Effect of chronic clenbuterol administration and exercise training on immune function in horses

Effects of longitudinal exercise training and acute intensive exercise (simulated race test) on immune function have not been reported in horses. Clenbuterol, a beta2-adrenergic agonist, is used to manage inflammatory airway disease in horses. This study investigated the interaction of 8 wk of exercise training with or without 12 wk of clenbuterol administration in horses. Twenty-three untrained standardbred mares (10 +/- 3 yr, Mean +/- SE) were used and divided into four experimental groups. Horses given clenbuterol plus exercise (CLENEX; n = 6) and clenbuterol alone (CLEN; n = 6) received 2.4 microg/kg BW of clenbuterol twice daily (in an average volume of 20 mL) on a schedule of 5 d on and 2 d off for 12 wk. The CLENEX group was also aerobically trained 3 d/wk. Mares given exercise alone (EX; n = 5) were aerobically trained for 3 d/wk, and the control group (CON; n = 6) remained sedentary. Both EX and CON horses were administered similar volumes (approximately 20 mL) of molasses twice daily. A simulated race test (SRT) resulted in an elevation in lymphocyte number postexercise (P < 0.05). There was no significant difference after acute exercise in either monocyte or granulocyte number. Acute exercise resulted in a decrease (P < 0.05) in the percentage of CD4+ and an increase (P < 0.05) in the percentage of CD8+ cells. The SRT resulted in a decreased lymphoproliferative response to pokeweed mitogen (P < 0.05). A SRT had no effect on antibody production in response to equine influenza vaccine. The EX group demonstrated greater cortisol concentrations at rest and at all other time points postexercise after completing the training regimen compared with CLENEX horses (P < 0.05). Preexercise (SRT) peripheral blood monocyte number was lower in CLENEX horses than in other treatment groups (P < 0.05). Clenbuterol and exercise training did not significantly affect post-SRT changes in leukocyte numbers. Exercise training resulted in a decrease (P < 0.05) in the percentage of CD8+ cells post-SRT compared with other groups, but the percentage of CD4+ cells was not altered by either clenbuterol or exercise conditioning. Lymphocyte proliferative response was not affected by clenbuterol or exercise treatment. Horses demonstrated responses to bouts of acute exercise as noted with other species, namely humans and rodents.     

Effects of exercise stress on various immune functions in horses.

Chemotactic locomotion and luminol-dependent chemiluminescence of neutrophils, mitogen-induced lymphocyte blastogenesis, serum cortisol concentration, immunoglobulin quantification, and leukocyte counts were determined to evaluate the effect of a single strenuous exercise in horses. Increased serum cortisol concentration (P less than 0.01) and an increased neutrophil-to-lymphocyte ratio (P less than 0.05) indicated that horses had been stressed. The chemotactic index and peak chemiluminescence production decreased significantly (P less than 0.05 and P less than 0.01, respectively) 1 day after exercise. Mitogen-induced blastogenesis of lymphocytes and serum immunoglobulin values remained unchanged in response to exercise. Results of this study indicated that a single bout of exercise may transiently impair neutrophil antimicrobial functions and nonspecific defense mechanisms, but not specific immunity in horses.     

Effects of treadmill exercise on cortical bone in the third metacarpus of young horses.

The effects of exercise and relative inactivity on cortical bone were compared in young horses. Two groups were used; one was given a 14-week programme of exercise (n = 6) and the other kept as unexercised controls (n = 6). The first nine weeks of exercise involved trotting and cantering (2 to 4 km d-1 at speeds up to 12 m s-1) on a treadmill set at an incline of 3 degrees. Over the next five weeks the horses were trained at near maximal speeds (that is, up to 14.5 m s-1) with no incline of the treadmill. At the end of the programme marked differences in cortical porosity and distribution of subperiosteal osteogenesis at the mid-shaft of the third metacarpal bone were found between the groups. Histomorphometrical examination of the dorsal cortex showed minimal bone remodelling in the exercised horses, but extensive modelling as evidenced by the large amount of subperiosteal bone formation. In contrast, the unexercised horses had significantly more bone remodelling and less formation of subperiosteal bone. The histomorphometric and microradiographic findings provided an explanation for changes in the non-invasive bone measurements that occurred during training. Bone mineral content of the mid-metacarpus was found to increase more in the exercised than the unexercised horses despite a lower overall growth in bodyweight. In those horses that completed the full training programme, ultrasound speed increased significantly by the end of the training programme. It remained unchanged in the horse that did not complete the full exercise programme and decreased slightly in the unexercised horses. The difference in ultrasound speed between the groups was considered to reflect differences in intracortical bone porosity, endosteal bone formation and alterations in skin thickness. The stiffness of cortical bone increased significantly in the exercised horses but remained unaltered in the unexercised horses.     

Endothelin response during and after exercise in horses

The objective of the present study was to measure plasma endothelin-1 (ET-1) at rest and during exercise in the horse. Six healthy, Standardbred and Thoroughbred mares (5.3+/-0.8 years; 445.2+/-13.1 kg) which were unfit, but otherwise accustomed to running on the treadmill, were used in the study. Plasma ET-1 concentrations were measured using a commercially available radioimmunoassay kit. Horses performed three trials: a standing control (CON) trial where blood was collected from the jugular vein every minute for 5 min; a graded exercise test (GXT) where blood samples were collected at the end of each 1 min step of an incremental exercise test; and a 15 min submaximal (60% VO(2max)) steady-state exercise test (SST) where blood samples were collected 1 min before, immediately after, and at 2 min, 10 min and 20 min post-exercise. Plasma ET-1 concentration did not change (P>0.05) during the CON trial where it averaged 0.18+/- 0.03 pg/mL (mean+/-SE). Surprisingly, plasma ET-1 concentration did not change during the GXT trial where it averaged 0.20+/-0.03 pg/mL. There were no differences between the mean concentrations obtained in either trial (P>0.05). Plasma ET-1 concentrations were, however, significantly elevated (P<0.05) immediately following exercise and at 2 min post-exercise in the SST. Post-exercise plasma ET-1 concentrations returned to baseline (P>0.05) by 10 min of recovery. Together, these data may suggest that ET-1 concentrations are altered in response to an exercise challenge.     

Effect of maternal immunity on the immune response to oral vaccination against rabies in young foxes.

OBJECTIVE: To determine effect of maternal antibodies on immune response to oral vaccination against rabies in young foxes. ANIMALS: 250 cubs from 48 vixens. PROCEDURE: Sera were obtained from cubs of 36 vaccinated (maternally vaccinated [MV+]) and 12 nonvaccinated (MV-) vixens between 23 and 71 days of age and tested for neutralizing antibodies. Seventy-one MV+ cubs and 33 MV-cubs were vaccinated orally with modified-live virus vaccine SAD B19. Geometric mean titer (GMT) was determined in these cubs approximately 21, 39, and 57 days after vaccination. In a subsequent experiment, 10 vaccinated MV+ cubs, 6 vaccinated MV- cubs, and 6 control cubs were challenge inoculated with virulent rabies virus approximately 100 days after vaccination. RESULTS: Serum GMT of nonvaccinated MV cubs (0.23 U/ml) was significantly greater than that of non-vaccinated MV- cubs (0.15 U/ml). The GMT of vaccinated MV+ cubs 21, 39, and 57 days after vaccination were 2.85, 2.11, and 0.79 U/ml, respectively, and were significantly less than those of vaccinated MV- cubs (12.19, 6.76, and 4.02 U/ml, respectively). All challenge-inoculated cubs with GMT < 0.5 U/ml succumbed to rabies. CONCLUSION AND CLINICAL RELEVANCE: Partially impaired immune response in cubs < 8 weeks old from vaccinated vixens causes insufficient protection against rabies. Inhibition of the immune response persists longer than the period during which maternal antibodies are detectable. Thus, oral vaccination campaigns for young foxes in areas where vaccination has been performed need to be reconsidered.     

The immune response of horses to tetanus toxoid

An intramuscular injection of 8-16 Lf tetanus toxoid in water-in-oil emulsion protected adult horses against tetanus for at least 128 weeks. A booster dose of 8 Lf toxoid in aqueous solution protected them for a further period of at least 3 1/2 years. Colostral immunity protected foals for at least 10 weeks. An intramuscular injection of 8 Lf toxoid in water-in-oil emulsion given to foals from immune dams when they were 10-18 weeks old did not elicit any antibody response. They did respond, however, to a booster injection of 8 Lf toxoid in aqueous solution given 12 weeks after the first dose. New-born foals were shown to be inherently unable to respond to an injection of tetanus toxoid.     

Influence of different exercise regimes on the proximal hoof circumference in young Thoroughbred horses

Reasons for performing study: Most lameness in horses relates to foot problems and may be associated with changes in hoof shape, but there is a lack of information on the influence of normal exercise on hoof shape. Objectives: To investigate the effect of training on proximal hoof circumference in young Thoroughbred racehorses being prepared for racing. Methods: Thirty‐seven young Thoroughbred racehorses were included in this study. Front hoof circumference immediately below the coronary band was measured weekly with a measuring tape in all horses present at the stable. Most horses accomplished a minimum of 2 training periods at the stable separated by periods of rest on a paddock. One sample t tests were used to evaluate if the mean change per week differed from zero. To estimate the repeatability coefficient, the left proximal hoof circumference of 25 horses was measured 3 times in a random order on one day. Results: Most horses showed a similar pattern of change. The proximal hoof circumference decreased during the training periods (P<0.0001) and increased when the horse was rested (P<0.0001). The decrease of the circumference during the first training period was ?0.66 mm/week on the left and ?0.64 mm/week on the right. During the second training period, this was ?0.58 mm/week on the left and ?0.57 mm/week on the right. During the rest period, the circumference increased by 1.03 mm/week on the left and 1.12 mm/week on the right. The repeatability coefficient for the left circumference was 1.8 mm. Conclusions: Horses showed a decrease in circumference during race training that reversed when they were rested. Potential relevance: Measurement of front hoof circumference is a simple method to assess change in hoof shape. It provides an opportunity to investigate the relationships between specific training, hoof shape and soundness.     

Alterations of cellular immune response during intensive training of event horses.

During strenuous exercise of horses that are prepared for international Three-Day-Events a significant decrease in the in vitro killing rate of phagocytosed yeast cells by the blood granulocytes has been observed. Other immunological parameters, such as the phorbolmyristate dependent chemiluminescence in granulocytes and the mitogenic stimulation of blood lymphocytes, remained unchanged.     

Effect of ivermectin on the immune response in mice.

To assess the effect of ivermectin on immune function (antibody production), male CD-1 mice were inoculated with an antigen the day after SC administration of ivermectin (0.2 mg/kg of body weight or 20 mg/kg). Responses were evaluated 5 days after inoculation of the antigen. Antibody production against sheep RBC, a T lymphocyte-, macrophage-dependent response, was enhanced by ivermectin treatment (P = 0.00049). In contrast, antibody production against dinitrophenyl-Ficoll, a T lymphocyte-independent, macrophage-dependent response, was not altered by ivermectin treatment. Results indicate that the immunostimulatory properties of ivermectin are associated with altered function of T lymphocytes, in particular, T-helper lymphocytes. The immunomodulating effects of ivermectin may provide an alternative approach for treatment of disease problems involving immunosuppression.     

Effect of exercise on age-related changes in collagen fibril diameter distributions in the common digital extensor tendons of young horses

OBJECTIVE: To determine whether specific treadmill exercise regimens would accelerate age-related changes in collagen fibril diameter distributions in the common digital extensor tendon (CDET) of the forelimbs of young Thoroughbreds. ANIMALS: 24 female Thoroughbreds. PROCEDURE: Horses were trained for 18 weeks (6 horses; short term) or 18 months (5 horses; long term) on a high-speed treadmill; 2 age-matched control groups (6 horses/group) performed walking exercise only. Horses were (mean +/- SD) 24 +/- 1 months and 39 +/- 1 months old at termination of the short-term and long-term regimens, respectively. Midmetacarpal CDET specimens were obtained and processed for transmission electron microscopy. Diameter and area of at least 1,000 collagen fibrils/specimen were measured by use of computerized image analysis. Mass-average diameter (MAD) of collagen fibrils and collagen fibril index were calculated for each horse. RESULTS: Collagen fibril MAD for the older horses was significantly less than that for the younger horses. Exercise did not significantly affect fibril diameter or distributions in either age group, and collagen fibril index did not differ significantly between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Age-related reduction in collagen fibril MAD agreed with findings for other tendons and species. Training did not accelerate age-related change in the CDET in contrast to a reported decrease in collagen fibril MAD in the superficial digital flexor tendon of horses trained long term. Our results support the concept that the functionally distinct nature of the CDET and superficial digital flexor tendon in horses results in fundamentally different responses to high-speed exercise regimens.     

Peripheral serotoninergic response to physical exercise in athletic horses

The purpose of this study was to evaluate the influence of exercise on plasma tryptophan (TRP) and free serotonin (f5-HT), whole blood-5-HT (WB-5-HT) and f5-HT/WB-5-HT ratio in Italian Saddle horses. Six clinically healthy Italian Saddle horses were subjected to a 450 meters obstacles course. Blood samples were collected from each horse by jugular venipuncture using vacutainer tubes with K₃-EDTA at rest, immediately after exercise, and after 30 min. TRP, f5-HT and WB-5-HT were analyzed by HPLC. Immediately after exercise, statistically significant increases of f5-HT (p<0.001) and WB-5-HT (p<0.001) were observed. After 30 min, f5-HT and WB-5-HT decreased compared to immediately after exercise, but were still significantly higher than rest values (p<0.01 and p<0.05, respectively). A significant linear regression between f5-HT and WB-5-HT was observed during experimental conditions. f5-HT and WB-5-HT modifications after exercise suggest an important role of peripheral serotoninergic markers in response to physical activity. The possible source of extra serotonin detected after show jumping should be clarified by further investigation.     

Effect of creatine supplementation on muscle metabolic response to a maximal treadmill exercise test in Standardbred horses

The aim of the present study was to investigate the effect of creatine (Cr) supplementation on muscle metabolic response in connection with a maximal treadmill exercise test, known to cause a marked anaerobic metabolic response and adenine nucleotide degradation. First, 6 Standardbred trotters performed a standardised maximal exercise test until fatigue (baseline test). The test used was an inclined incremental treadmill test in which the speed was increased by 1 m/s, starting at 7 m/s, every 60 s until the horse could no longer keep pace with the treadmill. After this baseline test, the horses were separated into 2 equal groups. One half received a dose of 25 g creatine monohydrate twice daily, and the other group were given the same dose of lactose (placebo). The supplementation period was 6.5 days, after which the maximal treadmill exercise test was performed again. A washout period of 14 days was allowed before treatments were switched between groups and a new supplementation period started. After this second supplementation period a new maximal exercise test was performed. After supplementation with creatine or placebo, horses were stopped after performing the same number of speed steps and duration of exercise as they had in the baseline test. Blood samples for analysis of plasma lactate, creatine (Cr), creatinine, hypoxanthine, xanthine and uric acid concentrations were collected at rest, during each speed step and during recovery. The total blood volume (TBV) was also determined. Muscle biopsies for analysis of muscle metabolites (adenosine triphosphate [ATP], adenosine diphosphate [ADP], adenosine monophosphate [AMP], inosine monophosphate [IMP], creatine phosphate [CP], lactate [La] and glycogen) were taken at rest, immediately post exercise and after 15 min recovery. The results showed no significant increase in plasma Cr or muscle total creatine concentration (TCr) after supplementation with Cr. At the end of exercise ATP and CP concentrations had decreased and IMP and lactate concentrations increased in muscle in all groups. Plasma lactate concentration increased during exercise and recovery and plasma uric acid concentration increased during recovery in all groups. No influence could be found in TBV after supplementation with creatine. These results show that creatine supplementation in the dosage used in this study had no influence on muscle metabolic response or TBV.     

The effect of initiation of exercise training in young horses on vitamin K status

Twelve Quarter Horses, 18 to 24 mo of age and having an average body weight of 460+/-12 kg, were randomly assigned to one of two exercise treatments (control and exercise) to study the effect of initiation of exercise training in young horses on vitamin K status. The control treatment consisted of hand-walking the horses 7 min/d and 5 d/wk for 180 d. Exercise consisted of the following treadmill exercise regimen: 2 min at 4.5 to 5.4 m/s, 3 min at 13.4 to 14.3 m/s, and 2 min at 4.5 to 5.4 m/s on 5 d/wk for 180 d. Both groups were allowed free access to brome grass hay (273 mg of phylloquinone/100 g) individually. Additionally, the exercise group was fed .45 kg of a grain-mix concentrate (40 mg of phylloquinone/100 g) on the days they were exercised. Jugular venous blood samples were collected at d 0, 30, 60, 90, 120, 150, and 180. Blood samples were analyzed for total serum osteocalcin and vitamin K status via the hydroxyapatite binding capacity of serum osteocalcin calculated as follows: Hydroxyapatite binding capacity of serum osteocalcin = [(total serum osteocalcin - serum osteocalcin following extraction of serum with hydroxyapatite)/total serum osteocalcin] x 100. All horses were killed with an overdose of sodium pentobarbital on d 180. Computed tomographic osteoabsorptiometry was used to measure relative bone density distribution on the surface of the distal radial carpal bone, proximal third carpal bone, and the distal third metacarpal condyle. Relative bone density distribution was measured in Hounsfield units and categorized as low-, medium-, and high-density bone corresponding to 800 to 1,199, 1,200 to 1,299, and 1,300 to 3,000 Hounsfield units, respectively. Carpal and metacarpophalangeal joints were assigned a score of 0 (normal) to 3 (severe) that reflected the presence and severity of joint lesions. Hydroxyapatite binding capacity of serum osteocalcin and serum osteocalcin were unchanged over the 180-d period in both groups. Exercised horses had a higher percentage of high density bone (P < .01) and a lower percentage of low density bone (P < .01) on the surface of the third metacarpal condyle and a higher cumulative gross lesion score (P < .01) in the metacarpophalangeal joint than controls. There were no differences between treatments in relative bone density distribution in the carpal bones, or cumulative gross lesion score of the carpal joints. No significant correlation was present between the serum measures, osteocalcin and hydroxyapatite binding capacity of serum osteocalcin, and the bone measures, relative bone density distribution and joint gross lesion score.     

Effect of lactation on the immune response to gastrointestinal nematodes.

The immune expulsion of gastrointestinal nematodes is impaired during lactation by the inhibition of the lymphocyte mediated component. As a result the population of worms may increase. At the same time, the fecundity of female worms in previously immune hosts may increase to approach that of worms in fully susceptible animals. There is good evidence that these events are primarily endocrinal in origin but although prolactin is probably involved, certain anomalies exist. These may reflect differences in the secretion pattern of the hormone in different phases of reproduction or they may indicate an interrelationship with a second hormone, for instance, adrenal glucocorticoids.     

Effects of branched-chain amino acids on immune status of young racing horses

High-intensity exercise and competition are associated with depressed immune function. Young horses, which participate in high-intensity exercise and competitions, are at increased risk for the development of infectious disease due to depression of immune function. The effects of branched-chain amino acid (BCAA) supplementation on the immune status of young racing horses were evaluated, determining whether BCAA might help to avoid or reduce immune suppression during exercise and competitions. Twenty horses (10 male and 10 female) were treated with BCAA supplementation; another twenty untreated horses (10 male and 10 female) constituted control group. Peripheral blood was collected from each animal and evaluated for lymphocyte subsets, phagocytosis analysis of monocytes and granulocytes, lymphocyte proliferative response, and expression of cytokine-encoding messenger ribonucleic acids (mRNAs). The numbers of CD4⁺, CD8⁺, and major histocompatibility complex (MHC) class II⁺ cells in females of the treated group were significantly higher than those in females of the control group. The lymphocyte proliferative response in female of the treated group also was significantly higher than that in females of the control group. In addition, expression of mRNAs encoding interleukin-1β (IL-1β) and interferon-γ (IFN-γ) in females of the treated group was significantly higher than that in females of the control group. There were no significant differences between males of the treated and control groups. The results of this study indicated the positive effects of BCAA supplementation in counteracting immunosuppression in young female racing horses during and following high-intensity exercise.     

Effect of gender and exercise on haematological and biochemical parameters in Holsteiner horses

The objective of this study was to assess the influence of exercise of average intensity in the haematological and biochemical values, as well as acidic resistance of erythrocytes in mares and stallions of Holsteiner breed. A total of seventeen horses of Holstein breed (seven mares and 10 stallions aged 6 years) were used in this study. The blood samples were assessed for haematocrit (HCT) value, haemoglobin concentration (HGB), the amount of red blood cells (RBC), white blood cells (WBC), platelets (PLT), leucogram, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), red cell distribution width (RDW) and platelet distribution width (PDW). Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) as well as oxidative stress biomarkers were analysed. Stallions showed a significant increase in leucocytes and granulocytes amount, as well as erythrocytes, haemoglobin and haematocrit levels after exercise test. Pre‐exercise level of mean corpuscular haemoglobin concentration was higher in stallions. At the same time, mares showed significant decrease in platelet volume after exercise test. Physical effort in stallions leads to significant increase in aspartate aminotransferase activity. Trained mares and stallions showed a decrease in lipid peroxidation after exercise. Exercise also caused increase in oxidative modified protein of erythrocytes in stallions indicating by exercise‐induced oxidative stress. The resistance of erythrocytes in 0.1 m HCl was similar between females and males. No statistically significant differences in the percentage of haemolysed erythrocytes before and after exercise were observed.     

Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1.

The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.     

Effect of ranitidine on gastric acid secretion in young male horses

Gastric cannulas were placed surgically in 5 young male horses. After a 2-week recovery period, horses were studied once a week. Horses were fasted for 24 hours, and gastric fluid output was collected for 5 continuous hours. Volumes were recorded every 15 minutes, and pH and hydrogen ion concentration were determined in an aliquot from each period. In 10 basal experiments, using 5 horses, volume, pH, and hydrogen ion concentration were continuously variable. Mean acid output was 45.1 +/- 2.02 microEq/15 min/kg (mean +/- SEM). In 6 experiments, using 3 horses, 0.5 mg of ranitidine/kg of body weight, given as an IV bolus after a 1-hour basal collection, significantly (P less than 0.02) inhibited hourly total acid output for 4 hours, but did not significantly change pH. The cannulation technique was done without complications, and horses tolerated the cannula for several months. Seemingly, the horse has a continuously variable gastric acid secretion, and histamine type-2 receptors have a role in this process.