Duration of ergovaline exposure influences serotonin-mediated vasoactivity of bovine mesenteric vasculature.

Ergovaline (ERV), produced in toxic endophyte-infected tall fescue, causes potent vasoconstriction of bovine peripheral and visceral vasculature. Ergovaline acts as both an agonist and an antagonist in bovine gut blood vessels through serotonin (5-HT) receptors and it appears that the type of action could be influenced by the extent of ERV exposure. Because it was unclear how the duration of ERV exposure influences 5-HT-mediated vasoactivity, experiments were designed to evaluate how simultaneous or prior ERV exposure influenced 5-HT-mediated vasoactivity of mesenteric artery (MA) and vein (MV) segments from Holstein steers (N = 10). Vessels were incubated in Krebs-Henseleit buffer containing 0, 0.01, or 0.1 µM ERV for 24 h prior to the 5-HT dose-response or exposed to fixed concentrations of 0, 0.01, or 0.1 µM ERV simultaneously during the 5-HT dose-response. Vessels were suspended in chambers of a multimyograph containing Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. Vessels were exposed to increasing concentrations of 5-HT (5 × 10-8 M to 1 × 10-4 M) every 15 min and contractile responses were normalized as a percentage of the maximum contractile response induced by 120 mM KCl reference addition. Two-way analysis of variance was used to separately analyze data for each vessel type and duration of exposure using the MIXED procedure of SAS. When 5-HT concentration increased from 5 × 10-8 to 1 × 10-6 M, simultaneous addition of 0.1 µM ERV increased (P < 0.01) the contractile response of MV compared with additions of 0 and 0.01 µM ERV. At 1 × 10-4 M 5-HT, the simultaneous presence of 0.01 and 0.1 µM ERV decreased (P < 0.01) the contractile response of both MA and MV compared with 0 µM ERV addition. As 5-HT concentrations increased, the contractile response increased (P < 0.01) in both MA and MV with no previous ERV exposure, but decreased in MA and MV with 24 h prior exposure to 0.01 and 0.1 µM ERV. These data demonstrate that the duration of ERV exposure influences 5-HT-mediated vasoconstriction and likely vasorelaxation in bovine mesenteric vasculature. If ERV and 5-HT exposure occur simultaneously, ERV can act as a partial agonist of 5-HT-mediated vasoconstriction. If 5-HT exposure occurs after blood vessels have had prior ERV exposure, it appears that 5-HT may induce vasorelaxation of blood vessels. More research is needed to identify cellular and molecular mechanisms involved with 5-HT-mediated vasoactivity.

Consumption of ergot alkaloids found in endophyte-infected tall fescue can lead to symptoms of fescue toxicosis, such as vasoconstriction, in ruminant livestock species. Ergovaline is one of the primary ergot alkaloids responsible for causing vasoconstriction when toxic varieties of fescue are consumed. It has been previously shown that ergovaline causes vasoconstriction by interacting with vascular serotonin receptors in cattle and sheep. Depending on when ergovaline exposure occurs, ergovaline can function as an agonist (stimulant) or antagonist (inhibitor) of vascular activity. However, it is unclear how the duration of ergovaline exposure affects vasoconstriction caused by serotonin. Experiments were conducted using the bovine mesenteric artery and mesenteric vein that were exposed to either 0, 0.01, or 0.1 µM ergovaline for 24-h prior to serotonin additions or simultaneously with serotonin additions. Maximum contractile response data were recorded using a multimyograph system and normalized as a percentage of the contractile response produced by the reference compound, KCl. The results of these experiments demonstrated that the duration of ergovaline exposure influences serotonin-mediated vasoconstriction and possibly vasorelaxation in bovine mesenteric vasculature. If ergovaline and serotonin exposure occur simultaneously, ergovaline can act as an agonist or antagonist of serotonin-mediated vasoconstriction. If serotonin exposure occurs after prior ergovaline exposure, serotonin can induce vasorelaxation of blood vessels. Understanding how complex interactions between ergovaline and serotonin occur and affect vascular function will aid in the development of strategies to mitigate sustained vasoconstriction caused during fescue toxicosis.

Influence of Prolonged Serotonin and Ergovaline Pre-Exposure on Vasoconstriction Ex Vivo.

Ergot alkaloid mycotoxins interfere in many functions associated with serotonergic neurotransmitters. Therefore, the objective was to evaluate whether the association of serotonin (5-hydroxytryptamine, 5-HT) and ergot alkaloids during a 24 h pre-incubation could affect the vascular contractile response to ergot alkaloids. To evaluate the effects of 24 h exposure to 5-HT and ergot alkaloids (ergovaline, ERV), two assays were conducted. The first assay determined the half-maximal inhibitory concentration (IC50) following the 24 h pre-exposure period, while the second assay evaluated the effect of IC50 concentrations of 5-HT and ERV either individually or in combination. There was an interaction between previous exposure to 5-HT and ERV. Previous exposure to 5-HT at the IC50 concentration of 7.57 × 10-7 M reduced the contractile response by more than 50% of control, while the exposure to ERV at IC50 dose of 1.57 × 10-10 M tended to decrease (p = 0.081) vessel contractility with a response higher than 50% of control. The 24 h previous exposure to both 5-HT and ERV did not potentiate the inhibitory response of blood vessels in comparison with incubation with each compound alone. These results suggest receptor competition between 5-HT and ERV. More studies are necessary to determine the potential of 5-HT to treat toxicosis caused by ergot alkaloids.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice.

The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Ergot alkaloid exposure during gestation alters: 3. Fetal growth, muscle fiber development, and miRNA transcriptome1.

The objective of this study was to assess how exposure to ergot alkaloids during 2 stages of gestation alters fetal growth, muscle fiber formation, and miRNA expression. Pregnant ewes (n = 36; BW = 83.26 ± 8.14 kg; 4/group; 9 groups) were used in a 2 × 2 factorial arrangement with 2 tall fescue seed treatments [endophyte-infected (E+) vs. endophyte-free (E-)] fed during 2 stages of gestation (MID, days 35 to 85 vs. LATE, days 86 to 133), which created 4 possible treatments (E-/E-, E+/E-, E-/E+, or E+/E+). Ewes were individually fed a total mixed ration containing E+ or E- fescue seed according to treatment assignment. Terminal surgeries were conducted on day 133 of gestation for the collection of fetal measurements and muscle samples. Data were analyzed as a 2 × 2 factorial with fescue treatment, stage of gestation, and 2-way interaction as fixed effects. Fetuses exposed to E+ seed during LATE gestation had reduced (P = 0.0020) fetal BW by 10% compared with E- fetuses; however, fetal body weight did not differ (P = 0.41) with E+ exposure during MID gestation. Fetuses from ewes fed E+ seed during MID and LATE gestation tended to have smaller (P = 0.058) kidney weights compared with E- fetuses. Liver weight was larger (P = 0.0069) in fetuses fed E- during LATE gestation compared with E+. Fetal brain weight did not differ by fescue treatment fed during MID (P = 0.36) or LATE (P = 0.40) gestation. The percentage of brain to empty body weight (EBW) was greater (P = 0.0048) in fetuses from ewes fed E+ fescue seed during LATE gestation, which is indicative of intrauterine growth restriction (IUGR). Primary muscle fiber number was lower (P = 0.0005) in semitendinosus (STN) of fetuses exposed to E+ during MID and/or LATE gestation compared with E-/E-. miRNA sequencing showed differential expression (P < 0.010) of 6 novel miRNAs including bta-miR-652_R+1, mdo-miR-22-3p, bta-miR-1277_R-1, ppy-miR-133a_L+1_1ss5TG, hsa-miR-129-1-3p, and ssc-miR-615 in fetal STN muscle. These miRNA are associated with glucose transport, insulin signaling, intracellular ATP, hypertension, or adipogenesis. This work supports the hypothesis that E+ tall fescue seed fed during late gestation reduces fetal weight and causes asymmetrical growth, which is indicative of IUGR. Changes in primary fiber number and miRNA of STN indicate that exposure to E+ fescue fed during MID and LATE gestation alters fetal muscle development that may affect postnatal muscle growth and meat quality.

Ergot alkaloid exposure during gestation alters. I. Maternal characteristics and placental development of pregnant ewes1.

Tall fescue [Lolium arundinaceum (Scheyreb.) Darbysh] is the primary cool season forage grass in the Southeastern United States. Most tall fescue contains an endophytic fungus (Epichloë coenophiala) that produces ergot alkaloids and upon ingestion induces fescue toxicosis. The objective of this study was to assess how exposure to endophyte-infected (E+; 1.77 mg hd-1 d-1 ergovaline and ergovalinine) or endophyte-free (E-; 0 mg hd-1 d-1 ergovaline and ergovalinine) tall fescue seed fed during 2 stages of gestation (MID, days 35-85/LATE, days 86-133) alters placental development. Thirty-six, fescue naïve Suffolk ewes were randomly assigned to 1 of 4 fescue treatments: E-/E-, E-/E+, E+/E-, or E+/E+. Ewes were individually fed the same amount of E+ or E- seed mixed into total mixed ration during MID and LATE gestation. Terminal surgeries were conducted on day 133 of gestation. Ewes fed E+ fescue seed had elevated (P < 0.001) ergot alkaloid excretion and reduced (P < 0.001) prolactin levels during the periods when fed E+ seed. Ewes switched on day 86 from E- to E+ seed had a 4% reduction (P = 0.005) in DMI during LATE gestation, which translated to a 2% reduction (P = 0.07) in DMI overall. Average daily gain was also reduced (P = 0.049) by 64% for E-/E+ ewes during LATE gestation and tended to be reduced (P = 0.06) by 33% overall. Ewes fed E+ seed during LATE gestation exhibited a 14% and 23% reduction in uterine (P = 0.03) and placentome (P = 0.004) weights, respectively. Caruncle weights were also reduced by 28% (P = 0.003) for E-/E+ ewes compared with E-/E- and E+/E-. Ewes fed E+ seed during both MID and LATE gestation exhibited a 32% reduction in cotyledon (P = 0.01) weights, whereas ewes fed E+ seed only during MID gestation (E+/E-) had improved (P = 0.01) cotyledon weights. The percentage of type A placentomes tended to be greater (P = 0.08) for E+/E+ ewes compared with other treatments. Other placentome types (B, C, or D) did not differ (P > 0.05). Total fetal weight per ewe was reduced (P = 0.01) for ewes fed E+ seed during LATE gestation compared with E-; however, feeding E+ seed during MID gestation did not alter (P = 0.70) total fetal weight per ewe. These results suggest that exposure to ergot alkaloids during LATE (days 86-133) gestation has the greatest impact on placental development by reducing uterine and placentome weights. This, in turn, reduced total fetal weight per ewe by 15% in ewes fed E+ seed during LATE gestation (E-/E+ and E+/E+).

Ergot alkaloid exposure during gestation alters: II. Uterine and umbilical artery vasoactivity1.

Previous research has shown that livestock exposed to ergot alkaloids results in decreased vasoactivity of gastrointestinal and peripheral vasculature. Little is known regarding the effect ergot alkaloid exposure during gestation may have on vasculature supporting the fetus. The objective of this study was to evaluate contractile responses of uterine and umbilical arteries collected from ewes consuming ergot alkaloids during gestation. On day 35 of gestation, 36 Suffolk ewes (78.24 ± 9.5 kg) were assigned to endophyte-infected (E+) or endophyte-free (E-) tall fescue seed treatments that were fed either throughout or switched on day 86 of gestation, creating four seed treatments E+E+, E+E-, E-E+, and E-E-. Ewes were fed E+ tall fescue seed to provide 1.77 mg of total ergovaline ⋅ hd-1 ⋅ d-1 with E- ewes receiving the same quantity of E- seed. Gestation was terminated on day 133, and sections of uterine artery and umbilical cord were surgically collected. Only collections from 28 ewes (n = 7/treatment) were of sufficient viability to proceed with the contractility experiments. Arteries were cleaned, sliced into 2-mm cross sections, and suspended in multi-myograph chambers containing 5 mL of continuously oxygenated Krebs-Henseleit buffer. Vessels were exposed to increasing concentrations (5 × 10-8 to 1 × 10-4 M) of norepinephrine, serotonin, ergotamine, and ergovaline (5 × 10-9 to 1 × 10-5M; extract of tall fescue seed) in 15-min intervals. Increasing concentrations of norepinephrine generated a contractile response by the uterine artery (P < 0.05), but no response in the umbilical artery. Increasing concentrations of serotonin resulted in negligible responses in uterine preparations, whereas umbilical artery preparations were responsive (P < 0.05) to serotonin. Ewes receiving E+E+ and E-E+ treatments had decreased vasoactivity in umbilical arteries to serotonin with a dextral shift in concentrations where the response curve initiated (P < 0.05). Interestingly, uterine arteries were not responsive to exposure to ergotamine or ergovaline, whereas umbilical arteries were responsive (P < 0.05). Umbilical arteries collected from ewes receiving E-E- and E+E- were more vasoactive to ergot alkaloids (P < 0.05) than other treatments. These findings indicate that maternal blood supply to the placenta appears protected from negative effects of ergot alkaloids; however, umbilical vasculature is not, and this could adversely influence fetal growth.

Effects of grazing different ergovaline concentrations on vasoactivity of bovine lateral saphenous vein.

Previous research has demonstrated that exposure to ergot alkaloids reduces vasoactivity of serotonin (5HT) receptors. Chemical suppression of tall fescue seedhead production is a tool to reduce the level of exposure to ergot alkaloids by a grazing animal. Therefore, the objective was to evaluate contractility of lateral saphenous veins biopsied from mixed breed steers following a 87- to 101-d grazing period on 3-ha pastures of bermudagrass (Cynodon dactylon; n = 5 steers; BW = 340 ± 9 kg), or toxic endophyte-infected tall fescue (Lolium arundinaceum) that was not treated (n = 5 steers; BW = 300 ± 6; 0.56 ppm ergovaline) or was treated (n = 5 steers; BW = 294 ± 9 kg; 0.24 ppm ergovaline) with herbicide containing aminopyralid and metsulfuron-methyl. To evaluate contractility, biopsied veins were mounted in a multimyograph and exposed to increasing concentrations of a tall fescue seed extract (EXT; ergovaline source) and 5HT1B (CP 93129), 5HT1D (L-694,247), and 5HT2A (TCB2) agonists. All contractility data were normalized to a maximal response of 1 × 10-4 M norepinephrine and were analyzed as a split plot treatment design using SAS for effects of pasture treatment, agonist concentration, and the interaction. There was no contractile response to any concentration of 5HT1B agonist in any of the pasture treatments. There were pasture × concentration interactions for contractile responses to 5HT2A agonist (P < 0.01) and EXT (P < 0.01). For both EXT and TCB2, veins from bermudagrass steers were more vasoactive to the higher concentrations of these compounds (P < 0.05), and there were no differences between veins collected from the unsuppressed or seedhead-suppressed treatments (P = 0.66). There was also a pasture × concentration interaction for the contractile responses to 5HT1D agonist (P < 0.01). However, these responses were not sigmoidal and reached a zenith at 5 × 10-7 and 1 × 10-6 M. At these concentrations, the response was greatest for veins from the unsuppressed treatment (P < 0.05) and did not differ between veins from suppressed and bermudagrass treatments (P = 0.41). Although reduced levels of ergovaline in seedhead-suppressed pastures did not alter vasoactivity of 5HT2A or 5HT1B receptors in the lateral saphenous vein, elevated vasoactivity of 5HT1D in veins from unsuppressed tall fescue pasture treatment suggests that lower ergovaline levels in seedhead-suppressed pastures can influence the vascular effects of ergot alkaloids.

Toxicity of endophyte-infected ryegrass hay containing high ergovaline level in lactating ewes.

The symbiotic association of var. (formerly named ) with perennial ryegrass () leads to the production of ergovaline (EV) and lolitrem B (LB) that are toxic for livestock. The objectives of this study were to determine the effects of feeding endophyte-infected ryegrass (SE+) hay on 16 lactating ewes (BW 80 ± 10 kg) in comparison with endophyte-free ryegrass (SE-) hay to investigate the putative mechanisms of action of EV and LB and to evaluate their persistence in milk and animal tissues. The mean EV and LB concentrations in SE+ hay were 851 and 884 µg/kg DM, respectively, whereas these alkaloids were below the limit of detection in SE- hay. No effect of SE+ was observed on animal health and skin temperature whereas prolactin decreased and significant differences between hays were observed from d 7 to 28 of the study ( < 0.03) but had no effect on milk production. Hematocrit and biochemical analyses of plasma revealed no significant difference between SE+ and SE-, whereas cortisol concentration differed significantly on d 28 ( = 0.001). Measurement of oxidative damage and antioxidant enzyme activities in plasma, liver, and kidneys revealed a slight increase in some enzyme activities involved in defense against oxidative damage in the SE+ fed ewes. Slight variations in the activities of hepatic and kidney flavin monooxygenase enzymes were observed, whereas in the kidney, glutathione -transferase activity decreased significantly ( = 0.002) in the SE+ fed ewes, whereas uridine diphosphate glucuronosyltransferase activity increased ( = 0.001). After 28 d of exposure of ewes to the SE+ hay, low EV and LB concentrations were measured in tissues. The highest concentration of EV was observed in the liver (0.68 µg/kg) whereas fat contained the highest concentration of LB (2.39 µg/kg). Both toxins were also identified at the trace level in milk.

Ergot alkaloids produced by endophytic fungi of the genus Epichloë.

The development of fungal endophytes of the genus Epichloë in grasses results in the production of different groups of alkaloids, whose mechanism and biological spectrum of toxicity can differ considerably. Ergot alkaloids, when present in endophyte-infected tall fescue, are responsible for "fescue toxicosis" in livestock, whereas indole-diterpene alkaloids, when present in endophyte-infected ryegrass, are responsible for "ryegrass staggers". In contrast, peramine and loline alkaloids are deterrent and/or toxic to insects. Other toxic effects in livestock associated with the consumption of endophyte-infected grass that contain ergot alkaloids include the "sleepy grass" and "drunken horse grass" diseases. Although ergovaline is the main ergopeptine alkaloid produced in endophyte-infected tall fescue and is recognized as responsible for fescue toxicosis, a number of questions still exist concerning the profile of alkaloid production in tall fescue and the worldwide distribution of tall fescue toxicosis. The purpose of this review is to present ergot alkaloids produced in endophyte-infected grass, the factors of variation of their level in plants, and the diseases observed in the mammalian species as relate to the profiles of alkaloid production. In the final section, interactions between ergot alkaloids and drug-metabolizing enzymes are presented as mechanisms that could contribute to toxicity.

The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Effects of supplementing endophyte-infected tall fescue with sainfoin and polyethylene glycol on the physiology and ingestive behavior of sheep.

Tannins in sainfoin (Onobrychis viciifolia) may bind to alkaloids in endophyte-infected tall fescue [E+; Lolium arundinaceum (Schreb.) Darbysh.] and attenuate toxicosis. If so, supplementing E+ with sainfoin will increase use of E+ by sheep, and polyethylene glycol (PEG)-a polymer that selectively binds to tannins-will reduce such response. To test these predictions, thirty-six 2-mo-old lambs were randomly assigned to 3 treatments (12 lambs/treatment). During exposure, all lambs were individually penned and fed E+ supplemented with beet pulp (CTRL), fresh-cut sainfoin and beet pulp (SAIN), or fresh-cut sainfoin plus PEG mixed in beet pulp (SAIN+PEG). Feed intake was measured daily. Rectal temperatures and jugular blood samples were taken at the beginning and end of exposure. After exposure, all lambs were offered choices between endophyte-free tall fescue (E-) and orchardgrass, and preference for E- was assessed. Then, all lambs were allowed to graze a choice of E+ and sainfoin or a monoculture of E+. The foraging behavior of lambs was recorded. When sainfoin was in mid-vegetative stage, lambs in SAIN ingested more E+ than lambs in CTRL (P = 0.05), but no differences were detected between lambs in SAIN+PEG and CTRL (P = 0.12). Sainfoin supplementation improved some physiological parameters indicative of fescue toxicosis. Lambs in SAIN had lower rectal temperatures (P = 0.02), greater numbers of leukocytes (P < 0.001) and lymphocytes (P = 0.03), and greater plasmatic concentrations of globulin (P = 0.009) and prolactin (P = 0.019) than lambs in CTRL. Some of these differences were offset by the SAIN+PEG treatment. When lambs were offered choices between E- and orchardgrass, only lambs in SAIN had greater intake of E- than lambs in CTRL (P < 0.001). When lambs were allowed to graze a choice of E+ and sainfoin, all treatments used E+ to the same extent (P > 0.05). On the other hand, when they grazed on a monoculture of E+, lambs in SAIN+PEG showed greater acceptance of E+ than lambs in SAIN or in CTRL (P < 0.05). In summary, sainfoin supplementation alleviated several of the classic signs of fescue toxicosis and increased intake of endophyte-infected tall fescue. Tannins in sainfoin partially accounted for this benefit since feeding a polymer that selectively binds to tannins (PEG) attenuated some these responses. However, sainfoin supplementation during initial exposure to E+ did not lead to an increased preference for E+ during grazing.

Alteration of fasting heat production during fescue toxicosis in Holstein steers.

This study was designed to examine alteration of fasting heat production (FHP) during fescue toxicosis. Six ruminally cannulated Holstein steers (BW = 348 ± 13 kg) were BW-matched into pairs and used in a 2 period crossover design experiment. Each period consisted of 2 temperature segments, one each at 22 and 30°C. During each period, 1 steer per pair was ruminally dosed twice daily with 0.5 kg of ground endophyte-infected fescue seed (E+) and the other with ground endophyte-free fescue seed (E-) for 7 d. Steers on E- treatment were pair-fed to E+ steers offered alfalfa cubes at 1.5 × NEm. On d 8 of each segment, steers were moved to individual metabolism stalls fitted with indirect calorimetry head boxes. Ruminal contents were removed, weighed, and subsampled for DM determinations. The reticulorumen was washed and filled with a buffer (NaCl = 96; NaHCO3 = 24; KHCO3 = 30; K2HPO4 = 2; CaCl2 = 1.5; MgCl2 = 1.5 mmol·kg buffer(-1)) that was gassed with a 75% N2 and 25% CO2 mixture before rumen incubation. During buffer incubation, an E+ or E- fescue seed extract was added at 12 h intervals to maintain treatment presentation to the animal. After a 12-h wait, heart rate, O2 consumption, CO2 production, and urinary output were recorded for 16 h. There was no difference (P = 0.931) in DMI/kg(0.75) between endophyte treatments by design; however, intake decreased (P = 0.004) at 30°C. Increased temperature had no effect (P > 0.10) on other measurements and there were no significant interactions (P > 0.11) of temperature and endophyte treatment. Heart rate was unaffected by fescue treatment or environmental temperature. Percent DM of ruminal contents as well as total rumen DM/kg(0.75) was increased (P < 0.0001) in E+ steers. Respiratory quotient was elevated (P = 0.02) in E+ steers. Oxygen consumption decreased (P = 0.04) and CO2 production tended to be reduced (P = 0.07) during E+ treatment. Calculated FHP (kcal/kg BW(0.75)) was also less (P = 0.006) in steers receiving E+ treatment. These data suggest that consumption of endophyte-infected tall fescue by cattle results in a reduction in basal metabolic rate.

Antagonism of lateral saphenous vein serotonin receptors from steers grazing endophyte-free, wild-type, or novel endophyte-infected tall fescue.

Pharmacologic profiling of serotonin (5HT) receptors of bovine lateral saphenous vein has shown that cattle grazing endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum) have altered responses to ergovaline, 5HT, 5HT2A, and 5HT7 agonists. To determine if 5HT receptor activity of tall fescue alkaloids is affected by grazing endophyte-free (EF), wild-type [Kentucky-31 (KY31)], novel endophyte AR542-infected (MAXQ), or novel endophyte AR584-infected (AR584) tall fescue, contractile responses of lateral saphenous veins biopsied from cattle grazing these different fescue-endophyte combinations were evaluated in presence or absence of antagonists for 5HT2A (ketanserin) or 5HT7 (SB-269970) receptors. Biopsies were conducted over 2 yr on 35 mixed-breed steers (361.5 ± 6.3 kg) grazing EF (n = 12), KY31 (n = 12), MAXQ (n = 6), or AR584 (n = 5) pasture treatments (3 ha) between 84 and 98 d (Yr 1) or 108 to 124 d (Yr 2). Segments (2 to 3 cm) of vein were surgically biopsied, sliced into 2- to 3-mm cross-sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O2/5% CO2; pH = 7.4; 37°C). Veins were exposed to increasing concentrations of 5HT, ergovaline, and ergovaline + 1 × 10(-5) M ketanserin or + 1 × 10(-6) M SB-269970 in Yr 1. In Yr 2, ergotamine and ergocornine were evaluated in presence or absence of 1 × 10(-5) M ketanserin. Contractile response data were normalized to a reference addition of 1 × 10(-4) M norepinephrine. In Yr 1, contractile response to 5HT and ergovaline were least (P < 0.05) in KY31 pastures and the presence of ketanserin greatly reduced (P < 0.05) the response to ergovaline in all pastures. However, presence of SB-269970 did not (P = 0.91) alter contractile response to ergovaline. In Yr 2, there was no difference in contractile response to ergotamine (P = 0.13) or ergocornine (P = 0.99) across pasture treatments, but ketanserin reduced (P < 0.05) the contractile response to both alkaloids. The 5HT2A receptor is involved in alkaloid-induced vascular contraction and alkaloid binding may be affected by exposure to different endophyte-fescue combinations.

Preference for tannin-containing supplements by sheep consuming endophyte-infected tall fescue hay.

Tannins may bind to alkaloids in endophyte-infected (E+) tall fescue and attenuate fescue toxicosis. To test this hypothesis, thirty-two 4-mo-old lambs were randomly assigned to 4 treatments (8 lambs/treatment) in a 2 by 2 factorial design that included a basal diet of tall fescue hay [E+ or endophyte-free (E-)] supplemented with (TS) or without (CS) bioactive Quebracho tannins. The concentration of ergovaline in E+ fed in 2 successive phases was 65 ± 21 µg/kg (Phase 1) and 128 ± 4 µg/kg (Phase 2). After exposure to hays and supplements, all lambs were offered choices between TS and CS and between E+ and E- hays. During Phase 1, lambs offered E+ consumed more hay than lambs offered E- (P = 0.03). Lambs on E+/TS displayed the greatest intake of hay and the least intake of TS (P < 0.05). During Phase 2, when the concentration of ergovaline increased, lambs offered E+ consumed less hay than lambs fed E- (P < 0.0001). Lambs on E+/CS consumed less hay than lambs on E-/CS (P = 0.02), but hay intake by lambs on E-/TS and E+/TS did not differ (P = 0.96). Lambs preferred CS to TS during preference tests (P < 0.0001) and lambs on E+/TS ingested the least amounts of supplement TS and the greatest amounts of supplement CS (P = 0.001). Lambs offered E+ displayed greater body temperatures than lambs offered E- in both phases (P < 0.05). When offered a choice among the 3 hays, lambs previously exposed to E+ preferred E+ (low content of ergovaline) > E- > E+ (greater content of ergovaline; P < 0.001). Thus, decreased concentrations of ergovaline increased rectal temperatures, and affected intake of and preference for tannins and fescue hay. Quebracho tannins did not attenuate the effects of E+ on body temperature and feed intake. Ingestion of E+ reduced intake of quebracho tannins, suggesting that alkaloids in E+ antagonized ingestion of condensed tannins.

Clinical expression of lolitrem B (perennial ryegrass) intoxication in horses.

REASONS FOR PERFORMING STUDY: Perennial ryegrass staggers is purported to be a common neurological mycotoxicosis of horses but the case description lacks detail and evidence. OBJECTIVE: To describe the clinical syndrome of lolitrem B intoxication in horses, limiting tests to those that are applicable to clinical practice, and to assess the potential value of enzyme-linked immunosorbent assay (ELISA) tests for lolitrem B in horse body fluids. METHODS: Seven horses in 2 separate groups were fed perennial ryegrass seed and hay containing 2 ppm lolitrem B. Paired data were collected prior to and after 2 weeks exposure to lolitrem B, including video-documented neurological examination and clinical examination. RESULTS: All horses developed a variable degree of tremor and ataxia when exposed to lolitrem B. Tremor depended on the level of activity and included a subtle, rapid tremor of the eyeball. Ataxia was exaggerated by blindfolding and primarily involved a truncal sway and irregular, but predictable, limb placements. No change was detected in urine lolitrem B levels and, although plasma lolitrem B increased during the treatment period, levels did not correlate with the severity of clinical signs displayed. Limb swelling, heel lesions and serous nasal discharge were also observed in horses most severely intoxicated. CONCLUSIONS: The clinical effects of lolitrem B intoxication in horses primarily involve action-related tremors and symmetrical vestibular ataxia. Ergovaline may have caused the limb swelling, heel lesions and serous nasal discharge. Plasma ELISA for lolitrem B may be of diagnostic use in the future. POTENTIAL RELEVANCE: This study provides a clearer appreciation of the clinical signs and variability of perennial ryegrass intoxication in horses.

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.

Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines.

l-Glutamate (Glu) is a major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50mM) Glu [Michaelis constant (measuring affinity), or K(m),=1 to 4mM] into synaptic vesicles (SV) for subsequent release into the synaptic cleft of glutamatergic neurons. Vesicular Glu transporter activity is dependent on vacuolar H(+)-ATPase function. Previous research has shown that ergopeptines contained in endophyte-infected tall fescue interact with dopaminergic and serotoninergic receptors, thereby affecting physiology regulated by these neuron types. To test the hypothesis that ergopeptine alkaloids inhibit VGLUT activity of bovine cerebral SV, SV were isolated from cerebral tissue of Angus-cross steers that were naive to ergot alkaloids. Immunoblot analysis validated the enrichment of VGLUT1, VGLUT2, synaptophysin 1, and vacuolar H(+)-ATPase in purified SV. Glutamate uptake assays demonstrated the dependence of SV VGLUT-like activity on the presence of ATP, H(+)-gradients, and H(+)-ATPase function. The effect of ergopeptines on VGLUT activity was evaluated by ANOVA. Inhibitory competition (IC(50)) experiments revealed that VGLUT-mediated Glu uptake (n=9) was inhibited by ergopeptine alkaloids: bromocriptine (2.83±0.59µM)

Hemodynamics are altered in the caudal artery of beef heifers fed different ergot alkaloid concentrations.

Doppler ultrasonography was used to compare blood flow characteristics in the caudal artery of heifers fed diets with endophyte (Neotyphodium coenophialum) noninfected (E-, 0 microg of ergovaline/g of DM), a 1:1 mixture of endophyte-infected and E- (E+E-; 0.39 microg of ergovaline/g of DM), or endophyte-infected (E+, 0.79 microg of ergovaline/g of DM) tall fescue (Lolium arundinaceum) seed. Eighteen crossbred (Angus x Brangus) heifers [345 +/- 19 kg (SD)] were assigned to individual pens and fed chopped alfalfa hay plus a concentrate that contained E- tall fescue seed for 7 d during an adjustment period. A 9-d experimental period followed with feeding treatments of chopped alfalfa hay plus a concentrate with E+, E-, or E+E- seed being assigned randomly to pens. Doppler ultrasound measurements (caudal artery luminal area, peak systolic velocity, end diastolic velocity, mean velocity, heart rate, and flow rate) and serum prolactin were monitored during the adjustment (3 baseline measures) and during the experimental period (7 measures). Statistical analyses compared proportionate differences between baseline and responses at 3, 27, 51, 75, 171, and 195 h from initial feeding of the experimental diets. Serum prolactin concentrations for E+ and E+E- diets were less (P < 0.001) than baseline concentrations beginning at 27 and 51 h, respectively, from initial feeding of the diets. Although baseline measures were taken when ambient temperatures were likely below thermoneutrality, caudal artery luminal cross-sectional area in E+ heifers had declined (P = 0.004) from baseline by 27 h and remained less (P < 0.02) until 195 h, and caudal artery luminal area declined (P = 0.004) in E+E- heifers from baseline by 51 h and remained less (P < 0.07) until 171 h. Blood flow rate was slower than the baseline rate at 51 h for E+ (P = 0.058) and E+E- (P = 0.02 heifers, but blood flow remained slower in E+E- heifers for 48 h, whereas it remained slower in E+ heifers for 96 h. Adjustments in artery luminal area and blood rate with the 3 diets appeared to parallel the increases in ambient temperature. Heifers fed a diet containing a larger amount of ergot alkaloids had less of a response to ambient temperature than heifers consuming the diet with less or no ergot alkaloids.

Effect of ergot alkaloids associated with fescue toxicosis on hepatic cytochrome P450 and antioxidant proteins.

Intake of ergot alkaloids found in endophyte-infected tall fescue grass is associated with decreased feed intake and reduction in body weight gain. The liver is one of the target organs of fescue toxicosis with upregulation of genes involved in xenobiotic metabolism and downregulation of genes associated with antioxidant pathways. It was hypothesized that short-term exposure of rats to ergot alkaloids would change hepatic cytochrome P450 (CYP) and antioxidant expression, as well as reduce antioxidant enzyme activity and hepatocellular proliferation rates. Hepatic gene expression of various CYPs, selected nuclear receptors associated with the CYP induction, and antioxidant enzymes were measured using real-time PCR. Hepatic expression of CYP, antioxidant and proliferating cell nuclear antigen (PCNA) proteins were measured using Western blots. The CYP3A1 protein expression was evaluated using primary rat hepatocellular cultures treated with ergovaline, one of the major ergot alkaloids produced by fescue endophyte, in order to assess the direct role of ergot alkaloids in CYP induction. The enzyme activities of selected antioxidants were assayed spectrophotometrically. While hepatic CYP and nuclear receptor expression were increased in ergot alkaloid-exposed rats, the expression and activity of antioxidant enzymes were reduced. This could potentially lead to increased oxidative stress, which might be responsible for the decrease in hepatocellular proliferation after ergot alkaloid exposure. This study demonstrated that even short-term exposure to ergot alkaloids can potentially induce hepatic oxidative stress which can contribute to the pathogenesis of fescue toxicosis.