Production of reactive oxygen species in neutrophils after repeated bouts of exercise in standardbred trotters.

Six trained Standardbred trotters exercised on a racetrack on 2 days with a 3-day interval. On both exercise days the horses trotted three different exercise bouts with increasing intensity with 60-min intervals. Exercise-induced stress was manifested as leucocytosis, an increase in the neutrophil:lymphocyte (N:L) ratio, and increased capacity to produce reactive oxygen species in the peripheral blood as indicated by an increase in whole blood chemiluminescence. The leucocytosis was mainly due to neutrophilia, which lasted for 6 h. Production of reactive oxygen species per single neutrophil showed no significant change during a day of exercise, but was lower on the second exercise day. The cortisol concentrations and N:L ratio, used as indicators of stress, behaved differently: Cortisol did not change significantly after exercise, whereas the N:L ratio increased. These results suggest that in trained horses, the N:L ratio is a sensitive indicator of stress of short duration, and an attenuated N:L response can be taken as an indicator of adaptation to exercise stress.

Resynthesis of glycogen in skeletal muscle from standardbred trotters after repeated bouts of exercise.

OBJECTIVE: To determine glycogen resynthesis rate and changes in plasma metabolite concentrations in horses before and after repeated exercise. ANIMALS: 6 clinically normal Standardbred trotters. PROCEDURE: Horses trotted distances of 3,000, 3,000, and 2,000 m (trial A) and 3 days later, trotted 2,100, 2,100, and 1,600 m (trial B). Horses had 1 hour rest periods between bouts of exercise. Trotting speed was increased with each exercise bout, up to a near maximal. Muscle biopsy specimens and venous blood samples were obtained before each trial and 0, 4, 24, 48, and 72 hours after the third bout. Blood samples were also taken between exercise bouts. Muscle glycogen content and plasma glucose, glycerol, nonesterified fatty acid, and triglyceride concentrations were determined. RESULTS: Muscle glycogen content was significantly decreased immediately after exercise from 473 +/- 45 to 329 +/- 79 mmol/kg of dry weight in trial A, and from 472 +/- 128 to 347 +/- 59 mmol/kg in trial B. Further decreases were measured 4 hours after exercise. Glycogen resynthesis was negligible 24 hours after exercise. Basal muscle concentrations of glycogen were obtained 72 hours after exercise in trial A (472 +/- 128 mmol/kg), but not in trial B (279 +/- 52 mmol/kg). Plasma concentrations of glucose were greater than or equal to before-exercise values. Plasma concentrations of lipid metabolites, glycerol, triglycerides, and nonesterified fatty acids, were less than before-exercise values 2 to 72 hours after exercising. CONCLUSIONS: Repeated bouts of exercise decrease glycogen repletion rate, which is not attributable to hypoglycemia, but may be influenced by limited availability of lipids for energy production.

Characterization of two lipopolysaccharide types isolated from Rhizobium galegae.

Lipopolysaccharides (LPS) of Rhizobium galegae, a symbiotically nitrogen-fixing species of root-nodule bacteria, were isolated by the phenol-water method from strain HAMBI 1461, the LPS of which resembled enterobacterial smooth type LPS, and from strains HAMBI 1174 and HAMBI 1208, the LPSs of which resembled rough type LPS. The results of PAGE analysis of LPSs, Bio-Gel P2 gel filtration of polysaccharide fractions and the presence of deoxysugars and 4-O-methyl-deoxysugar both in the rough and smooth LPSs suggested that rough LPS contained a short O-antigenic polysaccharide for which we propose the name short O-chain LPS. Accordingly, the smooth LPS is called long O-chain LPS. Despite of the differences in the structure of LPS of R. galegae, all strains were equally effective in nodulating their hosts. The short O-chain LPS of R. galegae showed many features similar to those of phylogenetically related agrobacteria.

Androgen receptors and skeletal muscle composition in trotters treated with nandrolone laureate.

To study the effects of nandrolone laureate (19-nortestosterone) on muscle hypertrophy and concentration of androgen receptors (AR), biopsy specimens were taken from the middle gluteal muscle of 6 Finnhorse trotters (geldings and mares) undergoing training before, immediately after, and 13 weeks after a 14-week treatment with nandrolone. Another 6 similarly trained horses served as controls. An additional 10 mares and 10 geldings were used to study annual variation in muscle concentration of AR. AR was immunohistochemically localized in the nuclei. AR concentration remained constant during the first 14 weeks of the study, but increased significantly during the 13-week follow-up period in both groups. This finding can be explained by the annual variation in AR. In the anabolic steroid (AS)-treated horses, but not in the controls (C), the cross-sectional area of the type I fibres increased significantly during the treatment period, and the percentage of type IIA fibres correlated positively with AR concentration at the end of nandrolone treatment. In the AS group, the concentration of DNA decreased during the 13-week follow-up period, and the percentage of H-chains in the isoenzymes of LDH increased. Protein concentration increased in both groups during the follow-up period. Glycogen content and the activity of citrate synthase in muscle during the study remained unchanged. It can thus be concluded that AS produce differing effects on type I and type II fibres, and the AR concentration in equine muscle may contribute to the change observed in the middle gluteal muscle.

Skeletal muscle characteristics of racing reindeer (Rangifer tarandus).

Samples from the middle gluteal muscle of 3-7-year-old racing reindeer were taken before (7 reindeer) and at the end of the racing season (12 reindeer) for determination of fibre type composition, activities of citrate synthase (CS), 3-hydroxyacyl-CoA-dehydrogenase (HAD) and the glycogen content. Muscle samples were also taken from 7 female and 18 male reindeer of same age for histochemical determination of muscle fibre composition. During the racing season the fibre type composition remained unchanged as did the activities of CS and HAD. The high activities of CS and HAD together with the high intensity in the NADH-dehydrogenase stain indicate that the reindeer muscle has a high oxidative capacity. The glycogen content increased during the winter which can explain the increasing tendency in the transverse fibre area. The racing reindeer had a lower percentage of type I fibres and a higher percentage of type IIA fibres than the ordinary male reindeer. The differences in the fibre type composition suggest that the racing reindeer represent a selected reindeer population.

Accumulation of uric acid in plasma after repeated bouts of exercise in the horse.

Plasma concentration of uric acid, total peroxyl radical-trapping antioxidative parameter (TRAP), blood lactate concentration and plasma activity of xanthine oxidase (XO) were measured in six Standardbreed trotters after six bouts of exercise with increasing intensity on two separate days three days apart. Blood samples were taken immediately, 5, 10, 15, 30 and 60 min after each heat and 2, 4, and 6 hr after the last heat. Exercise caused an increase in TRAP and in the concentrations of lactate and uric acid. Plasma uric acid concentration increased exponentially with respect to time after the last heat performed maximal speed, indicating a rapid increase in the rate of purine degradation. Plasma XO activity increased during exercise, but the intensity of exercise had only a minor effect on the level of XO activity. In conclusion, these data suggest that a threshold for the plasma accumulation of uric acid in terms of the intensity of exercise may exist and that XO may play a role in the formation of uric acid in horse plasma. Intense exercise causes an increase in the plasma antioxidant capacity that in the horse is mainly caused by the increase in the plasma uric acid concentration.

Exercise-induced changes in the activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase in plasma and muscle of standardbred trotters.

The activities of lysosomal enzymes, such as beta-glucuronidase and N-acetyl-beta-D-glucosaminidase, have been shown to increase in muscle after endurance exercise. We examined whether measurable activities of lysosomal enzymes are present in equine plasma and whether the exercise-induced changes in the muscle are reflected in plasma. Six trained Standardbred trotters performed three exercise bouts with 1 h intervals and the same procedure was repeated 3 days later. Venous blood samples and muscle biopsies from the middle gluteal muscle were taken before and after exercise. The activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase were measured both from plasma and muscle specimens. Cell infiltration into the muscle after exercise was evaluated by the DNA content and histochemically by haematoxylin stain. The activity of N-acetyl-beta-D-glucosaminidase in plasma was increased immediately after exercise, but had returned to the basal level at 4 h. N-acetyl-beta-D-glucosaminidase in muscle and beta-glucuronidase in muscle and plasma increased 2 days after exercise and returned to the basal level on day 3. A similar pattern was seen when the exercise protocol was repeated 3 days later, except that the activities continued to increase during the 3 days after exercise. The DNA content in muscle correlated with beta-glucuronidase in muscle and plasma and with the N-acetyl-beta-D-glucosaminidase in muscle indicating that the activities reflect the infiltration of phagocytes into the exercise-injured muscle. It can be concluded that the activities of the lysosomal enzymes in plasma increase after exercise and that the changes are mainly due to a simultaneous increase in the number of neutrophils. Therefore, plasma activities of the lysosomal enzymes are poor indicators of exercise-induced muscle damage.

Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR.

Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.

Responses of blood and plasma lactate and plasma purine concentrations to maximal exercise and their relation to performance in standardbred trotters.

OBJECTIVE: To study whether end products of 2 pathways of anaerobic energy metabolism, lactate and purines, that accumulate in the blood after intense exercise indicate any relation to exercise performance. DESIGN: Venous blood samples were taken within 1 and 15 minutes after a trotting race of 2,100 m. ANIMALS: 16 Clinically healthy Standardbred trotters. PROCEDURE: Blood and plasma lactate concentrations were measured by enzymatic analyzer, and purines, uric acid and allantoin, were determined by high-performance liquid chromatography. The concentrations of metabolites were then correlated to racing time and individual performance indexes that are annually calculated from the percentage of winnings, placings, and starts rejected, average earnings per start, and the racing record. RESULTS: Blood lactate concentration immediately and calculated cell lactate concentration immediately and 15 minutes after the race correlated positively (P < 0.05 to P < 0.01) with the individual performance indexes. Plasma lactate concentration was not correlated to the individual performance indexes. Uric acid concentration, immediately and 15 minutes after the race, was negatively correlated (P < 0.05) to the individual performance indexes, and a positive relation (P < 0.05) was found between the highest concentration of uric acid and the racing time. Concentration of allantoin immediately or 15 minutes after the race did not have any significant correlation to the individual performance indexes. CONCLUSIONS: Accumulation of lactate in the blood, which was greater in the superior performing horses, may prove to be an useful predictor of anaerobic capacity. The results also indicate that the loss of purine nucleotides was less in the superior performing horses, although further studies are needed to confirm this.

Plasma atrial natriuretic peptide in standardbred and Finnhorse trotters during and after exercise.

To study the exercise-induced changes in atrial natriuretic peptide (ANP), a hormone with cardiovascular and renal effects, an incremental submaximal exercise test on a high-speed treadmill was carried out with Standardbred and Finnhorse trotters, the former bred for speed and the latter originally for heavy work. Standardbreds performed the 2 min exercise intervals at speeds of 6, 7, 8, 9 m s-1 and Finnhorses, according to their training status, at 5, 6, 7, 8 m s-1, 4, 5, 6, 7 m s-1 or 5, 6, 7 m s-1. Steady-state heart rate (HR) was reached within each 2 min interval. The increase in HR was linear and proportional to work intensity and physical condition and it peaked, average 204 beats min-1, during the last speed of the treadmill. Plasma ANP increased significantly and equally, by 27 +/- 4 pg mL-1, in both breeds and peaked at 5 min post-exercise. The rise in ANP during exercise showed good linearity with HR and increasing work intensity. The decrease of ANP after exercise was slow, which may be connected to the regulation of water and electrolytes. Interbreed differences in plasma ANP were not observed. The results suggest a role of ANP in cardiovascular control and fluid balance during and after exercise. In addition to other possible releasing factors during exercise, the increase in HR explains about 40% of the variability in the plasma ANP values.

Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress.

The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaCl; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaCl. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCl or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2°C.

Accumulation of allantoin and uric acid in plasma of exercising trotters.

Plasma concentrations of hypoxanthine, uric acid, and allantoin, which are breakdown products of adenine nucleotides, were measured in Standardbred and Finnhorse trotters during and after an exercise test on a high-speed treadmill, after an incremental exercise test performed on a racetrack, and after a racing competition. Fiber-type composition of the middle gluteal muscle and the muscle concentrations of adenine nucleotides and inosine monophosphate were measured after the racetrack test. Changes in the concentration of hypoxanthine were not observed in any of the tests. Peak concentration of uric acid was measured between 5 and 30 minutes after exercise, and it was three- to tenfold higher than the value at rest. The variability can be explained by intensity of the exercise test and variation among horses. The concentration of allantoin after exercise was 2 to 3 times as high as that at rest, depending on the intensity of the exercise, although the absolute increase was about 10 times as high as the increase in the concentration of uric acid. Peak values of allantoin for the treadmill and the racetrack tests were obtained 4 to 6 minutes after exercise and < 30 minutes after the races. Peak concentration of allantoin correlated positively with the percentage of type-II (IIA+IIB) fibers in the middle gluteal muscle. Significant correlations were not observed between plasma concentration of uric acid or allantoin and muscle concentrations of adenosine triphosphate (ATP) or inosine monophosphate. It can be concluded that in horses, breakdown of ATP during and after exercise continues until allantoin is produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of xanthine dehydrogenase mRNA in horse skeletal muscle by in situ hybridization with digoxigenin-labelled probe.

In situ hybridization was used to localize xanthine dehydrogenase (XDH) mRNA in horse skeletal muscle. Capillary endothelial cells were found to express XDH, but muscle cells did not give any signal. The digoxigenin-labelled probe was produced by PCR with primers based on the cDNA sequence of mouse XDH and horse lung cDNAs. A 4.3 kb mRNA was detected in a Northern blot.

Expression of Rhizobium galegae common nod clones in various backgrounds.

The cosmid clone pRg30, carrying common nodulation genes of Rhizobium galegae HAMBI 1174, and pRg33, a subclone of pRg30 that contains a 5.7-kb ClaI insert carrying nodDABC were conjugated into various Rhizobium nod- mutant strains and into a Ti plasmid-cured Agrobacterium tumefaciens. Complementation and expression of the nodABC genes of R. galegae were studied by following microscopically the infection process and the nodulation on different test plants. The nodABC genes of R. galegae complemented the nod- strains of other Rhizobium species. The presence of extra copies of common nod genes in the homologous R. galegae nodABC- strain induced an increased nodulation on Galega orientalis. However, the inserts of R. galegae in pRg30 and pRg33 do not carry sufficient genetic information for normal nodulation of test plants in an Agrobacterium background, because the Agrobacterium transconjugants induced root hair deformation on Galega plants, but no infection threads were detected and nodulelike structures developed only at low frequency. The Agrobacterium carrying the nodDABC of R. galegae did not cause the root hairs of Medigo sativa to deform.