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The present studies were conducted to determine the effects of obesity on insulin resistance, inflammatory state and risk for laminitis, and the effectiveness of exercise training to reduce obesity and insulin resistance in equids. Practical methods of assessing adiposity were developed and verified, including a condition scoring system for neck crest adiposity and morphometric measurements for generalized (girth:height) and localized (neck circumference:height) adiposity. Evaluation of 74 and 57 pony mares in March of two consecutive years resulted in the identification of predictive tests for incipient pasture-associated laminitis, including hyperinsulinemia (> 32 mU/L), hyperleptinemia (> 7.3 ng/mL), and generalized (body condition score ≥ 7) and localized (cresty neck score ≥ 4) obesity. Induction of obesity in 13 Arabian geldings by 4 months of overfeeding resulted in compensated insulin resistance (minimal model analysis) with hyperinsulinemia and hyperleptinemia. Although lipid concentrations (nonesterified fatty acids and triglyceride) decreased on a high concentrate diet, they did not differ before and after weight gain. The resulting obesity-induced insulin resistance was accompanied by an increase in chemokine (monocyte chemoattractant protein [MCP]-1, MCP-2, interleukin [IL]-8) but not inflammatory cytokine (tumor necrosis factor [TNF]α, IL-1β, IL-6) mRNA expression in subcutaneous adipose tissue. Additionally, there was a decrease in plasma TNFα protein concentration with weight gain. By using 12 of the obese, insulin resistant Arabian geldings (8 exercised, 4 control) it was demonstrated that 8 weeks of moderate intensity exercise training reduced adiposity (4% reduction in body weight, 35% reduction in fat mass) without affecting glucose and insulin dynamics or plasma hormone and lipid concentrations. Collectively, these studies demonstrate the impact obesity has on metabolism and risk for laminitis in equids, and that exercise training may provide an effective countermeasure for the reduction of obesity. / Ph. D.

Two times are critical for the horse ¾ the first few days of its life and the last few moments of a race. Vitamin E is critical in regard to immune competence in the first and antioxidant status in the latter. Studies conducted at the Middleburg Agricultural Research and Extension (MARE) Center include the development of horse feeds that replace sugar and starch with fat and fiber. The previous fat source of the pasture supplement under development was corn oil, which contains much vitamin E, was replaced with a cereal by product, which contains relatively little. Vitamin E has been studied in horses to a limited degree but not in grazing Thoroughbreds, thus the MARE Center gave me the opportunity to study vitamin E in Thoroughbred mares and foals. Middleburg is located in the Blue Ridge Mountains on Northern Virginia close to the site of one of the toughest endurance races in the world. This allowed me the opportunity to study vitamin E and antioxidant status in the horse during endurance racing. Initial studies of vitamin E supplementation to mares during the last trimester of gestation were disconcerting with no changes in serum concentrations of a-tocopherol (vitamin E). Studies conducted during the post-partum period revealed evidence of responses to vitamin E supplementation, as increased a-tocopherol concentrations were observed in mares' milk and in foal serum. Foals are born with virtually no circulatory antibodies and the supplementation of a synthetic form of vitamin E to mares demonstrated an increased passive transfer of immunoglobulins to foals. Natural vitamin E has shown a greater bioavailability than synthetic forms, in other species and was tested here at the MARE Center on mares. A greater passive transfer of immunoglobulins was observed with natural vitamin E supplementation compared with the synthetic forms, with immunoglobulin M concentrations in foal serum remaining higher for a longer period after birth compared to foals of non-supplemented mares. The transfer of a-tocopherol via the milk was also increased in concentration and duration in mares supplemented with natural vitamin E. Bioavailability of five oral forms of vitamin E (3 natural and 2 synthetic) were tested and one natural form was also administered intravenously so that clearance of vitamin E could be used to calculate the efficiency of absorption of the oral forms. Efficiency of absorption for oral treatments was not determined because of the slow turnover time of the intravenously administered vitamin E, which confounded all subsequent baseline serum a-tocopherol concentrations. Of the salvageable data, serum a-tocopherol concentrations were higher in grouped treatments at 9 and 12 h post dosing. Lipid fractions revealed possible insufficient absorption of the oral doses of vitamin E and possibly tissue saturation following intravenous doses of vitamin E. Serum concentrations of a-tocopherol were generally higher following natural forms of oral vitamin E administration. As vitamin E is the most important antioxidant in cells, it is often supplemented to endurance horses competing in 80 and 160 km races. Vitamin E protects lipid cell membranes from peroxidation by free radicals, which are increased during strenuous exercise resulting in oxidative stress. The antioxidant status of horses is severely tested during endurance racing and so a study was conducted to monitor changes in circulating antioxidants during three endurance races. Interesting novel findings in the horse were the maintenance of serum a-tocopherol and the depletion of erythrocyte glutathione and plasma ascorbate during two 80 km and one 160 km races. Associations were found between increased muscle cell enzyme leakage and decreased antioxidant status during endurance exercise and although associations do not prove a causation of oxidative stress, they do provide motivation to search for a cause and it is tempting to propose that oxidative stress damaged muscle cell membranes in endurance horses. Further, these findings propose a connection between muscle cell damage and a new form of exertional rhabdomyolysis (ER) that has been observed in endurance horses, where oxidative fibers are damaged compared to the typical glycolytic fiber damage associated with known forms or ER. An increased understanding of vitamin E utilization in the horse will improve the health and welfare of all horses, but especially newborn foals and the athletic endurance horses. / Ph. D.

Computed tomography (CT) is becoming more available as a diagnostic tool in the evaluation of the equine skull and brain. Objectives of this study were: 1) refine a CT protocol for evaluating the equine pituitary gland, 2.) define the CT anatomy of the pituitary region, 3.) determine a set of normal values for the pituitary dimensions (length, width, height, volume and weight), 4.) refine CT techniques for measuring pituitary size. Horses were scanned using 10x10mm, 10x5mm, 4x4mm and 4x2mm slice thickness and interval combinations. The pituitary glands were removed immediately after CT and gross measurements were performed. CT measurements were compared with gross pituitary measurements using analysis of variance (ANOVA) in a randomized block design. Accuracy percentages were also calculated using gross measurements as the known value. Mean dimensions of the histologically normal pituitary glands were: length 21.07mm, width 21.62mm, height 9.78mm and volume 2.66cm³. The weights ranged from 1.7g to 3.4g with a mean of 2.6g. Computed tomographic measurement analysis demonstrated that the 10mm slices were the most accurate way to estimate the length of the gland. The 4mm slices yielded the highest accuracy values for width, height and volume of the pituitary gland. The volume was underestimated by all interval and slice thickness combinations performed by CT. No evidence of an overlap effect was identified for any of the dimensions. Our findings indicated that contrast-enhanced CT is an accurate technique for estimating pituitary linear dimensions. Three-dimensional CT volumetry may not be an accurate method for estimating pituitary volume. / Master of Science

The acceptability and digestibility of mixtures containing selected fats of animal, vegetable and blended sources were studied. Ten fat containing mixtures were fed to Saddlebred horses in cafeteria style palatability trials and ranked according to preference. Preference ranking of the fats from highest to lowest is as follows: corn oil, blend no. 1, cottonseed oil, inedible tallow and fancy bleached tallow. When the most preferred fats from each classification (animal, vegetable and blended) were offered in single mixture palatability trails, no significant difference was observed in consumption. In Digestion Experiment I three fats, corn oil, blend no. 3 and inedible tallow, were added to an alfalfa and grain diet at the 102 level. The apparent digestibility of fatty acids in corn oil when calculated by difference, was higher than an inedible tallow or blend no. 3. The apparent digestibility of energy in corn oil calculated by difference, was 95.4%. However, this value was not different from that of inedible tallow and blend no. 3. The addition and kind of fat did not affect the apparent digestibility of acid detergent fiber or apparent absorption of minerals. Blood serum levels of total fatty acids, calcium, inorganic phosphorus, magnesium and glucose were not affected by the addition of the fats. However, serum cholesterol levels were increased (P <.05) by addition of fat. In Digestion Experiment II, feeding four fats (corn oil, peanut oil, inedible tallow and blend no. 3) at two levels (7.5 and 152%) was studied. When the apparent digestibility of fatty acids in the diet was calculated by difference, the average values were higher for corn oil and peanut oil, but the differences were not significant. The true digestibility of fatty acids showed all fat sources and levels were digested similarly. The apparent digestibility of diet energy was increased by the addition of the fats. The energy of diets containing corn oil had higher (P <.05) digestibilities than those containing inedible tallow or blend no. 3. As in Experiment I, there was no difference in energy digestibility of the added fats when calculated by difference. There was no depression of calcium absorption due to kind or level of added fat. Apparent absorption of phosphorus was not affected by type of level of added fat. Apparent absorption of magnesium was higher (P <.05) for the basal diet and those diets containing blend no. 3. Blood serum levels of total fatty acids, calcium, inorganic phosphorus, magnesium, cholesterol and glucose were not affected by the addition or type of fat. / Ph. D.

Carolina Rinse (CRS) has been shown to be effective in decreasing vascular permeability and neutrophil infiltration in reperfused equine small intestine. The objective of this study was to show that CRS applied topically and intraluminally could prevent immediate reperfusion injury after low flow ischemia or distention in the equine jejunum. Materials & Methods: Two groups of 5 horses were used to evaluate CRS treatment after low-flow ischemia (Group 1) and intraluminal distention (Group 2) of distal jejunum. Mesenteric blood flow, osmotic reflection coefficient (ORC), wet weight to dry weight ratios (WW/DW), and neutrophil accumulation in the serosa were measured. ORC is defined as the lymph protein concentration to plasma protein concentration ratio subtracted from one (1- Cl / Cp) at maximal lymph flow. The ORC from baseline values and at 60 minutes after initiating reperfusion was compared between Groups 1 and 2. Pair wise comparisons were made for mesenteric blood flow, tissue volume, neutrophil number, and WW/DW proximal control and CRS treated jejunal segments were made using a Mann Whitney U test (P< 0.05). Results: The mean ORC of bowel treated topically and intraluminally with CRS was similar to that recorded in normal bowel or ischemic intestine treated with CRS by arterial perfusion. The ORC after distention and decompression increased and was similar to that reported in untreated intestine. The WW/DW after both ischemia and distention increased compared to the proximal control segments. There was no difference in neutrophil number in either ischemic or distended intestine compared to the proximal control segments. Discussion: Carolina CRS was effective in preventing alterations in microvascular permeability during reperfusion afterischemia but not distention. Neutrophil migration curtailed in both groups suggesting that combined topical and intraluminal application of CRS to ischemic intestine may reduce the acute inflammatory responses during reperfusion thereby decreasing complications after ischemia or distention. / Master of Science

The objective of these studies was to contribute to the understanding and assessment of thyroid function in horses. The first study evaluated methods of assessing thyroid function in horses, including validation of an enzyme immunoassay (EIA) for measuring equine thyroid hormones and development and assessment of a thyrotropin releasing hormone (TRH) response test. Our data indicated that EIA is an acceptable method for the measurement of total (T) and free (F) thyroxine (T4) and triiodothyronine (T3) in equine plasma. Its measurements are not equivalent to values obtained by radioimmunoassay (RIA), but they can be calibrated to predict corresponding RIA values. A protocol was developed for TRH response tests involving administration of 1 mg TRH intravenously, with blood sample collection immediately before, 2.5, 5.0, and 24 h after administration. Analysis of plasma TT4, FT4, TT3, and FT3 revealed that the magnitude of hormone response was best approximated by the area under the curve of hormone plotted against time and by the absolute change in thyroid hormone concentration. Baseline concentrations, peak concentrations, and percent of baseline values were not as well able to predict the magnitude of hormone response. The second study assessed the effects of exercise and feed composition on thyroid status. Thirteen mature Arabian geldings, adapted to either a high sugar and starch (SS) or high fat and fiber (FF) feed, underwent 15 wk of exercise training followed by a treadmill exercise test. The TRH response tests performed before training, after training, and the morning after the exercise test revealed that the exercise test decreased the TT4 and FT4 response, whereas feeding of high levels of sugars and starches increased the response of TT3 and FT3. During the first four weeks of training, increased TT4 and FT4 concentrations occurred simultaneously with increased nonesterified fatty acid concentrations, decreased triglyceride concentrations, and increased insulin sensitivity. The increase in TT4 and FT4 may have provided the cellular signaling necessary for increased lipolysis and insulin sensitivity. These metabolic changes facilitate the increases in lipid and carbohydrate metabolism that are needed to fulfill the additional energy requirements of regular exercise. The third study assessed thyroid status in ponies with different laminitic histories. Total T4, FT4, TT3, and FT3 were measured during March and May 2004 in 126 ponies that were categorized as either previously laminitic (PL; n = 54) or never laminitic (NL; n = 72) and evaluated for current laminitis in May (CL; n = 13). Decreased concentrations of TT4 and FT4 were found in PL ponies when compared to NL ponies in March (P = 0.018, 0.020) and May (P = 0.018, 0.001). However, TT4 and FT4 concentrations in CL ponies were not different than concentrations in NL ponies in May (P = 0.82, 0.72), and when retrospectively separated out in March, were not different than NL ponies (P = 0.90, 0.84). Therefore, basal thyroid hormone concentrations are not useful as a predictor or hormonal characteristic of pasture-associated laminitis. The decreased TT4 and FT4 in PL ponies may be an indication of a response or compensation to laminitis and may facilitate the metabolic changes necessary to cope with the disease. / Master of Science

Repeatabilities (t) and heritabilities (h²) for racing time were estimated from data on 7206 2- and 3-year-old standardbred racehorses competing in charted races at six tracks between 1975 and 1976. There were a total of 38,487 records from 2367 sire progeny groups. The data were divided according to gait into two subsets. Trotters only were used to test alternative models proposed to characterize horse’s racing time. Initially, class of race was recognized as a subjective categorization reflective of non-random assignment of horses to races. After extensive investigation, it was concluded that adjustment of horse’s time should include regression on pace (time of the winner). Data were analyzed within track-age-gait subclasses. Intra-horse regression coefficients of time on pace (sec/sec) ranged from .61 ± .08 to .79 ± .05. When records were adjusted for pace, repeatability and heritability estimates ranged from .32 ± .08 to .55 ± .04 and from -.25 ± .22 to .66 ± .15, respectively. Pooled estimates of t and h² were .44 ± .01 and .29 ± .03, respectively. When records were not adjusted for pace, sire, horse and error variance components were inflated by environmental bias (t_pooled = .62: h² pooled = .36). When records were pre-adjusted for pace, the permanent environmental variance appeared smaller compared with the total phenotypic variance than if there was no preadjustrnent for pace. Since genetic progress is based on selection of genetically superior sires and dams, time adjusted for pace should be used for estimation of breeding values and for objective evaluation of an individual horse's real ability to compete against horses having similar abilities. Thus, resulting breeding values should be more accurate dnd biased to a lesser degree than if unadjusted racing times were utilized. / Ph. D.

Equine neutrophils (PMN) play a critical role in inflammatory processes in horses. The objective of this study was to characterize equine PMN function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigger different branches of the PMN activation process that occurs in vivo. Equine PMN were isolated from the whole blood of six clinically normal geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion production were evaluated in assay systems which had previously been established to guantitate PMN function. Dose-response curves for A23187 and PMA were derived for the three functions. Results indicate that equine PMN aggregation and superoxide anion production are more responsive to activation by PMA as the maximum change in percent transmittance and maximum nanomoles of superoxide anion produced following PMA stimulation (60.82% and 10.4nmols/10⁶cells, respectively) were greater than those values stimulated by A23187 (41.5% and 5.2nmols/10⁶cells, respectively). However, degranulation was found to be more responsive to A23187 stimulation (maximum percent degranulation= 56.1%) than to PMA stimulation (maximum percent degranulation= 30.7%). Dose-response curves following A23187 and PMA stimulation revealed that superoxide anion production had the lowest threshold concentration among the three functions. Degranulation had the highest threshold concentration among the three functions for both stimulants. Results indicate that equine PMN functions differ in their dependence on second messengers in the activation pathway. These functions also occur in a dose-dependent manner and differ in the threshold concentrations required for their stimulation. / Master of Science

A marker model of fecal kinetics using chromic oxide (Cr) or ytterbium chloride (Yb) is being developed for grazing horses. The model consists of removal of feces at a constant rate from a single compartment, the prefecal mass. It was tested in experiments on stall-fed horses in the context of digestion balance trials. Following the preliminary work of Holland et al., (1998), three improvements in experimental design were tested. First, the rate constants were determined from both the administration and post-administration curve of the one-compartment model. Second, markers were administered three times a day to reduce diurnal variation in fecal marker concentration. Third, yttrium (Y) and Yb were tested were tested as internal markers, for the estimation of digestibility of hay and supplements, respectively. Eight horses were fed Diet 1 (orchardgrass/alfalfa mixed, OG) or Diet 2 (tall fescue/alfalfa mixed, TF) in Exp.1, and Diet 3 (OG plus fat-and-fiber supplement, OGFF) or Diet 4 (OG plus sugar-and-starch supplement, OGSS) in Exp.2. Balance-marker experiments were conducted for 17 and 20 d, with 7 and 10 d of dietary accommodation in Exp.1 and 2, respectively. Chromic oxide and Yb were administered orally and fecal samples were collected every 8 h for 8 d. Dry matter, Cr, Yb and Y were measured in feeds and feces. In balance experiments, estimates of DMD (D_E) using Y, were determined precisely (SE 1 to 3 %) for hay and hay and supplement diets. Linear relationships, correlations and calibration curves were determined, validating Y as a marker. Mean daily fecal Cr data (C_t) at time t (days) including a delay (d) were fitted to a single exponential, with one rate constant (k), rising to an asymptote (C_a): C_t = C_a - C_a·e^-k(t-d) Diets 1 and 2 had two sets of C_tdata, total collection (a) and fecal grab data (b), and each set was used in model development. Diets 3 and 4 had two sets of C_t data (both using fecal grab data), Cr marker dilution (3Cr and 4Cr) and Yb marker dilution (3Yb and 4Yb). For pooled data, delays of 3 to 6 h (Diets 1a, 1b, 2a and 2b) and delays of 5 to 7 h (Diets 3Cr, 4Cr, 3Yb and 4Yb) gave best fits (highest estimates of R²). The delays introduced to the Cr model for both 3Cr and 4Cr diets did not correspond to the preliminary study (Holland et al., 1998), where a 2 h delay gave the best fit in the model for horses fed hay and supplement. The present estimates may more realistically relate to mouth-to-cecum transport times, because the marker was administered three times a day instead of once, and the initial part of the tracer curve was more precisely defined. The results showed that fecal Cr kinetics could be calibrated precisely (SE 1 to 3 %) to predict fecal DM output of horses fed Diets 1b, 2b, 3a but not 4a. Similarly, fecal Yb kinetics could be calibrated to predict fecal DM output of horses fed Diet 3b but not 4b. The rate constants yielded turnover times (TT) that were longer with hay and supplement diets, than with hay alone, and which contrast with previous findings in the horse. However, the longer TT were similar to slower rates of marker excretion in sheep fed concentrates instead of all-roughage diets, suggesting that the lower fiber content retarded the rate of propulsion of digesta through the digestive tract. For two of the eight models of fecal kinetics, the rate constants of the post-administration curve were not well determined by the data, and rate constants from the administration curve were used. In future experiments, more frequent fecal sample collection during the post-administration period may improve rate constant determination. Improvements in diurnal variation of fecal marker concentration were obtained by dosing three times a day. But discrepancies between Cr and Yb concentration means of diurnal samples and combined samples showed incomplete mixing, the major source of tracer error. Therefore more frequent marker administration and fecal samples should be tested in future experiments to achieve more thorough mixing in the prefecal mass for modeling fecal kinetics, and in the small intestine for estimating digestibility. / Master of Science