The effect of pergolide mesylate on adrenocorticotrophic hormone responses to exogenous thyrotropin releasing hormone in horses.

Thyrotropin releasing hormone (TRH) stimulation testing is often used to support a diagnosis of pituitary pars intermedia dysfunction (PPID) in horses although it is unclear whether or not repeat TRH stimulation testing post-treatment is a valid means of assessing response to medical therapy. Laboratory submissions from 64 suspected equine PPID cases were examined including the initial pre-treatment TRH stimulation test and a follow up test within 100 days of starting medical therapy with pergolide. In a subset of cases, further follow-up tests were examined beyond 100 days of starting treatment. Results from tests conducted between 1 July and 30 November were excluded. Significant improvements were seen in both the baseline and TRH-stimulated adrenocorticotrophic hormone (ACTH) concentrations within 100 days with no further improvements seen in the subset of cases examined thereafter. Although 88% (n = 56/64) of all cases showed a decreased response to TRH post-treatment, only 24% (n = 9/38) of horses with positive pre-treatment TRH stimulation tests normalised following treatment, with a further 34% (n = 13/38) improving into an equivocal test outcome category. Most commonly (42%; n = 16/38), horses with positive pre-treatment TRH stimulation tests remained positive following treatment, although 75% (n = 12/16) of these showed a numerically lower post-treatment response to TRH. These results will help inform practitioners of expected changes in TRH stimulation test results when assessing response of horses with PPID to medical therapy with pergolide.

The effect of month and breed on plasma adrenocorticotropic hormone concentrations in equids.

Use of plasma adrenocorticotropic hormone (ACTH) concentrations for the diagnosis of PPID in equids requires awareness of other factors that might influence ACTH concentrations, including breed and time of year. This study was designed to investigate effects of breed on plasma ACTH concentrations and potential interactions between breed and time of year. Ten breeds were selected from a laboratory database containing data on plasma ACTH concentrations. Breeds represented were Arabians, Cobs, Connemara ponies, donkeys, Irish Sports horses, New Forest ponies, Shetland ponies, Thoroughbreds, Warmbloods and Welsh breeds. Multivariable analysis was used to determine the effects of month and breed on plasma ACTH concentrations. A circannual pattern of ACTH concentration was documented, with a nadir in April and a peak in September. Arabian horses and donkeys had significantly higher ACTH concentrations than some other breeds, primarily from May to November, whereas in Shetland ponies and Welsh breeds, relatively higher ACTH concentrations only occurred from July to November. The annual increase in plasma ACTH appeared to commence in late April/early May in response to increasing daylight and decreased again promptly after the late September equinox as daylength fell below 12 h per day. Inter-breed differences in plasma ACTH were substantial and indicated that breed and time of year should be considered when interpreting plasma ACTH concentrations in equids suspected for PPID.

Stability of common biochemistry analytes in equine blood stored at room temperature.

REASONS FOR PERFORMING STUDY: Time delays between collection of blood samples and biochemical analysis of equine blood are unavoidably common in equine practice. The effect that delays may have on the accuracy of results of blood biochemical analyses is not well established. HYPOTHESIS: Delays in processing of blood of up to 72 h results in alterations in measured levels of common biochemical analytes that are of potential clinical relevance. Separation of serum prior to storage is protective against the effects of time delays. METHODS: Samples of clotted blood, separated serum and oxalate fluoride plasma from 20 horses were stored and analysed at 0, 24, 48 and 72 h. Graphical exploration of each analyte was undertaken. General linear models with fixed effects were fitted for the whole blood data. The mean bias and 95% limits of agreement were calculated, using bootstrapped data, to assess agreement between pairs of samples analysed at 0 h and other time points. Bland-Altman plots were used to explore general trends in the data. Paired t tests were used to compare the results from whole blood and separated serum. RESULTS: Delays in processing equine blood resulted in significant increases in measured concentrations of aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total bile acids and magnesium. A significant decrease in concentration was identified for glucose (serum and oxalate fluoride preserved plasma). Separation of serum immediately following clot formation resulted in nonsignificant increases in accuracy for some analytes. CONCLUSIONS AND PRACTICAL SIGNIFICANCE: Delays in processing of blood samples may result in biochemical changes of clinical relevance in individual cases; however, in the majority of cases, where delays are only a few days and a number of analytes are assessed concurrently, delays are unlikely to have an effect on the interpretation of results. Separation of serum following clot formation is of limited benefit. Clinical samples in which a delay in processing has occurred may be interpreted with reference to the data presented.

Type 2 diabetes mellitus with pancreatic beta cell dysfunction in 3 horses confirmed with minimal model analysis.

REASONS FOR PERFORMING STUDY: Type 2 diabetes mellitus (T2DM) is diagnosed rarely in equine practice although it may be under-recognised. A greater awareness of the condition and therapeutic considerations would be to the benefit of such cases presenting in practice. More investigation into the pharmacological management of these cases is needed. OBJECTIVES: Three cases of diabetes mellitus were investigated using a specific test for insulin sensitivity and pancreatic beta cell function in order to define accurately and characterise the existence of T2DM in all 3 subjects. METHODS: The insulin-modified frequently sampled i.v. glucose tolerance test was performed in each case and the data so obtained were subject to minimal model analysis of insulin-glucose dynamics. Cases were then monitored following treatment using a combination of dietary modification, metformin, glibenclamide and pergolide. RESULTS: Marked insulin resistance was identified in each case and, furthermore, severe pancreatic beta cell dysfunction was present therefore classifying each case as end stage T2DM. Treatment was nevertheless associated with restoration of normoglycaemia in all cases. CONCLUSIONS: T2DM in horses may be more common than generally considered.In some cases individuals may respond to therapy aimed at restoring insulin sensitivity and pancreatic function. Drugs used in other species for the treatment of T2DM have not yet been adequately tested in horses. POTENTIAL RELEVANCE: T2DM should be considered as an important differential diagnosis in mature to elderly horses and ponies suffering from weight loss, polydipsia and polyuria. Clinicians should be encouraged to offer treatment and management advice when such cases are encountered.

The effect of metformin on measurements of insulin sensitivity and beta cell response in 18 horses and ponies with insulin resistance.

REASONS FOR PERFORMING STUDY: Laminitis in equids is a very common debilitating disease, and insulin resistance (IR) and hyperinsulinaemia are increasingly recognised as important predisposing factors. Pharmacological modification of IR and hyperinsulinaemia might reduce the risk of laminitis. HYPOTHESIS: Metformin, a drug commonly prescribed for treatment of human IR, may also decrease IR in equids. METHODS: Eighteen horses and ponies with IR and recurrent laminitis were treated with 15 mg/kg bwt metformin per os q. 12 h. Each animal served as its own control by comparing pre- and post treatment proxies for IR, insulin sensitivity (IS) and pancreatic beta cell function while controlling for possible dietary and managemental influences on IR. RESULTS: Evidence of significantly improved IS and decreased pancreatic beta cell secretion was found following metformin treatment. The magnitude of effect was greater at earlier resampling (6-14 days) than at later times (23-220 days). Apparent subjective clinical benefits were good but less favourable than effects on IR. CONCLUSIONS: Metformin is safe and appears to increase IS in equids. POTENTIAL RELEVANCE: Metformin may be indicated as a treatment for IR in equids. Further studies are required to define appropriate selection of subjects warranting therapy, dosing schedule and pharmacokinetics.

Long-term management of sabulous cystitis in five horses.

Five horses with sabulous cystitis were managed for up to three years. They were treated by emptying the bladder through a urinary catheter and saline lavage with cytoscopic guidance to remove residual sabulous material. The cystitis was treated with antimicrobial and anti-inflammatory medications, and bethanechol chloride was also administered. Frequent catheterisation and emptying of the bladder was an alternative to regular cystoscopic examination with saline lavage but it resulted in the development of a urethral stricture in one case. Four of the horses returned to work and one was retired owing to persistent incontinence.

A study of the environmental survival of Streptococcus equi subspecies equi.

BACKGROUND: Streptococcus equi represents a common hazard to equids worldwide. Environmental contamination with bacteria shed from an infected horse may represent a significant source of contagion and further knowledge of ex vivo bacterial survival under different conditions is important for disinfection and isolation protocols. OBJECTIVES: To determine the potential duration of survival and vigour of growth of S. equi inoculated onto surfaces relevant to equine veterinary practice and stabling in summer and winter. STUDY DESIGN: Repeat sampling of environmental inocula of S. equi. METHODS: Cultures of S. equi were inoculated onto wood, a shoe sole, cotton overalls, inside a nasogastric tube, inside a dental rasp, in a wet plastic bucket and onto a fence post both in the summer and winter seasons. Frequent resampling and culture from the inoculated sites was conducted until no viable bacteria were found. Bacterial viability was determined by both duration (time to first negative culture) and vigour of growth (growth score over the first 3 days of culture) and compared between inoculated sites and times of year. RESULTS: Bacterial viability was enhanced by a wet local environment and by the winter season. Survival tended to be short in the summer (up to 9 days in wet sites and up to 2 days in dry sites) but much longer in the winter (up to 34 days in wet sites and up to 13 days in dry sites). Vigour of bacterial growth was also greater in the winter than in the summer as judged by 3-day-growth scores. MAIN LIMITATIONS: Direct comparison with the variable size and nature of naturally shed infectious material is difficult. CONCLUSIONS: Veterinarians and personnel handling horses should be aware that S. equi may survive in an equine environment for longer than previously found, especially when protected by wet and cold conditions.

Clinical, immunophenotypic and functional characterisation of T-cell leukaemia in six horses.

REASON FOR PERFORMING STUDY: Lymphoid leukaemia (LL) is rare in equids. In man, immunophenotypic classification identifies distinct leukaemic types with different treatment strategies. Improved understanding and classification of equine LL may allow similar advances. OBJECTIVES: To document the clinical, immunophenotypic and functional characteristics in 6 cases of equine LL of T-cell origin. METHODS: The clinical records and pathological findings from 6 cases of equine LL were analysed. Immunohistochemistry to identify T or B lymphocytes was performed on paraffin embedded tissues in 4 cases. Peripheral blood mononuclear cells (PBMC) were phenotyped for expression of CD4, CD8, MHC class I and II and B-cell antigens in 4 cases using monoclonal antibodies (mAbs) and flow cytometry. Neoplastic lymphocytes from 4 horses were stimulated with mitogens. RESULTS AND CONCLUSIONS: Six horses of various breeds were identified with LL of T-cell origin. The clinical course and presenting signs varied. Neoplastic lymphocytes were identified in peripheral blood samples from all horses and tissue invasion was confirmed at examination post mortem in 4 horses. Immunophenotyping identified a predominance of CD3+ T-cells in lymphoid tissues and CD4+ T-cells in circulating peripheral blood mononuclear cells (PBMC) in the affected horses. Neoplastic lymphocytes from the 4 cases that were tested failed to proliferate in response to mitogens. POTENTIAL RELEVANCE: Characterisation of the clinical, pathological and immunological findings in 6 horses with LL has added to reports of this rare condition, characterised it in greater detail and therefore provides a starting point for further investigations.

Effect of storage time and temperature on the results of analysis of synovial and mesothelial fluids.

REASONS FOR PERFORMING STUDY: Delays between collection and laboratory analysis of equine body fluid samples are common in practice; however, the effects of delays on the accuracy of results and diagnostic interpretation are unknown. OBJECTIVES: To assess the effects of storage time and temperature combination on protein and cell parameters of equine synovial and mesothelial cavity fluids and determine whether any changes affect clinicopathological interpretation. STUDY DESIGN: In vitro experiment. METHODS: Body fluid samples obtained from horses during diagnostic investigation were divided into 7 aliquots and total protein concentration (TP), total nucleated cell count (TNCC) and neutrophil morphology were analysed immediately (T0 ) and at 24 (T24 ), 48 (T48 ) and 72 h (T72 ) after storage at 4 or 22°C. Linear mixed models were used to analyse effects of fluid type and storage conditions on TP, TNCC and neutrophil morphology grade. Changes in interpretation of samples over time and diagnostic performance at each analysis point were recorded. RESULTS: A total of 32 samples were collected from 23 horses. Storage had no effect on TP. Cell count was influenced by fluid type and was significantly reduced at T72 for storage at 4°C and T24 , T48 and T72 for 22°C (P<0.001). Neutrophil morphology grade was significantly greater at T24 , T48 and T72 than at T0 for both 4 and 22°C (P<0.001). For 9 samples, the diagnostic interpretation changed over time. Specificity and positive predictive value at each analysis point was 100%; however, sensitivity and negative predictive value decreased with greater storage duration and temperature. CONCLUSIONS: Alterations in the TNCC and neutrophil morphology of body fluid samples occur when analysis is delayed, especially with higher storage temperatures, and may influence interpretation and clinical decision-making. Body fluid samples should be analysed as soon as possible after collection to minimise preanalytical errors due to storage.

Clinical application of parenteral nutrition in the treatment of five ponies and one donkey with hyperlipaemia.

Five ponies and one donkey with hyperlipaemia that occurred secondarily to a variety of primary clinical conditions were treated with lipid-free partial parenteral nutrition comprising equal volumes of 50 per cent glucose and 15 per cent amino acids. The infusion supplied energy and protein at rates of 2.6 kJ/kg per hour and 34.3 mg/kg per hour, respectively. In all six cases there was a prompt and sustained decrease in serum concentrations of triglyceride. In four of the six cases a good response to treatment of the primary condition was also seen and the subjects were discharged successfully. In the remaining two cases, poor clinical response of the primary condition resulted in euthanasia, although hyperlipaemia was nevertheless resolved. The main complication of parenteral nutrition was hyperglycaemia.

Quantification of immune cell populations in the lamina propria of equine jejunal biopsy specimens.

The histological diagnosis of inflammatory bowel disease (IBD) in horses and other species is subjective, and pathological assessments vary considerably as a result. One important criterion is increased infiltration of the lamina propria by eosinophils, plasma cells, lymphocytes or macrophages, but this is difficult to assess without a knowledge of the normal immune cell populations and potential for individual variation. Retrospective jejunal specimens were analysed from 14 horses aged 13-15 years which had not shown clinical or post-mortem signs of gastrointestinal disease. Populations of plasma cells, T lymphocytes (CD3+), B lymphocytes (CD79a+ cytoplasmic membranes), eosinophils, macrophages and neutrophils were counted in 9000-microm2 areas of the villous lamina propria and intercryptal lamina propria for each horse. There were significantly higher counts of plasma cells, B lymphocytes and eosinophils in the intercryptal than in the villous region, which accords with previous findings in dogs. This information will be used as control data for future quantitative morphometrical analysis of immune cells in small intestinal specimens from horses in which IBD has been diagnosed.

Investigation of single and paired measurements of adrenocorticotropic hormone for the diagnosis of pituitary pars intermedia dysfunction in horses.

BACKGROUND: Paired measurement of ACTH concentration may be more reliable than a single measurement. HYPOTHESIS/OBJECTIVES: To determine whether the mean of 2 measurements of ACTH concentration is more reliable in assessing pituitary pars intermedia dysfunction (PPID) than a single measurement. ANIMALS: Paired ACTH measurements were performed on (1) 148 occasions from 124 horses being investigated for PPID, (2) 90 occasions from 76 horses with PPID that were receiving treatment with pergolide, and (3) 63 occasions from 50 horses in which there was no clinical suspicion of PPID. Histologic examination of the pars intermedia was performed in 67 of the untreated horses. METHODS: Outcome of testing using single and the mean of paired samples was compared directly and both methods were compared against histology, which was considered the gold standard. RESULTS: Paired ACTH measurement altered binary classification as healthy or diseased in 6 of 211 cases, all off which had equivocal initial ACTH concentrations between 20 and 39 pg/mL. Using histology as the gold standard, optimal sensitivity and specificity for diagnosing PPID were 69.4 and 80.9%, respectively, for a single measurement and 72.2 and 76.2%, respectively, for paired measurements. The area under the receiver operating characteristic curve was 0.72 and 0.73 for single and paired measurements compared with histopathologic diagnosis, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Paired measurement of ACTH concentration offers no advantage over a single measurement.

An evaluation of diagnostic data in comparison to the results of liver biopsies in mature horses.

REASONS FOR PERFORMING STUDY: The diagnostic value of several investigative procedures commonly used during the evaluation of suspected equine hepatopathy cases has not been specifically quantified in previous studies. HYPOTHESIS: No noninvasive procedures would clearly discriminate between horses with and without significant liver disease. METHODS: Histopathology of biopsy samples was used as the 'gold standard' technique for definitive diagnosis of the presence or absence of significant liver disease. Clinical, ultrasonographic and clinicopathological data obtained during the investigation of 82 suspected cases of hepatopathy in mature horses were compared with the results of subsequent biopsy in order to quantify the diagnostic value of each test. RESULTS: Of the 82 cases, 61 were confirmed to have significant liver disease and 21 were not. Only serum concentrations of gamma-glutamyltransferase (gammaGT), globulins and alkaline phosphatase (AP) were found to be significantly different between the 2 groups of horses. Clinical and ultrasonographic abnormalities were found, when present, to be good indicators of the presence of liver disease. Certain single serum biochemical tests and combinations thereof were found to have high values for sensitivity (SN), specificity (SP), positive predictive value (PPV) and/or negative predictive value (NPV), but none showed high values for all 4 parameters leading to important limitations in their practical diagnostic application. Of all serum biochemical tests studied, the PPVs of only gammaGT and globulins could be clearly improved by considering especially high positive results. CONCLUSIONS: The single positive test results of greatest diagnostic value were presence of hepatic encephalopathy (HE), increased gammaGT, hyperglobulinaemia, hypoalbuminaemia, increased AP, increased total bile acids (TBA) and increased total bilirubin (TBil). Increased aspartate aminotransferase (AST) and increased glutamate dehydrogenase (GLDH) were also of good diagnostic value, but only when used in combination with the above tests. However, no single, combination or sequential test was able to discriminate fully between horses with and without biopsy-confirmed liver disease and reliance on the use of noninvasive tests for the prediction of the presence or absence of significant liver disease may lead to frequent diagnostic errors. Although certain positive results did reliably predict the presence of liver disease, negative test results were invariably poor predictors of the absence of liver disease. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, the results may help clinicians with their selection and interpretation of appropriate test results in cases of suspected liver disease.

Equine hepatic disease: the effect of patient- and case-specific variables on risk and prognosis.

REASONS FOR PERFORMING STUDY: Liver disease is fairly common in horses and, although previous literature suggests that fatality rates are high, impressions gained from current clinical practice suggest that this is no longer the case. OBJECTIVES: A case-control study was undertaken in 2 hospitals to investigate whether age, gender or breed type predisposed horses to developing hepatic disease, with the additional aim of determining whether these factors and disease category had any effect on outcome. METHODS: Eighty-eight cases were identified by review of computerised archives at 2 hospitals. Three cases admitted immediately before and after the cases were used as controls. Multivariant models were developed to explore the relationship between age, breed type and gender and the development of liver disease, and within the clinical cases, to identify whether any of these factors or specific forms of liver disease were associated with fatality rate. RESULTS: Ponies were more likely to develop hepatic disease than light riding horses, but neither age nor gender were significant risk factors. Overall, the fatality rate was fairly low; horses with unclassified hepatopathies had the lowest fatality rate and those with cholangiohepatitis, pyrrolizidine alkaloid toxicity and chronic active hepatitis had significantly higher fatality rates in comparison. However, age, breed type and gender had no detectable effect on outcome. CONCLUSIONS AND POTENTIAL RELEVANCE: Such data could be of particular value to equine practitioners, as the cases were drawn from both primary and referral practice, and were all seen within the last 5 years.

Nutritional and clinicopathological effects of post operative parenteral nutrition following small intestinal resection and anastomosis in the mature horse.

REASONS FOR PERFORMING STUDY: There is an absence of data describing the nutritional requirements and nutritional status of horses following surgery for colic; furthermore, the potential effect of parenteral nutrition (PN) on improving nutritional status in such cases is unknown. HYPOTHESIS: Post operative colic cases suffer from a potentially detrimental negative energy balance and the PN formulation developed in this study would lead to clinicopathologically detectable improvements in the subjects' nutritional status. METHODS: Several clinicopathological variables, some known to be associated with nutritional status, were compared in 2 groups of horses in the post operative period following colic surgery; Group N (n = 15) were treated with PN and Group C (n = 15) were starved routinely. RESULTS: Group N had significantly lower serum concentrations of triglycerides, total bilirubin, albumin and urea and significantly higher serum concentrations of glucose and insulin compared with Group C in the post operative period. CONCLUSIONS: The control group of horses demonstrated significant clinicopathological evidence of starvation and the described PN protocol resulted in a demonstrably improved nutritional status in the treated horses. POTENTIAL RELEVANCE: Further study is required to investigate clinical benefits and possible harmful side effects of post operative parenteral nutrition before the technique can be advocated for widespread use in practice.

Development and application of a scoring system for prognostic evaluation of equine liver biopsies.

REASONS FOR PERFORMING STUDY: The study was designed to investigate the prognostic value of liver biopsy during investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: That liver biopsy is the most prognostically useful technique in common usage in the investigation of suspected liver disease. METHODS: This study examined the prognostic value of liver biopsy during the investigation of suspected liver disease in 73 mature horses. Histopathological variables comprising fibrosis, irreversible cytopathology, inflammatory infiltration, haemosiderin accumulation and biliary hyperplasia were found to be significant predictors of nonsurvival and were used to formulate a weighted biopsy score representing a prognostically useful broad comparative index of histopathological severity. Minimum and maximum possible scores were 0 and 14 points, respectively. RESULTS: Retrospective application of the biopsy scoring system to the study population indicated that horses with scores of 0 or 1 were equally likely to survive to 6 months with a combined mortality of 4%. Horses with biopsy scores between 2 and 6 had a combined mortality of 33% and were at a 12-fold increased risk of nonsurvival within 6 months (hazard ratio = 12.04, 95% CI 134-107.81, P = 0.026) compared to horses with a biopsy score of 0. Horses with biopsy scores between 7 and 14 had a combined mortality of 86% and were at a 46-fold increased risk of nonsurvival (hazard ratio = 46.01, 95% CI 5.92-357.5, P < 0.001) compared to horses with biopsy score 0. CONCLUSIONS: Application of the biopsy scoring system to histopathological findings in liver biopsy specimens was very useful in predicting survival of cases of suspected liver disease. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, liver biopsy appears to be the most valuable prognostic technique employed during the investigation of suspected cases of liver disease in mature horses.

Retrospective analysis of historical, clinical, ultrasonographic, serum biochemical and haematological data in prognostic evaluation of equine liver disease.

REASONS FOR PERFORMING STUDY: Results of noninvasive tests of liver disease do not always correlate with the degree of hepatic disease nor outcome of the case. OBJECTIVE: To investigate the prognostic value of data collected using noninvasive tests during the investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: Much of the data gathered during the investigation of suspected hepatopathy cases offers little prognostic guidance and interpretation of such data can be misleading. METHODS: The results from a range of common and noninvasive diagnostic techniques applied in 116 mature horses with suspected liver disease, were assessed for their ability to predict survival within a 6 month period. RESULTS: A significantly poorer prognosis was found in association with clinical signs suggestive of liver disease, presence of hepatic encephalopathy, ultrasonographic abnormalities, increased serum globulins, increased total bile acids (TBA), increased alkaline phosphatase (AP), increased gamma-glutamyl transferase (gammaGT), erythrocytosis, leucocytosis, low serum albumin and low serum urea. Additional significant novel findings of interest included an association between increased plasma fibrinogen and low serum creatinine concentrations with nonsurvival in cases of liver disease, an association between raised serum concentrations of AP and gammaGT with biliary hyperplasia and also an association between hepatic fibrosis, haemosiderosis and biliary hyperplasia with ultrasonographically detected hepatic abnormalities. CONCLUSIONS: The most useful noninvasive prognostic test in cases of suspected liver disease in mature horses is the severity of clinical signs. Other data may be of some limited prognostic value. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, the findings should at least be considered when prognosis is based on similar criteria.

Study of the clinical effects of postoperative parenteral nutrition in 15 horses.

Several clinical variables were compared in two groups of 15 horses recovering from resection and anastomosis of a strangulated small intestine; 15 were treated with parenteral nutrition and 15 were starved routinely. There was some evidence that parenteral nutrition had a short-lived adverse effect on both the catheter sites and gastric emptying, but there were no marked adverse clinical effects and no evidence of any improvement in the horses' condition.