Search

Horses have a slow rate of muscle glycogen repletion relative to other species for unknown reasons. Our aim was to determine the expression of glucose transporters ( GLUT ) and genes impacting GLUT4 expression and translocation in the gluteal muscle. Five fit Thoroughbred horses performed glycogen-depleting exercises on high-starch (HS, 2869 g starch/day) and low-starch, high-fat diets (LS-HF, 358 g starch/d) with gluteal muscle biopsies obtained before and after depletion and during repletion. Muscle glycogen declined by ≈30% on both diets with little increase during repletion on LS-HF. Transcriptomic analysis identified differential expression (DE) of only 2/12 genes impacting GLUT4 translocation (two subunits of AMP protein kinase) and only at depletion on LS-HF. Only 1/13 genes encoding proteins that promote GLUT4 transcription had increased DE ( PPARGC1A at depletion LS-HF). GLUT4 comprised ≈30% of total GLUT mRNA expression at rest. Remarkably, by 72 h of repletion expression of GLUT3 , GLUT6 and GLUT10 increased to ≈25% of total GLUT mRNA. Expression of GLUT6 and GLUT10 lagged from 24 h of repletion on HS to 72 h on LS-HF. Lacking an increase in GLUT4 gene expression in response to glycogen-depleting exercise, equine muscle increases GLUT3 , GLUT6 and GLUT10 expression potentially to enhance glucose transport, resembling responses observed in resistance trained GLUT4-null mice.

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 ( p = 0.07), GSH ( p = 0.75), and malondialdehyde ( p = 0.47), as well as activities of superoxide dismutase ( p = 0.16) and catalase ( p = 0.66), did not differ, whereas glutathione peroxidase activity ( p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (E CII ) was greater, and the contribution of complex I to maximal electron transfer capacity (FCR PCI and FCR PCIG ) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.

Objective: To assess the relationship between plasma and RBC fatty acid composition and incidence and severity of squamous gastric ulcers when altered by short-chain (SC) or long-chain (LC) polyunsaturated fatty acid (PUFA) supplementation., Animals: 13 fit Thoroughbred horses in training., Procedures: Horses were evaluated by gastroscopy for squamous ulcer score, gastric pH, and blood fatty acid composition prior to supplementation (UNSUPP) and after 3 months of supplementation with a corn-flax oil blend of alpha-linolenic acid and linoleic acid (SC-PUFA) or a gamma-linolenic acid (GLA)-fish oil blend of GLA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA; LC-PUFA) in a crossover design. Prior to gastroscopy and blood collection, horses performed a 4,600-m standardized exercise test on the racetrack as a stressor., Results: Three months of supplementation with LC-PUFAs increased RBC levels of GLA, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), EPA, and DHA, and reduced severe ulcer prevalence (38% UNSUPP vs 8% LC-PUFA with a severe ulcer score of grade 3 to 4). Short-chain PUFA supplementation did not effectively elevate RBC GLA, DGLA, AA, EPA, or DHA and severe ulcer incidence was not different (38% UNSUPP vs 23% SC-PUFA with a severe ulcer score of grade 3 to 4). Lower levels of RBC GLA, DGLA, AA, and EPA correlated with severe squamous gastric ulceration (grade 3 to 4)., Clinical Relevance: Equine gastric ulcer syndrome is prevalent in high-performance horses and is a concern to owners and trainers. Long-chain PUFA supplementation increased levels of GLA, DGLA, AA, EPA, and DHA, unlike SC-PUFA supplementation, and was associated positively with prevention or resolution of severe squamous gastric ulceration. Further studies are needed to evaluate different management styles and exercise intensities.

Horses have one of the highest skeletal muscle oxidative capacities amongst mammals, which, combined with a high glycolytic capacity, could perturb redox status during maximal exercise. We determined the effect of 30 d of oral coenzyme Q10 and N-acetyl-cysteine supplementation (NACQ) on muscle glutathione (GSH), cysteine, ROS, and coenzyme Q10 concentrations, and the muscle proteome, in seven maximally exercising Thoroughbred horses using a placebo and randomized cross-over design. Gluteal muscle biopsies were obtained the day before and 1 h after maximal exercise. Concentrations of GSH, cysteine, coenzyme Q10, and ROS were measured, and citrate synthase, glutathione peroxidase, and superoxide dismutase activities analyzed. GSH increased significantly 1 h post-exercise in the NACQ group ( p = 0.022), whereas other antioxidant concentrations/activities were unchanged. TMT proteomic analysis revealed 40 differentially expressed proteins with NACQ out of 387 identified, including upregulation of 13 mitochondrial proteins (TCA cycle and NADPH production), 4 Z-disc proteins, and down regulation of 9 glycolytic proteins. NACQ supplementation significantly impacted muscle redox capacity after intense exercise by enhancing muscle glutathione concentrations and increasing expression of proteins involved in the uptake of glutathione into mitochondria and the NAPDH-associated reduction of oxidized glutathione, without any evident detrimental effects on performance.

Generation of high-affinity IgG is essential for defense against infections and cancer, which is the intended consequence of many vaccines, but can cause autoimmune and inflammatory diseases when inappropriately directed against self. The interplay of T follicular helper (T FH ) cells and T follicular regulatory (T FR ) cells is critical for the production of high-affinity IgG of a specific subclass. In this study, we sought to improve Ag-specific IgG responses with two interventions intended to transiently diminish T FR cell influence. First, adult mice were administered an antibiotic mixture (ABX) for an extended period to deplete the immunoregulatory intestinal microbiota. This intriguingly increased T FH cell and reduced T FR cell numbers. 2,4,6-Trinitrophenyl hapten conjugated to keyhole limpet hemocyanin immunization resulted in higher affinity 2,4,6-trinitrophenyl hapten-specific IgG1 in ABX mice compared with controls. In a model of IgG-driven inflammatory nephritis, ABX mice had significantly worse nephritis accompanied by higher affinity Ag-specific IgG2b and enriched T FH cells compared with controls. Second, we sought to functionally manipulate T FH and T FR cells, which both express the checkpoint inhibitory molecule, PD-1, by administration of anti-PD-1 during immunization. This intervention enhanced the affinity of Ag-specific IgG of the appropriate subclass and increased in T FH cells following 2,4,6-trinitrophenyl hapten conjugated to keyhole limpet hemocyanin immunization and nephritis induction. These results suggest that altering T FH and T FR cell ratios during immunization is an appealing strategy to qualitatively improve Ag- and subclass-specific IgG responses., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Proton pump inhibitors such as omeprazole reduce nutrient digestibility in humans. This study determined the effect of omeprazole on the digestibility of diets containing limestone or marine-derived calcium (BMC) and to assess changes in blood parameters associated with gastric acid production and calcium status in horses. Thoroughbreds were used to evaluate the digestibility of diets containing different calcium sources with or without omeprazole over four 21-day periods. Each 21-day period had a 15-day diet adaptation phase followed by a 6-day collection phase, consisting of a 5-day total fecal collection period and a final day for gastroscopy and blood sampling. Horses were fed the same diet with either 60 g/d BMC or 50 g/d limestone, so the total diet provided ∼45 g calcium. Horses on omeprazole were given GastroGard once daily for the final 14 day of each 21-day period, which supplied 3.91 ± 0.17 mg/kg BW/d of omeprazole. On day 21, blood samples were taken and gastric fluid pH was measured 8 hour after omeprazole administration. Omeprazole had a profound effect on gastric fluid pH in omeprazole-treated horses compared with nontreated horses. Serum gastrin doubled in omeprazole-treated horses compared with nontreated horses. Omeprazole and calcium source did not affect digestibility of phosphorus, magnesium, potassium, sodium, iron, copper, zinc, or manganese but did affect calcium digestibility. Omeprazole reduced apparent calcium digestibility from 52.0% to 41.4% in limestone and from 55.1% to 46.5% in BMC, equalling a 20.3% and 15.6% decrease in calcium digestibility in the limestone and BMC, respectively. Mineral source had a significant effect on calcium digestibility with BMC at 50.8% and limestone at 46.7%., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Observations of microbial dysbiosis in patients with rheumatoid arthritis (RA) have raised interest in studying microbial-mucosal interactions as a potential trigger of RA. Using the murine collagen-induced arthritis (CIA) model, we undertook this study to test our hypothesis that microbiota modulate immune responses leading to autoimmune arthritis., Methods: CIA was induced by immunization of mice with type II collagen (CII) in adjuvant on days 0 and 21, with arthritis appearing on days 23 and 24. Intestinal microbiota were profiled by 16S ribosomal RNA sequencing every 7 days during the course of CIA, and intestinal mucosal changes were evaluated on days 14 and 35. Then, microbiota were depleted either early (7 days before immunization) or late (day 21 after immunization) by administration of broad-spectrum antibiotics. Disease severity, autoantibody and systemic cytokine production, and intestinal mucosal responses were monitored in the setting of microbial reduction., Results: Significant dysbiosis and mucosal inflammation occurred early in CIA, prior to visible arthritis, and continued to evolve during the course of disease. Depletion of the microbiota prior to the induction of CIA resulted in an ~40% reduction in disease severity and in significantly reduced levels of serum inflammatory cytokines and anti-CII antibodies. In intestinal tissue, production of interleukin-17A (IL-17A) and IL-22 was delayed. Unexpectedly, microbial depletion during the late phase of CIA resulted in a >50% decrease in disease severity. Anti-CII antibodies were mildly reduced but were significantly impaired in their ability to activate complement, likely due to altered glycosylation profiles., Conclusion: These data support a model in which intestinal dysbiosis triggers mucosal immune responses that stimulate T and B cells that are key for the development of inflammatory arthritis., (© 2018, American College of Rheumatology.)

Self-reactive IgGs contribute to the pathology of autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. Paradoxically, IgGs are used to treat inflammatory diseases in the form of high-dose intravenous immunoglobulin (IVIG). Distinct glycoforms on the IgG crystallizable fragment (Fc) dictate these divergent functions. IgG anti-inflammatory activity is attributed to sialylation of the Fc glycan. We therefore sought to convert endogenous IgG to anti-inflammatory mediators in vivo by engineering solubilized glycosyltransferases that attach galactose or sialic acid. When both enzymes were administered in a prophylactic or therapeutic fashion, autoimmune inflammation was markedly attenuated in vivo. The enzymes worked through a similar pathway to IVIG, requiring DC-SIGN, STAT6 signaling, and FcγRIIB. Importantly, sialylation was highly specific to pathogenic IgG at the site of inflammation, driven by local platelet release of nucleotide-sugar donors. These results underscore the therapeutic potential of glycoengineering in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A growing body of evidence supports the integration of palliative care with standard cancer treatments. In these situations, patients often experience a better quality of life, better quality of care, decreased cost, and, in some cases, improved survival with the addition of palliative care services to traditional treatment pathways. In this manuscript, we explore the integration of radiation oncology at palliative care. First, we discuss the impetus for change at Vanderbilt University and the inception of Vanderbilt's inpatient Palliative Radiation Oncology Service at Vanderbilt. Second, we discuss the growth of palliative care and how this invites innovative collaborative care delivery models. As you will see, this mutually beneficial relationship expands new service lines, brings radiation oncology interventions and expertise to more patients seen by palliative care specialists, and improves overall care for some of the sickest, most vulnerable patients in the health care system. As we move away from fee-for-service and toward bundled and global-based strategies, there will be further emphasis on supportive and palliative care services at the end of life. Understanding how radiation oncology can evolve is ever more relevant., (Copyright © 2016 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

Epigenetic "readers" that recognize defined posttranslational modifications on histones have become desirable therapeutic targets for cancer and inflammation. SP140 is one such bromodomain- and plant homeodomain (PHD)-containing reader with immune-restricted expression, and single-nucleotide polymorphisms (SNPs) within SP140 associate with Crohn's disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unclear. We show that SP140 is critical for transcriptional programs that uphold the macrophage state. SP140 preferentially occupies promoters of silenced, lineage-inappropriate genes bearing the histone modification H3K27me3, such as the HOXA cluster in human macrophages, and ensures their repression. Depletion of SP140 in mouse or human macrophages resulted in severely compromised microbe-induced activation. We reveal that peripheral blood mononuclear cells (PBMCs) or B cells from individuals carrying CD-associated SNPs within SP140 have defective SP140 messenger RNA splicing and diminished SP140 protein levels. Moreover, CD patients carrying SP140 SNPs displayed suppressed innate immune gene signatures in a mixed population of PBMCs that stratified them from other CD patients. Hematopoietic-specific knockdown of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)-induced colitis, and low SP140 levels in human CD intestinal biopsies correlated with relatively lower intestinal innate cytokine levels and improved response to anti-tumor necrosis factor (TNF) therapy. Thus, the epigenetic reader SP140 is a key regulator of macrophage transcriptional programs for cellular state, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by ineffective innate immunity, normally critical for intestinal homeostasis., (Copyright © 2017, American Association for the Advancement of Science.)

Background: We sought to validate the consensus recommendation and assess dosimetric significance of selective omission of nodal level V from intensity-modulated radiotherapy (IMRT) clinical target volume (CTV) for oropharyngeal cancer., Methods: IMRT plans and clinical outcomes for 112 patients with oropharyngeal cancer (nodal classification N0-N2b) were analyzed for coverage of ipsilateral and contralateral nodal level V. Additionally, new IMRT plans were generated in 6 randomly selected patients to assess its dosimetric impact., Results: With median follow-up of 3.4 years, there were no failures identified in nodal level V with or without nodal level V omission. Upon dosimetric evaluation, significant reduction in integral dose, V10 Gy , V20 Gy , V30 Gy , V40 Gy , and V50 Gy was observed by excluding unilateral and bilateral level V from the CTV., Conclusion: We clinically validate the consensus recommendation for selective omission of level V nodal coverage in IMRT planning of patients with oropharyngeal cancer and demonstrate significant dosimetric advantages., (© 2016 Wiley Periodicals, Inc.)

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology., (© 2015 Shade et al.)

Objective: To evaluate effects of fats with odd and even numbers of carbon atoms on muscle metabolism in exercising horses with polysaccharide storage myopathy (PSSM)., Animals: 8 horses with PSSM (6 females and 2 males; mean +/- SD age, 6.3 +/- 3.9 years)., Procedures: Isocaloric diets (grain, triheptanoin, corn oil, and high-fat, low-starch [HFLS] feed) were fed for 3 weeks each; horses performed daily treadmill exercise. Grain was fed to establish an exercise target, and HFLS feed was fed as a negative control diet. Daily plasma samples were obtained. For each diet, a 15-minute exercise test was performed, and gluteus medius muscle specimens and blood samples were obtained before and after exercise., Results: Feeding triheptanoin, compared with the corn oil diet, resulted in exercise intolerance; higher plasma creatine kinase (CK) activity and concentrations of C3:0- and C7:0-acylcarnitine and insulin; and lower concentrations of nonesterified fatty acids (NEFA) and C16:0-, C18:1-, and C18:2-acylcarnitine, without changes in concentrations of plasma glucose or resting muscle substrates and metabolites. Feeding grain induced higher CK activity and insulin concentrations and lower NEFA concentrations than did corn oil or HFLS feed. Feeding grain induced higher glucose concentrations than did triheptanoin and corn oil. In muscle, feeding grain resulted in lower glucose-6-phosphate, higher citrate, and higher postexercise lactate concentrations than did the other diets., Conclusions and Clinical Relevance: Triheptanoin had detrimental effects, reflecting decreased availability of NEFA, increased insulin stimulation of glycogen synthesis, and potential inhibition of lipid oxidation. Long-chain fats are the best dietetic for PSSM.

Objective: To compare effects of corn oil or a 7-carbon fat (triheptanoin) on acylcarnitine, lipid, and carbohydrate metabolism in plasma or muscle of exercising horses., Animals: 8 Thoroughbred geldings., Procedures: Horses received isocaloric diets containing 650 mL of oil (triheptanoin or corn oil)/d for 18 or 25 days in a crossover design with a 26-day washout period. On day 17 or 24 of each feeding period, the respective oil (217 mL) was nasogastrically administered; 120 minutes later, horses performed a 90-minute submaximal exercise test (SET). Blood and muscle samples were obtained before oil administration and immediately before (blood only), during (blood only), immediately after, and 24 hours after SETs., Results: Compared with values before oil administration, triheptanoin administration increased plasma insulin and C7:0-, C5:0- and C3:0-acylcarnitine concentrations, whereas corn oil administration increased plasma NEFA concentrations. During SETs, plasma C7:0-, C5:0-, and C3:0-acylcarnitine concentrations were higher when triheptanoin, rather than corn oil, was administered to horses. Plasma glucose, NEFA, and C2:0-, C18:1-, and C18:2-acylcarnitine concentrations increased during SETs similarly for both oils. Respiratory quotient and muscle lactate, citrate, malate, glycogen, and ATP concentrations changed similarly from before to after SETs for both oils. Compared with muscle concentrations immediately after SETs, those for glucose-6-phosphate and citrate 24 hours after SETs were lower and for glycogen were similar to values before SETs., Conclusions and Clinical Relevance: Fatigue was not associated with depletion of citric acid cycle intermediates for either oil. Triheptanoin induced a significantly higher insulin secretion and did not appear to enhance muscle glycogen repletion.

The effect of dietary starch and fat content on serum creatine kinase (CK) activity and substrate availability was evaluated in 4 mares of Quarter Horse-related breeds with polysaccharide storage myopathy (PSSM). Four isocaloric diets ranging in digestible energy (DE) from 21.2% (diet A), 14.8% (B), 8.4% (C), to 3.9% (D) for starch, and 7.2% DE (diet A), 9.9% (B), to 12.7% DE (diet C and D) for fat were fed for 6-week periods (4 weeks with exercise) using a 4 X 4 Latin square design. Postprandial glucose and insulin responses were measured, and 4 hours postexercise, serum CK activity, glucose, insulin, free fatty acids (FFA), and beta-hydroxybutyrate (beta-HBA) were analyzed. Glycogen, glucose-6-phosphate, citrate synthase, 3-hydroxy-acyl-CoA dehydrogenase, lactate dehydrogenase as well as abnormal polysaccharide and lipid content were measured in middle gluteal muscle samples. Postprandial insulin and glucose response was higher for diet A versus D. Log CK activity was higher with diets A, B, and C versus D. Daily insulin was higher and FFA lower on diet A versus B, C, and D, whereas glucose varied only slightly with diet. Muscle oxidative capacity and lipid stores were low in PSSM horses and muscle glycogen and abnormal polysaccharide content high on both diets A and D. Individual variation occurred in the response of PSSM horses to diets differing in starch and fat content. However, for those horses with clinical manifestations of PSSM, a diet with <5% DE starch and >12% DE fat can reduce exertional rhabdomyolysis, potentially by increasing availability of FFA for muscle metabolism.

To determine the effect of dietary starch, bicarbonate, and fat content on metabolic responses and serum creatine kinase (CK) activity in exercising Thoroughbreds with recurrent exertional rhabdomyolysis (RER), 5 RER horses were fed 3 isocaloric diets (28.8 Mcal/d [120.5 MJ/d]) for 3 weeks in a crossover design and exercised for 30 minutes on a treadmill 5 days/wk. On the last day of each diet, an incremental standardized exercise test (SET) was performed. The starch diet contained 40% digestible energy (DE) as starch and 5% as fat: the bicarbonate-starch diet was identical but was supplemented with sodium bicarbonate (4.2% of the pellet): and the fat diet provided 7% DE as starch and 20% as fat. Serum CK activity before the SET was similar among the diets. Serum CK activity (log transformed) after submaximal exercise differed dramatically among the diets and was greatest on the bicarbonate-starch diet (6.51 +/- 1.5) and lowest on the fat diet (5.71 +/- 0.6). Appreciable differences were observed in the severity of RER among individual horses. Postexercise plasma pH, bicarbonate concentration, and lactate concentration did not differ among the diets. Resting heart rates before the SET were markedly lower on the fat diet than on the starch diet. Muscle lactate and glycogen concentrations before and after the SET did not differ markedly among the diets. A high-fat, low-starch diet results in dramatically lower postexercise CK activity in severely affected RER horses than does a low-fat, high-starch diet without measurably altering muscle lactate and glycogen concentrations. Dietary bicarbonate supplementation at the concentration administered in this study did not prevent increased serum CK activity on a high-starch diet.

Objective: To determine daily variation in urinary clearance and fractional excretion (FE) of electrolytes and minerals within and between horses and to compare volumetric and single-sample urine collection for determining FE values of diets with a range of dietary cation-anion balance (DCAB)., Animals: 5 Thoroughbred and 6 mixed-breed mares., Procedure: 3 isocaloric diets with low, medium, and high DCAB values (85, 190, and 380 mEq/kg of dry matter, respectively) were each fed for 14 days. Daily blood samples, single urine samples collected by using a urinary catheter (5 mares), and volumetric urine collections (6 mares) were obtained during the last 72 hours of each diet., Results: Urine and plasma pH values, plasma concentrations, and FE values of sodium, chloride, potassium, magnesium, phosphorus, and calcium were altered by varying the DCAB. Noticeable variation in clearance and FE values was detected within horses from day-to-day on the same diet as well as between horses. Fractional excretion values were not significantly different between single-sample and volumetric methods, except for magnesium in the high DCAB diet. Volumetric and single-sample collections revealed similar patterns of change in urinary FE values with varying DCAB, except for calcium and magnesium., Conclusions and Clinical Relevance: Substantial variation in clearance and FE of electrolytes and minerals are evident within horses between 24-hour periods as well as between horses fed a specific diet. Three daily urine samples provide similar information regarding dietary-induced changes in clearance and FE values (excluding calcium and magnesium) as that obtained by volumetric urine collection.

This study was designed to determine the effects of fat adaptation on carbohydrate and fat oxidation in conditioned horses during low-intensity exercise. Five mature Arabians were studied. The study was conducted as a crossover design with 2 dietary periods, each of 10 week's duration: a) a control (CON) diet, and b) a fat-supplemented (FAT) diet. The total amount of digestible energy (DE) supplied by the fat in the CON and FAT diets was 7% and 29%, respectively. During each period, the horses completed exercise tests at the beginning of the period (Week 0) and after 5 and 10 weeks on the diet. Tests consisted of 90 min of exercise at a speed calculated to elicit 35% VO2max on a treadmill inclined to 3 degrees. Oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were measured at 15-min intervals. For determination of glucose kinetics, a stable isotope ([6-6-d2] glucose) technique was used. Compared to the CON diet, FAT diet consumption for 5-10 weeks was associated with an altered metabolic response to low-intensity exercise, as evidenced by a more than 30% reduction in the production and utilisation of glucose; a decrease in RER; a decrease in the estimated rate of whole-body carbohydrate utilisation; and an increase in the whole-body rate of lipid oxidation during exercise.

Objective: To determine whether plasma, urine, and fecal electrolyte and mineral concentrations differ between clinically normal horses and Thoroughbreds with recurrent exertional rhabdomyolysis (RER) after consumption of diets varying in cation-anion balance., Animals: 5 Thoroughbred mares with RER and 6 clinically normal mixed-breed mares., Procedure: Each of 3 isocaloric diets designated as low, medium, and high on the basis of dietary cation-anion balance (DCAB) values of 85, 190, and 380, respectively, were fed to horses for 14 days. During the last 72 hours, 3 horses with RER and 3 control horses had daily urine and fecal samples obtained by total 24-hour collection. Remaining horses had urine samples collected daily by single catheterization., Results: For each diet, no differences existed between horses with RER and control horses in plasma pH, electrolyte concentrations, and creatine kinase activity or in urine pH and renal fractional excretion (FE) values. Plasma pH, strong ion difference, bicarbonate and total carbon dioxide concentrations, and base excess decreased and plasma chloride and ionized calcium concentrations increased with decreasing DCAB. Urine pH decreased with decreasing DCAB. The FE of chloride and phosphorus were greatest for horses fed the low diet. The FE values for all electrolytes exept magnesium did not differ between urine samples obtained by single catheterization and total 24-hour collection. Daily balance of calcium, phosphorus, sodium, chloride, and potassium did not differ significantly among horses fed the various diets., Conclusions: In clinically normal horses and in horses with RER, the DCAB strongly affects plasma and urine pH and the FE of sodium, potassium, chloride, and phosphorus.

The purpose of this study was to examine the effects of an external nasal strip (NS), frusemide (FR) and a combination of the 2 treatments (NS + FR) on exercise-induced pulmonary haemorrhage (EIPH) in Thoroughbred horses. It was hypothesised that both the NS and FR would attenuate EIPH as assessed by red blood cell count in bronchoalveolar lavage fluid. In random order, 8 horses completed each of 4 sprint exercise tests on a treadmill: 1) NS; 2) FR (0.5 mg/kg bwt i.v., 4 h pre-exercise); 3) NS + FR; and 4) control (C; no treatment). After a 5 min warm-up (4.5 m/s), horses completed 2 min running at 120% maximum oxygen consumption (VO2max) with the treadmill set at 3 degrees incline. Mean +/- s.d. running speed was 14.2+/-0.2 m/s. In the FR and NS + FR trials, horses carried weight equal to that lost as a result of frusemide administration. During exercise at 120% Vo2max, oxygen consumption (Vo2) and carbon dioxide production (Vco2) were measured at 15 s intervals. Plasma lactate concentration was measured in samples collected before exercise, at the end of the sprint and after 5 min cool-down at the trot. Thirty minutes after the run, bronchoalveolar lavage (BAL) was performed and the red cell count in the fluid quantified. Vo2 and Vco2 were significantly lower in NS and NS + FR trials than in the C and FR trials at the end of the sprint exercise protocol. However, plasma lactate concentrations did not differ among treatments. Compared with the C trial (61.1+/-30.5 x 10(6) red blood cells/ml BAL fluid), pulmonary haemorrhage was significantly (P<0.05) decreased in both the NS (15.9+/-4.0 x 106 RBC/ml) and FR (12.2+/-5.8 x 10(6) RBC/ml) trials. EIPH in the NS + FR trial (7.9+/-1.0 x 10(6) RBC/ml) was further diminished (P<0.05) compared to the NS trial, but not different from the FR trial. We conclude that both the external nasal strip and frusemide attenuate pulmonary haemorrhage in Thoroughbred horses during high-speed sprint exercise. The external nasal strip appears to lower the metabolic cost of supramaximal exertion in horses. Given the purported ergogenic effects of frusemide, the external nasal strip is a valuable alternative for the attenuation of EIPH.

Objective: To determine the effects of 3 rations (low grain, fat, high grain) on plasma creatine kinase (CK) activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolysis (RER)., Animals: 5 Thoroughbreds with RER and 3 healthy Thoroughbreds (control horses)., Procedures: Rations were formulated to meet (low-grain and fat rations) or exceed (high-grain ration) daily energy requirements. Each ration was fed to horses in a crossover design for 3 weeks. Horses were exercised on a treadmill Monday through Friday; maximum speed on Monday and Friday was 11 m/s (6% slope), on Tuesday and Thursday was 9 m/s, and on Wednesday was 4.5 m/s. Plasma CK activity and lactate concentration were determined before and after exercise., Results: Horses with RER fed the high-grain ration had significantly greater CK activity and change in CK activity 4 hours after exercise, compared with those fed the low-grain ration. Horses with RER exercised at the trot or canter had significantly greater increases in CK activity, compared with those exercised at the gallop. Plasma lactate concentrations after exercise were similar in control and affected horses. Lactate concentration and CK activity were not correlated in horses with RER., Conclusions and Clinical Relevance: Rations high in grain and formulated to exceed daily energy requirements may increase episodes of rhabdomyolysis in thoroughbred horses susceptible to RER.

To delay the onset of fatigue, endurance horses are often fed at rest stops during races. The resulting increase in blood insulin may adversely inhibit lipolysis. In humans, ingestion of fructose produces a smaller insulin rise than glucose. This study compared glucose and fructose as carbohydrate supplements for endurance horses. Three Arabian geldings were given 300 g of fructose (F), glucose (G) or 50% glucose: 50% fructose (GF), in 1.5 L water, by stomach tube. In the Resting Test, carbohydrate was administered at rest. Following treatment, blood samples were taken every 30 min for 8 h, and feces were collected for 24 h. Treatment did not affect fecal weight or water content. Plasma glucose and insulin responses did not differ among treatments. Post-treatment (60 min), plasma L-lactate tended to be higher (P = 0.06) after the F and GF treatments than after the G treatment. In the Exercise Test, two treadmill exercise bouts at 0 degrees incline (Bout 1: 90 min; Bout 2: 120 min) were separated by a 1-h rest period. A total distance of 36.84 km was covered at a mean speed of 2.9 m/s. Carbohydrate was administered 45 min before Bout 2. Plasma glucose and insulin at the start of Bout 2 were higher (P = 0.02 and 0.03, respectively) with the GF treatment than with the F treatment. However, during exercise, plasma glucose concentrations did not differ among treatments. We conclude that fructose is well-absorbed by horses and rapidly converted to glucose.

This study looked at the effect of feeding diets supplemented with either a predominantly saturated or unsaturated vegetable oil over a prolonged period to exercising horses. Eight Thoroughbred horses were assigned to 2 diet treatments and for 10 months were fed Timothy hay and oats, together with a fortified sweet feed supplemented with either a predominantly unsaturated (Un) or a saturated (S) vegetable oil so that approximately 19% DE (Digestible Energy) came from dietary fat and approximately 12% from either the Un or S source (AC). An increased amount of Un or S fortified sweet feed, replacing the oats, was then fed for a further 6 months (HF) so that approximately 27% DE came from fat and approximately 20% from the Un or S vegetable oil. Standardised incremental treadmill exercise (8-12 m/s) tests (STEP) and duplicate oral glucose tolerance tests (TOL) were carried out after 3, 6 and 9 months of the AC diet and after 3 and 6 months on the HF diet. There was no significant effect of dietary treatment or when the tests were undertaken (time) on the insulin or lactate responses to the STEP tests. Overall there was a significant (P < 0.05) effect of time and treatment on the glucose response, but there was no difference between treatments at the first and last tests or between the results for these tests or between the endAC and endHF tests. No significant effect of treatment or time was seen on the TOL glucose response (% change from Time '0') although there was a trend for the glucose concentrations to be lower and the insulin responses higher (nonsignificant) in the S treatment group. No significant effect of treatment on haematological parameters, monitored monthly, was found. Total protein and gamma glutamyl transferase remained within the normal range throughout. There was a significant effect of treatment (P < 0.05) on cholesterol and triglycerides with higher concentrations in the S group from the first (1 month) sample. Linoleic acid was the main fatty acid in all the 4 plasma lipid classes with slightly, but significant (P < 0.05), higher concentrations in Un for the cholesterol ester and phospholipid classes. There was no effect of time. Overall, the total resting plasma fatty acid content was significantly higher (P < 0.05) with S at the sample points (endAC and endHF). No adverse effects of feeding either diet on apparent coat condition or hoof appearance were seen apart from an apparent increase in the grease score. Many of the parameters assessed showed significant improvements with time (P < 0.05). In conclusion, no apparent adverse effects of feeding a diet supplemented with either an unsaturated or saturated vegetable oil for 6 months at approximately 20% DE after 10 months at approximately 12% DE were identified and there were no apparent disadvantages of feeding a saturated vegetable oil supplemented diet compared with an unsaturated one.

Previous studies have associated recurrent exertional rhabdomyolysis (RER) with a diet high in soluble carbohydrate (CHO). The purpose of this study was to investigate the effect of 3 diets on clinical and metabolic parameters in 5 Thoroughbred horses with RER and 3 healthy Thoroughbreds performing a standardised exercise test (SET). Two diets were formulated to meet energy requirements for the amount of exercise being performed in the form of CHO or fat (21.4 Mcal DE/day). The third diet was formulated to provide 135% of the DE of the other 2 diets in the form of an excessive amount of carbohydrate (28.8 Mcal DE/day). Diets were fed in a crossover design for 3 week blocks and then horses performed a near maximal SET. Changes in heart rate (HR), plasma lactate, plasma glucose, total plasma solids, packed cell volume (PCV), muscle lactate and muscle glycogen concentration were measured immediately prior to, during, and 5 min after exercise. Serum creatine kinase (CK) activity was measured prior to and 4 h post SET. A 2-way ANOVA was used to examine the effect of group and dietary treatment. When dietary treatments were compared, horses fed the high-CHO diet had a mean pre-SET PCV and pre-SET HR that was higher than horses fed the fat diet (P = 0.06 and P = 0.07, respectively). Pre-SET heart rates were highest in RER horses consuming the high-CHO diet compared to RER horses consuming the low-CHO and fat diets (P = 0.02). Horses with RER had 4 h post SET CK activity greater than 400 u/l in 7/14 (50%) measurements compared to control horses which had CK activity greater than 400 u/l in 2/7 (29%) measurements. This study did not demonstrate a significant effect of diet on rhabdomyolysis, indicated by CK activity, or on the metabolic response to exercise. However, diet may have a calming effect on Thoroughbred horses with RER as manifested by decreased pre-exercise heart rates and decreased pre-exercise PCV in horses fed the fat diet.

High-grain diets for the exercising horse were compared with diets which provided 15% of the total caloric intake from either vegetable oil or a highly fermentable fibre source (beet pulp). Six Thoroughbreds age 3 years were fed one of 3 diets or 5 weeks in a replicated 3 x 3 Latin square. The CONTROL diet was 3.65 kg of sweet feed (SF), 0.9 kg of a protein/vitamin/mineral pellet and 5.45 kg of hay cubes. The FAT diet replaced 1.15 kg of SF with 0.45 kg of soybean oil and the FIBRE diet replaced 1.15 kg of SF with 1.36 kg of beet pulp. Horses were exercised 3 times per week on a high-speed treadmill. During the last week of each period, the horses performed a standardised exercise test (SET). A series of blood samples was drawn immediately before feeding and every 0.5 h for 3 h after feeding, throughout the exercise bout and 30 min post exercise. Plasma was analysed for lactate, glucose, cortisol, insulin, packed cell volume, total protein and triglycerides. Water intake was measured at regular intervals during SET day. Blood glucose was lower (P < 0.05) in the FAT-fed horses during the 3 h post feeding as compared to either CONTROL or FIBRE-fed horses. Insulin was lower (P < 0.05) in the FAT-fed both post feeding and throughout exercise. Cortisol was lower (P < 0.05) in the FAT than the CONTROL-fed during exercise. Following exercise, the FAT-fed drank more water (P < 0.01) than either CONTROL or FIBRE-fed. Substituting 15% of DE as vegetable oil had a greater effect on metabolic response to exercise than a 15% substitution of beet pulp.

There is considerable debate among horsemen about how to feed horses before exercise. Should horses be fed or fasted before work and when should hay be fed relative to grain and/or exercise? Three experiments were conducted to evaluate if feeding hay with and without grain affects glycaemic and haematological responses in Thoroughbred (TB) horses at rest and during a simulated competition exercise test (CET) on a high-speed treadmill. In Experiment 1, 6 TB horses were fed hay at 3 different times relative to a grain meal. Time of feeding hay affected glycaemic response, plasma protein and water intake post grain feeding. During Experiment 2, 4 TB horses were used in a 4 x 4 Latin square design to determine whether feeding grain with or without hay prior to a CET would affect substrate utilisation and exercise. Feeding grain reduced free fatty acid (FFA) availability and increased blood glucose disappearance during exercise (P < 0.05). Feeding hay either along with grain or ad libitum the night before exercise resulted in reduced plasma volume (P < 0.05) and higher lactate production (P < 0.05) and heart rates (P < 0.05) during exercise. During Experiment 3, 4 TB horses were used in a 4 x 4 Latin square design experiment to determine whether feeding forage but no grain prior to CET would affect substrate utilisation and performance. Feeding only forage before exercise did not adversely affect performance. It was concluded that grain should be withheld from horses before exercise, but that small quantities of hay should be fed to ensure proper gastrointestinal tract function.

Six Thoroughbreds were used to evaluate time of feeding on changes in exercise response in horses receiving either a textured feed or a fat-supplemented textured feed. Using a crossover design, 3 horses were fed a fat-supplemented diet while 3 horses received a control ration of textured feed. Horses performed a standardised exercise test (SET) on a high speed treadmill. The SET was performed at 3 different times: 1) following an overnight 12 h fast, 2) 3 h after feeding and 3) 8 h after feeding. The SET consisted of a 2 min walk at 1.4 m/s, 800 m trot at 4.2 m/s, 800 m gallop at 7.7 m/s, 1600 m gallop at 11 m/s, 800 m trot at 4.2 m/s and 2 min walk at 1.4 m/s. Jugular blood samples were taken before feeding, hourly until the beginning of the SET, at the end of each exercise step, 15 min post exercise and 30 min post exercise. During the SET, heart rate was measured and blood samples collected for analysis of glucose, lactate, insulin and nonesterified fatty acids (NEFA). Feeding horses 3 h prior to exercise resulted in elevated concentrations of plasma glucose and insulin (P < 0.01) at rest. Elevated concentrations of insulin in horses fed 3 h prior to exercise decreased plasma glucose (P < 0.01) during exercise and appeared to have suppressed fat oxidation during exercise because horses that were either fasted or fed 8 h post prandial had a net disappearance of NEFA in the plasma during exercise. This study indicates that beginning exercise with elevated plasma insulin appeared to be of no benefit during the exercise conducted in this experiment.

Energy balance studies using indirect calorimetry were conducted with four mature equids ranging in weight from 125 to 856 kg. Each animal was fed three different levels of intake of the same diet. The amounts of digestible and metabolizable energy required for zero energy balance were determined by calculating regression equations for energy balance against energy intake. It was concluded that the maintenance requirements of equids vary linearly with body weight. No advantage was found for the use of weight for the comparison of equids within the range studied. The digestible energy (DE) requirement for equids confined to metabolism stalls was DE (kcal/d) = 975 + 21.28 X W where W equals body weight in kg. If a factor for activity similar to that suggested by the National Research Council was included, the requirement could be calculated by the equation: DE (kcal/d) = 1,375 + 30.0 X W.

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (μmol C2H4g(-1) nodule fr. wt. h(-1)), were both affected by the added nitrate.

Nitrogen fixation was induced in a strain of "cowpea" rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C2H2 reduction, was maximized at an O2 concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH4 (+) and NO3 (-) repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C2H2 in association with tobacco cells; the highest levels of activity were found among cowpea strains.

Energy expenditure was measured in four geldings (433 to 520 kg) during submaximal exercise on a racetrack using a mobile open-circuit indirect respiration calorimeter. A total of 304 5-min measurements of O2 consumed and CO2 produced were taken. Measurements were made with and without riders. The amount of energy expended by the horses was exponentially related to speed and was proportional to the body weight of the riderless horse or the combined weight of the horse plus rider and tack. Total energy expended by the four horses walking, trotting, cantering was best described by the equation: Y = e3.02 + .0065X where Y = energy expended (cal X kg-1 X min-1) and X = speed (m/min). Digestible energy (DE) required above maintenance was calculated as (Formula: see text).

Nutritional and physical conditions affecting nitrogenase activity in the strain of "cowpea" rhizobia, 32H1, were examined using cultures grown on agar medium. Arabinose in the basic medium (CS7) could be replaced by ribose, xylose, or glycerol, but mannitol, glucose, sucrose, or galactose only supported low nitrogenase (C2H2 reduction) activity. Succinate could be replaced by pyruvate, fumarate, malate, or 2-oxoglutarate, but without any carboxylic acid, nitrogenase activity was low or undetectable unless a high level of arabinose was provided. Inositol was not essential. Several nitrogen sources could replace glutamine including glutamate, urea, (NH4)2SO4 and asparagine. The maximum nitrogenase activity of cultures grown in air at 30 degrees C was observed under assay conditions of pO2=0.20-0.25 atm and 30 degrees C incubation. Greatest activity occurred after a period of rapid bacterial growth, when viable cell count was relatively constant. Compared with results obtained on the CS7 medium, nitrogenase activity could be substantially increased and/or sustained for longer periods of time by using 12.5 MM succinate and 100 mM arabinose, by increasing phosphate concentration from 2 to 30-50 mM, or by culturing the bacteria at 25 degrees C.

Twenty-two pony mares were fed one of three diets that provided 93.0, 74.8 or 57.2 kcal of digestible energy (DE) per kg body weight per day. Milk samples were taken at 14 day intervals. A total of five samples were taken from each mare. The samples were analyzed for total solids, crude protein, lactose, total lipids, ash, calcium and phosphorus. Gross energy was calculated from composition data. Increases in energy intake decreased the concentration of total solids, protein, fat and gross energy of mare's milk. Energy intake had a greater influence on the mare's body condition than on milk energy production. It was concluded the objective of a feeding program for a lactating mare should be to keep the mare in a desirable body condition rather than to influence milk composition or production.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.