremia and abortion published before January 19, 2021. Randomized controlled trials and observational studies were assessed for risk of bias or publication quality. RESULTS: A total of 189 unique studies were identified, of which 34 met the inclusion criteria. Thirty studies evaluated viremia and neurologic outcomes including 4 observational studies. Eight experimental studies examined viremia and abortion, which used the Ab4 and OH03 virus strains or recombinant Ab4 derivatives. Incidence rates for both EHM and abortion in experimental studies varied among the studies as did the level of evidence. Viremia was generally detectable before the onset of either EHM or abortion. Risk of bias was generally low to moderate, sample sizes were small, and multiple studies reported negative outcome data. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study support that viremia is regularly present before EHM or abortion occurs. However, no inferences could be made about the relationship between the occurrence of either neurological signs or abortion and the magnitude or duration of viremia.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) infection is associated with upper respiratory disease, EHM, abortions, and neonatal death. RESEARCH QUESTIONS: Are nasal secretions a more sensitive biological sample compared to blood for the detection of EHV-1 infection? How long is EHV-1 detectable after primary infection by PCR? METHODS: MedLine and Web of Science searches identified original peer-reviewed reports evaluating nasal shedding and viremia using virus isolation methods or PCR published in English before October 9, 2023. RESULTS: Sixty experimental and 20 observational studies met inclusion criteria. EHV-1 detection frequency by qPCR in nasal secretions and blood from naturally-infected horses with fever and respiratory signs were 15% and 9%, respectively; qPCR detection rates in nasal secretions and blood from horses with suspected EHM were 94% and 70%, respectively. In experimental studies the sensitivity of qPCR matched or exceeded that seen for virus isolation from either nasal secretions or blood. Detection of nasal shedding typically occurred within 2 days after EHV-1 inoculation with a detection period of 3 to 7 days. Viremia lasted 2 to 7 days and was usually detected ≥1 days after positive identification of EHV-1 in nasal secretions. Nasal shedding and viremia decreased over time and remained detectable in some horses for several weeks after inoculation. CONCLUSIONS AND CLINICAL IMPORTANCE: Under experimental conditions, blood and nasal secretions have similar sensitivity for the detection of EHV-1 when horses are sampled on multiple consecutive days. In contrast, in observational studies detection of EHV-1 in nasal secretions was consistently more successful.

Equines have been used for utilized for manual labor, recreation, and companionship amongst many other valuable conveniences since their domestication. As the modern horse progressed from livestock to athlete, attention was paid to the body conformation to be used as an indicator of biomechanics and can dictate equine performance. Poor conformation can put physical limitations on the body and predispose the horse to injury and chronic disease. When not managed properly, these flaws can lead to injury, lameness, and premature retirement in sport horses. The distal limb is composed of tendons and ligaments that are all susceptible to tear or rupture. Protective wraps or boots are typically applied to the distal limb prior to exercise to prevent superficial injury from the environment or interference. However, these preventatives can trap heat against the skin which can have detrimental effects on the fibroblasts which can lead to failure. It was not until the early twentieth century that the idea of equine physiotherapy was adopted, and practices changed to meet remedial needs and create a sustainable, healthy equine athlete. Equine physiotherapy is a broad-spectrum term used to describe the therapeutic efforts made to keep the body in good health by means of prevention of injury to improve or maintain performance. Traditionally, therapeutics are administered by a veterinarian or trained professional in the event of an existing injury. In recent years therapeutics have been commercialized and are readily available for everyday preventative use. The most common readily available treatments being variations of pulsating electromagnetic fields (PEMF), vibration therapy, cryotherapy, and thermotherapy. When used prior to or after exercise, the therapeutics are designed to prepare the body for exercise and improve recovery by increasing circulation and down regulating the inflammatory response. The studies performed evaluate the efficacy of Rambo®Ionic (Horseware, Dundalk

LGD-3303 is a Selective Androgen Receptor Modulator (SARM) that is prohibited in both equine and human sports due to its anabolic properties. The aim of this study was to investigate the equine in vivo metabolite profile of LGD-3303 and identify drug metabolites that can be suitable as new and improved analytical targets for equine doping control. This was performed by an oral administration of 0.05 mg.kg(-1) LGD-3303 to horses, where blood and urine samples were collected up to 96 h after administration. The in vivo samples consisting of plasma, urine and hydrolyzed urine were analyzed utilizing ultra-high performance liquid chromatography hyphenated to a Q Exactive (TM) Orbitrap (TM) high resolution mass spectrometer with a heated electrospray ionization source. A total of eight metabolites of LGD-3303 were tentatively identified, including one carboxylated and several hydroxylated metabolites in combination with glucuronic acid conjugates. A monohydroxylated metabolite is suggested as an analytical target for doping control analysis of plasma and urine after hydrolysis with beta-glucuronidase, due to the high intensity and prolonged detection time in comparison to parent LGD-3303.

LGD-3303 is a Selective Androgen Receptor Modulator (SARM) that is prohibited in both equine and human sports due to its anabolic properties. The aim of this study was to investigate the equine in vivo metabolite profile of LGD-3303 and identify drug metabolites that can be suitable as new and improved analytical targets for equine doping control. This was performed by an oral administration of 0.05 mg.kg(-1) LGD-3303 to horses, where blood and urine samples were collected up to 96 h after administration. The in vivo samples consisting of plasma, urine and hydrolyzed urine were analyzed utilizing ultra-high performance liquid chromatography hyphenated to a Q Exactive (TM) Orbitrap (TM) high resolution mass spectrometer with a heated electrospray ionization source. A total of eight metabolites of LGD-3303 were tentatively identified, including one carboxylated and several hydroxylated metabolites in combination with glucuronic acid conjugates. A monohydroxylated metabolite is suggested as an analytical target for doping control analysis of plasma and urine after hydrolysis with beta-glucuronidase, due to the high intensity and prolonged detection time in comparison to parent LGD-3303.

LGD-3303 is a Selective Androgen Receptor Modulator (SARM) that is prohibited in both equine and human sports due to its anabolic properties. The aim of this study was to investigate the equine in vivo metabolite profile of LGD-3303 and identify drug metabolites that can be suitable as new and improved analytical targets for equine doping control. This was performed by an oral administration of 0.05 mg.kg(-1) LGD-3303 to horses, where blood and urine samples were collected up to 96 h after administration. The in vivo samples consisting of plasma, urine and hydrolyzed urine were analyzed utilizing ultra-high performance liquid chromatography hyphenated to a Q Exactive (TM) Orbitrap (TM) high resolution mass spectrometer with a heated electrospray ionization source. A total of eight metabolites of LGD-3303 were tentatively identified, including one carboxylated and several hydroxylated metabolites in combination with glucuronic acid conjugates. A monohydroxylated metabolite is suggested as an analytical target for doping control analysis of plasma and urine after hydrolysis with beta-glucuronidase, due to the high intensity and prolonged detection time in comparison to parent LGD-3303.

Infections of fracture fixation implants and synovial structures are a primary cause of complications, increased treatment costs, and mortality in people and horses. Treatment failure is often due to biofilms that are communities of bacteria that are adhered to a surface or to each other and are surrounded in a self-secreted extracellular matrix. The biofilm matrix protects the indwelling bacteria from being killed by antibiotics and the immune system. Biofilms also stimulate chronic inflammation and tissue destruction, including peri-implant osteolysis and subsequent implant failure and chondromalacia with subsequent osteoarthritis. In horses, the resulting lameness, reduced athletic potential, and poor quality of life may necessitate euthanasia. Equine bone marrow-derived mesenchymal stromal cells (MSC) reduce inflammation and promote healing in musculoskeletal injuries and have recently been discovered to have antimicrobial properties. Equine MSC kill planktonic (free-floating) bacteria and prevent biofilm establishment in laboratory models. MSC from mice and people also promote the transition from acute inflammation to tissue regeneration (resolution of inflammation) by secretion of specialized pro-resolving lipid mediators (SPM). Whether equine MSC can disrupt established biofilms of orthopedic pathogens and modulate the inflammatory response to orthopedic biofilms is unknown. Using a novel biofilm-MSC co-culture model, our objectives were two-fold. We investigated whether MSC alone or with amikacin sulfate, an antibiotic used to treat equine orthopedic infections, could reduce biomass, pellicle size, and live bacteria of biofilms of orthopedic infectious agents S. aureus and E. coli. Next, we investigated whether MSC could modulate immune response to S. aureus biofilms by reducing secretion of pro-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) and by secreting SPM. MSC demonstrated partial ability to reduce biofilms but performed different

Unaccustomed, strenuous exercise can cause skeletal muscle damage that subsequently induces an acute inflammatory response in the tissue which is marked by an infiltration of leukocytes into the damaged muscle. To try and suppress the initial pro-inflammatory response in skeletal muscle of horses performing a single exercise stress test, a commercial sodium hyaluronate (HA) treatment was administered and tested for anti-inflammatory properties. Unfit, adult Thoroughbreds were intravenously injected three times with HA or received no injection at all (CON) over a 3-week period before performing a single submaximal exercise test. Gluteal muscle biopsies were collected before and 1 h after the completion of exercise for RNA-Seq and staining. The results indicated that HA treatment in horses down regulated genes associated with lymphocyte activation and cytokine production (Il17RA, OSCAR, LYL1, TLR1, TLR2, TLR8, TLR10) but did not irreversibly down regulate these genes with the addition of exercise. Exercise as a stressor did cause an acute inflammatory response in muscle which was seen through global expression of macrophage and neutrophil surface markers (NCF2, ELANE, CD168I). These results determine that HA treatment does act as an anti-inflammatory in equine skeletal muscle but does not possess prolonged effects with the initiation of inflammation.

Disorders of sexual development (DSD) are associated with atypical chromosomal, gonadal, or phenotypic sex. It is likely that the number of cases of DSD are underestimated in the equine population. Monorchidism in the horse is very rare. This case report describes the clinical assessment of a phenotypic mare with stallion-like behavior which led to the diagnosis of a DSD. A 4-year-old Quarter Horse mare presented in good body condition, with normal external genitalia for a mare, and normal mammary glands with two bilaterally symmetric teats. No uterus, cervix, or gonads were detected on transrectal palpation. Transrectal ultrasonography revealed a single gonad in the right dorsal abdomen with the morphologic appearance of a testicle. Presurgical hormonal evaluation revealed elevated serum testosterone and anti-Müllerian hormone (AMH) concentrations. The right gonad was successfully removed via standing exploratory laparoscopy and submitted for histopathology. No gonad was identified on the left side during laparoscopy. Histopathologic examination confirmed that the excised gonad was a testicle. Cytogenetic and molecular analysis revealed a 64,XY, SRY-positive chromosomal constitution. Hormonal evaluation 5 weeks after surgery revealed low serum testosterone and AMH levels. A diagnosis of monorchidism was based on ultrasound examination, laparoscopic exploration of the abdomen, removal of a single gonad, and a subsequent decrease in serum testosterone and AMH concentrations to basal levels. In summary, a combination of clinical signs, endocrine evaluation, chromosomal and molecular analysis, and histopathology can be used in the diagnosis of DSD conditions.

Small intestinal ileus affects up to half of all horses undergoing small intestinal surgery, leading to prolonged gastrointestinal (GI) transit time which can be life-threatening. Various prokinetic medications have been associated with varying side effects, questionable efficacy, and increased cost. Gum chewing as a form of sham feeding is used as a safe, effective, well-tolerated, and inexpensive way to ameliorate ileus following GI surgery in humans. Bit chewing for horses, an analogous activity, has been shown to significantly decrease GI total transit time (TTT); however, a direct effect of bit chewing on gastric emptying time (GET), small intestinal transit time (SITT), and total orocecal transit time (OCTT) has not been investigated. Our objective was to determine whether bit chewing increased small intestinal motility and decreased GE, SITT, and OCTT in clinically normal horses. Gastrointestinal motility was compared in horses that were bit chewing compared to control conditions (no bit chewing) in a prospective crossover design study using acetaminophen as a marker for GET and video endoscopy (ALICAM) capsules to determine GET, SITT, and OCTT. Bit chewing was well tolerated by all horses with no side effects noted. Bit chewing led to a shorter GET, SITT, and significantly shorter OCTT when compared to the control group (P = 0.015). Median times for bit chewing conditions were as follows: GE 2.86 hr, SITT 3.65 hr, and OCTT 6.15 hr whereas the median times for control conditions were as follows: GE: 5 hr, SITT 4.4 hr, and OCTT 9.92 hr. In summary, bit chewing proves to be a potential tool to hasten the motility of the oral GIT. It is safe, inexpensive, and potentially effective prokinetic treatment to horses suffering from postoperative ileus and further investigation is warranted.