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Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:16Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:16Z : No. of bitstreams: 1 2-s2.0-84873867256.pdf: 796974 bytes, checksum: 64f172274307a2abd3e5977743eb1671 (MD5) Made available in DSpace on 2014-05-27T11:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-30 Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. FST calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection. © 2013 Petersen et al. University of Minnesota College of Veterinary Medicine, St Paul, MN Texas AandM University College of Veterinary Medicine and Biomedical Science, College Station, TX Swedish University of Agricultural Sciences Department of Animal Breeding and Genetics, Uppsala University of Kentucky Department of Veterinary Science, Lexington, KY University of California Davis School of Veterinary Medicine, Davis, CA Equine Analysis, Midway, KY University Estadual Paulista Department of Veterinary Clinical Science, Botucatu-SP University College Dublin School of Veterinary Medicine, Dublin University of Azores Institute for Biotechnology and Bioengineering Biotechnology Centre of Azores, Angra do Heroísmo University of Veterinary Medicine Hannover Institute for Animal Breeding and Genetics, Hannover University of Perugia Faculty of Veterinary Medicine, Perugia Animal Health Trust, Lanwades Park, Newmarket, Suffolk French National Institute for Agricultural Research-Animal Genetics and Integrative Biology Unit, Jouy en Josas University of Sydney Veterinary Science, New South Wales Nihon Bioresource College, Koga, Ibaraki University of Helsinki Faculty of Veterinary Medicine, Helsinki University College Dublin College of Agriculture, Food Science and Veterinary Medicine, Belfield, Dublin University of Bern Institute of Genetics, Bern Institute of Cancer Research Breakthrough Breast Cancer Research Centre, London Royal Veterinary College Comparative Neuromuscular Diseases Laboratory, London Swiss National Stud Farm Agroscope Liebefeld-Posieux Research Station, Avenches Norwegian School of Veterinary Science Department of Basic Sciences and Aquatic Medicine, Oslo Animal DNA Diagnostics Ltd, Cambridge Laboratory of Racing Chemistry Department of Molecular Genetics, Utsunomiya, Tochigi

Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332-764) U/L for homozygotes, 301 (222-377) U/L for heterozygotes and 260 (216-320) U/L for controls, and mean (+/- SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/-92) U/L and for controls, 311 (+/-64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses.

Reasons for performing study Equine cervical osteoarthritis is a common disease known to contribute to both neck pain and cervical vertebral compressive myelopathy. There are no published studies demonstrating the usefulness of latero-oblique radiography as a diagnostic tool for cervical osteoarthritis. Objectives To determine the sensitivity, specificity and positive and negative predictive values of latero-oblique radiography as a diagnostic tool for cervical articular process joint osteoarthritis. Study design Prospective cadaver study. Methods Latero-oblique radiographs and CT images were collected, and post mortem examinations performed, on 27 cadaver necks from Thoroughbred-type horses of various ages and sexes. Two equine clinicians independently reviewed each set of images for the presence of osteoarthritis. The authors, under the guidance of a veterinary pathologist, reviewed all articular process joints for bony changes indicative of osteoarthritis. Results The prevalence of osteoarthritis identified on CT images was 57.9% (Assessor A) and 23% (Assessor B). The prevalence of lesions identified on post mortem examination was 25.6%. Latero-oblique radiography showed a low sensitivity for identifying osteoarthritis when compared to both CT imaging (6.3–16.1%) and post mortem examination (2.2–4.4%). However, it showed a high specificity when compared to both CT imaging (92.1–97.8%) and post mortem examination (87.2–95.0%). The positive predictive value for identifying osteoarthritis was moderate (40.9–80.0%) when compared to CT imaging and poor (4.2–18.2%) when compared to post mortem examination. The negative predictive value was moderate when compared to both CT imaging (42.9–76.3%) and post mortem examination (78.0–79.8%). Conclusions Latero-oblique radiography has low sensitivity, but high specificity for the detection of cervical osteoarthritis when compared to both CT imaging and post mortem examination. Further investigation comparing the sensitivity, specificity and positive and negative predictive values of latero-oblique radiography vs. laterolateral radiography would determine whether taking latero-oblique radiographs, which can be more difficult to obtain and interpret, is necessary for the diagnosis of cervical osteoarthritis in practice. Ethical animal research: Ethical committee oversight not currently required by this congress: The study was performed on material obtained from an abattoir. Sources of funding: None. Competing interests: None.

SPINAL ataxia is a common presenting sign in horses with neurological disease in the UK and in most cases the signs are attributed to spinal cord compression. This report describes an unusual, but perhpas overlooked degenerative cause of spinal ataxia in horses in which diagnosis was confirmed at

Objective—To investigate the influence of simulated contraction of the cricoarytenoideus dorsalis (CAD) muscle on the 3-D motion of the arytenoid cartilage. Sample Population—5 larynges from equine cadavers. Procedures—Serial computed tomographic scans of each larynx were conducted at 7 incremental forces simulating contraction of medial, lateral, and combined bellies of the left CAD muscle. Three-dimensional reconstruction of radiopaque markers placed at anatomic landmarks on the left arytenoid and cricoid cartilages enabled quantification of marker displacement according to a Cartesian coordinate system. Rotation (roll, pitch, and yaw) of dorsal and ventral arytenoid planes was calculated relative to a plane formed by the coordinates of 3 markers on the cricoid cartilage by use of Euler angles. Results—Displacement and rotational data showed that rocking motion occurs throughout arytenoid abduction and most of the rotational component is attributable to pitch; greater pitch was associated with action of the lateral belly. Roll of the ventral arytenoid plane was principally associated with action of the medial belly, which counteracted the tendency of the arytenoid cartilage to rotate medially into the rima glottidis lumen. The distance between markers on the arytenoid cartilage was not constant during contraction because of slight deformation of the corniculate process of the arytenoid cartilage, therefore indicating that the arytenoid cartilage is not a rigid body during abduction. Conclusions and Clinical Relevance—Arytenoid cartilage abduction was dependent on the rocking motion elicited by the lateral belly of the CAD muscle; therefore, laryngoplasty suture placement should mimic the action of the lateral, rather than the medial, muscle belly. (Am J Vet Res 2010;71:1003–1010)

BACKGROUND\ud \ud Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot".\ud \ud METHODOLOGY/PRINCIPAL FINDINGS\ud \ud Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression.\ud \ud CONCLUSIONS/SIGNIFICANCE\ud \ud Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

HERNIATION through the inguinal ring is an uncommon complication of castration. Herniation has been reported to occur up to 12 days after castration but usually occurs within hours ([Hutchins and Rawlinson 1972][1], [Boussauw and Wilderjans 1996][2]). Draught breeds and Andalusian horses may be more

Reasons for performing study: The extent to which variability affects endoscopic grading of arytenoid cartilage movement is uncertain. Objective: To determine the observer and within horse variability of grading arytenoid cartilage movement in horses during resting endoscopic examination, using a 7-grade system. Methods: Endoscopic recordings of the upper respiratory tract made at rest in 270 draught horses were reviewed independently by 2 veterinarians to assess interobserver variability when scoring horses' laryngeal function with a 7-grade system. Grading was repeated by both examiners in 80 randomly selected recordings in order to assess intraobserver variability. In 120 horses, endoscopy was repeated after 24-48 h, with videos graded by both veterinarians to assess intrahorse variability. Results: The mean weighted K statistic for concordance within examiners was 0.867, with a mean intraobserver agreement of 76.3%. The weighted kappa statistic for concordance between the 2 examiners was 0.765, with an interobserver agreement of 63.1%. Of the horses receiving 2 endoscopic examinations, the same grade was assigned to 57.1% of horses at the second examination, when effects resulting from interobserver variability were removed. The mean weighted K statistic for concordance between the grade assigned at first vs. second examinations was 0.588, indicating only moderate agreement. Conclusions and potential relevance: Intra- and interobserver reliability of resting endoscopic grading of arytenoid cartilage movement using a 7-grade system was high when examinations were conducted by experienced veterinarians. However, there was moderate daily intrahorse variability, suggesting that results of resting endoscopic examinations performed on a single day should be interpreted with caution.

REASONS FOR PERFORMING STUDY Muscle biopsy is increasingly used in equine veterinary practice for investigating exertional, inflammatory or immune mediated myopathies and unexplained muscle atrophy. Although formalin-fixed samples are often used, for complete evaluation, fresh-frozen tissue is required. Freezing muscle in veterinary practice is impractical: samples sent to specialist laboratories for processing are therefore susceptible to delays, potentially leading to artefact and compromising histological interpretation. HYPOTHESIS Altered temperature, duration and hydration status influence the severity of storage-induced artefact in equine muscle. METHODS Skeletal muscle obtained immediately post euthanasia was divided into 6 independent samples from each of 8 horses. One sample per horse was frozen immediately in isopentane precooled in liquid nitrogen. Additional samples were stored in conditions designed to mimic possible situations encountered in practice, including increased storage times, temperature and hydration status. Following storage, stored samples were frozen as before. Cryosections were stained using haematoxylin and eosin and ranked for artefact on 2 occasions by 2 blinded observers. The best samples were processed subsequently with a panel of routine stains and immunolabelled for collagen V to enable the measurement of minimum fibre diameters. RESULTS Both prolonged storage and increased hydration resulted in more storage-associated artefact. Samples stored for 24 h chilled on dry gauze were ranked higher than those stored on damp gauze; however, a panel of routinely-used histochemical staining techniques was unaffected by chilled 24 h storage. There was no significant effect of storage on mean fibre diameter; however, both chilled dry and damp storage for 24 h caused a significant increase in fibre-size variability. CONCLUSION AND POTENTIAL RELEVANCE Caution should be exercised when interpreting fibre size profiles in shipped samples. Equine muscle biopsy samples are optimally shipped in dry gauze, sealed in plastic containers and shipped on ice packs to be processed within 24 h and can thus be interpreted by the receiving laboratory with minimal artefact.

Objective—To develop a reliable method for converting cultured equine skin–derived fibroblasts into muscle cells. Sample Population—Equine skin–derived fibroblasts. Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining. Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen. Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) is one of a number of allelic disorders caused by mutations in the nuclear lamina proteins, lamins A and C. The disorder is characterised by the early onset of skeletal muscle weakness and joint contractures and later, by dilated cardiomyopathy and cardiac arrythmias. Although the pathophysiology is not understood, one theory suggests that disordered structural organisation at weakened nuclei in contractile cells may underlie the disease. Previous work shows that mice deficient in lamin A/C develop similar skeletal and cardiac muscle signs to patients with AD-EDMD and ultrastructural examination of muscle from these mice shows abnormal localisation of desmin. We hypothesised therefore that desmin localisation may be abnormal in muscle or cells from patients with AD-EDMD and/or in cells expressing mutant lamins. In order to evaluate this, desmin immunolocalisation was determined in skeletal muscle biopsy sections from patients with AD-EDMD and cell lines including MyoD-transfected fibroblast-derived myotubes from AD-EDMD patients and murine embryonic stem cell-derived cardiomyocytes stably transfected with mutant human lamin A. Ultrastructural examination of patient muscle was also performed. Desmin was expressed and localised normally in patient muscle and cell lines and ultrastructural examination was similar to controls. These results fail to provide any evidence that dominant mutations in lamin A/C lead to a disorganisation of the desmin associated cytoskeleton.

Individuals with the same genetic disorder often show remarkable differences in clinical severity, a finding generally attributed to the genetic background. We identified two patients with genetically proven Emery-Dreifuss muscular dystrophy (EDMD) who followed an unusual course and had uncommon clinicopathological findings. We hypothesized digenic inheritance and looked for additional molecular explanations. Mutations in additional separate genes were identified in both patients. The first patient was a member of a family with molecularly proven X-linked EDMD. However, the clinical features were unusually severe for this condition in the propositus: he presented at 2.5 years with severe proximal weakness and markedly elevated serum creatine kinase. Muscle weakness rapidly progressed, leading to loss of independent ambulation by the age of 12. In addition, the patient developed cardiac conduction system disease requiring pacing at the age of 11 and severe dilated cardiomyopathy in the early teens. Despite pacing, he had several syncopal episodes attributed to ventricular dysrhythmias. As these resemble the cardiac features of patients with the autosomal dominant variant of EDMD, we examined the lamin A/C gene, identifying a de-novo mutation in the propositus. The second patient had a cardioskeletal myopathy, similar to his mother who had died more than 20 years previously. Because of the dominant family history, a laminopathy was suspected and a mutation in exon 11 of the LMNA gene was identified. This mutation, however, was not present in his mother, but instead, surprisingly, was identified in his virtually asymptomatic father. Unusual accumulations of desmin found in the cardiac muscle of the propositus prompted us to examine the desmin gene in this patient, and in so doing, we identified a desmin mutation, in addition to the LMNA mutation in the propositus. These cases suggest that separate mutations in related proteins that are believed to interact, or that represent different parts of a presumed functional pathway, may synergistically contribute to disease severity in autosomal dominant EDMD. Furthermore, digenic inheritance may well contribute to the clinical severity of many other neuromuscular disorders.

Mutations of the LMNA gene, encoding the nuclear envelope proteins lamins A and C, give rise to Emery-Dreifuss muscular dystrophy and to limb-girdle muscular dystrophy 1B (EDMD and LGMD1B). With one exception, all the reported EDMD and LGMD1B mutations are confined to the first 10 exons of the gene. We report four separate cases, with mutations in the same codon of LMNA exon 11, characterized by remarkable variability of clinical findings, in addition to features not previously reported. One patient had congenital weakness and died in early childhood. In two other patients, severe cardiac problems arose early and, in one of these, cardiac signs preceded by many years the onset of skeletal muscle weakness. The fourth case had a mild and late-onset LGMD1B phenotype. Our cases further expand the clinical spectrum associated with mutations in the LMNA gene and provide new evidence of the role played by the C-terminal domain of lamin A.

Reasons for performing study Recurrent laryngeal neuropathy (RLN) is a common equine distal axonopathy associated with neurogenic atrophy of intrinsic laryngeal muscles (particularly the left) causing laryngeal paresis and poor performance. An objective, reliable method of assessing changes in structure that correlates with laryngeal muscle function in vivo is necessary to determine response to novel treatments, including functional electrical stimulation. This study aimed to determine whether laryngeal muscle and nerve morphology (determined by CT and histopathology) correlated with laryngeal function. Study design Prospective cohort study. Methods Resting (grades 1–4) and exercising (grades A–C) laryngeal function was graded in adult Thoroughbred horses. Standing CT of the larynx was performed to compare volume, cross-sectional area (XSA) and tissue density between left and right cricoarytenoideus dorsalis (CAD) muscles in 22 horses. CAD muscles and recurrent laryngeal nerves were collected in a subgroup of 10 horses and analysed for fibrosis, fat infiltration and nerve fibre density (axons/μm2). Results Horses with grade C RLN on exercising endoscopy had significantly (P

Reasons for performing study Advanced imaging modalities enable assessment of the equine skull and brain. Horses are susceptible to neurological dysfunction of the cranial nerves; however, our understanding of these structures’ imaging anatomy is limited. Hypothesis Magnetic resonance imaging (MRI) will be superior to computed tomography (CT) for the identification of cranial nerves, but optimal assessment may depend on both modalities. Objectives Identify cranial nerves and compare and contrast the utility of MRI and CT images of cadaver and clinical material. Interpret images from both modalities and determine the cranial nerve imaging anatomy. Study design Prospective cadaver anatomical study combined with retrospective clinical case study. Methods The head of a neurologically normal 9-year-old Thoroughbred gelding was scanned immediately following euthanasia (performed for reasons unrelated to this study). High resolution MRI (1.5 Tesla) and CT examinations were conducted over a 12 h interval following euthanasia. Images obtained were compared with selected clinical cases which were scanned during anaesthesia (MRI; approximately 30–60 min) or standing sedation (CT; approximately 30 s). Results On a high resolution MRI scan of a cadaver equine skull, each of the 12 cranial nerves and their topographic location was readily appreciated. Cranial nerves 1, 2, 3, 5, 7 and 8 were more easily identified in clinically relevant MRI scans. CT allowed visualisation of the stylomastoid foramen, the inner and middle ear and cranial nerves 2, 3, 5 and 7. Conclusions High field MRI allows for excellent visualisation of equine cranial nerves. CT allows for detailed visualisation of the osseous canals and foramina. This study advances anatomical knowledge of the normal equine cranial nerves to aid interpretation in horses that display neurological dysfunction localising to the brain and brainstem. Ethical animal research: Horse owners gave their consent for their animals to be included in the study. Sources of funding: Institutional. Competing interests: None.

Introduction Equine Type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense, gain-of-function mutation in the equine skeletal muscle glycogen synthase gene (GYS1). Affected horses have excessive glycogen storage, amylopectate-like inclusions in their muscle and higher muscle glycogen synthase activities (GS) than controls. GS is normally allosterically-regulated by glucose-6-phosphate and enzyme activity is reduced by phosphorylation. We hypothesised that the higher GS activity in PSSM1-affected horses might be due either to 1) increased expression or reduced degradation of the mutant enzyme or 2) due to reduced phosphorylation. Methods Semimembranosus muscle samples were biopsied from 4 homozygotes (HH), 13 heterozygotes (RH) and 12 control (RR) Belgian Draught horses. Samples were analysed for glycogen content and GS activity. Western immunoblotting was used to examine expression of total and phosphorylated forms of GS, GSK3β and GLUT4. Data was analysed using non-parametric ANOVA statistical tests with post-hoc Dunn's multiple comparison tests. Results PSSM1 affected heterozygotes and homozygotes had significantly higher muscle glycogen contents than controls (RR vs. RH P = 0.005, RR vs. HH P = 0.041) and GS activity was significantly higher in PSSM1-affected homozygotes than in heterozygotes and control horses (P = 0.03). Although there was no difference in total GS expression between groups, there was significantly more expression of GS phosphorylated at site 2 + 2a in PSSM1-affected horses in comparison with controls (P = 0.009). Conclusions Our data suggest that increased GS activity in PSSM1-affected horses is not associated with increased expression or degradation of the mutant enzyme and occurs despite increased phosphorylation of GS at site 2 + 2a. Alternative mechanisms, including aberrant ligand binding might account for the increased GS activity in PSSM1-affected muscle. Ethical Animal Research Muscle biopsy samples were collected according to ethical approval from the local Institutional Animal Care and Use Committee. Explicit owner informed consent for participation in this study is not stated. Sources of funding: Petplan Charitable Trust and The Royal Veterinary College. Competing interests: none.

Introduction Measurement of incorporation of stable isotope-containing amino acids into muscle is the gold-standard technique for measuring muscle protein synthesis (MPS), but its use in assessing MPS in horses has not been evaluated. Determination of MPS requires steady-state tracer plasma enrichment and serial muscle biopsy. Given that MPS is often assessed in response to exercise and that muscle biopsy in the standing horse typically requires sedation, the influence of exercise and sedation on tracer kinetics was determined. Methods Three mature Thoroughbred geldings received 0.1 mg/kg D5-phenylalanine (D5-Phe) IV and plasma D5-Phe enrichment was measured over 3 hrs. Pharmacokinetic modeling determined the rate of appearance and phenylalanine pool size, from which a loading dose and infusion rate that would achieve a steady 5–10% enrichment were calculated. Subsequently, steady-state was confirmed by measuring plasma enrichment during 6 hours of infusion after loading. Horses then received the same dose and completed a 12-minute exercise test and were sedated with xylazine 0.4 mg/kg IV. Plasma D5-Phe enrichment was determined every 5–15 minutes for 2 hours after both exercise and sedation. Results The mean rate of phenylalanine appearance in the horse was 56 umol/kg/hr (SD 24) and mean phenylalanine pool size 41 umol/kg (SD 8.8). A loading dose of 3.3 umol/kg and an infusion rate of 4.5 umol/kg/hr D5-phenylalanine was calculated and subsequently, confirmed to achieve steady state tracer concentration at 5.6–9.1% APE in sedentary horses. Exercise caused a transitory, and sedation a more prolonged reduction in plasma enrichment (up to 20%). Conclusions D5-phenylalanine is a suitable metabolic tracer in the horse as a steady-state can be achieved, however interventions should be taken into account. Ethical Animal Research The study was performed in in accordance with the local Ethics Committee approval and Home office project license (PPL 70/7523) under the UK A(SP)A Act 1986. Sources of funding: Boehringer Ingelheim. Competing interests: R Naylor is completing a PhD studentship sponsored by Boehringer Ingelheim.

PIERCY, R. J., K. W. HINCHCLIFF, P. S. MORLEY, R. A. DISILVESTRO, G. A. REINHART, S. L. NELSON, JR., K. E. SCHMIDT, and A. M. CRAIG. Association between vitamin E and enhanced athletic performance in sled dogs. Med. Sci. Sports Exerc., Vol. 33, No. 5, 2001, pp. 826-833. Purpose: To determine the association between prerace plasma vitamin E concentration and performance in sled dogs competing in the 1998 Iditarod Race. Methods: Prerace blood samples were collected from 670 dogs. Samples were analyzed for plasma vitamin E concentration while controlling for selected hematological and biochemical variables and signalment. Starting in teams of 16, exercise consisted of running up to 1159 miles pulling a laden sled and musher via checkpoints. The records of dogs that were withdrawn from the race for health reasons, fatigue, or strategic or technical reasons, and those of dogs that finished the race were analyzed. Multiple logistic regression and Cox proportional hazards analysis were used to determine factors associated with endurance. Multiple linear regression analysis was used to determine factors associated with team speed. Results: A total of 323 dogs (48%) were withdrawn from racing at various distances from the start. Median time to finish for 39 teams was 11.5 d d and the winning time was 9.2 d. Dogs with prerace plasma vitamin E concentrations > 40.7 μg.mL -1 were 1.9 times more likely to finish (P = 0.0006) and had 1.8 times less of a risk of being withdrawn for every mile ran (P = 0.03) than were dogs with plasma vitamin E concentrations between 16.3 and 40.7 μg.mL -1 . Neither a team's mean prerace vitamin E concentration, nor the proportion of dogs within a team with high (>40.7 μg.mL -1 ) vitamin E concentration was associated with team speed. Conclusions: Dogs with higher plasma vitamin E concentrations have enhanced endurance compared with dogs with lower plasma vitamin E concentrations, but the plasma vitamin E status of a team is not associated with team speed.

Exertional rhabdomyolysis (ER) is common in sled dogs, animals with high energy expenditures that consume high fat (60% of ingested calories) diets. Associations between pre-race plasma [vitamin E] and total antioxidant status (TAS) and risk of developing ER were examined in dogs competing in the 1998 Iditarod race. Pre-race blood samples were collected from 750 dogs and a second sample was collected from 158 dogs withdrawn from the race at various times. Plasma creatine kinase activity was used to identify withdrawn dogs with ER. There was no association between pre-race plasma [vitamin E] and risk of development of ER. Dogs that developed ER started the race with higher TAS, but when withdrawn, had lower TAS than unaffected dogs and had similar pre-race [vitamin E] but higher [vitamin E] at time of withdrawal. Hence, the risk of ER in sled dogs is not affected by plasma [vitamin E] before the race.

Objective—To determine whether dietary antioxidants would attenuate exercise-induced increases in plasma creatine kinase (CK) activity in sled dogs. Animals—41 trained adult sled dogs. Procedure—Dogs, randomly assigned to 2 groups, received the same base diet throughout the study. After 8 weeks on that diet, 1 group (21 dogs) received a daily supplement containing vitamins E (457 U) and C (706 mg) and β-carotene (5.1 mg), and a control group (20 dogs) received a supplement containing minimal amounts of antioxidants. After 3 weeks, both groups performed identical endurance exercise on each of 3 days. Blood samples were collected before and 3 weeks after addition of supplements and after each day of exercise. Plasma was analyzed for vitamins E and C, retinol, uric acid, triglyceride, and cholesterol concentrations, total antioxidant status (TAS), and CK activity. Results—Feeding supplements containing antioxidants caused a significant increase in vitamin E concentration but did not change retinol or vitamin C concentrations or TAS. Exercise caused significantly higher CK activity, but did not cause a significant difference in CK activity between groups. Exercise was associated with significantly lower vitamin E, retinol, and cholesterol concentrations and TAS but significantly higher vitamin C, triglyceride, and uric acid concentrations in both groups. Conclusions and Clinical Relevance—Use of supplements containing the doses of antioxidants used here failed to attenuate exercise-induced increases in CK activity. Muscle damage in sled dogs, as measured by plasma CK activity, may be caused by a mechanism other than oxidant stress. (Am J Vet Res 2000;61:1438–1445)

Multisystemic, eosinophilic, epitheliotropic disease and intestinal lymphosarcoma were diagnosed in a Paso Fino mare that presented with anorexia and weight loss. The stomach, ileum, cecum, colon, pancreas, and lungs were infiltrated by large numbers of eosinophils forming prominent eosinophilic granulomas, as well as lymphocytes and plasma cells. Two jejunal masses composed of solid sheets of neoplastic lymphocytes were present. In contrast to the regions of inflammation, the infiltrates in these masses did not contain plasma cells, eosinophils, and eosinophilic granulomas. Immunohistochemically, the neoplastic lymphocytes expressed CD3 but not CD20 or kappa and lambda light chains, supporting a diagnosis of T-cell lymphosarcoma. Concurrent diagnoses of hypereosinophilic syndrome and lymphosarcoma in this horse and several humans suggest that the multisystemic eosinophilic and lymphoplasmacytic infiltrates were caused by the clonal proliferation of T-lymphocytes that secreted interleukin-5 triggering differentiation and activation of eosinophils.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse., Author Summary A breed of the horse typically consists of individuals sharing very similar aesthetic and performance traits. However, a great deal of variation in traits exists between breeds. The range of variation observed among breeds can be illustrated by the size difference between the Miniature horse (0.74 m and 100 kg) and draft horse (1.8 m and 900 kg), or by comparing the optimum racing distance of the Quarter Horse (1/4 mile) to that of the Arabian (100 miles or more). In this study, we exploited the breed structure of the horse to identify regions of the genome that are significantly different between breeds and therefore may harbor genes and genetic variants targeted by selective breeding. This work resulted in the identification of variants in the Paint and Quarter Horse significantly associated with altered muscle fiber type proportions favorable for increased sprinting ability. A strong signature of selection was also identified in breeds that perform alternative gaits, and several genomic regions identified are hypothesized to be involved in the determination of size. This study has demonstrated the utility of this approach for studying the equine genome and is the first to show a functional consequence of selective breeding in the horse.

CHOLESTEROL granulomata – benign choroid plexus masses often detected at equine postmortem examinations – are sometimes associated with central neurological signs (Ivoghli and others 1977, Johnson and others 1993, Jackson and others 1994, King 1997). Antemortem diagnosis was impossible until advent of magnetic resonance imaging (Maulet and others 2008) and CT (Vink-Nooteboom and others 1998, Vanschandevijl and others 2008). These two cases are the first to illustrate diagnosis using standing (unanaesthetised) CT (CT Lightspeed QX/i; GE medical systems) and describe the long-term follow-up of one case, revealing that the prognosis for some cases is fair. Case 1: A 12-year-old Irish Draught cross gelding was presented with a 10-day history of intermittent lethargy and abnormal head carriage. On presentation, episodes of generalised muscle rigidity and ataxia were noticed by the owner; the horse was obtunded with reduced tongue tone and drooping of the lower lip and reluctant to flex or extend the neck and back. Neurological signs had improved following administration of methylprednisolone, penicillin and flunixin meglumine. On presentation to the Royal Veterinary College, the horse was obtunded; menace responses were reduced bilaterally, but pupillary light reflexes were intact. Mild superficial gluteal muscle fasciculations were evident. The horse had an exaggerated guarding response to deep palpation of the neck at C5 to C7. The neuroanatomical localisation suggested forebrain involvement, but based on cervical discomfort on …

BORNA disease is a sporadically occurring neurological disease caused by Borna disease virus (BDV), a RNA virus of the Bornaviridae family (Rott and Becht 1995, Staeheli 2002). BDV infection has been reported in a growing number of species, although sheep and horses are the main natural hosts; in horses, clinical disease is invariably fatal (Herden and Richt 2009), although more recently, milder forms of the disease have been reported by some researchers (Dieckhofer and others 2004, Bode 2008). Since the 1700s, classical clinical disease due to BDV infection has occurred only in geographically restricted areas of Germany, Austria, Switzerland and Lichtenstein (Rott and Becht 1995, Staeheli and others 2000, Durrwald and others 2006, Herden and Richt 2009). There is serological evidence for BDV infection of horses and other species in an increasing number of countries, including several other European countries, Israel, Japan, Iran, Australia and the USA (Herden and Richt 2009). However, whether natural Borna disease exists outside the areas in Europe where it is endemic, and whether BDV infections might be associated with other clinical manifestations, are still matters of considerable debate (Staeheli and others 2000, Durrwald and others 2006, Herden and Richt 2009). In the UK, serological studies of BDV in horses have not been reported; however, antibodies have been detected in cats and human beings without any clear association with disease (Reeves and others 1998, Thomas and others 2005). Moreover, until now, there have been no confirmed cases of clinically manifest Borna disease in horses or sheep in Great Britain. Seroepidemiological surveys from Germany have indicated that BDV infection of susceptible hosts often runs an inapparent course, since the average …

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p=0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. This article is protected by copyright. All rights reserved.