Abstract
Objective—To characterize clinical findings, outcomes, muscle characteristics, and serum or muscle concentrations of α-tocopherol for horses with vitamin E–responsive signs of muscle atrophy and weakness consistent with signs of equine motor neuron disease (EMND).
Design—Retrospective case-control study.
Animals—8 affected (case) adult horses with acute (n = 3) or chronic (5) gross muscle atrophy that improved with vitamin E treatment and 14 clinically normal (control) adult horses with adequate (within reference range; 8) or low (6) muscle concentrations of α-tocopherol.
Procedures—Medical records were reviewed, serum and muscle concentrations of α-tocopherol were measured, and frozen biopsy specimens of sacrocaudalis dorsalis medialis muscle and gluteal muscle were histologically evaluated for pathological changes. Fiber type composition and fiber diameters were assessed in gluteal muscle specimens.
Results—A myopathy that was histologically characterized by redistribution of mitochondrial enzyme stain (moth-eaten appearance) and anguloid atrophy of myofibers was evident in sacrocaudalis dorsalis medialis muscle fibers of the 8 affected horses that had low serum (6/8) or skeletal muscle (5/5) concentrations of α-tocopherol; these histopathologic changes were not found in muscle specimens of control horses with low or adequate muscle concentrations of α-tocopherol. All affected horses regained strength and muscle mass within 3 months after initiation of vitamin E treatment and dietary changes.
Conclusions and Clinical Relevance—A vitamin E–deficient myopathy characterized histologically by a moth-eaten appearance in the mitochondria and anguloid myofiber atrophy in frozen sections of sacrocaudalis dorsalis medialis muscle biopsy specimens was found in horses with clinical signs of EMND that were highly responsive to vitamin E treatment. This myopathy may be a specific syndrome or possibly precede the development of neurogenic muscle fiber atrophy typical of EMND.
Equine motor neuron disease is an acquired neurodegenerative disorder of the somatic lower motor neurons.1,2 Dysfunction or death of parent motor neurons results in secondary degenerative lesions in peripheral myelinated motor axons and neurogenic atrophy of highly oxidative type 1 muscle fibers.1 When 30% of motor neurons become diseased or dysfunctional,2 clinical signs of weight loss, muscle atrophy, and weakness occur. Weakness manifests clinically as muscle fasciculation, excessive recumbency, frequent shifting of body weight between the hind limbs, and abnormally low head and neck carriage.3,4
Diagnosis of EMND is made on the basis of relevant clinical signs, low serum concentration of α-tocopherol (< 1 μg/mL), and presence of neurogenic angular atrophy of predominantly type 1 and some type 2 myofibers with adjacent myofiber hypertrophy on histologic evaluation.5 The diagnostic sensitivity of neurogenic atrophy in the sacrocaudalis dorsalis medialis muscle for EMND is reported to be > 90%.4,6,7 Limb muscles in EMND may also have fiber size variation, centrally located nuclei, and alterations in mitochondrial staining, such as dense peripheral rims and redistribution of mitochondrial enzyme stains (moth-eaten appearance). These findings have been considered occasional and nonspecific for EMND.5
Clinical signs and neuropathologic lesions of EMND have been produced experimentally by feeding a vitamin E–deficient diet for 44 months.2 Thus, it is believed that EMND is related to vitamin E deficiency, although < 45% of affected horses are reported to respond to vitamin E treatment, with 69% euthanized within 0 to 3 months following onset of clinical signs and diagnosis.8 Other studies1–4,9 suggest that factors such as genetics, environment, management, and diet may also play a role. We identified a specific subset of vitamin E–deficient horses with clinical signs of EMND that lacked angular neurogenic atrophy in sacrocaudalis dorsalis medialis muscle typical of EMND but rather had characteristic myopathic changes in mitochondrial staining and anguloid myofiber atrophy. These horses responded dramatically to vitamin E treatment, and sacrocaudalis dorsalis medialis muscle biopsy findings did not have antemortem diagnostic features of EMND. The purpose of the study reported here was to describe the signalment, history, clinical signs, and myopathic features of sacrocaudalis dorsalis medialis muscle and gluteal muscle biopsy specimens from vitamin E–deficient horses with clinical signs of muscle atrophy and weakness with favorable response to vitamin E treatment.
Materials and Methods
Case selection—Medical records of horses with acute onset or chronic progressive muscle atrophy and weakness from April 23, 2001, to March 10, 2010, at the University of Minnesota Veterinary Medical Center and Oregon State University Lois Bates Acheson Veterinary Teaching Hospital were reviewed. Inclusion criteria for affected (case) horses were as follows: weight loss, generalized muscle atrophy and weakness, low serum or sacrocaudalis dorsalis medialis muscle tissue concentrations of α-tocopherol (< 2 or < 3.2 μg/mL, respectively), absence of remarkable neurogenic myofiber atrophy in frozen sections of sacrocaudalis dorsalis medialis muscle, and a favorable response to vitamin E treatment. Eight horses met these inclusion criteria (7 at the University of Minnesota Veterinary Medical Center and 1 at the Oregon State University Lois Bates Acheson Veterinary Teaching Hospital). Affected horses were on regular vaccination and deworming protocols, had good appetites, consumed an appropriate amount of feed to maintain body weight, and had no other identifiable cause for weight loss.
Data were extracted from the review of medical records, and owners of affected horses were contacted by telephone for long-term follow-up (mean, 1.6 years; median, 2 years; range, 3 months to 3 years). Data collected included signalment, history, physical examination findings, clinicopathologic abnormalities, serum concentrations of α-tocopherol, prescribed treatment, and outcome. On the basis of history and physical examination findings, there were 2 clinically distinct groups of affected horses. Acutely affected horses had weight loss and muscle atrophy < 3 weeks in duration and acute (2 days to 3 weeks) onset of severe weakness characterized by whole body muscle fasciculation, excessive shifting of body weight between the hind limbs, difficulty standing with excessive recumbency, a camped under stance, and abnormally low head carriage. Chronically affected horses had a > 2-month history of progressive weight loss, muscle atrophy, and weakness with a progressive decrease in performance.
Control horses—The limited prospective aspects of the study protocol involving control horses were reviewed and approved by an animal use and care committee and performed in accordance with institutional guidelines for research on animals. Where applicable, muscle biopsy specimens and serum samples were obtained from client-owned horses following owner consent.
adequate muscle concentrations of α-tocopherol
Control horses with muscle concentrations of α-tocopherol within reference range consisted of 8 client-owned healthy adult Quarter Horses (6 mares and 2 geldings) ranging in age from 7 to 12 years (mean, 9.1 years; median, 9.0 years) with an adequate (within reference range) muscle concentration of α-tocopherol (8/8 horses) and, where available, adequate serum concentration of α-tocopherol (2/8 horses). Six of these 8 control horses were selected retrospectively on the basis of grass pasture access > 6 mo/y, absence of gross evidence of muscle atrophy or weight loss, and sufficient snap-frozen stored gluteal muscle available for α-tocopherol concentration analysis. Gluteal muscle samples were obtained at a standardized site. Serum was not available retrospectively for α-tocopherol analysis in these 6 control horses. The remaining 2 control horses were selected prospectively on the basis of grass pasture access > 6 mo/y, absence of gross evidence of muscle atrophy or weight loss, and serum concentrations of α-tocopherol (3.0 and 2.9 μg/mL) within reference range (2 to 4 μg/mL). Gluteal and sacrocaudalis dorsalis medialis muscle biopsy specimens were prospectively obtained from these 2 control horses, snap frozen, and stored for muscle α-tocopherol concentration analysis and histologic evaluation.
Low muscle concentrations of α-tocopherol
Control horses with low muscle concentrations of α-tocopherol consisted of 6 healthy Quarter Horses (3 mares and 3 geldings ranging in age from 4 to 10 years; mean, 5.8 years; median, 6.0 years) that were clinically normal with adequate body condition (body score, > 5/9) and used as research horses at the University of Minnesota. These clinically normal horses were selected prospectively on the basis of deficient serum concentrations of α-tocopherol (< 2 μg/mL; reference range, 2 to 4 μg/mL) and deficient skeletal muscle concentrations of α-tocopherol (< 3.0 μg/g). Both sacrocaudalis dorsalis medialis muscle and gluteal muscle biopsy specimens were obtained.
Muscle biopsy procedure—Percutaneous needle biopsy technique was used to obtain muscle specimens.10 Gluteus medius muscle biopsy specimens were obtained at a standard site from all control horses and 5 of 8 affected horses (ie, all chronically affected horses).10 Sacrocaudalis dorsalis medialis muscle biopsy specimens were obtained 1 to 2 cm off of midline, 4 cm cranial to the tail head. Sacrocaudalis dorsalis medialis muscle biopsy specimens were obtained from all affected horses, 2 control horses with adequate muscle concentrations of α-tocopherol, and 6 control horses with low muscle concentrations of α-tocopherol. Biopsy specimens were oriented in cross-section on moist cork and frozen in isopentane chilled in liquid nitrogen.
Analysis of muscle biopsy specimens—Frozen specimens of sacrocaudalis dorsalis medialis muscle and gluteal muscle were cut at a thickness of 10 μm and stained with H&E, periodic acid-Schiff, NADH-Tr, and ATPase (pH, 4.6). Sacrocaudalis dorsalis medialis muscle specimens were also stained with modified Gomori trichrome and oil red O.11
Neurogenic atrophy was defined as angular atrophy of both type 1 and type 2 fibers (≥ 2 sides of muscle fibers concave creating points). Myogenic atrophy was defined as the presence of anguloid atrophy (< 3 slightly concave sides) with ≤ 2 angular atrophied fibers present in the biopsy specimen. Fibers with a moth-eaten mitochondrial staining pattern were defined by disruption of mitochondrial staining with the NADH-Tr stain.11 To confirm that abnormalities in NADH-Tr were mitochondrial in origin, staining of cytochrome C oxidase and succinate dehydrogenase was performed in muscle biopsy specimens from 1 affected horse and 1 control horse.
Scoring system—One author (SJV) scored samples for the presence of anguloid and angular myofiber atrophy myofiber hypertrophy and a moth-eaten mitochondrial staining pattern in muscle fibers at 20× magnification (0, not present; 1, present in 1 to 10 fibers/random microscopic field; 2, present in 11 to 50 fibers/random microscopic field; and 3, present in > 50 fibers/random microscopic field).12 Muscle fiber type compositions were determined by typing > 250 fibers/biopsy specimen and calculating a percentage composition. Maximum skeletal muscle fiber diameters were determined in 25 gluteal fibers of each type with a computer image analysis systema in 5 affected horses, 8 control horses with adequate muscle concentrations of α-tocopherol, and 5 control horses with low muscle concentrations of α-tocopherol. Adenosine triphosphatase staining did not produce 3 fiber types for 1 control horse with a low muscle concentration of α-tocopherol, and it was not analyzed.
Measurement of α-tocopherol concentrations—Serum concentration of α-tocopherol was assessed in venous blood samples from all affected horses, all control horses with low muscle concentrations of α-tocopherol, and 2 control horses with adequate muscle concentrations of α-tocopherol by high-performance liquid chromatography at Michigan State University Diagnostic Center for Population and Animal Health. Approximately 250 mg of gluteal muscle tissue snap frozen in liquid nitrogen and stored at −80°C was submitted to the Michigan State University Diagnostic Center for Population and Animal Health for determination of wet-weight muscle concentration of α-tocopherol by high-performance liquid chromatography where gluteal muscle was available (5 chronically affected horses, 8 control horses with adequate muscle concentrations of α-tocopherol, and 6 control horses with low muscle concentrations of α-tocopherol). Sacrocaudalis dorsalis medialis muscle was used for determination of wet-weight muscle concentration of α-tocopherol when sample size permitted (> 100 mg; 2 control horses and 3 affected horses).
Statistical analysis—A 1-way ANOVA was used to compare fiber type compositions and fiber diameters in the gluteal muscle between control horses and affected horses, with significance set at P < 0.05.
Results
Signalment and history—Of the 8 affected horses, 3 were acutely affected (horses A, B, and C); they had a 2-day to 3-week history of excessive recumbency, severe muscle fasciculation, hind limb stiffness, and acute weight loss despite a normal appetite. All 3 acutely affected horses had seasonal access to pasture for at least 6 h/d and were fed grass (orchard or timothy) or alfalfa hay. Two of the 3 acutely affected horses (B and C) were fed a commercial concentrate; the remaining horse (A) was fed a locally milled grain mixture (pellets, rolled corn, and sweet feed) until 2 weeks prior to referral. None of the acutely affected horses were given supplements containing vitamin E. The amount of feed provided was deemed adequate to maintain an ideal body condition, and other horses on 2 of the 3 farms (that of horses B and C) did not have weight loss. Following the introduction of newly purchased round hay bales on the remaining farm (that of horse A), however, a Quarter Horse stallion had died with similar clinical signs to those of the acutely affected horses. On that same farm, there were also 2 old cars in the pasture to which all horses had access. Diagnostic testing was not performed prior to referral, but all acutely affected horses had been treated with either flunixin meglumine or phenylbutazone; 1 horse (B) also was treated with prednisone (0.7 mg/kg [0.32 mg/lb], PO, q 24 h) followed by a taper in dose over 1 week. All 3 horses had been dewormed with either moxidectin (horses A and C) or ivermectin (horse B) within 3 weeks (horse C), 2 months (horse B), and 5 months (horse A) prior to referral.
Of the 8 affected horses, 5 were chronically affected (horses D, E, F, G, and H); they had chronic weight loss and muscle atrophy most pronounced over the hindquarters of > 2 to 3 months’ duration. Two of the 5 chronically affected horses were privately owned and used as performance horses (1 hunter-jumper horse [horse D] and 1 western pleasure and trail horse [horse E]), and both had a gradual decrease in performance level. Historically, both of these horses were described as easy keepers (ie, horses that can maintain a good body weight on relatively little food) and were overweight prior to onset of clinical signs. One horse (E) weighed 577 kg (1,269 lb), with a previous body weight of 693 kg (1,525 lb) and body condition score of 8 of 9 that had been documented 1 year previously. Three of the 5 chronically affected horses (F, G, and H) were part of the University of Minnesota teaching herd; these 3 horses had daily turnout limited to dirt paddocks and were fed free choice grass hay and a commercial concentrate. One of the 2 chronically affected performance horses (D) was fed 1.8 kg (4 lb) of a commercial senior feedb (150 U of vitamin E/lb feed) top dressed with corn oil (3/4 cup) twice a day. The other performance horse (E) was fed a commercial 14% protein sweet feed (1 kg [2 lb]) a day. The 3 chronically affected horses that were part of the University of Minnesota teaching herd were fed 0.5 kg (1 lb) of a commercial grass hay ration balancerc (700 U of vitamin E/lb) once a day. None of these 5 horses were fed supplements containing vitamin E. All 5 chronically affected horses were dewormed at least twice per year with PO administration of ivermectin or ivermectin-praziquantel paste. One of the chronically affected performance horses (D) was given daily pyrantel. Apart from these chronically affected horses, no other horses on the premises had evidence of weight loss, and the amount of feed provided was deemed sufficient to maintain an ideal body weight.
Physical examination findings—All 3 acutely affected horses (A, B, and C) were underweight with low body condition scores. Acutely affected horses had severe whole body muscle fasciculations, shifting of body weight between the hind limbs, difficulty standing, a camped-under stance, and abnormally low head carriage (Figure 1). One acutely affected horse (C) sweated profusely, and another horse (A) walked with a stiff, stilted gait; both horses had increased digital pulses in all 4 limbs. All 3 acutely affected horses were tachycardic (heart rate range, 48 to 72 beats/min) with normal rectal temperatures and tachypnea. Neurologic examinations did not identify abnormalities in mentation, cranial nerve function, tail and anal tone, cutaneous trunci, cervicofacial reflexes, and proprioception. On the basis of results of history and physical examination, EMND was suspected; however, other differential diagnoses considered for weight loss, muscle atrophy, muscle fasciculation, and weakness were neurologic, gastrointestinal, or metabolic disease; botulism; and myopathy. Additional differential diagnoses considered for one of the acutely affected horses (A) were lead toxicity because of access to old cars in the pasture and organophosphate toxicity because of acute onset following introduction of newly purchased round hay bales. Laminitis was also considered in 2 acutely affected horses (A and C) because of the increased digital pulses in all 4 limbs, with a stiff, stilted gait in 1 of these horses.
Photographs of 1 of the 3 acutely affected horses (horse C) prior to (A) and after (B) 6 months of vitamin E treatment. In panel A, notice the marked generalized muscle atrophy, camped-under stance, and elevated tail head. In panel B, notice the normal body condition, muscle mass, and stance.
Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1127
All 5 chronically affected horses (D, E, F, G, and H) were considered thin, with low body condition scores; no abnormalities were detected on physical and neurologic examination findings (normal mentation, cranial nerve function, tail and anal tone, cutaneous trunci, cervicofacial reflexes, and proprioception) except for moderate symmetric hind limb weakness in response to tail pull. One of the 5 chronically affected horses (F) had slight bilateral circumduction and slight toe drag of both hind limbs when turned in tight circles, which was suspected as being secondary to hind limb weakness in the absence of any further evidence of abnormal proprioception. Another chronically affected horse (G) had a historical episode of an acute gait abnormality that involved protracted flexion of both hind limbs prior to walking forward or backward; this disappeared after several steps but recurred upon standing. The gait abnormality had resolved within 24 hours without any treatment. Differential diagnoses for weight loss and hind limb weakness included gastrointestinal disease or malabsorptive syndromes, myopathy, EMND, other neurologic or metabolic disease, and neoplasia.
Clinicopathologic findings—All 3 acutely affected horses (A, B, and C) had initial CBC and serum biochemical analyses performed. For one of the acutely affected horses (C), no abnormalities were detected on CBC. The 2 remaining acutely affected horses (A and B) had leukocytosis (15.4 × 103 cells/L and 14.6 × 103 cells/L, respectively; reference range, 4.6 × 103 cells/L to 11.6 × 103 cells/L) secondary to mature neutrophilia (13.4 × 103 cells/L and 9.7 × 103 cells/L, respectively; reference range, 1.5 × 103 cells/L to 8.5 × 103 cells/L). On serum biochemical analyses, high serum creatine kinase activities (781 and 5,805 U/L; reference range, 82 to 303 U/L) were found for 2 acutely affected horses (A and B, respectively), high aspartate transaminase activities (419, 2,328, and 442 U/L; reference range, 162 to 316 U/L) were found for all 3 acutely affected horses (A, B, and C, respectively), and mild hyperglycemia (128 mg/dL; reference range, 71 to 106 mg/dL) was detected in 1 acutely affected horse (B). Food was withheld from 1 acutely affected horse (C) for 12 hours prior to general anesthesia for CSF collection, at which time blood sample collection was performed; only slight abnormalities of hyperbilirubinemia (2.8 mg/dL; reference range, 0.8 to 2.6 mg/dL) and hypoproteinemia (5.6 g/dL; reference range, 5.9 to 7.3 g/dL) with serum albumin concentration within reference limits were found.
Fecal egg counts were recommended in all acutely affected horses to rule out parasitism as a cause of poor body condition but were declined by the owners of 2 horses (B and C) because of recent deworming prior to referral (3 and 8 weeks, respectively). A quantitative fecal egg count was performed in 1 acutely affected horse (A) because the last deworming occurred 5 months prior, and results were a low positive (100 strongyle eggs/g of feces), ruling out heavy parasitism as a cause of weight loss in this horse.
Colic as a cause of recumbency, shifting of body weight between the hind limbs, muscle fasciculation, and tachycardia was considered initially in 1 acutely affected horse (A); a rectal examination and nasogastric intubation were performed, and no abnormalities were found. Laminitis was also considered in 2 acutely affected horses (A and C). One of these horses (C) underwent a brief lameness examination and application of hoof testers, and results were negative for solar pain. In this horse, bilateral medial and lateral abaxial sesamoidean nerve blocks did not improve the observed stiff, stilted gait. For both horses, no abnormalities were observed on lateral radiographic views of the third phalanx in both hind limbs (horse A) or all 4 limbs (horse C), ruling out laminitis as the cause of muscle fasciculation, shifting of body weight between the hind limbs, and recumbency.
A CSF analysis was performed for all 3 acutely affected horses to further investigate the neurologic disease. On cytologic analysis of CSF samples, the RBC count and nucleated cell counts were within reference range in all horses. The CSF glucose and total protein concentrations and CSF creatine kinase activity were determined for 1 acutely affected horse (C) and were within reference range. Two of the acutely affected horses (A and C) also had Western blot analysis of CSF for the detection of Sarcocystis neurona antibodies, results of which were negative, suggesting clinical signs were not likely due to equine protozoal myeloencephalitis. A CSF sample for Western blot analysis for the detection of S neurona antibodies was collected and saved for submission at a later date for the third acutely affected horse (B), but sample submission was declined by the owner because of rapid favorable clinical response to vitamin E treatment. A serum capture ELISA titer for West Nile virus was also performed for 1 acutely affected horse (A), and the results were negative; whole blood lead concentration and serum cholinesterase activity were also measured in this horse, and both values were within reference range. Botulism is a differential diagnosis for muscle weakness, but all acutely affected horses had a normal swallow reflex and never had any signs of dysphagia.
Chronically affected horses had initial CBC and serum biochemical analysis performed. The CBC results were within reference range for 3 chronically affected horses (D, E, and F). Serum biochemical analysis results were also within reference range in these horses with the following exceptions: mild hyperglycemia (horse D, 118 mg/dL; horse E, 119 mg/dL; reference range, 75 to 116 mg/dL), mildly high serum total bilirubin concentration (horse D, 3.1 mg/dL; reference range, 0.8 to 2.6 mg/dL), serum alkaline phosphatase activity (horse E, 185 U/L; reference range, 48 to 148 U/L), and serum aspartate transaminase activity (horse F, 354 U/L; reference range, 162 to 316 U/L). Serum creatine kinase activity, which was within reference range, was measured in 1 chronically affected (horse G) following an acute temporary gait abnormality. A CBC and serum biochemical analysis were not performed for 2 chronically affected horses (G and H) because of budgetary constraints for the University of Minnesota teaching herd; however, their clinical signs, diet, and management mirrored that of the other chronically affected horse (F) that was also part of the teaching herd. Although all horses were dewormed regularly, quantitative fecal egg counts were performed for chronically affected horses (D and E) to rule out parasitism as a cause of weight loss; results were negative for 1 horse (D) and low positive for the other (E; 100 strongyle eggs/g feces). Fecal egg counts were considered for the 3 chronically affected horses that were part of the University of Minnesota teaching herd; however, with a regular deworming program and limited budget, the expense could not be justified.
The 2 chronically affected performance horses (D and E) underwent testing for gastrointestinal disease as a cause of weight loss. An oral glucose tolerance test was performed in 1 of the 2 horses (D) and revealed a peak in blood glucose concentration within 60 minutes at 134 mg/dL, which was only 74% of the 120-minute peak value for clinically normal horses in a previous study.12 With only slight hypoproteinemia and a serum albumin concentration within reference range, intestinal malabsorption as a sole cause of major weight loss in this horse (D) was unlikely; however, decreased plasma glucose curves after oral glucose tolerance testing have been reported in 50% of horses with EMND.3 The other chronically affected horse (E) underwent abdominal ultrasonography and abdominocentesis to rule out gastrointestinal disease or possible abdominal neoplasia. No abnormalities were detected on abdominal ultrasonography, results of peritoneal fluid analysis (total protein, RBC count, and nucleated cell counts) were within reference range, and no abnormalities were found on cytologic evaluation of peritoneal fluid. In 1 chronically affected horse (E), CSF was collected; cytologic evaluation, RBC count, nucleated cell counts, total protein concentration, glucose concentration, and creatine kinase activity of the CSF were all within reference ranges, and results of indirect fluorescent antibody test for S neurona and Neospora hughesii were negative. The owner of the other chronically affected performance horse (D) declined CSF collection pending the findings on histologic evaluation of a muscle biopsy specimen.
Vitamin E and selenium concentrations—Serum concentrations of α-tocopherol in the 3 acutely affected horses (A, B, and C) were low (0.20, 0.56, and 0.04 μg/mL, respectively; reference range, 2 to 4 μg/mL). Three chronically affected horses (F, G, and H) had low serum concentrations of α-tocopherol (1.63, 0.66, and 0.26 μg/mL, respectively; reference range, 2 to 4 μg/mL); 2 chronically affected horses had values within (horse D; 2.7 μg/mL) or slightly above (horse E; 4.5 μg/mL) the reference range. Control horses with low muscle concentrations of α-tocopherol had serum concentrations of α-tocopherol less than the reference limit of 2 μg/mL.
For control horses with adequate muscle concentrations of α-tocopherol, the mean ± SEM value was 6.26 ± 0.59 μg/g (reference range, 3.6 to 8.1 μg/g). Biopsy specimens for 2 control horses with adequate muscle concentrations of α-tocopherol appeared to have adipose tissue contamination with high α-tocopherol concentrations of 22 and 35 μg/g and were not included in calculation of the mean. For control horses with low muscle concentrations of α-tocopherol, the mean ± SEM α-tocopherol concentration was 2.10 ± 1.51 μg/g (reference range, 3.6 to 8.1 μg/g). Muscle concentrations of α-tocopherol were only determined in chronically affected horses (D, E, F, G, and H) because of tissue availability; values (1.14, 0.56, 1.10, 0.43, and 0.66 μg/g, respectively) were similar to those of control horses with low muscle concentrations of α-tocopherol and less than half of values for horses with adequate muscle concentrations of α-tocopherol.
Whole blood selenium concentration was measured in 3 affected horses as follows: 1 acutely affected horse (C [218 ng/mL]) and 2 chronically affected horses (D [222 ng/mL]; E [211 ng/mL]). Whole blood selenium concentrations were within the reference range (160 to 275 ng/mL) for all 3 horses.
Muscle histopathologic findings—Rare angular atrophied type 1 myofibers were present in sacrocaudalis dorsalis medialis muscle of 2 affected horses (Table 1). Mild to moderate fiber hypertrophy was present in sacrocaudalis dorsalis medialis muscle of 5 of 8 affected horses. Anguloid atrophy of type 1 and, when present, type 2 muscle fibers was the most common feature of the sacrocaudalis dorsalis medialis muscle of affected horses. Myofibers of 3 chronically affected horses (D, E, and G) had centrally located nuclei, and 1 acutely affected horse (B) had a few degenerating fibers with macrophage infiltration. Two chronically affected horses (F and H) had abnormal periodic acid-Schiff–positive material in a few myofibers, typical of polysaccharide storage myopathy. The most striking histochemical abnormality present in all sacrocaudalis dorsalis medialis muscle biopsy specimens of affected horses was the presence of a moth-eaten mitochondrial staining pattern in the oxidative type 1 fibers with the NADH-Tr stain (Figure 2). The largest number of fibers with a moth-eaten mitochondrial staining pattern, which had a high degree of morphological alterations, was found in 2 chronically affected horses (D and E). The moth-eaten mitochondrial staining pattern was apparent in cytochrome C oxidase (all fibers stained positive) and succinate dehydrogenase stains of sacrocaudalis dorsalis medialis muscle, confirming that the abnormal NADH-Tr staining involved mitochondria.
Photomicrographs of cross-sections of sacrocaudalis dorsalis medialis muscle biopsy specimens from a clinically normal control horse (panel A) and from a horse with chronic vitamin E–responsive muscle atrophy (horse E; panel B). Notice that > 50% of fibers in the chronically affected horse in panel B have disruption of mitochondrial staining (ie, moth-eaten mitochondrial staining pattern). M = Disruption in the normal distribution of mitochondria. NADH-Tr stain; bars = 100 μm.
Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1127
Individual and mean ± SEM histopathologic scores* for sacrocaudalis dorsalis medialis muscle biopsy specimens from 8 (case) adult horses with acute (n = 3) or chronic (5) gross muscle atrophy and weakness that improved with vitamin E treatment and from 8 clinically normal (control) adult horses with adequate (2) or low (6) muscle concentrations of α-tocopherol.
| Horses | No. of horses | Anguloid atrophy | Angular atrophy | Hypertrophy | Amount of MEMSP | Degree of MEMSP | PAS positive | Total score |
|---|---|---|---|---|---|---|---|---|
| A (acute) | NA | 2 | 0 | 1 | 3 | 2 | 0 | 8 |
| B (acute) | NA | 2 | 1 | 1 | 3 | 2 | 3 M | 12 |
| C (acute) | NA | 2 | 1 | 1 | 3 | 2 | 0 | 9 |
| D (chronic) | NA | 2 | 0 | 0 | 3 | 3 | 1 CN | 9 |
| E (chronic) | NA | 2 | 0 | 0 | 3 | 3 | 1 CN | 9 |
| F (chronic) | NA | 1 | 0 | 0 | 2 | 2 | 1 AP | 6 |
| G (chronic) | NA | 2 | 1 | 2 | 2 | 1 | 1 CN | 9 |
| H (chronic) | NA | 2 | 0 | 1 | 2 | 1 | 1 AP | 7 |
| All affected | 8 | 1.9 ± 0.1 | 0.4 ± 0.2 | 0.8 ± 0.3 | 2.5 ± 0.2 | 2.0 ± 0.3 | 1.0 ± 0.3 | 8.8 ± 0.6 |
| Adequate-control† | 2 | 1.5 ± 0.4 | 0 ± 0 | 0 ± 0 | 0.5 ± 0.4 | 0.5 ± 0.4 | 0 ± 0 | 2.5 ± 0.4 |
| Low-control‡ | 6 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0.1 ± 0.2 | 0.1 ± 0.2 |
0 = Not present. 1 = Mild. 2 = Moderate. 3 = Severe.
Clinically normal horses with adequate (within reference range) muscle concentrations of α-tocopherol.
Clinically normal horses with low muscle concentrations of α-tocopherol.
AP = Abnormal polysaccharide. CN = Central nuclei. M = Macrophages. MEMSP = Moth-eaten mitochondrial staining pattern. PAS = Periodic acid–Schiff.
The sacrocaudalis dorsalis medialis muscle from 1 of 2 control horses with adequate muscle concentrations of α-tocopherol had mild anguloid atrophy (grade 1) and a few fibers with a slight moth-eaten mitochondrial staining pattern. Sacrocaudalis dorsalis medialis muscle from low muscle α-tocopherol–control horses had no histopathologic abnormalities apart from abnormal amylase-resistant polysaccharide consistent with polysaccharide storage myopathy in a few muscle fibers of 1 horse.
The gluteal muscle histochemical appearance was normal in 3 of 5 chronically affected horses, 8 of 8 control horses with adequate muscle concentrations of α-tocopherol, and 5 of 6 control horses with low muscle concentrations of α-tocopherol. Two chronically affected horses (F and H) and 1 control horse with low muscle concentrations of α-tocopherol had normal muscle histologic morphology apart from the presence of subsarcolemmal vacuoles and periodic acid–Schiff-positive, amylase-resistant material, consistent with a diagnosis of polysaccharide storage myopathy.
Muscle fiber type composition—The sacrocaudalis dorsalis medialis muscle of 2 acutely affected horses (A and C) and 2 chronically affected horses (E and G) consisted exclusively of type 1 myofibers, whereas type 1 fiber type composition ranged from 54% to 68% and type 2 fiber type composition from 32% to 46% for other acutely affected horses. A wide range of fiber type compositions were seen in sacrocaudalis dorsalis medialis muscle specimens of control horses, ranging from 6% to 85% for type 1 fibers and 15% to 94% for type 2 fibers.
There was no difference in muscle fiber type composition for gluteal muscle among chronically affected horses, control horses with adequate muscle concentrations of α-tocopherol, and control horses with low muscle concentrations of α-tocopherol. Percentage of type 1 fibers for chronically affected horses, control horses with low muscle concentrations of α-tocopherol, and control horses with adequate muscle concentrations of α-tocopherol was 20.8 ± 2.7%, 17.6 ± 3.4%, and 15.2 ± 1.5%, respectively. Percentage of type 2A fibers for chronically affected horses, control horses with low muscle concentrations of α-tocopherol, and control horses with adequate muscle concentrations of α-tocopherol was 26.0 ± 4.2%, 24.0 ± 2.2%, and 27.0 ± 2.7%, respectively. Percentage of type 2B fibers for chronically affected horses, control horses with low muscle concentrations of α-tocopherol, and control horses with adequate muscle concentrations of α-tocopherol was 53.2 ± 3.3%, 58.2 ± 2.6%, and 57.8 ± 2.7%, respectively.
Muscle fiber diameter—The mean maximal myofiber diameter for type 2B fibers in gluteal muscle was significantly smaller in chronically affected horses versus control horses with adequate muscle concentrations of α-tocopherol. Type 2B fiber diameters of control horses with low muscle concentrations of α-tocopherol were also significantly smaller than those of control horses with adequate muscle concentrations of α-tocopherol and similar to those of all affected horses (Figure 3).
Mean ± SEM maximum fiber diameter measured by fiber type in affected horses (black bars), control horses with low muscle concentrations of α-tocopherol (striped bars), and control horses with adequate muscle concentrations of α-tocopherol (white bars). Horizontal bars indicate significantly (P < 0.05) smaller fiber diameters in type 2B muscle fibers of affected horses and control horses with low muscle concentrations of α-tocopherol, compared with control horses with adequate muscle concentrations of α-tocopherol.
Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1127
Treatment and outcome—All acutely affected horses received vitamin E. One acutely affected horse (A) had clinical signs of recumbency, muscle fasciculation, frequent shifting of body weight between the hind limbs, and tachycardia until IM injections of vitamin E and seleniumd and PO administration of vitamin E were initiated on day 3 of hospitalization. By day 4, muscle fasciculations were considerably decreased and tachycardia had resolved. Additional treatments for this acutely affected horse (A) included flunixin meglumine (1.1 mg/kg [0.5 mg/lb], IV q 12 h) on day 1, IV fluid administration at 2.5 mL/kg/h (1.1 mL/lb/h) on days 1 and 2, and PO administration of 1% ivermectin paste for ectoparasites and endoparasites on day 3.
On day 2 of hospitalization, another acutely affected horse (B) received vitamin E PO and a single IM injection of vitamin E.e There was remarkable improvement in clinical signs on day 3, and the horse was discharged from the hospital.
The third acutely affected horse (C) received a vitamin E and selenium injection IMd as well as vitamin E IMe and PO on day 1. Oral and IM administration of vitamin Ee was continued on day 2, and the horse was discharged from the hospital with much clinical improvement on day 3. Recommendations were made for continued PO administration of vitamin E.f Additional treatment for this horse (C) included turnout on grass pasture, a complete vitamin supplementg (60 g/d, PO), and provision of a low starch feedh with higher vitamin E (125 U/lb of feed) concentrations than previously fed (12.5 U/lb of feed).
At 3 months after hospital discharge, 1 acutely affected horse (A) was clinically normal; longer term follow-up was not available. Within 2 months after hospital discharge, another acutely affected horse (B) had regained muscle mass and was reported to be clinically normal. This horse (B) remained clinically normal and was sold 3 years after initial diagnosis. At 6 months of follow-up, the third acutely affected horse (C) had regained muscle mass and was clinically normal. Follow-up serum α-tocopherol concentration determination was not performed for any acutely affected horses.
All chronically affected horses received vitamin Ei PO. Additional treatments for one of the chronically affected performance horses (D) included grass pasture 3 to 4 h/d seasonally and a commercial low-starch, high-fat feed (5 kg [11 lb] of feed/d) with a higher vitamin E concentration (440 U/lb of feed) than fed previously The other chronically affected performance horse (E) received rehabilitation treatment during 3 weeks of hospitalization, consisting of 15 minutes of hand walking twice daily that included walking over cavaletti ground poles and walking with a resistance band that wrapped around the hindquarters and attached to a surcingle to further engage the hindquarter muscle development. This horse (E) also received grass pasture turnout for 24 h/d after hospital discharge and was fed increasing amounts (up to 4 kg [8.8 lb]) of a feedj that contained a low amount of starch, high amount of fat, and high amount of vitamin E (440 U/lb of feed).
Within 3 to 6 months, all 5 chronically affected horses regained muscle mass and strength. One chronically affected performance horse (D) returned to previous body condition and performance levels by 12 months, and the other chronically affected performance horse (E) returned to previous body condition and performance levels by 6 months. At ≥ 2 years of follow-up, all 5 chronically affected horses were clinically normal.
Serum α-tocopherol concentration was determined on follow-up for 1 chronically affected performance horse (D), and concentrations were high (8.7 μg/mL; reference range, 2 to 4 μg/mL) after 3 months of treatment. The amount of dietary vitamin E provided was reduced gradually, and the horse continued to receive vitamin E powderk PO (5,000 U/d during winter and fall and 2,500 U/d during spring and summer), resulting in serum α-tocopherol concentrations of 7.0 μg/mL. For 3 chronically affected horses (F, G, and H), follow-up muscle α-tocopherol concentrations were increased from before treatment (3.18, 4.88, and 3.08 μg/g, respectively), which were lower than values for clinically normal horses. Two of these horses (F and H) continued to receive vitamin El PO, and the third horse (G) was relocated and given free access to pasture.
Discussion
Results of the present investigation indicate that some horses with clinical signs of EMND and a deficiency of vitamin E may not be determined to have EMND before death because they lack evidence of neurogenic atrophy in the sacrocaudalis dorsalis medialis muscle. Such horses may account for the reported 90% diagnostic sensitivity of sacrocaudalis dorsalis medialis muscle biopsy for EMND. We suggest that many such undiagnosed cases may be the result of a specific myogenic presentation of vitamin E deficiency that can only be identified in mitochondrial stains of frozen sacrocaudalis dorsalis medialis muscle. A diagnosis of vitamin E–deficient myopathy has likely been missed previously because formalin-fixed biopsy specimens are most often submitted, and mitochondrial staining is not possible with this fixative. Vitamin E–deficient myopathy may be an entity unto itself or a predecessor to development of EMND. This distinction could not be evaluated in the present study because all horses successfully responded to vitamin E treatment, precluding postmortem examination for subclinical EMND. Rather than a paucity of motor neurons, as seen with EMND, the observed generalized weakness in the horses in the present study may have been due to a reversible manifestation of skeletal muscle or mitochondrial oxidative stress associated with vitamin E deficiency.
Vitamin E deficiency has been shown to independently produce pathological changes in muscle and nervous tissue, including necrotizing myopathy in rats, humans, dogs, and Vietnamese potbellied pigs.13–15 Degenerative lesions of the myocardium and skeletal muscles caused by vitamin E and selenium deficiency have been documented in young calves16,17 and foals18 but only rarely in adult horses.19,20 In adult horses, vitamin E deficiency has been associated with equine degenerative myeloencephalopathy,21 neuroaxonal dystrophy,22 and EMND.2 In the present study, whole blood selenium concentration was measured in 3 affected horses (1 acutely affected horse and 2 chronically affected horses), and results were within reference range. In addition, degenerative lesions and calcification were rare or absent in muscle tissues of all affected horses but were characterized instead by a moth-eaten mitochondrial staining pattern, indicating that this myopathy was distinct from nutritional myodegeneration.
The abnormal mitochondrial staining pattern (moth-eaten appearance) represents multifocal zones that lack mitochondria as represented by the activity of the mitochondrial enzyme NADH-Tr and likely indicates disturbed muscle fiber oxidative metabolism.23,24 Muscle fibers with a moth-eaten mitochondrial staining pattern have been reported in rats with vitamin E–deficient myopathy25 and 2 previous studies of EMND with NADH-Tr stains of frozen limb muscles5 and succinate dehydrogenase stains of deep gluteal muscle biopsy specimens (depth, 7 to 8 cm).26 However, muscle fibers with a moth-eaten mitochondrial staining pattern are not specific to vitamin E deficiency and have been reported in an equine congenital myopathy and in several human myopathies, sometimes associated with defects in the ryanodine receptor 1 gene.27–29 Normally, mitochondria contain a large amount of vitamin E to counteract the generation of superoxide radicals arising from normal respiratory chain function30 and the high proportion of polyunsaturated fatty acids in their membranes.30–33 A deficiency of vitamin E may damage mitochondrial membranes, resulting in altered oxidative capacity, abnormal mitochondrial staining patterns in muscle biopsy specimens, and signs of generalized muscle weakness.
There was a clear relationship between clinical signs of weakness, low muscle concentration of α-tocopherol, and a moth-eaten mitochondrial staining pattern in sacrocaudalis dorsalis medialis muscle fibers. Clinically normal horses with low muscle concentration of α-tocopherol did not have abnormal sacrocaudalis dorsalis medialis muscle biopsy findings on histologic examination. Thus, it is unlikely that vitamin E deficiency alone was responsible for the presence of mitochondrial abnormalities in horses in the present study. After several months of treating affected horses with vitamin E, the moth-eaten mitochondrial staining pattern of sacrocaudalis dorsalis medialis muscle fibers in biopsy specimens did not resolve, despite resolution of clinical signs. It is possible that the development of mitochondrial alterations in sacrocaudalis dorsalis medialis muscle might be dependent on the duration of vitamin E deficiency, which was unknown in the affected horses and control horses with low muscle concentrations of α-tocopherol. Alternatively, development of muscle fibers with a moth-eaten mitochondrial staining pattern may depend on individual susceptibility of mitochondria to this type of oxidant stress or the percentage of oxidative type 1 muscle fibers in a given muscle.
Where measured in the present study, both sacrocaudalis dorsalis medialis muscle and gluteal muscles of affected horses had low α-tocopherol concentrations; however, histologic mitochondrial abnormalities were only present in the sacrocaudalis dorsalis medialis muscle. This might be explained by the higher percentage of type 1 oxidative fibers in the sacrocaudalis dorsalis medialis muscle versus gluteal muscle, making it more susceptible to oxidant stress. Muscle fibers with a moth-eaten mitochondrial staining pattern have been reported in deep gluteal specimens of horses with EMND,26,34 which contain a higher portion of type 1 fibers than the more superficial middle gluteal muscle biopsy specimens obtained in the present study. Clinically, muscle mass was decreased in most major muscle groups of affected horses in the present study, suggesting that most muscles, including the gluteal muscles, were affected to some degree, not just the sacrocaudalis dorsalis medialis muscle. Generalized muscle atrophy might be explained by the smaller type 2B myofiber sizes in gluteal muscle in affected horses, compared with those in control horses with adequate muscle concentrations of α-tocopherol. Loss of muscle mass and weakness is a feature of mitochondrial myopathies in horses35 and humans,36 and a recent study37 in mice suggests that a mitochondrial stress response may induce gene expression that mimics starvation in a normal nutritional state. A full explanation for the morphological alterations in sacrocaudalis dorsalis medialis muscle and gluteal muscle in affected horses and the relationship to vitamin E requires more extensive investigation. Starvation did not explain the muscle atrophy and weakness observed in affected horses in this study because all horses were on a good plane of nutrition and received vitamins and minerals from a commercial grain mixture or ration balancer. Detailed analysis of each ration was not performed, so other subtle nutrient deficiencies could have been present.
The low muscle concentrations of α-tocopherol in the horses in the present study could have resulted from low amounts of dietary vitamin E or from an individual abnormality in assimilating vitamin E into muscle tissue. Dietary analysis for vitamin E was not determined, and it is unclear whether horses had a major dietary deficiency in vitamin E. A sedentary to lightly exercising 500-kg (1,100-lb) horse requires 500 to 800 U of α-tocopherol/d.38 Almost half of this need could be supplied by a good-quality grass hay fed at 1.5% of body weight/d (30 U/kg of hay × 7.5 kg of hay = 225 U/d). However, the quality of the grass hay fed to the horses in the present study was unknown. For acutely affected horses, the remaining 275 U of vitamin E could have been provided, depending on the quantity available, by pasture, which has high vitamin E content,39 and grain.40 In chronically affected horses, the remaining 275 U may have been provided by the concentrate or ration balancer. Once a diagnosis was established, to further increase dietary vitamin E, feed changes were implemented in all horses, except for 2 chronically affected horses (horses F and H), because of the inability to alter management. Two of the chronically affected horses (horses D and E) were fed commercial concentratej specially formulated for horses with muscle disease that contained higher concentrations of vitamin E than previously fed. One acutely affected horse (horse A) was fed a commercial grain mixture. All affected horses, except 2 of the chronically affected horses (horses F and H), were allowed free access to grass pasture; 3 chronically affected horses (horses D, E, and G) were relocated to farms with grass pasture available. Some of the improvement in muscle mass in these horses could have occurred because of the overall change in diet and higher vitamin E content in pasture grasses.
Serum α-tocopherol concentration alone did not consistently identify vitamin E deficiency in affected horses. Vitamin E status has been routinely determined by serum concentration, but accuracy of results may be questionable as a result of the effects from exposure to light,41 season,42,43 breed,44 and frequency of blood sample collection (ie, single value vs mean of multiple values).45 All affected horses in the present study were deficient in vitamin E on the basis of serum α-tocopherol concentration, except 2 chronically affected horses that had a serum α-tocopherol concentration within reference range (horse D) or had a high serum α-tocopherol concentration (horse E) despite low muscle α-tocopherol concentrations. Incongruence between serum and muscle α-tocopherol concentrations may be due to impaired tissue uptake of vitamin E from blood.46 Determination of muscle rather than serum α-tocopherol concentration may be a more valuable method of assessing vitamin E status in horses suspected of this vitamin E–deficient myopathy. This method requires at least 250 mg of snap-frozen tissue.
In the present study, the amount of vitamin E given to affected horses varied according to clinician preference and the formulation of vitamin E available. Recommendations for vitamin E treatment in horses with EMND are 5,000 to 7,000 U/d.1 All horses in the present study received vitamin E within or above the treatment recommendations, except 3 chronically affected horses (horses F, G, and H) that were part of the University of Minnesota teaching herd, because of financial limitations. However, vitamin El was provided to those horses in a natural water-soluble form that has enhanced absorption and tissue uptake, compared with synthetic or natural acetate formulations. In a recent study,47 horses fed the water-soluble natural vitamin E had higher plasma vitamin E concentrations than those of horses fed either synthetic vitamin E or natural vitamin E acetate at dosages of 500 to 8,000 U/horse/d.
In conclusion, although horses in the present study had clinical signs similar to EMND, they lacked the requisite evidence of substantial neurogenic angular atrophy in the sacrocaudalis dorsalis medialis muscle to establish a diagnosis of EMND. Rather, these horses appeared to have a myopathy characterized by abnormal mitochondria in sacrocaudalis dorsalis medialis muscle that is highly responsive to vitamin E treatment. Fresh, rather than formalin-fixed, muscle biopsy specimens stained with mitochondrial stains are necessary to identify characteristic abnormalities in mitochondrial staining in this vitamin E–deficient myopathy.
ABBREVIATIONS
| EMND | Equine motor neuron disease |
| NADH-Tr | Nicotinamide adenine dinucleotide tetrazolium reductase |
iSolution Lite, IMT, North Hollywood, Calif.
Equine Senior, Purina Mills LLC, Gray Summit, Mo.
Assurance Grass Hay Ration Balancer, Farmer's Mill and Elevator Inc, Castle Rock, Minn.
E-SE, Merck/Schering-Plough, Whitehouse Station, NJ.
Vital E-500, Stuart Products Inc, Bedford, Tex.
Horse Guard Vitamin E 5000, Equine Nutrition Inc, Redmond, Ore.
Horse Guard Maintenance, Equine Nutrition Inc, Redmond, Ore.
SafeChoice, Purina Mills LLC, Gray Summit, Mo.
Elevate liquid suspension, Kentucky Equine Research, Versailles, K y.
Re-leve, Kentucky Equine Research, Versailles, Ky.
Vitamin E 5000, Med-Vet Pharmaceuticals, Eden Prairie, Minn.
Elevate Vitamin E Powder, Kentucky Equine Research, Versailles, K y.
References
1. Divers TJMohammed HOCummings JF. Equine motor neuron disease. Vet Clin North Am Equine Pract 1997 13 97–105.
2. Mohammed HODivers TJSummers BAet al. Vitamin E deficiency and risk of equine motor neuron disease. Acta Vet Scand 2007 49 17.
3. Divers TJMohammed HOCummings JFet al. Equine motor neuron disease: findings in 28 horses and proposal of a pathophysiological mechanism for the disease. Equine Vet J 1994 26 409–415.
4. Rua-Domenech RMohammed HOCummings JFet al. Association between plasma vitamin E concentration and the risk of equine motor neuron disease. Vet J 1997 154 203–213.
5. Valentine BAde Lahunta AGeorge Cet al. Acquired equine motor neuron disease. Vet Pathol 1994 31 130–138.
6. Divers TValentine BJackson CAet al. Simple practical muscle biopsy test for equine motor neuron disease Proceedings1996 180–181.
7. Jackson CADe Lahunta ACummings JFet al. Spinal accessory nerve biopsy as an antemortem diagnostic test for equine motor neuron disease. Equine Vet J 1996 28 215–219.
8. Divers TMohammed H. Equine motor neuron disease. In: Robinson NESprayberry KA, eds. Current therapy in equine medicine 6th ed. St Louis: Saunders Elsevier, 2009 615–617.
9. Divers TJCummings JEde Lahunta Aet al. Evaluation of the risk of motor neuron disease in horses fed a diet low in vitamin E and high in copper and iron. Am J Vet Res 2006 67 120–126.
10. Lindholm APiehl K. Fibre composition, enzyme activity and concentrations of metabolites and electrolytes in muscles of Standardbred horses. Acta Vet Scand 1974 15 287–309.
11. Cumming WJKFulthorpe JJHudgson Pet al. Color atlas of muscle pathology London: Mosby-Wolfe, 1994.
12. De La Corte FDValberg SJMacLeay JMet al. Glucose uptake in horses with polysaccharide storage myopathy. Am J Vet Res 1999 60 458–462.
13. Green SLBouley DMPinter MJet al. Canine motor neuron disease: clinicopathologic features and selected indicators of oxidative stress. J Vet Intern Med 2001 15 112–119.
14. Kleopa KAKyriacou KZamba-Papanicolaou Eet al. Reversible inflammatory and vacuolar myopathy with vitamin E deficiency in celiac disease. Muscle Nerve 2005 31 260–265.
15. Pillai SRTraber MGKayden HJet al. Concomitant brainstem axonal dystrophy and necrotizing myopathy in vitamin E-deficient rats. J Neurol Sci 1994 123 64–73.
16. Kennedy SRice DA. Histopathologic and ultrastructural myocardial alterations in calves deficient in vitamin E and selenium and fed polyunsaturated fatty acids. Vet Pathol 1992 29 129–138.
17. Lofstedt J. White muscle disease of foals. Vet Clin North Am Equine Pract 1997 13 169–185.
18. Ferrans VJVan Vleet JF. Cardiac lesions of selenium-vitamin E deficiency in animals. Heart Vessels Suppl 1985 1 294–297.
19. Wilson TMMorrison HAPalmer NCet al. Myodegeneration and suspected selenium/vitamin E deficiency in horses. J Am Vet Med Assoc 1976 169 213–217.
20. Owen RRMoore JNHopkins JBet al. Dystrophic myodegeneration in adult horses. J Am Vet Med Assoc 1977 171 343–349.
21. Mayhew IGBrown CMStowe HDet al. Equine degenerative myeloencephalopathy: a vitamin E deficiency that may be familial. J Vet Intern Med 1987 1 45–50.
22. Baumgartner WFrese KElmadfa I. Neuroaxonal dystrophy associated with vitamin E deficiency in two Haflinger horses. J Comp Pathol 1990 103 114–119.
23. Banker BQEngel AG. Basic reactions of muscle. In: Engel AGFranzini-Armstrong C, eds. Myology 2nd ed. New York: McGraw-Hill, 1994 832–888.
24. Larsson BBjork JKadi Fet al. Blood supply and oxidative metabolism in muscle biopsies of female cleaners with and without myalgia. Clin J Pain 2004 20 440–446.
25. Thomas PKCooper JMKing RHet al. Myopathy in vitamin E deficient rats: muscle fibre necrosis associated with disturbances of mitochondrial function. J Anat 1993 183 451–461.
26. Palencia PQuiroz-Rothe ERivero JL. New insights into the skeletal muscle phenotype of equine motor neuron disease: a quantitative approach. Acta Neuropathol (Berl) 2005 109 272–284.
27. Ferreiro AEstournet BChateau Det al. Multi-minicore disease—searching for boundaries: phenotype analysis of 38 cases. Ann Neurol 2000 48 745–757.
28. Mitsuhashi SNonaka IWu Set al. Distal myopathy in multi-minicore disease. Intern Med 2009 48 1759–1762.
29. Pietrini VMarbini AGalli Let al. Adult onset multi/minicore myopathy associated with a mutation in the RYR1 gene. J Neurol 2004 251 102–104.
30. Buttriss JLDiplock AT. The relationship between alpha-tocopherol and phospholipid fatty acids in rat liver subcellular membrane fractions. Biochim Biophys Acta 1988 962 81–90.
31. Oliveira MMWeglicki WBNason Aet al. Distribution of alpha-tocopherol in beef heart mitochondria. Biochim Biophy Acta 1969 180 98–113.
32. Bonetti ENovello F. Distribution of H3-tocopherol in rat tissues and subcellular particles. Int J Vitam Nutr Res 1976 46 244–247.
33. Boveris AOshino NChance B. The cellular production of hydrogen peroxide. Biochem J 1972 128 617–630.
34. Sewell DAHarris RCMarlin DJ. Skeletal muscle characteristics in 2 year-old race-trained Thoroughbred horses. Comp Biochem Physiol Comp Physiol 1994 108 87–96.
35. Valberg SJCarlson GPCardinet GH IIIet al. Skeletal muscle mitochondrial myopathy as a cause of exercise intolerance in a horse. Muscle Nerve 1994 17 305–312.
36. DiMauro SBonilla E. Mitochondrial encephalomyopathies. In: Engel AGFranzini-Armstrong C, eds. Myology: basic and clinical 3rd ed. New York: McGraw-Hill, 2004 1623–1662.
37. Tyynismaa HCarroll CJRaimundo Net al. Mitochondrial myopathy induces a starvation-like response. Hum Mol Genet 2010 19 3948–3958.
38. National Research Council Nutrient requirements of horses 6th ed. Washington, DC: National Academies Press, 2007.
39. Thafvelin BOksanen HE. Vitamin E and linolenic acid content of hay as related to different drying conditions. J Dairy Sci 1966 49 282–286.
40. Lennox M. Hard to find nutrients for ration evaluations: filling in the holes Nottingham, Nottinghamshire, England: Nottingham University Press, 2001 135–141.
41. Craig AMBlythe LLRowe KEet al. Variability of alpha-tocopherol values associated with procurement, storage, and freezing of equine serum and plasma samples. Am J Vet Res 1992 53 2228–2234.
42. Maenpaa PHKoskinen TKoskinen E. Serum profiles of vitamins A, E and D in mares and foals during different seasons. J Anim Sci 1988 66 1418–1423.
43. Blackley BRBell RJ. The Vitamin A and E status of horses raised in Alberta and Saskatchewan. Can Vet J 2011 35 297–300.
44. Steiss JETraber MGWilliams MAet al. Alpha tocopherol concentrations in clinically normal adult horses. Equine Vet J 1994 26 417–419.
45. Craig AMBlythe LLLassen EDet al. Variations of serum vitamin E, cholesterol, and total serum lipid concentrations in horses during a 72-hour period. Am J Vet Res 1989 50 1527–1531.
46. Mardones PStrobel PMiranda Set al. Alpha-tocopherol metabolism is abnormal in scavenger receptor class B type I (SRBI)-deficient mice. J Nutr 2002 132 443–449.
47. Fiorellino NMLamprecht EDWilliams CA. Absorption of different formulations of natural vitamin E in horses. J Equine Vet Sci 2009 29 100–105.
