Sleep time following anesthesia in mouse lines selected for resistance or susceptibility to fescue toxicosis

In previous work, a mouse line selected for resistance (R) to fescue toxicosis had higher activities of two hepatic Phase II detoxification enzymes than a mouse line selected for fescue toxicosis susceptibility (S). The primary objective of the present study was to determine whether those same lines also differed in hepatic Phase I enzyme activity, estimated from sleep time (ST) following sodium pentobarbital anesthesia. Additional objectives were to determine whether ST differences between lines were modulated by endophyte-infected fescue in the diet (with or without an enzyme inducer) and whether ST of individual mice was correlated with the effect of a toxin-containing diet on the postweaning growth of those mice. In Exp. I, 24 males from each line were randomly assigned to each of five diets: control (commercial rodent food meal); E+ (50% endophyte-infected fescue seed, 50% control); E+P (the E+ diet supplemented with 1,000 ppm phenobarbital); E- (50% endophyte-free fescue seed, 50% control); and E-P (the E- diet supplemented with 1,000 ppm phenobarbital). After 4 wk on these diets, ST was measured on all the mice. A second ST was recorded on each mouse by randomly sampling one-fourth of the population after 1, 2, 3, or 4 wk on a pelleted rodent food diet. Regardless of diet, R mice had shorter first and second ST than S mice (P < 0.01), suggesting higher hepatic Phase I microsomal enzyme activity. Mice on both phenobarbital-supplemented diets had shorter first ST than mice whose diets did not include that microsomal enzyme inducer (P < 0.01). In Exp. II, ST was measured on male and female R and S mice (n = 280) after they had been fed the E- diet for 2 wk, then the E+ diet for 2 wk, and then a pelleted rodent food diet for 2 wk. Growth response to the E+ diet was the percentage of reduction in gain on the E+ diet compared to gain on the E- diet the previous 2 wk. As in Exp. I, S mice slept longer than R mice (P < 0.01). The residual correlation between ST and gain reduction associated with the E+ diet equaled 0.04. Thus, an animal's apparent Phase I enzyme activity did not predict its growth rate depression on the toxin-containing diet. Based on these and previous studies, divergent selection for toxicosis response in mice was successful partially by causing divergence in activities of hepatic Phase I and II detoxification enzymes.     

A model of fescue toxicosis: responses of rats to intake of endophyte-infected tall fescue

A study was conducted to develop a model for fescue toxicosis using rats fed a diet containing endophyte-infected tall fescue seed (E+). Rats implanted with telemetric transmitters to continuously monitor core body temperature (Tc) and activity were housed at thermoneutrality (21 degrees C) and were fed a diet containing endophyte-free fescue seed (E-). After 2 wk, they were assigned to either E+ or E- diets and initially maintained at thermoneutrality (preheat) for 8 d. They were then exposed to heat stress (31 degrees C) for 22 d, followed by 1 wk of recovery at thermoneutrality (post-heat). Body weight and feed intake were measured daily. Rats receiving the E+ diet showed decreased feed intake (P = 0.001) and weight gains (P = 0.003) during the preheat period. The decrease in Tc from the pre-treatment level was greater in E+ than in E- rats during the preheat (P = 0.001) and postheat (P = 0.001) periods. With heat stress, both groups showed parallel decreases in feed intake. The increase in Tc from pre-heat to heat conditions was greater in E+ vs. E- rats (P = 0.001). Activity level was lower in E+ than in E-rats during heat stress (P = 0.009) and postheat (P = 0.037) periods. These results show that the rat model for fescue toxicosis is extremely useful because many of the observed responses to E+ diet are similar to those noted for cattle, and additional variables that are difficult to measure in cattle, such as activity, can be easily evaluated.     

Effects of feeding ergovaline on lamb performance in a heat stress environment

Lambs exposed to a heat-stressed environment (33 degrees C, 50% relative humidity) were used in three experiments to determine whether ergovaline (EV) is the primary toxin involved in fescue toxicosis. The first study evaluated the effects of feeding diets containing increasing levels of endophyte-infected tall fescue seed (E+) and decreasing levels of endophyte-free tall fescue seed (E-). The second and third study evaluated the response to a diet that contained synthetic EV added to an E- diet and the response to a diet containing endophyte-infected ryegrass seed (R+) with an elevated concentration of EV. In Exp. 1, lambs were fed diets of: 1) 10% E- and 0% E+, 2) 5% E- and 5% E+, or 3) 0% E- and 10% E+. Increasing the percentage of E+ in the diet resulted in a linear decrease (P < 0.01) in feed intake (as-fed basis), skin temperature, thermocirculation index (TCI), and serum prolactin. Body weight gain also decreased (P < 0.06). Respiratory rate and core body temperature were not affected by the 5 or 10% E+ diets. In Exp. 2, lambs were fed diets that contained: 1) 10% E-, 2) 10% E- with synthetic EV added at a level equivalent to the 10% E+ diet, or 3) 10% E+. Feed intake (as-fed basis), body weight gain, and skin temperature did not differ for lambs fed the E- and EV diets. The EV diet elicited a decrease (P < 0.05) in TCI and prolactin compared with the E- diet. The TCI for lambs fed EV did not differ (P > 0.10) from the E+ lambs; however, serum prolactin was lower (P < 0.05) for lambs on the E+ diet than for those fed EV. Core body temperature was not affected (P > 0.10) by feeding EV or E+ fescue seed in Exp. 2. In Exp. 3, lambs were fed diets that contained: 1) 10% E-, 2) 3.24% R+ and 6.76% E-, which added an equivalent amount of EV to E+ diets but reduced concentrations of other ergot alkaloids, or 3) 10% E+. Lambs fed the E+ diet and maintained at 33 degrees C had lowered feed intake (as-fed basis), skin temperature, and TCI compared with lambs fed the E- or R+ diets (P < 0.05). Lambs fed the E+ diet had increased rectal temperatures and lowered serum prolactin compared with lambs on the R+ diet (P < 0.05). Lambs on the R+ diet had a greater rectal temperature and lower serum prolactin than lambs on the E- diet (P < 0.05). These results suggest that EV is a fescue toxin; however, other alkaloids might work synergistically with EV, causing the full expression of fescue toxicosis.     

Use of nonergot alkaloid-producing endophytes for alleviating tall fescue toxicosis in sheep

Nonergot alkaloid-producing endophytes from New Zealand were inserted into tall fescue (Festuca arundinacea) cultivars in an attempt to address the problem of fescue toxicosis in grazing sheep. A 3-yr grazing study was conducted to determine lamb performance and to evaluate toxicosis in lambs grazing nonergot alkaloid-producing endophyte-infected (AR542 or AR502), endophyte-free (E-), or wild-type toxic endophyte-infected (E+) Jesup tall fescue or nonergot alkaloid-producing endophyte-infected (AR542) Georgia-5 tall fescue. Replicated 0.11-ha tall fescue paddocks were established at the central Georgia Branch Station during September 1997 and stocked with lambs from spring 1998 through autumn 2000. Mean ergot alkaloid concentrations were higher (P < 0.01) in E+ forage than in AR542, AR502, and E- tall fescue, and ergot alkaloid concentrations in E- plants and plants infected with AR542 and AR502 were low. Forage availability did not differ (P = 0.92) across treatments during autumn and was higher (P < 0.05) in Georgia-5 AR542 than in Jesup AR502 and E+ pastures. Initial serum prolactin (PRL) concentrations did not differ (P = 0.58) across treatments during autumn, but were higher on Jesup AR542 than E+ during spring. Post-treatment serum PRL concentrations were depressed (P < 0.01) on E+ compared with AR542, AR502, and E- in both spring and autumn. Signs of heat stress were observed in E+ lambs during periods of high ambient temperatures. Mean post-treatment rectal temperature and mean stocking rate exhibited treatment x year interactions (P < 0.05). Lamb ADG was higher (P < 0.05) on AR542, AR502, and E- than on E+ tall fescue. Similarly, gain/hectare was higher (P < 0.015) on AR542, AR502, and E- than on E+. Tall fescue pastures containing AR542 and AR502 endophytes yielded lamb performance that did not differ from that on E- tall fescue and which was superior to performance on E+ tall fescue. Depressed PRL concentrations and elevated rectal temperatures as indicators of toxicosis were evident only in lambs grazing E+ tall fescue, suggesting that nonergot alkaloid-producing endophyte-infected tall fescue is a viable alternative for alleviating tall fescue toxicosis.     

Effects of hydrated sodium calcium aluminosilicate on fescue toxicosis and mineral absorption.

The possibility of supplementing livestock diets with an aluminosilicate to protect them from fescue toxicosis was investigated. An in vitro study showed that hydrated sodium calcium aluminosilicate (HSCAS) removed greater than 90% of the ergotamine from aqueous solutions at pH 7.8 or lower, indicating a high affinity of ergotamine for HSCAS in vitro. Rats fed diets containing tall fescue seed infested (E+) with the endophytic fungus Acremonium coenophialum had lower (P less than .05) feed intakes and weight gains than did rats fed diets containing uninfested (E-) tall fescue seed. When feed intake by rats fed the E- seed diet was limited to that of rats fed the E+ seed diet, weight gains did not differ, but testes weights and serum prolactin (PRL) concentrations were lower (P less than .05 and .10, respectively) in rats receiving E+ seed. Supplementing E+ seed diets with HSCAS did not eliminate effects of E+ seed on intake, PRL, or testes weights. Sheep fed E+ tall fescue hay had higher (P less than .05) rectal temperatures than did sheep fed an equal amount of E- tall fescue hay, but OM and N digestion coefficients did not differ between the two hays. Supplementing E+ hay diets with HSCAS did not eliminate the effect of E+ hay on rectal temperatures. Addition of 2% HSCAS to tall fescue hay diets did not affect apparent absorption by sheep of OM, N, Ca, P, Na, K, or Cu, but it reduced (P less than .05) the apparent absorption of Mg, Mn, and Zn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of nonergot alkaloid-producing endophytes for alleviating tall fescue toxicosis in stocker cattle

Grazing studies were conducted to determine cattle growth performance, evaluate toxicosis, and compare grazing behavior in stocker cattle grazing nonergot alkaloid-producing endophyte-infected (AR542 or AR502), endophyte-free (E-), or wild-type toxic endophyte-infected (E+) Jesup, Georgia-5, and Kentucky-31 tall fescue. Replicated 0.81-ha tall fescue paddocks were established at the Central Georgia Branch Station at Eatonton and the Northwest Georgia Branch Station at Calhoun during October 1998 and were stocked with beef cattle for autumn and spring periods from fall 1999 through spring 2002. Mean ergot alkaloid concentrations were higher (P < 0.01) on E+ pastures than the other treatments at both locations. At Calhoun and Eatonton, post-treatment serum prolactin concentrations were decreased (P < 0.01) on E+ compared with AR542, AR502, and E- tall fescue. Cattle on AR542, AR502, and E- pastures had lower (P < 0.05) post-treatment rectal temperatures than cattle grazing E+ tall fescue during spring at Eatonton and Calhoun. Calf ADG was higher (P < 0.05) on AR542, AR502, and E- as compared with E+ tall fescue during autumn and spring grazing at Eatonton, and at Calhoun, cattle on E+ pastures had lower (P < 0.05) ADG in both autumn and spring. Gain/hectare was higher (P < 0.05) on AR542, AR502, and E- than on E+ during autumn at Eatonton and during spring at both locations. In autumn at Calhoun, gain/hectare was greater (P < 0.05) on AR502 and E- compared with E+ tall fescue. During April, May, and June, cattle grazing E+ pastures at Eatonton spent more (P < 0.01) time idling, more (P < 0.01) time standing, and used more (P < 0.01) water than cattle on AR542 and E- tall fescue. Daily prehensions and biting rate were each higher (P < 0.01) on AR542 and E- tall fescue than E+ tall fescue in both grazing seasons. There were no differences among pasture treatments for bite size in either spring (P = 0.50) or autumn (P = 0.34). Steers grazing E+ pastures had lower DMI than steers grazing AR542 and E- pastures during spring (P < 0.10) and lower DMI than steers grazing E- pastures during autumn (P < 0.05). Daily steer water usage was decreased (P < 0.10) in E+ pastures compared with AR542 and E- pastures during late fall. These results indicate that nonergot alkaloid-producing endophyte technology is a promising option for alleviating tall fescue toxicosis in stocker cattle.     

The effect of the endophyte (Acremonium coenophialum) and associated toxin(s) of tall fescue on serum titer response to immunization and spleen cell flow cytometry analysis and response to mitogens

Experiments were conducted with rats and mice to evaluate the effect of the consumption of endophyte (Acremonium coenophialum) and associated toxin(s) infected tall fescue on humoral and cellular aspects of immune function. Treatment diets were: (1) rodent chow (RC) or (2) rodent chow mixed 1:1 (w/w) with endophyte infected (E+) or (3) non-infected (E-) tall fescue seed. Rats fed the E+ diet in experiment 1 (43 days) exhibited a lower (P less than 0.05) serum titer to sheep red blood cell (SRBC) immunization than those fed the E- diet (38.4 vs 131.3). The E+ rats also had lower (P less than 0.01) white cell counts than either RC or E- groups (5225 vs 8959 and 7491/mm3). Spleen cells from mice fed the E+ diet for 37 days exhibited a reduced (P less than 0.05) response to the mitogens Concanavalin A and lipopolysaccharide. Flow cytometric analysis revealed a significant (P less than 0.01) 42% increase in T suppressor cell numbers in spleens of mice fed the E+ vs RC diets.     

Urinary alkaloid excretion as a diagnostic tool for fescue toxicosis in cattle.

Fescue toxicosis research studies have often included serum prolactin as a physiologic index of the disorder. Serum prolactin has not been used as a clinical measure of fescue toxicosis because of variation associated with sex and physiologic condition of the animal and climatic and seasonal factors. The primary excretory route of the alkaloids responsible for this toxicosis is the urine. Three pasture experiments were conducted to examine serum prolactin and urinary ergot alkaloid variability among steers continuously grazing endophyte-infected (E+) or endophyte-free (E-) tall fescue and among steers that were switched from one pasture form to the other. A fourth grazing experiment was used to examine how to best to manage the steers prior to sampling for urinary ergot alkaloid excretion. Coefficients of variability for urinary alkaloid excretion were consistently lower (46-65%) than serum prolactin (64-142%). Urinary alkaloid excretion patterns changed within 12 hours following switching steers from E+ to E- pasture or vice versa, but serum prolactin was recalcitrant to change. Because it is less variable and more dynamic than serum prolactin, urinary alkaloid excretion can be used for health assessment of steers grazing E+ and E- pastures. Regression analysis established a quadratic relationship between alkaloid excretion and average daily weight gain, with a regression coefficient of 0.86. Urinary alkaloid analysis was useful in determining whether cattle were consuming toxic tall fescue.     

Evaluation of a ruminally dosed tall fescue seed extract as a model for fescue toxicosis in steers

Tall fescue (Lolium arundinaceum) toxicosis research is often complicated by a reduction in intake of infected forage or seed, making treatment comparisons difficult. This study was conducted to develop a fescue toxicosis model that would allow for variations in DMI without altering the quantity of alkaloids consumed over the course of the experiment. Ground tall fescue seed and a tall fescue seed extract were used in two 2-period crossover experiments to determine the effectiveness of ruminal dosing of a tall fescue seed extract to induce fescue toxicosis. This experiment used 4 growing Holstein steers (BW = 337 ± 24 kg) surgically fitted with ruminal cannulas. Steers were maintained on a diet of endophyte-free fescue hay fed ad libitum throughout the experiment. Endophyte-infected (E+; 4.1 mg/kg of ergovaline) and uninfected (E-; 0.0 mg/kg of ergovaline) KY-31 tall fescue seed was ground and dosed or extracted with ethanol, concentrated, and lyophilized before ruminal dosing. Ergovaline concentration of the final extract was 102 mg/kg. Animals were given a minimum of a 3-wk washout period between treatments. Physiological indicators were measured over 7 d at 22°C (d 1 to 3) and 32°C (d 4 to 7) during both seed and extract dosing. Seed and extract E+ dosing reduced serum prolactin concentrations such that they were not different from zero (P < 0.10). Treatment with E+ reduced feed intake (P < 0.05) and heart rate (P < 0.001), and increased respiration rate (P < 0.01) and core temperature (P < 0.05) during both seed and extract dosing. Increasing environmental temperature from 22 to 32°C reduced total intake (P < 0.05) and increased core temperature (P < 0.001) and respiration rate (P < 0.001) during both seed and extract dosing. Diastolic blood pressure tended (P < 0.09) to be increased during E+ extract dosing and reduced during heat stress. These physiological alterations are consistent with those reported for cattle grazing or consuming seed from endophyte-infected tall fescue. These data indicate that a ruminally dosed ethanol extract of tall fescue seed is efficacious in inducing fescue toxicosis in cattle.     

Influence of consumption of endophyte-infested tall fescue hay on performance of heifers and lambs

Two experiments were conducted to evaluate performance and physiological responses of heifers and lambs to Neotyphodium coenophialum-infested tall fescue hay fed under European rearing conditions. Endophyte-free (E-) or 100% endophyte-infested (E+) hay was derived from the same cultivar (cv. Clarine) so that the effect of the endophytic fungus could be clearly separated from a possible cultivar effect. In Exp. 1, starting in June 1996, 20 age- and body weight-paired Holstein dairy heifers were assigned for 97 d to one of two treatments consisting of ad libitum access to either E- or E+ hay, corresponding to 0 and .41 mg/kg ergovaline, respectively. During the experimental period, no significant difference (P>.20) in forage consumption, rectal temperature, or behavioral status of the animals was observed between the two treatments. The E+ diet induced a 10% apparent decrease in ADG and a clear reduction in prolactin (PRL) plasma concentration compared to the E- diet. When animals were all reassigned to a common endophyte-free diet, the E+ group recovered body weight and PRL to levels similar to those in animals fed E- after 7 wk. In Exp. 2, 30 Texel ram lambs were assigned to two treatments consisting of dietary E- or E+ tall fescue hay. The E- and E+ hays were harvested from the same plots as used in Exp. 1 and contained 0 and .96 mg/kg ergovaline, respectively. No effect of the endophyte was found on intake or carcass or testicle weight (P>.20) after the 95-d feeding period. The E+ treatment resulted in a slight reduction in BW at slaughter, mainly explained by a lower ruminal fill (P<.01). In E+ treated animals, prolactin concentrations dropped significantly (P<.001) from d 27. Hay assessment in both experiments showed no difference in chemical composition and IVDMD. The endophytic fungus strongly lowered the palatability of the E+ hay, although there was no effect on intake with heifers (Exp. 1) or with lambs (Exp. 2). The potential of severe heat stress, as expressed by the temperature humidity index, was not high in our experimental conditions, although they were considered rather unusually stressful for the western part of northern Europe. Yet, no economic effect on cattle was observed, in disagreement with results obtained in many previous U.S. studies.     

Hemodynamic responses of the caudal artery to toxic tall fescue in beef heifers

Color Doppler ultrasonography was used to compare blood flow characteristics in the caudal artery of heifers fed diets with endophyte (Neotyphodium coenophialum) infected (E+) or noninfected (E-) tall fescue seed. Eighteen crossbred (Angus x Brangus) heifers were assigned to 6 pens and were fed chopped alfalfa hay for 5 d and chopped alfalfa hay plus a concentrate that contained E-tall fescue seed for 9 d during an adjustment period. An 11-d experimental period followed, with animals in 3 pens fed chopped alfalfa hay plus a concentrate with E+ seed and those in the other 3 pens fed chopped hay plus concentrate with E E- seed. Color Doppler ultrasound measurements (caudal artery area, peak systolic velocity, end diastolic velocity, mean velocity, heart rate, stroke volume, and flow rate) and serum prolactin were monitored during the adjustment (baseline measures) and during the experimental period. Three baseline measures were collected on d 3, 5, and 6 during the adjustment period for comparison to post E+ seed exposure. Statistical analyses compared the proportionate differences between baseline and response at 4, 28, 52, 76, 100, 172, and 268 h from initial feeding of E+ seed. Serum prolactin concentrations on both diets were lower (P <0.001) than baseline beginning at 4 h from the start of the experimental period. However, trends in serum prolactin concentrations for heifers on the E- diet suggested ambient temperature was affecting these concentrations. Caudal artery area in E+ heifers had declined (P <0.10) from baseline by 4 h and was consistently lower (P <0.05) for the remainder of the period. Heart rates for E+ heifers were lower than the baseline rate from 4 (P <0.10) to 100 (P <0.001) h, but were similar (P >0.10) to the baseline for 172 and 268 h measures. Blood flow in E+ heifers was consistently lower than the baseline from 4 (P <0.05) to 172 (P <0.001) h, but was similar to the baseline at 268 h when heart rate was similar to the baseline rate. Caudal artery areas for the E- diet were similar to baseline areas except at 100 h when it was greater than baseline. Heart rates and flow rates for E- heifers did not differ (P >0.10) from baseline measures during the experimental period. Results indicated that onset of toxicosis was within 4 h of cattle exposure to E+ tall fescue and is related to vasoconstriction and reduction in heart rate.     

Impacts of an endophyte-infected fescue seed diet on traits of mouse lines divergently selected for response to that same diet

In previous work, mouse lines were selected for eight generations for resistance (R) or susceptibility (S) to endophyte-infected fescue toxicosis using depression in postweaning gain caused by a toxin-containing diet as the selection criterion. Characterizing biological changes associated with resistance or susceptibility in those mice might suggest genetic or therapeutic approaches to alleviate fescue toxicosis in cattle. The first objective of the current experiment was to determine whether the toxin-containing diet depressed reproduction and mature size more severely in S than in R mice. The second was to investigate line and diet effects on hepatic glutathione-S-epoxytransferase (GST) and uridine diphosphate glucuronosyl-transferase (UDPGT) activities and to relate enzyme activities to reproduction within line by diet groups. Twenty-eight pairs per line (S or R) x diet (toxin-containing [+] or toxin-absent [-]) group cohabitated for 36 wk. The + diet depressed the number of pups born and weaned and litter weight weaned (P < .01) within the first two litters produced. Diet effects were greatest early in the experiment. Percentage changes in reproduction caused by the + diet for R and S pairs, respectively, were -13 and -28 for total pups born, -10 and -25 for total pups weaned, -13 and -14 for total litters produced, and -30 and -42 for total litter weight weaned. The S line mice were heavier than R line mice on both diets, but the + diet had a larger depressing effect on mature size of S line than of R line males (line x diet interaction, P = .09) and females (interaction not significant). Averaged across diets, GST activity was higher in R than in S dams (P = .05) at 44 wk of age but was not affected by diet or line x diet. Activity of GST was correlated with number of pups born (-.50), number of litters produced (-.44), and survival percentage (.40) within the R- group; in the R+ group, GST activity was correlated only with survival percentage (.37). In the S- and S+ groups, GST activity was not correlated with any reproductive trait. Line, diet, and their interaction did not affect UDPGT activity, and UDPGT activity was not correlated with any reproductive trait in any line x diet group. Selected lines differed in response to a toxin-containing diet as measured by its effect on reproduction and mature size. The R and S mice also differed in GST activity, but GST activity was correlated with reproductive traits only in R-line mice.     

Effects of initial and extended exposure to an endophyte-infected tall fescue seed diet on faecal and urinary excretion of ergovaline and lysergic acid in mature geldings

Abstract

AIM: To determine the amount of ergovaline and lysergic acid retained or excreted by geldings fed endophyte-infected seed containing known concentrations of these alkaloids, and the effects of exposure time on clinical expression of toxicosis.

METHODS: Mature geldings (n=10) received diets containing either endophyte-free (E-) or endophyte-infected (E+) tall fescue seed during three experimental phases. The first phase (Days ?14 to ?1) was an adaptation phase, to allow all horses to adapt to a diet containing E- tall fescue seed. The second (Days 0 to 3) was the initial exposure phase to E+ tall fescue seed, used for the delivery of ergovaline and lysergic acid at 0.5 and 0.3 mg/kg of diet, respectively, to test the initial effects of exposure on routes and amounts of elimination of alkaloid. During this phase, half the geldings were exposed to an E+ diet while the rest served as controls by remaining on the E- diet. Once assigned to treatments, geldings remained on the same diet through the third phase (Days 4 to 21), which served as the extended exposure phase. Total outputs of faeces and urine were collected within each phase, to determine retention of ergovaline and lysergic acid and nutrient digestibility. Serum was collected weekly and analysed for activities of enzymes and concentrations of prolactin. Bodyweights (BW) and rectal temperatures were recorded weekly.

RESULTS: BW, rectal temperature, enzyme activities and concentrations of prolactin in serum, and nutrient digestibility were not affected by treatment. Total intake of ergovaline by geldings on the E+ diet was 3.5 and 3.6 (SE 0.20) mg/day, and 2.1 and 2.3 (SE 0.11) mg/day were not accounted for in initial and extended phases, respectively. Lysergic acid was excreted in the urine (4.0 and 4.9 (SE 0.97) mg/day) and faeces (2.5 and 2.7 (SE 0.35) mg/day) at greater amounts than that consumed (2.0 and 1.9 (SE 0.09) mg/day) during the initial and extended exposure phases, respectively. Animals exposed to E+ seed for a period of 20 days appeared to excrete more (1.5 vs 1.2 mg/day; SE 0.08; p=0.03) ergovaline in the faeces than those exposed for only 4 days.

CONCLUSIONS: Exposure time to the ergot alkaloids had a limited effect on the route of elimination or the amounts of ergovaline or lysergic acid excreted by horses. The primary alkaloid excreted was lysergic acid, and urine was the major route of elimination. These data will aid future research to improve animals′ tolerance to toxic endophyte-infected tall fescue.     

Effect of dietary phosphorus and its interaction with genetic background on global gene expression in porcine muscle

Environmental concerns and costs associated with dietary phosphorus (P) supplementation have lead to attempts to minimize the amount of P added to swine diets. In addition to its requirement for bone growth, dietary P is also necessary for muscular growth. To examine the effects of genetic background and dietary P on global gene expression in the muscle of young pigs, we utilized muscle tissue from 36 gilts sired from two different sire lines. These animals were fed either a P adequate, P deficient or P repletion diets for 14 days and showed differences in growth performance and bone integrity in response to the interaction of genetic background and dietary P. Total RNA from the loin muscle of these animals was obtained for microarray analysis. Significant differences (p < 0.01) in gene expression were seen based on the effect of sire line (339 genes), dietary P (18 genes) and the interaction between sire line and dietary P (31 genes). The microarray data were validated by semi-quantitative real-time PCR. These results support our hypothesis that genetic background and dietary P treatment can affect the homeorhetic control of P metabolism in pigs. Genes identified as differentially expressed in this study may be excellent candidate genes for additional work to elucidate genotype specific P requirements as well as to identify a genetic background that can maintain superior growth in a more environmentally friendly manner.     

Genomic analysis of the impact of fescue toxicosis on hepatic function

Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodium coenophialum, in tall fescue [Lolium arundinaceum (Schreb.) Darbysh]. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes to identify potential targets for future pharmacological/toxicological intervention. Male rats (n = 24) were implanted with temperature transmitters, which measure core temperature every 5 min. After an 8-d recovery, the rats were fed an endophyte-free diet for 5 d. During the following 5-d treatment period, rats were fed either an endophyte-free or an endophyte-infected (91.5 microg of ergovaline.kg of BW(-1).d(-1)) diet. At the end of treatment, rats were euthanized and a sample of the liver was obtained. Feed conversion efficiency was calculated for both treatment groups. Serum prolactin concentrations were measured using ELISA. Liver tissue RNA was reverse transcribed and hybridized to an oligonucleotide microarray chip. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR. Significant reductions in mean core temperature, feed intake, feed conversion efficiency, BW, liver weight per unit of BW, and serum prolactin concentrations were observed in endophyte-infected rats. There was downregulation (P < 0.05) of various genes associated with energy metabolism, growth and development, and antioxidant protection, as well as an upregulation of genes associated with gluconeogenesis, detoxification, and biotransformation. This study demonstrated that even short-term exposure of rats to tall fescue endophytic toxins under thermoneutral conditions can result in physiological responses associated with altered gene expression within the liver.     

Effects of initial and extended exposure to an endophyte-infected tall fescue seed diet on faecal and urinary excretion of ergovaline and lysergic acid in mature geldings

AIM: To determine the amount of ergovaline and lysergic acid retained or excreted by geldings fed endophyte-infected seed containing known concentrations of these alkaloids, and the effects of exposure time on clinical expression of toxicosis. METHODS: Mature geldings (n=10) received diets containing either endophyte-free (E-) or endophyte-infected (E+) tall fescue seed during three experimental phases. The first phase (Days -14 to -1) was an adaptation phase, to allow all horses to adapt to a diet containing E- tall fescue seed. The second (Days 0 to 3) was the initial exposure phase to E+ tall fescue seed, used for the delivery of ergovaline and lysergic acid at 0.5 and 0.3 mg/kg of diet, respectively, to test the initial effects of exposure on routes and amounts of elimination of alkaloid. During this phase, half the geldings were exposed to an E+ diet while the rest served as controls by remaining on the E- diet. Once assigned to treatments, geldings remained on the same diet through the third phase (Days 4 to 21), which served as the extended exposure phase. Total outputs of faeces and urine were collected within each phase, to determine retention of ergovaline and lysergic acid and nutrient digestibility. Serum was collected weekly and analysed for activities of enzymes and concentrations of prolactin. Bodyweights (BW) and rectal temperatures were recorded weekly. RESULTS: BW, rectal temperature, enzyme activities and concentrations of prolactin in serum, and nutrient digestibility were not affected by treatment. Total intake of ergovaline by geldings on the E+ diet was 3.5 and 3.6 (SE 0.20) mg/day, and 2.1 and 2.3 (SE 0.11) mg/day were not accounted for in initial and extended phases, respectively. Lysergic acid was excreted in the urine (4.0 and 4.9 (SE 0.97) mg/day) and faeces (2.5 and 2.7 (SE 0.35) mg/day) at greater amounts than that consumed (2.0 and 1.9 (SE 0.09) mg/day) during the initial and extended exposure phases, respectively. Animals exposed to E+ seed for a period of 20 days appeared to excrete more (1.5 vs 1.2 mg/day; SE 0.08; p=0.03) ergovaline in the faeces than those exposed for only 4 days. CONCLUSIONS: Exposure time to the ergot alkaloids had a limited effect on the route of elimination or the amounts of ergovaline or lysergic acid excreted by horses. The primary alkaloid excreted was lysergic acid, and urine was the major route of elimination. These data will aid future research to improve animals' tolerance to toxic endophyte-infected tall fescue.     

Effects of short-term early gestational exposure to endophyte-infected tall fescue diets on plasma 3,4-dihydroxyphenyl acetic acid and fetal development in mares

Consumption of wild-type (toxic) endophyte-infected tall fescue (E+) by horses during late gestation is known to adversely affect pregnancy outcome; however, little is known of the potential disruptive consequences of E+ consumption by mares during the critical phases of placentation and fetal development in early pregnancy. The objective of this study was to evaluate the detrimental effects of feeding E+ to mares during early gestation. Mares (n = 12) paired by stage of gestation (d 65 to 100) were assigned to diets (six per diet) consisting of endophyte-free (E-) or E+ tall fescue seed (50% E- or E+ tall fescue seed, 45% sweet feed, and 10% molasses fed at 1.0% of BW/d). Mares also had ad libitum access to E+ or E- annual ryegrass hay, and were fed diets for 10 d. Following removal from the tall fescue diet on d 11, mares were placed on common bermudagrass pasture and monitored until d 21. Morning and evening rectal temperatures were recorded and daily blood samples were collected for progesterone and prolactin (PRL) analyses, whereas samples for 3,4-dihydroxyphenyl acetic acid (a catecholamine metabolite) analysis were collected on alternate days. For clinical chemistry analysis, blood samples were collected on d 0, 5, 10 and 21. Daily urine samples were collected for ergot alkaloid analysis, and ultrasonography was performed for presence of echogenic material in fetal fluids. Rectal temperatures (E+ 37.76+/-0.03; E- 37.84+/-0.03 degrees C) and serum PRL concentrations (E+ 14.06< or =0.76; E- 12.11+/-0.76 ng/mL) did not differ (P = 0.96) between treatments. Measuring the change in basal serum concentration from d 0 over time, progesterone concentrations did not differ (-0.64 +/-1.49 and -0.55+/-1.47 ng/mL for E+ and E- mares, respectively). There was no negative pregnancy outcome, and ultrasonography indicated no increase in echogenic material in fetal fluids. Plasma 3,4-dihydroxyphenyl acetic acid concentrations decreased (P < 0.05) in E+ compared with E- mares (2.1+/-0.14 and 4.4+/0.43 ng/mL, respectively). Urinary ergot alkaloid concentration was greater (P < 0.01) in mares consuming E+ compared with E- (532.12+/- 52.51 and 13.36+/-2.67 ng/mg of creatinine, respectively). Although no fetal loss was observed during the current study, elevated concentrations of urinary ergot alkaloid were consistent with depressed endogenous catecholamine activity, suggestive of an endocrine disruptive effect of hypothalamic origin.     

The impact of endophyte-infected fescue consumption on digital circulation and lameness in the distal thoracic limb of the horse

This experiment was conducted to evaluate if consumption of endophyte-infected fescue alters digital circulation in the distal thoracic limb of the horse and to assess if soundness of the hooves of horses is affected by consumption of endophyte-infected fescue. Twelve American Quarter Horses (mean initial BW 459 ± 31 kg), 6 mares and 6 geldings, were used in this 90-d study that comprised high-endophyte (E+) and low-endophyte (E-) treatment groups. Fescue seed was integrated into the E+ diet at a rate sufficient to bring total ergovaline to 200 μg/kg, and endophyte-free fescue seed was incorporated into the E- diet from d 0 to 90. From d 30 to 60, native prairie hay was replaced with high- or low-endophyte fescue hay, bringing total dietary ergovaline to 280 μg/kg (E+) and 18 μg/kg (E-). From d 61 to 90, fescue seed was ground to decrease particle size. On d 0, 30, 60, and 90, Doppler ultrasonography and thermographic imaging were used to measure the diam. of the medial palmar artery, velocity of blood flow, and surface temperature of the hoof as indicators of digital circulation. Lameness examinations were conducted on the same days. There were no consistent treatment differences observed when evaluating measurements of digital circulation. On d 60, horses in the E+ treatment group showed increased hoof sensitivity in the left limb (P = 0.02). These horses tended to have increased hoof sensitivity when both thoracic limbs were averaged (P = 0.06), and they demonstrated increased lameness during longeing (P = 0.08). Data indicated that mares may have increased digital circulation, regardless of treatment, compared with geldings (P ≤ 0.05). Heavier horses also had greater arterial diam., velocity of blood flow, and hoof temperature than lighter BW horses (P ≤ 0.05) on d 30, 60, and 90 at time points that ranged from 90 to 180 min after feeding. Although horses consuming the E+ diet demonstrated increased lameness, especially on d 60, compared with horses consuming the E- diet, the measures of digital circulation did not support the hypothesis that digital circulation was reduced. Because of observed lameness issues, limiting the access of horses to endophyte-infected fescue may be prudent.