Eight Thoroughbred or Thoroughbred-cross horses (5 geldings, 3 mares; aged 5-20 years; weight 463-558 kg) were used. Each horse was instrumented with a permanent gastric cannula placed for the purpose of other experiments. Mares were used during behavioral diestrus. There was at least 1 week between the end of one study period and the beginning of the next, during which horses were maintained on grass pasture with concentrate and grass hay available. All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee.
Horses were stalled 12-18 hours prior to each study period for acclimation. Horses were studied in groups of 2-3 to reduce the effects of social isolation on study outcome. Body weight was measured following acclimation and at the end of each study. Horses were offered free choice grass hay (in pre-weighed bins) and water (in graduated buckets) and twice-daily concentrate during the course of the study. Hay and water consumption was measured and recorded every 4 hours throughout the study period.
Each study period began between 0800 and 1000 hours. Each horse received each treatment in an orthogonal Latin square design; investigators were blinded as to treatment. Treatments were 0.9% sodium chloride (Placebo, P; 60 mL bolus, 0.15 mL/kg/hr), lidocaine hydrochloride (Lidocaine hydrochloride injectable-2%, Phoenix Pharmaceutical, Inc., St. Joseph, MO, USA) (L; 1.3 mg/kg bolus, 3 mg/kg/hr), ketamine hydrochloride (KetaVed®, Vedco, St. Joseph, MO, USA) (K; 0.55 mg/kg bolus, 0.5 mg/kg/hr), butorphanol tartrate (Torbugesic®, Fort Dodge Animal Health, Fort Dodge, IA, USA) (B; 0.018 mg/kg bolus, 0.013 mg/kg/hr) administered individually and in combination (P, L, B, K, LK, LB, KB, and LBK) for a total of 8 treatments. No diluent was needed for treatments including lidocaine; all other drugs and combinations were diluted in sodium chloride. Infusions were prepared in 1-L increments. The bolus was administered over 15 minutes and the CRI administration began immediately following the bolus with a computerized infusion pump for 96 hours.
Prior to the live animal study, stability of the drugs in combination was assessed. Ketamine and butorphanol were added to lidocaine (100 mL total volume) at the dosage indicated for LBK infusion. The drug combination was maintained inside in a climate controlled environment at room temperature (approximately 20-22ºC) in ambient light. A 2mL sample was removed and frozen at -20ºC every 6 hours for 72 hours. The concentrations of butorphanol, lidocaine and ketamine were determined by liquid chromatography (Shimadzu Prominence, Shimadzu Scientific Instruments, Columbia, MD, USA) and mass spectrometry (API 2000, Applied Biosystems, Foster City, CA, USA). The internal standards were fentanyl (Cerilliant Corporation, Round Rock, TX, USA), mepivacaine (Sigma-Aldrich, St Louis, MO, USA), and ketamine d4 (Cerilliant Corporation, Round Rock, TX, USA) for butorphanol, lidocaine and ketamine respectively. The qualifying and quantifying ions for butorphanol, lidocaine and ketamine were mass to charge ratio (m/z) 328.21→157.2, 235.22→86.2, and 238.09→124.9, respectively. The qualifying and quantifying ions for fentanyl, mepivacaine and ketamine d4 were (m/z) 337.14→105.3, 247.21→98.2, and 242.16→129.1, respectively. Standard curves for each of the analytes were accepted if they were linear with calculated concentrations within 15% of the actual concentration and the correlation coefficient was at least 0.99. The standards and the tested solutions were diluted in 0.1% formic acid in water. The mobile phase consisted of A: acetonitrile and B: 0.1% formic acid in deionized water at a flow rate of 0.4 mL/min. The mobile phase started at 90% B with a linear gradient to 40% B at 4 minutes and back to 90% B at 5 minutes with a total run time of 6.5 minutes. Separation was achieved with a C18 column (ACE C18AR, 150 mm x 3.0 mm x 5 μm) (MAC-MOD Analytical, Chadds Ford, PA, USA) maintained at 40°C.
For each trial, a 14-g intravenous catheter was placed aseptically into each jugular vein and horses were fitted with a fecal collection device. A nasogastric tube was placed and 200 3x5-mm plastic beads administered in 1 L water by gravity flow using a funnel. Different color beads were used for each trial to avoid any carryover from previous trials. After bead administration was complete, treatment bolus then CRI began. Vital signs (heart rate, respiratory rate, and rectal temperature), behavior scores, and gastrointestinal borborygmi scores were recorded as previously described  every 6 hours for the study duration. Feces were collected, weighed, and beads manually retrieved every 2 hours. Following retrieval of 180 beads, the fecal collection device was removed and feces collected from the stall floor were weighed every 2 hours. Hay (kg) and water (L) consumption was recorded every 6 hours.
Thermal threshold (TT) testing was performed using a wireless device as previously described . Briefly, an area on one side of the withers (alternating randomly) was shaved and the TT probe placed in direct contact with the skin. The probe was secured using an adjustable nylon strap around the thorax. Consistent pressure between the probe and skin was ensured by inflating a modified blood pressure cuff and pressure monitored by a sensor within the device. Skin temperature was recorded following an equilibration period of at least 5 minutes. The device was activated by a wireless hand-held toggle switch by an investigator positioned outside of the horse’s stall. The device was activated when the horse was not visibly interacting with the investigator. Heating was discontinued either when the horse displayed a response or at 55ºC, whichever occurred first. Responses included a skin twitch, looking at the flank, or an abrupt lifting of the head. TT was performed twice prior to treatment bolus (baseline), 15 minutes into the drug infusion then every 12 hours for the duration of the study. TT was not performed on one horse due to its failure to respond to the stimulus at the baseline measurement. For time points reaching the cut-off value (55ºC), responses were recorded as 55.5 for data analysis.
Unless indicated otherwise, all data are expressed as least square mean ± standard error of the mean. For TT, baseline was considered the mean of two measurements. The data for each response variable conformed to a split-plot ANOVA according to the model: Y = Treatment + Period + Horse + Error1 + Time + Treatment*Time + Error2. When period was not a significant factor for a given response variable, period was eliminated from each model and data were then analyzed by a three-factor ANOVA with the fixed factors of Time and Treatment and the random factor of Horse. When significant main effects or interactions occurred, post hoc comparisons were made by the Bonferroni t test. A commercial software package (SAS/STAT, SAS Institute Inc., Cary, NC, USA) was used for all analyses and a p <0.05 was considered significant. For post hoc comparisons, the critical p was considered 0.05/number of comparisons.
Mean fecal bead passage fit to a four-parameter logistic equation (SigmaPlot 11.0, Systat Software, Inc., San Jose, CA, USA) that was then used to calculate the time to passage of 1, 25, 50, and 75% of beads for each treatment via regression.