Added effects of acute heat-stress on the clinical signs associated with fescue toxicosis

Intake of ergot alkaloids associated with endophyte-infected fescue (E+) has adverse effects on animal health and productivity, which is collectively, termed fescue toxicosis. These effects are exacerbated under hot and humid (summer) conditions. A rat model for this condition was used to evaluate the effect of endophytic toxins on hepatic gene expression under acute (three days) heat stress (HS). Core temperature (Tc) was monitored continuously in rats (n=12) implanted with telemetric transmitters. Rats were fed ad libitum an E+ diet and maintained under thermoneutral (TN) conditions (21 ˚C) for five days, followed by TN or HS conditions (31 ˚C) for three days. Feed intake (FI) and body weight (BW) were measured daily. Both E+ and HS induced alterations in hepatic genes were evaluated using DNA microarrays. Intake of E+ reduced FI and BW under TN and HS conditions from pretreatment level, with greater reduction occurring during HS period. Core temperature at TN did not change from pretreatment level, but increased during HS. Genes associated with ATP synthesis, immune function, chaperone activity, and antioxidant function were reduced in E+HS vs E+TN. Present findings suggest that rats respond to E+ during heat stress by inducing hepatic CYP3A4 expression and suppressing chaperone, antioxidant and immune systems, which could ultimately increase the stress resulting in various pathological abnormalities. Keywords: Stress response The experimental schedule consisted initially of an 8 day recovery period after i.p. implantation of telemetric temperature transmitters (Model VM-FH; Mini-Mitter Company, Inc., Bend, OR). This was followed by a 5 day pretreatment period, during which Tc, FI and BW were collected to establish a baseline for statistical analysis (Spiers et al., 2005). During recovery and pretreatment periods, rats were fed the E- diet, whereas during the 8 day treatment period, the rats were fed the E+ (91.5 µg of EV.kg of BW-1.day-1) diet. At the end of 5 days of treatment, rats were divided into two groups, one maintained under continuous HS (31 °C) and the other continuing at TN for three additional days. During treatment Tc was collected at 10 minute intervals, with daily measurement of FI and BW at 0800 hour. At the end of the study, rats were anesthetized by i.m. injection of a xylazine (13 mg/mL)-ketamine (87 mg/mL) cocktail (0.1 mL/100g) and exsanguinated, and liver was collected and weighed. A transverse section from the middle lobe of liver (~5 g) was fixed in 10% neutral buffered formalin for immunohistochemistry (IHC) and terminal dUTP nick-end labeling (TUNEL), and another portion (~5 g) was snap-frozen in liquid nitrogen for microarray, real time-PCR, and antioxidant enzyme activity analysis. The data, as submitted, was entered into JMAANOVA. In JMAANOVA, intensity based LOWESS normalization was performed on the data. The analysis was performed, using array as random effect and dye and treatment as fixed effect and ANOVA was performed using 500 permutations. Step Up FDR was used for the analysis.