Development of KVO treatment strategies for chronic pain in a rat model of Gulf War Illness.

We examined whether combinations of Kv7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (-55 to -40 mV; Vh = -60 mV; 1500 ms) and Kv7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5-100 µM) and/or Diclofenac (50-140 µM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED50: 62.7 ± 3.18 µM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED50: 93.2 ± 8.99 µM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 µM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 µM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors.

DEET potentiates the development and persistence of anticholinesterase dependent chronic pain signs in a rat model of Gulf War Illness pain.

Exposure to DEET (N,N-diethyl-meta-toluamide) may have influenced the pattern of symptoms observed in soldiers with GWI (Gulf War Illness; Haley and Kurt, 1997). We examined how the addition of DEET (400mg/kg; 50% topical) to an exposure protocol of permethrin (2.6mg/kg; topical), chlorpyrifos (CP; 120mg/kg), and pyridostigmine bromide (PB;13mg/kg) altered the emergence and pattern of pain signs in an animal model of GWI pain (Nutter et al., 2015). Rats underwent behavioral testing before, during and after a 4week exposure: 1) hindlimb pressure withdrawal threshold; 2) ambulation (movement distance and rate); and 3) resting duration. Additional studies were conducted to assess the influence of acute DEET (10-100µM) on muscle and vascular nociceptor Kv7, KDR, Nav1.8 and Nav1.9. We report that a 50% concentration of DEET enhanced the development and persistence of pain-signs. Rats exposed to all 4 compounds exhibited ambulation deficits that appeared 5-12weeks post-exposure and persisted through weeks 21-24. Rats exposed to only three agents (CP or PB excluded), did not fully develop ambulation deficits. When PB was excluded, rats also developed rest duration pain signs, in addition to ambulation deficits. There was no evidence that physiological doses of DEET acutely modified nociceptor Kv7, KDR, Nav1.8 or Nav1.9 activities. Nevertheless, DEET augmented protocols decreased the conductance of Kv7 expressed in vascular nociceptors harvested from chronically exposed rats. We concluded that DEET enhanced the development and persistence of pain behaviors, but the anticholinesterases CP and PB played a determinant role.

Calcium supplementation of hen drinking water.

Two experiments with White Leghorn hens (42 and 56 wk old, respectively) examined the effects of calcium supplementation through drinking water in the presence of adequate and inadequate dietary calcium. Each experiment was of 28 d duration with six replicate pens of five individually caged hens in each treatment. Treatments were a combination of either 2.25 or 3.5% dietary calcium coupled with tap water or water supplemented with .2% calcium from calcium lactate. In both studies, specific gravity of eggs was significantly improved when low dietary calcium was supplemented with .2% calcium in the drinking water. Egg production and egg weight were not influenced by waterborne calcium. Daily water intake was reduced by calcium lactate in all cases. Feed consumption was also depressed by waterborne calcium in both studies when 3.5% dietary calcium was given, and in Experiment 1 when 2.25% was fed. Waterborne calcium as calcium lactate was found to be effectively utilized for eggshell quality improvement when dietary sources were inadequate.

Effects of dietary boron supplementation on broilers.

Two experiments were conducted with a total of 432 broiler chicks to investigate the influence of supplementing different amounts of boron to practical corn-soybean meal diets. The birds were housed in batteries and had free access to feed and water. In Experiment 1, 144 1-day-old broiler chicks were fed a basal diet supplemented with 0, 5, 40, 80, or 120 ppm boron for 21 days. Female body weight was not influenced by the dietary treatments. However, males supplemented with 5 ppm boron were heavier and their tibias resisted more load than the control birds. Overall feed conversion was not influenced by boron. In Experiment 2, 288 1-day-old broiler chicks were fed a basal diet supplemented with 0, 60, 120, 240, or 300 ppm boron for 22 days. Male and female body weights of the 300-ppm group were lower than those of the control birds. Percentage tibia ash was highest with 300 ppm boron. No significant differences were found in intestinal tract weight (grams of intestine per 100 g body weight) among treatments. Boron concentration in the breast muscle and liver increased as dietary concentration of boron increased. Data collected in these two experiments indicated that consumption of diets containing up to 240 ppm boron from hatch to 21 days of age was not detrimental to broiler performance. Data were not conclusive on the need for supplemental boron in broiler diets based on corn and soybean meal.

Broiler chick and laying hen tolerance to sodium hypochlorite in drinking water.

Two broiler and two laying hen experiments were conducted to establish threshold levels for sodium hypochlorite use in poultry drinking water. Both broiler experiments were of 21-days duration, and the first and second hen experiments ran for 8 and 4 wk, respectively. A 5.25% sodium hypochlorite solution was used to provide chloride levels of 0, 10, 100, or 500 ppm in the first broiler study and 0, 150, 200, 250, 300, 350, 400, or 450 ppm in the second study. Comparable sodium levels from sodium bicarbonate were additional treatments in the first broiler experiment. The initial hen study had drinking water treatments of 0, 50, or 100 ppm chloride whereas in the second study, treatments varied from 0 to 70 ppm chloride at 10-ppm intervals. In chick experiments, water intake was reduced by 100 ppm chloride and body weight by 300 ppm. Hen experiments demonstrated the adverse effect level to be 40 ppm for water consumption and 60 ppm for egg production when the application was to pullets producing eggs at a high rate in warm weather. Water consumption of older hens in cooler weather was adversely affected by 50 ppm chloride, but egg production, egg weight, or daily feed intake was not reduced by 100 ppm.

2-Hydroxy-4 (methylthio) butanoic acid as a drinking water supplement for broiler chicks.

Two experiments were conducted to study the effects on chick performance of a commercially available liquid methionine analog [2-hydroxy-4 (methylthio) butanoic acid; HMB] supplied through drinking water. Methionine activities of .025 and .05% were added to drinking water and compared with levels of .03, .06, or .09% included in a low-methionine basal diet from 0 to 21 days of age. Mortality was not significantly altered by any dietary treatment. Neither feed nor water intake was affected adversely by HMB inclusion in drinking water. Only male body weight in Experiment 1 did not respond to HMB feed supplementation. Equivalent levels of total sulfur amino acid intake among water treatments were associated with significantly heavier body weights than the control. In both experiments, body weight and feed efficiency of birds receiving .05% methionine activity as HMB in water were equal or superior to those of groups that received supplemented feed. The results indicate that, under the study conditions, HMB provided in drinking water can be effectively assimilated by broiler chicks, at least through 21 days of age.

Supplementation of broiler drinking water with liquid ammonium polyphosphate.

1. In each of two experiments lasting 21 days a commercially available ammonium polyphosphate solution was provided in the drinking water as the sole source of supplemental phosphorus for day-old chicks housed in batteries. 2. In both experiments, similar daily phosphorus intakes either from dicalcium phosphate in the food or from ammonium polyphosphate in the drinking water supported equivalent responses in body weight gains. The same was true for the tibia ash results in experiment 2. 3. Liquid ammonium polyphosphate, while marketed for addition to diets, appears to be also very effective as a waterborne source of phosphorus for broilers.

Research note: Feeding various levels of ground Sesbania macrocarpa Muhl. seed to bobwhite quail.

Two 28-day experiments were conducted to determine the effects of various levels of ground Sesbania macrocarpa Muhl. seed on mature bobwhite quail. In Experiment 1, S. macrocarpa Muhl. seed levels of 0, 1, 2, 3, 4, and 5% were added to a basal diet at the expense of filler and fed to five replicate groups of six 58-wk-old paired quail (one male and one female). Average daily feed consumption, hen-day egg production, average BW change, mortality, fertility, and hatchability were monitored. Four groups of eight individually caged females, 63 wk of age, were each given a diet containing 0, 2, 4, 6, 8 or 10% ground S. macrocarpa Muhl. seed in Experiment 2. A seventh treatment was added that consisted of 10% ground S. macrocarpa Muhl. from an older seed shipment used previously in work with White Leghorn hens. Increasing S. macrocarpa Muhl. seed levels in Experiment 1 did not cause significant deviations from the control treatment for average daily feed consumption, BW change, hen-day egg production, fertility, total hatchability, or hatchability of fertile eggs. In Experiment 2 neither average daily feed consumption nor hen-day egg production were affected by seed level or source. Quail given the 10% seed level using the older seed shipment had a significantly greater weight loss than the control birds. With the exception of this greater weight loss and in contrast with work involving chickens, dietary levels of ground S. macrocarpa Muhl. seed of up to 10% were acceptable to bobwhite quail.

Tolerance to ground Sesbania macrocarpa seed by broiler chicks and White Leghorn hens.

Two 21-day experiments using day-old male broiler chicks and a 28-day experiment using 35-wk-old White Leghorn hens were conducted to determine the deleterious effects of ground Sesbania macrocarpa seed in the diet. Seed levels of 0, .10, .25, .50, and 1% were used in both experiments, with a 2% level of inclusion added to Experiment 2. The hens were fed diets containing 0, 1, 2, 3, 4, 5, 6, or 7% seed. In each experiment, four replicate pens of 8 chicks or of 5 hens received each treatment diet. In both chick studies, a seed inclusion of 1% caused a significant reduction in body weight at 21 days of age. Total feed intake was significantly lowered by the inclusion of 1% seed in the first study and by 2% seed in the second study. Feed efficiency, excreta moisture, and mortality were not affected by dietary level of seed in either trial. Seed inclusion at 3% or more depressed the daily feed consumption of the hens; at 6% seed inclusion, egg production was reduced. Egg weight, egg specific gravity, mortality, and body weight change were not affected by treatment. Grains containing sufficient S. macrocarpa seed to produce finished feed with 1 or 3% seed would appear to be detrimental to broiler growth and feed intake by hens, respectively. The blending of such grains with uncontaminated stock could be a possible method of utilizing the contaminated grain.

Response of White Leghorn hens to various dietary levels of Cassia obtusifolia and nutrient fortification as a means of alleviating depressed performance.

Two 28-day experiments with White Leghorn hens were conducted to determine the adverse effect level of ground Cassia obtusifolia seed (coffeeweed) in the diet and to examine the nutrient fortification required to improve depressed performance due to the presence of the seed. In both experiments, five individually caged hens were used for each of the four treatment replications. Treatments for Experiment 1 consisted of a control and six diets with ground C. obtusifolia seed substituted for a filler at levels of 2, 3, 4, 5, 6, and 7%. In Experiment 2, a series of nutritionally fortified (15 to 20% higher nutrient levels) and unfortified diets coupled with seed levels of 0, 4, 6, and 8% for a total of eight treatments was used. Average daily feed intake, hen-day egg production, egg weight, egg specific gravity, and body weight change were determined during each experiment. For Experiment 1, average daily feed consumption was measured every 24 h for the first 3 days. Egg production and feed consumption were significantly reduced by 2% seed in Experiment 1. Within the first 24 h, feed intake was decreased to 37% below that of controls by increasing to the highest seed level. Egg specific gravity dropped significantly below that of controls when hens were fed 5% seed. In Experiment 2, fortification improved feed consumption, hen-day egg production, body weight change, and egg specific gravity over that of birds fed unfortified diets with the same seed level. Finished diets contaminated with 2%, or more, C. obtusifolia seed can cause reduced performance in laying hens, but nutritionally fortifying these diets can help to alleviate depressed performance.

Precipitated bone phosphate in broiler chick diets.

Day-old Cobb x Cobb broiler chicks were housed in battery brooders for 21-day feeding periods during two experiments. Dietary treatments consisted of a corn-soybean starter feed (.37% total; .14% nonphytin phosphorus) supplemented with 0, .05, .10, .15, or .28% phosphorus in the form of dicalcium phosphate, precipitated bone phosphate, or a mixture of soft and precipitated phosphates (each providing 50% of the phosphorus addition). Total dietary calcium was held constant at .90%. Comparable dietary phosphorus from precipitated bone and the soft-precipitated mixture supported body weights statistically equivalent to those of dicalcium phosphate treatments. Tibia ash results at suboptimal phosphorus levels, with one exception, indicated statistically equal utilization from the soft-precipitated combination. Slope-ratio techniques using tibia ash and total nonphytin phosphorus intake established the bioavailability of phosphorus from precipitated bone was 120% when dicalcium phosphate was the standard. The data indicate that precipitated bone phosphate for broilers was fully equivalent in performance to dicalcium phosphate. Providing one-half the supplemental phosphorus from both soft phosphate and precipitated bone seemed to be an acceptable method of meeting practical phosphorus requirements.