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Laminitis is a common and painful condition of the equine foot and approximately 90% of cases are associated with insulin dysregulation (ID) that is a central feature of the common endocrine disorder equine metabolic syndrome (EMS) and occurs in a subset of animals with pituitary pars intermedia dysfunction. Additional features of EMS include obesity, altered circulating concentrations of adipokines (particularly adiponectin and leptin) and hypertriglyceridaemia. Obesity, ID, hypoadiponectinaemia, hyperleptinaemia and an altered plasma lipid profile are also features of human metabolic syndrome (HMS) alongside hyperglycaemia. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a novel class of oral hypoglycaemic agents used in combination with lifestyle changes in the management of HMS. SGLT2 receptors are responsible for 90% of the renal glucose reabsorption that occurs in the proximal convoluted tubule. Thus, these drugs increase urinary glucose excretion by suppressing glucose reabsorption from the glomerular filtrate resulting in urinary calorie loss with consequent weight loss and improvements in ID, hyperglycemia, hypoadiponectinaemia and hyperleptinaemia. There are no licenced veterinary drugs available for treating ID and preventing insulin-associated laminitis in horses. Thus, the use of SGLT2i for the control of equine hyperinsulinaemia with the goal of improving recovery from associated active laminitis or preventing future laminitis has recently been advocated. There are a small number of published studies reporting the use of the SGLT2i canagliflozin, ertugliflozin and velagliflozin to aid the management of equine ID. However, the doses used are largely extrapolated from human studies with limited consideration of species-specific variations. In addition, there is limited evaluation of the fundamental differences between ID in horses and humans, particularly the fact that most horses with ID remain hyperinsulinaemic but normoglycaemic such that increased urinary loss of glucose may not explain the beneficial effects of these drugs. Further study of the potential deleterious effects of treatment-associated hypertriglyceridaemia is required together with the effect of SGLT2i therapy on circulating concentrations of adipokines in horses., (© 2024 The Author(s). Journal of Veterinary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.)

Background: Hypoadiponectinaemia is a risk factor for endocrinopathic laminitis, but the directionality and nature of its association with insulin dysregulation is unclear., Objectives: To investigate the effects of short-term induced hyperinsulinaemia and dexamethasone challenge on circulating [total adiponectin] and whole blood expression of adiponectin (AdipoR1 and AdipoR2), insulin, and insulin-like growth factor 1 (IGF-1) receptors in insulin-sensitive ponies., Study Design: In vivo experiment., Methods: Six never-laminitic, insulin-sensitive, native-breed UK ponies first underwent a dexamethasone challenge (0.08 mg/kg i.v.) with blood samples collected every 15 min over 3 h. After a 14-day washout period, hyperinsulinaemia was induced for 9 h via a euglycaemic-hyperinsulinaemic clamp (EHC), with blood samples collected every 30 min. Serum [insulin], plasma [total adiponectin], and plasma [IGF-1] were measured using validated assays and receptor gene expression was assessed via quantitative polymerase chain reaction (qPCR). Finally, whole blood was incubated with 10-1000 ng/mL dexamethasone for 3 h at 37°C to investigate its direct effects on gene expression., Results: There were no adverse effects observed during either protocol. Dexamethasone challenge did not alter circulating [insulin] or [total adiponectin] at any time-point, but significantly upregulated AdipoR1 and IGF-1R expression at 150 and 180 min. Ex vivo incubation of whole blood with dexamethasone did not alter expression of the genes examined. There was no change in [total adiponectin] or expression of the genes examined associated with EHC-induced hyperinsulinemia., Main Limitations: This was a small sample size that included only native-breed ponies; total adiponectin was measured rather than high-molecular-weight adiponectin., Conclusions: Short-term induced hyperinsulinaemia and dexamethasone challenge did not affect circulating [total adiponectin] in insulin-sensitive ponies. However, dexamethasone administration was associated with upregulation of two receptors linked to adiponectin signalling, suggesting that a physiological response occurred possibly to counteract dexamethasone-associated changes in tissue insulin sensitivity., (© 2023 MARS Horsecare and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: Pituitary pars intermedia dysfunction (PPID) is a prevalent, age-related chronic disorder in equids. Diagnosis of PPID can be challenging because of its broad spectrum of clinical presentations and disparate published diagnostic criteria, and there are limited available treatment options., Objectives: To develop evidence-based primary care guidelines for the diagnosis and treatment of equine PPID based on the available literature., Study Design: Evidence-based clinical guideline using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework., Methods: Research questions were proposed by a panel of veterinarians and developed into PICO or another structured format. VetSRev and Veterinary Evidence were searched for evidence summaries, and systematic searches of the NCBI PubMed and CAB Direct databases were conducted using keyword searches in July 2022 and updated in January 2023. The evidence was evaluated using the GRADE framework., Results and Recommendations: The research questions were categorised into four areas: (A) Case selection for diagnostic testing, pre-test probability and diagnostic test accuracy, (B) interpretation of test results, (C) pharmacological treatments and other treatment/management options and (D) monitoring treated cases. Relevant veterinary publications were identified and assessed using the GRADE criteria. The results were developed into recommendations: (A) Case selection for diagnostic testing and diagnostic test accuracy: (i) The prevalence of PPID in equids aged ≥15 years is between 21% and 27%; (ii) hypertrichosis or delayed/incomplete hair coat shedding provides a high index of clinical suspicion for PPID; (iii) the combination of clinical signs and age informs the index of clinical suspicion prior to diagnostic testing; (iv) estimated pre-test probability of PPID should be considered in interpretation of diagnostic test results; (v) pre-test probability of PPID is low in equids aged <10 years; (vi) both pre-test probability of disease and season of testing have strong influence on the ability to diagnose PPID using basal adrenocorticotropic hormone (ACTH) or ACTH after thyrotropin-releasing hormone (TRH) stimulation. The overall diagnostic accuracy of basal ACTH concentrations for diagnosing PPID ranged between 88% and 92% in the autumn and 70% and 86% in the non-autumn, depending on the pre-test probability. Based on a single study, the overall diagnostic accuracy of ACTH concentrations in response to TRH after 30 minutes for diagnosing PPID ranged between 92% and 98% in the autumn and 90% and 94% in the non-autumn, depending on the pre-test probability. Thus, it should be remembered that the risk of a false positive result increases in situations where there is a low pre-test probability, which could mean that treatment is initiated for PPID without checking for a more likely alternative diagnosis. This could compromise horse welfare due to the commencement of lifelong therapy and/or failing to identify and treat an alternative potentially life-threatening condition. (B) Interpretation of diagnostic tests: (i) There is a significant effect of breed on plasma ACTH concentration, particularly in the autumn with markedly higher ACTH concentrations in some but not all 'thrifty' breeds; (ii) basal and/or post-TRH ACTH concentrations may also be affected by latitude/location, diet/feeding, coat colour, critical illness and trailer transport; (iii) mild pain is unlikely to have a large effect on basal ACTH, but caution may be required for more severe pain; (iv) determining diagnostic thresholds that allow for all possible contributory factors is not practical; therefore, the use of equivocal ranges is supported; (v) dynamic insulin testing and TRH stimulation testing may be combined, but TRH stimulation testing should not immediately follow an oral sugar test; (vi) equids with PPID and hyperinsulinaemia appear to be at higher risk of laminitis, but ACTH is not an independent predictor of laminitis risk. (C) Pharmacologic treatments and other treatment/management options: (i) Pergolide improves most clinical signs associated with PPID in the majority of affected animals; (ii) Pergolide treatment lowers basal ACTH concentrations and improves the ACTH response to TRH in many animals, but measures of insulin dysregulation (ID) are not altered in most cases; (iii) chasteberry has no effect on ACTH concentrations and there is no benefit to adding chasteberry to pergolide therapy; (iv) combination of cyproheptadine with pergolide is not superior to pergolide alone; (v) there is no evidence that pergolide has adverse cardiac effects in horses; (vi) Pergolide does not affect insulin sensitivity. (D) Monitoring pergolide-treated cases: (i) Hormone assays provide a crude indication of pituitary control in response to pergolide therapy, however it is unknown whether monitoring of ACTH concentrations and titrating of pergolide doses accordingly is associated with improved endocrinological or clinical outcome; (ii) it is unknown whether monitoring the ACTH response to TRH or clinical signs is associated with an improved outcome; (iii) there is very weak evidence to suggest that increasing pergolide dose in autumn months may be beneficial; (iv) there is little advantage in waiting for more than a month to perform follow-up endocrine testing following initiation of pergolide therapy; there may be merit in performing repeat tests sooner; (v) timing of sampling in relation to pergolide dosing does not confound measurement of ACTH concentration; (vi) there is no evidence that making changes after interpretation of ACTH concentrations measured at certain times of the year is associated with improved outcomes; (vii) evidence is very limited, however, compliance with PPID treatment appears to be poor and it is unclear whether this influences clinical outcome; (viii) evidence is very limited, but horses with clinical signs of PPID are likely to shed more nematode eggs than horses without clinical signs of PPID; it is unclear whether this results in an increased risk of parasitic disease or whether there is a need for more frequent assessment of faecal worm egg counts., Main Limitations: Limited relevant publications in the veterinary scientific literature., Conclusions: These findings should be used to inform decision-making in equine primary care practice., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: The hormones insulin and adiponectin are commonly measured in equids because of their involvement in endocrinopathic laminitis. These are currently measured in serum/plasma, although jugular venipuncture can cause stress in some animals and may be impossible in needle-shy individuals. However, both hormones can be measured in saliva in other species., Objectives: To determine whether [insulin] and [total adiponectin] are detectable in equine saliva using automated assays and whether saliva collection is associated with changes in stress indicators. Additionally, the correlation between serum and salivary [insulin] was investigated., Study Design: In vivo experiment., Methods: Paired blood and saliva samples were collected from eight adult ponies at multiple time-points (n = 45 paired samples). [Insulin] and [total adiponectin] were measured using automated assays validated for equine serum/plasma. Blink rates and heart rates were determined, using video recordings and a wearable heart rate monitor respectively, to assess the effects of sample collection on stress indicators compared with a control situation without a stressful stimulus., Results: [Total adiponectin] was undetectable in saliva. However, salivary [insulin] was measurable with acceptable inter-assay (1.3 ± 0.9%) and intra-assay (1.1 ± 0.6%) variability. Blink and heart rates during saliva collection did not differ significantly from those in the control setting. Serum and salivary [insulin] were not significantly correlated., Main Limitations: Small sample size comprising native UK ponies; potential sampling bias as animals were recruited to the study partly based on their behaviour during blood sampling; saliva collected from unfasted animals., Conclusions: Insulin is measurable in equine saliva using an automated assay currently available in the UK, but further validation and the determination of specific diagnostic thresholds are required. Saliva collection was not associated with changes in stress indicators. Further research is therefore needed to determine the potential of equine saliva as a non-invasive alternative to blood for insulin determination., (© 2023 MARS Horsecare and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: Equine metabolic syndrome is a collection of risk factors associated with an increased risk of endocrinopathic laminitis. All affected animals display insulin dysregulation and some may show adiponectin dysregulation and/or excessive adiposity. However, the relationship between obesity and hypoadiponectinaemia in equids remains unclear., Objectives: To investigate the relationship between obesity and circulating plasma total adiponectin (TA) concentrations in native-breed ponies in England., Study Design: Secondary data analysis., Methods: Data collected for three previous studies were retrospectively analysed and cohorts were pooled where possible (maximum sample size: n = 734 ponies). Correlations between [TA], age, and morphometric measures were assessed using Spearman's correlation coefficient. [TA] was compared between animals of different body condition score (BCS) classification (ideal-weight, overweight, and obese), breed, and body shape using Kruskal-Wallis with Dunn's post hoc tests, and sex using Mann-Whitney U test. The proportions of obese and ideal-weight ponies with basal hyperinsulinaemia and/or hypoadiponectinaemia were compared using a Chi-square test of homogeneity and post hoc z-test. Logistic regression was used to identify factors that may discriminate ponies with hypoadiponectinaemia., Results: [TA] was weakly positively correlated with BCS, height, weight, and weight:height ratio (Spearman's ρ = 0.14-0.29, p < 0.05). There were significant differences in [TA] in ponies with different BCS group classification, body shape, and breed. A greater percentage of obese (54.6%) than ideal-weight ponies (33.1%, p < 0.001) had both normal [TA] and [basal insulin], and a greater percentage of ideal-weight (38.6%) than obese ponies (16.5%, p < 0.001) showed hypoadiponectinaemia. Weight:height and BCS group were significant variables in a logistic regression of hypoadiponectinaemia but model fit and predictive accuracy were poor., Main Limitations: Retrospective study design, only native-breed ponies included., Conclusions: Morphometric measures such as BCS do not closely reflect [TA]. Circulating [TA] and [basal insulin] should be determined in all animals with predisposing factors, regardless of obesity status., (© 2023 MARS Horsecare and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: Serum insulin concentration at 60 min (InsulinT60) during an oral sugar test (OST) indicates future laminitis risk and insulin dysregulation (ID). Associations between InsulinT60 and physical and owner-reported variables may help clinicians select individuals for ID testing. Associations between InsulinT60 and other metabolic markers may help elucidate ID pathophysiology., Objectives: To describe associations between (A) season, physically-apparent and owner-reported factors and binary InsulinT60 interpretation (initial models) and (B) variables included in the initial models, other metabolic markers and continuous InsulinT60 (full models)., Study Design: Prospective longitudinal., Methods: Non-laminitic ponies were examined and OSTs (0.3 mL/kg Karo syrup) performed every 6 months (autumn and spring) for ≤4 years. Factors associated with InsulinT60 were determined using mixed-effects models with physical, owner-reported, season and serum/plasma markers as fixed effects and pony and premises identifiers as random effects. Autumn and spring data were analysed separately for full models., Results: One thousand seven hundred and sixty-three OSTs from 367 ponies were included. High-risk InsulinT60 (>153 μIU/mL) was independently associated with (odds ratio, 95% confidence interval [CI]): age (1.36, 1.16-1.60), body condition score (BCS) (2.38, 1.21-4.69), and bulging supraorbital fatpads (7.25, 2.1-24.98). However, the initial models provided little explanatory power (Nakagawa R 2  = 0.1-0.27). LoginsulinT60 was independently associated with (effect estimate, 95% CI): age (0.02, 0.01-0.04), Welsh/Welsh X breed (0.22, 0.05, 0.39), sex (gelding = -0.2, -0.34 to 0.06), BCS (0.16, 0.08-0.23), plasma adiponectin (-0.02, -0.02 to 0.01) and basal insulin (0.01, 0.01-0.01) in spring, and: age (0.03, 0.02-0.04), BCS (0.17, 0.08-0.26), bulging supraorbital fatpads (0.37, 0.2-0.54), turnout score (0.05, 0.02-0.09), plasma adiponectin (-0.01, -0.02 to 0.01), ACTH (per 10 pg/mL) (0.01, 0.00-0.01), triglycerides (0.28, 0.07-0.49) and InsulinT0 (0.01, 0.01-0.01) in autumn., Main Limitations: Only non-laminitic ponies in one region were included., Conclusions: Owner-reported and physically-apparent data were limited InsulinT60 predictors. InsulinT60 is a complex trait, independently associated with numerous variables, some with seasonal interactions., (© 2023 Mars Horsecare and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Feedstuffs are often recommended to mitigate potential damage from acid associated with equine squamous gastric disease (ESGD). In acidic conditions, pectin alters its structure to one like mucus and binds the stomach mucosa, whilst alfalfa has a strong intrinsic acid buffering capacity. The study aimed to determine whether feeding a commercial beet pulp/alfalfa/oat fibre mix aids ESGD healing and/or prevention of recurrence. Ten adult horses with naturally occurring ESGD were included. All animals were treated with omeprazole as per the attending veterinarian's recommendation and randomly allocated to also be fed a commercial beet pulp/alfalfa/oat fibre mix (1Kg/horse divided into 2 meals/day; n=5) or no additional feed (n=5) for one month. Gastroscopy was then repeated to assess response to therapy. If the ESGD had healed, omeprazole therapy was discontinued, and the commercial feed given to all horses for a further month. Gastroscopy was repeated to determine ESGD recurrence. The mean (±SD) age of the horses was 11.6 (±3.8) years; 4 mares and 6 geldings; various breeds were represented; and the median (range) initial ESGD grade was 2 (2-4). ESGD had healed (grade 0/4) in all animals after one month. After a further month, ESGD had recurred in significantly (p=0.04) more animals that did not receive the commercial feed initially (3/5; 60%; mean [range] ESGD grade 3 [0,4]) compared to those that did (0/5; 0%; mean [range] ESGD grade 0 [0,0]). Thus, the commercial beet pulp/alfalfa/oat fibre mix aided prevention of ESGD recurrence when fed during the healing and prevention phases., Competing Interests: Declaration of competing interest Tom Shurlock is an equine nutrition consultant for the study funder, British Horse Feeds. Nicola Menzies-Gow does not have any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: High concentrations of adrenocorticotropic hormone (ACTH) are used as an indicator of pituitary pars intermedia dysfunction (PPID), but other factors that may influence ACTH need to be understood, if diagnostic reference ranges for ACTH are to be used with confidence. Insulin dysregulation (ID) could be one such factor, as insulin affects pituitary hormones in other species., Objectives: To test the hypothesis that a relationship exists between high insulin and high ACTH in aged (>15-year-old) animals with no clinical signs of PPID., Study Design: A cohort study., Methods: Thirteen horses and eleven ponies (17-25 years-old; mares and geldings) were clinically examined for signs of PPID in the spring (November 2020) and autumn (April 2021). On the same day, blood samples were taken before and 2 h after an oral glucose test (OGT). Concentrations of insulin, glucose, ACTH and cortisol were measured., Results: There was no association between ACTH and cortisol. However, there was a positive linear correlation between ACTH and post-OGT (insulin in the autumn (r = 0.427, p = 0.04). Two horses and six ponies had ACTH above the cut-off value for PPID diagnosis, and of these eight animals, six also had insulin concentrations above the cut-off value for ID., Main Limitations: The cohort was small and thyrotropin-releasing hormone (TRH) stimulation tests were not performed., Conclusions: In autumn, high ACTH was associated with ID, when no clinical signs of PPID were present. Because ACTH is used in PPID diagnosis, further work is required to understand this interaction., (© 2023 Mars Horsecare UK and The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH, α-melanocyte stimulating hormone (α-MSH, ACTH 1-17) and CLIP. Synthetic ACTH and CLIP bound to the capture antibody of the chemiluminescent ACTH assay, but α-MSH did not. Equine endogenous CLIP was detected by LCMS in pony plasma taken in the autumn and could be eluted from the capture antibody of the ACTH chemiluminescent immunoassay. Further research is required to enable quantification of CLIP. Equine CLIP may alter measured ACTH concentrations in vivo., (Copyright © 2023. Published by Elsevier Inc.)

Background: Emergency laparotomies in donkeys are infrequently performed and there is limited literature on the subject., Objectives: To determine findings and associated outcomes of exploratory laparotomies in donkeys., Study Design: Descriptive retrospective study., Methods: Donkeys undergoing emergency exploratory laparotomy for investigation and treatment of colic at seven UK referral hospitals between 2005-2017 were included. Data were retrieved from available hospital records. Descriptive statistics and inferential statistical analysis of outcomes of interest was performed in three steps., Results: Thirty-three cases fulfilled the inclusion criteria. Clinical signs on presentation were available for 32 donkeys, of which 53.1% (17/32) presented for investigation of colic while in 46.9% (15/32) the presenting complaint was non-specific. Primary lesion location included small intestine (42.4%, 14/33), large colon (39.3%, 13/33), caecum (6.1%, 2/33), stomach (6.1%, 2/33) and 6.1% (2/33) had multiple abnormal findings without a clear primary lesion. Overall survival to discharge was 54.5% (18/33). Five donkeys (15.2%, 5/33) were euthanased at surgery and of those recovering from general anaesthesia a further 35.7% (10/28) were euthanased or died prior to discharge. Six donkeys (21.4%, 6/28) required a second laparotomy of which 4 (66.7%, 4/6) survived. Post-operative complications occurred in 82.1% (23/28) of cases and included hyperlipaemia (42.9%, 12/28), incisional complications (21.4%, 6/28), ileus (21.4%, 6/28) and persistent colic (17.9%, 5/28). When adjusted for other complications, donkeys with primary gastric lesions were less likely to have presented with severe colic compared with those with primary small intestinal lesions (OR: 0.07, 95% CI 0.01-0.95, p = 0.05). Only age was positively associated with death prior to discharge (OR: 1.18, 95% CI 1.03-1.36, p = 0.02)., Main Limitations: Small sample size and retrospective design., Conclusion: Donkeys with abdominal lesions may present with a range of signs often not including colic. Surgical findings were diverse and survival to discharge appears to be lower than in horses., (© 2022 EVJ Ltd.)

Background: Quantifying risk factors for laminitis development requires improvement., Objectives: To identify the most useful physical examination, metabolic and management factors to predict laminitis development in client-owned, nonlaminitic ponies., Study Design: Prospective cohort study., Methods: Physical examination, metabolic and management data were collected from a pony cohort 6 monthly for up to 4 years. Ponies were monitored for the development of laminitis. Metabolic data included basal plasma concentrations of ACTH ([ACTH]), adiponectin ([adiponectin]), triglycerides and glucose. Serum insulin concentrations ([insulin]) were measured in the unfasted basal state ([insulin]T0) and 60 minutes ([insulin]T60) after administration of corn syrup (0.3ml/kg). Separate multivariable Cox proportional-hazards models were developed for physical, management/signalment and metabolic data and later combined into two final models. Low-, medium- and high-laminitis risk categories were defined based on basal or T60 [insulin]., Results: Overall, 374 ponies (age 5-32 years) and 891 pony-years were included in the main analysis. Laminitis incidence (95% confidence interval (CI)) was 4.8 (3.5-6.5) cases/100 pony-years. Laminitis development was associated with numerous univariable factors. Significant (P < .05) factors retained in the final multivariable models included [insulin]T0, [insulin]T60, [adiponectin] and divergent hoof growth. [ACTH] was not independently associated with laminitis. Based on [Insulin]T0, low- (<21.6 µIU/ml), medium- (21.6-45.2 µIU/ml) and high-risk (>45.2 µIU/ml) categories encompassed 70, 20 and 10% of the population and had estimated 4-year laminitis incidences (95%CI) of 6 (2-9)%, 22 (10-33)% and 69 (48-82)% respectively. Based on [Insulin]T60 the low- (<53.4 µIU/ml), medium- (53.4-153 µIU/ml) and high-risk (≥153 µIU/ml) categories comprised 60, 30 and 10% of the population and had estimated 4-year laminitis incidences (95%CI) of 3 (0-6)%, 20 (10-29)% and 73 (52-84)% respectively., Main Limitations: Results may not apply to different insulin assays, geographical regions, breeds or management types., Conclusions: [Insulin]T0 or [insulin]T60 best quantify the risk of future laminitis development in nonlaminitic ponies., (© 2022 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Previously laminitic (PL) ponies are reported to have higher blood pressure than non laminitic (NL) ponies. This relative hypertension may be related to endothelial cell dysfunction, similar to humans with metabolic syndrome. To investigate the relationship between laminitis predisposition and endothelial dysfunction, the effect of nitric oxide synthase (NOS) inhibition on the change in circulating nitric oxide (NO) concentrations and systemic blood pressure (BP) was determined. An intravenous NO sensor was used to measure changes in blood NO concentration during and after intravenous infusion of the NOS inhibitor L-nitroarginine methyl ester (L-NAME; 4 mg/kg IV) in PL and NL ponies. NO concentrations decreased and BP increased in response to L-NAME infusion, with a significantly (p = 0.02) greater rate of increase in systolic BP in PL (14.62 ± 1.88 mmHg/h) compared to NL (8.54 ± 1.88 mmHg/h) ponies. This greater effect on BP in PL compared to NL ponies is consistent with higher basal NO production in PL ponies, suggesting that the relative hypertension previously seen in PL ponies results in increased basal NO production, rather than being caused by reduced NO production as hypothesised. Up-regulation of the NO system may be a compensatory mechanism stimulated by the higher resting BP in PL ponies. Further investigation of the mechanism underlying the relative hypertension seen in PL ponies is required., (Copyright © 2022 Mars Horsecare UK Limited, The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This work aimed to determine the effect of strip grazing on physical activity in ponies using behavioral observations alongside accelerometers positioned at the poll. In study one, ten British native breed ponies were randomly assigned to paddock A (50 × 110 m) or B (50 × 110 m divided into seven equal strips with access to one additional strip per day) for seven days (n = 5/paddock). In study two, ten different British native breed ponies were randomly assigned for 14 days individually to (1) a control field where the animal was allowed complete access to their allotted area (n = 4); (2) a field that increased in size daily by moving a lead fence (n = 2); and (3) a field that was strip grazed using lead and back fences moved the same distance daily (n = 4). Accelerometer data were sorted into twenty-four-hour periods; each 10-second epoch was categorized as standing, grazing or locomoting using previously validated cut-off points; and time spent in each category for each day calculated. Behavioral monitoring was undertaken by direct observation on days 12-14 (study two only). Accelerometer and behavioral data were compared between grazing methods within each study. Strip grazing had no significant effect on the time spent in each physical activity category in either study. Behavioral observation revealed all ponies spent most time grazing ≤4 hours after fence moving and strip grazed ponies spent significantly more time grazing the newly available grass than elsewhere. Thus, strip grazing did not alter physical activity in ponies, but did result in preferential grazing of new grass., (Copyright © 2021. Published by Elsevier Inc.)

Background: Laminitis causes lameness in donkeys, but its prevalence and factors associated with disease remain uncertain., Objectives: To determine the prevalence of and identify factors associated with laminitis in donkeys., Study Design: Retrospective cross-sectional study., Methods: All donkeys at the Donkey Sanctuary, UK, October 2015 to March 2019 were included. For animals that had laminitis during this period, age, sex, weight, body condition score, and the onset date and type of each episode (first or recurrent, acute or chronic) were recorded. Additionally, management data, foot lesion score, endocrine data, other medical conditions, occurrence of foot trimming, surgical procedures, diagnostic imaging, behavioural modification therapy or movement between farms within the month prior were noted. Controls were animals that did not experience laminitis during this period and similar data were recorded. Multivariable logistic regression modelling assessed the differences between the control group and laminitis outcome groups (first, all laminitis, acute and chronic episodes)., Results: Altogether, 707 animals were included; 364 were control animals; 343 had a first episode of laminitis during the study period, of which 200/343 had no further episodes and 143/343 had recurrent episodes resulting in a total of 512 laminitis episodes and the period prevalence was 48.5% over 42 months. Overall, 180/512 (35%) laminitis episodes were acute and 332/512 (65%) were chronic. Compared with control animals, the laminitic outcome groups were significantly (P < .05) more likely to be younger (first episode), less likely to get extra feed (all four groups) or have an additional medical problem (first episode), and less likely to have undergone dental work, movement, imaging (all four groups) or surgery (first; all laminitis, chronic episodes) in the month preceding the episode., Main Limitations: These results may not be applicable to the wider donkey population., Conclusions: Laminitis commonly affects donkeys, but factors associated with donkey laminitis differ from those reported in horses., (© 2021 EVJ Ltd.)

Background: Equine pituitary pars intermedia dysfunction (PPID) is treated with daily pergolide therapy. Owner compliance and its effect on PPID control have not been previously investigated., Methods: Clinical records were searched to identify the sample of animals with PPID treated with pergolide from 2016 to 2019. The signalment was noted and the dose of pergolide received calculated. Animals were classified as compliant (receiving ≥90% of the veterinarian recommended dose of pergolide) or non-compliant, and as controlled (follow-up basal adrenocorticotrophic hormone concentrations within the reference range) or not., Results: In total, 110 animals were included. The majority (85%) were ≥16 years (mean ± SD 19.8 ± 4.4 years); the most common breeds were Cob (18%), Thoroughbred (16%) and Welsh (15%); 37% were female and 63% male. Overall, 48% were compliant and 52% non-compliant. There was no significant effect of compliance on laboratory control. Of those that were compliant, 74% were controlled, while 67% of non-compliant animals were controlled. Univariable analysis revealed a significant (p < 0.001) effect of age and breed on compliance and control, and of sex on control. On multivariable analysis, only age (compliance) and breed (compliance and control) were retained in the final model., Conclusion: Only half of animals received the recommended pergolide dose; however, this did not affect laboratory control of PPID., (© 2021 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Background: Blood l-lactate concentrations are higher in people with developing or established diabetes mellitus and insulin resistance., Objectives: To investigate whether blood l-lactate concentrations are positively correlated with measures of insulin dysregulation (ID) or increased autumnal ACTH concentrations in ponies., Animals: Systemically healthy client-owned ponies (n = 101)., Methods: Prospective case-control study. Blood samples were obtained from 101 clinically healthy ponies. Breed, weight, height, and subjective and objective measures of body condition were recorded. Blood l-lactate, glucose, triglyceride, total adiponectin, and ACTH concentrations were measured and an oral sugar test (OST) was carried out. Correlations between blood l-lactate and variables of endocrine health were determined., Results: Using a seasonal cutoff point of ACTH concentrations ≥47 pg/mL, 55 ponies had increased autumnal ACTH concentrations and 45 did not. Using a basal insulin concentration of >50 μiU/mL, 42 ponies were diagnosed with ID and 58 were not. Using a 60 minutes after OST cutoff point of >45 μiU/mL, 57 ponies had ID and 37 did not. Blood l-lactate concentrations were significantly lower in obese (average body condition score ≥ 7/9) compared to nonobese ponies (0.6 mmol/L; range, 0.0-1.9 mmol/L vs 0.8 mmol/L; range, 0.3-2.7 mmol/L; P = .01). No other significant correlations were detected. No differences were detected between ponies with and without increased autumnal ACTH concentrations (0.7 mmol/L; range, 0.0-2.7 mmol/L vs 0.7 mmol/L; range, 0.3-1.8 mmol/L; P = .84) and with and without ID (0.7 mmol/L; range, 0.3-2.7 mmol/L vs 0.8 mmol/L; range, 0.0-1.6 mmol/L; P = .63)., Conclusions and Clinical Importance: Results do not support an effect of endocrine status on l-lactate concentrations in blood of ponies., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: The chemiluminescence (CL) and immunofluorescence (IF) assays yield different results for basal adrenocorticotropin hormone concentrations [ACTH] in pony plasma. It is unclear whether this difference also occurs in basal samples from horses or samples from ponies following thyrotropin-releasing hormone (TRH) stimulation., Objectives: To compare the results of [ACTH] analysis by CL and IF methods in basal samples from horses and pony samples following TRH stimulation., Study Design: Method comparison., Methods: Plasma [ACTH] was measured concurrently using CL and IF methods in 12 ponies (basal and post-TRH stimulation) in November and basal samples from horses (n = 45; November and May)., Results: CL and IF methods yielded different results (P < .01). The median difference (CL-IF) (95% CI) for ponies was 5.9 (0.1-7.5) pg/mL at baseline and 227.9 (61-1001) pg/mL post TRH; and horses 1.9 (1.1-5.4) pg/mL in November and 9.4 (8.2-11.5) pg/mL in May, at baseline. Correlation was good in ponies at baseline (R = 0.80, P = .003) but not post-TRH, and good in horses in November and May (R = 0.68 and 0.71, P < .001). Bland-Altman analysis demonstrated moderate bias and wide 95% limits of agreement (95% LOA) in ponies at baseline (bias 5.5 pg/mL; 95% LOA -9.9 to 20.9 pg/mL) and horses in May (bias 10.6 pg/mL; 95% LOA -9 to 30.3 pg/mL) and very large bias and wide 95% LOA in ponies post-TRH (bias 477 pg/mL; 95% LOA -633 to 1587 pg/mL). Using CL cut-offs of >29 and >110 pg/mL, agreement was moderate (ƙ = 0.67) and very good (ƙ = 0.82) for binary classification of PPID in ponies at baseline and post-TRH; and good (ƙ = 0.73) for horses in November, but poor (ƙ = 0.40) in May., Main Limitations: Limited numbers of horses with [ACTH] above threshold values., Conclusions: The assays yielded different absolute values, particularly in post-TRH samples from ponies, suggesting TRH stimulates secretion of cross-reacting peptides other than ACTH. Agreement for binary classification for PPID was moderate to good, except in basal samples from horses in May., (© 2020 EVJ Ltd.)

Background: Blood l-lactate and glucose concentrations were higher in ponies with gastrointestinal disease than in horses, possibly because of differences in body condition (BC)., Objectives: To investigate whether l-lactate and glucose concentrations correlate with BC and differ between healthy horses and ponies., Animals: Systemically healthy client-owned ponies (n = 101) and horses (n = 51)., Methods: Prospective observational study. Breed, weight, height, and subjective and objective measures of BC were recorded and l-lactate and glucose concentrations were measured. Correlations between l-lactate and glucose concentrations and BC were established. The association between l-lactate concentrations, equid type (pony or horse), BC, age, and glucose concentrations was investigated using a multivariable model., Results: Weak but significant (P = .001) negative correlations were detected between l-lactate concentration and average BC score (r = -0.29), heart girth:height ratio (r = -0.27), and age (r = -0.27). Glucose concentrations were significantly (P < .001) positively correlated with neck length:heart girth ratio (r = 0.37) and heart girth:height ratio (r = 0.31). l-lactate and glucose concentrations were weakly correlated (r = 0.15; P = .04). In the final multivariable model, age (-0.02 ± 0.006; P = .001) and heart girth:height ratio (-1.74 ± 0.53; P = .001) were significantly associated with the natural logarithm of l-lactate concentration (Lnl-Lactate). This represents a 2% decrease in l-lactate concentration per year increase in age and 10% decrease in l-lactate concentration per 0.06 unit increase in heart girth:height ratio., Conclusions and Clinical Importance: In healthy horses and ponies, age and BC significantly influence l-lactate concentrations., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Equine metabolic syndrome (EMS) is a widely recognized collection of risk factors for endocrinopathic laminitis. The most important of these risk factors is insulin dysregulation (ID). Clinicians and horse owners must recognize the presence of these risk factors so that they can be targeted and controlled to reduce the risk of laminitis attacks. Diagnosis of EMS is based partly on the horse's history and clinical examination findings, and partly on laboratory testing. Several choices of test exist which examine different facets of ID and other related metabolic disturbances. EMS is controlled mainly by dietary strategies and exercise programs that aim to improve insulin regulation and decrease obesity where present. In some cases, pharmacologic aids might be useful. Management of an EMS case is a long-term strategy requiring diligence and discipline by the horse's carer and support and guidance from their veterinarians., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Circulating adiponectin concentrations were lower in ponies with a history of endocrinopathic laminitis and in nonlaminitic ponies that subsequently developed laminitis. The assays used in these studies have been discontinued or are no longer valid., Objectives: (1) to determine the validity of immunoturbidimetric (IT) and enzyme linkedimmunosorbent (ELISA) assays for equine total and high molecular weight (HMW) [adiponectin] measurement and (2) to investigate the association between [adiponectin] measured using these assays and endocrinopathic laminitis., Study Design: Method validation and cohort study., Methods: Accuracy and precision of IT and ELISA assays for measuring total (TAC) and HMW (HMWAC) [adiponectin] were determined. Using the IT assay, the effects of anti-coagulant and storage temperature were assessed, TAC was measured in previously laminitic (PL) and never laminitic (NL) ponies (n = 6/group). Comparison with a previously validated radioimmunoassay was made in NL ponies (n = 223). Association between TAC and subsequent laminitis development in NL ponies was investigated using univariable logistic regression and ROC curve analysis., Results: The IT assay was precise and demonstrated good agreement with the previously validated radioimmunoassay. TAC was significantly (P<0.01) lower in PL (mean ± s.d. 8.9 ± 2.9 μg/mL) compared to NL (24.2 ± 11.8 μg/mL) ponies and in NL ponies that developed laminitis within 12 months (median 4.8 μg/mL; IQR 2.65-13.4 μg/mL) compared to those that remained nonlaminitic (19.9 μg/mL; 9.95-31.5 μg/mL). TAC was significantly (P = 0.01) associated with laminitis occurrence within 12 months. Use of the area under the ROC curve to distinguish animals that did and did not develop laminitis showed good accuracy (0.76). None of the ELISA methods validated satisfactorily., Main Limitations: Laminitis risk is based on data from ponies in one region., Conclusions: The IT method is suitable for measurement of equine TAC. TAC is lower in ponies with previous or future laminitis. The ELISA methods are not suitable for measurement of equine HMWAC or TAC., (© 2018 EVJ Ltd.)

Background: The oral sugar test (OST) is used to identify equine insulin dysregulation (ID); however only a dose of 0.15 mL/kg bwt corn syrup has been evaluated., Objectives: To determine the effect of varying the dose of corn syrup on insulin and glucose response to the OST and the test's ability to distinguish between ponies with a history of laminitis (PL) and without laminitis (NL)., Study Design: Randomised crossover experiment., Methods: After an overnight fast, in a three-way randomised crossover study with a 7-day washout, 0.15, 0.3 or 0.45 mL/kg bwt corn syrup was administered orally to eight ponies (5 PL and 3 NL) and blood obtained between 0 and 120 min. Serum [insulin] and [glucose] were measured using previously validated radioimmunoassay and colorimetric assays respectively. The repeatability of and the effect of continued pasture access on the dose that best distinguished PL and NL ponies were then assessed. The effect of dose, laminitis history and fasting on serum [insulin] and [glucose] responses were assessed using mixed-effects models., Results: The serum [insulin] following 0.15 mL/kg bwt were not significantly different from 0.3 mL/kg bwt at any time point, while serum [insulin] following 0.45 mL/kg bwt significantly (P<0.01) differed from 0.15 and 0.3 mL/kg bwt at all time points apart from 0 min. The serum [insulin] concentration significantly (P<0.01) differed between NL (mean 86 [95% CI 59, 113] μiu/mL) and PL (146 [95% CI 124, 167] μiu/mL) only following 0.45 mL/kg bwt at 60 min. Repeatability of serum [insulin] at 60 min following 0.45 mL/kg bwt dose under fasted conditions was 0.51. Using AUC insulin improved repeatability to 0.83. There was no significant difference between the fasted and at pasture results., Main Limitations: The OST was performed in small numbers of ponies on limited occasions., Conclusions: A dose of 0.45 mL/kg bwt corn syrup may be preferable to differentiate PL and NL ponies., (© 2018 EVJ Ltd.)

Background: Analysis of plasma adrenocorticotropic hormone concentration [ACTH] aids diagnosis of pituitary pars intermedia dysfunction (PPID). Comparisons of the validated chemiluminescent-immunoassay (CI) and immunofluorescent (IF) assays are limited., Objectives: To compare the results of [ACTH] analysis by CI and IF methods of samples collected in autumn and spring and assess cross-reactivity., Study Design: Method comparison., Methods: Plasma from nonlaminitic ponies was analysed concurrently using the IF and CI methods in autumn and the following spring. Diagnostic thresholds for the IF method were derived using ROC curves and Youden indices to correspond with CI thresholds. Assay specificity was assessed using commercially available ACTH fragments and degradation products of endogenous ACTH., Results: CI and IF methods yielded different results (P<0.001); mean differences (CI-IF), (95% confidence intervals): Autumn (n=99) 38.6 (30.6-46.5) pg/ml, Spring (n=88) 5.1 (3.9-6.3) pg/ml. The association between CI and IF results differed in autumn and spring, consistent with seasonally dependent cross-reactivity or interference. Good (κ=0.66-0.74) agreement was obtained for binary interpretation in spring between IF and CI using thresholds of >24 and >29 pg/ml respectively and in autumn between IF and CI using thresholds of >27 and >47 pg/ml respectively or >33 and >77 pg/ml respectively. Of 88 ponies with both spring and autumn samples, 56 (64%) exceeded a published autumn CI threshold (>47 pg/ml), of which 39 (70%) were below the equivalent threshold (<29 pg/ml) the following spring without treatment. The CI assay showed apparent increases in [ACTH] following addition of CLIP (ACTH 18-39). Degradation of ACTH during storage affected the assays differently., Main Limitations: Limited numbers of PPID cases were included. Immunoreactivity of commercially available peptides may differ from their endogenous equivalents., Conclusions: The methods yielded different absolute values but the agreement for binary classification was good. An altered pituitary secretome in autumn that affects apparent [ACTH] values is likely. The Summary is available in Spanish - see Supporting information., (© 2017 EVJ Ltd.)

Microvesicles are small (up to 1 μm) vesicles found in plasma and other bodily fluids. They are recognised as part of the normal system of inter-cellular communication but altered numbers are also used as biomarkers of disease. Microvesicles have not been studied in detail in the horse but may be relevant to diseases such as laminitis. Identification of equine cell specific microvesicles was performed by developing a panel of cross reactive antibodies to use in flow cytometry to detect microvesicles of platelet, leucocyte and endothelial origin in plasma from healthy ponies and those predisposed to laminitis. The total number and proportion of microvesicles from the different cell types varied with season and there were more annexin V positive endothelial MV in non laminitic ponies compared to previously laminitic ponies. Development of this antibody panel and the technique for measuring microvesicles in the horse opens a new field for further investigation of these important structures in equine health and disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Birthweight of Thoroughbred foals has increased in recent years. It is unknown whether this is associated with increased broodmare obesity or endocrine dysfunction., Objectives: To determine insulin, leptin and triglyceride concentrations in Thoroughbred mares throughout gestation and investigate their association with obesity and foal birthweight., Study Design: Cohort study., Methods: A total of 66 mares were included from 40 days post-breeding. Body condition score (BCS), weight and blood samples were obtained every 60 days throughout gestation. Serum/plasma insulin, leptin and triglyceride concentrations and foal birthweight were recorded. Associations between hormone/triglyceride concentration with BCS, stage of gestation and birthweight were analysed using a linear mixed effects model., Results: Serum insulin concentrations were greater at 1-60 days (4.31 μiu/mL) compared with 241-300 days (3.13 μiu/mL) and 61-120 days (5.33 μiu/mL) compared with 181-240, 241-300 and 301-360 days (3.78, 3.13, 3.37 μiu/mL) gestation (P<0.05). There was no significant hyperinsulinaemia and no association of insulin concentration with BCS. Leptin concentration was greater at 181-240 days (2.28 μg/L, P<0.0001) compared with all other time points and correlated with BCS (P<0.0003). Triglyceride concentration was greater at 241-300 days (0.245 mmol, P<0.02) compared with earlier time points, but was not associated with BCS. Foal birthweight was weakly positively correlated with BCS (r = 0.13, P<0.001) and inversely correlated with leptin concentrations at 61-120 and 241-300 days gestation (r = -0.64, P<0.05)., Main Limitations: Reduction in sample size over the study and tight clustering of BCS., Conclusions: Mare BCS correlated with foal birthweight; obese mares had heavier foals. Significant hyperinsulinaemia was not identified in this population. Increased leptin concentration in early and late gestation was associated with decreased foal birthweight. Further work is required to establish whether leptin concentration in late gestation could predict foal birthweight., (© 2016 EVJ Ltd.)

Background: It is recommended that the oral sugar test (OST) for insulin dysregulation (ID) be performed after an overnight fast, but fasting is impractical in ponies kept solely at pasture. There are few data on OST repeatability and reliability in ponies., Objectives: To report 1) whether OST results obtained in the morning after an overnight fast or without fasting in the afternoon (FASTING/FED) can be used interchangeably, 2) time of highest insulin concentration T max [insulin], repeatability and reliability of insulin response to the OST when FASTING or FED and 3) dichotomous agreement (ID/normal) within a small sample when FASTING or FED., Study Design: Method comparison study., Methods: Oral sugar tests were performed on four occasions in 10 adult native British ponies, twice FASTING and twice FED. Insulin concentrations were measured by radioimmunoassay at 0-120 min (T 0,30,60,75,90,120 ). Differences between FASTING and FED results were assessed using mixed effects models. Indices of repeatability and reliability were calculated; dichotomous agreement was reported using kappa statistics., Results: Serum [insulin] was significantly (P≤0.05) higher at T 60 -T 90 with prior fasting (estimated differences [95% confidence intervals]): T 60 : 23.5 μiu/ml (8.7-38.4 μiu/ml), T 75 : 27.1 μiu/ml (12.3-41.8 μiu/ml), T 90 : 15.1 (0.36-29.9 μiu/ml). Most frequently, T max [ins] occurred at T 30 . At any single time point, within-subject coefficients of variation were: FASTING: 40% and FED: 31%. The 95% limits for repeatability were FASTING: 29-340%, FED: 41-240%. Test reliabilities were FASTING: 0.70 and FED: 0.67. For dichotomous interpretation similar results (kappa = 0.7) were obtained using cut-offs of [Insulin] >60 μiu/ml at T 60 or T 90 for FASTING and [Insulin] >51 μiu/ml at T 30 or T 60 for FED samples., Main Limitations: Oral sugar tests were performed on a small number of animals on one pasture during one season (spring)., Conclusions: Clinicians should beware of interpreting changes in absolute OST results owing to poor repeatability. When stabling is unavailable, OSTs of ponies at pasture may yield similar dichotomous results without prior fasting., (© 2016 EVJ Ltd.)

Background: Certain individuals appear to be predisposed to recurrent pasture-associated laminitis. Previous studies have predominantly investigated risk factors only after disease occurrence., Objectives: To investigate risk factors for pasture-associated laminitis prior to disease occurrence., Study Design: Prospective cohort study., Methods: Non-laminitic ponies aged ≥7 years were recruited. Body condition score (BCS), height, weight, crest height and thickness were measured and an overnight dexamethasone suppression test performed. Plasma or serum adiponectin, leptin, triglyceride, basal insulin, insulin post-dexamethasone, insulin-like growth factor 1 (IGF-1), IGF binding protein 1 (IGFBP-1), IGFBP-3, C-reactive protein, von Willebrand's factor, soluble E-selectin and P-selectin concentrations were assayed. Follow-up data were obtained from owners annually for 3 years to ascertain occurrences of veterinarian-diagnosed pasture-associated laminitis. Data were analysed by multivariate logistic regression. Receiver operating characteristic (ROC) curves analysis was performed for significant risk factors and cut-off values determined., Results: A total of 446 animals with a median (interquartile range) age of 15 (10-20) years were recruited. Of these, 50.4% were mares and 49.6% were geldings. The most common breeds were Welsh (36.4%), Shetland (17.0%) and cob (9.4%). Overall, 72.2% of animals were overweight/obese (BCS 7-9/9), 27.3% were of ideal weight (BCS 4-6/9) and 0.5% were underweight (BCS 1-3/9). After 1, 2 and 3 years, respectively, 18 (4.0%), 30 (6.7%) and 44 (9.9%) animals were reported to have had laminitis. Plasma adiponectin, and serum basal (insulin) and (insulin) post-dexamethasone levels were significantly (P ≤ 0.05) associated with laminitis occurrence cumulatively after 1, 2 and 3 years. Use of the area under the ROC curves to distinguish animals that did and did not develop laminitis showed good (basal [insulin] after 1 year), fair (all others) or poor ([insulin] post-dexamethasone) levels of accuracy., Main Limitations: Animals were evaluated at a single time point and biomarkers were assayed using single assays., Conclusions: Risk factors for future laminitis prior to disease occurrence include low plasma adiponectin and high serum basal insulin or insulin post-dexamethasone concentrations., (© 2016 EVJ Ltd.)

Reasons for Performing Study: The in-feed oral glucose test (OGT) and oral sugar test (OST) are advocated as field tests of insulin sensitivity in horses and ponies but have not been directly compared., Objectives: To compare the insulin response to OGT and OST in 8 ponies and 5 horses of unknown insulin sensitivity., Study Design: Experimental, randomised crossover study., Methods: Animals were fasted for 8 h overnight before and throughout testing. They were fed 1 g/kg bwt glucose powder with chaff (OGT) or 0.15 ml/kg bwt corn syrup (Karo™ Light Syrup; OST) was administered per os in a randomised crossover study with 48 h between tests. Blood samples were obtained at 0, 30, 60, 75, 90, 120 and 180 min. The maximal insulin concentration (Cmaxi ), time to maximal insulin concentration (Tmaxi ) and area under the curve of insulin concentration over time (AUCi ) for the tests were compared using Student's paired t test. The effect of individual subject, horse or pony and test were analysed using a linear mixed model., Results: The OGT Cmaxi (mean ± s.d.; 154 ± 116 μiu/ml), Tmaxi (136 ± 52 min) and AUCi (15,308 ± 9886 μiu/ml/min) were significantly (P<0.05) greater compared with the OST Cmaxi (72 ± 55 μiu/ml), Tmaxi (63 ± 25 min) and AUCi (5980 ± 4151 μiu/ml/min). The Cmaxi , Tmaxi and AUCi varied significantly between individual subjects. The Tmaxi was significantly different between horses and ponies during OGT and OST. Using previously defined criteria of insulin dysregulation, OGT identified 7/13 animals as insulin resistant, whereas OST identified 5/13 animals as insulin resistant., Conclusions: The OGT and OST showed agreement in identification of insulin dysregulation in 85% of equine subjects. Results of the OGT and OST are not comparable in all cases. Further work is required to establish which test more accurately diagnoses insulin dysregulation in horses and ponies., (© 2015 EVJ Ltd.)

Reasons for Performing Study: The birthweight of Thoroughbred foals has increased in recent years. It is unknown whether this is associated with increased broodmare obesity or endocrine dysfunction., Objectives: To determine insulin, leptin and triglyceride concentrations in obese and non-obese Thoroughbred mares throughout gestation and to investigate their association with foal birthweight., Study Design: Prospective, cohort study., Methods: Fifty-seven pregnant Thoroughbred mares were included in the study. From 40 days post-breeding, body condition score (BCS), weight and venous blood samples were obtained every 60 days throughout gestation. Feed was withheld for 6 h before blood sampling. Serum/plasma insulin, leptin and triglyceride concentrations were measured using validated/standard methods. Foal birthweight was recorded. Association of hormone or triglyceride concentration with time, BCS and birthweight were analysed using a linear mixed effects model. A Pearson correlation co-efficient was calculated between hormone or triglyceride concentration, BCS and birthweight., Results: Serum insulin concentrations were significantly greater (P<0.05) at 0-59 days compared with 240-299 days and at 60-119 days compared with 180-359 days gestation. 55% of mares had BCS ≥ 7. There was 1.5% incidence of fasting hyperinsulinaemia throughout gestation and no association of fasting insulin concentration with BCS. Leptin concentration was significantly (P<0.0001) greater at 180-239 days compared with all other time points and was significantly (r = 0.29, P<0.0003) correlated with BCS. Triglyceride concentration was significantly (P<0.02) greater at 240-299 days compared with earlier time points but was not associated with BCS. Foal birthweight was significantly positively correlated with BCS (r = 0.13, P<0.001) and inversely correlated with leptin concentration at 60-119 days and 240-299 days gestation (r = -0.64, P<0.05)., Conclusions: Mare BCS correlated with foal birthweight such that obese mares had heavier foals. Significant fasting hyperinsulinaemia was not identified in this population. Increased leptin concentration in early and late gestation was associated with decreased foal birthweight and may be useful to predict foal birthweight. Ethical animal research: Informed client consent was obtained for all animals used in the study., Source of Funding: Private donor. Competing interests: None declared., (© 2015 The Author(s). Equine Veterinary Journal © 2015 EVJ Ltd.)

Objective and Design: Insulin and inflammatory cytokines may be involved in equine laminitis, which might be associated with digital vascular dysfunction. This study determined the effects of TNF-α and insulin on the endothelial-dependent relaxant responses of equine digital blood vessels and on equine digital vein endothelial cell (EDVEC) cGMP production., Material: Isolated rings of equine digital arteries (EDAs) and veins (EDVs) were obtained and EDVECs were cultured from horses euthanized at an abattoir., Methods: The effect of incubation with TNF-α (10 ng/ml) and/or insulin (1,000 μIU/ml) for 1.5 h or overnight under hyperoxic and hypoxic conditions on carbachol (endothelium-dependent) induced relaxation was assessed. The time course and concentration dependency of the effect of TNF-α and the effect of insulin (1,000 μIU/ml) on EDVEC cGMP production was determined., Results: Incubation of EDAs overnight with TNF-α under hypoxic conditions resulted in endothelial-dependent vascular dysfunction. EDVs produced a more variable response. TNF-α increased EDVEC cGMP formation in a time- and concentration-dependent manner. Insulin had no significant effects., Conclusions: There is a mismatch between the results obtained from isolated vessel rings and cultured endothelial cells suggesting TNF-α may reduce the biological effect of NO by reducing its bioavailability rather than its formation, leading to endothelial cell dysregulation.

Reasons for Performing Study: The mechanisms underlying predisposition to pasture-associated laminitis remain unclear; chronic inflammation is implicated, and this may be exacerbated by physical inactivity., Objectives: To determine whether exercise affects the inflammatory profile of normal and previously laminitic ponies., Study Design: Prospective case-control study., Methods: The short (1 day) and longer term (14 days) effects of low intensity (10 min walking and 5 min trotting) exercise on plasma inflammatory marker concentrations in normal (NL) and previously laminitic (PL) nonobese ponies (n = 6/group) was determined. Plasma concentrations of TNF-α, serum amyloid A (SAA), haptoglobin, insulin, adiponectin and fibrinogen were assayed by validated/standard methods. Data were analysed using a linear mixed effects model., Results: Before exercise, plasma [adiponectin] was significantly (P = 0.0001) lower in PL (mean ± s.d. 2.4 ± 0.1 ng/l) than in NL (4.03 ± 0.2 ng/l), but exercise had no effect. Previous laminitis and exercise had no effect on plasma [TNF-α] or [fibrinogen]. Serum amyloid A concentrations in all ponies were significantly (P = 0.00001) reduced after longer term exercise compared to Day 1 values. Plasma [haptoglobin] was significantly (P = 0.00001) higher in PL compared to NL on Day 1. This difference was no longer apparent after longer term exercise, such that [haptoglobin] in PL had decreased to concentrations similar to NL. Following short-term exercise, all ponies had an initial decrease in serum [insulin] immediately after exercise, followed by an increase peaking 10 min after exercise cessation, before returning to pre-exercise values. On Day 14 these fluctuations were significantly (P = 0.001) reduced in all ponies., Conclusions: Fourteen days of low intensity exercise significantly decreased [SAA] in all ponies and plasma [haptoglobin] in PL such that it was no longer increased compared to NL. Regular low intensity exercise appears to have an anti-inflammatory effect, which is possibly greater in PL and so may be beneficial in reducing this putative risk factor in pasture-associated laminitis., (© 2013 EVJ Ltd.)

Reasons for Performing Study: Plasma fructosamine concentration ([fructosamine]) is believed to reflect medium term, average blood glucose concentration and in a previous study was higher in horses with active laminitis than in normal horses. Pituitary pars intermedia dysfunction (PPID) is associated with hyperglycaemia and laminitis., Objectives: To test the hypotheses that: [fructosamine] is higher in PPID cases than normal animals; furthermore, that within cases of PPID [fructosamine] is higher in those with active laminitis than nonlaminitic cases and in cases that have been affected by active laminitis in the preceding year than those that have not., Study Design: Observational, case-control/cross-sectional study., Methods: [Fructosamine] was measured in cases of PPID (n = 46) and normal animals (n = 139). A normal range was calculated; values were compared between the 2 groups and within the PPID group, between cases with and without active laminitis and between cases that had and had not been affected by active laminitis in the preceding year., Results: In normal animals mean [fructosamine] was 248.7 μmol/l; the normal range (mean ± 2 s.d.) was 195.5-301.9 μmol/l. Plasma [fructosamine] was not higher in PPID cases than in normal animals. In PPID cases, [fructosamine] was significantly (P = 0.006) higher in cases with active laminitis (mean ± s.d. 261.2 ± 39.2 μmol/l) compared with those without active laminitis (234.5 ± 32.9 μmol/), but [fructosamine] was not higher in cases that had been affected by active laminitis in the preceding year than those that had not., Conclusions: In horses affected by PPID, [fructosamine] is higher in cases with active laminitis than in cases without active laminitis., Potential Relevance: Clinical use of [fructosamine] is limited due to overlap with the normal range. Further studies into glucose and protein metabolism in cases of PPID are warranted., (© 2013 EVJ Ltd.)

Reasons for Performing Study: A simple, accurate test for identifying individual animals at increased risk of laminitis would aid prevention. Laminitis-prone ponies have a greater serum insulin response to dexamethasone administration than normal ponies in the summer, but the response during different seasons is unknown., Objective: To test the hypothesis that previously laminitic ponies have a greater insulin response to dexamethasone than normal ponies, which is present during all seasons., Study Design: Prospective longitudinal study., Methods: Overnight dexamethasone suppression tests were performed on 7 normal ponies and 5 previously laminitic ponies in spring 2009 and 2010, summer 2008 and 2010, autumn 2009 and winter 2008, while the ponies were at pasture. In spring 2010, a dexamethasone suppression test was performed after the ponies had been fed only hay for 3 weeks. Serum cortisol and insulin concentrations pre- and post dexamethasone were measured. Linear mixed models were used to analyse the data., Results: Insulin concentrations pre- and post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies during spring 2009 and summer 2008, but there was no difference between groups in spring 2010, summer 2010, autumn 2009 or winter 2008. Insulin concentration varied significantly with season. Diet had no apparent effect on insulin concentration pre- or post dexamethasone in spring 2010. Cortisol concentrations post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies in autumn 2009, with concentrations above the reference range (<25 nmol/l) in both groups in summer 2008 and autumn 2009. Individual ponies had insufficient cortisol suppression in all seasons., Conclusions: There were significant differences between groups in insulin and cortisol concentrations post dexamethasone during some seasons, but this was not present in all years. Wide interindividual variation in response limits the usefulness of a dexamethasone suppression test for predicting the susceptibility of an individual animal to laminitis., Potential Relevance: Abnormal insulin and cortisol responses to dexamethasone must be interpreted in the light of the individual animal, seasonal and annual variation reported here., (© 2013 EVJ Ltd.)

Reasons for Performing Study: The mechanisms underlying individual animal predisposition to pasture-associated laminitis remain unclear; however, chronic inflammation is implicated., Objectives: To identify differences in the inflammatory profile of a group of previously laminitic ponies compared with control animals at pasture in late spring and winter., Methods: Previously laminitic (PL; n = 38 and 42) and nonlaminitic control ponies (NL; n = 41 and 39) were sampled in late spring and winter. Body condition score, height, weight and crest height and thickness were measured. Plasma concentrations of tumour necrosis factor-α, serum amyloid A, haptoglobin, insulin, adiponectin, triglyceride, fibrinogen, interleukin-17, interleukin-4 and interferon-γ were assayed by validated/standard methods. Factors independently associated with each cytokine were determined by multivariate analysis., Results: Plasma [adiponectin] was significantly influenced by laminitis status, being lower in PL (median [interquartile range] 2.1[1.4-3.2] μg/l) than in NL ponies (3.4 [2.6-4.1] μg/l; P<0.0001). No other cytokines or inflammatory markers were associated with laminitis status. Plasma fibrinogen and serum amyloid A concentrations were significantly (P = 0.04 and P = 0.01) higher in geldings (3.5 [3.0-4.0] g/l; 2.2 [0.5-3.6] mg/l) than in mares (3.0 [3.0-4.0] g/l; 1.5 [0.4-2.1] mg/l) and significantly (P = 0.04 and P<0.001) higher in winter (3.5 [3.0-4.0] g/l; 2.5 [0.9-3.6] mg/l) than in late spring (3.0 [3.0-3.5] g/l; 1.1 [0.3-1.9] mg/l). Serum haptoglobin concentration showed the same significant (P<0.001) seasonal difference (winter 2.1 [1.6-2.6 g/l; late spring 1.8 1.4-2.4 g/l) and was significantly (P = 0.01) inversely associated with weight. Serum interleukin-4 concentration was significantly (P<0.0001) higher in winter (2.0 [1.2-3.0] ng/l) than in late spring (0.0 [0.0-0.0] ng/l). Serum insulin concentration was significantly (P = 0.02) influenced by season (winter 31.7 [9.6-43.5] miu/l; late spring 84.0 [7.0-131.0] miu/l). Plasma triglyceride concentration was significantly (P = 0.02) higher in PL (0.5 [0.3-0.7] mmol/l) than in NL ponies (0.4 [0.2-0.5] mmol/l)., Conclusions: There were significant effects of season, gender and bodyweight on a number of proinflammatory mediators or markers of inflammation. The only marker influenced by laminitis status was adiponectin, and concentrations of this anti-inflammatory marker were lower in previously laminitic animals., Potential Relevance: Recurrent laminitis may be associated with reduced anti-inflammatory capacity rather than a proinflammatory state., (© 2012 EVJ Ltd.)

Digital vasoconstriction, ischaemia and hypoxia may predispose to acute laminitis. Laminitis incidence varies seasonally, peaking in spring and summer. Direct seasonal influences on equine digital artery (EDA) contractility have not been investigated. This study assessed seasonal variation in maintenance of phenylephrine (PHE)-induced tone in isolated EDAs under hypoxic (95% nitrogen) and hyperoxic (95% oxygen) conditions. The objective was to measure change in arterial tone over time after constriction to a plateau with PHE. Tone was measured at plateau and over time and percentage change calculated. Hyperoxic EDAs maintained PHE-induced tone over 1 h with no seasonal variation. Hypoxic EDAs relaxed in fall (median [inter-quartile range] 59% [44-77%] decrease from plateau; P=0.008), contracted in spring (65% [20-192%] increase from plateau; P=0.03) and did not significantly change tone in winter (18% [0-28%] decrease; P=0.13). Continued contraction under hypoxic conditions in spring may contribute to digital vasoconstriction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To evaluate a human radioimmunoassay (RIA) and equine and high-range porcine (hrp) species-specific ELISAs for the measurement of high serum insulin concentrations in ponies., Samples: Serum samples from 12 healthy nonobese ponies (7 clinically normal and 5 laminitis prone; 13 to 26 years of age; 11 mares and 1 gelding) before and after glucose, insulin, and dexamethasone administration., Procedures: Intra-and interassay repeatability, freeze-thaw stability, dilutional parallelism, and assay agreement were assessed., Results: Assay detection limits were as follows: RIA, < 389 μU/mL; equine ELISA, < 175 μU/mL; and hrp ELISA, 293 to 8,775 μU/mL. Mean ± SD intra- and interassay repeatability were respectively as follows: RIA, 6.5 ± 5.1 % and 74 ± 3.4%; equine ELISA, 10.6 ± 11.0% and 9.0 ± 4.6%; and hrp ELISA, 19.9 ± 172% and 173 ± 16.6%. Freezing and thawing affected measured concentrations. Dilutional parallelism in the RIA was only evident when insulin-depleted equine serum was used as a diluent (percentage recovery, 95.7 ± 274%); in the ELISAs, dilutional parallelism was observed when a zero calibrator was used. Agreement between RIA and equine ELISA results was good for samples containing concentrations < 175 μU of insulin/mL (bias, -18.5 ± 25.5 μU/mL; higher in RIA). At higher concentrations, assay agreement was poor between RIA and equine ELISA results (bias, -185.3 ± 98.7 μU/mL) and between RIA and hrp ELISA results (bias, 25.3 ± 183.0 μU/mL)., Conclusions and Clinical Relevance: Agreement among results of the 3 assays was variable, and dilutional parallelism was only evident with the RIA when insulin-depleted equine serum was tested. Caution is recommended when evaluating high insulin concentrations measured with the RIA or ELISAs.

Identification of ponies (Equus caballus) at increased risk of pasture-associated laminitis would aid in the prevention of the disease. Insulin resistance has been associated with laminitis and could be used to identify susceptible individuals. Insulin resistance may be diagnosed by feeding supplementary water-soluble carbohydrate (WSC) and measuring blood glucose and insulin concentrations. The aim of this study was to assess the glycemic and insulinemic responses of 7 normal (NP) and 5 previously laminitic (PLP), mixed breed, native UK ponies fed glucose, fructose, and inulin [1 g/(kg·d) for 3 d] or no supplementary WSC (control) in spring and fall after a 7-d adaptation to a pasture or hay diet. Blood samples were taken for 12 h after feeding on each day, and baseline and peak concentrations and area under the curve (AUC) for glucose and insulin were recorded. Linear mixed models were used for statistical analysis. Differences between PLP and NP groups were most marked after glucose feeding with differences in peak glucose (P = 0.02) and peak insulin (P = 0.016) concentrations. Season and diet adaptation also affected results. Peak concentrations of glucose and insulin occurred 2 to 4 h after WSC feeding. Peak insulin concentration was greater and more variable in fall, particularly in PLP adapted to fall pasture. Baseline glucose and insulin concentrations varied between individuals and with season and diet adaptation but were not greater in PLP than NP. Insulin AUC was greater in PLP than NP after feeding both glucose and fructose (P = 0.017), but there were no differences between PLP and NP in glucose AUC. Glycemic and insulinemic changes were less (P ≤ 0.05) after feeding fructose than glucose, although differences between PLP and NP were still evident. Minimal changes in glucose and insulin concentrations occurred after inulin feeding. Measurement of peak insulin 2 h after feeding of a single dose of glucose (1 g/kg) may be a simple and practical way to aid identification of laminitis-prone ponies before the onset of clinical disease, particularly when ponies are adapted to eating fall pasture.

Objective: To determine between-pony and within-pony variations and interobserver and intraobserver agreements of a technique for measurement of flow-mediated vasodilation (FMD) in healthy ponies., Animals: 6 healthy pony mares (weight range, 236 to 406 kg; body condition score range, 3/9 to 7/9; age range, 14 to 25 years)., Procedures: In each pony, the left median artery was occluded with a blood pressure cuff (inflated to > 300 mm Hg for 5 minutes). Two-dimensional ultrasonographic images of the artery were recorded for 30 seconds before cuff inflation and for 2 minutes after cuff deflation. Maximum luminal diameters of arteries were compared with their baseline diameters to calculate FMD (relative percentage increase in luminal size). Images were obtained from 6 ponies 1 time and from 1 pony 6 times. Independent analysis of images was performed by 2 investigators, 1 of whom analyzed images on 2 occasions., Results: Mean ± SD FMD in 6 ponies (1 time) was 12.57 ± 4.28% and in 1 pony (6 times) was 7.30 ± 2.11%. Between-pony and within-pony coefficients of variation were 34.09% and 28.84%, respectively. Interobserver agreement was fair (intraclass correlation coefficient, 0.47); intraobserver agreement was poor (intraclass correlation coefficient, 0.30)., Conclusions and Clinical Relevance: FMD was identified and measured in ponies. Measurement of FMD is used to assess endothelial function in humans and has been investigated in dogs. Measurement of FMD in ponies appeared to be feasible and could be used to assess endothelial function (to determine predisposition for development of laminitis or cardiovascular diseases).

Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A retrospective study of laminitis was carried out to identify risk factors associated with this disease on an East Anglian farm with approximately 1000 animals living in an area of 1000 acres. Medical records between January 1997 and May 2000 and between April 2005 and March 2008 were reviewed, and the age, sex, weight (kg), height (inches [in] and hands [H]) and weight-to-height ratio (kg/in) was recorded. The prevalence, incidence and seasonality of laminitis were determined and their relationship to the monthly temperature, rainfall and hours of sunshine was evaluated. Averaged over the six years, the highest prevalence (2.6 per cent) and incidence (16 cases/1000 animals) of laminitis occurred in May. The findings of a multivariate analysis revealed that females (P=0.007, odds ratio [OR] 1.46, 95 per cent confidence interval [CI] 1.1053 to 1.9646) and light animals (P ≤ 0.001, OR=0.995, 95 per cent CI =0.9932 to 0.9963) had the greatest risk of developing laminitis. A positive association was found between hours of sunshine and incidence (P=0.007, relative risk [RR] 1.009, 95 per cent CI 1.001 to 1.012) and prevalence (P=0.002, RR 1.008, 95 per cent CI 1.003 to 1.012) of laminitis. The data suggest that there is a relationship between season, sex of the animal and the development of laminitis.

Data from 107 cases of pasture-associated laminitis were obtained from first opinion practices to study factors associated with severity, survival and return to ridden exercise. There were 43 mares and 64 geldings, with a median age of 11 years. Of the 107 animals, 33 were small ponies, 45 were large ponies/cobs, 17 were small horses and 12 were large horses. Ninety-seven animals were categorised as having laminitis as defined by Cripps and Eustace (1999): 76 had mild (Obel grade 1 or 2) laminitis and 31 had severe (Obel grade 3 or 4) laminitis. Forty-three animals had previously had laminitis, and were significantly less likely (P=0.02) to have severe laminitis than those that had not. Eighty-nine animals were overweight, and there was a trend (P=0.09) towards severe laminitis cases having a higher body mass index. Eight weeks after disease onset, 102 animals were alive. Lower bodyweight, optimal body condition, mild laminitis and category of acute/chronic founder as defined by Cripps and Eustace (1999) were significantly associated with survival. There was a trend (P=0.06) towards treatment with acepromazine being associated with survival. Of the 81 animals that were used for riding, 48 were being ridden again; this was 2.6 times more likely in animals without previous laminitis. The clinical outcome was judged by a panel of three veterinarians as 'good' in 77 of 107 of cases. Clinical outcome was significantly associated (P=0.03) with horse type: the outcome was 'bad' in none of the small horses, compared with 15 of 45 large ponies/cobs, 11 of 33 small ponies and three of 12 large horses.

Certain individual animals appear predisposed to recurrent pasture-associated laminitis, but the exact mechanisms underlying their predisposition remain a fundamental question in laminitis research. The risk of endocrinopathic laminitis can be reduced if steps are taken to improve insulin sensitivity and reduce inflammation using strategies based on exercise and diet. Exercise has been shown to reduce insulin resistance and suppress inflammation, and dietary manipulation can significantly affect insulin sensitivity., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate both the repeatability (intraobserver agreement) and reproducibility (interobserver agreement) of Obel grading, the most widely accepted method for describing the severity of equine laminitis. The study comprised two parts. In study A, to test intraobserver agreement, 25 ponies were graded twice by a single blinded experienced equine veterinary surgeon, on the first occasion via clinical examination and on the second occasion via a video recording of the initial clinical examination. In study B, to test interobserver agreement, video recordings of 13 ponies were graded independently by 58 blinded experienced equine veterinary surgeons. A weighted kappa (kappa(w)) statistic was used to determine the measure of agreement between gradings. Intraobserver agreement of Obel grading was shown to be moderate (kappa(w)=0.54), and interobserver agreement was found to be substantial (kappa(w)=0.65). In addition, intraobserver agreement improved from moderate to substantial (kappa(w) increased from 0.54 to 0.70) when the Obel grades were grouped and horses were categorised as either sound, or as having mild (grade 1 or 2) or severe (grade 3 or 4) lameness.

The endothelium plays a major role in the pathogenesis of endotoxemia. Binding of endotoxin (lipopolysaccharide; LPS) to endothelium initiates a range of signalling events, including activation of p38 mitogen activated protein kinases (MAPKs) that are involved in the initiation of inflammatory responses. In the present study we have examined whether clinically relevant concentrations of LPS can activate p38 MAPK in equine endothelial cells and have investigated the role of the kinase in neutrophil adhesion and mediator release. Cultured equine digital vein endothelial cells (EDVEC) were exposed to LPS and phosphorylation of p38 MAPK was assessed by Western blotting using phospho-specific antibodies. Neutrophil adhesion was quantified by assaying myeloperoxidase and the release of prostacyclin (PGI(2)) was measured by radioimmunoassay of its stable breakdown product 6-keto-PGF(1alpha). The effects of the p38 MAPK inhibitors, SB203580 and PD169316, on neutrophil adhesion and 6-keto-PGF(1alpha) release were examined, as was the effect of an anti-E-selectin antibody on neutrophil adhesion to LPS-activated EDVEC. LPS treatment significantly increased p38 MAPK phosphorylation, which was maximal after a 1h LPS incubation using a concentration of 10(-5)g/ml (EC(50) = 2 x 10(-7)g/ml). Neutrophil adhesion to LPS-stimulated endothelial cells (maximal at 10(-6)g/ml; EC(50) = 6 x 10(-10)g/ml) was significantly inhibited in the presence of p38 MAPK inhibitors (reduced from a maximum of 33+/-6% to 13+/-4% adhesion at 10(-6)M SB203580 and to 21+/-4% adhesion at 10(-6) M PD169316), or an anti-E-selectin antibody (reduced from 17+/-1% adhesion to 6+/-1% adhesion). 6-keto-PGF(1alpha) release was increased in a concentration-dependent manner following LPS exposure (maximal at 10(-6)g/ml; EC(50) = 1 x 10(-9)g/ml), and was significantly inhibited by p38 MAPK blockade (reduced from 1.6+/-0.3 x 10(-9)g/ml to 4+/-1 x 10(-10)g/ml and 4+/-2 x 10(-10)g/ml with 10(-6) M SB203580 or 10(-6) M PD169316, respectively). This study has demonstrated that clinically relevant concentrations of LPS activate p38 MAPK in equine endothelial cells and that both neutrophil adhesion to LPS-activated EDVEC and PGI(2) release are dependent upon p38 MAPK phosphorylation. These results reveal a key role for p38 MAPK in the response of the endothelium to LPS and suggest that inhibition of this kinase may reduce inflammatory events in the endotoxemic horse.

Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 +/- 8% reduction; n = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCdelta inhibitor, rottlerin: 69 +/- 13%, 63 +/- 14% and 97 +/- 1%, respectively; n = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 +/- 3% and 88 +/- 2%, respectively; n = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.