Skeletal Muscle Fiber Type Composition and Citrate Synthase Activity in Fit and Unfit Warmbloods and Quarter Horses.

Selective breeding and discipline specific training has led to equine breeds adept at various athletic disciplines. Breed-specific skeletal muscle adaptations have been studied in many breeds but not Warmbloods (WB). We evaluated gluteal muscle contractile muscle fiber types and citrate synthase activity (CS), a marker for mitochondrial volume density, in WB trained for dressage (second level-Grand Prix) contrasted with Quarter Horses (QH). Gluteus medius muscle biopsies from 14 unfit/18 fit dressage-trained WB and 20 unfit/16 fit reining/working cow QH were analyzed fluorometrically and fiber types determined by ATPase activity. Comparisons were made by one-way ANOVA. Unfit and fit WB had significantly higher % type 1 and lower % type 2X fibers than QH. Unfit WB had significantly higher CS than unfit QH but CS did not differ between fit WB and fit QH. CS was only significantly higher in fit versus unfit QH, not fit versus unfit WB. In conclusion, WB gluteal muscle has an inherently high % type 1/low % type 2X fibers and high mitochondrial content whether unfit or trained for dressage, contrasting QH with an inherently low % type 1/high % type 2X and low mitochondrial content, that was enhanced in fit QH. Similar CS activity in fit WB versus QH despite a two-fold difference in % type 2X fibers indicates that mitochondrial volume density cannot accurately be predicted from contractile fiber type composition.

The use of prolonged administration of low-dose intravenous lipid emulsion to treat ivermectin toxicosis in goats.

CASE DESCRIPTION: 2 Nigerian Dwarf goats (a doe [goat 1] and a wether [goat 2]) with coughing and nasal discharge since they were purchased at an auction 6 days prior were empirically treated for suspected pneumonia and intestinal parasitism. An ivermectin dosing error (intended dose, 0.4 mg/kg, PO; administered dose, 10 mg/kg, PO) was retrospectively discovered, and the owner was urged to return the goats for hospitalization and treatment. CLINICAL FINDINGS: On admission 19 hours after iatrogenic ivermectin overdose, both goats had tachycardia, tachypnea, and absent menace responses. Goat 1 also had vomited in transit, was lethargic and febrile, had slow pupillary light reflexes, and walked into walls and obstacles. Goat 2 was quiet but responsive, not ataxic or febrile, and had pale mucous membranes and a prolonged capillary refill time. TREATMENT AND OUTCOME: Each goat received 20% IV lipid emulsion (2 mL/kg, IV bolus over 15 minutes, followed by 0.008 mL/kg/min, IV) and immediately improved. Activated charcoal was administered by orogastric tube, and 6 hours later, mineral oil was similarly administered. Goat 1 had complete resolution of signs and was discharged by 48 and 72 hours, respectively, after admission. Goat 2 improved but developed progressive respiratory distress after the second orogastric intubation and was euthanized. Necropsy findings were consistent with acute renal tubular necrosis, acute respiratory distress syndrome of unknown cause, ruminal tympany, and mesenteric caseous lymphadenitis. CLINICAL RELEVANCE: Results indicated that IV lipid emulsion could be used to successfully treat ivermectin toxicosis in goats. Treatment early in the course of ivermectin toxicosis is advisable to avoid severe clinical signs and secondary complications.

Black oil sunflower seed ingestion associated with renal azotemia, gastroesophageal ulceration, and a high mortality rate in four alpacas and two llamas.

CASE DESCRIPTION: 4 alpacas and 2 llamas (11 months to 11 years old) from 2 properties were examined for lethargy (6/6), salivation and regurgitation (4/6), and recumbency (3/6). Signs developed approximately 48 to 72 hours after accidental access to black oil sunflower seeds. CLINICAL FINDINGS: 3 alpacas died suddenly prior to treatment and were necropsied. One llama survived, and 1 alpaca and 1 llama died after days of medical treatment. All 3 treated animals had systemic inflammatory signs including tachycardia, fever, and hematologic changes. Biochemical anomalies included azotemia, hyperglycemia, hyponatremia, hypochloremia, and hypoalbuminemia. Necropsy identified numerous sunflower seeds in the gastrointestinal tract of all 5 animals that died, with pulmonary congestion (5/5 animals), mild centrilobular vacuolar hepatic degeneration (4/5), and erosions of the esophagus (3/5) and first (3/5) and third (1/5) compartments of the forestomach. Renal tubular necrosis was found in the 2 animals that died on day 4 of treatment. TREATMENT AND OUTCOME: One llama responded successfully to intensive medical management including supplemented IV fluid therapy, oral and partial parenteral nutrition, and administration of antimicrobials, furosemide, and insulin and was clinically normal with plasma biochemical analysis values within reference range 12 weeks later. Vitamin D, oxalates, heavy metals, and mycotoxins were excluded as the cause of clinical signs on the basis of screening of uneaten seeds and tissue samples and gastric content from the treated llama that died. CLINICAL RELEVANCE: Inadvertent large volume black oil sunflower seed ingestion resulted in a high mortality rate in camelids. A specific toxic principle was not identified. Feeding this product to camelids is not recommended to avoid the risk of accidental overingestion and subsequent disease.

Lymphoplasmacytic and eosinophilic enteritis with or without globule leukocyte hyperplasia in 4 goats.

Four, mature, client-owned goats were presented to referral hospitals for recurrent diarrhea despite treatment for intestinal parasitism. Common clinical findings included diarrhea, poor condition, neutrophilia, and hypoalbuminemia. Testing for common infectious causes of diarrhea in goats was negative. Ultrasonography and computed tomography in 2 cases was suggestive of enteritis, including thickened intestinal walls and fluid filled, dilated small intestines, respectively. Lymphoplasmacytic and eosinophilic enteritis (LEE) was ultimately diagnosed on intestinal biopsy histopathology based on the presence of small intestinal villous blunting and increased numbers of lymphocytes and eosinophils predominantly within the lamina propria. Numerous globule leukocytes were also noted on histopathology in 3 cases. All goats responded favorably to corticosteroid treatment with weight gain and resolution of diarrhea and clinicopathologic abnormalities. Relapses occurred, and complete cure was difficult to achieve. Reported in other species, this series describes the clinical presentation, diagnosis, and treatment of LEE in adult goats.

Nutritional Influences on Skeletal Muscle and Muscular Disease.

Skeletal muscle comprises 40% to 55% of mature body weight in horses, and its mass is determined largely by rates of muscle protein synthesis. In order to support exercise, appropriate energy sources are essential: glucose can support both anaerobic and aerobic exercise, whereas fat can only be metabolized aerobically. Following exercise, ingestion of nonfiber carbohydrates and protein can aid muscle growth and recovery. Muscle glycogen replenishment is slow in horses, regardless of dietary interventions. Several heritable muscle disorders, including type 1 and 2 polysaccharide storage myopathy and recurrent exertional rhabdomyolysis, can be managed in part by restricting dietary nonstructural carbohydrate intake.

Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis.

BACKGROUND: Commercial genetic tests for type 2 polysaccharide storage myopathy (PSSM2) and myofibrillar myopathy (MFM) have not been validated by peer-review, and formal regulation of veterinary genetic testing is lacking. OBJECTIVES: To compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323), FLNC (P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Warmblood (WB) and Arabian (AR) horses diagnosed with PSSM2/MFM by muscle histopathology, and phenotyped breed-matched controls. To quantify variant frequency in public repositories of ancient and modern horse breeds. STUDY DESIGN: Cross sectional using archived clinical material and publicly available data. METHODS: We studied 54 control-WB, 68 PSSM2/MFM-WB, 30 control-AR, 30 PSSM2/MFM-AR and 205 public genotypes. Variants were genotyped by pyrosequencing archived DNA. Genotype and allele frequency, and number of variant alleles or loci were compared within breed between controls, PSSM2/MFM combined and MFM or PSSM2 horses considered separately using additive/genotypic and dominant models (Fisher's exact tests). Variant frequencies in modern, early domestic and Przewalski horses were determined from a public data repository. RESULTS: There was no significant association between any P locus and a histopathological diagnosis of PSSM2/MFM, and no difference between control and myopathic horses in total loci with alternative alleles, or total alternate alleles when PSSM2/MFM was considered combined or separately as PSSM2 or MFM. For all tests, sensitivity was <0.33. Allele frequencies in WB (controls/cases) were: 8%/15% (P2), 5%/6% (P3a/b) and 9%/13% (P4); in AR, frequencies were: 12%/17% (P2), 2%/2% (P3a/b) and 7%/12% (P4). All P variants were present in early domestic (400- to 5500-year-old) horses and P2 present in the Przewalski. CONCLUSIONS: Because of the lack of significant association between a histopathological diagnosis of PSSM2 or MFM and the commercial genetic test variants P2, P3 and P4 in WB and AR, we cannot recommend the use of these variant genotypes for selection and breeding, prepurchase examination or diagnosis of a myopathy.

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle.

Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed.

Influence of specific management practices on blood selenium, vitamin E, and beta-carotene concentrations in horses and risk of nutritional deficiency.

BACKGROUND: Selenium or alpha-tocopherol deficiency can cause neuromuscular disease. Beta-carotene has limited documentation in horses. OBJECTIVE: To evaluate the effect of owner practices on plasma beta-carotene concentration and risk of selenium and alpha-tocopherol deficiencies. ANIMALS: Three-hundred and forty-nine adult (≥1 year), university and privately owned horses and mules. METHODS: Cross-sectional study. Whole blood selenium, plasma alpha-tocopherol, and plasma beta-carotene concentrations were measured once. Estimates of daily selenium and vitamin E intake, pasture access, and exercise load were determined by owner questionnaire. Data were analyzed using t tests, Mann-Whitney tests, parametric or nonparametric analysis of variance (ANOVA), Kruskal-Wallis test, Spearman's correlation and contingency tables (P < .05). RESULTS: Nearly 88% of the horses received supplemental selenium; 71.3% received ≥1 mg/d. Low blood selenium concentration (<80 ng/mL) was identified in 3.3% of horses, and 13.6% had marginal concentrations (80-159 ng/mL). Non-supplemented horses were much more likely to have low blood selenium (odds ratio [OR], 20.2; 95% confidence interval [CI], 9.26-42.7; P < .001). Supplemental vitamin E was provided to 87.3% of horses; 57.7% received ≥500 IU/d. Deficient (<1.5 µg/mL) and marginal (1.5-2.0 µg/mL) plasma (alpha-tocopherol) occurred in 15.4% and 19.9% of horses, respectively. Pasture access (>6 h/d) and daily provision of ≥500 IU of vitamin E was associated (P < .001) with higher plasma alpha-tocopherol concentrations. Plasma beta-carotene concentration was higher in horses with pasture access (0.26 ± 0.43 versus 0.12 ± 0.13 µg/mL, P = .003). CONCLUSIONS AND CLINICAL IMPORTANCE: Suboptimal blood selenium and plasma alpha-tocopherol concentrations occurred in 16.7% and 35.5% of horses, respectively, despite most owners providing supplementation. Inadequate pasture access was associated with alpha-tocopherol deficiency, and reliance on selenium-containing salt blocks was associated with selenium deficiency.

Viral testing of 10 cases of Theiler's disease and 37 in-contact horses in the absence of equine biologic product administration: A prospective study (2014-2018).

BACKGROUND: A novel equine parvovirus (EqPV-H) was recently discovered in the equine liver with Theiler's disease. OBJECTIVES: To determine the prevalence of EqPV-H infection in naturally occurring Theiler's disease cases and in-contact horses in the absence of historical equine biologic product administration. ANIMALS: Ten cases of Theiler's disease from 6 separate properties were included in the study, based on the criteria of acute onset of clinical signs of liver failure with laboratory or histopathologic findings characteristic of Theiler's disease and no history of receiving an equine biologic product within the preceding 4 months. In addition, 37 in-contact horses from 4 of the 6 properties were screened for EqPV-H infection and hepatitis. METHODS: In prospective case series, cases were diagnosed with Theiler's disease by the attending veterinarian and were tested for EqPV-H by PCR of liver or serum. In-contact horses were assessed via serum chemistry and PCR at the attending veterinarian's discretion. Hepatitis was defined as serum gamma-glutamyltransferase activity above reference interval. The association of EqPV-H with hepatitis was determined by Fisher's exact test. RESULTS: Nine of 10 (90%) Theiler's disease cases and 54% of tested in-contact horses were EqPV-H positive. Hepatitis was significantly associated with EqPV-H infection (P = .036). CONCLUSIONS AND CLINICAL IMPORTANCE: Although further study is required to identify EqPV-H as the causative agent of Theiler's disease, EqPV-H appears strongly associated with cases of fatal Theiler's disease and subclinical hepatitis in horses in contact with those cases. The prevalence of EqPV-H infection on affected properties can be high.

Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways.

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase ( CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter ( SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin ( ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin ( DSP ↑2.4 MFM), and basement membrane usherin ( USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.