Assessment of exercise-induced alterations in neutrophil function in horses

OBJECTIVE: To evaluate the effects of a standardized exercise test to exhaustion in horses on leukocyte function ex vivo. ANIMALS: 6 Thoroughbred geldings. PROCEDURES: Blood samples were obtained from each horse before exercise; at exhaustion (termed failure); and at 2, 6, 24, 48, and 72 hours after exercise to evaluate hematologic changes, rate of leukocyte apoptosis, and leukocyte production of reactive oxygen species (ROS) ex vivo. To assess leukocyte function, leukocyte ROS production in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, and phorbol myristate acetate was evaluated. Apoptosis was evaluated via assessment of caspase activity in leukocyte lysates. RESULTS: In response to lipopolysaccharide, production of ROS by leukocytes was significantly increased at 2 hours and remained increased (albeit not significantly) at 6 hours after exercise, compared with the preexercise value. In the absence of any stimulus, leukocyte ROS production was significantly increased at 6 and 24 hours after exercise. In contrast, ROS production in response to phorbol myristate acetate was significantly decreased at 6, 24, and 72 hours after exercise. Leukocyte ROS production induced by zymosan or peptidoglycan was not altered by exercise. Leukocytosis was evident for 24 hours after exercise, and neutrophilia was detected during the first 6 hours. A significant increase in the rate of leukocyte apoptosis was detected at failure and 72 hours after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that strenuous exercise undertaken by horses causes alterations in innate immune system functions, some of which persist for as long as 72 hours after exercise.     

Exercise-induced alterations in pro-inflammatory cytokines and prostaglandin F2alpha in horses

Using an established standardized exercise test on a high-speed treadmill, thirteen Thoroughbred racehorses were exercised to fatigue (failure); blood samples were obtained before exercise, at failure, and at 2, 6, 24, 48, and 72 h after exercise. The exercise test induced a systemic inflammatory response characterized by a mild transient endotoxemia, leukocytosis, increased leukocyte expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, and IL-6, and increased circulating concentrations of TNF-alpha and prostaglandin F2 alpha (PGF 2 alpha), with the most pronounced changes being evident at failure and 2h after exercise. Expression of mRNA for IL-6, TNF-alpha, and IL-1 beta was increased by 120-fold, three-fold, and four-fold, respectively, when compared to pre-exercise values. Plasma concentrations of 6-keto-PGF1alpha and PGE2 did not change in response to the exercise test. Collectively, these findings indicate that brief, strenuous exercise induces endotoxemia and a systemic pro-inflammatory response in horses that persists for at least 2h.     

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses

OBJECTIVE: To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. ANIMALS: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). PROCEDURE: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. RESULTS: Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.     

Intravenous and sublingual buprenorphine in horses: pharmacokinetics and influence of sampling site

ObjectiveTo describe the pharmacokinetics and adverse effects of intravenous (IV) and sublingual (SL) buprenorphine in horses, and to determine the effect of sampling site on plasma concentrations after SL administration.Study designRandomized crossover experiment; prospective study.AnimalsEleven healthy adult horses between 6 and 20 years of age and weighing 487–592 kg.MethodsIn the first phase; buprenorphine was administered as a single IV or SL dose (0.006 mg kg^?1) and pharmacokinetic parameters were determined for each route of administration using a noncompartmental model. In the second phase; the jugular and lateral thoracic veins were catheterized for simultaneous venous blood sampling, following a dose of 0.006 mg kg^?1 SL buprenorphine. For both phases, plasma buprenorphine concentrations were measured using ultra-performance liquid chromatography with mass spectrometry. At each sampling period, horses were assessed for behavioral excitement and gastrointestinal motility.ResultsFollowing IV administration, buprenorphine mean ± SD half-life was 5.79 ± 1.09 hours. Systemic clearance (Cl) following IV administration was 6.13 ± 0.86 mL kg^?1 minute^?1 and volume of distribution at steady-state was 3.16 ± 0.65 L kg^?1. Following IV administration, horses showed signs of excitement. Gastrointestinal sounds were decreased following both routes of administration; however, none of the horses exhibited signs of colic. There was a significant discrepancy between plasma buprenorphine concentrations measured in the jugular vein versus the lateral thoracic vein following phase 2, thus pharmacokinetic parameters following SL buprenorphine are not reported.Conclusions and clinical relevanceBuprenorphine has a long plasma half-life and results in plasma concentrations that are consistent with analgesia in other species for up to 4 hours following IV administration of this dose in horses. While buprenorphine is absorbed into the circulation following SL administration, jugular venous sampling gave a false measurement of the quantity absorbed and should not be used to study the uptake from SL administration.     

Systemic and colonic venous plasma biochemical alterations in horses during low-flow ischemia and reperfusion of the large colon.

The purpose of this study was to determine the effects of low-flow ischemia and reperfusion (I-R) of the large colon on 16 systemic venous (SV) and colonic venous (CV) plasma biochemical variables in horses. Horses (n = 24) were randomly allocated to 3 groups: sham-operated (n = 6), 6 h ischemia (n = 9), and 3 h ischemia followed by 3 h reperfusion (n = 9). SV and CV heparinized blood was collected at 0, 1, 3, 3.25, 4, and 6 h. The SV-CV difference was calculated for each variable. The SV, CV, and SV-CV difference for albumin, total protein, and calcium decreased significantly (P < 0.05) across time in horses of all groups, but there were no differences among groups. SV phosphorous was significantly increased from baseline (BL) at 1 to 6 h in horses of all groups, but there were no differences among groups. CV phosphorous was significantly greater than BL from 1 to 6 h in group-2 horses and from 1 to 3 h in group-3 horses. SV potassium was not different among groups, but was significantly higher at 6 h, compared with BL in horses of all groups. CV potassium was significantly greater than BL from 1 to 6 h in horses of groups 2 and 3. SV glucose was greater at 6 h compared with all previous times in horses of all groups, but there were no difference among groups. CV glucose was significantly lower than BL and group-1 values in horses of groups 2 and 3 during ischemia, but returned to BL during reperfusion in group-3 horses. CV anion gap was significantly greater and SV-CV anion gap was significantly more negative in horses of groups 2 and 3, compared with group-1 horses during ischemia. The biologic relevance of these alterations is unknown, but they may contribute to histopathologic, hemodynamic, and metabolic alterations characteristic of low-flow I-R. Alternatively, these alterations may simply reflect colonic injury sustained during I-R. Results suggest that the colon utilizes glucose as a fuel and generates acid anions during low-flow ischemia. Increased CV phosphorous and potassium during I-R likely occurs as a result of leakage of intracellular stores subsequent to cellular damage.     

Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.     

Changes in digital venous pressures of horses moving at the walk and trot

Blood pressures from the catheterized lateral digital vein of the fore-limbs of 6 clinically normal horses were measured at rest, at the walk, and at the trot. Digital venous pressures were compared with the phases of the stride and weight-bearing forces, using electrogoniometry and a force platform. Rapid increases in digital venous pressures to maximal values were observed immediately before maximal forces during the support period of the stride. At the trot, increases in peak vertical forces were paralleled by increases in peak digital venous pressures. Seemingly, the hydrodynamics of the digital circulatory system help to dissipate the initial impact of hoof strike at the walk and the trot.     

Cardiopulmonary,blood and peritoneal fluid alterations associated with abdominal insufflation of carbon dioxide in standing horses

REASONS FOR PERFORMING STUDY: Abdominal insufflation is performed routinely during laparoscopy in horses to improve visualisation and facilitate instrument and visceral manipulations during surgery. It has been shown that high-pressure pneumoperitoneum with carbon dioxide (CO2) has deleterious cardiopulmonary effects in dorsally recumbent, mechanically ventilated, halothane-anaesthetised horses. There is no information on the effects of CO2 pneumoperitoneum on cardiopulmonary function and haematology, plasma chemistry and peritoneal fluid (PF) variables in standing sedated horses during laparoscopic surgery. OBJECTIVES: To determine the effects of high pressure CO2 pneumoperitoneum in standing sedated horses on cardiopulmonary function, blood gas, haematology, plasma chemistry and PF variables. METHODS: Six healthy, mature horses were sedated with an i.v. bolus of detomidine (0.02 mg/kg bwt) and butorphanol (0.02 mg/kg bwt) and instrumented to determine the changes in cardiopulmonary function, haematology, serum chemistry and PF values during and after pneumoperitoneum with CO2 to 15 mmHg pressure for standing laparoscopy. Each horse was assigned at random to either a standing left flank exploratory laparoscopy (LFL) with CO2 pneumoperitoneum or sham procedure (SLFL) without insufflation, and instrumented for measurement of cardiopulmonary variables. Each horse underwent a second procedure in crossover fashion one month later so that all 6 horses had both an LFL and SLFL performed. Cardiopulmonary variables and blood gas analyses were obtained 5 mins after sedation and every 15 mins during 60 mins baseline (BL), insufflation (15 mmHg) and desufflation. Haematology, serum chemistry analysis and PF analysis were performed at BL, insufflation and desufflation, and 24 h after the conclusion of each procedure. RESULTS: Significant decreases in heart rate, cardiac output and cardiac index and significant increases in mean right atrial pressure, systemic vascular resistance and pulmonary vascular resistance were recorded immediately after and during sedation in both groups of horses. Pneumoperitoneum with CO2 at 15 mmHg had no significant effect on cardiopulmonary function during surgery. There were no significant differences in blood gas, haematology or plasma chemistry values within or between groups at any time interval during the study. There was a significant increase in the PF total nucleated cell count 24 h following LFL compared to baseline values for LFL or SLFL at 24 h. There were no differences in PF protein concentrations within or between groups at any time interval. CONCLUSIONS: Pneumoperitoneum with CO2 during standing laparoscopy in healthy horses does not cause adverse alterations in cardiopulmonary, haematology or plasma chemistry variables, but does induce a mild inflammatory response within the peritoneal cavity. POTENTIAL RELEVANCE: High pressure (15 mmHg) pneumoperitoneum in standing sedated mature horses for laparoscopic surgery can be performed safely without any short-term or cumulative adverse effects on haemodynamic or cardiopulmonary function.     

Metalloproteinases and tumor necrosis factor-alpha activities in synovial fluids of horses: correlation with articular cartilage alterations

Early detection of osteoarthritis in horses represents a challenge for equine practitioners. Several biological markers have been implicated in the pathological processes involved in articular cartilage destruction. To further document cartilage matrix proteases production, synovial fluid was collected from 14 horses (90 joints) before they were subjected to euthanasia. Growth macroscopic examination of the joints gave information on cartilage alterations. Samples were analyzed for matrix metalloproteinase (MMPs) activities by gelatin zymography and tumor necrosis factor alpha (TNF-alpha) cytotoxicity using L929 cells. Significant increase of MMP-9 monomer and dimer were found in synovial fluids of joints with severe cartilage alterations. On the contrary, the activity of TNF-alpha was not correlated to the degree of joint damage. The levels of MMP-9 monomer and dimer in the synovial fluid could reflect cartilage alteration in arthritis in the horse.     

Comparative biokinematic study of young and adult Andalusian horses at the trot

The purpose of this study was to determine basic kinematic parameters (linear, temporal and angular) in young and adult Andalusian horses (P.R.E.) at the trot, using a normal computer-aided videography system. The trotting gaits of 16 horses were analysed: seven young horses (3.7 +/- 0.2 years old, height at withers 167.1 +/- 4.1 cm) and nine adult stallions (12.3 +/- 2.9 years old, height at withers 162.9 +/- 3.6 cm) were recorded at least 6 times at the trot using a 25-Hz video-camera filming from the side. Video images were processed with a real-time digital system (SMVD). Speeds averaged 3.84 and 3.75 m/s for young and adult horses, respectively. Differences between age groups for speed and linear and temporal parameters of the stride were not significant. However, variations in angular parameters were detected: adults showed a greater ARM than younger horses for most forelimb joints. In the hind limb, hip, stifle, and, to a lesser degree, the tarsal joint, a smaller degree of extension during the stance phase was observed in adult horses.     

Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-aged horses

The objective of this study was to compare bone marrow (BM) aspirates from the sternum and the tuber coxae of middle-aged horses. Bone marrow was obtained from the sternum and both tubera coxae of 12 healthy, 13-year-old geldings. Two different puncture techniques were used for the tuber coxae. The 2 syringes used for sternal sampling were evaluated separately. The mononuclear cell (MNC) fraction of the BM was isolated and the mesenchymal stem cells (MSCs) were culture-expanded. At the sternum, BM aspiration was always possible. Bone marrow aspiration at the tuber coxae required straight and deep needle penetration combined with high negative pressure. With this technique a median sample amount of 11.0 mL with large individual variation was obtained. A median of 3.06 × 10(6) MNC/mL BM (1st syringe) and 2.46 × 10(6) MNC/mL BM (2nd syringe) was isolated from sternal samples. In contrast, the tuber coxae yielded a median of 0.27 × 10(6) MNC/mL BM. The first passage yielded a median of 2.19 × 10(6) MSC (1st syringe) and 1.13 × 10(6) MSC (2nd syringe) from sternal samples, compared to a significantly lower median number of MSC from tuber coxae BM (0.06 × 10(6) MSC). The number of MNC and MSC obtainable from the BM aspirates taken from the tuber coxae is significantly lower than that obtained from the sternal BM aspirates. Autologous BM for the equine athlete is particularly clinically relevant at an advanced age. Based on our findings, the tuber coxae cannot be recommended for BM aspiration in middle-aged horses.     

Medical examination of horses at auction sales.

The role of the veterinarian at auction sales begins long before the consignment is assembled. A number of veterinarians are called upon by sales companies to evaluate the candidates before acceptance into the consignment. At sales time, the number of veterinarians involved grows to large numbers as the prospective buyers employ them to evaluate the animals to suit their individual needs. Professional ethics must be considered at all times when evaluating animals before auction. The findings must be kept confidential. The consignor allows veterinarians to examine his or her animals with the understanding that the opinions are kept in confidence with the individual employing the veterinarian. Frequently, more than one buyer will request radiographs of an animal that the veterinarian has previously examined for another buyer. This is best resolved by sharing the fee and the information found in the examination. Unfortunately, it is impossible to prevent a client from revealing information to another buyer, and the consignor often blames the veterinarian for disclosing findings. It is obvious that the veterinarian's' ethics are on trial at auction sales, and a veterinarian must guard his or her confidential opinion at all times.     

Regional differences in biochemical,biomechanical and histomorphological characteristics of the equine suspensory ligament

Reasons for performing study: Desmopathies of the suspensory ligament are relatively frequent and vary in prevalence over different anatomical regions. This variation may be because of regional differences in tissue characteristics. Objective: To characterise different regions of healthy forelimb suspensory ligaments (SLs) by means of biochemical, biomechanical and histomorphological techniques. Hypothesis: There is substantial topographical heterogeneity in the SL with respect to structural, compositional and functional characteristics. Methods: SLs were harvested from 38 limbs and used for biochemical (n = 20), biomechanical (n = 14) and histomorphological (n = 4) evaluation. Sulphated glycosaminoglycan (S‐GAG), DNA and collagen content, degree of lysyl hydroxylation and numbers of enzymatic and nonenzymatic cross‐links were determined in 7 regions of the SL: lateral and medial part of the origin (OM, OL), mid‐body (MB), axial and abaxial parts of the lateral and medial branches (ILAX, ILAB, IMAX and IMAB, respectively). Passive resistance to tensile loading was measured in 5 regions of the SL (all except OL and OM). Results: DNA content was lower in OL and OM than in all other parts. GAG content was also lower in OL and OM and highest in ILAB and IMAB. Collagen content was lower in OL/OM and highest in ILAX/IMAX. Pentosidine levels were highest in OL and significantly lower in the lateral insertion (ILAX/ILAB). There were no differences in hydroxylysylpyridinoline (HP) cross‐links or lysyl hydroxylation. Stiffness (P<0.01) and modulus of elasticity (P<0.01) were substantially higher in the MB region than in all other regions except for IMAB. Strain at failure was lower in the MB region (P<0.0001), resulting in a comparable force at rupture as in the other regions. Conclusions: Matrix composition differs to a relatively limited extent between different regions of the SL. The mid‐body of the ligament is stiffer than the branches, which have similar properties and relevance and mechanical differences between mid‐body and branches/origin may explain some use‐related differences in the prevalence of lesions.     

Alternating one-letter symbols for the representation of codon variations at the level of amino acid sequences

An idea is presented to use different letter types (e.g. L. 1, L, 1, L, l) for the alternating triplets coding the same amino acids. Such compact and demonstrative amino acid sequence comparisons can make easy the identification of differences at the DNA level if not seen in amino acid sequences (e.g. for choosing specific or non-specific primers for polymerase chain reaction).     

Acute myopathy in horses at grass in east and south east Scotland

A myopathy of horses at grass in east and south east Scotland was recognised in the autumn and winter of 1984 and the spring of 1985. The clinical signs resembled those of paralytic myoglobinuria. Grossly increased creatine kinase activities and the passage of dark brown urine were consistent features. However, the horses were not in training, most of them died and the muscles affected were those of posture and respiration rather than movement. The condition may be unrelated to nutritional myopathy because all the cases had adequate levels of alpha-tocopherol although their selenium status varied from normal to deficient. The clinical and pathological findings in 12 cases are presented and the differential diagnosis and possible aetiologies discussed.     

Microbiological and serological study of leptospirosis in horses at slaughter: first isolations

A bacteriological survey of kidneys from 145 abattoir horses was performed, which resulted in the isolation of two Leptospira strains. The isolates were serologically typed as belonging to serogroups Australis and Pomona, and REA identified them as L. interrogans serovar Bratislava and L. kirschneri serovar Tsaratsovo, respectively. These are the first Leptospira isolates obtained from horses in Portugal and the Bratislava strain is the first serogroup Australis strain to be isolated in this country. The 145 horses were also serologically tested for leptospiral antibodies, and 37% had MAT titres #10878;1:10.