Toxic Endophyte-infected Tall Fescue Research Articles

5-hydroxytryphophan mitigates ergot alkaloid-induced suppression of serotonin and feed intake in cattle.

The impact of ergot toxicosis on livestock industries is detrimental and treatments are needed in many countries. The objective of this study was to evaluate the effects of acute exposure to ergot alkaloids and 5-hydroxytryptophan (5-HTP) supplementation on feed intake, serotonin metabolism, and blood metabolites in cattle. Eight Holstein steers (538 ± 18kg) fitted with ruminal cannulas were used in a replicated 4 × 4 Latin Square design experiment with a 2 × 2 factorial treatment structure. The treatments were the combination of 0 (E-) or 15 µg ergovaline/kg BW (E+) and 0 (5HTP-) or 0.5mg of 5-hydroxy-l-tryptophan/kg BW (5HTP+) administered daily for 6 d. Toxic endophyte-infected tall fescue seed was used to supply the daily dose of ergovaline. Endophyte-free seed was used to equalize seed intake between treatments. Ground seed was placed into the rumen immediately before feeding. The 5-HTP was dissolved in water and infused into the abomasum via the reticulo-omasal orifice. Blood was collected from a jugular vein catheter at 0, 1, 2, 4, 8, and 24h after treatment administration. Ergovaline without 5-HTP (E+/5HTP-) decreased dry matter intake (DMI) in comparison to steers without ergovaline and 5-HTP (E-/5HTP-). However, 5-HTP infusion in association with ergovaline (E+/5HTP+) normalized the DMI. Although E + did not affect (P > 0.05) the area under the curve (AUC) of serum 5-HTP, 5-hydroxyindoleacetic acid, tryptophan, and kynurenine, serum and plasma serotonin concentrations were decreased (P < 0.05). The infusion of 5-HTP increased (P < 0.05) the AUC of serum 5-HTP, serum and plasma serotonin, and serum 5-hydroxyindoleacetic acid. In conclusion, acute exposure to ergot alkaloids reduced DMI and circulating serotonin in cattle but 5-HTP administration showed potential to normalize both circulating serotonin and feed intake.

412 Awardee Talk: New Insights into Mechanisms of Serotonin Receptor-Mediated Vasoactivity and Complex Interactions with Ergot Alkaloids in Bovine Vasculature

Abstract Ergovaline (ERV), produced in toxic endophyte-infected tall fescue, causes potent vasoconstriction of bovine vasculature by interacting with serotonin (5-HT) receptors. Ergovaline acts as a partial agonist of 5-HT2A-mediated vasoconstriction. The duration of ERV exposure affects 5-HT-mediated vasoactivity of mesenteric blood vessels. If ERV and 5-HT exposure occur simultaneously, ERV can increase 5-HT-mediated vasoconstriction. If 5-HT exposure occurs after blood vessels have had prior ERV exposure, increasing levels of 5-HT can induce vasorelaxation of blood vessels. To better understand mechanisms of 5-HT-mediated vasorelaxation, lateral saphenous veins from steers (n = 5) were assessed for vasoactivity in response to increasing concentrations of selective 5-HT receptor agonists Isolated vessel segments were suspended in chambers of a multi-myograph containing 5 mL of continuously oxygenated Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. Vessels were pre-contracted with 1×10-4 M phenylephrine and exposed to increasing concentrations of 5-HT or 5-HT receptor agonists that were selective for 5-HT1B, 5-HT2B, 5-HT4, and 5-HT7. Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre-contraction. Increasing concentrations of 5-HT resulted in divergent vasoconstriction and vasorelaxation responses that were dose-dependent. Serotonin caused an 86.3% increase (P &amp;lt; 0.01) in relaxation activity at 1×10-7 M and 5-HT caused a 166% increase (P &amp;lt; 0.01) in contraction activity at 1x10-4 M. Increasing concentrations of the selective 5-HT1B agonist did not result in any vasoactive response. Increasing concentrations of agonists selective for 5-HT2B, 5-HT4, or 5-HT7 resulted in a 27.3%, 91.6%, or 43.9% (P &amp;lt; 0.01) increase in maximal vasorelaxation activity. Of these 5-HT receptor agonists, the selective 5-HT4 receptor agonist resulted in the lowest -log IC50 value (6.30) compared with 5-HT2B and 5-HT7 receptor agonists (4.21 and 4.66, respectively). To confirm the involvement of 5-HT4 in 5-HT-mediated vasorelaxation, blood vessels were exposed to either DMSO (control), a selective 5-HT2A antagonist (1×10-5M), or selective antagonists of 5-HT2A and 5-HT4 simultaneously for 5-min before the pre-contraction with phenylephrine and additions of 5-HT. Antagonism of the 5-HT2A receptor did not result in increased vasorelaxation activity compared with control. Antagonism of the 5-HT2A receptor and 5-HT4 receptor simultaneously inhibited vasorelaxation activity and increased (P &amp;lt; 0.01) the maximal contractile response occurring at 1×10-4 M by 47% compared with control. Approximately 84% of the maximal vasorelaxation activity occurring in response to 5-HT could be accounted for through 5-HT4. These data provide strong evidence that 5-HT4 is the primary receptor involved with 5-HT-mediated vasorelaxation in bovine lateral saphenous vein. More research is needed to determine if 5-HT4-mediated vasorelaxation is responsible for relaxation responses observed in blood vessels with prior ERV exposure. This will aid in the development of strategies for alleviating symptoms of sustained vasoconstriction that occur during fescue toxicosis in ruminants.

285 Duration of Ergovaline Exposure Influences Serotonin-Mediated Vasoconstriction and Vasorelaxation of Bovine Vasculature

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

Gene expression profiling indicates a shift in ammonia assimilation capacity along the hepatic acinus induced by different forms of selenium in vitamin–mineral mixes fed to beef steers grazing on toxic endophyte-infected tall fescue

Recently, we demonstrated that supplementing (with 3 mg Se per day) the diets of growing beef steers grazing on Se-deficient toxic endophyte-infected tall fescue-based forage with either organic Se (OSe, SEL-PLEX) or a 1:1 blend (MIX) of OSe and inorganic Se (ISe, sodium selenite) in vitamin–mineral mixes, rather than inorganic forms of Se, ameliorated several classic serum symptoms of fescue toxicosis. Importantly, higher levels of hepatic glutamine synthetase activity were observed in MIX and OSe steers. Accordingly, transcriptome level and targeted mRNA expression analyses were conducted on the same liver tissue to determine if Se treatments affected other hepatic metabolic pathways, especially those that are responsible for supplying substrates of glutamine synthetase. The effect of ISe, OSe, and MIX treatments (n = 8/treatment) on the relative abundances of mRNA [determined using microarray and real-time reverse-transcription PCR (RT-PCR)] and protein (determined using Western blotting) in liver tissue was assessed by ANOVA. Fisher’s protected LSD procedure was used to separate treatment means, with significance being declared at p ≤ 0.05. Microarray analysis identified (p&amp;lt; 0.01, false discovery rate of&amp;lt; 33%) 573 annotated differentially expressed gene (DEG) transcripts. Canonical pathway analysis identified the DEGs that are central to glutamine and glutamate biosynthesis/degradation and proline biosynthesis. Targeted RT-PCR analyses found that MIX and OSe steers had lower periportal ammonia-assimilation and urea-synthesizing capacities (lower glutaminase 2, key ornithine cycle enzymes, and mitochondrial ornithine/citrulline exchanger mRNA) than ISe steers. In addition, MIX and OSe steers had a higher capacity for pericentral ammonia assimilation (higher glutamine synthetase activity) and a higher capacity for the production of glutamate in pericentral hepatocytes from α-ketoglutarate (higher levels of glutamine dehydrogenase, and decreased levels of arginase 2 and ornithine aminotransferase and mRNA). The form of supplemental Se also affected steers’ capacity for hepatic proline metabolism, with OSe steers having a higher capacity for proline synthesis and MIX steers having a higher capacity for pyroline-5-carboxylate synthesis. In conclusion, supplementing the diets of growing beef steers grazing on toxic endophyte-infected tall fescue with MIX and OSe in vitamin–mineral mixes, rather than with inorganic forms of Se, shifts hepatic ammonia assimilation from periportal urea production to pericentral glutamine production, thus potentially increasing whole-animal N efficiency by increasing the supply of hepatic-synthesized glutamine.

Interactions between animal temperament and exposure to endophytic tall fescue: Effects on cell-mediated and humoral immunity in beef heifers

Two experiments (n=12 Angus heifers/experiment) investigated influences of animal temperament, as indicated by exit velocity (EV; determined at weaning) and consumption of toxic endophyte-infected tall fescue seed on peripheral lymphocyte production of interferon-γ. Heifers were selected from calves born on the University of Kentucky’s C. Oran Little Research Center. In experiment 1, calves were randomly selected from 50 heifers within a single calf crop. In experiment 2, calves with the 6 fastest and 6 slowest EV in the subsequent year’s calf crop were selected. In both experiments, heifers were assigned to either high or low EV treatments based on relative ranking, and endophyte treatments (toxic endophyte-infected, E+, or endophyte-free, E-, fescue seed) were balanced by body weight. Rations were restricted to 1.8 x NEm and common diet was top-dressed with fescue seed each morning. Experiment 1 had four phases (pre-endophyte treatment/thermoneutral, increased room temperature, increased room temperature/endophyte treatment, and post-endophyte/thermoneutral) and experiment 2 had two phases (increased room temperature/endophyte treatment and thermoneutral). During endophyte treatment phases, heifers were fed their respective treatment seed. During all other phases, all heifers received E- seed. In experiment 1, proportions of lymphocytes producing interferon-γ were decreased in E+ heifers during the heat/endophyte phase (P=0.03) whereas during the subsequent thermoneutral period this response was greater in high, compared with low, EV heifers on E- treatment, with no difference observed among E+ heifers (interaction P=0.08). Also during the recovery phase, average lymphocyte production of interferon-γ was higher in E+ heifers (P=0.01). Consistent with experiment 1 findings, during the recovery period of experiment 2, endophyte exposure increased the per cell production of interferon-γ (P&amp;lt;0.01). In this experiment, the difference was of sufficient magnitude to result in a concomitant increase (P=0.03) in total interferon-γ production during that period. These results indicate peripheral lymphocyte production of interferon-γ can be influenced by both EV and endophyte exposure following periods of increased ambient temperature humidity indices, though there was minimal indication of interactions between temperament and alkaloid exposure. This may imply that cattle with high exit velocities and those previously exposed to toxic endophyte-infected tall fescue are better poised against cell-mediated challenges.

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

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

Using supplemental condensed tannin to mitigate tall fescue toxicosis in non-pregnant, non-lactating ewes consuming tall fescue silage

Tall fescue [Schedonorus arundinaceus (Shreb.)], the primary grazing forage in the mid-south, and has a symbiotic relationship with an endophytic fungus (Neotyphodium coenophialum) that produces ergot alkaloids that cause fescue toxicosis in grazing livestock. Some evidence indicates that condensed tannins (CT) can bind to ergot alkaloids and render them ineffective, thereby preventing symptoms of fescue toxicosis. Non-pregnant, non-lactating ewes [n = 20; 57 ± 1.3 kg initial body weight (BW)] were housed in individual pens (1 × 1.5 m) with metal grate flooring. Ewes were offered ad libitum access to either novel endophyte (NE) or toxic endophyte-infected tall fescue silage with no CT (E0), or CT at 10 (E10) or 30 (E30) g/kg of silage dry matter (DM). The CT used was Quebracho tannin (Schinopsis quebracho-colorado). All ewes received 4 g/kg BW liquid molasses, and this served as the carrier for the CT treatments. The study consisted of 2, 21-d periods with a 30-d washout between periods. A 14-d adaption was followed by 7-d of total fecal and urine collection in each period. Blood samples for prolactin analysis were taken on the last day of each period. Data were analyzed using individual ewe within period as the experimental unit, treatment was a fixed effect, and period and pen were random effects. Prolactin was lesser (P < 0.01) in endophyte infected silage treatments. Intake (DMI; g/kg BW) and neutral detergent fiber (aNDF) intake (g/kg BW) were greater (P = 0.05) in NE compared to other treatments. Dry matter digestibility (DMD; g/kg DMI) and aNDF digestibility (g/kg NDF) were greater (P = 0.01) in NE compared to other treatments and organic matter (OM) digestibility [(OMD, g/kg OM intake (OMI)] did not differ between NE and E10, but NE was greater (P = 0.01) than E0 and E30. Digestible DMI and OMI (g/kg BW) were greater (P = 0.01) from NE compared to other treatments. Apparent N absorbed (g/kg N intake) was greater (P = 0.03) in NE, E0, and E10 compared with E30. Fecal N (g/kg N intake) was greater in E30 compared to E0 and E10, but not different from NE. Nitrogen retained (g/kg N intake and N absorbed) did not differ (P ≥ 0.31) among treatments. Condensed tannin supplementation at 30 g/kg DM had negative impacts on DM and OM digestibility and did not prevent decreases in prolactin associated with tall fescue toxicosis.

Pituitary and liver selenoprotein transcriptome profiles of grazing steers and their sensitivity to the form of selenium in vitamin-mineral mixes

Many supplemental Se-dependent metabolic effects are mediated through the function of selenoproteins. The full complement and relative abundance of selenoproteins expressed by highly metabolic cattle tissues have not been characterized in cattle. The complement and number of selenoprotein mRNA transcripts expressed by the pituitary and liver of healthy growing beef steers (n = 7 to 8) was characterized using NanoString methodology (Study 1). Of the 25 known bovine selenoproteins, 24 (all but SELENOH) were expressed by the pituitary and 23 (all but SELENOH and SELENOV) by the liver. Transcript abundance ranged (P ≤ 0.05) over 5 orders of magnitude in the pituitary (&amp;gt; 10,000 for GPX3, &amp;lt; 10 for DIO1 and GPX2) and liver (&amp;gt; 35,000 for SELENOP, &amp;lt; 10 for DIO2). Also unknown is the sensitivity of the selenoprotein transcriptome to the form of supplemental Se. The effect of form of supplemental Se on the relative content of selenoprotein mRNA species in the pituitary and liver of steers grazing a Se-deficient (0.07 ppm Se) tall fescue pasture and consuming 85 g/d of a basal vitamin-mineral mix that contained 35 ppm Se as either ISe (n = 6), organically-bound Se (SELPLEX; OSe, n = 7 to 8), or a 1:1 blend of ISe and OSe (MIX, n = 7 to 8) was determined by RT-PCR after sequence-validating the 25 bovine selenoprotein cDNA products (Study 2). In the pituitary, Se form affected (P &amp;lt; 0.05) the relative content of 9 selenoprotein mRNAs and 2 selenoprotein P receptor mRNAs in a manner consistent with a greater capacity to manage against oxidative damage, maintain cellular redox balance, and have a better control of protein-folding in the pituitaries of OSe and MIX versus ISe steers. In the liver, expression of 5 selenoprotein mRNA was affected (P ≤ 0.05) in a manner consistent with MIX steers having greater redox signaling capacity and capacity to manage oxidative damage than ISe steers. We conclude that inclusion of 3 mg Se/d as OSe or MIX versus ISe, forms of supplemental Se in vitamin-mineral mixes alters the selenoprotein transcriptome in a beneficial manner in both the pituitary and liver of growing steers consuming toxic endophyte-infected tall fescue.

PSXVI-27 Using tannin to mitigate tall fescue toxicosis

Abstract Fescue toxicosis has a negative impact on livestock production. Tannins could bind to tall fescue toxins rendering them ineffective. The objective was to improve health and well-being of sheep offered tall fescue infected with the endophytic fungus (Neotyphodium coenophialum) by binding toxins in the rumen with condensed tannins. Non-pregnant, non-lactating ewes (n=20; 57±1.3 kg initial BW) were housed in individual 1×1.5-m pens with grate flooring and were offered ad libitum access to either non-toxic novel-endophyte infected tall fescue haylage (NE), or toxic endophyte infected tall fescue haylage with no condensed tannin (E0), or condensed tannin at 10 (E10), or 30 g/kg haylage DM (E30). Additionally, ewes received 4 g/kg BW liquid molasses, which served as the carrier for the condensed tannins in the E10 and E30 treatments. The study consisted of 2, 21-d periods with a 30-d wash out between periods. In each period, a 14-d adaption was followed by 7 d of total fecal and urine collection. Temperature was maintained at 27 to 29 °C. Dry matter and OM intake (g/kg BW; DMI and OMI, respectively) were not affected (P ≥ 0.15) by treatment, but DM and OM digestibility were greater (P &amp;lt; 0.05) from NE vs E30. Digestibility of NDF was greater (P &amp;lt; 0.05) from NE vs E0, E10, and E30. Digestible DMI was greater (P &amp;lt; 0.05) from NE and E0 vs E30, and digestible OMI was greater (P &amp;lt; 0.05) from NE vs E30. Apparent N absorbed (g/kg N intake) was greater (P &amp;lt; 0.05) by lambs offered E0 and E10 compared with E30. Nitrogen retained (g/kg N intake and N absorbed) did not differ (P≥0.29). Therefore, overall impacts of ensiled toxic fescue on lambs were inconsistent and the impact of adding condensed tannins at 30 g/kg DM was negative on most of the DM and OM digestibility measurements.

178 Grazing Toxic Endophyte-infected Tall Fescue Alters Monocyte Abundance and White Blood Cell Expression of Prolactin and NF-kB Pathway Genes in Beef Steers

Abstract The hypotheses tested were that (a) summer-long (84 d) grazing of endophyte-infected tall fescue (TE) would alter the count of circulating leukocytes (WBC) and WBC expression of key prolactin and NF-kB pathway genes and (b) that ad libitum intake of an 1:1 blend (MIX) sodium selenite (ISe) and SELPLEX vs ISe in a vitamin-mineral mix (V-M, 27 ppm Se) would ameliorate negative TE effects. Sixteen TE-naïve beef steers depleted of Se were randomly assigned to ad libitum consumption of ISe vs MIX for 35 d. Within Se-form treatments, 2 steers (ISe = 316±37 kg, MIX = 314±39 kg) were randomly assigned to graze each of 4, 2-acre TE paddocks for 84 d and had ad libitum access to their respective V-M. Paddock was the experimental unit (n = 4). The MIXED procedure of SAS was used to assess effect of grazing TE (d 0 vs 84), Se-form (ISe vs MIX), and their interaction on clinical parameters WBC, and relative mRNA content (RT-PCR) in blood. Whole blood Se was 24% greater (P &amp;lt; 0.01) for MIX steers and decreased (P &amp;lt; 0.01) 34% by d 84. Serum prolactin decreased 88% by d 84 and was 98% greater (P &amp;lt; 0.01) for MIX steers on d 84. Monocytes decreased (P &amp;lt; 0.01) 47% with TE and were 67% lower (P = 0.04) in MIX steers. Grazing of TE altered mRNA content of CISH, STA5B, PPIB, AGAP2, and SPRLR; and IL-8, RELA, NFKBIA, TLR, IKBKG, ZAP70, and LCK; of the prolactin (P ≤ 0.05) and NF-kB (P ≤ 0.04) pathways, respectively. In contrast, Se form had no effect (P ≥ 0.07) on mRNA content. Summer-long consumption of TE by steers altered monocyte numbers and expression of prolactin and NF-kB genes, but ad libitum intake of MIX Se forms did not ameliorate these effects.

Determining the Rate of Ergot-alkaloid Degradation in Toxic Endophyte-infected Tall Fescue Hay and Haylage During Long Term Storage

Abstract The objectives of this experiment were to determine changes in the nutritive value and ergot alkaloid concentrations of endophyte-infected tall fescue hay and haylage during a 180-d storage period. Forage from a single field of Kentucky-31 tall fescue was cut for hay in late June and allowed to dry in the field. The dry matter (DM) of the windrow of cut forage was measured every 2 h after clipping. Forage was sampled from the windrow in 6 location blocks once forage DM reached target levels for haylage and hay treatments. Haylage and hay samples were taken when the DM of the windrow reached 50% and 80%, respectively. Seven subsamples of each treatment within block were chopped to 1.91 cm in length with a lettuce chopper and vacuum sealed in oxygen-excluding bags. Sample bags were stored indoors and opened at 30 d intervals over the 180-d storage period. Samples were analyzed for pH, nutritive value, and individual ergot alkaloid concentrations using high-performance liquid chromatography. Within each storage day, treatment within block was considered the experimental unit. Data were analyzed in SAS using the MIXED procedure with fixed effects of treatment, day, and the treatment by day interaction. Location block was considered a random effect. As expected, pH was decreased for haylage compared to hay at all time points (P &amp;lt; 0.01) and DM was greater (P &amp;lt; 0.01) for hay compared to haylage. Neutral detergent fiber values were greater (P &amp;lt; 0.01) for hay compared to haylage and declined during storage (P &amp;lt; 0.01). Total ergot alkaloid concentrations did not differ by treatment (P = 0.61), but ergovaline concentrations declined (P &amp;lt; 0.01) during storage. Collectively, these results indicate minimal differences in nutritive value and ergot alkaloid concentrations between hay and haylage during storage, and that ergovaline concentrations decline during storage.

PSIX-7 Grazing toxic endophyte-infected tall fescue does not influence pancreatic or small intestinal digestive enzyme activities in beef steers

Abstract The objective of this experiment was to determine the influence of toxic endophyte-infected tall fescue exposure on pancreatic and small intestinal digestive enzyme activities in steers. Twelve fescue-naive beef steers [693 ± 42.7 kg of body weight (BW)] were stratified by BW and randomly assigned within BW ranks to graze non-toxic endophyte-infected tall fescue (NTE, n = 6; 0.01 ± 0.009 mg ergovaline + ergovalinine/kg) or toxic endophyte-infected tall fescue (TE, n = 6; 0.50 ± 0.048 mg ergovaline + ergovalinine/kg) pastures for 84 d. After the grazing period, steers were weighed and then slaughtered in three groups for tissue collection. A 1-m segment of the small intestine was collected 5-m distal to the pyloric sphincter. Subsamples of the small intestinal mucosa and pancreas were collected, homogenized in saline, and assayed for protein concentration. Pancreatic and small intestinal digestive enzyme activities were assayed. Data were analyzed using the GLM procedure of SAS as a randomized complete block design for effects of treatment and slaughter group (blocking factor). Steers grazing NTE pastures had a greater (P &amp;lt; 0.001) average daily gain (0.282 vs -0.631 kg/d) and final BW at slaughter (739 vs 620 kg) than steers grazing TE pastures. Small intestinal segment mass and mucosal density (P ≥ 0.16) were not influenced by treatment. Grazing TE pasture did not influence (P ≥ 0.21) pancreatic or small intestinal protein concentrations. Pancreatic α-amylase and trypsin activity per gram pancreas (P ≥ 0.21) or per gram pancreatic protein (P ≥ 0.30) were not influenced by fescue treatment. Small intestinal glucoamylase, isomaltase, and maltase activities did not differ between steers grazing NTE or TE pastures when expressed per gram of intestinal tissue (P ≥ 0.47) or per gram of intestinal protein (P ≥ 0.60). These data indicate that decreased gains observed in cattle consuming TE are not a result of decreased pancreatic or small intestinal digestive enzyme activities.

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

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

Forms of selenium in vitamin-mineral mixes differentially affect serum prolactin concentration and hepatic glutamine synthetase activity of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. an 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially affect metabolic parameters and performance of growing steers grazing toxic endophyte-infected tall fescue mixed forage (E+) pasture. Predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, and MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of an E+ pasture (0.51 ppm total ergovaline per ergovalinine; 10.1 ha). Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The PROC MIXED procedure of SAS was used to assess effect of Se-form treatments on whole blood Se (ng/mL) and serum prolactin (ng/mL) at day 0, 22, 43, 64, and 86, and caudal arterial area (mm2) at day -7, 43, and 86. The effect of Se treatment on ADG (day 86), and liver glutamine synthetase (GS) mRNA, protein, and activity (nmol/mg wet tissue/min) were assessed using the PROC GLM procedure of SAS. Fisher's protected LSD procedure was used to separate treatment means. Whole blood Se increased (P < 0.01) for all treatments from day 0 to 22 and then did not change (P ≥ 0.17), and was greater (P ≤ 0.04) for MIX and OSe steers. Serum prolactin decreased (P < 0.01) over time and was greater (P < 0.05) for MIX and OSe steers. Liver GS mRNA content was 66% and 59% greater (P < 0.05) in MIX and OSe steers, respectively, than ISe steers. Liver GS protein content in MIX steers was 94% more (P < 0.01) than ISe steers. Moreover, MIX and OSe steers had 99% and 55% more (P ≤ 0.01) liver GS activity, respectively, than ISe steers. ADG was not affected (P = 0.36) by Se treatments. We conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in VM mixes increased 1) whole blood Se content, an indicator of greater whole-body Se assimilation; 2) serum prolactin, the reduction of which is a hallmark of fescue toxicosis; and 3) hepatic GS activity, indicating greater hepatic assimilation of acinar ammonia. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.

Case Study: Recovery from ergot alkaloid-induced vasoconstriction for steers conditioned to grazing seedhead-suppressed and unsuppressed pastures of toxic endophyte-infected tall fescue

<h2>ABSTRACT</h2> Chemical seedhead suppression of toxic endophyte–infected (E+) tall fescue (<i>Lolium arundinaceum</i>) can enhance steer performance and mitigate the adverse effects of ergot alkaloids on cattle physiology; however, it is not known if seedhead suppression can mitigate alkaloid-induced vasoconstriction and improve postgraze performance. A 2-yr experiment was conducted with Angus crossbred steers using a pasture phase to precondition steers to grazing seedhead-suppressed E+ tall fescue; unsuppressed E+ fescue; or a bermudagrass (<i>Cynodon dactylon</i>), white clover (<i>Trifolium repens</i>), and Kentucky bluegrass (<i>Poa pratensis</i>) control. A pen phase followed to monitor luminal areas of the caudal artery for assessing alkaloid-induced vasoconstriction and BW to compare the E+ treatments with the nontoxic treatment. During the pen phase, luminal areas of caudal arteries in steers preconditioned on suppressed E+ were comparable (<i>P</i> > 0.10) with those for nontoxic preconditioning on and after 28 and 13 d on the nontoxic diets (DNTD) in the first and second years, respectively. Caudal arteries in steers preconditioned on unsuppressed E+ were constricted compared (<i>P</i> > 0.10) with the nontoxic preconditioned steers over all DNTD and from 0 to 34 DNTD in the first and second years, respectively. Body weights of steers preconditioned on suppressed pastures were similar (<i>P</i> > 0.10) to the nontoxic steers, except for 0 and 8 DNTD in the first year and 6 DNTD in the second year. Body weights for steers on unsuppressed pastures were less (<i>P</i> < 0.10) than those of nontoxic steers over all DNTD in each year. Results indicated that chemical seedhead suppression of E+ fescue can relieve alkaloid-induced vasoconstriction and improve postgraze performance.

Rumen-protected arginine alters blood flow parameters and luteinizing hormone concentration in cyclic beef cows consuming toxic endophyte-infected tall fescue seed.

The objective of this study was to determine the effect of rumen-protected arginine on median caudal artery blood flow and LH dynamics in cows fed toxic endophyte-infected tall fescue seed. Four ruminally cannulated nonlactating beef cows (539 ± 30 kg) were used in a 2 × 2 factorial arrangement of treatments utilizing a 4 × 4 Latin square design with 4 periods of 31 d each. Each cow was assigned to individual pens and fed orchardgrass hay (10.3% CP and 85% NDF; OM basis) during a 10-d adaptation period, followed by a 21-d collection period in which each cow was assigned 1 of 4 treatments: 1) rumen-protected Arg (180 mg/kg of BW) and 1.0 kg/d of toxic endophyte-infected fescue seed (AE+), 2) rumen-protected Arg (180 mg/kg of BW) and 1.0 kg/d of endophyte-free fescue seed (AE-), 3) 1.0 kg/d of toxic endophyte-infected fescue seed (E+) alone, or 4) 1.0 kg/d of endophyte-free fescue seed (E-) alone. In each period, Doppler ultrasound measurements for blood flow parameters were quantified on d 1, 5, 10, 15, and 20. On d 20 of each period, blood samples were collected every 10 min for 6 h and then once every hour for 12 h for LH response following exogenous GnRH. There was an Arg × fescue seed type interaction ( = 0.05) for median caudal artery blood flow due to an increase in blood flow in cows fed rumen-protected Arg with endophyte-free fescue seed. In addition, mean blood flow velocity in the artery was greater ( = 0.01) with the inclusion of rumen-protected Arg in the diet. Median caudal artery area ( = 0.03) and diameter ( = 0.01) were decreased in cows consuming E+ compared to those consuming E- with no effect ( ≥ 0.38) by Arg inclusion. Circulating nitric oxide (NO) concentrations tended to be influenced ( = 0.09) by the interaction of Arg × fescue seed type with E+ alone decreasing NO concentrations. Circulating NO concentrations were unaffected by rumen-protected Arg ( = 0.48). Mean serum LH concentration exhibited ( = 0.02) an Arg × fescue seed type interaction. Cows consuming E+ had decreased ( < 0.05) LH concentrations compared to all other treatments. However, cows consuming AE+ had ( ≥ 0.67) LH concentrations similar to those of cows consuming AE- and E-. Thus, supplementing rumen-protected Arg to cows consuming toxic endophyte-infected fescue seed has the potential to increase reproductive performance and peripheral blood flow.

Vasoactivity and Vasoconstriction Changes in Cattle Related to Time off Toxic Endophyte-Infected Tall Fescue.

Previous research has indicated that serotonergic and α-adrenergic receptors in peripheral vasculature are affected by exposure of cattle grazing toxic endophyte-infected (E+; Epichlöe coenophialia) tall fescue (Lolium arundinaceum). The objective of this experiment was to determine the period of time necessary for the vascular effects of ergot alkaloids to subside. Two experiments were conducted to investigate changes in vascular contractile response and vasoconstriction over time relative to removal from an ergot alkaloid-containing E+ tall fescue pasture. In Experiment 1, lateral saphenous vein biopsies were conducted on 21 predominantly Angus steers (357 ± 3 kg body weight) at 0 (n = 6), 7 (n = 6), 14 (n = 5), or 28 days (n = 4) after removal from grazing pasture (3.0 ha; endpoint ergovaline + ergovalinine = 1.35 mg/kg DM) for 126 days. In Experiment 2, lateral saphenous veins were biopsied from 24 Angus-cross steers (361 ± 4 kg body weight) at 0, 21, 42, and 63 days (n = 6 per time point) following removal from grazing tall fescue pastures (3.0 ha; first 88 days endpoint ergovaline + ergovalinine = 0.15 mg/kg DM; last 18 days endpoint ergovaline + ergovalinine = 0.57 mg/kg DM) for 106 total days. Six steers (370 ± 18 kg body weight) off of bermudagrass pasture for the same time interval were also biopsied on Day 0 and Day 63 (n = 3 per time point). Additionally, in Experiment 2, cross-sectional ultrasound scans of caudal artery at the fourth coccygeal vertebra were taken on Days 0, 8, 15, 21, 29, 36, 42, and 45 to determine mean artery luminal area to evaluate vasoconstriction. In both experiments, steers were removed from pasture and housed in a dry lot and fed a corn silage diet for the duration of biopsies and ultrasound scans. Biopsied vessels used to evaluate vasoactivity were cleaned, incubated in a multimyograph, and exposed to increasing concentrations of 4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide (TCB2; 5HT2A agonist), guanfacine (GF; α2A-adrenergic agonist), and (R)-(+)-m-nitrobiphenyline oxalate (NBP; α2C-adrenergic agonist) in both experiments and ergovaline (ERV) and ergotamine (ERT) in Experiments 1 and 2, respectively. In Experiment 1, days off pasture × agonist concentration was not significant (p > 0.1) for all four compounds tested. In Experiment 2, GF, NBP, TCB2 and ERT were significant for days off pasture × agonist concentration interaction (p < 0.02) and vasoactivity increased over time. Vasoactivity to agonists was reduced (p < 0.05) when steers were initially removed from E+ tall fescue pasture compared to bermudagrass, but did not differ by Day 63 for any variable. Luminal areas of caudal arteries in steers grazed on E+ tall fescue relaxed and were similar to steers that had grazed bermudagrass for 36 days on non-toxic diet (p = 0.15). These data demonstrate changes in peripheral vasoactivity and recovery from vasoconstriction occur beyond five weeks off toxic pasture and 5HT2A receptors appear to be more dramatically affected in the lateral saphenous vein by grazing E+ tall fescue pasture than adrenergic receptors.

Gene expression profiling indicates an increased capacity for proline, serine, and ATP synthesis and mitochondrial mass by the liver of steers grazing high vs. low endophyte-infected tall fescue.

Grazing -infected forages results in a variety of reduced animal performance parameters, collectively known as "fescue toxicosis." The initial, limited evaluations of hepatic mechanisms affected by fescue toxicosis have used transcriptomic expression profiling of experimental phenotypes developed by short-term feeding of concentrated ergot alkaloids or fescue seeds to rodents and steers. To assess the effects of fescue toxicosis in growing cattle using a commercially relevant phenotype, we induced fescue toxicosis in beef steers by summer-long grazing (89 to 105 d) of a single high toxic endophyte-infected tall fescue pasture (HE; 0.746 μg/g ergot alkaloids; 5.7 ha; = 10; BW = 267 ± 14.5 kg) vs. a low toxic endophyte tall fescue-mixed pasture (LE; 0.023 μg/g ergot alkaloids; 5.7 ha; = 9; BW = 266 ± 10.9 kg). High toxic endophyte tall fescue-mixed pasture steers had decreased BW (313 vs. 338 kg) and an increased potential for hepatic gluconeogenesis from AA-derived carbons. To gain a greater perspective into fescue toxicosis-induced hepatic metabolism and identify candidate regulatory mechanisms, the goal of the current research was to examine liver samples for changes in gene (mRNA) expression profiles using a Bovine Affymetrix microarray and selected reverse-transcription PCR and immunoblot analyses. The expression (false discovery rate < 10%; < 0.01) of 147 genes was increased (7 to 268%) and that of 227 was decreased (4 to 87%) in livers of HE vs. LE steers. The top (1) functional gene category was cell-mediated immune response (33 genes; ≤ 0.012), (2) canonical cell signaling pathway was primary immunodeficiency signaling (8 genes; ≤ 0.0003), and (3) canonical metabolic pathways were oxidative phosphorylation (5 genes; ≤ 0.016) and purine metabolism (8 genes; ≤ 0.029). High toxic endophyte tall fescue-mixed pasture steers had increased ( ≤ 0.022) expression of genes critical for increased (1) Pro () and Ser () synthesis, (2) shunting of AA carbons into pyruvate () and ATP synthesis (, , , COX4, , and ), and (3) mitochondrial mass (COX4). Targeted reverse-transcribed PCR or immunoblot assays corroborated ( ≤ 0.035) these latter microarray findings for , , , , and COX4. Moreover, network analysis identified glucocorticoid receptor-mediated signaling as the most probable mechanism to coordinate the above findings. These results greatly extend our knowledge of the consequences of summer-long grazing of endophyte-infected tall fescue to the hepatic metabolism of growing steers.

The Effects of Steroid Implant and Dietary Soybean Hulls on Estrogenic Activity of Sera of Steers Grazing Toxic Endophyte-Infected Tall Fescue Pasture.

Soybean hulls (SBHs) have been fed to cattle pasturing on endophyte-infected tall fescue in attempts to increase rate of gain. Literature reports indicated some symptoms associated with fescue toxicosis were ameliorated by the use of steroidal implants containing estradiol (E2) and progesterone [implantation (IMP)], feeding SBHs, or the combination of the two. While the mechanism for amelioration was unclear, the SBHs were postulated as acting as a diluent of the toxic factors of the fescue. Alternatively, estradiol and phytoestrogens of SBHs might be acting through relaxation of the persistent vasoconstriction found in animals ingesting ergot alkaloids of endophyte-infected fescue. If so, estrogenic activity of serum of steers receiving SBHs, IMP, or a combination of the two should be elevated. Using the cellular proliferation assay of estrogenicity (E-Screen), estradiol equivalents (E2Eqs) were determined on both SBHs and the serum of steers from a previously reported study. Range of SBHs was 5.0–8.5 ng Eqs g−1 DM (mean 6.5, n = 4 from different commercial sources of SBHs). At the rate fed, theoretically calculated blood E2Eq could be physiologically relevant (~80 pg mL−1, based on 2.3 kg SBHs d−1, 300 kg steer, 5.7% blood volume, and 10% absorption). Serum E2Eqs did increase in steers (P ≤ 0.05) with steroidal implants or fed SBHs by 56 and 151% over control, respectively, and treatments were additive (211% increase). Serum prolactin was also greatest for the SBH + IMP group (188 ng mL−1, P < 0.05), concentrations comparable to values reported for steers grazing endophyte-free fescue. Prolactin in the SBH group was higher than IMP or control groups (146 versus 76 and 60 ng mL−1, respectively). Still unknown is if additional E2Eqs from dietary phytoestrogens or exogenous sources of estradiol can further reduce symptoms of fescue toxicosis. The E-Screen assay was an effective tool in monitoring serum for estrogenic effects of dietary supplementation with SBHs or estrogenic implants.

Fungal endophyte symbiosis alters nitrogen source of tall fescue host, but not nitrogen fixation in co-occurring red clover

Infection of tall fescue with the common toxic fungal endophyte Epichloe coenophiala harms livestock via toxic alkaloid production; therefore, non-toxic ‘novel’ strains of the endophyte have been developed and released. How different endophyte strains impact biological nitrogen fixation (BNF) in mixed species pastures is unknown. We asked whether novel endophyte or common toxic endophyte-infected (NE+; CTE+) tall fescue affects symbiotic and non-symbiotic BNF, and utilization of biologically-fixed nitrogen in tall fescue. Tall fescue was planted either endophyte-free (E-), infected with CTE, two non-toxic strains AR542 NE, AR584 NE, or a blend of endophyte treatments. We measured natural abundance of 15 N in plant and soil samples, and conducted soil acetylene reduction assays. Endophyte presence and strain significantly affected the δ15N of tall fescue. Near red clover, CTE+ and AR584 NE+ tall fescue were most 15 N-depleted; but away, E- tall fescue was most 15 N-depleted. Endophyte strain significantly influenced N concentration in red clover, but not symbiotic or non-symbiotic BNF. Endophyte strains produce different effects on tall fescue’s competitive ability and nitrogen utilization. In mixed pastures, deployment of NE strains for decreased alkaloid toxicity will differentially impact use of biologically fixed nitrogen in tall fescue and nitrogen concentration in red clover.