Alkaloid Quantities in Endophyte-Infected Tall Fescue are Affected by the Plant-Fungus Combination and Environment.

Many grass species are symbiotic with systemic, vertically-transmitted, asymptomatic Epichloë endophytic fungi. These fungi often produce alkaloids that defend the host against herbivores. We studied how environmental variables affect alkaloids in endophyte-infected tall fescue (Schedonorus phoenix) from three Northern European wild origins and the widely planted US cultivar 'Kentucky-31' (KY31). The plants were grown in identical common garden experiments in Finland and Kentucky for two growing seasons. Plants were left as controls (C) or given water (W), nutrient (N) or water and nutrient (WN) treatments. For 8-10 replications of each plant origin and treatment combination in both experiments, we analyzed ergot alkaloids, lysergic acid, and lolines. In Finland, tall fescue plants produced 50 % more ergot alkaloids compared to plants of the same origin and treatments in Kentucky. Origin of the plants affected the ergot alkaloid concentration at both study sites: the wild origin plants produced 2-4 times more ergot alkaloids than KY31, but the ergot alkaloid concentration of KY31 plants was the same at both locations. Overall lysergic acid content was 60 % higher in plants grown in Kentucky than in those grown in Finland. Nutrient treatments (N, WN) significantly increased ergot alkaloid concentrations in plants from Finland but not in plants from Kentucky. These results suggest that the success of KY31 in US is not due to selection for high ergot alkaloid production but rather other traits associated with the endophyte. In addition, the environmental effects causing variation in alkaloid production of grass-endophyte combinations should be taken into account when using endophyte-infected grasses agriculturally.

Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism.

Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichloë or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichloë festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism.

Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media.

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epichloë (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC-MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15-30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes.

Mortality of horn fly larvae (Diptera: Muscidae) in bovine dung supplemented with ergotamine and N-formyl loline.

Dung-dwelling larvae of ectoparasites of livestock such as the horn fly, Hematobia irritans (L.), may be exposed to > or = 1 different alkaloid species in dung from animals ingesting herbage of the tall fescue (Festuca arundinacea Schreb.)--endophyte association (Neotyphodium coenophialum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.). First-instar horn flies were exposed to bovine dung supplemented with up to 50 microM each of N-formyl loline and ergotamine tartrate in factorial combination. In the absence of ergotamine tartrate, N-formyl loline caused a linear decline in the number of pupae recovered, and probit analysis indicated an LC50 of 36 microM. In the absence of N-formyl loline, significant quadratic responses of larvae to ergotamine tartrate were established, and probit analysis indicated a LC50 of 34 microM. An interaction (P < 0.001) was found between the 2 alkaloids for larval survival. This interaction showed that ergotamine tartrate moderated the toxicity of N-formyl loline and indicates that a membrane-bound receptor may be involved. There was no evidence of carryover of effects of alkaloids on subsequent stages of development or expressed as abnormalities of pupae or adults. Interactions between alkaloids probably are involved in other plant-herbivore relationships of endophyte-infected grasses.

Mortality of horn fly (Diptera: Muscidae) larvae in bovine dung supplemented with loline alkaloids from tall fescue.

Larvae of arthropod ectoparasites of livestock, such as the horn fly, Haematobia irritans (L.), may be exposed to acyl-loline alkaloids in dung of ruminant livestock ingesting herbage of the tall fescue (Festuca arundinacea Schreb.)-endophyte association [Neotyphodium coenophialum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.]. Biological activity of alkaloid-supplemented bovine dung was assayed by growth, development, and survival of 1st instars of horn fly. An extract from tall fescue seed, containing N-formyl loline (NFL), N-acetyl loline (NAL), and loline (59:21:20 by mass, respectively) caused 100% mortality of horn fly larvae when dung was supplemented at > or = 100 micrograms/g. Probit analysis of data corrected for natural mortality indicated a LD50 of 30 micrograms/g (95% fidicial limits: 20-49 micrograms/g). When horn fly larvae were introduced to dung supplemented with up to 50 microM of acyl-loline derivatives, mortality of larvae varied significantly between alkaloids (P < 0.0001). Probit analysis indicated that NFL [LD50: 34 microM (95% fidicial limits: 3-53 microM)] was more toxic than NAL [LD50: 46 microM (0-83 microM)], and that loline hydrochloride was not toxic.

Fertilization rates and embryonic development in CD-1 mice fed fungal endophyte-infected tall fescue seed.

The objective of this study was to assess the effects of endophyte-infected tall fescue (KY-31) seed (80% infected, containing Acremonium coenophialum ) on the fertilization rates and embryonic development of CD-1 mice. Twenty-four pairs of mature CD-1 mice were randomly allocated to two dietary treatments containing 40% mouse chow and 1) 60% noninfected tall fescue seed or 2) 60% infected tall fescue seed (w/w). The mice were preconditioned on their respective diets for 60 d prior to 96 h of cohabitation between pairs of males and females. Following the removal of the males, equal numbers of females (n = 4) from each dietary treatment were sacrificed at 80 h, 6 and 10 d after vaginal plug establishment. The female reproductive tracts were excised and flushed to recover eggs and embryos or dissected to identify the number and status of the implanted fetuses. The number of eggs-embryos and/or fetuses per mouse present at the time of sacrifice were 9.8, 8.8 and 11.5 vs 8.2, 5.3 and 4.8 for the mice fed the noninfected and infected fescue seeds, respectively. Similarly, the conception rates (%) were 100, 100 and 100 vs 73, 50 and 50 for the two treatments. The mice sacrificed at 80 h and fed the noninfected fescue had 9.8 expanded blastocysts per animal, whereas those fed the infected fescue had only 4.0 expanded blastocysts. The results suggest that the infected fescue seed consumed by these mice had a significant effect on fertilization rates. More importantly, the infected fescue seed influenced negatively the development of these embryos during the embryonic-preimplantation period.

Lactation in mice fed endophyte-infected tall fescue seed.

This study was conducted to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY-31) seed (80% infected) on lactation in CD-1 dams and suckling performance of pups as measured by pup survival and growth rates. Twenty-four pairs of mature CD-1 mice were randomly allocated to four dietary treatments: 1) 100% mouse chow ad libitum; 2) 40% endophyte-infected tall fescue seed and 60% mouse chow (w/w); 3) reduced intake (100% chow), adjusted daily to the intake level of Treatment 2; and 4) 60% infected tall fescue seed and 40% chow. The mice were preconditioned on their respective diets for 100 d prior to 96 h of cohabitation between pairs of males and females. At parturition the litters were removed, and each dam was given a litter of six pups of equal weight, size and sex ratio to suckle for 15 days. All pups given to all the dams were born to other mice that were not part of the study and had not been exposed to endophyte-containing diets. Dams and litter weights were measured daily for 15 consecutive days. The combined body weight measurements of litters from dams fed the tall fescue containing diets (Treatments 2 and 4) were significantly lower (2.07 +/- 0.41 g/d) than that of litters from dams fed the chow containing diets (Treatments 1 and 3) during the suckling trial (P<0.05). Similarly, nine of ten (90%) dams fed the chow containing diets maintained five or more pups (5.5 +/- 0.2) throughout the study as compared to five of nine (55.6%) dams fed the tall fescue containing diets that maintained less than five pups (4.5 +/- 0.2).

Effects of feeding endophyte-infected tall fescue seed on the reproductive performance in male and female CD-1 mice by combination crosses.

The present study was undertaken to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY 31) seed (80% infected) on the reproductive performance of male and female CD-1 mice by combination crosses. Forty-eight male and 48 female 30-d-old mice were randomly allocated to two diet treatments. Twenty-four males and 24 females were fed Diet 1, consisting of 50% non-infected fescue and 50% mouse chow; the remaining animals were fed Diet 2, containing 50% infected fescue. At the end of 50 d, the animals were paired in groups (n = 12) as follows: 1) Diet 1 males and females; 2) Diet 2 males and Diet 1 females; 3) Diet 1 males and Diet 2 females; and 4) Diet 2 males and females. The pairs were maintained on the diets of their female partners and were allowed to cohabitate for 96 h. After this period the males were removed and the females were carried to term. Pregnancy rates among the four treatment groups were 100, 83.3, 75.0 and 54.5%, respectively. Similarly, the average number of pups born per litter among the four groups were 11.5, 8.0, 9.7, and 7.5, respectively. Also, the weights (grams) of pups born per litter were 17.5, 12.6, 12.4, and 9.8, respectively. The results point out that the reproductive capacity of both male and female mice are affected (P < 0.05) by the ingestion of endophyte-infected fescue seeds. Such negative effects were more severe in the female than the male (P < 0.05).

Effect of feeding fungal endophyte (Acremonium coenophialum )-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding.

This study was designed to assess the effect of endophyte-infected (Acremonium coenophialum ) tall fescue (KY-31) seed (80% infected) on reproductive performance in CD-1 mice by continuous breeding. Twenty-four pairs of 70-d-old CD-1 mice were randomly allocated to four diets: 1) mouse chow ad libitum; 2) 40% infected fescue seed and 60% chow (w/w); 3) reduced intake (100% chow) similar to the intake, adjusted daily, in Diet 2; and 4) 60% infected fescue seed and 40% chow. Males and females were randomly paired (six pairs/treatment) and placed on the above diets. The mice were fed the corresponding diets for 80 d, although the pairs were separated on Day 60 (prior to the birth of the 3rd litter) and the females were monitored for one additional gestation period (20 d). The pregnancy data (litters produced) among the four treatments were 100.0 (18), 77.8 (14), 100.0 (18) and 80.0% (12) respectively. Similarly, the average number of pups born per litter among the four treatments was 11.8, 9.3, 10.1, and 9.8. When the chow treatment (1 and 3) and the fescue treatments (2 and 4) were pooled and compared, the percent pregnancy was 100.0 (n = 36) and 78.8 (n = 26), and the pups born per litter (means +/- SEM) were 11.0 +/- 0.5 and 9.5 +/- 0.6, respectively. Also the intervals between the three litters born during the 60-d cohabitation period were 21.6 +/- 1.1 and 24.5 +/- 0.9 d for the chow and fescue treatments, respectively. The results point out that 40 and 60% infected fescue seed in the diet of mice does influence (P < 0.05) their reproductive capacity as measured by percent pregnancy and litter size.

Sulfur and nitrogen metabolism in the bovine fed different forms of supplemental sulfur.

Twelve growing Angus heifers averaging 282 kg in weight were fed a tall fescue hay-based diet in a completely randomized design experiment to determine sulfur (S) and nitrogen (N) metabolism and bioavailability of different forms of supplemental S:elemental S (E), sodium sulfate (SU) and DL-methionine (M). The study included a 72-h adaptation phase that was followed immediately by a 5-d retention phase. Control (C) heifers were fed a ground fescue hay diet twice daily throughout the study. Supplementation with .15% S from E, SU or M to the basal diet began at h 0. Differences in total blood S, plasma S and ruminal NH3 N were dependent on sampling time in the 72-h adaptation phase. Urinary S excretion during 0 to 72 h was greater for S-supplemented calves than for C. Mean molar proportions of acetate, butyrate, isobutyrate and valerate were affected by form of S supplementation. No treatment differences (P greater than .10) were noted in total VFA concentrations. Sulfur supplementation increased daily urinary sulfate (P less than .01), total urinary S (P less than .01) and fecal S (P less than .01) excretion during the retention phase. Methionine and SU heifers had the greatest urinary S losses, and fecal S was highest for E (P less than .01). No differences (P greater than .10) in DM digestion occurred among treatments. N retention (g/d) and N retention as a percentage of intake averaged 5.9, 8.5; 13.2, 18.7; 13.8, 20.5; and 11.3, 19.2, respectively, for C, E, SU and M. Sulfur retention (g/d) was increased (P less than .10) by S supplementation. Sulfur supplementation of a basal fescue hay diet increased S retention transitorily, although much of the added S was lost through excretory routes. Supplementing S as E, SU or M appeared to be equally beneficial, despite differences in route and extent of S excretion among the three S sources.

Alkaloid binding and activation of D2 dopamine receptors in cell culture.

Ergot and pyrrolizidine alkaloids, either extracted from endophyte-infected tall fescue, synthesized, or purchased commercially, were evaluated in cultured cells to estimate their binding to the D2 dopamine receptor and subsequent effects on cyclic AMP production in GH4ZR7 cells, transfected with a rat D2 dopamine receptor. Ergopeptide alkaloid (alpha-ergocryptine, bromocryptine, ergotamine tartrate, and ergovaline) inhibition of the binding of the D2-specific radioligand, [3H]YM-09151-2, exhibited inhibition constants (K(I)) in the nanomolar range, whereas dopamine was less potent (micromolar). The lysergic acid amides (ergine and ergonovine) were 1/100th as potent as the ergopeptide alkaloids. Ergovaline and ergotamine tartrate were equally effective in inhibiting vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production, with consistent nanomolar effective concentration (EC50) values. The remaining ergopeptide alkaloids (alpha-ergocryptine and bromocryptine), lysergic acid amides (ergonovine and ergine), and dopamine were 1/100th as potent. Two representative pyrrolizidines, N-formylloline and N-acetylloline, exhibited no binding activity at the D2 dopamine receptor or effects on the cyclic AMP system within the concentration ranges of nanomolar to millimolar. Our results indicate that the commercially available ergot alkaloids ergotamine tartrate and ergonovine may be used interchangeably in the D2 dopamine receptor system to simulate the effects of extracted ergovaline and ergine and to examine responses in receptor binding and the inhibition of cyclic AMP.

Summer fescue toxicity in dairy steers fed tall fescue seed.

Seeds of the tall fescue ( Festuca arundinacea Schreb .) cultivars Kentucky 31 and an experimental ryegrass X tall fescue hybrid derivative strain (G1-307), and orchard grass (OG) seed were fed in a carrier diet to calves in controlled environmental rooms (31 to 32 C). Both tall fescue varieties produced symptoms of summer toxicosis in dairy steers. Total feed intake (P less than .01) and water intake (P less than .01) of calves were reduced by the tall fescue seed diets when compared with orchard grass. Steers fed G1-307 and Kentucky 31 tall fescue lost (NS) 17.5 and 7.8 kg of body weight, respectively, while those consuming orchard grass gained (P less than .01) 6.2 kg during the experiment. Rectal temperatures were lower (P less than .05) in the calves fed OG (39.4 C) when compared with those fed G1-307 (40.6 C) and Kentucky 31 (40.8 C) tall fescue seed, respectively. In a second trial Kentucky 31 seed was fed in a carrier diet to Holstein steers at graded levels of 0, 350, 700 and 1,050 g seed/d. Consumption of 700 and 1,050 g seed/d adversely affected performance of steers. Total feed intake was lower and water intake was reduced (P less than .05), with rectal temperatures being elevated (P less than .01) in these two groups when compared with 0- or 350-g treatments. No significant differences were detected in body weight changes in this trial. Respiration rates were not significantly affected in either trial. Results of these trials with the young bovine indicate that a toxic substance(s) is present in tall fescue seed.

Interaction of environmental temperature and anti-quality factors on the severity of summer fescue toxicosis.

Two experimental strains of tall fescue grass (Festuca arundinacea Schreb) that had previously been shown to produce differences in animal performance during the summer period were fed to Holstein calves in temperature controlled rooms. In the first year, GI-306 (less toxic) and GI-307 (more toxic) tall fescues were fed during July in rooms maintained at 10 to 13, 21 to 23 and 34 to 35 C. In the second year, the same tall fescues and orchardgrass were fed at a high temperature during May (32 to 33 C), July (34 to 35 C) and October (31 to 32 C). A comparison at a lower temperature (16 to 18 C) was also included in the July and October trials, On both years, calves consuming GI-307 tall fescue consistently had lower dry matter intakes, higher rectal temperatures, higher respiration rates and lower weight gains than those fed GI-306 tall fescue or orchardgrass at temperatures above 31 C. At lower ambient temperatures, differences in dry matter intake, rectal temperature and respiration rates between animals on the two strains of forage were not statistically different. The results demonstrate the poor performance of cattle grazing fescue is related to environmental temperature and that the toxic substance(s) is present throughout the growing season.

Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers.

Factors associated with fat necrosis in cows and tall-fescue summer toxicosis in steers were studied. In the cow study, fescue pastures were fertilized, using 3 rates of N: high N (703 to 483 kg and 0 kg of N/ha/year from broiler litter in 1972 to 1974 and 1975, respectively), moderate N, and low N (224 and 74 kg of N/ha/yr from NH4NO3, 1972 to 1975, respectively). Bermuda grass pastures were fertilized at 2 rates of N: moderate N and low N (280 and 20 kg of N/ha/year from NH4NO3, 1972 to 1975, respectively). Fat necrosis developed only in cows grazing tall fescue, with an occurrence of 60%, 8%, and 3% for high-N, moderate-N, and low-N pastures, respectively. Cows grazing the high-N fescue, and to some extent those grazing the moderate-N fescue, had clinical signs of summer fescue toxicosis. Plasma cholesterol concentrations were lowest in cattle grazing the high-N fescue, averaging 114 mg/dl, followed by 134 and 127 mg/dl in cattle grazing the moderate-N and low-N fescue, respectively. In the steer grazing study, 24 paddocks of 0.49 ha each were seeded with tall-fescue lines G1-307 or G1-306 or with tall-fescue cultivars, KY-31 or Kenhy. All paddocks were fertilized with 170 kg of N/ha/year. Serum cholesterol concentrations were lower in steers grazing on G1-307 than in steers grazing on G1-306 or cultivars. Serum total lipids followed a similar trend, with a positive correlation (r = 0.49) between cholesterol and total lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute exposure to ergot alkaloids from endophyte-infected tall fescue does not alter absorptive or barrier function of the isolated bovine ruminal epithelium.

Ergot alkaloids in endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum) have been shown to cause a reduction in blood flow to the rumen epithelium as well as a decrease in volatile fatty acids (VFA) absorption from the washed rumen of steers. Previous data also indicates that incubating an extract of endophyte-infected tall fescue seed causes an increase in the amount of VFA absorbed per unit of blood flow, which could result from an alteration in the absorptive or barrier function of the rumen epithelium. An experiment was conducted to determine the acute effects of an endophyte-infected tall fescue seed extract (EXT) on total, passive or facilitated acetate and butyrate flux across the isolated bovine rumen as well as the barrier function measured by inulin flux and tissue conductance (G t ). Flux of ergovaline across the rumen epithelium was also evaluated. Rumen tissue from the caudal dorsal sac of Holstein steers (n=6), fed a common diet, was collected and isolated shortly after slaughter and mounted between two halves of Ussing chambers. In vitro treatments included vehicle control (80% methanol, 0.5% of total volume), Low EXT (50 ng ergovaline/ml) and High EXT (250 ng ergovaline/ml). Results indicate that there is no effect of acute exposure to ergot alkaloids on total, passive or facilitated flux of acetate or butyrate across the isolate bovine rumen epithelium (P>0.51). Inulin flux (P=0.16) and G t (P>0.17) were not affected by EXT treatment, indicating no alteration in barrier function due to acute ergot alkaloid exposure. Ergovaline was detected in the serosal buffer of the High EXT treatment indicating that the flux rate is ~0.25 to 0.44 ng/cm2 per hour. Data indicate that specific pathways for VFA absorption and barrier function of the rumen epithelium are not affected by acute exposure to ergot alkaloids from tall fescue at the concentrations tested. Ergovaline has the potential to be absorbed from the rumen of cattle that could contribute to reduced blood flow and motility and lead to reduced growth rates of cattle.

Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography.

The hypotheses that endophyte (Neotyphodium coenophialum)-infected tall fescue (TF) seed causes vasoconstriction in horses in vivo and that ground seed would cause more pronounced vasoconstriction than whole seed were tested. Ten horses each received 1 of 3 treatments: endophyte-free ground (E-G; n = 4 horses) seed, endophyte-positive whole (E+W; n = 3) seed, or endophyte-positive ground (E+G; n = 3) seed. There were two 14-d periods, P1 and P2. During P1, animals were adapted to a concentrate (0.2% BW, as fed, twice daily) and alfalfa cubes. During P2, the seed was mixed into the concentrate portion of the diet and alfalfa cubes were offered ad libitum. Fescue seed was fed in increasing amounts ranging from 0.02% BW on d 1 (averaging 76 ug/kg ergovaline + ergovalinine) to 0.22% BW on d 11 to 14 (averaging 713 ug/kg ergovaline + ergovalinine). The distal palmar artery of the left foreleg of each horse was scanned via Doppler ultrasonography for 4 d during each period, with 5 replicate scans performed on each scanning day. The measurements taken at each scan included artery luminal diameter, area, and circumference, peak systolic velocity, end diastolic velocity and blood flow variables. Animal temperature, heart rate, and respiration rate and ambient temperature and humidity were also recorded. Blood samples were taken on each scanning day to measure inflammatory cytokine mRNA abundances, and blood samples were collected on d 0, 4, 8, and 14 of P2 to measure prolactin concentrations. Consumption of E+G TF seed caused decreased artery lumen diameter (P = 0.0033), area (P = 0.0406), and circumference (P = 0.0480) compared with E-G seed, and E+W seed produced an intermediate response. Blood flow volume was reduced (P < 0.05) during P2 in horses receiving E+G seed compared with horses receiving E-G seed. Other ultrasound variables were not different (P > 0.05) among treatment groups, and neither were cytokine mRNA or prolactin concentrations. Treatment did not alter (P > 0.05) animal temperature, heart rate, or respiration rate, and neither ambient temperature nor relative humidity was consistently correlated with any response variable measured. Taken together, these data confirm that consumption of E+G fescue seed caused vasoconstriction in horses, which could be readily measured by Doppler ultrasonography. Use of Doppler ultrasound to monitor the diameter of the palmar artery of horses grazing endophyte-infected (E+) fescue pastures may provide a convenient and satisfactory biomarker to determine premonitory signs of fescue toxicosis.

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.

Ergot alkaloids from endophyte-infected tall fescue decrease reticuloruminal epithelial blood flow and volatile fatty acid absorption from the washed reticulorumen.

An experiment was conducted to determine if ergot alkaloids affect blood flow to the absorptive surface of the rumen. Steers (n=8) were pair-fed alfalfa cubes and received ground endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum; E+) seed (0.015 mg ergovaline·kg BW(-1)·d(-1)) or endophyte-free tall fescue (E-) seed via the rumen cannula 2x daily for 7 d at thermoneutral (TN; 22°C) and heat stress (HS; 32°C) conditions. On d 8, the rumen was emptied and rinsed. A buffer containing VFA was incubated in the following sequence: control (CON), 15 µg ergovaline·kg BW(-1) (1×EXT) from a tall fescue seed extract, and 45 µg ergovaline·kg BW(-1) (3×EXT). For each buffer treatment there were two 30-min incubations: a 30-min incubation of a treatment buffer with no sampling followed by an incubation of an identical sampling buffer with the addition of Cr-EDTA and deuterium oxide (D2O). Epithelial blood flow was calculated as ruminal clearance of D2O corrected for influx of physiological water and liquid outflow. Feed intake decreased with dosing E+ seed at HS but not at thermoneutral conditions (TN; P<0.02). Dosing E+ seed decreased serum prolactin (P<0.005) at TN. At HS, prolactin decreased in both groups over the 8-d experiment (P<0.0001), but there was no difference in E+ and E- steers (P=0.33). There was a seed treatment×buffer treatment interaction at TN (P=0.038), indicating that E+ seed treatment decreased reticuloruminal epithelial blood flow at TN during the CON incubation, but the two groups of steers were not different during 1×EXT and 3×EXT (P>0.05). Inclusion of the extract in the buffer caused at least a 50% reduction in epithelial blood flow at TN (P=0.004), but there was no difference between 1×EXT and 3×EXT. There was a seed × buffer treatment interaction at HS (P=0.005), indicating that the reduction of blood flow induced by incubating the extract was larger for steers receiving E- seed than E+ seed. Volatile fatty acid flux was reduced during the 1×EXT and 3×EXT treatments (P<0.01). An additional experiment was conducted to determine the effect of time on blood flow and VFA flux because buffer sequence could not be randomized. Time either increased (P=0.05) or did not affect blood flow (P=0.18) or VFA flux (P>0.80), indicating that observed differences are due to the presence of ergot alkaloids in the rumen. A decrease in VFA absorption could contribute to the signs of fescue toxicosis including depressed growth and performance.

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.