Clinical implications of imprecise sampling time for 10- and 30-min thyrotropin-releasing hormone stimulation tests in horses.

BACKGROUND: The thyrotropin-releasing hormone (TRH) stimulation test is used to diagnose pituitary pars intermedia dysfunction (PPID) using 10- or 30-min protocols. Imprecise sampling time for the 10-min protocol can lead to misdiagnoses. OBJECTIVES: To determine the effect of imprecise sampling time for the 30-min protocol of the TRH stimulation test. STUDY DESIGN: In vivo experiment. METHODS: Plasma immunoreactive adrenocorticotropin (ACTH) concentrations were measured 9, 10, 11, 29, 30 and 31 min after intravenous administration of 1 mg of TRH in 15 control and 12 PPID horses. Differences in ACTH concentrations between sampling times, variability in ACTH concentrations between protocols, and diagnostic classification of PPID were assessed using Friedman's test, Bland-Altman plots, and Fisher's exact test, respectively, with 95% confidence intervals reported and significance set at p < 0.05. RESULTS: Imprecise sampling time resulted in variable ACTH concentrations, but significant differences in absolute ACTH concentrations were not detected for imprecise sampling within each protocol or between protocols. Imprecise sampling changed PPID diagnostic classification for 3/27 (11 [4-28] %) horses for both protocols. Using the 30-min protocol as a reference, 1/12 (8 [1-35] %) horses returned a negative test result and 5/12 (42 [19-68] %) horses returned equivocal test results that would be considered positive in practice due to the presence of supportive clinical signs. MAIN LIMITATIONS: Limited sample size and inter-horse variability reduced the ability to detect small but potentially relevant differences. CONCLUSIONS: Overall, the impact of imprecise sampling was not significantly different between the 10- and 30-min TRH stimulation test protocols. However, diagnostic classification for PPID would have varied between the 10- and 30-min protocols in this population, if clinical signs had been ignored. Precise timing during TRH stimulation tests and contextual interpretation of ACTH concentrations remain fundamental for the diagnosis of PPID.

Physiological and metabolic effects of short-term dopamine reduction in healthy horses using a tyrosine hydroxylase inhibitor (alpha-methyl-para-tyrosine).

Alpha-methyl-para-tyrosine (AMPT) is a reversible inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This study aimed to determine whether AMPT could reduce dopamine concentrations in horses. Six healthy adult Standardbred geldings were administered AMPT (40 mg/kg BW, orally) or placebo in a randomised crossover study design. Clinical examination findings were recorded, and blood samples were collected for up to 6 h after administration of AMPT or placebo, for measurement of blood glucose, plasma ACTH and cortisol concentrations, and plasma metabolomic analysis. Plasma prolactin concentration was determined as a proxy index of central dopamine reduction. No adverse clinical effects were detected after oral administration of AMPT, with heart rate, mean arterial pressure and blood glucose concentration not differing between AMPT treatment or placebo. Plasma prolactin concentration peaked 1 h after AMPT administration before returning to baseline at 2 h (for five horses) or 6 h (for one horse). Metabolomic analysis demonstrated a reduction in plasma dopamine (0.72-fold change; P=0.016) 1 h after AMPT treatment. Plasma ACTH and cortisol concentrations were not different between AMPT and placebo over time. A few metabolites associated with ketogenesis were increased, and certain amino acids decreased, at 1 h compared with baseline, for both AMPT treatment and placebo. Therefore, AMPT was effective in reducing both central and circulating dopamine concentrations in healthy horses following a single oral dose. Further pharmacokinetic and pharmacodynamic studies are warranted to optimise the dose and duration of AMPT treatment to achieve longer-term dopamine reduction. Plasma metabolomic findings suggested an interruption to energy flux at the time of sample collection, which may be relevant to nutritional studies in horses and warrants further investigation.

Effect of short-term dopamine reduction on insulin sensitivity and post-prandial insulin and glucose responses in Standardbred horses.

The role of dopamine in the regulation of insulin secretion in horses is poorly understood and requires further investigation. Pituitary pars intermedia dysfunction (PPID) is associated with decreased activity of dopaminergic neurons which normally suppress peptide hormone secretion from the pituitary pars intermedia. A high proportion of horses with PPID also have insulin dysregulation (ID), characterised by post-prandial hyperinsulinaemia and/or tissue insulin resistance, which are risk factors for the development of laminitis. The aim of this study was to investigate the effects of alpha-methyl-para-tyrosine (AMPT), a tyrosine hydroxylase inhibitor that reduces dopamine production, on insulin sensitivity and the post-prandial insulin response to a glucose-containing meal. Six healthy Standardbred horses were enrolled in a placebo-controlled randomised crossover study, in which one dose of AMPT (40 mg/kg BW) or placebo was administered orally, prior to performing an in-feed oral glucose test (OGT) and a frequently sampled intravenous glucose tolerance test (FSIGTT). Dopamine reduction by AMPT was confirmed by an increase in plasma prolactin concentration and the lack of post-prandial increase in plasma dopamine concentration compared to placebo. Post-prandial insulin responses, both peak and AUCi, were increased after AMPT compared to placebo (P=0.048 and P=0.005, respectively), without affecting blood glucose concentrations. However, one dose of AMPT did not appear to affect tissue sensitivity as assessed by the FSIGTT. This study confirmed that dopamine plays a role in the regulation of insulin secretion in horses, as it does in other species, whereby the post-prandial release of dopamine into the circulation may inhibit pancreatic insulin secretion. Further studies are required to evaluate different dosing protocols for AMPT, and to further investigate the links between PPID, ID and laminitis risk in horses.

Strongyle egg shedding and egg reappearance periods in horses with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling, using a gamma distribution, was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required, in line with sustainable parasite management practices.

Evidence for dopamine production and distribution of dopamine D2 receptors in the equine gastrointestinal mucosa and pancreas.

Insulin dysregulation in horses is characterised by hyperinsulinaemia and/or tissue insulin resistance and is associated with increased risk of laminitis. There is growing evidence in other species that dopamine attenuates insulin release from the pancreas; however, this has yet to be examined in horses. The present study aimed to identify whether there are cells capable of producing or responding to dopamine within the equine gastrointestinal mucosa and pancreas. Tissue samples were collected from the stomach, small and large intestines, and pancreas of six mature horses following euthanasia. Samples of stomach contents and faeces were also collected. Immunohistochemistry was performed to identify tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine production, and dopamine D2 receptors in tissue sections. Additional immunostaining for glucagon, insulin and chromogranin A was performed to identify α cells, ß cells and enteroendocrine cells, respectively. Gastric parietal cells expressed both TH and D2 receptors, indicating that they are capable of both producing and responding to dopamine. Dopamine was quantified in stomach contents and faeces by high-performance liquid chromatography with electrochemical detection, with similar concentrations found at both sites. Dopamine D2 receptors were expressed in duodenal epithelial cells but not more distally. A subset of enteroendocrine cells, located sporadically along the gastrointestinal tract, were found to be immunopositive for the D2 receptor. In pancreatic islets, TH was present in α cells, while D2 receptors were strongly expressed in ß cells and variably expressed in α cells. These findings are consistent with studies of other species; however, dynamic studies are required to further elucidate the role of dopamine in the modulation of insulin and glucagon secretion in horses. This descriptive study provides preliminary evidence for a potential role of dopamine to act as a paracrine messenger in the gastrointestinal mucosa and endocrine pancreas of horses.

Epidemiological investigation of insulin dysregulation in Shetland and Welsh ponies in Australia.

BACKGROUND: Insulin dysregulation (ID) is central to equine metabolic syndrome. There are limited epidemiological studies investigating dynamic testing of ID in ponies. OBJECTIVES: To evaluate prevalence and risk factors for ID through dynamic testing of hyperinsulinaemia (DHI) and insulin resistance (IR). STUDY DESIGN: Cross-sectional. METHODS: Sex, age, breed, height, cresty neck score (CNS), body condition score (BCS), laminitis, HMGA2:c.83G>A genotype and pituitary pars intermedia dysfunction (PPID) status were documented. Dynamic hyperinsulinaemia was diagnosed with an oral sugar test (OST) and IR with an insulin tolerance test (ITT). Owners completed surveys reporting activity, laminitis history and perception of body condition using a (1-9) visual analogue scale (VASo). Ordinal scores were converted to binary outcomes for CNS (≤2/5 or ≥3/5), BCS and VASo (≤6/9 or ≥7/9). Variables associated with insulin concentrations, glucose reduction after the ITT and laminitis were evaluated with mixed effects regression models accounting for random effects of farms. RESULTS: Among 167 ponies tested, median (range) age was 9 (4-21) years and BCS was 6 (4-8). Prevalence (95% confidence interval [CI]) of ID was 61 (53-68)%. Factors associated with insulin concentrations (estimate [95% CI]; µIU/mL) 60 min post-OST were: age (1.07 [1.02-1.11]), CNS (≥3/5, 1.52 [1.04-2.23]) and VASo (≥7/9, 1.75 [1.09-2.79]); and 90 min post-OST were: age (1.08 [1.03-1.12]), CNS (≥3/5, 1.80 [1.22-2.64]), VASo (≥7/9, 2.49 [1.52-4.08]) and sex (male, 0.64 [0.45-0.91]). Factors associated with glucose reduction after the ITT (estimate [95% CI]; %) were: age (-1.34 [-2.01 to -0.67]), sex (female, -6.21 [-11.68 to -0.74]) and VASo (≥7/9, -1.74 [-18.89 to -4.78]). Factors associated with laminitis (odds ratio [95% CI]) were DHI (4.60 [1.68-12.58]), IR (3.66 [1.26-10.61]) and PPID (11.75 [1.54-89.40]). MAIN LIMITATIONS: Single time-point sampling, laminitis definition and diet analysis. CONCLUSIONS: Ageing, being female and owner-perceived obesity were associated with ID.

Development of a body condition index to estimate adiposity in ponies and horses from morphometric measurements.

BACKGROUND: There is a high prevalence of obesity in ponies and pleasure horses. This may be associated with equine metabolic syndrome and an increased risk of laminitis. Body condition scoring (BCS) systems are widely used but are subjective and not very sensitive. OBJECTIVES: To derive a body condition index (BCI), based on objective morphometric measurements, that correlates with % body fat. STUDY DESIGN: Retrospective cohort study. METHODS: Morphometric measurements were obtained from 21 ponies and horses in obese and moderate body condition. Percentage body fat was determined using the deuterium dilution method and the BCI was derived to give the optimal correlation with body fat, applying appropriate weightings. The index was then validated by assessing inter-observer variation and correlation with % body fat in a separate population of Welsh ponies; and finally, the correlation between BCI and BCS was evaluated in larger populations from studies undertaken in Australia, the United Kingdom and the United States. RESULTS: The BCI correlated well with adiposity in the ponies and horses, giving a Pearson r value of 0.74 (P < 0.001); however, it was found to slightly overestimate the % body fat in leaner animals and underestimate in more obese animals. In field studies, the correlation between BCI and BCS varied particularly in Shetlands and miniature ponies, presumably due to differences in body shape. MAIN LIMITATIONS: Further work may be required to adapt the BCI to a method that is more applicable for Shetlands and miniature ponies. CONCLUSIONS: This BCI was able to provide an index of adiposity which compared favourably with condition scoring in terms of accuracy of estimating adiposity; and was more consistent and repeatable when used by inexperienced assessors. Therefore, this may be a useful tool for assessing adiposity; and may be more sensitive than condition scoring for tracking weight gain or weight loss in individual animals.

Evaluation of an HMGA2 variant contribution to height and basal insulin concentrations in ponies.

BACKGROUND: The HMGA2:c.83G>A variant was identified in Welsh ponies having pleiotropic effects on height and insulin concentration. OBJECTIVE: Determine whether the HMGA2:c.83G>A variant is associated with decreased height and higher basal insulin concentrations across pony breeds. ANIMALS: Two hundred thirty-six ponies across 6 breeds. METHODS: Cross-sectional study. Ponies were genotyped for the HMGA2:c.83G>A variant and phenotyped for height and basal insulin concentrations. Stepwise regression was performed for model analysis using a linear regression model for height and mixed linear model for insulin with farm as a random effect. Coefficient of determination, pairwise comparison of the estimated marginal means and partial correlation coefficients (parcor) were calculated to assess the relationship between HMGA2 genotype and height or insulin. RESULTS: Breed and genotype accounted for 90.5% of the variation in height across breeds, and genotype explained 21% to 44% of the variation within breeds. Breed, genotype, cresty neck score, sex, age, and farm accounted for 45.5% of the variation in insulin, with genotype accounting for 7.1%. The HMGA2 A allele frequency was 62% and correlated with both height (parcor = -0.39; P < .001) and insulin (parcor = 0.22; P = .02). Pairwise comparisons found A/A ponies were >10 cm shorter than other genotypes. Compared with G/G individuals, A/A and G/A individuals had 4.3 µIU/mL (95% confidence interval [CI]: 1.8-10.5) and 2.7 µIU/mL (95% CI: 1.4-5.3) higher basal insulin concentrations, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: These data demonstrate the pleiotropic effects of the HMGA2:c.83G>A variant and its role in identifying ponies at increased risk for insulin dysregulation.

Comparison of Feed Digestibility between Ponies, Standardbreds and Andalusian Horses Fed Three Different Diets.

Ponies and some horse breeds such as Andalusians exhibit an 'easy keeper' phenotype and tend to become obese more readily than other breeds such as Standardbreds. Various hypotheses have been proposed, including differences in appetite or metabolic efficiency. This study aimed to investigate the effect of breed on nutrient digestibility. Ponies, Standardbreds and Andalusian horses were adapted to consuming either a control fibre-based diet (n = 9), a hypercaloric cereal-rich diet (n = 12) or a hypercaloric fat-rich diet (n = 12) over 20 weeks. Total faecal collection was performed over 24 h to determine apparent total tract digestibility of gross energy, dry matter (DM), neutral detergent fibre (NDF), starch, crude protein and crude fat. There was no effect of breed on apparent digestibility for any of the nutrients studied (all p > 0.05). However, there was a significant effect of diet, with animals consuming the cereal-rich or fat-rich diets demonstrating higher digestibility of gross energy, DM, NDF and crude protein compared with those consuming the control diet (all p < 0.05). Animals adapted to the cereal-rich diet demonstrated higher digestibility of starch (p < 0.001) and animals adapted to the fat-rich diet demonstrated higher digestibility of fat (p < 0.001). This study found that horses and ponies had similar nutrient digestibility when adapted to the same diets and management conditions. Limitations included the relatively small number of animals from each breed per diet group and the short period of total faecal collection. The tendency towards increased adiposity in ponies and Andalusian-type horse breeds is more likely to reflect differences in metabolism, rather than differences in feed digestibility.

Ross River Virus Infection of Horses: Appraisal of Ecological and Clinical Consequences.

Ross River virus (RRV) is a mosquito-borne arbovirus of the genus Alphavirus that causes disease in humans and horses in Australia. A temporal association of RRV infection in horses with clinical signs including pyrexia, malaise, and polyarthralgia has been reported, along with reduced athletic performance, often for extended periods. Despite these reports, disease due to RRV remains somewhat controversial as experimental infection of horses has resulted in obvious viraemia yet minimal signs of clinical disease. The relatively high viraemia demonstrated by horses infected with RRV has led to speculation that they could act as an important reservoir host of the virus, although this remains unclear. This review sought to appraise the existing literature relating to RRV infection of horses and to summarize the ecological and clinical consequences of RRV of relevance to the equine industry and to public health more broadly.

Influence of dietary restriction and low-intensity exercise on weight loss and insulin sensitivity in obese equids.

BACKGROUND: The importance of including exercise with dietary modification for the management of obese equids is not clearly understood. OBJECTIVES: To evaluate the effect of a practical low-intensity exercise regimen, in addition to dietary restriction, on indices of insulin sensitivity (SI) and plasma adipokine concentrations in obese equids. ANIMALS: Twenty-four obese (body condition score [BCS] ≥ 7/9) horses and ponies. METHODS: Over a 12-week period, animals received either dietary restriction only (DIET) or dietary restriction plus low-intensity exercise (DIET+EX). All animals were provided with a restricted ration of grass hay at 1.25% body weight (BW) on a dry matter basis, providing 82.5% estimated digestible energy requirements. The DIET+EX group undertook low-intensity exercise 5 days per week on an automated horse walker. Before and after weight loss, total body fat mass (TBFM) was determined, indices of SI were calculated using minimal model analysis of a frequently sampled IV glucose tolerance test, and adipokines plus inflammatory biomarkers were measured using validated assays. RESULTS: Decreases in BCS, BW, and TBFM were similar between groups (all P > .05). After weight loss, animals in both groups had decreased basal insulin and leptin concentrations, and increased adiponectin concentrations (all P < .001). Furthermore, animals in the DIET+EX group had significantly improved SI and decreased serum amyloid A concentrations relative to animals in the DIET group (both P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Regular low-intensity exercise provided additional health benefits compared with dietary restriction alone in this population of obese equids.

Peer Feedback on Collaborative Learning Activities in Veterinary Education.

Collaborative learning activities are an increasingly prominent feature of veterinary curricula that have been redesigned to achieve competency-based graduate learning outcomes. This evolution challenges the traditional individualistic approach to veterinary education and necessitates revisions to assessment and feedback practices to ensure constructive alignment. Peer feedback has been widely reported in the medical education literature as a teaching intervention in collaborative learning settings, with learning gains reported for students who receive and provide peer feedback. In this setting, peer feedback has been demonstrated to provide valuable formative feedback on professional behaviors and skills. However, there are very few such reports in the veterinary education literature to date. Barriers to the introduction of this approach can include teacher and student perceptions, and concerns around validity and reliability. This review aimed to provide an overview of current evidence regarding peer feedback on collaborative learning activities in higher education, and to explore opportunities and challenges for the introduction of peer feedback in the context of veterinary education. We contend that early and repeated provision of formative peer feedback can provide an opportunity to scaffold the development of crucial core competencies within veterinary education, including the self-regulated learning skills required to work in collaborative teams, and interpret and act on feedback.

Comparison and validation of ELISA assays for plasma insulin-like growth factor-1 in the horse.

Insulin-like growth factor-1 (IGF-1) plays several important physiological roles, and IGF-related pathways have been implicated in developmental osteochondral disease and endocrinopathic laminitis. This factor is also a downstream marker of growth hormone activity and its peptide mimetics. Unfortunately, previously used assays for measuring equine IGF-1 (radioimmunoassays and ELISAs) are no longer commercially available, and many of the kits on the market give poor results when used on horse samples. The aim of the present study was to compare three different ELISA assays (two human and one horse-specific). Plasma samples from six Standardbreds, six ponies and six Andalusians were used. The human IGF-1 ELISA kit from Immunodiagnostic Systems (IDS) proved to be the most accurate and precise of the three kits; the other two assays gave apparently much lower concentrations, with poor recovery of spiked recombinant human IGF-1 and unacceptably poor intra-assay coefficients of variation (CV). The IDS assay gave an intra-assay CV of 3.59 % and inter-assay CV of 7.31%. Mean percentage recovery of spiked IGF-1 was 88.82%, and linearity and dilutional parallelism were satisfied. The IGF-1 plasma concentrations were 123.21 ±8.24 ng/mL for Standardbreds, 124.95 ±3.69 ng/mL for Andalusians and 174.26 ±1.94 ng/mL for ponies. Therefore of the three assays assessed, the IGF-1 ELISA manufactured by IDS was the most suitable for use with equine plasma samples and may have many useful applications in several different research areas. However, caution should be used when comparing equine studies where different analytical techniques and assays may have been used to measure this growth factor.