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 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.     

Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue

Four wethers and 14 steers (environmentally heat stressed, 32 degrees C, 60% relative humidity) were evaluated for changes in blood flow induced by endophyte-infected tall fescue. Concentration of the ergopeptide ergovaline was used as an indicator of diet toxicity due to the endophytic fungus Acremonium coenophialum. Blood flow to specific tissues was measured using radiolabeled microspheres. Wethers received one of two dietary treatments for 30 d before determination of tissue blood flow: 1) a low-endophyte diet (less than .05 ppm ergovaline) or 2) a high-endophyte (1.18 ppm ergovaline) diet. Blood flows to the adrenal glands and skin covering the inner hind leg were less (P less than .10) in wethers consuming the high-endophyte diet than in those consuming the low-endophyte diet. Tissue blood flows in steers were determined on two occasions: 1) after steers had received a low- (less than .01 ppm ergovaline) or high-endophyte (.52 ppm ergovaline) fescue diet for 14 d and 2) 8 d after steers had been switched to a common, fescue-free diet. Blood flows to skin covering the ribs, cerebellum of the brain, duodenum, and colon were less (P less than .10) in steers consuming the high-endophyte diet. However, 8 d after consuming fescue-free diets, steers that had previously consumed the high-endophyte diet had greater (P = .08) blood flow to the coronary bands of the front hooves than steers that had consumed the low-endophyte diet. Blood flows to all other tissues were similar between treatments. We inferred from these experiments that the toxin(s) associated with endophyte-infected tall fescue caused decreased blood flow to peripheral and core body tissues and that this effect was abated within 8 d of removing the toxin(s).     

Assessing the Association Between Hoof Thermography and Hoof Doppler Ultrasonography for the Diagnosis of Lameness in Horses

The objectives of this study were to assess the correlation between hoof surface temperature and ultrasonographic measurements of digital blood vessels in horses and to evaluate the measurements' potential as predictors for clinical lameness. Twelve 3-year-old American Quarter Horses, 6 geldings and 6 mares, with average initial body weight of 459 ± 31 kg were used. On days 0, 30, 60, and 90 of the study, horses were weighed and subjected to clinical lameness examinations. Doppler ultrasonography was used to measure diameter of the medial palmar artery in the distal left forelimb and velocity of blood flow through that artery, starting at 60 minutes after morning feeding and repeated at 30-minute intervals. Temperature measurements on the hoof were collected at 15-minute intervals beginning 75 minutes after feeding, using a digital thermographic camera. A series of bivariate linear mixed models were fitted to estimate the correlation between Doppler and temperature measurements. The within-horse and between-horse correlations between hoof surface temperature and velocity of blood flow in the distal limb through the medial palmar artery was estimated at 0.40 (P > .50) and 0.99 (P < .001), respectively. These results indicate that at the horse level, the correlation between hoof temperature and velocity of blood flow in the distal limb was very high but that the within-horse correlation was not significantly different from 0. Velocity of blood flow at 60 minutes after feeding improved model fit to the lameness data, so it was included as a model predictor for lameness.     

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.     

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.     

Thermoregulatory ability of beef heifers following intake of endophyte-infected tall fescue during controlled heat challenge

Consumption of endophyte-infected tall fescue during summer months can result in severe hyperthermia in cattle. Six heifers (296+/-8.3 kg BW) were used to determine changes in body temperature control that occur with consumption of an endophyte-infected tall fescue diet during controlled heat challenge. All animals were exposed in five separate periods to a step increase in ambient temperature (Ta) from 21 to 31 degrees C while fed E+ (5 microg ergovaline x kg(-1) x d(-1)) or endophyte-free (E-) diets. Core body temperature (Tcore) was monitored continuously using implanted, telemetric temperature transmitters. Heat production and heat loss were also measured at selected times to identify primary effects of E+ on thermal balance. Pretreatment Tcore exhibited a diurnal rhythm at a constant Ta of 21 degrees C, with high and low values at 2300 and 1300, respectively. An increase in daily averaged Tcore (P < 0.001) occurred with an increase in Ta from 21 to 31 degrees C. Likewise, all phases of the daily cycle increased equally during this challenge. This increase at 31 degrees C was associated with higher levels of respiration rate, skin temperature, respiratory vaporization, and skin vaporization (P < 0.05) and lower blood levels of thyroxine (P < 0.05). Intake of the E+ diet further elevated Tcore in heifers during the short-term heat challenge (2 d), and the effect was most pronounced at 0000 to 0300 and declined thereafter. The increase in Tcore during E+ treatment was associated with an increase in respiration rate (P < 0.05), whereas metabolic heat production, skin temperature, skin vaporization and respiratory vaporization were unaffected. These results show that consumption of an E+ diet during continuous heat challenge results in a marked increase in core body temperature, especially during nighttime exposure to heat stress, due primarily to a reduction in cutaneous heat transfer, with no effect on heat production or other measured avenues of heat loss.     

Physiological and digestive effects of Neotyphodium coenophialum-infected tall fescue fed to lambs

The digestive responses and degradation of ergovaline and production of lysergic acid in the rumen of sheep offered Neotyphodium coenophialum-infected tall fescue straw at 2 ergovaline levels were investigated. Six crossbred wethers (56 +/- 3.0 kg of BW) were used in a randomized crossover design involving 2 treatments, for a total of 6 observations per treatment. The experiment consisted of two 28-d feeding periods with a 14-d washout period between them. The treatments were 1) tall fescue straw containing <0.010 mg of ergovaline/kg (E-), and 2) tall fescue straw containing 0.610 mg of ergovaline/kg (E+). Feed, orts, and feces were measured and analyzed for DM, ADF, and CP, and used to determine digestibilities. Feed and water intake were monitored throughout the feeding periods. Body weight and serum prolactin levels were measured at the beginning and end of each feeding period. Ruminal fluid was sampled 3 times (d 0, 3, and 28) during each 28-d feeding period for determination of ergovaline, lysergic acid, ammonia, and pH. Samples were collected before feeding (0 h) and at 6 and 12 h after feeding. Total fecal and urine collection commenced on d 21 and continued until d 25 of each feeding period. Ruminal ammonia, ruminal pH, and rectal temperature were not influenced by ergovaline concentration (P > 0.10). Digestion of DM, ADF, and CP was not different between treatments (P > 0.10). Daily water intake was less for the E+ diet (2.95 vs. 2.77 L/d; P < 0.05) as was serum prolactin (22.9 vs. 6.4 ng/mL; P < 0.05). Ergovaline concentration in ruminal fluid increased over sampling days at each sampling time (P < 0.05). Lysergic acid concentration in ruminal fluid increased over time from d 0 to 3 (P < 0.05) but was not different between d 3 and 28 (P > 0.10). In the E+ treatment, ergovaline was not detectable in the urine, whereas the concentration in the feces was 0.480 mg/kg. Lysergic acid was detected in the diet of the E+ treatment at 0.041 g/kg, lysergic acid in the urine was 0.067 mg/kg and in the feces was 0.102 mg/kg. The apparent digestibility of the alkaloids was 64.2% for ergovaline and -12.5% for lysergic acid. Approximately 35% of dietary ergovaline and 248% of dietary lysergic acid were recovered in the feces and urine. The appearance of lysergic acid in the feces, urine, and ruminal fluid is likely due to microbial degradation of ergovaline in the rumen and further breakdown in the lower digestive tract.     

Thyroid function in mature horses ingesting endophyte-infected fescue seed

OBJECTIVE: To determine whether ingestion of fescue seed infected with the endophyte Neotyphodium coenophialum would alter thyroid function in adult horses. DESIGN: Original study. ANIMALS: 4 adult mares that were not pregnant and 6 adult geldings. PROCEDURE: Thyrotropin releasing hormone stimulation tests were performed while horses received a standard diet and after infected seed (2.3 kg/d [5 lb/d]) had been fed for 1 and 2 months. Serum prolactin concentrations were measured to verify endophyte absorption. RESULTS: Serum prolactin concentrations indicated that at least 8 of 10 horses absorbed the endophyte. Baseline concentrations of thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxine and the change in hormone concentrations in response to administration of thyrotropin releasing hormone (1 mg, i.v.) were not altered by ingestion of endophyte-infected fescue seed. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that ingestion of fescue seed infected with the endophytic fungus N. coenophialum for 2 months has little effect on thyroid function in adult horses that are not pregnant.     

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.     

Effect of fescue toxicosis on hepatic gene expression in mice

Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endophytic fungus Neotyphodium coenophialum. Signs of fescue toxicosis include increased core body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis, as indicated by reduced growth rate and reproductive performance. Mice were used to study the effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+; 6 ppm ergovaline) or endophyte-free (E-) fescue seed for 2 wk under thermoneutral conditions. Liver genes differentially expressed due to fescue toxicosis were identified using DNA microarray. A 2-stage ANOVA of microarray data identified 36 differentially expressed genes between mice fed E+ and E- diets. Another analysis method, significance analysis of microarray, identified 9 genes as differentially expressed between treatment groups, and some genes overlapped with genes identified by ANOVA. Hierarchical clustering of 36 genes identified by ANOVA clearly separated the mice by diet, with 100% confidence as computed by bootstrap analysis. Expression of 11 genes was verified using quantitative real-time PCR. The E+ diet resulted in downregulation of genes involved in the sex-steroid metabolism pathway and genes involved in cholesterol and lipid metabolism. Genes coding for ribosomes and protein synthesis were upregulated by the E+ diet. Genes identified in the present analysis indicate some of the mechanisms by which fescue toxicosis occurs in animals.     

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 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.     

Effect of Ergovaline Ingestion on Recovery of Horses Subjected to an Anaerobic Standard Exercise Test

The objective of this study was to determine whether consumption of tall fescue seed which contained high levels of ergovaline would affect post-exercise recovery of horses subjected to an anaerobic exercise test. Ten Quarter horses were separated into two groups and fed alfalfa hay, commercial sweet feed, and either endophyte-infected (E+) or endophyte-free (E−) ground tall fescue seed. Diets contained 11.8% seed, which resulted in 459 ppb of ergovaline for the E+ diet. During the first 28 days, horses in group A received E+ and group B received E−. Diets were reversed during the next 28 days (crossover design). Horses were ridden for 5 days per week. On day 14, 28 (period 1), 42, and 56 (period 2), they were subjected to a standardized exercise test (SET) that consisted of 41 turns in 4 minutes and was designed to raise the horse’s heart rate (HR) beyond the anaerobic threshold (150 bpm). There was a horse effect (P < .05) on post-SET respiration rate, HR, and post-exercise whole blood lactate level, but not rectal temperature (RT). For these variables or period effects, there was no horse × treatment, period × treatment, or period × horse interactions (P < .05). Treatment had no effect on whole blood lactate level, RT, or HR at any time measured. Respiration rate did not vary by treatment at rest or 1 minute after SET but was higher for the E+ treatment at 5 and 10 minutes after SET (P < .005). Consumption of diets that averaged 450 ppb of ergovaline caused the horses expend more respiratory effort in an attempt to recover to resting RT.     

Constriction of bovine vasculature caused by endophyte-infected tall fescue seed extract is similar to pure ergovaline

Ergovaline has been extensively used to study vasoactive effects of endophyte- (Neotyphodium coenophialum) infected tall fescue (Lolium arundinaceum). However, initial results indicated that an extract of toxic tall fescue seed (E+EXT) is more potent than ergovaline alone in a right ruminal artery and vein bioassay. The E+EXT induced a greater contractile response than an equal concentration of ergovaline alone in the ruminal artery of heifers (P = 0.018). This led to a hypothesis that other compounds in the seed extract contribute to vasoconstriction. Thus, experiments were conducted to determine if vasoactivity of an E+EXT is different from a mixture of ergot alkaloids (ALK; ergovaline, ergotamine, ergocristine, ergocryptine, ergocornine, ergonovine, and lysergic acid) of similar concentrations and to determine if the vasoactivity of an E+EXT differs from an endophyte-free tall fescue seed extract (E-EXT). Segments of lateral saphenous vein and right ruminal artery and vein were collected from Holstein steers (n = 6) shortly after slaughter. Vessels were cleaned of excess connective tissue and fat and sliced into segments that were suspended in a multimyograph chamber with 5 mL of continually oxygenated Krebs-Henseleit buffer, equilibrated for 90 min, and exposed to a reference compound (120 mM KCl for ruminal vessels and 0.1 mM norepinephrine for saphenous vein). Increasing concentrations of each treatment (E+EXT, E-EXT, ALK, and ergovaline) were added to the respective chamber every 15 min after buffer replacement. Data were normalized as a percentage of maximal contractile response of the reference compound and fit to a sigmoidal concentration response curve. Ergovaline, ALK, and E+EXT induced similar responses in the saphenous vein, ruminal artery, and ruminal vein. The E+EXT displayed a smaller EC(50) (half maximal effective concentration) than ergovaline or ALK in the saphenous vein and ruminal vein (P < 0.008), but not the ruminal artery (P = 0.31). Extrapolated maximum response was greatest in the saphenous vein for ergovaline, least for E+EXT, and intermediate for ALK (P < 0.0001). The E-EXT did not induce a contractile response in any vessel tested (P > 0.1). Data from this study indicate that ergovaline is largely responsible for the locally induced vasoconstriction of bovine vasculature observed with endophyte-infected tall fescue.     

Growth rate, diet digestibility, and serum proclactin of yearling horses fed non-infected and infected tall fescue hay

To determine to effect of feeding tall fescue hay infected with endophyte on growth rate, diet digestibility, and serum prolactin, 17 Quarter Horse yearlings were fed a diet of 60% concentrate and 40% tall fescue hay for 5 months. Diets were formulated to meet 1989 NRC recommendations for moderate growth. The dietary treatments included either non-infected (E-, n=9) or infected (E+, n=8) tall fescue hay. Field sampling determined a 52% infection rate of the E+ hay with the endophyte Acremonium coenophialum. Initially and at 29-day intervals, yearlings were weighed and measured for skeletal growth including: (1) height at the withers, (2) length of bone between knee and fetlock, (3) length of bone between hock and fetlock, and (4) the circumference of fore and rear cannon bones. Total fecal collection from the 10 geldings during the last month of the trial permitted the calculation of diet digestibility. Serum samples were taken throughout the trial to determine prolactin response to dietary treatments.The E+ fescue hay did not affect (P>.05) growth rate of yearling horses. Yearlings gained .40 and .38 kg/d when consuming the E− and E+ based diets, respectively. Geldings gained more (P<.05) than fillies. Changes in height at the wither and length and circumference of the fore and rear cannon were positive but did not differ between hay treatments or sexes.E+ fescue hay diets tended to be lower in dry matter, apparent energy, and crude protein digestibility, but values were not statistically different. Fiber digestibility was lower (P<.01) for the diet based on E+ hay. Neutral detergent fiber digestibility averaged 66.2 and 54.4% for E− and E+ diets, respectively.Acid detergent fiber digestibility averaged 78.9 and 64.5% for E− and E+ diets, respectively. Although fiber digestibility of E+ tall fescue hay was lower than E-hay, grain supplementation supportive of a moderate growth rate produced equal growth among yearling horses. Also, no differences in prolactin were apparent between the yearlings fed E− and E+ tall fescue hay.     

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.     

Tall fescue utilization by exercised - yearling horses

Little information has been published concerning the effects of endophyte-infected tall fescue consumption on performance of young growing horses. Therefore, this study evaluated exercise performance and growth by yearlings fed endophyte-infected fescue. Twelve Quarter Horse yearlings (12-16 months of age) were blocked by age and sex and randomly assigned to one of three groups. Group I received native prairie hay (P), Group II received endophyte-free fescue hay (EF) and, Group III received endophyte infected fescue hay (EI). Rectal temperatures were monitored daily and growth parameters (weight, hip and wither heights, and body fat) were measured at the start of the study (d 0) and every 2 weeks throughout the 106 d study. Additionally, blood samples were collected on d 0 and every 28 d for the duration of the study to monitor blood concentrations of calcium, phosphorus, prolactin (PRL), thyroxine (T₄), and triiodothyronine (T₃). Horses were exercised twice a week for 10-30 min. Respiration, heart rate, and rectal temperature were measured at the start and 5, 30, and 60 min postexercise. Growth parameters and daily rectal temperatures were not different (P >.05) among grdups. No differences (P >.05) occurred among groups in concentrations of serum calcium, phosphorus, PRL, T₃, and T₄. Respiration rates were lower (P <.05) at 30 and 60 rain postexercise for horses consuming fescue than for horses consuming prairie hay. Postexercise heart rates and rectal temperatures showed no difference (P <.05) among groups. These data suggest that young growing horses being exercised can utilize endophyte-infected fescue efficiently on a short-term basis.     

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.     

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.