Maternal obesity increases insulin resistance, low-grade inflammation and osteochondrosis lesions in foals and yearlings until 18 months of age.

INTRODUCTION: Obesity is a growing concern in horses. The effects of maternal obesity on maternal metabolism and low-grade inflammation during pregnancy, as well as offspring growth, metabolism, low-grade inflammation, testicular maturation and osteochondrotic lesions until 18 months of age were investigated. MATERIAL AND METHODS: Twenty-four mares were used and separated into two groups at insemination according to body condition score (BCS): Normal (N, n = 10, BCS ≤4) and Obese (O, n = 14, BCS ≥4.25). BCS and plasma glucose, insulin, triglyceride, urea, non-esterified fatty acid, serum amyloid A (SAA), leptin and adiponectin concentrations were monitored throughout gestation. At 300 days of gestation, a Frequently Sampled Intravenous Glucose Tolerance Test (FSIGT) was performed. After parturition, foals' weight and size were monitored until 18 months of age with plasma SAA, leptin, adiponectin, triiodothyronine (T3), thyroxine (T4) and cortisol concentrations measured at regular intervals. At 6, 12 and 18 months of age, FSIGT and osteoarticular examinations were performed. Males were gelded at one year and expression of genes involved in testicular maturation analysed by RT-qPCR. RESULTS: Throughout the experiment, maternal BCS was higher in O versus N mares. During gestation, plasma urea and adiponectin were decreased and SAA and leptin increased in O versus N mares. O mares were also more insulin resistant than N mares with a higher glucose effectiveness. Postnatally, there was no difference in offspring growth between groups. Nevertheless, plasma SAA concentrations were increased in O versus N foals until 6 months, with O foals being consistently more insulin resistant with a higher glucose effectiveness. At 12 months of age, O foals were significantly more affected by osteochondrosis than N foals. All other parameters were not different between groups. CONCLUSION: In conclusion, maternal obesity altered metabolism and increased low-grade inflammation in both dams and foals. The risk of developing osteochondrosis at 12 months of age was also higher in foals born to obese dams.

Sensitivity and specificity of blood leukocyte counts as an indicator of mortality in horses after colic surgery.

The objectives of this study were to describe and relate perioperative changes in blood leukocyte counts to the outcome of surgical colic horses, determine a cut-off value in the early postoperative period to obtain an indicator of the outcome, and compare the obtained value to a validation population of horses. Fifty-three horses undergoing colic surgery were included in the descriptive part of the study. Total leukocyte counts were performed before, during and serially after surgery. A receiver operating characteristic analysis was performed on the leukocyte counts of 45 of these horses to determine a cut-off value for the outcome. The results obtained were validated on a second set of 50 horses that underwent colic surgery in similar conditions. The kinetics of blood leukocytes in survivors was higher than in non-survivors during the first days. Non-survivor horses were more likely to have at least one blood leukocyte count ≤ 3.9 × 10(3)/mm(3) between 28 and 60 hours after surgery than survivor horses. This cut-off value was confirmed in the validation population. These results suggest that routine values of blood leukocyte counts can be used as an additional prognostic indicator after colic surgery alongside other predictors previously associated with the outcome.

Association of breeding conditions with prevalence of osteochondrosis in foals.

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet elucidated, but it is accepted that OC lesions are the result of intrinsic genetic and external factors. The aim of the present work was to evaluate the relationship between breeding management and OC. Breeding conditions were recorded, and a radiological examination was performed in 223 foals. Feeding practice and housing management were analysed in a multivariate model to determine risk factors for OC in three periods: gestation, birth to weaning and weaning to one-year-old. The major breakthrough of this study is the significant relationship between OC development and (1) the maternal nutrition during gestation and (2) the type of housing of the foals during their first year. It appears that mares fed with concentrates during gestation are more likely to produce foals that are subsequently affected by OC compared with other mares (P<0.05). Foals housed exclusively at pasture until one year of age are significantly less affected than foals exclusively housed in box or, alternatively, in box and at pasture (P<0.05). These results underline the role of the energy metabolism and the level of exercise in the aetiologic process of the disease, and help to develop preventive strategies during the crucial period of gestation to one year of age of the foal.

Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses.

REASONS FOR PERFORMING STUDY: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. OBJECTIVE: To compare plasmatic NE concentrations before and after different forms of intensive exercise. MATERIALS AND METHODS: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. RESULTS: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P < 0.05). NE was significantly (P < 0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P < 0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. CONCLUSIONS: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.

Heel effects on joint contact force components in the equine digit: a sensitivity analysis.

REASONS FOR PERFORMING STUDY: Whereas the effect of heel configuration on the tension of the suspensory apparatus is well documented in the literature, there are few reports of joint contact force components in the equine distal forelimb. OBJECTIVES: To improve understanding of the effect of heel configuration on equine digit joint loading, a sensitivity analysis was performed to compare the effect of a raised heel on joint contact force components in the coffin and fetlock joints during the stance phase of the trot. MATERIALS AND METHODS: Four Warmblood horses were used. An inverse dynamic analysis was carried out using kinematic and kinetic data. Taking into account the tendon wrapping forces (WF) around the sesamoid bones in the calculations, the joint contact forces (CF) were estimated for the coffin and fetlock joints during the trot stance phase (4 m/s). To test the sensitivity of the results to heel configuration changes, calculations were performed repeatedly for different heel configurations (raised by 0, 6 and 12°). A one-way ANOVA with repeated measures was used to test the effect of heel configuration (at the 3 levels) (α = 0.05; P < 0.05; post hoc testing: Bonferroni). RESULTS: For heel configurations raised from 0-12°: whereas the tension of the deep digital flexor tendon decreased and the tension of the superficial digital flexor tendon increased, for the coffin joint the peak WF (1.4 ± 0.25 bwt; 1.2 ± 0.2 bwt; 0.95 ± 0.1 bwt) and the peak CF (2.45 ± 0.25 bwt; 2.2 ± 0.2 bwt; 2 ± 0.1 bwt) decreased significantly (P < 0.05). For the fetlock joint, the peak WF (3.8 ± 0.7 bwt; 4.1 ± 0.3 bwt; 4.4 ± 0.25 bwt) and the peak CF (4.35 ± 0.7 bwt; 4.7 ± 0.35 bwt; 5 ± 0.3 bwt) increased, but not significantly. CONCLUSION: This analysis suggests that the coffin joint loading and fetlock joint loading are strongly connected. The heel configuration may influence both coffin joint and fetlock joint contact force components.

Radiographic evolution of the forelimb digit in Ardenner horses from weaning to 28 months of age and its clinical significance.

Ossification of collateral cartilages is a common radiographic finding in draught horses and indication of a high frequency of interphalangeal osteo-arthropathy have been found in Ardenner horses. This study first aimed at following the evolution of the digits of the forelimbs on radiographic images in a group of 32 Ardenner horses from weaning to 852 +/- 19 days and, secondly, at evaluating the clinical significance of the observed radiographic changes. At 250 +/- 21 days, only 23% of the horses presented a small separate ossification centre of collateral cartilages above the level of the distal sesamoid bone or at the level of the proximal half of the middle phalanx. At 852 +/- 19 days, 100% of the horses presented an ossification of collateral cartilages but at different stages and grades. The presence and the extent of the ossification of the lateral cartilages were not significantly associated with lameness. Phalanges changes appeared at the average age of 1 year and they were represented by entheseophytes on the dorsal surface of the middle and distal phalanges. The frequency of affected horses increased with age as well as the size of the entheseophytes. This may reflect mechanical stresses applied to the interphalangeal joints. A significant association with the presence of lameness was pointed out. The presence and the severity of phalanges changes could be of importance, in combination with a clinical examination, in improving the selection of horses able to assume work and/or leisure activities.

Pancreatic injury in equine acute abdomen evaluated by plasma trypsin activity and histopathology of pancreatic tissue.

In cases of equine acute abdominal disease, where pancreatic damage is suspected, pancreatic damage can be assessed by measuring increased trypsin activity in the plasma of horses suffering intestinal obstruction and severe shock. The pancreas is particularly vulnerable to splanchnic hypoperfusion because it is a highly active tissue. In this study, 10 horses undergoing abdominal surgery for intestinal obstruction were assayed for trypsin activity on admission and, because of extensive intestinal lesions that were not amenable to surgery, euthanasia was selected; the pancreas was removed before euthanasia. Trypsin activity in the plasma of these horses was significantly higher than in healthy horses (196 ng/ml +/- 128.2 versus 28.5 ng/ml +/- 19.2; P = 0.0026). Light and transmission electron microscopy revealed slight to severe lesions of vacuolar degeneration, a few zymogen granules, dilation of the endoplasmic reticulum, and swelling of mitochondria in the exocrine pancreas. The activation of an inflammatory cascade occurring during strangulating intestinal obstruction could increase pancreatic anoxic lesions caused by severe shock and hypoperfusion in the horse. Further studies will show the significance of pancreatic lesions and the ensuing damage in equine acute intestinal obstruction and shock.

Plasma trypsin level in horses suffering from acute intestinal obstruction.

Gastrointestinal disorders in horses leading to endotoxic shock could have further consequences on other splanchnic organs such as the pancreas, as can be seen in humans suffering from septic shock. In this study, the range of enzymatically active trypsin (EAT) in healthy horses was established and is similar to the range observed in healthy humans. EAT values were determined in horses with acute abdominal crises on admission as well as during anaesthesia and in the postoperative phase. A significant increase in plasma EAT was found in 59% of the horses with surgical colic when compared to our established reference range. Significantly higher values were found in severe shock cases. When separated in groups according to the duration of colic before referral, significantly higher EAT values were observed in the non-survivor group compared to the survivor group of colics of short duration. EAT plasma values increased significantly during the postoperative phase, and were significantly higher in small intestine obstructions than in large bowel disorders. In human medicine, hypovolaemic or septic shock patients show an increase in pancreatic proteases. Splanchnic hypoperfusion during shock could lead to pancreatic damage resulting in trypsin liberation into the peritoneal space and an increase in plasma levels. Trypsin is able to activate inflammatory cascades and leucocytes and could play a role in multiple organ failure. Further studies are needed to evaluate the implications of changes in plasma trypsin in the disease process of equine acute abdomen and to demonstrate possible pancreatic damage.

Determination of a gravity and shock score for prognosis in equine surgical colic.

A retrospective study evaluated 200 surgical colic cases. A gravity score (GS) based on four clinical parameters estimating intestinal obstruction (rectal palpation, borborygmi, abdominal distension, pain) and classified into three categories was established and tested to determine if it could evaluate prognosis. A shock score (SS) based on six parameters was also attributed to each case. The overall survival rate was 54%. The statistical analysis showed a significant (P < 0.01) difference in the survival rate in the different categories of the GS, as well as in the categories of the SS. A multivariate logistic regression model showed that horses with GS 3 are 10.6 times more likely to die than those with GS 1. A model combining the two scores showed an odds ratio of 7.1 for GS 3 versus GS 1, and for SS 3 versus SS 1, the odds ratio was 7.2.

Comparison of thiopentone/guaifenesin, ketamine/guaifenesin and ketamine/midazolam for the induction of horses to be anaesthetised with isoflurane.

Forty-eight horses subjected to elective surgery were randomly assigned to three groups of 16 horses. After premedication with 0.1 mg/kg acepromazine intramuscularly and 0.6 mg/kg xylazine intravenously, anaesthesia was induced either with 2 g thiopentone in 500 ml of a 10 per cent guaifenesin solution, given intravenously at a dose of 1 ml/kg (group TG), or with 100 mg/kg guaifenesin and 2.2 mg/kg ketamine given intravenously (group KG), or with 0.06 mg/kg midazolam, and 2.2 mg/kg ketamine given intravenously (group KM). Anaesthesia was maintained with isoflurane. The mean (sd) end tidal isoflurane concentration (per cent) needed to maintain a light surgical anaesthesia (stage III, plane 2) was significantly lower in group KM (0.91 [0.03]) than in groups TG (1.11 [0.03]) and KG (1.14 [0.03]). The mean (sd) arterial pressure (mmHg) was significantly lower in group KG (67.4 [2.07]) than in groups TC (75.6 [2.23]) and KM (81.0 [2.16]). There were no significant differences in the logarithm of the heart rate, recovery time or quality of recovery between the three induction groups. However, pronounced ataxia was observed in the horses of group KM, especially after periods of anaesthesia lasting less than 75 minutes.

Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture.

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.

Plasma myeloperoxidase level and polymorphonuclear leukocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results.

Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3). The mean peak values of MPO for these phases were 121.6 ng/mL (P1), 168.6 ng/mL (P2), and 107.0 ng/mL (P3) for the non-strangulating group, and 242.6 ng/mL (P1); 426.0 ng/mL (P2), and 379.5 ng/mL (P3) for the strangulation group. The variations of the mean peak values of plasma MPO were significantly different between the 2 groups and between the different phases. A significant increase of the least square means of MPO was observed between P1 and P2. A significant decrease of the least square means of the number of circulating leukocytes was observed between P1 and P3. Polymorphonuclear neutrophil activation could play a major role in the pathogenesis of acute abdominal disease and endotoxic shock.

In-shoe foot force sensor to assess hoof balance determined by radiographic method in ponies trotting on a treadmill.

Adaptation of an in-foot shoe force sensor and the gait analysis system 'Fscan' makes it possible to monitor the distribution of the vertical forces under the equine foot in motion. The aim of this study is to investigate the effects of two different trimmings on forces under the foot during the trot. The first one increased the height of the lateral hoof wall and the second one restored the mediolateral balance of the foot. These two trimmings were examined by using a radiographical method that quantifies the interphalangeal articular asymmetries due to asymmetrical bearing. The location of the centre of force of the weight-bearing foot and the distribution of the forces applied to the lateral and medial solar surfaces during a stride were analyzed. After optimal trimming, the centre of force of the weight-bearing foot tended to approach the centre of the palmar figure, perpendicular to the distal interphalangeal joint centre. The sum of the forces recorded under the lateral and medial parts respectively of the foot during one stride tended to balance out after corrective trimming.

Interactions between lipopolysaccharides and blood factors on the stimulation of equine polymorphonuclear neutrophils.

In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (> or = 99% neutrophils; viability > or = 98%). Increasing concentrations of Escherichia coli (E. coli) LPS (from 0.01-10 microg ml(-1)) were used to activate the neutrophils. When LPS was used directly, without another stimulator, the respiratory burst of neutrophils was not activated (N=12 horses; n=5 assays per horse). On the contrary, when LPS was added to whole blood, the neutrophils isolated from this blood were stimulated in a LPS dose-dependent manner, but polymyxin B added to whole blood suppressed this stimulation (N=2; n=6). LPS dissolved in autologous equine plasma stimulated the isolated neutrophils in a dose-dependent manner from 0.1-10 microg ml(-1) (N=5; n=12). Heat inactivation of the plasma abolished this CL increase (N=2; n=5). LPS added to equine albumin did not stimulate the isolated neutrophils (N=2; n=5). On the contrary, the addition of gamma-globulins (1 mg ml(-1)) to LPS (10 microg ml(-1)) led to the stimulation of neutrophils (N=2; n=5). We concluded that LPS did not directly stimulate the isolated equine neutrophils, but that plasmatic factors are needed for the stimulation of these cells by LPS.

Radiographic assessment of equine interphalangeal joints asymmetry: articular impact of phalangeal rotations (Part I).

This study is part of a work to develop a radiographic method that defines objectively the individual conformation of an equine digit and its appropriate trimming. The authors used isolated distal limbs on a rotation support to study the influence of induced foot rotations on several angles measured from specific radiographs. The results of this work enabled the authors to quantify the rotation of the proximal phalanx compared to the distal, and to determine exactly the possible rotation in the distal interphalangeal joint. It was also observed that, faced with a forced rotation of the foot, the digit responded by a hoof plastic deformity, a distal interphalangeal intra-articular rotation and asymmetrical interphalangeal compressions.

Radiographic assessment of equine interphalangeal joints asymmetry: articular impact of asymmetric bearings (Part II).

This study is part of a work to develop a radiographic method that defines objectively the individual conformation of an equine digit and its appropriate trimming. The authors used isolated distal limbs fastened on a rotation support with variable inclinations to study the influence of induced asymmetric bearings on various angles measured from specific radiographs. The digit responded to asymmetric bearings by 1. deformation of the hoof; 2. rotation of the phalanges; and 3. joint asymmetry or local articular space narrowing. The two last situations occurred mainly in the distal interphalangeal joint. Some of the angles mentioned above allow quantification of interphalangeal articular asymmetry, angular deformity and rotational deviation of a distal limb.

Purification of myeloperoxidase from equine polymorphonuclear leucocytes.

Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).

Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml).

Failure of lipopolysaccharides to directly trigger the chemiluminescence response of isolated equine polymorphonuclear leukocytes.

Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN). We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary. PMN were isolated from citrated blood on a discontinuous density gradient of Percoll. The luminol (10(-3) mol/L)-enhanced chemiluminescence (CL) of 1.25 x 10(6) cells was measured after addition of Escherichia coli LPS (0.001-10 micrograms/ml) alone or after incubation in autologous plasma (1 h, 37 degrees C). After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse. LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56 degrees C). These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein. Studies using specific antibodies against these factors are needed to clarify this question.