G. E. Aiken

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-naïve 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.

Bioaccumulation of ergovaline in bovine lateral saphenous veins in vitro.

Ergot alkaloids have been associated with vasoconstriction in grazing livestock affected by the fescue toxicosis syndrome. Previous in vitro investigations studying how ergot alkaloids caused vasoconstriction have shown that ergovaline has a distinct receptor affinity and sustained contractile response. A similar contractile response has not been noted for lysergic acid. The objectives of this study were to determine if repetitive in vitro exposure of bovine lateral saphenous vein to lysergic acid or ergovaline would result in an increasing contractile response and if a measurable bioaccumulation of the alkaloids in the vascular tissue occurs over time. Segments of vein were surgically biopsied from healthy, Angus x Brangus cross-bred, fescue-naïve yearling heifers (n = 16) or collected from healthy mixed breed and sex cattle immediately after slaughter (n = 12) at a local abattoir. Veins were trimmed of excess fat and connective tissue, sliced into cross-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). Contractile responses to repetitive additions of ergovaline (1 x 10(-9) and 1 x 10(-7) M) and lysergic acid (1 x 10(-5) and 1 x 10(-4) M) were evaluated using the biopsied veins. For the bioaccumulation experiments, veins collected at the abattoir underwent repetitive additions of 1 x 10(-7) M ergovaline and 1 x 10(-5) M lysergic acid and the segments were removed after every 2 additions and media rinses for alkaloid quantification via HPLC/mass spectrometry. Contractile data were normalized as a percentage of contractile response induced by a reference dose of norepinephrine (1 x 10(-4) M). Repetitive additions of 1 x 10(-9) M ergovaline and 1 x 10(-5) and 1 x 10(-4) M lysergic acid resulted in contractile response with a negative slope (P < 0.02). In contrast, repetitive addition of 1 x 10(-7) M ergovaline resulted in a contractile response that increased with each addition (P < 0.01). Lysergic acid and ergovaline were detected at all 4 exposure levels (2x to 8x), but only the 1 x 10(-7) M ergovaline treatment resulted in increased tissue content as the number of exposures increased (P < 0.05). These data indicate that ergovaline, but not lysergic acid, bioaccumulates with repetitive exposure in vitro. These results suggest that ergovaline may have a greater potential for inducing toxicosis in grazing animals than lysergic acid because of its potential to bioaccumulate at the cellular site of action.

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.

Contractile response of fescue-naive bovine lateral saphenous veins to increasing concentrations of tall fescue alkaloids.

Various alkaloids found in endophyte-infected tall fescue have been shown to elicit different effects in the grazing animal. As part of an ongoing characterization of vascular response generated by different alkaloids, the objective of this study was to examine the vasoconstrictive potentials of ergonovine (a simple lysergic acid derivative) and alpha-ergocryptine, ergocristine, and ergocornine (all ergopeptine alkaloids) using bovine lateral saphenous veins (cranial branch) biopsied from fescue-naïve cattle. Segments (2 to 3 cm) of vein were surgically biopsied from healthy crossbred yearling cattle (n = 18; 274 +/- 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). Tissue was allowed to equilibrate at 1 g of tension for 90 min before initiation of treatment additions. Increasing doses of each alkaloid (1 x 10(-10) to 1 x 10(-4) M) were administered every 15 min after buffer replacement. Data were normalized as a percentage of contractile response induced by a reference dose of norepinephrine (1 x 10(-4) M). Exposure of vein segments to increasing concentrations of ergocryptine, ergocristine, and ergonovine did not result in a contractile response until 1 x 10(-7) M, and ergocornine was even less potent (P < 0.05). Ergonovine had a greater maximal contractile intensity than ergocristine and ergocryptine (P < 0.05), with the 1 x 10(-4) M responses of ergonovine, ergocristine, ergocryptine, and ergocornine reaching maximums of 68.5 +/- 4.1, 45.5 +/- 4.5, 42.9 +/- 4.1%, and 57.2 +/- 9.9% of the norepinephrine maximum, respectively. The contractile response to increasing concentrations of ergonovine vs. ergocryptine, ergocristine, and ergocornine were opposite from previous evaluations of ergoline (e.g., lysergic acid) and ergopeptine (e.g., ergovaline) alkaloids using this bioassay, where the ergopeptine generated the greater contractile intensity. These data indicate that ergopeptines structurally different only at a single position of the peptide moiety do not exhibit differing contractile responses when considering contractile intensity. This difference may alter the potency when considering ergocornine was less potent than ergocryptine or ergocristine. These alkaloids may need to be considered when evaluating causative agents vasoconstriction associated with tall fescue-induced toxicosis.

Influence of toxic endophyte-infected fescue on sperm characteristics and endocrine factors of yearling Brahman-influenced bulls

Sixteen (mean age = 1.1 +/- 0.1 yr; mean BW = 478 +/- 34 kg) Brahman-influenced bulls were used to determine the influence of fescue type on sperm characteristics and serum concentrations of prolactin, cortisol, and testosterone. Bulls were blocked by BW, scrotal circumference (SC), and pregrazing sperm characteristics and randomly assigned to graze toxic endophyte-infected (EI; 4 bulls/pasture; 2 pastures) or novel endophyte-infected (NE; 4 bulls/pasture; 2 pastures) tall fescue for 121 d. Semen was collected by electroejaculation, and SC was measured and blood samples collected monthly. Sperm were evaluated for motility and morphology with an integrated visual optical system. Overall mean concentration of prolactin was decreased more (P < 0.01) in EI bulls than NE bulls from May to August. Scrotal circumference was not affected by fescue type (P = 0.58); overall SC averaged 36.7 +/- 2.3 cm. Percentage of live sperm was not different (P = 0.24) between NE bulls (80%) than EI bulls (67%) in July and August. Bulls grazing NE fescue had more (P < 0.06) motile sperm than EI bulls in July and August. Percentages of progressive (57 vs. 38%, NE and EI, respectively; P < 0.06) and rapid (67 vs. 46%, NE and EI, respectively; P = 0.04) sperm were greater from bulls grazing NE than EI bulls in July and August. Average velocity of the smoothed sperm path and progressive velocity in a straight line from the beginning to the end of the sperm track were slower (P < 0.09) in EI bulls than NE bulls and were slower (P = 0.04) in August compared with July. Mean width of head oscillation as the sperm swims was less (P < 0.06) in August than July. Concentrations of cortisol and testosterone were not (P > 0.10) influenced by fescue type. Semen from bulls grazing EI had reduced motility and morphology than bulls grazing NE. Detrimental effects of toxic fescue may not be mediated by cortisol, testosterone, or both. Semen quality of bulls grazing toxic EI tall fescue was decreased with increased maximum ambient temperatures.

Effects of hops (Humulus lupulus L.) extract on volatile fatty acid production by rumen bacteria

Aims:  To determine the effects of hops extract on in vitro volatile fatty acid (VFA) production by bovine rumen micro‐organisms.

Effects of fescue toxicosis on bull growth, semen characteristics, and breeding soundness evaluation.

Tall fescue possesses heat, drought, and pest resistance conferred to the plant by its mutualistic relationship with the ergot alkaloid producing fungal endophyte, Neotyphodium coenophialum. The objective of this study was to evaluate the impact of ergot alkaloid consumption on growth, scrotal circumference (SC), and semen quality. The SC measurement and percentage of motile and normal sperm were used to determine if a bull passed the breeding soundness exam (BSE) requirements. Bulls (n = 14) between 13 and 16 mo of age exhibiting ≥32 cm SC and having passed a BSE were assigned to 1 of 2 dietary treatments accounting for BCS and BW. Bulls were fed the treatment diet containing toxic tall fescue seed (E+; 0.8 μg of ergovaline and ergovalanine/g DM) or the control diet containing endophyte-free nontoxic tall fescue seed (E-) for 126 d. Blood samples were collected and BSE and BCS accessed at the start of the test (d 0) and every 21 d to the end of test (d 126). Weights were obtained on d 0 and d 126. Serum prolactin (PRL) concentrations were affected by treatment × day interactions (P = 0.04) verifying the effectiveness of the E+ diet. Bulls consuming the E+ diet exhibited declining PRL concentrations from 250 ± 52.1 ng/mL on d 0 to 30.6 ± 46.9 ng/mL by d 126 whereas bulls receiving the E- ration maintained serum PRL concentrations greater than or equal to 226.7 ± 50.4 ng/mL across the 126-d study. Body condition score (P = 0.4) and BW (P = 0.4) were not different between treatments. No difference due to treatment was observed for the percentage of bulls passing a standard BSE exam (P = 0.6) and no treatment effect was observed for any semen characteristic measured by computer-assisted semen analysis (CASA; P ≥ 0.2). The SC was negatively affected by treatment × day interaction (P = 0.04) with E- bulls exhibiting a larger SC at d 126 compared with E+ bulls of 36.7 ± 0.8 versus 34.3 ± 0.8 cm, respectively. Within treatment, E+ bulls exhibited a decrease in SC (P = 0.0001) with a d 0 SC of 37.3 ± 0.8 cm and dropping to 34.3 ± 0.8 by d 126. Theoretically, reduced SC would negatively impact semen quality, but this was not observed. However, CASA and BSE evaluation data are consistent with recent reports indicating that bulls grazing E+ tall fescue exhibited only subtle, if any, differences on semen characteristics.

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.

Technical note: Comparison between two tracing methods with ultrasonography to determine lumen area of the caudal artery in beef cattle

Two experiments were conducted to compare variation between 2 tracing methods in measuring cross-sectional lumen area of the caudal artery in 5 beef heifers on 3 different dates (Exp. 1) and to compare tracing methods in detecting changes in artery lumen area after 5 heifers were switched from a diet containing nonendophyte-infected tall fescue [Schedonorus arundinaceus (Schreb.) Dumort] seed to one containing endophyte-infected tall fescue seed (Exp. 2). Lumen area determined by tracing the Doppler flow signal was 25% less than that determined by tracing the intima of the connective tissue, but there was no difference (P = 0.90) in the variation of measures between the 2 methods in Exp. 1. Declines in lumen area were detected at the same level of significance (P < 0.01) for both tracing methods after cattle in Exp. 2 were switched from noninfected to infected tall fescue diets. Variation in lumen areas was different between noninfected and infected diets with tracing the Doppler flow signal (P < 0.05) or the intima of the connective tissue (P < 0.01). Results indicated that lumen area of the caudal artery can be measured with similar precision by tracing the intima of the connective tissue in the artery wall or the outer boundary of the Doppler flow signal.

Prevalence of Escherichia coli O157:H7 and Salmonella in beef steers consuming different forage diets*

Aims:  To compare the prevalence of faecal shedding of Escherichia coli O157:H7 and Salmonella in growing beef cattle consuming various forages.