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.

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

Master of Science / Department of Biomedical Sciences / Warren Beard / Reasons for performing study: Caecal acidosis is a central event in the metabolic cascade that occurs following grain overload. Buffering the caecal acidosis by enterally administered sodium bicarbonate may be beneficial to affected horses. Objectives: To determine the effect and duration of enterally administered sodium bicarbonate (NaHCO[subscript]3) on caecal pH in healthy horses. Study design: Prospective controlled study using normal horses with caecal cannulas Methods: 9 horses previously fitted with a caecal cannula. 6 horses received 1.0 g/kg bwt NaHCO[subscript]3 via nasogastric tube and 3 control horses were given 3 L of water via nasogastric tube. Clinical parameters, water consumption, venous blood gases, caecal pH, faecal pH and faecal water content were measured at 6 hour intervals over a 36 hour study period. Results: Horses that received enterally administered NaHCO[subscript]3 had a significantly increased caecal pH that lasted the duration of the study. Treated horses increased their water intake, developed metabolic alcalemia, significantly increased sodium concentrations and significantly decreased potassium concentrations. Conclusions and potential relevance: Enterally administered NaHCO[subscript]3 may be beneficial in buffering the caecal acidosis that occurs following an acute carbohydrate overload

Laminitis is one of the most common multifactorial diseases of horse limbs. Recent studies show that 90% of this disease is due to endocrinological diseases such as Cushing's syndrome or equine metabolic syndrome. In available literature, there is not much relevant information on the relationship of laminitis to hematological and biochemical blood parameters. This diploma thesis "Changes in selected blood parameters of horses" evaluates biochemical and haematological blood parameters in connection with laminitis in 27 sick horses. It statistically compares the differences in blood parameters between healthy and sick horses.

Orientador: José Corrêa de Lacerda Neto / Coorientador: Rita de Cássia de Lima Sampaio / Banca: Lina Maria Wehrle Gomide / Banca: Deborah Penteado Martins Dias / Resumo: Diferentes estudos foram realizados buscando relacionar a ocorrência de distúrbios gastrointestinais e a manifestação da laminite. Neste contexto, destacou-se a participação de processos inflamatórios e sua relação com a expressão de metaloproteinases (MMP), que sabidamente promovem a degradação da membrana basal no tecido laminar do casco. O presente estudo objetivou avaliar a expressão das MMP-2 e MMP-9 no tecido laminar, bem como sua integridade celular por avaliação histológica, em equinos submetidos à obstrução intraluminal do cólon menor. Oito equinos adultos hígidos foram avaliados. Realizou-se laparotomia e obteve-se obstrução do cólon menor utilizando-se balão de vinil inserido no lúmem intestinal. O balão foi inflado à pressão de 80 mmHg e a obstrução foi mantida por 4 horas, simulando a presença de um enterólito. Foram realizadas biópsias pelo acesso transmural-dorsal para obtenção de tecido laminar podal em três momentos (T): antes da obstrução (T0), imediatamente após desobstrução (T4) e após 72 horas da desobstrução (T72). As amostras foram submetidas à análise zimográfica e histológica. Segundo a zimografia, não observou-se alterações nos valores de MMP-2 e MMP-9 em T4 e T72. Entretanto, observou-se o surgimento de lesões no tecido laminar analisado microscopicamente (T4 e T72). Concluiu-se que as alterações inflamatórias decorrentes da obstrução do cólon menor levaram ao surgimento de lesões no tecido laminar podal. Porém, a inflamação promovida não foi suficiente para ocasionar alteração na expressão das MMP-2 e MMP-9 no tecido avaliado / Abstract: A number of studies have been conducted in order to relate gastrointestinal disorders and laminitis occurrence in horses. In this issue, special attention has been given to the role of inflammatory process and its relation with metalloproteinase (MMP) expression, which may lead to basement membrane degradation in the hoof laminar tissue. The purpose of the present study was to investigate MMP-2 and MMP-9 expression in the laminar tissue, as well as its cellular integrity assessed histologically, in horses subjected to intraluminal obstruction of the small colon. Eight healthy horses were evaluated. A laparotomy was performed and the small colon obstruction was achieved by the use of a vinyl ball inserted into the intestinal lumen. The ball was insufflated at 80 mmHg and the obstruction was sustained for 4 hours, mimicking an enterolithiasis. Through a dorsal-transmural access, podal laminar tissue samples were biopsied at three time-points: before obstruction (T0), immediately after desobstruction (T4) and after 72 hours from desobstruction. Samples were subjected to zymographic and histological analysis. The zymography did not reveal changes in MMP-2 and MMP-9 values at T4 and T72. However, microscopic evaluation showed laminar tissue lesions (T4 and T72). In conclusion, the inflammatory changes followed by the small colon obstruction led to podal laminar tissue damage. However, the inflammation was not sufficient to induce changes in MMP-2 and MMP-9 expression in the evaluated tissue / Mestre

Orientador: Celso Antonio Rodrigues / Banca: José Renato Junqueira Borges / Banca: Ana Liz Garcia Alves / Resumo: Este estudo objetivou correlacionar a acidose ruminal subaguda com a ocorrência de laminite subclínica em vacas leiteiras de alta produção, provenientes de um rebanho comercial. Foram avaliadas 200 vacas holandesas de alta produção leiteira, divididas em quatro grupos, sendo estes estabelecidos a partir da produtividade diária. Os animais foram submetidos ao exame clínico, coleta de líquido ruminal por meio de sondagem oral, hemogasometria, termografia digital e pedometria. Os valores do pH e prova de redução pelo azul de metileno do líquido ruminal, produtividade diária, número de passos, temperatura dos dígitos e membros, hemogasometria (PCO2, TCO2, HCO3, SO2 e EB) foram determinados e correlacionados estatisticamente. Nenhum animal avaliado apresentou alterações no pH ruminal e hemogasometria. A análise termográfica revelou maior temperatura nos dígitos mediais dos membros torácicos direito (59,4%) e esquerdo (55,2%). Enquanto que nos membros pélvicos, observou-se que a temperatura foi maior nos dígitos laterais direito (65,9%) e esquerdo (64,9%). Notou-se que 84,9% dos animais apresentaram os membros pélvicos mais quentes que os membros torácicos. Observou-se ainda que quanto maior foi à temperatura registrada, menor foi a produção de leite (p<0,01). As alterações encontradas pela termografia digital permitiram a triagem das vacas acometidas por afecções podais. Contudo, a identificação destas afecções somente foi possível mediante exame clínico específico dos dígitos. A pedometria foi efetiva, pois se observou redução no número de passos devido à dor, correlacionada a menor produção leiteira. Os fatores de riscos como o concreto abrasivo, instalações inadequadas, associados à acidose ruminal subaguda, não diagnosticada pela metodologia utilizada, foram os principais causadores das afecções podais. A laminite subclínica acometeu primariamente as vacas ... / Abstract: This study aimed to correlate the subacute ruminal acidosis with the occurrence of subclinical laminitis in high producing dairy cows, of a commercial herd. We assessed 200 Holstein cows with high milk production, they were divided into four groups, which was established high milk production from the daily productivity. The animals were submitted to clinical examination, collection of rumen fluid by oral gavage, blood gas analysis, thermography and digital pedometrics. Values of pH and evidence of reduction of methylene blue rumen, daily productivity, number of animals steps, the temperature digits and limbs, blood gas (PCO2, TCO2, HCO3, SO2 and EB) all those had been determined and statistically correlated. No have assessed animals that showed changes in ruminal pH and blood gas analysis. The thermographic analysis revealed higher temperature in the medial forelimb digits right (59.4%) and left (55.2%). While in the hind limbs was observed that the temperature was bigger in the right side digits (65.9%) and left (64.9%). It had been noted that 84.9% of the animals had pelvic limbs warmer than the forelimbs. It had been also observed that the higher the temperature recorded was, the lower was the milk yield (P <0.01). The abnormalities were found by digital thermography allowed in cows triage affected by foot problems affections. Therefore, these disorders identification were only possible through clinical examination of specific digits. The pedometrics was effective, because we had observed a reduction number of animals steps because of the pain, and then correlated with lower milk production. Risk factors as abrasive concrete, inadequate accommodation, subacute ruminal acidosis associated with undiagnosed by the methodology used, they were the main causes of affections podal. The subclinical laminitis occurred primarily cows from the herd, considering the multifactorial etiology from this affections, ... / Mestre

Background: A number of conditions are associated with laminitis in horses, such as corticosteroid administration, equine Cushing’s syndrome and equine metabolic syndrome. In common to these conditions are disturbed glucose and insulin metabolism and importantly, the development of insulin resistance. Insulin resistance is seen when the insulin-responsive glucose transport proteins (GLUTs) that are largely responsible for glucose disposal in tissues such as skeletal muscle begin to fail. Aims: 1. The aim of this thesis was to determine the relationship between disturbed carbohydrate metabolism and laminitis in horses and test the hypothesis that impaired glucose uptake in the hoof lamellae is involved in the pathogenesis of laminitis, by investigating the mechanisms that control glucose uptake in hoof lamellae. 2. Having determined that glucose uptake in the hoof occurs independently of insulin, the hypothesis was re-examined, with the aim of determining the effects of hyperinsulinaemia, in the absence of cortisol manipulation, dietary modification, or hyperglycaemia on lamellar integrity in the hooves of healthy ponies. Methods: 1. An in vitro lamellar explant model was used to investigate the effects of insulin on glucose uptake in hoof lamellae. The β-adrenoceptor (β-AR) populations in equine lamellae were characterised using the radioligand binding technique and the glucose uptake response to stimulation with a potent β-AR agonist (l-isoprenaline) in the hoof was investigated. Further, the mRNA expression of both GLUT1 and GLUT4 in lamellar tissue was determined via PCR analysis. 2. Clinically healthy ponies were randomly allocated to either treatment (n = 5) or control (n = 4) groups. Treatment involved a prolonged (72 h) euglycaemic-hyperinsulinaemic clamp technique while control ponies received an equivalent volume infusion of 0.9% saline. Ponies were euthanased at the onset of Obel grade 2 laminitis (treatment) or at 72 hours (controls). Lamellar tissue was obtained from all ponies and analysed via gelatin zymography, histopathology and immunohistochemistry. Results: 1. The predominant β-AR subtype in lamellae was the β2-AR (90%), with β1-AR expression less abundant (10%). Furthermore, stimulation with l-isoprenaline inhibited glucose uptake by up to 30% in lamellae. This is consistent with the known effects of isoprenaline in other species and tissues, and supports the hypothesis that stimulation with adrenaline could result in reduced glucose uptake in hoof lamellae, suggesting a possible mechanism by which impaired glucose metabolism may be involved in laminitis. Glucose uptake in lamellar explants was not affected by either acute (10-120 min) or long-term (24 h) stimulation with porcine insulin. These results do not support a glucose deprivation model for laminitis, in which reduced insulin sensitivity results in impaired glucose uptake in the hoof. Further, exposing lamellar explants to increasing concentrations of glucose resulted in a GLUT saturation point indicative of predominantly insulin-independent GLUT1 proteins. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue and weak in skeletal muscle in control animals and was similar in ponies with insulin-induced laminitis. In contrast, mRNA expression of the insulin-dependent GLUT4 was strong in skeletal muscle and was either absent or barely detectable in coronary band and lamellar tissue. These results are consistent with a predominantly GLUT1-mediated glucose transport system, and suggest that it is unlikely that the GLUT4 gene plays a substantial role in glucose uptake in the hoof. 2. Treated ponies all developed laminitis within 55.4  5.5 hours, while no laminitis occurred in control ponies. Insulin-induced laminitis indicated elongated, collapsed secondary epidermal lamellae (SEL), as well as enlarged and increased numbers of basal cell nuclei, mitotic figures and increased keratinisation in SELs. However, in contrast to the histological appearance of tissue obtained from oligofructose-induced laminitis, basement membrane disintegration was not a major finding. There was no increase in either active or latent forms of MMP-2 or -9 in lamellar homogenates obtained from ponies with insulin-induced laminitis compared with controls, except in one pony that demonstrated increased MMP-2 activity, which was euthanased five days after developing laminitis. Conclusions: Collectively, the research outlined in this thesis indicates that glucose uptake in the equine hoof is independent of insulin, utilising a predominantly GLUT1-mediated glucose transport system. However, a more complete understanding of the metabolic processes within equine hoof lamellae would involve further characterising the effects of other hormones, such as cortisol and IGF-1 on glucose transport, as these results indicate a possible role for adrenaline in reducing glucose uptake in lamellar tissue. Nevertheless, the results presented in this thesis do not support a glucose deprivation model for laminitis, in which reduced insulin sensitivity results in impaired uptake of glucose into hoof lamellar tissue. This research demonstrates that prolonged hyperinsulinaemia induces laminitis in normal ponies, independent of changes in blood glucose concentration. Preliminary studies investigating the pathophysiology of insulin-induced laminitis suggest the possible involvement of increased cellular proliferation and inflammation, rather than MMP activation. However, the exact mechanism by which hyperinsulinaemia induces laminitis awaits further investigation.

No description available.

It is well established that failure of the lamellar basement membrane (BM) occurs during the development of equine laminitis. This is due to loss of the crucial BM components; laminins and collagens along with loss of attachment complex, the hemidesmosome, of the basal cell to the underlying BM. Previous studies have suggested that Ln-332 may be the primary protein involved in lamellar failure. However, the details of the progression and mechanism involved in this pathology are not currently fully known. This thesis aimed to refine the proteolytic processes and mechanisms occurring during the development of oligofructose induced laminitis. Through the use of novel temporal lamellar biopsies obtained during the development of laminitis induction, it was determined that loss of both Ln-332 and collagen type IV occurs as early as 12 hours post induction. This loss of reactivity initially occurred in a focal pattern with increasing loss as the disease progressed in severity. At the later stages of laminitis, separation of the basal epithelial cell from the dermal tissue was also observed, however at these points the BM still appeared intact. This suggests that more than one mechanism may be involved in disease pathology; one resulting in fragmentation of the BM while a second results in loss of the cell attachment allowing the intact BM to slip away. Immunohistochemical analysis of lamellar tissue revealed a unique pattern of reactivity for the Ln-332 γ2 antibody D4B5, in which no reactivity was observed in normal lamellar tissue, yet the epitope recognized by this antibody becomes apparent during disease development. This initially led to the hypothesis that cleavage of the γ2 subunit and the release of biologically active fragments may occur. However, at the molecular level, no γ2 fragments were detected by western blotting. In vitro cleavage of partially purified equine Ln-332 revealed that both MMP-2 and MT1-MMP were able to process the molecule to produce fragments corresponding to the biologically active counterparts. This suggests that the change in reactivity with this antibody may be due to other mechanisms such as decreased interaction of Ln-332 with other BM components resulting in loss of structural stability of the BM allowing for a change in the orientation of Ln-332. Increased MMP-2, MMP-9 and MT1-MMP expression has been demonstrated in laminitis and this was assumed to be the causative agent resulting in tissue destruction and failure. However, work in this thesis found no increase in gene expression of MMP- 2 and MT1-MMP, as well as no activation of pro MT1-MMP. Increased pro MMP-9 gene and protein expression was observed early in the disease progression yet no MMP- 9 activation occurred. Additionally, activation of MMP-2 was found to occur late in laminitis progression at least 12 hours following BM degradation, thus MMP-2 activation is a secondary effect of laminitis development. Thus, other proteases are expected to result in BM processing. Gene expression of the metalloprotease ADAMTS-4, was observed to increase early during laminitis development, suggesting this is a putative factor involved in intensifying the degradation of the lamellar BM. Work in this thesis also revealed that both Ln-332 and collagen type IV are widely distributed throughout organs in the equine body and localized primarily to BM structures. A novel finding of this thesis is that not only does BM degradation occur in the lamellar BM, it also occurs in organs remote from the hoof. At both the onset of lameness and the acute phase of laminitis, fragmentation of both Ln-332 and collagen type IV also occurs in both the skin and stomach. Recent studies have indicated that both leukocyte emigration and increased cytokine expression occurs in the lamellar tissue during laminitis. Work in this thesis added to this knowledge as leukocyte infiltration into the lamellar tissue occurs early during oligofructose laminitis induction as does increased IL-6 gene expression. Overall, work conducted in this thesis has added to the knowledge of the events occurring during laminitis development. Even though the complete mechanism of tissue destruction and lamellar failure was not established, the progression of events is now more clear in that BM degradation is one of the first events to occur, while MMP-2 activation occurs secondarily. Thus, other mechanisms must be at work early during laminitis development and discovering what they are must remain a research priority for the realization of effective therapeutic strategies.

No description available.