Jean-Philippe Lejeune

Effects of moderate amounts of barley in late pregnancy on growth, glucose metabolism and osteoarticular status of pre-weaning horses

In stud management, broodmares are commonly fed concentrates in late pregnancy. This practice, however, was shown to correlate with an increased incidence of osteochondrosis in foals, which may be related to insulin sensitivity. We hypothesized that supplementation of the mare with barley in the last trimester of pregnancy alters the pre-weaning foal growth, glucose metabolism and osteoarticular status. Here, pregnant multiparous saddlebred mares were fed forage only (group F, n=13) or both forage and cracked barley (group B, n=12) from the 7th month of pregnancy until term, as calculated to cover nutritional needs of broodmares. Diets were given in two daily meals. All mares and foals returned to pasture after parturition. Post-natal growth, glucose metabolism and osteoarticular status were investigated in pre-weaning foals. B mares maintained an optimal body condition score (>3.5), whereas that of F mares decreased and remained low (<2.5) up to 3 months of lactation, with a significantly lower bodyweight (-7%) than B mares throughout the last 2 months of pregnancy. B mares had increased plasma glucose and insulin after the first meal and after the second meal to a lesser extent, which was not observed in F mares. B mares also had increased insulin secretion during an intravenous glucose tolerance test (IVGTT). Plasma NEFA and leptin were only temporarily affected by diet in mares during pregnancy or in early lactation. Neonatal B foals had increased serum osteocalcin and slightly increased glucose increments and clearance after glucose injection, but these effects had vanished at weaning. Body measurements, plasma IGF-1, T4, T3, NEFA and leptin concentrations, insulin secretion during IVGTT, as well as glucose metabolism rate during euglycemic hyperinsulinemic clamps after weaning, did not differ between groups. Radiographic examination of joints indicated increased osteochondrosis relative risk in B foals, but this was not significant. These data demonstrate that B or F maternal nutrition has very few effects on foal growth, endocrinology and glucose homeostasis until weaning, but may induce cartilage lesions.

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.

Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils.

In neutrophils (PMNs), superoxide anion (O2*-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10(-4) to 10(-6) M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2(-) production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10(-5)M, while resveratrol appeared to be an O2*- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production.

Effect of a 120 km endurance race on plasma and muscular neutrophil elastase and myeloperoxidase concentrations in horses.

REASONS FOR PERFORMING STUDY Intense physical exercise can induce the degranulation of neutrophils leading to an increase in plasma concentration of the neutrophil marker enzymes myeloperoxidase (MPO) and elastase (ELT). These enzymes have pro-oxidative and pro-inflammatory properties and may play a role in the exercised-induced muscular damage. OBJECTIVES To measure MPO and ELT concentrations in plasma and muscles of endurance horses and to correlate them to the extent of exercise-induced muscular damage. METHODS Seven endurance horses qualified on 120 km races were tested in this study. Neutrophil count, serum creatine kinase (CK), plasmatic and muscular MPO and ELT concentrations were measured before and 2 h after a 120 km endurance race. RESULTS The race produced a significant increase of neutrophils, CK, and plasma MPO and ELT levels. A significant correlation was observed between the MPO and ELT values in plasma (r(2) = 0.92, P < 0.01) and in muscles (r(2) = 0.89, P < 0.01) while plasmatic concentrations of MPO and ELT were not significantly correlated to muscular ones. An increase of mean concentrations (± s.e.) of MPO (T0: 9.85 ± 3.9, T1: 228.9 ± 95.9 ng/mg proteins) and ELT (T0: 8.4 ± 2.4, T1: 74.5 ± 39.7 ng/mg proteins) in the muscles were observed after the race. Interestingly, the individual data showed large differences between the horses. Muscular MPO and ELT concentrations were significantly correlated to plasma CK levels. The coefficient of correlation (r(2)) was 0.69 (P < 0.01) for MPO and 0.66 (P < 0.01) for ELT, respectively. CONCLUSIONS Results underline the possible role of MPO and ELT in exercise-induced muscular damage. POTENTIAL RELEVANCE Further studies should investigate the effect of exercise type and intensity, as well as the role of the training state on MPO and ELT involvement in muscular damage. The assessment of the intensity of exercise-induced neutrophilic degranulation may have a potential role in the monitoring of the athletic career.

Gene expression profiling from leukocytes of horses affected by osteochondrosis.

Osteochondrosis (OC) is a developmental disease that affects growing horses and that severely affects their ability to perform. The genetic basis of its pathogenesis is poorly understood. The aim of the study was to analyze the transcript profile of leukocytes from horses affected with OC. Two transcriptome libraries were constructed from leukocytes of OC‐affected and non–OC‐affected horses using digital gene expression analysis (DGE) and real‐time PCR. Statistical analysis allowed selection of 1,008 tags upregulated in the non–OC‐affected group and 1,545 tags upregulated in the OC‐affected group. Among these genes, 16 regulated genes and 5 housekeeping genes were selected. Metabolic pathways analysis showed an obvious dysregulation of several signaling pathways related to cartilage formation or cartilage repair, including Wnt, Indian hedgehog, and TGF‐beta signaling. Other genes, including ISG, ApoB, MGAT4, and TBC1D9, showed a significantly different expression between groups. These genes may play a role in high carbohydrate diet, abnormal insulin metabolism, or inflammation, mechanisms suspected to be involved in OC. This DGE analysis of the transcript profile of leukocytes from OC‐affected horses demonstrated significant differences in comparison to the control library. These results open new perspectives for the understanding of equine OC.

Relationship between biochemical markers and radiographic scores in the evaluation of the osteoarticular status of Warmblood stallions

Establishing the osteoarticular status of the horse is often performed by means of radiological screening of the animals. Widespread blood sampling could reveal to be an interesting alternative to this procedure which is time consuming and sometimes technically difficult. The aim of this study was to investigate the relationship between the radiological status of the horses and the levels of biochemical markers of cartilage degradation and synovial inflammation. A specific radiological scoring and classification system was therefore developed and applied on 63 stallions presented for studbook admission. Additionally, groups of horses were established according to the occurrence of osteochondrosis, degenerative joint disease and distal interphalangeal joint effusion. Insulin growth factor-I, myeloperoxidases, Coll2-1 and Coll2-1NO(2) were used as blood markers. The combination of the blood parameters did not seem to correlate with the used scoring system. Coll2-1NO(2) levels however tended to increase with poorer radiological class and this could therefore potentially be a useful predictor of the osteoarticular status in the horse. Coll2-1 levels were significantly higher in the degenerative joint disease group. A high percentage of horses with distal interphalangeal joint effusion was present in this study and was associated with decreased IGF-I and increased Coll2-1 levels.

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O2) uptake of equine articular chondrocytes to assess their reactions to anoxia/re‐oxygenation. They were cultured under 5% or 21% gas phase O2 and at glucose concentrations of 0, 1.0 or 4.5 g/L in the culture medium (n = 3). Afterwards, the O2 consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25 min. The glucose consumption and lactate release were measured at the end of the re‐oxygenation period. The chondrocytes showed a minimal O2 consumption rate, which was hardly changed by anoxia. Independently from the O2 tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5 g/L glucose (n= 3). Lactate release was also independent from O2 tension, but lower for cells at 4.5 g/L glucose (n= 3). Our observations indicated that O2 consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re‐oxygenation. But the chondrocytes metabolism was modified by an excess of O2 and glucose.

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.

Plasma Concentration of Insulin-like Growth Factor I (IGF-I) in Growing Ardenner Horses Suffering from Juvenile Digital Degenerative Osteoarthropathy

Degenerative osteoarthropathy resulting in a reduced active lifespan was observed in Ardenner horses. In the context of joint biology, insulin-like growth factor I (IGF-I) is a potential candidate to affect the anabolism of cartilage matrix molecules. A group of 30 Ardenner horses reared under standardized conditions from weaning were evaluated periodically from 15 to 28 months of age to detect the early manifestations of the disease. At the end of this period, horses were classified in two pathological groups related to the degree of interphalangeal degenerative osteoarthropathy based on clinical and radiographic evaluations: healthy (46.7%) and pathological (53.3%) horses. Seven sequential blood samples were taken from each horse (during the evaluation period) to study the variation of IGF-I plasma concentration. We tested the variations of the IGF-I plasma concentration during growth, and the effect of sex and of pathological classes. Significant variations were observed during the research period, with a maximum value corresponding to spring and a minimum in autumn. A significant reduction of the IGF-I plasma concentration was also observed in the pathological horses (433.5 ± 19.5 ng/ml) compared to the healthy horses (493.9 ± 18.2 ng/ml). An alteration in the level of this growth factor could induce a disregulation of the mechanisms involved in the local control of joint and bone tissue development.

Variations of plasmatic concentrations of Insulin-like Growth Factor-I in post-pubescent horses affected with developmental osteochondral lesions.

Developmental osteochondral lesions are often encountered in the equine population and are a major cause of lameness. Different growth factors that act systemically as well as locally regulate the growth of cartilage. Among them is Insulin-like Growth Factor I that has been demonstrated to promote chondrocyte growth and differentiation and that has been shown to influence cartilage repair. The aims of this study were to investigate differences in circulating plasma levels of Insulin-like Growth Factor-I in post-pubescent horses affected with developmental osteochondral lesions compared to unaffected ones. Significantly higher values of circulating Insulin-like Growth Factor-I levels were found in the affected group (n = 82) compared to controls (n = 86). This result may still reflect an earlier imbalance in IGF-I levels from horses with developmental osteochondral lesions considering the aetiopathological link which has been made between IGF-I and the occurrence of osteochondrosis. However, other studies have shown increased expression of IGF-I after cartilage damage. The higher levels found in this study could be due to a healing response of the cartilage to the damage caused by the osteochondral lesions.