Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hard remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material. (C) 2012 Elsevier B.V. All rights reserved.Swedish Foundation for Equine Research, SwedenSwedish Foundation for Equine Research, Swede

Variation in training regimens in professional showjumping yards

REASONS FOR PERFORMING STUDY: Training regimens of showjumping horses under field conditions are largely undocumented. OBJECTIVES: The aims of this study were to quantify and compare training regimens used in professional-level showjumping yards, with respect to time exercised and type of activity. STUDY DESIGN: Prospective cohort study. METHODS: A prospective 6-month cohort study of showjumping horses in 4 European countries (The Netherlands, Sweden, Switzerland, Great Britain) was designed to analyse training and health data, in yards with several horses in training and riders competing at professional level. Riders documented the daily frequency and duration of all physical activities of the horses. Variation in training routines were compared between riders, location and time. Mixed-models analysis was used to examine factors associated with total time exercised and time spent in flatwork. RESULTS: In 4 countries, the 31 participating riders trained 263 European Warmbloods. The total days at risk (e.g. days in which the horses were considered fit for exercise) was 39,262. Mean time spent in daily exercise, including ridden work, lungeing and treadmill exercise, varied between riders from 19-52 min/day at risk. There was considerable variation in activities and level of heavy work and light exercise, i.e. turnout. Total time exercised and time spent in flatwork differed with month, country and proportion of days lost to training. Low variation of activities was associated with decreased total time trained and increased time spent in flatwork. CONCLUSIONS: Riders at this elite professional level of showjumping used training regimens that vary substantially in time spent training and other physical activities and showjumping horses are challenged differently during training despite competing at the same level. Whether all training regimens prepare the horses equally for the demands of competition remains to be determined