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.

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.

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.

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.

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.

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.

Physiological responses in rats fed extracts of endophyte-free and endophyte-infected tall fescue seed relative to some known ergot alkaloids.

Ethanol and ethanol-water extracts of endophyte-infected tall fescue seed were effective for extracting toxin(s) responsible for feed intake and average daily weight gain (ADG) depression in Sprague Dawley rats. Although the ethanol extract of endophyte-infected seed depressed serum prolactin (Prl) concentrations, the data were less reflective of overall toxicity than feed intake and weight gain. Inclusion of commercially available ergonovine maleate, ergocryptine and ergotamine tartrate had no effect on rat feed intake, ADG or serum Prl at the levels tested. Dietary addition of a recombination of hexane, ethanol and ethanol-water extracts caused a toxicity response (depressed feed intake, ADG, and serum Prl) equivalent to that of the whole seed. The toxic factor(s) were extracted primarily in more polar solvents such as ethanol and ethanol-water. Rat assays can be used successfully in lieu of cattle assays as a routine evaluation of toxic components in endophyte-infected tall fescue. However, species differences dictate that eventually all suspected toxins be evaluated in the bovine.

Reproductive responses in male rats fed extracts of fescue seed infected by the endophytic fungus Acremonium coenophialum.

This study was undertaken to determine whether a methanol extract of endophyte-infected tall fescue seed contained the factor(s) that were responsible for the decrease in male reproductive potential when rats were fed toxic tall fescue seed. Twenty-four Sprague-Dawley male rats (ca. 225 g.) were randomly allocated to one of three dietary treatments containing 50% (w/w) rodent chow and 50%: (1) noninfected tall fescue seed; (2) noninfected tall fescue seed treated with concentrated methanol effluent from serial methanol extraction of infected seed; and (3) the extracted seed residue of seed that was greater than 95% infected by Acremonium coenophialum. Parameters assessed were testicular weight, epidydimal weight, parenchymal weight, progressive sperm motility, sperm motility, sperm count and daily sperm production potential. Significant reduction (P less than 0.05) for feed intake, average daily gain, testicular weight, epidydimal weight and daily sperm production potential were observed for treatment 2. These data suggest that the serial methanol extract contains the factor(s) responsible for the lowered reproductive potential in male rats. The extract contained 15,578 micrograms/ml of pyrrolizidine alkaloids and only trace amounts of ergopeptides. These results suggest that N-formyl and N-acetyl loline may play an important role in the fescue toxicity noted in the exposed rats.