Semiquantitative determination of ergot alkaloids in seed, straw, and digesta samples using a competitive enzyme-linked immunosorbent assay.

Ergot alkaloids present in endophyte-infected (E+) tall fescue cause fescue toxicosis and other toxic effects in livestock that consume infected plant tissue, leading to significant financial losses in livestock production each year. The predominant method currently in use for quantifying ergot alkaloid content in plant tissue is through high-performance liquid chromatography (HPLC), which quantifies the amount of ergovaline, one of many ergot alkaloids in E+ plant tissue. The enzyme-linked immunosorbent assay (ELISA) method used in this study detects quantities of nonspecific ergot alkaloids and therefore accounts for greater amounts of the total ergot alkaloid content in E+ tissue than does HPLC. The ELISA can also be used to more expediently analyze a larger number of forage samples without sophisticated and costly analytical equipment and therefore could be more desirable in a diagnostic setting. The purpose of this study was to evaluate the between-day and within-run variability of the ELISA and to determine the binding efficiency of 6 ergot alkaloids to the 15F3.E5 antibody used in the competitive ELISA to ascertain its feasibility as a quick analysis tool for ergot alkaloids. Straw samples had an average coefficient of variation (CV) for concentration of 10.2% within runs and 18.4% between runs, and the seed samples had an average CV for concentration of 13.3% within runs and 24.5% between runs. The grass tissue-based lysergic acid standard curve calculated from the ELISA had an average r2 of 0.99, with a CV of 2.1%. Ergocryptine, ergocristine, ergocornine, and ergotamine tartrate did not bind strongly to the 15F3.E5 antibody because of the presence of large side groups on these molecules, which block their binding to the antibody, whereas ergonovine and ergonovine maleate were bound much more efficiently because of their structural similarity to lysergic acid. Clarified rumen fluid was tested as an additional matrix for use in the ergot alkaloid competitive ELISA to determine whether future livestock metabolism experiments on the postingestion fate of ergot alkaloids in ruminants could utilize this assay as a quick screening tool for the presence of nonspecific ergot alkaloids in rumen fluid. HPLC and ELISA procedures were compared for their ability in determining ergot alkaloid toxicity based on the repeatability of the procedures and on the specific compounds they measure. The ratio of ELISA concentration to HPLC concentration (ergovaline) varied from 2.00 to 2.81 in seed samples and from 0.62 to 8.66 in straw samples, showing no consistent pattern between the 2 methods. Based on the lack of data at present for the identity of the toxin causing endophyte toxicosis and the lack of agreement between the ergovaline HPLC and ELISA analyses for ergot alkaloids, each method is equally valid as an indicator of toxicityand is the best means for determining the quantity of the specific toxin(s) they measure.     

Effect of ergot alkaloids on contractility of bovine right ruminal artery and vein

Ergot alkaloids produced by the endophyte (Neotyphodium coenophialum) associated with tall fescue (Lolium arundinaceum) are implicated in the clinical signs of fescue toxicosis. These compounds were hypothesized to correspondingly affect foregut vasculature. The objective of this study was to determine vasoconstrictive potentials of ergovaline, ergotamine, ergocryptine, ergocristine, ergonovine, ergocornine, and lysergic acid on right ruminal artery and vein. Segments of right ruminal artery and vein were collected from the ventral coronary groove of predominantly Angus heifers (n = 10) shortly after slaughter and placed in a modified Krebs-Henseleit buffer on ice. Vessels were cleaned of excess connective tissue and fat, sliced into 2- to 3-mm segments, and suspended in a multi-myograph chamber with 5 mL of continuously oxygenated Krebs-Henseleit buffer (95%O(2)/5% CO(2); pH 7.4; 37°C). Arteries and veins were equilibrated to 1.0 and 0.5 g, respectively, for 90 min followed by the reference addition of 120 mM KCl. Increasing concentrations of each alkaloid were added to the respective chamber every 15 min after buffer replacement. Data were normalized as a percentage of the contractile response induced by KCl. Alkaloid (P < 0.0001), concentration (P < 0.0001), and vessel type (artery or vein; P = 0.004) affected contractility. No arterial response was observed until 10(-6) M for ergovaline and ergotamine; 10(-5) M for ergocryptine, ergocornine, and ergonovine; and 10(-4) M for ergocristine. Lysergic acid did not induce a contractile response in the ruminal artery. No venous contractile response was observed until concentrations of 10(-6) M for ergovaline, 10(-5) M for ergotamine, and 10(-4) M for ergocryptine and ergocristine were achieved. Lysergic acid, ergonovine, and ergocornine did not induce a contractile response in the ruminal vein. A greater arterial maximal response was observed for ergovaline (P < 0.0001), whereas the arterial and venous responses were not different for ergotamine (P = 0.16), ergocryptine (P = 0.218), and ergocristine (P = 0.425). These results indicate that ergot alkaloids associated with toxic endophyte-infected tall fescue are vasoactive and can potentially alter arterial blood supply and venous drainage from the bovine foregut.     

Ergocryptine and other ergot alkaloids stimulate the release of [3H]dopamine from rat striatal synaptosomes.

Ergocryptine is an ergot alkaloid that affects dopaminergic activity principally by interacting with D2-type receptors. In this study the ability of ergocryptine and several other ergot alkaloids to release [3H]dopamine from isolated nerve endings was demonstrated using in vitro superfusion of rat striatal synaptosomes. Ergocryptine, ergocristine, and bromocryptine produced an elevation in baseline dopamine release of approximately 400% with effective concentrations (EC50) of approximately 30 microM. Ergotamine, ergonovine, ergovaline, and ergocornine were devoid of activity. The time-course of the ergocryptine-stimulated release was relatively slow compared with amphetamine, nicotine, or K+-stimulated [3H]dopamine release; the maximal increase in release required a 5-min treatment. A number of receptor antagonists were examined for their ability to block ergocryptine-stimulated release. Of the dopaminergic, adrenergic, serotonergic, GABA-ergic, and cholinergic antagonists examined, only phentolamine produced a moderate attenuation in evoked release. Omission of Ca++ from the medium did not affect ergocryptine-evoked release. Following ergocryptine treatment, the synaptosomes were fully responsive to other stimulant. The results indicate that, in addition to interacting with dopamine receptors, several ergot alkaloids may produce dopaminergic effects by increasing the release of dopamine from central nerve endings. Several mechanisms to account for the evoked neurotransmitter release are discussed.     

Effects of selected combinations of tall fescue alkaloids on the vasoconstrictive capacity of fescue-naive bovine lateral saphenous veins

Vasoconstriction is a response associated with consumption of toxic endophyte-infected tall fescue. It is not known if endophyte-produced alkaloids act alone or collectively in mediating the response. Therefore, the objective of this study was to examine the vasoconstrictive potentials of selected ergot alkaloids, individually or in paired combinations, using bovine lateral saphenous veins biopsied from fescue-na?ve cattle. Segments (2 to 3 cm) of vein were surgically biopsied from healthy crossbred yearling heifers (n = 22; 330 +/- 8 kg of BW). Veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O(2)/5% CO(2); pH = 7.4; 37 degrees C). Increasing doses of ergovaline, lysergic acid, and N-acetylloline individually or in combination were evaluated. Contractile data were normalized as a percentage of the contractile response induced by a reference dose of norepinephrine (1 x 10(- 4) M). Increasing concentrations of lysergic acid did not result in an appreciable contractile response until the addition of 1 x 10(- 4) M lysergic acid. In contrast, the vascular response to increasing concentrations of ergovaline was apparent at 1 x 10(- 8) M and increased to a maximum of 104.2 +/- 6.0% with the addition of 1 x 10(- 4) M ergovaline. The presence of N-acetylloline did not alter the onset or magnitude of vascular response to either lysergic acid or ergovaline. The presence of 1 x 10(- 5) M lysergic acid with increasing concentrations of N-acetylloline and ergovaline generated an increased contractile response during the initial additions compared with the responses of N-acetylloline and ergovaline alone. In the presence of 1 x 10(- 7) M ergovaline, the contractile response increased with increasing concentrations of N-acetylloline and lysergic acid. Neither N-acetylloline nor lysergic acid elicited an intense contractile response individually (maximum contractile responses of 1.9 +/- 0.3% and 22.6 +/- 4.1%, respectively), suggesting that this was the result of the repetitive addition of 1 x 10(- 7) M ergovaline. These data indicate that ergovaline is a more potent vascular toxicant than lysergic acid or N-acetylloline. The contractile responses of the ergovaline and lysergic acid combinations appeared to differ from the individual dose responses. These data support the possibility that an additive alkaloid exposure effect may exist and should be considered during evaluations of ergot alkaloids.     

麦角生物碱在醉马草内生真菌共生体中的空间分布

本研究检测了2007年9月采自新疆哈密(HM)、青海铁卜加(TBJ)、甘肃肃南(SN)、甘肃天祝(TZ)、甘肃夏河(XH)5个不同地理种群的醉马草内生真菌共生体的麦角生物碱含量,结果表明,醉马草种群内不同器官中麦角新碱及麦角酰胺含量均不同,且麦角新碱的含量均显著高于麦角酰胺的含量(P<0.05)。5个种群间麦角酰胺、麦角新碱及总生物碱的含量除叶鞘外,均以HM最高,分别为53.656,117.784和160.816mg/kg干重。相关性分析表明,植株叶片、茎秆和果穗麦角酰胺、麦角新碱及总生物碱的含量均与种群生长的年均温度呈正相关关系。     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

Ergot alkaloid transport across ruminant gastric tissues

Ergot alkaloids cause fescue toxicosis when livestock graze endophyte-infected tall fescue. It is generally accepted that ergovaline is the toxic component of endophyte-infected tall fescue, but there is no direct evidence to support this hypothesis. The objective of this study was to examine relative and potential transport of ergoline and ergopeptine alkaloids across isolated gastric tissues in vitro. Sheep ruminal and omasal tissues were surgically removed and placed in parabiotic chambers. Equimolar concentrations of lysergic acid, lysergol, ergonovine, ergotamine, and ergocryptine were added to a Kreb's Ringer phosphate (KRP) solution on the mucosal side of the tissue. Tissue was incubated in near-physiological conditions for 240 min. Samples were taken from KRP on the serosal side of the chambers at times 0, 30, 60, 120, 180, and 240 min and analyzed for ergot alkaloids by competitive ELISA. The serosal KRP remaining after incubation was freeze-dried and the alkaloid species quantified by HPLC. The area of ruminal and omasal tissues was measured and the potential transportable alkaloids calculated by multiplying the moles of transported alkaloids per square centimeter of each tissue type by the surface area of the tissue. Studies were conducted to compare alkaloid transport in reticular, ruminal, and omasal tissues and to determine whether transport was active or passive. Ruminal tissue had greater ergot alkaloid transport potential than omasal tissue (85 vs 60 mmol) because of a larger surface area. The ruminal posterior dorsal sac had the greatest potential for alkaloid transport, but the other ruminal tissues were not different from one another. Alkaloid transport was less among reticular tissues than among ruminal tissues. Transport of alkaloids seemed to be an active process. The alkaloids with greatest transport potential were lysergic acid and lysergol. Ergopeptine alkaloids tended to pass across omasal tissues in greater quantities than across ruminal tissues, but their transport was minimal compared to lysergic acid and lysergol.     

Cardiovascular, respiratory, and body temperature responses of sheep to the ergopeptides ergotamine and ergovaline

OBJECTIVE: To compare the effects of the ergot alkaloid ergovaline with effects of ergotamine on blood pressure, heart rate, respiratory rate, and body temperature in conscious sheep. ANIMALS: 3 sheep with indwelling arterial catheters. PROCEDURE: Ergotamine and ergovaline were injected IV (20 nmol/kg), and their effects on arterial blood pressure, heart rate, respiratory rate and pattern, body temperature, and skeletal muscle electromyographic activity were compared with control values obtained following injections of saline (0.9% NaCI) solution or acetone. RESULTS: Both ergopeptides caused immediate and significant increases in blood pressure (50 to 75 mm Hg) without concomitant increases in heart rate. Ergovaline but not ergotamine significantly increased pulse pressure (35 mm Hg). Both ergopeptides resulted in decreased respiratory rate and increased respiratory depth within the first hour of administration. Body temperature was decreased slightly upon ergopeptide administration but continued to increase thereafter, with greater increases developing with ergovaline than with ergotamine. Increased body temperatures of 3.0 to 3.5 C were maintained for at least 10 hours. Respiratory rate was increased to rates as high as 210 to 220 breaths/min in association with hyperthermia. Ergopeptides had no effect on skeletal muscle electromyographic activity. CONCLUSIONS AND CLINICAL RELEVANCE: In sheep, ergovaline has similar effects to ergotamine on cardiovascular and pulmonary function and body temperature but is more potent. These effects are consistent with clinical signs observed in the toxicoses developed when ruminants ingest grass with high concentrations of ergopeptides.     

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.     

Complex interaction of ergovaline with 5-HT2A, 5-HT1B/1D, and alpha1 receptors in isolated arteries of rat and guinea pig

Vascular effects of ergovaline mediated by 5-hydroxytryptamine(HT)2A, 5-HT1B/1D, and alpha1 receptors were studied in isolated arterial preparations of rat and guinea pig. In rat tail artery ergovaline behaved as a potent contractile partial agonist showing an agonist potency (pEC50) of 8.86 +/- 0.03, a maximum response (Emax) of 59 +/- 2% with respect to 5-HT, and a partial agonist affinity (pK(P)) of 8.51 +/- 0.06. Ergovaline was equipotent with ergotamine (pEC50, 8.69 +/- 0.07; Emax, 52 +/- 4%; pK(P), 8.36 +/- 0.11). Contractile responses to ergovaline and ergotamine were surmountably antagonized by the 5-HT2A receptor antagonist ketanserin (3 nM). Antagonist affinity (apparent pA2) for ketanserin against ergovaline and ergotamine was 9.19 +/- 0.08 and 9.36 +/- 0.17, respectively. Ergovaline showed extremely slow on-set and off-set kinetics in rat tail artery. The construction of cumulative concentration-response curves required about 4 h, and the contractile response to ergovaline (30 nM), which completely abolished the subsequent contractile response to 5-HT (10 nM to 1 mM), could not be reversed by wash-out. In guinea pig iliac artery moderately precontracted with prostaglandin F2alpha (0.05 to 0.5 microM) ergovaline behaved as an agonist (pEC50, 7.71 +/- 0.10) with a potency similar to that of 5-HT (pEC50, 7.60 +/- 0.05). The contractile response to ergovaline was inhibited by the 5-HT1B/1D receptor antagonist GR127935 (10 nM). The apparent pA2 value for GR127935 against ergovaline was 8.90 +/- 0.12. Ergovaline (10 nM) produced no contractile response in guinea pig iliac artery when added before the PGF2alpha-induced precontraction but caused insurmountable blockade of the contractile response to the 5-HT1B/1D receptor agonist 5-carboxamidotryptamine (5-CT). The apparent pA2 value for ergovaline against 5-CT was 8.56 +/- 0.18. In rat thoracic aorta ergovaline (2 microM) activated alpha1 adrenoceptors only with low efficacy (Emax, 12 +/- 3%) but surmountably antagonized norepinephrine-induced contractions with a pK(P) of 7.07 +/- 0.12. It is concluded that the powerful constrictor effect of ergovaline mediated by activation of vascular 5-HT2A and 5-HT1B/1D receptors may explain the vascular symptoms of fescue toxicosis observed in livestock grazing tall fescue pastures infected with the endophytic fungus Neotyphodium coenophialum.     

Effects of ergotamine and ergovaline on the electromyographic activity of smooth muscle of the reticulum and rumen of sheep

OBJECTIVE: To investigate the effects of IV administration of ergotamine and ergovaline and intraruminal administration of ergotamine on electromyographic (EMG) activity of reticuloruminal smooth muscle in conscious sheep. ANIMALS: 3 sheep with indwelling electrodes in the musculature of the reticulum and rumen. PROCEDURE: In a crossover design study, reticuloruminal motility before and after IV administration of ergotamine (5, 10, 20, and 40 nmol/kg) or ergovaline (2.5, 5, and 10 nmol/kg) was evaluated; EMG effects were compared with those of corresponding control treatments (IV administration of saline [0.9% NaCl] solution or acetone, respectively) in sheep. Ergotamine (800 nmol/kg) or water was also administered intraruminally and their effects compared. RESULTS: After IV administration of ergopeptides, vagally dependent cyclical A and B sequences of contraction of the reticulorumen were immediately inhibited, preceding increases in baseline EMG activity (tonus). The return of cyclical contractions was associated with an increase in contraction amplitude. The effects were dose dependent; administration of 40 nmol of ergotamine/kg resulted in responses that continued for 3 to 4 hours. The effects of intraruminal administration of ergotamine were variable; after 8 hours, EMG activity was increased from baseline for < 2 hours in 1 sheep, 10 hours in another, and > 15 hours in the third. CONCLUSIONS AND CLINICAL RELEVANCE: In sheep, the effects of ergotamine and ergovaline on reticuloruminal motility after IV administration and the duration of responses following intraruminal administration suggest that disruption of digestion may occur in animals grazing endophyte-infected pasture that has a high ergopeptide content.     

Technical note: epimerization of ergopeptine alkaloids in organic and aqueous solvents

Purified ergopeptine alkaloids are often used in studies related to tall fescue toxicosis without regard to epimerization that occurs when ergopeptines are solvated. The objectives of this study were to measure the rates of alpha-ergocryptine epimerization to alpha-ergocryptinine at room temperature and at -40 degrees C, and to measure the rate of ergovaline epimerization to ergovalinine at 37 degrees C. Alpha-ergocryptine tartrate was stable (< 0.5% epimerization) in protic or aprotic solvents when stored at -40 degrees C for 20 to 52 d. At room temperature, alpha-ergocryptine epimerization in chloroform did not occur; epimerization was modest in acetone and acetonitrile (< 5%) but was substantial in methanol (78% by 38 d) and in a 70:30 water methanol mix (47% by 42 d). Ergovaline epimerization to ergovalinine occurred at 37 degrees C in 0.1 M phosphate buffers (pH 3, 7.5, and 9) in 9% aqueous solutions of fetal bovine serum (FBS), and in water, methanol, and acetonitrile. The degree of epimerization at 37 degrees C was solvent-dependent. Epimerization rates with respect to time were roughly linear in phosphate buffer (pH 3 only), water, methanol, and acetonitrile; epimerization rates resembled first-order kinetics in phosphate buffers (pH 7.5 and 9) and in the presence of FBS (pH 3, 7.5 and in Dulbecco's culture media). Epimerization equilibria (48 to 63% ergovaline) were reached within approximately 1 to 19 h. Results from this study indicate that researchers conducting studies with purified ergopeptines should carefully control the storage conditions of solvated ergopeptines and measure isomeric composition under the actual experimental conditions used in experiments.     

色氨酸对醉马草内生真菌共生体麦角生物碱含量的影响

本研究以被甘肃内生真菌(Neotyphodium gansuense)侵染的醉马草(Achnatherum inebrians)为材料,在温室条件下,研究了不同浓度(0、250、500、750 mg·L-1)色氨酸对醉马草幼苗麦角酰胺和麦角新碱两类麦角生物碱含量的影响。结果表明,不同浓度的色氨酸均能显著降低醉马草幼苗的鲜质量、株高、分蘖数(P0.05),显著增加植株中麦角酰胺和麦角新碱含量(P0.05)。     

Ergovaline-induced vasoconstriction in an isolated bovine lateral saphenous vein bioassay

Ergovaline has been proposed as a toxic component of endophyte-infected tall fescue. As many of the symptoms of fescue toxicosis are a result of compromised circulation, the objective of this study was to examine the vasoconstrictive potentials of ergovaline and a more documented ergopeptine, ergotamine, using a bovine, lateral (cranial branch) saphenous vein bioassay. Segments of the cranial branch of the lateral saphenous vein (2 to 3 cm) were collected from healthy, mixed breed cattle (n = 12 and n = 5 for the ergovaline and ergotamine experiments, respectively) at local abattoirs. The veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm cross sections, and suspended in a myograph chamber containing 5 mL of a modified Krebs-Henseleit, oxygenated buffer (95% O2 + 5% CO2; pH = 7.4; 37 degrees C). The tissue was allowed to equilibrate at 1 g of tension for 90 min before of the addition of treatments. Increasing doses of ergovaline (1x10(-11) to 1 x10(-4) M) or ergotamine (1 x10(-11) to 1 x 10(-5) M) were administered every 15 min after buffer replacement. Contractile response data were normalized to a percentage induced by a reference dose of norepinephrine (1 x10(-4) M). Contractile responses of saphenous veins were similar for ergovaline and ergotamine. Initial contractile responses began at 1 x10(-8) M for both ergovaline and ergotamine (4.4 +/- 0.8% and 5.6 +/-1.1%, respectively). Vascular tension continued to increase as the alkaloid concentrations increased (maximums: 43.7 +/-7.1% at 1 x10(-5) M ergotamine; 69.6 +/- 5.3% at 1 x10(-4) M ergovaline). Interestingly, ergovaline-induced contractions (1 x10(-4) M) were not reversed by repeated buffer replacement over a 105-min period. As previously shown with ergotamine, these results confirm that ergovaline is a potent vasoconstrictor. The resistance of an ergovaline-induced contraction to relaxation over an extended period of time suggests a potential for bioaccumulation of this ergopeptine alkaloid and may aid in understanding its toxicity within the animal.     

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.     

披碱草内生真菌共生体中麦角生物碱的组织分布与季节动态

采用高效液相色谱(HPLC)检测了2009年4,6,8和10月采集的雾灵山、沁源、五台山和百望山等4个侵染了Neotyphodium属内生真菌的披碱草种群的叶片、茎、叶鞘和根以及10月采集的种子中2种麦角生物碱(麦角酰胺和麦角新碱)的含量。结果表明,在4,6和8月,4个披碱草种群的麦角酰胺主要分布在叶片(5.037~ 30.037 mg/kg)和叶鞘(6.259~22.255 mg/kg),10月,麦角酰胺主要集中在种子(5.045~8.971 mg/kg)和叶片(3.119~6.658 mg/kg);各种群的叶片、茎秆和叶鞘中麦角酰胺在4或6月含量(13.831~30.037 mg/kg)最高,8或10月含量(3.119~6.658 mg/kg)显著减小(P<0.05),根中8月麦角酰胺(0.552~0.576 mg/kg)显著低于其他月份(P<0.05)。麦角新碱含量的分布与麦角酰胺相似,4,6和8月主要分布于叶片(2.206~8.226 mg/kg)和叶鞘(0.268~5.490 mg/kg)中,10月主要集中于种子(1.647~5.664 mg/kg)和叶片(1.353~2.251 mg/kg)中;各种群的叶片、茎秆和叶鞘中麦角新碱在4或6月(5.102~8.226 mg/kg)最高,8或10月含量(1.353~2.251 mg/kg)显著减小(P<0.05),根中麦角新碱在8月含量(0.128~0.283 mg/kg)低于其他月份。不同生长时期,4个披碱草种群整株中麦角酰胺和麦角新碱的含量均不同。麦角生物碱的总含量中麦角酰胺显著高于麦角新碱(P<0.05),麦角酰胺和麦角新碱呈极显著线性正相关(P<0.01)关系,相关系数为r=0.827。     

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.     

乙烯利及水杨酸对醉马草幼苗生长及生物碱含量的影响

本研究主要通过对醉马草(Achnatherum inebrians)幼苗进行水杨酸(Salicylic acid,SA)和乙烯利(Ethephon)处理,测定了醉马草幼苗的形态指标及其生物碱含量,探讨了醉马草幼苗在以上两种处理条件下麦角类生物碱含量的变化情况。结果表明,在Ethephon和SA处理下,与对照相比,醉马草幼苗的生物量、株高有所增加,但无显著差异(P0.05),而带菌醉马草幼苗麦角酰胺和麦角新碱的含量显著增加(P0.05)。     

The effect of alkaloids and seed extracts of endophyte-infected tall fescue on prolactin secretion in an in vitro rat pituitary perfusion system.

The objective of this research was to measure the effects of endophyte-infected tall fescue seed extract and various alkaloids associated with the endophyte on in vitro prolactin secretion by rat hemipituitaries. Rat anterior pituitaries (AP) were dissected into halves and placed in temperature-controlled culture chambers (37 degrees C). The tissue was perfused with culture media at a flow rate of 12 mL/h. After perfusion for at least 90 min with control media, AP halves were exposed to their respective treatments for 15 min before they were returned to the control media. The treatments for Exp. 1 were .01 micrograms of alpha-ergocryptine/mL of culture medium, .01 microgram of ergonovine/mL of culture medium, .01 gram-equivalents of endophyte-infected tall fescue seed/mL of culture medium, and .01 gram-equivalents of endophyte free tall fescue seed/mL of culture medium. Treatments for Exp. 2 consisted of 10(-4), 10(-6), and 10(-8) M concentrations of perloline, N-formyl loline, N-acetyl loline, N-methyl loline, and alpha-ergocryptine. alpha-Ergocryptine suppressed (P less than .10) prolactin secretion in both experiments. Ergonovine and perloline both stimulated (P less than .10) prolactin secretion. The loline alkaloids (N-formyl loline, N-acetyl loline, N-methyl loline) had no effect on prolactin secretion. The endophyte-infected seed extract treatment suppressed (P less than .10) prolactin secretion. The endophyte-free seed extract treatment had no effect on prolactin secretion. In Exp. 2, prolactin secretion from AP responded to alpha-ergocryptine treatment in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)